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CONTRIBUTORS

John A. Cooper Department of Cell Biology, Washington University, St. Louis, MO, 63110 Rodney J. Devenish Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Microbial Structural and Functional Genomics, Monash University, Clayton Campus, Victoria, 3800, Australia Birthe Fahrenkrog M.E. Mu¨ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Sheng T. Hou Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada Susan X. Jiang Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada Irina M. Konstantinova Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia Roderick Y. H. Lim M.E. Mu¨ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Alexey G. Mittenberg Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia Mark Prescott Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Microbial Structural and Functional Genomics, Monash University, Clayton Campus, Victoria, 3800, Australia Mary Ann Rempel Department of Environmental Sciences, University of California, Riverside, CA 92521

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Contributors

Francisco Rivero The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, United Kingdom Andrew J. W. Rodgers Industrial Biotechnology Group, CSIRO Division of Molecular and Health Technologies, Clayton, Victoria, 3168, Australia Daniel Schlenk Department of Environmental Sciences, University of California, Riverside, CA 92521 David Sept Department of Biomedical Engineering and Center for Computational Biology, Washington University, St. Louis, MO, 63130 Robert A. Smith Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, G12 8QQ, Scotland Anna S. Tsimokha Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia Katharine S. Ullman Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112 Mirjam Zegers Department of Surgery, University of Chicago, Chicago, IL 60637

C H A P T E R

O N E

The Structure and Function of Mitochondrial F1F0-ATP Synthases Rodney J. Devenish,* Mark Prescott,* and Andrew J. W. Rodgers† Contents 1. Introduction 2. Mitochondrial ATP Synthase 2.1. Overview of the structure and subunit composition 2.2. Rotary catalysis 2.3. The F1 sector 2.4. The peripheral/‘‘Stator’’ stalk 2.5. The F0 sector 3. Supramolecular ATP Synthase 3.1. Introduction 3.2. Dimers and oligomers 3.3. Subunits relevant to dimer formation 3.4. The arrangement of mtATPase in mitochondrial membranes 3.5. The role of mtATPase oligomerisation 3.6. Is oligomerization regulated in vivo? 3.7. Supramolecular structures involving other respiratory complexes? 4. Extra-Mitochondrial Expression of F1F0-ATP Synthase 4.1. Introduction 4.2. How might F1F0 ATP synthase get to the plasma membrane? 4.3. The function of coupling factor 6 as a vasoconstrictor: Detachment and reattachment of an F0 component of eAS? 4.4. Multiple receptor functions of subunit b 4.5. The inhibitor action of IF1 can be demonstrated for eAS complexes 5. Concluding Remarks References

* {

2 3 3 5 7 12 18 23 23 23 25 29 31 34 35 36 36 37 39 40 42 42 43

Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Microbial Structural and Functional Genomics, Monash University, Clayton Campus, Victoria, 3800, Australia Industrial Biotechnology Group, CSIRO Division of Molecular and Health Technologies, Clayton, Victoria, 3168, Australia

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00601-1

#

2008 Elsevier Inc. All rights reserved.

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Rodney J. Devenish et al.

Abstract We review recent advances in understanding of the structure of the F1F0-ATP synthase of the mitochondrial inner membrane (mtATPase). A significant achievement has been the determination of the structure of the principal peripheral or stator stalk components bringing us closer to achieving the Holy Grail of a complete 3D structure for the complex. A major focus of the field in recent years has been to understand the physiological significance of dimers or other oligomer forms of mtATPase recoverable from membranes and their relationship to the structure of the cristae of the inner mitochondrial membrane. In addition, the association of mtATPase with other membrane proteins has been described and suggests that further levels of functional organization need to be considered. Many reports in recent years have concerned the location and function of ATP synthase complexes or its component subunits on the external surface of the plasma membrane. We consider whether the evidence supports complete complexes being located on the cell surface, the biogenesis of such complexes, and aspects of function especially related to the structure of mtATPase. Key Words: Cristae, Dimers, External ATP synthase (eAS), Mitochondrial ATP synthase (mtATPase), Mitochondrial inner membrane, Peripheral or ‘‘stator’’ stalk. ß 2008 Elsevier Inc.

1. Introduction F1F0-ATP synthases are enzyme complexes found in eubacterial plasma membranes, chloroplast thylakoid membranes and the inner membranes of mitochondria. Their function is to harness energy from a gradient of protons (or sodium ions, in some bacteria) across the membrane to synthesise ATP from ADP and Pi. This chapter will review recent advances in the structure and function of mitochondrial ATP synthase (mtATPase). We focus on three areas: (i) the emerging understanding of the structure of the peripheral, or stator, stalk and the subunits comprising it; (ii) the relationship between oligomeric ATP synthase complexes and mitochondrial cristae; and (iii) the extra-mitochondrial location and function of enzyme complexes apparently equivalent to ATP synthase. We draw principally on studies in yeast and mammalian cells (bovine and rat), but refer to relevant studies of the bacterial enzyme particularly in relation to structure and function of the complex. The assembly of mtATPase is not considered here as it has been a major focus of a recent review (Ackerman and Tzagoloff, 2005).

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Structure and Function of mtATPases

2. Mitochondrial ATP Synthase 2.1. Overview of the structure and subunit composition F1F0-ATP synthases are traditionally viewed as consisting of a soluble portion (the F1 sector), where the sites catalysing the formation and hydrolysis of ATP are located, and a membrane-bound portion (the F0 sector), which functions as a proton channel (Fig. 1.1). Table 1.1 shows the subunit a

OSCP

b

b

d

F6 g e d

C10

b

a +e,f,g, A6L

Figure 1.1 The subunit organization in mtATPase. Subunits are labelled. F1 is the globular domain made of subunits a, b and the three central stalk subunits, g, d and e.The F0 domain is comprised of the subunit c ring (10 copies in yeast), subunit a, and the peripheral stalk subunits b, d, F6(h) and OSCP. The so-called minor subunits [e, f, g, and A6L(8)] are not shown individually, but they all span the membrane and are probably present in a 1:1:1:1 stoichiometry.The rotor is made up of the central stalk and the c-ring. The remainder of the subunits make up the stator. F1 is shown with one a subunit removed for clarity.The inhibitor protein (IF1) is also not shown; it binds in a catalytic a/b interface near the bottom of (ab)3. [This article was published in Biochimica et Biophysica Acta,Vol. 757,Walker, J. E. and Dickson,V. K.,The peripheral stalk of the mitochondrial ATP synthase, 286^296, Copyright Elsevier (2006).]

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Table 1.1 Subunit composition, genetic specification and stoichiometry of mtATPase from yeast and mammalian cells Mitochondria

Bacteria (E. coli) Subunit

Genea

Subunitb

ATP1 ATP2 ATP3

a

a b g (see OSCP) d e Su6

a b g (see OSCP) d e Su6m

1 1 1

b c

B Su9

b Su9

1 10

A6Lm

1

OSCP d e f g F6

1 1 1 (2d ) 1 1 1 1 ? 1 1

Sector Subunit

F1

a b g d e

F0

Mammalian Stoichiometryc

Yeast

Su8 OSCP D E F G H i/j K INH STF1 (9 kDa) STF2 (15 kDa) STF3

ATP16 ATP15 ATP6 m (oli2) ATP4 ATP9m (olil) ATP8m (aap1) ATP5 ATP7 ATP21 ATP17 ATP20 ATP14 ATP18 ATP19 INH1 STF1

IF1

3 3 1

STF2

1

STF3

?

Subunits are aligned horizontally based on sequence or functional homology. The bacterial (E. coli) subunits are shown for comparison with mtATPase subunits. a Genes are in nuclear DNA of Saccharomyces cerevisiae, except those marked with m, which are in mitochondrial DNA. References for individual subunits are as follows: ATP1, Takeda et al., 1986; ATP2, Takeda et al., 1985; ATP3, Paul et al., 1994; ATP16, Giraud and Velours, 1994; ATP15, Guelin et al., 1993; ATP6 (oli2), Macino and Tzaqgoloff, 1980; ATP4, Velours et al., 1988; ATP9 (oli1), Hensgens et al., 1979; Macino and Tagoloff, 1979; ATP8 (aap1), Macreadie et al., 1983; ATP5, Uh et al., 1990; ATP7, Norais et al., 1991; ATP21, Arnold et al., 1997; ATP17, Spannagel et al., 1997; ATP20, Boyle et al., 1999; ATP14, Arselin et al., 1996; ATP18, Arnold et al., 1999 and Vaillier et al., 1999; ATP19, Arnold et al., 1998; INH1, Ichikawa et al., 1990; STF1, Akashi et al., 1988; STF2, Yoshida et al., 1990; STF3, Hong and Pedersen, 2002.

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composition of mtATPase. In the mitochondrial enzyme, F1 is composed of three copies of each of subunits a and b, and one each of subunits g, d and e. The inhibitor protein (IF1) is not traditionally considered as an F1 subunit, but we will consider it here in this context because the available evidence suggests that when bound to mtATPase it associates principally with F1 subunits. F1 subunits g, d and e constitute the ‘‘central’’ stalk of mtATPase. F0 consists of a subunit c ring (comprising 10 copies in the case of the yeast enzyme, but varying to up to 14 copies in other organisms) and one copy each of subunits a, b, d, h (F6) and OSCP. Subunits b, d, F6 (h) and OSCP form the peripheral stalk which lies to one side of the complex. A number of additional subunits (e, f, g, i/j, k and A6L) are associated with F0, although their precise locations within the complex remain uncertain (see discussion below). The F0 and F1 sectors of the enzyme, and the two stalks connecting them, are clearly visible in electron cryomicroscopy images of mtATPase (Rubinstein et al., 2003).

2.2. Rotary catalysis Synthesis of ATP by F1F0-ATPase is achieved by coupling the activities of two rotary motors; one in F0, for which a rotational mechanism was first proposed by Cox et al. (1984), and the other in F1 (Boyer and Kohlbrenner, 1981). The presence of a proton motive force drives protons through a channel in F0 at the interface between subunit a and the subunit c ring. In the case of the mitochondrial enzyme, protons pass from the intermembrane space into the matrix. This releases energy which causes rotation of the ring (relative to subunit a), along with subunits g, d, and e, to which it is attached. In turn, rotation of subunit g within the F1 a3b3 hexamer provides energy for ATP synthesis at the catalytic sites (located in each of the three b subunits, at the interface with an adjacent a subunit). The rotary mechanism of F1-ATPase was proved in remarkable singlemolecule experiments carried out by Noji et al. (1997). The a3b3 hexamer of bacterial F1 was immobilised on a flat surface, and ATP-dependent rotation of a fluorescently labelled actin filament attached to the g subunit was directly observed under the fluorescence microscope. A similar technique was used to show that the hydrolysis of one molecule of ATP causes a

b a, b, g, d, e, Walker et al., 1985; Su6, A6L, Fearnley and Walker, 1986; Su9, Sebald and Hoppe, 1981; b, d, Walker et al., 1987b; OSCP, F6, INH, Walker et al., 1987a; e, Walker et al., 1991; f,g, Collinson et al., 1994a. c Compilation of data for both yeast and bovine systems presented in Arnold et al., 1998; Fronzes et al., 2003; Gregory and Hess, 1981; Hekman et al., 1991; Muraguchi et al., 1990; Okada et al., 1986; Paumard et al., 2000; Stephens et al., 2003; Stutterheim et al., 1981; Todd et al., 1980; and Walker et al., 1985. A question mark indicates subunits for which no reliable stoichiometric data are available. d Arakaki et al., 2001.

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rotation of 120
(Yasuda et al., 2001), as would be expected from the existence of three catalytic sites. Binding of ATP causes a rapid (<0.1 ms) 80
rotation, which is followed by hydrolysis (during the 2 ms ‘‘interim dwell’’), and another rapid 40
movement (Shimabukuro et al., 2003). Recent experiments have revealed the sequence of catalytic and rotational steps in even more detail (Adachi et al., 2007). The interim dwell is divided into a 1 ms ‘‘catalytic’’ dwell, in which cleavage of ATP occurs, and a second phase, the length of which is dependent on Pi concentration. The second of the 1 ms dwells at the 80
point marks Pi release, and energy from the Pi release drives the last 40
substep (Adachi et al., 2007). Single-molecule experiments have also shown that rotational catalysis occurs in F1F0-ATP synthase (Sambongi et al., 1999), and that ATP synthesis by F1 can be driven by externally applied rotational force (Itoh et al., 2004; Rondelez et al., 2005). A framework for understanding rotary catalysis in F1 is provided by the ‘‘binding-change’’ mechanism, first proposed by Boyer (1975). According to this mechanism, the three catalytic sites undergo cyclical interconversion between three states with markedly different nucleotide-binding affinities. When the enzyme is operating in the direction of ATP synthesis, each site switches cooperatively through conformations in which ADP and phosphate bind, ATP is formed, and then released. It is now accepted that these transitions are brought about by rotation of the g subunit. The catalytic mechanism of the enzyme when operating in the opposite direction (ATP hydrolysis) appears likely to use essentially the same pathway, but in reverse (Adachi et al., 2007). The most detailed picture yet of the binding and catalytic events taking place in each of the b subunits over the full 360
rotation of the g subunit has emerged from recent experiments by Ariga et al. (2007). These authors studied ATP hydrolysis and rotation by a hybrid F1 containing one or two mutant b subunits with altered catalytic kinetics, and showed that all three b subunits participate in driving each 120
rotation of the g subunit, with a 120
phase difference. In any particular b subunit, ATP bound when g is positioned at 0
is cleaved when g has rotated 200
(Ariga et al., 2007). For proton-driven ATP synthesis by F1F0-ATPase to occur, the a3b3 hexamer must remain fixed relative to subunit a during catalysis; this occurs by virtue of a physical bridge (the peripheral stalk) formed by subunits b, d, F6 (h) and OSCP. From a mechanistic point of view, the enzyme can therefore be divided into ‘‘rotor’’ (c10-14, g, d, e) and ‘‘stator’’ [a3b3, a, b, d, h (F6), OSCP] components (Fig. 1.1). The binding-change mechanism as presented by Boyer (1993) predicted that during steady-state catalysis, only two of the three catalytic sites would be occupied (on time average) with nucleotide (‘‘bi-site’’ catalysis). This reaction scheme appears unlikely, however, as it would require (when the reaction proceeds in the direction of ATP hydrolysis) that ATP bind to the lowest affinity site in preference to the medium affinity site (Weber and

Structure and Function of mtATPases

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Senior, 2000). In addition, Weber and Senior (1997) have used a sensitive assay based on quenching of fluorescence from tryptophan residues introduced into the active sites to show that in bacterial (E. coli) F1, the Kd for binding of MgATP to the lowest-affinity site corresponds to Km (MgATP), and that all three catalytic sites must be filled to obtain Vmax (and physiologically relevant) rates of ATPase activity (‘‘tri-site’’ catalysis). Similar techniques have been employed to show that this is also the case with F1 from the thermophilic bacterium Bacillus PS3 (Ren et al., 2006) and yeast mitochondrial F1 (Corvest et al., 2005). Experiments carried out with mitochondrial F1 using indirect methods of measuring nucleotide binding appear to favor ‘‘bi-site’’ catalysis (Milgrom and Cross, 2005). However, the enzyme preparation used in these studies hydrolyzed ATP at a rate which was about 50% of that reported in other work ( Jault and Allison, 1993; Milgrom et al., 1998), suggesting that it had defective catalytic properties (Ren et al., 2006). To accommodate the wealth of data supporting ‘‘tri-site’’ catalysis, alternative reaction schemes for ATP synthesis and hydrolysis have been proposed in which three catalytic sites are filled, on time average, over the course of the reaction cycle (Weber and Senior, 2003). A recent structure of yeast F1 (Kabaleeswaran et al., 2006) showed two molecules of AMP-PNP and a phosphate bound to the three catalytic sites. This structure appears to support ‘‘tri-site’’ catalysis as this arrangement is predicted to be an intermediate state during the tri-site (Weber and Senior, 2003), but not bi-site (Milgrom and Cross, 2005) reaction schemes. Singlemolecule experiments in which both rotation and nucleotide binding/ release were monitored simultaneously in real time (Adachi et al., 2007) have provided the most detailed picture yet of the relationship between the rotational angle of the g subunit and nucleotide occupancy of the catalytic sites, and strongly support a ‘‘tri-site’’ mechanism similar to that proposed by Weber and Senior (2003).

2.3. The F1 sector 2.3.1. Structure and subunit composition The landmark crystal structure of bovine mitochondrial F1 (Abrahams et al., ˚ , provided many valuable insights into 1994), solved at a resolution of 2.4 A the function of the enzyme. The protein was crystallized in the presence of Mg2þ, ADP, azide and an ATP analogue, AMP-PNP. The three a subunits and three b subunits were shown to pack alternately, like segments of an orange, around a cental spindle formed by the N- and C-terminal helices of subunit g. The presence of six nucleotide-binding sites was confirmed; three catalytic sites, located in the three b subunits at the interface with an adjacent a subunit, and three noncatalytic sites in the three a subunits. Each of the a and b subunits features an N-terminal b-barrel, a central mixed a/b domain (where the nucleotide-binding sites are located) and a

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Rodney J. Devenish et al.

C-terminal helical bundle. The structures of the three a subunits were very similar, with AMP-PNP bound to each of the three noncatalytic nucleotide binding sites. By contrast, an important feature of the structure was the asymmetry of the three b subunits. They were reported to contain ADP (bDP), AMP-PNP (bTP), or no nucleotide (bE) (Abrahams et al., 1994). The structure of bDP and bTP were very similar to each other with the bound nucleotide being the only major difference. However, the structure of bE was significantly different, being referred to as the open conformation. Here a large displacement of the C-terminal domain was observed (some portions ˚ ), that caused a disruption in a small b-sheet of the displaced by 20 A nucleotide binding domain. The existence of three conformations provided clear support for binding-change models of ATP synthesis/hydrolysis, which predict that each b subunit cycles through conformations with markedly different nucleotide-binding affinities (Boyer, 1993; Weber and Senior, 2003). In addition, both the tip of the C-terminal helix of subunit g, and the sleeve around it formed by regions of subunits a and b immediately below the b-barrel domains, are predominantly composed of hydrophobic residues. This assembly was suggested to act as a molecular bearing, facilitating rotation of subunit g within the F1 a3b3 hexamer during catalysis (Abrahams et al., 1994). Several other high-resolution structures of F1 have been solved since 1994 with an array of different inhibitors bound, and with various states of nucleotide occupancy (Abrahams et al., 1996; Bianchet et al., 1998; Bowler et al., 2006, 2007; Braig et al., 2000; Chen et al., 2006; Gibbons et al., 2000; Kabaleeswaran et al., 2006; Menz et al., 2001; Orriss et al., 1998; van Raaij et al., 1996). However, a high-resolution structure with all three catalytic sites filled with either MgATP or MgAMP-PNP has yet to be solved, despite their presence in crystallization buffers at concentrations greater than their respective Kd values for the lowest affinity site. This would indicate that, in these cases at least, the process of crystal formation tends to dissuade nucleotide occupancy of the bE site, in a manner not yet understood. The sole example of a structure in which all three sites are filled with nucleotide (Menz et al., 2001) is of F1 crystallized in the presence of ADP and fluoroaluminate. The presence of the very tight-binding inhibitor ADP-fluoroaluminate in two of the catalytic sites allows binding of ADP and a sulfate ion (probably mimicking phosphate) in the third site (bE). This structure is proposed to represent a post-hydrolysis, pre-product release step on the catalytic pathway (Menz et al., 2001). In a more recent structure of yeast F1 (Kabaleeswaran et al., 2006), a phosphate ion is bound to the bE site. Comparison of the position of this phosphate ion with the position of the g-phosphate of AMP-PNP in previous structures provides a description of the path followed by the phosphate as it binds to the enzyme, is moved into position ready for reaction with MgADP, and subsequent catalysis. Two recent structures of bovine F1 (Bowler et al., 2006, 2007)

Structure and Function of mtATPases

9

reveal the binding site for azide and explain how it inhibits ATPase activity. In the structure from crystals grown in the presence of ADP, AMP-PNP and azide (Bowler et al., 2006), the azide anion occupies a position in the ADP-binding catalytic subunit, bDP, very similar to the site occupied by the g-phosphate in the AMP-PNP (ATP) binding subunit, bTP. The structure from crystals grown in the absence of azide (Bowler et al., 2007) has AMPPNP bound in both bDP and bTP. Azide therefore appears to inhibit the enzyme by sterically blocking the pocket where the g-phosphate of ATP would bind. The presence of azide in bDP also tightens the binding of several amino acid side chains to the ADP, enhancing its affinity and thereby stabilizing the ‘‘ADP-inhibited’’ state of the enzyme (Bowler et al., 2006). 2.3.2. The ‘‘Central’’ stalk Although the F1 structures published prior to 2000 provided a nearly complete picture of the architecture of the a3b3 hexamer, and of the Nand C-terminal a-helices of the g subunit, crystal disorder in the central stalk proteins of F1 (g, d and e) resulted in a lack of detailed structural information on this portion of the enzyme. NMR (Wilkens et al., 1995; Wilkens and Capaldi, 1998a) and crystal (Uhlin et al., 1997) structures of the isolated bacterial e subunit (equivalent to the d subunit in mtATPase) showed that this protein consists of a 10-stranded b-sandwich at the N-terminus, followed by a pair of antiparallel coiled-coil a-helices which fold back to make contact with the b-sandwich. The crystal structure of bovine F1 bound with the inhibitor dicyclohexylcarbodiimide (DCCD) (Gibbons et al., 2000) revealed the arrangement of central stalk proteins in the mitochondrial enzyme for the first time. The long C-terminal helix of subunit g extends to the bottom of F1. A short loop connects it to an a/b domain featuring a Rossman fold, and this domain is connected to the N-terminal a-helix. The d subunit folds in a way very similar to that seen in the isolated bacterial subunit e, with a b-sandwich at the N-terminus and a helical hairpin at the C-terminus. The protein is packed against a face formed by both the N- and C-terminal helices of subunit g. The third component of the central stalk in mitochondrial F1, subunit e, has no equivalent in bacterial and chloroplast ATP (Table 1.1). This protein is wedged into a cleft between the two domains of the d subunit, thereby forming a connection between them. The crystal structure of the central stalk protein complex from bacterial (E. coli) F1 (comprising the central domain of subunit g plus subunit e) (Rodgers and Wilce, 2000) highlighted differences in subunit organization and in the mechanism of regulation between the bacterial and mitochondrial complexes. Although the folds of subunit g and the N-terminal b-sandwich domain of bacterial subunit e are very similar to their mitochondrial equivalents, the arrangement of the C-terminal helices of bacterial subunit e is markedly different. In the bacterial structure, the helices are separated from one another and wrap around subunit g. Cross-linking studies carried

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Rodney J. Devenish et al.

out on the bacterial complex show that the subunit e-helices can adopt both this conformation and the contracted arrangement seen in the mitochondrial structure (Tsunoda et al., 2001), as well as an even more extended conformation in which the e-helices point directly up and deeply penetrate the F1 a3b3 hexamer (Suzuki et al., 2003). Recent chemical labelling experiments (Ganti and Vik, 2007) confirm that the C-terminal helices of bacterial subunit e have a high degree of flexibility in the intact enzyme. Nucleotide occupancy of the catalytic sites and the presence of a proton motive force across the membrane influence movement of the helices between these conformations (Feniouk et al., 2007; Suzuki et al., 2003; Tsunoda et al., 2001). Importantly, ATP synthesis and hydrolysis activities are affected differently by the conformation of the subunit e-helices (Tsunoda et al., 2001). When they lie close to F0, the enzyme is active in both ATP hydrolysis and synthesis; when they point toward F1, ATP hydrolysis is inhibited, yet the enzyme is fully functional in ATP synthesis. The bacterial e subunit therefore acts via a ‘‘ratchet’’ mechanism to switch the enzyme to different gears depending on whether rotation of the subunit ge complex is in the direction of synthesis or hydrolysis (Tsunoda et al., 2001). This mechanism, which involves interaction between the C-terminal helix of subunit e and the ‘‘DELSEED’’ region of subunit b (Hara et al., 2001) is believed to be important for survival in bacteria, by preventing depletion of intracellular ATP under starving conditions (Feniouk et al., 2007; Suzuki et al., 2003; Tsunoda et al., 2001). Conformational changes in subunit e have also been implicated in regulation of chloroplast ATP synthase ( Johnson and McCarty, 2002) and may involve a ‘‘ratchet’’ mechanism similar to that seen in bacteria (Richter et al., 2005). 2.3.3. Inhibitor protein (IF1) In contrast to the bacterial enzyme, mtATPase is regulated to carry out ATP synthesis exclusively. Wasteful hydrolysis of ATP is not desirable, and must be prevented when oxygen supply to the electron transport chain is limited (such as in the case of ischaemia). The involvement of a ‘‘ratchet’’ mechanism similar to that seen in the bacterial enzyme is very unlikely, given that the C-terminal helices of the mitochondrial subunit d are packed securely at the foot of the central stalk (Gibbons et al., 2000) in such a way that would tightly constrict their movement. Rather, this regulation is carried out by the inhibitor protein IF1, which potently inhibits mitochondrial F1-ATPase activity (Pullman and Monroy, 1963) in a pH-dependent manner (Van Heeke et al., 1993). IF1 can exist in at least two conformations (active and inactive), with the active form predominating at pH values <6.5 (Cabezon et al., 2000b). In its active form, bovine IF1 exists as a dimer, stabilized by formation of an antiparallel coiled-coil by a-helices in the C-terminal portion of the

Structure and Function of mtATPases

11

monomers (Cabezon et al., 2001). In the presence of ATP, the N-terminal portion of each of the monomers is able to bind to F1 (Milgrom, 1991), causing formation of F1 dimers in solution (Cabezon et al., 2000a), and stabilization of mtATPase dimers which occur in the mitochondrial inner membrane (Garcia et al., 2006). IF1 probably contributes to dimerization by forming part of a protein cross-bridge at the F1-F1 interface of the dimeric structure, observed for the first time in transmission EM images of the bovine heart mtATPase (Minauro-Sanmiguel et al., 2005). At higher pH values, a-helices in the N-terminal portion of IF1 associate with one another, causing formation of tetramers (Cabezon et al., 2001); the masking of the inhibitory N-terminal portions prevents binding to F1 and thus renders IF1 inactive as an inhibitor. In the event of a collapse in the proton gradient across the inner membrane, the mitochondrial matrix becomes more acidic, favoring formation of the active form of IF1. The inhibitor protein therefore appears to play two crucial roles in the cell. Firstly, it acts as a pH-dependent sensor that prevents mtATPase from wastefully hydrolysing ATP in the mitochondrial matrix under anoxic conditions. Secondly, IF1 stabilizes the dimer structure, which plays a part in determining the morphology of mitochondrial cristae of the inner membrane (see Section 3.3.2 for detailed discussion). Cross-linking experiments showed that IF1 makes contact with both the F1 a3b3 hexamer and the rotor subunits of the central stalk (MinauroSanmiguel et al., 2002). These results were confirmed by crystal structures of dimeric IF1 associated with two F1-ATPase moieties [(F1-IF1)2] (Cabezon et al., 2003) and of F1 bound to a truncated (residues 1-60), monomeric form of IF1 (Gledhill et al., 2007). These structures showed that IF1 binds mainly at the interface between subunits aDP and bDP (using the notation of Abrahams et al., 1994), and also makes contact with subunit g. Bound IF1 therefore appears to inhibit enzyme activity by interfering with the conformational changes in the nucleotide-binding sites (at the a/b interface) required for catalysis, and possibly by blocking rotation of the central stalk subunits (Cabezon et al., 2003; Garcia et al., 2006; Gledhill et al., 2007; Minauro-Sanmiguel et al., 2002). The binding of IF1 to F1 requires ATP to be bound first (de GomezPuyou et al., 1980; Milgrom, 1991). Interestingly, the (F1-IF1)2 crystal structure (Cabezon et al., 2003) features a molecule of ATP (or its analogue AMP-PNP) in the bDP catalytic site, rather than the ADP molecule present in the F1 structure with no IF1 bound (Abrahams et al., 1994). This property is shared with the ‘‘ground-state’’ F1 structure derived from crystals grown in the absence of azide (Bowler et al., 2007). This would suggest that binding of IF1 traps ATP in this site, and that the (F1-IF1)2 structure represents a prehydrolysis state in the catalytic cycle (Cabezon et al., 2003; Gledhill et al., 2007). Corvest et al. (2005) investigated the effect of nucleotide occupancy of the catalytic sites on the rate of inhibition by IF1, and

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found that at least two of the sites must be filled to allow binding of IF1. Subsequent hydrolysis of ATP was proposed to lock IF1 in place (Corvest et al., 2005). The yeast complex has an inhibitory protein associated with it, Inh1p, homologous to IF1. Additionally, in yeast other small proteins are involved. STF1 is reported as having similar functional properties and sensitivity to the same effectors as Inh1p, but different affinity for mtATPase. Despite much detailed investigation the role of STF1 in vivo remains unclear (Venard et al., 2003). While both yeast proteins can form dimers there is no evidence that they cannot function as monomers. Furthermore, the role of dimeric yeast Inh1p has been questioned (Ichikawa et al., 2002). Two other small proteins, STF2 and STF2, share some 65% homology and are proposed to contribute to regulation of yeast mtATPase through modulation of Inh1p and STF1 function. How this modulation is effected remains unclear (Hong and Pederson, 2002).

2.4. The peripheral/‘‘Stator’’ stalk 2.4.1. Overall structure and subunit composition Although early EM images of bacterial (E. coli ) ATP synthase reconstituted into phospholipid vesicles (Gogol et al., 1987) clearly showed the presence of a central stalk linking F1 to F0, these pictures did not reveal the presence of proteins linking the two sectors at the periphery of the complex. Rather, information on the location and structure of the peripheral stalk was initially built up from cross-linking and reconstitution experiments. Collinson et al. (1994b) combined purified bovine subunits d, OSCP, F6 and the membraneextrinsic portion of subunit b and found that these proteins formed a 1:1:1:1 complex. Moreover, this sub-complex was able to bind with purified F1 in a 1:1 ratio (Collinson et al., 1994b, 1996). The crystal structure of most of the peripheral stalk from bovine mtATPase has recently been solved (Dickson et al., 2006; see below). Proving that the peripheral stalk acts as a ‘‘stator’’ in ATP synthase required functional studies on catalytically active enzyme. This has largely involved disulfide cross-linking experiments carried out with the bacterial (E. coli) enzyme, which offers the advantage that site-directed mutant enzymes with introduced cysteine residues can be readily constructed and purified, and the effect of the zero-length cross-link on enzyme activity easily measured. Cross-links from either subunit g or e to either subunit a or b abolish activity completely (Aggeler et al., 1995; Aggeler and Capaldi, 1996), as expected if rotation of subunits g and e relative to subunits a and b is required for catalysis. The bacterial d subunit (Ogilvie et al., 1997) and the C-terminus of subunit b (Rodgers and Capaldi, 1998) can be cross-linked to the N-terminal domain of an a subunit, subunit d can be cross-linked to subunit b (McLachlin and Dunn, 2000) and subunit g can be cross-linked

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to the subunit c ring (Watts et al., 1995). Crucially, these cross-links have little effect on enzyme activity. After it had been established experimentally that the a-b, a-d and g-c cross-links had no effect on rotational catalysis, the peripheral stalk could be properly designated as a ‘‘stator’’ that holds subunit a and the F1 a3b3 hexamer in position as the rotor component of the complex rotates. Once the composition and function of the stator stalk of the bacterial enzyme had emerged, it became clear that the b:d:OSCP:F6 complex (Collinson et al., 1994b) was very likely to play an equivalent role in bovine mtATPase. The peripheral stalk may also contribute to transient accumulation of elastic energy during catalysis (Cherepanov et al., 1999) that would arise from a mismatch in symmetry between the rotary motors of F1 (containing 3 catalytic sites) and F0 (with 1014 copies of the proton-translocating c subunit). In the late 1990s, a number of EM studies were carried out on single particles of negatively stained ATP synthase complexes, and were successful in visualizing the peripheral stalk. This technique, which relies on averaging of many images, revealed the presence of protein mass at the periphery of the enzyme purified from bovine mitochondria (Karrasch and Walker, 1999), chloroplast (Bottcher et al., 1998) and E. coli (Wilkens and Capaldi, 1998b). More recently, EM images with higher resolution have been obtained from single unstained bovine mitochondrial F1F0 particles embedded in vitreous ice (Rubinstein et al., 2003). In these pictures, electron density outside the central F1/c-ring complex is more clearly defined, and appears large enough to accommodate the subunits predicted to make up the peripheral stalk [b (extramembrane domain), d, OSCP and F6] and F0 components outside the subunit c ring [subunits a and A6L (8), along with the membrane spanning regions of subunits b, e, f and g] (Dickson et al., 2006; Rubinstein et al., 2003). EM of negatively stained bacterial (E. coli) F1F0 complexes that had been decorated with monoclonal antibodies against the bacterial subunit d (equivalent to mitochondrial OSCP) showed that this protein is located at the top of F1 (Wilkens et al., 2000). EM has also been used to probe the positions of subunits h and OSCP within the yeast mitochondrial stator stalk. By tagging these subunits with biotin and avidin and examining the position of the avidin molecule in negatively stained images, subunit h appeared to reside close to the membrane surface (Rubinstein et al., 2005), while OSCP was located toward the top of F1 (Rubinstein and Walker, 2002). This result is consistent with cross-linking studies in the bacterial (E. coli) (Ogilvie et al., 1997) and chloroplast (Lill et al., 1996) enzymes, which place subunit d in close proximity to the N-terminal domain of subunit a. The crystal structure of most of the peripheral stalk from bovine ATP synthase has recently been solved at 2.8 A˚ resolution (Dickson et al., 2006). The crystallized complex contains most of the extramembranous

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portion of subunit b [residues 79184 (missing residues 185214)], most of subunit d [residues 1124 (missing 125160)] and all of subunit F6 (h). All of the subunits are predominantly a-helical (structures of the individual proteins are discussed below). Importantly, the structure fits well into a region of electron density at the periphery of the low-resolution structure of the whole complex derived from EM studies (Rubinstein et al., 2003), see Figure 1.2. This density in the structure is that which is unaccounted for once the F1 (Abrahams et al., 1994; Gibbons et al., 2000) and the subunit c ring (Stock et al., 1999) structures are docked into it and is very likely to be occupied by the peripheral stalk subunits [OSCP, b, d, and F6 (h) plus membrane-bound subunits outside the subunit c ring (a, e, f, g and A6L] (Dickson et al., 2006; Rubinstein et al., 2003). 2.4.2. Structures of individual subunits 2.4.2.1. OSCP The NMR solution structure of the N-terminal domain for both bacterial (E. coli) subunit d (Wilkens et al., 1997) and bovine OSCP (Carbajo et al., 2005) have been determined. The C-terminal regions (residues 112190 in OSCP and 134177 in subunit d) are highly susceptible to proteolysis, and appear from NMR studies to be mostly disordered (Carbajo et al., 2005; Wilkens et al., 1997). The N-terminal domains of both proteins consist of a bundle of six a-helices, and their folds are extremely similar. The N-terminal domain of OSCP appears to fit well into a region of density in the EM-derived low-resolution structure which lies directly above the top of F1 (Rubinstein et al., 2003), while the C-terminus appears to be located in a region of the peripheral stalk further away from the central axis of the F1 a3b3 hexamer (Dickson et al., 2006); see Figure 1.2. The F1-binding surface on bacterial subunit d has been studied by mutagenesis and tryptophan fluorescence (Weber et al., 2003b), and found to be localized to helices 1 and 5 of the N-terminal domain. A synthetic peptide consisting of the first 22 residues of bacterial subunit a was shown to bind to the N-terminal domain of subunit d specifically and with high affinity (Weber et al., 2003a). The binding interface within this complex was analyzed using NMR (Wilkens et al., 2005). Similar techniques were used by Carbajo et al. (2005) to demonstrate binding of the N-terminal residues of bovine subunit a to the N-terminal domain of bovine OSCP, and also to rule out any involvement of N-terminal residues of subunit b in binding OSCP. These and later studies indicate that a stretch of residues at the N-terminus of subunit a is likely to form an a-helix which binds to OSCP/d by packing into a groove between helices 1 and 5 (Carbajo et al., 2005, 2007; Wilkens et al., 2005). Weber et al. (2002) used a fluorimetric assay to determine a Kd value of 1.4 nM for the binding affinity of bacterial subunit d to F1, suggesting a standard free energy of binding of 50.2 kJ/mol. This would be roughly equivalent with the strain demands placed upon the

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Figure 1.2 The composite structure of mtATPase. Detailed structures of the F1c10 subcomplex (gray), the N-terminal domain of the OSCP (cyan) and the peripheral stalk subcomplex (magenta, orange and green) were introduced by eye into an electron density map determined by averaging single particles of the intact bovine complex observed by electron cryomicroscopy. (A) Side view and (B) residual density corresponding to the peripheral stalk and the second domain of F0 (Rubinstein et al., 2003). Dotted lines represent the lipid bilayer. (C) View looking down onto the ‘‘crown’’ of the F1 catalytic ˚ . [Adapted by permission from Macmillan Publishers Ltd. domain. The scale bar is 50 A EMBO Journal (Dickson, V. K., Silvester, J. A., Fearnley, I. M., Leslie, A. G., Walker, J. E. On the structure of the stator of the mitochondrial ATP synthase. EMBO J. 25: 2911^2918) Copyright 2006.]

stator by the torque energy generated by the rotor during catalysis (Hasler et al., 1999), although the direct binding of subunit b to F1 may also contribute to the stability of the stalk/F1 connection in the bacterial enzyme (Weber et al., 2004).

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2.4.2.2. Subunit b of the bacterial, chloroplast and mitochondrial ATP synthases are predicted to play similar roles in their respective enzymes (Velours et al., 1988; Walker et al., 1987b), in that they constitute a continuous link from the membrane-integral F0 components to near the top of F1, and provide a scaffold for assembly of other subunits of the peripheral stalk. However, there are marked differences in stoichiometry and topology between the subunits from the different sources. The bacterial enzyme features a homodimer of two b subunits, while in some other bacteria and chloroplasts, there is a heterodimer of two versions of b subunits (referred to as b and b’ in bacteria, or CF0I and CF0II in chloroplasts). However, only a single copy of subunit b is present in mtATPase (Bateson et al., 1999; Collinson et al., 1994a, 1996). This subunit features two membrane-spanning domains at the N-terminus, presumed to be a-helices, with the N-terminal residue on the matrix side of the inner membrane (Velours et al., 1989; Soubannier et al., 2002). The hydrophilic C-terminal portion extends into the matrix, and binds subunits d, F6 (h) and OSCP in 1:1:1:1 ratio (Collinson et al., 1994b, 1996). Given that OSCP is located near the top of F1 (Carbajo et al., 2005) the b subunit must extend to this region. In yeast, two-hybrid and cross-linking (Soubannier et al., 1999; Velours et al., 1998) as well as FRET (Gavin et al., 2003) studies provide evidence for interaction between the C-terminus of OSCP with the C-terminus of subunit b. These findings are consistent with disulfide cross-linking experiments in bacterial (E. coli) ATP synthase, which place the C-terminus of subunit b close to the N-terminus of subunit a (Rodgers and Capaldi, 1998) and to bacterial subunit d (McLachlin and Dunn, 2000; Rodgers et al., 1997). The crystal structure of the peripheral stalk from bovine mtATPase (Dickson et al., 2006) contains residues 79184 of the b subunit, which ˚ long. The N-terminal amino acid of form a continuous a-helix about 160 A the fragment (residue 79 in the full-length protein) is predicted from hydropathy profiles to lie at the interface between the mitochondrial inner membrane and the matrix (Walker et al., 1987b). It would therefore be expected that in the intact mtATPase, the helix in the b subunit would extend away from the membrane surface towards the top of F1 in the N- to C-terminal direction (Dickson et al., 2006). If the peripheral stalk crystal structure is docked into the electron density map of the F1F0 EM structure (Rubinstein et al., 2003), with residue 79 of the b subunit positioned at the predicted membrane surface, the length of the b subunit helix fits very well with the distance from the membrane to the top of F1 (Dickson et al., 2006). Part of the density unaccounted for by the peripheral stalk proteins may be due to a segment of subunit b which is missing from the crystallized complex (residues 185214). The position of this density suggests that there is a turn in subunit b around residues 185187, followed by another a-helix (residues 188204) which lies antiparallel to the long a-helix (Dickson et al., 2006; see Fig. 1.2).

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The structure of the membrane-bound segment of subunit b (residues 179) is presently unknown. Secondary structure and hydropathy analyses of the sequence (Walker and Dickson, 2006) predict that the long a-helix continues uninterrupted across the inner mitochondrial membrane to the intermembrane space. A turn, followed by a second transmembrane a-helix (residues 3347), results in the N-terminal region (residues 130) being exposed to the mitochondrial matrix (Walker and Dickson, 2006). 2.4.2.3. Subunit d is unique to mtATPases being characterized first in mammals by Walker et al. (1987b), and in yeast by Norais et al. (1991). The protein is predominantly hydrophilic, and has been shown to be essential for enzyme function (Norais et al., 1991). Reconstitution experiments showed that subunit d makes contact with all of the other three components of the peripheral stalk (b, F6 and OSCP) and is present in one copy per ATP synthase complex (Collinson et al., 1994b, 1996). The portion of subunit d (residues 3123) which is resolved in the crystal structure of the bovine peripheral stalk (Dickson et al., 2006) is composed of 5 a-helices separated by extended linker regions. The protein has an extensive interface with subunit b, interacting predominantly via a parallel and two antiparallel coiled-coil interactions in the region of residues 99162 of subunit b, placing the protein approximately halfway along the extramembranous portion of the peripheral stalk (Dickson et al., 2006; see Fig. 1.2). 2.4.2.4. Subunit h (F6 ) Subunit h of yeast mtATPase is an acidic watersoluble protein, present in one copy per complex (Fronzes et al., 2003). Yeast mutants lacking this protein are deficient in oxidative phosphorylation, showing that subunit h plays an essential role in assembly and/or catalysis (Velours et al., 2001). The bovine subunit F6 can replace subunit h in such mutants, indicating that the two proteins are functionally homologous, despite sharing just 14.5% sequence identity (Velours et al., 2001). Reconstitution experiments with purified bovine subunits demonstrated that F6 binds directly with the b subunit, and interacts with subunit d either directly or through subunit b (Collinson et al., 1994b). Disulfide and chemical crosslinking experiments with yeast ATP synthase place subunit h in close proximity to subunits b and d, and also to the membrane bound subunit f (Fronzes et al., 2003). This would imply that the location of subunit h within the peripheral stalk is in a region close to the mitochondrial inner membrane. EM images of yeast F1F0 particles containing subunit h labelled with biotin/avidin also suggested that the protein is close to the F0 sector of the enzyme (Rubinstein et al., 2005). In contrast, the crystal structure of the bovine peripheral stalk (Dickson et al., 2006) shows that F6 is likely to be located in a region close to OSCP and the N-terminal portions of the a and b subunits (at the top of F1) and that the C-terminus of the protein would lie about 70 A˚ from the membrane surface (Dickson et al., 2006; see Fig 1.2).

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Even when the slightly greater length of the yeast subunit h is taken into account (16 amino acids) it appears unlikely that this protein would extend as far as the membrane surface. The solution structure of isolated bovine subunit F6 has been determined by NMR, and shows two loosely-packed a-helices separated by an unstructured linker (Carbajo et al., 2004). In this form, the protein is highly flexible and shows a high degree of structural heterogeneity. However, the crystal structure of the bovine peripheral stalk (Dickson et al., 2006) revealed that this was due to the subunit being studied in isolation. When bound to other components of the peripheral stalk, F6 adopts a more elongated conformation, and the observed interactions between the helices within isolated F6 are replaced by other inter-subunit contacts. In the peripheral stalk structure, the helices (residues 824 and 3451) are linked by an extended region; extended regions are also present at the N- and C-termini of the protein. Hydrophobic patches on the helices and portions of the extended regions make specific interactions with the b subunit, and a segment of the C-terminal helix of F6 makes a parallel interaction with a helix from subunit d (Dickson et al., 2006).

2.5. The F0 sector 2.5.1. Overall structure, function and subunit composition The F0 sector of mtATPase consists of three subunits with homologues in the bacterial enzyme (subunits a, b and c), whose roles are clear, along with at least six others (e, f, g, i/j, k and A6L) the functions of which are less wellcharacterized. All complexes contain a single copy of subunit a, while the stoichiometry of subunit c appears to vary between organisms. Yeast mtATPase contains 10 copies (Stock et al., 1999), as does the bacterium E. coli ( Jiang et al., 2001). By contrast, the bacterium Ilyobacter tartaricus (Meier et al., 2005) and spinach chloroplast (Seelert et al., 2003) complexes possess 11 and 14 copies, respectively. The function of F0 is to couple proton translocation across the membrane with rotation of the subunit c ring relative to subunit a. An acidic amino acid residue present in subunit c from all known sources (Glu58 in the human mitochondrial enzyme), which lies in the middle of the membrane (Meier et al., 2005; Stock et al., 1999), plays a critical role in proton movement. Alternative models have been proposed to explain how protons access this residue. Dimroth et al. (2000) suggested that the subunit c ring contains an aqueous channel through which protons (or Naþ ions in the case of the I. tartaricus enzyme) would gain access to the conserved glutamic acid side-chain. However, the crystal structure of the I. tartaricus subunit c ring (Meier et al., 2005) did not reveal the presence of an aqueous access channel. More likely, residues in subunit a enable access of protons to the

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conserved glutamic acid residue from both sides of the membrane, via two spatially distinct ‘‘half-channels’’ (Angevine et al., 2007; Junge et al., 1997). F0 is thought to operate as a ‘‘Brownian ratchet’’ in which directional motion is generated based on stochastic thermal fluctuations of the relative positions of subunit a and the subunit c ring, with rotation biased to proceed in a particular direction based on the polarity of the prevailing proton electrochemical gradient ( Junge and Nelson, 2005). Fluctuations are constrained by the need for the essential acidic acid side-chain of subunit c to be negatively charged when facing a positively charged residue in subunit a, but neutralized by protonation when facing hydrophobic membrane lipids. The presence of a proton gradient across the inner mitochondrial membrane would favor a proton entering the lower ‘‘half-channel’’ (from the mitochondrial intermembrane space) and binding to the essential acidic residue in subunit c at the interface with subunit a, disrupting electrostatic interactions between it and residues in subunit a. This would allow the subunit c ring (and the attached subunit g) to rotate one step counterclockwise. Simultaneously, the acidic residue of the adjacent subunit c would move into the subunit a interface and release a proton into the matrix through the upper ‘‘half-channel’’ ( Junge and Nelson, 2005). Due to the lack of high-resolution structures of F0 subunits from mtATPases, our present understanding of the architecture of this portion of the enzyme has come mainly from studies on their bacterial equivalents. In the case of subunit a, genetic and biochemical studies in E. coli have defined the likely topology and packing arrangement of the transmembrane helices. With regards to subunit c, an electron density map of modest resolution, obtained from crystals of a subcomplex of yeast mtATPase, was sufficiently detailed to show the presence of a ring of 10 subunits (Stock et al., 1999). However, a clearer picture of how the subunit ring is ˚ ) crystal structure of assembled was provided by the high-resolution (2.4 A þ the unadecameric ring of the Na -translocating ATP synthase from I. tartaricus (Meier et al., 2005). The structure and arrangement of the individual proteins present in F0 are discussed below. 2.5.1.1. Subunit a (Subunit 6) Cox et al. (1984) first suggested that proton translocation through F0 is accompanied by rotation of subunit a relative to the subunit c ring. It was also proposed that amino acid side chains from both of these proteins contribute to the proton channel across the membrane (Cox et al., 1986). Based on hydropathy profiles and secondary structure predictions, these authors suggested a model for folding of the bacterial a subunit in which there are five transmembrane a-helices, with the N-terminus on the periplasmic side of the membrane (equivalent to the intermembrane space in the case of the mitochondrial subunit a) and the C-terminus on the cytoplasmic side (mitochondrial matrix). Subsequent work involving site-directed mutagenesis, analysis of second-site revertants,

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cross-linking and labelling has confirmed this topology (Fillingame et al., 2003; Hatch et al., 1995; Vik and Ishmukhametov, 2005). Cox et al. (1986) proposed that conserved residues on the fourth transmembrane helix, including Arg210 (Arg159 in human mtATPase) could form a proton translocation pathway, along with the conserved acidic residue on the second transmembrane helix of subunit c (Asp61 in the bacterial protein, Glu59 in yeast mtATPase). Site-directed mutagenesis showed that Arg210 is required absolutely for proton translocation (Cain and Simoni, 1989; Lightowlers et al., 1987). Recent cross-linking experiments with the bacterial (E. coli) enzyme, in which Cys residues were introduced into subunit a, indicated that the packing arrangement for helices 2, 3, 4 and 5 is a four-helix bundle (Schwem and Fillingame, 2006). Chemical modification studies indicated that protons may access the critical residues (subunit a Arg210 and subunit c Asp61) from the periplasmic (intermembrane space) side of the membrane via an aqueous channel in the middle of the helical bundle, and that access to the cytoplasm (matrix) is provided by residues on the external faces of helices 4 and 5, which contact subunit c (Angevine et al., 2007). These authors propose that gating of the two half-channels would be achieved as follows. During proton-transport driven rotation of the subunit c ring relative to subunit a, a protonated Asp61 of subunit c would release its proton to the cytoplasmic half-channel due to its interaction with Arg210 of subunit a; the acidic side-chain would be neutralized by protons sourced from the periplasmic half channel and moved from the centre of the helical bundle to the outside by means of swivelling of one or both of helices 4 and 5 (Angevine et al., 2007). Several debilitating human mitochondrial diseases are caused by point mutations in subunit a of mtATPase (Schon et al., 2001). These include maternally inherited Leigh syndrome (MILS) (Tatuch et al., 1992), neurogenic ataxia and retinitis pigmentosa (NARP) (Holt et al., 1990), and some cases of Leber hereditary optic neuropathy (LHON) (Majander et al., 1997). 2.5.1.2. Subunit c (Subunit 9) Structural information on subunit c has come from NMR experiments on the isolated subunit in organic solvent (Girvin et al., 1998; Nakano et al., 2006; Rastogi and Girvin, 1999) along with studies on the oligomeric subunit c ring involving cryo-electron microscopy (Vonck et al., 2002), atomic force microscopy (Seelert et al., 2000; Stahlberg et al., 2001) and x-ray crystallography (Meier et al., 2005; Stock et al., 1999). The protein folds as a hairpin comprised of two transmembrane helices, with the critical acidic residue (Asp61 in E. coli, Glu58 in human mitochondria) situated approximately halfway between the two sides of the membrane. The NMR-derived structures of the isolated bacterial (E. coli) subunit c, determined at pH 5 (Girvin et al., 1998) and pH 8 (Rastogi and Girvin

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et al., 1999) revealed different conformations of the protein, with Asp61 in protonated and deprotonated states, respectively. The deprotonated form (likely to represent subunit c at the interface with subunit a) differed from the protonated form (likely to represent subunit c facing the lipid bilayer) in that the C-terminal helix was rotated by 140
(Girvin et al., 1999). Fillingame and Dmitriev (2002) suggested a model for a 10-mer subunit c ring in which the individual subunits pack in a ‘‘front-to-back’’ manner with the first transmembrane helix (TM1) facing inside the ring, and the second helix (TM2) on the outside. On the basis of the protonated and deprotonated conformations seen in the NMR structures (Girvin et al., 1998, 1999), it was proposed that access of protons to Asp61 during the protonation/deprotonation cycle would require TM2 to rotate, such that Asp61 moves from an occluded position (protonated state) to a position on the outside of the ring (deprotonated state, in the interface with the a subunit). This swivelling motion was proposed to drive the rotation of the subunit c ring relative to subunit a (Fillingame and Dmitriev, 2002). The high-resolution structure of the 11-mer subunit c ring from I. tartaricus (Meier et al., 2005) confirmed that the subunits pack with TM1 on the inside and TM2 on the outside, but did not support an ion-translocating mechanism involving swivelling of TM2. In this structure, the side-chains of all of the critical acidic residues (each with bound cation) face toward the outside surface of the ring, eliminating the need for swivelling of TM2 during the protonation/deprotonation cycle. Meier et al. (2005) also noted that the highly compact structure of the subunit c ring would impose steric hindrance which would be likely to severely restrict the proposed swivelling. A more detailed understanding of the conformational changes undergone by subunit c during ion translocation, and of the access pathways to the conserved acidic residues await a high-resolution structure of F0 in which both subunit a and the subunit c ring are packed together in their native arrangement. 2.5.1.3. Subunits e, g and k The ability of mtATPase to form a homodimer was first observed by native gel electrophoresis of solubilized yeast mitochondria (Arnold et al. 1998). Three proteins, e, g and k, were found associated with the dimeric form of the enzyme, but not with the monomer, and were not essential for enzyme function as an ATP synthase (see Section 3). 2.5.1.4. Subunit 8 (A6L) This small hydrophobic subunit is unique to the mtATPase complex (Table 1.1), having a stoichiometry of one per complex (Stephens et al., 2003b). It is a membrane-embedded protein oriented with the C-terminus facing into the mitochondrial matrix (Stephens et al., 2000). The protein has no clear function within the complex, but yeast mutants failing to express subunit 8 are unable to assemble subunit 6 into the enzyme

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complex (Hadikusumo et al., 1988) and therefore have nonfunctional mtATPase. The yeast subunit has been shown by cross-linking to be proximal to several other subunits of the peripheral stalk. The N-terminus of subunit 8 forms chemical cross-links with subunits b, d, f and subunit 6 in the intermembrane space, and the C-terminus interacts with subunits b, d and f within the mitochondrial matrix (Stephens et al., 2000, 2003a). Based on its proximity to the other component protein of the peripheral stalk as well as its interaction with subunit 6, it was postulated that subunit 8, in combination with subunit d, may complement the single copy of subunit b as a component of the stator stalk (Bateson et al., 1999). 2.5.1.5. Subunit f Another yeast subunit lacking a bacterial homologue, its presence is required for the stable assembly of the three mitochondrially encoded subunits, 6 (a), 8, and 9 (c) into mtATPase (Spannagel et al., 1997). The C-terminus domain of this subunit possesses a hydrophobic transmembrane domain which is not essential for enzyme function, but does seem to play a role in functional coupling and structural stabilization of the enzyme (Roudeau et al., 1999). The N-terminal domain of the subunit resides within the mitochondrial matrix (Belogrudov et al., 1996; Roudeau et al., 1999). The 66 N-terminal residues within the matrix (Roudeau et al., 1999) would extend two-thirds of the way up the side of F1 if in a-helical confirmation. Interactions of subunit f with subunits b, i, h and 8 have been demonstrated (Fronzes et al., 2003; Spannagel et al., 1998b; Stephens et al., 2003a; Velours et al., 2000). 2.5.1.6. Subunit i/j This small membrane integral protein was identified concurrently by two research groups, who named this protein subunit i (Vaillier et al., 1999) and j (Arnold et al., 1999). It has a single hydrophobic transmembrane domain orientated with its N-terminus inside the mitochondrial matrix (Paumard et al., 2000). Functional assessments of the protein differed between the two groups. One group found that a null mutant strain, while having reduced ATP catalytic activity and retarded proton pumping, was still able to grow on a nonfermentable carbon source (Vaillier et al., 1999). The null strain of the other group, however, displayed no oligomycin-sensitive ATPase activity, was unable to grow in medium containing a nonfermentable carbon source, and adopted a petite phenotype (Arnold et al., 1999). Subunit i/j forms cross-links with subunits d and f of the stator stalk, as well as subunit 6 and subunit g (Paumard et al., 2000). Its presence is required also for the stable expression of subunit 6 and subunit f (Arnold et al., 1999). Interactions between proximal mtATPase complexes can occur through this subunit, indicating it has a peripheral location within the complex (Paumard et al., 2002a), and may be involved in mtATPase dimerization (Fronzes et al., 2006).

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3. Supramolecular ATP Synthase 3.1. Introduction The mitochondrial inner-membrane (IM) contains one of the highest protein concentrations of cellular membranes. Thus, one might expect these proteins would be highly organised and, possibly arranged into large supramolecular complexes. Such a view of the mitochondrial IM is now generally accepted. In this section we will focus on supramolecular organization of mtATPase. In the last decade it has become clear that strong, stable interactions exist between individual mtATPase complexes that have important ramifications for cristae structure/function and mitochondrial bioenergetics. The significant advances in our knowledge of protein complex organization in mitochondrial membranes can be attributed, in-part, to the development and application of new high-performance analytical approaches. Gel based electrophoretic approaches, in particular blue-native PAGE (BNPAGE) and clear native PAGE (CN-PAGE), have been important for the high-resolution separations and visualising the complexes. These techniques and their application to protein complexes in different membrane systems are the subject of recent comprehensive reviews (Krause, 2006; Wittig and Schagger, 2007). BN-PAGE is a charge-shift method relying on the binding of an anionic dye to membrane proteins and complexes which allows separation according to size with superior resolution compared to more traditional techniques such as gel filtration or density gradients. Although constrained to separating proteins with a native negative charge, CN-PAGE performed without the addition of dye, allows weaker interactions to be preserved and enzyme activity to be more efficiently monitored in situ. Of equal importance to the success of such studies has been the selection of the best detergent that upon solubilization of membranes preserves both enzymatic activity and supramolecular organization. In the case of mitochondrial membranes the use of nonanionic detergents, in particular digitonin, has been found to be very effective in preserving many supramolecular structures from mitochondrial membranes.

3.2. Dimers and oligomers First identified in mitochondrial membranes isolated from yeast cells (Arnold et al., 1998), the existence of dimers of mtATPase (d-mtATPase) was initially met with some scepticism even though such an arrangement had been hinted by the remarkable EM images of Paramecium multimicronucleatum mitochondria (Allen, 1989; Allen et al., 1995) (see below). The existence of such dimers and indeed other oligomeric forms is now well accepted.

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Indeed, d-mtATPase can be recovered from a number of sources including mammals, fungi and plants (Table 1.2). Oligomeric forms of mtATPase have to date not been identified in bacteria presumably due to the absence of those F0 subunits believed to be involved in promoting oligomer formation (Schagger, 2002). In contrast to the rather unstable nature of d-mtATPase from most mitochondrial sources requiring careful use of the gentle detergents such as digitonin or Triton X-100, Chlamydomonas reinhardtii mitochondria contained a particularly stable d-mtATPase that was readily recovered using dodecylmaltoside (van Lis et al., 2003), a detergent that disrupts d-mtATPase from other sources. Indeed m-mtATPase could not be recovered under these conditions. The initial reports of d-mtATPase were soon followed by those showing that tetramers (Arselin et al., 2004; Paumard et al., 2002b) could be recovered in small amounts from yeast mitochondria through the careful selection of alternative detergent/protein ratios. More recently hexamers and even octamers of mtATPase, in addition to dimers, have been recovered from rat Table 1.2 Dimers and oligomers of ATP synthase identified by BN-PAGE or CN-PAGE Organism and/or cell type a

Human fibroblast and osteosarcoma Bovine heart

Liver, skeletal muscle, brain kidney, brain Rat liver Higher plants (Arabidopsis) Yeast Saccharomyces cerevisiae

Filamentous fungus Podospora anserina Chlamydomonad Algae C. reinhardtii chloroplasts Polytomella sp. a

Dimer

Oligomers

References

X

X

X

X

X

X

Cortes-Hernandez et al., 2007 Schagger and Pfeiffer 2000; Pfeiffer et al., 2003 Krause et al., 2005

X

X

Garcia et al., 2006; Krause et al., 2005 Eubel et al., 2003, 2004

X

Arnold et al., 1998; Gavin et al., 2005; Paumard et al., 2002b

X

X

X

Krause et al., 2004

X X X

van Lis et al., 2003 Rexroth et al., 2004 Dudkina et al., 2006a,b

Unless otherwise indicated, listings relate to mitochondria.

Structure and Function of mtATPases

25

heart mitochondrial membranes through careful titration with digitonin combined with analysis by CN-PAGE (Wittig et al., 2005). These oligomeric forms of mtATPase are evidently more labile, but readily observed by CN-PAGE rather than BN-PAGE where the binding of the blue dye to proteins presumably weakens protein–protein interactions (Wittig et al., 2006). Interestingly in this study only even numbered oligomers were recovered, a finding that has relevance to the organization of mtATPase complexes within the membrane (see Section 3.4). The evidence for the existence of oligomeric forms of mtATPase is largely based on their analysis after detergent extraction from mitochondrial membranes leaving the possibility that they might arise from nonspecific aggregation of mtATPase monomers under the high protein concentrations used in BN-PAGE or CN-PAGE analysis. However, this possibility now seems unlikely. Recovery of d-mtATPase is dependent on the presence of specific subunits in mitochondrial membranes (see below). Moreover, d-mtATPase can be stabilized upon cross-linking in membranes of isolated yeast mitochondria (Paumard et al., 2002a). The close proximity of mtATPase monomers in intact yeast cells could be inferred using a noninvasive fluorescent protein cross-linking approach (Gavin et al., 2004).

3.3. Subunits relevant to dimer formation 3.3.1. The dimer specific subunits e, g and k Use of BN-PAGE resulted in the identification of proteins now considered subunits of mtATPase. Two of these subunits, e and g, first identified as being associated with bovine heart mtATPase (Collinson et al., 1994a; Walker et al., 1991) and subsequently found to exist in yeast (Arnold et al., 1997, 1998; Boyle et al., 1999) are often referred to as the dimer specific subunits. An analysis of the subunit composition of mtATPase complexes solubilized from yeast with different detergents revealed these subunits to be associated with d-mtATPase but not m-mtATPase. The importance of these subunits was underscored by the finding that d-mtATPase could only be recovered only in very small amounts, or not at all, from yeast mitochondria lacking expression of subunit g or e, respectively (Arnold et al., 1998; Schagger, 2002). A third protein, subunit k, was also found to be associated with d-mtATPase, but is not considered important to the formation of dimers as d-mtATPase was readily recovered from cells lacking expression of this subunit. Although both subunits e and g are involved in the formation of d-mtATPase, it is subunit e that appears to play a more central role as its presence is required for the stability of subunit g, whereas subunit e is stable, albeit present in lower amounts, in the absence subunit g. Furthermore, in mitochondria from the strain lacking subunit e small amounts (5%) of dimer could be isolated, and subunit g can in some cases be found bound to m-mtATPase complexes (Schagger, 2002). However, a recent report indicates that phosphorylation of

26

Rodney J. Devenish et al.

a serine residue located in the matrix domain of subunit g regulates formation of d-mtATPase (Reinders et al., 2007). Both subunit g and e are small transmembrane hydrophobic proteins with orientation Nin- Cout relative to the mitochondrial matrix. In rat liver mitochondria the stoichiometry of subunit e was found to be two per mmtATPase (Arakaki et al., 2001). In yeast two copies of the protein are in close proximity as homodimers were observed upon oxidation of its unique cysteine (Everard-Gigot et al., 2005). However, cross-linking studies suggest that there are two populations of subunit e that exist in different environments (see model below). Homodimerization and heterodimerization of subunits e and g has been shown through the cross-linking of both endogenous and introduced cysteines (Brunner et al., 2002). The subunit e/g heterodimer is specific to d-mtATPase; the g/g and e/e homodimers are found in oligomeric forms (Arselin et al., 2003; Bustos and Velours, 2005). The 95-amino acid subunit e has a unique transmembrane span containing a conserved GXXXG dimerization motif essential for the stability of the supramolecular structure (Arselin et al., 2003). Substitutions within this motif abolish the recovery of d-mtATPase from yeast mitochondria. In such mitochondria subunit e associated only weakly with mtATPase and subunit g was absent indicating the mutual dependence of these two subunits. Subunit g also contains a GXXXG motif in its single transmembrane segment that has been demonstrated to be important for function and stability of the subunit within mtATPase (Saddar and Stuart, 2005). However, these authors also showed that an intact GXXXG motif in subunit g was not essential for its interaction with subunit e. Subunit e has a C-terminal coiled-coil motif projecting into the intermembrane space that is also involved increasing the stability of d-mtATPase (Everard-Gigot et al., 2005). Removal of one or more of the coils from yeast subunit e resulted in decreased stability of d-mtATPase and oligomers as determined by detergent solubilization and BN-PAGE analysis (Bornhovd et al., 2006). Under these conditions subunit g was subject to increased degradation. Clearly subunit e has an important role in the formation or stability of d-mtATPase and oligomers. However, it is not clear if the absence of subunit e alone is sufficient to abolish interactions between monomers. This issue was addressed in intact yeast cells in which the single copy subunit b was expressed fused in equal amounts to blue fluorescent protein and green fluorescent protein (Gavin et al., 2005). Analysis of Fo¨rster resonance energy transfer (FRET) between the subunit b fusion proteins in cells lacking expression of subunit e indicated that in vivo interactions between the subunits b were maintained suggesting that subunit e was not involved in the interface stabilized by the b–b interactions. This result was in keeping with the presence of two distinct interfaces between the monomers in d-ATPase, as previously suggested by Paumard et al. (2002b). Subsequently it has been found that subunit e

Structure and Function of mtATPases

27

is not involved in driving formation of the b–b interface, but rather helps stabilise it on solubilization of membranes in detergents (Fronzes et al., 2006). 3.3.2. Other proteins that promote/stabilise dimerization and oligomerization The finding that chloroplast d-mtATPase can be extracted from Chlamydomonas reinhardtii where there is, as yet, no evidence for subunits e and g (or homologous proteins) suggests other proteins are involved in dimer formation in chloroplasts (van Lis et al., 2003). Despite the interactions mediated through subunits e and g, data exist to suggest other proteins play an important role in dimerization. Although d-mtATPase cannot be isolated by the use of detergents from yeast mitochondria lacking subunit e or g there is convincing evidence to indicate that d-mtATPase exists in the membranes of such mutant mitochondria. Thus, it was still possible to recover d-mtATPase from mitochondrial membranes of cells expressing subunit i/j modified by introduced cysteines in place of the native subunit and under conditions which facilitate formation of cross-links (Fronzes et al., 2006). The stator stalk appears to play an important role in formation or stabilization of d-mtATPase. Homodimers of subunit b can be isolated by SDS-PAGE after cross-linking of introduced cysteines in the hydrophilic loop of this protein (Spannagel et al., 1998a). However, in this case crosslinked d-mtATPase could not be solubilized from the membranes using detergents suggesting that the mtATPase had in some way been altered. In a different study, d-mtATPase or oligomers could not be recovered from mitochondria expressing subunit b with a truncation of the N-terminal portion of the loop traversing the inner membrane (Soubannier et al., 2002). Such a truncation did not affect the function or assembly of monomeric mtATPase (m-mtATPase). By contrast, subunit h, in cross-linking studies was able to form homo-dimers, and in this case d-mtATPase could be solubilized with digitonin and visualized by BN-PAGE (Fronzes et al., 2006). Importantly these same cross-linked dimers could be isolated in the absence in mitochondria lacking subunit e or g indicating the importance of stator stalk interactions for mtATPase dimerization. There have been suggestions that IF1 may play a role in dimer formation. It has been demonstrated that bovine IF1 self-associates as an inhibitory dimer (Cabezon et al., 2000b) that can induce the dimerization of F1 particles (Cabezon et al., 2000a). A crystal structure F1 reconstituted with IF1 shows a dimeric structure (Cabezon et al., 2003). However, dimeric complexes could be isolated from the mitochondria of yeast cells lacking expression of Inh1p, the homologue of IF1 (Dienhart et al., 2002), or bovine heart submitochondrial particles in which were depleted of IF1 (Tomasetig et al., 2002). Although the role of IF1 in this respect remains controversial more recently depletion of IF1 from rat liver and bovine heart

28

Rodney J. Devenish et al.

sub-mitochondrial particles was found to decrease the dimer to monomer (D/M) ratio of mtATPase whereas reconstitution with recombinant IF1 restored partially the D/M ratio as determined using BN-PAGE (Garcia et al., 2006). Furthermore, 2-fold increased expression of IF1 in AS30D hepatoma mitochondria led to a 1.4-fold increase in D/M ratio, compared to mitochondria from normal liver cells having a sub-stoichiometric amount of IF1 (Garcia et al., 2006). A working model for IF1 in dimerization has been proposed accommodating data accumulated from crytallographic, cross-linking and EM studies. In this model, depicting the arrangement of IF1 in rat liver d-mtATPase, the C-termini of the two IF1 molecules are proposed to bend and cross at the dimer interface to interact with OSCP (Garcia et al., 2006). 3.3.3. Associations with non-mtATPase proteins We have discussed the homodimerization of mtATPase. Are there other proteins associated with the complex? This certainly appears to be the case based on the isolation of the ATP synthasome from rat liver mitochondria, a monomeric ATPase complex together with the adenine nucleotide carrier (ANC) and the phosphate carrier (PC) in a ratio of 1:1:1 (Ko et al., 2003). Subsequent EM analysis of the ATP synthasome indicated an increased dimension of F0 in the plane of the membrane sufficient to allow the docking of a putative heterodimer of ANC and PC. It is intriguing that the components of the ATP synthasome have not been recognized in the EM analyses of d-ATPase (see discussion above). The reasons for this discrepancy are not clear but may be related to differences in the properties of the detergents used in each study. CHAPS, and not digitonin, was used to solubilise the ATP synthasome. Nevertheless, it will be important to ultimately define the relationship of the components of the ATP synthasome to oligomeric forms of mtATPase. If the d-mtATPase interface involves the stator stalk, perhaps components of the ATP synthasome are located on an alternative interface. The stator stalk was not visualized in the ATP synthsome structure determined by EM (Chen et al., 2004). In a recent report BN-PAGE was used to identify two small proteins, denoted MLQ and AGP, associated with bovine mtATPase (Meyer et al., 2007). Interestingly, both proteins have been identified in different contexts: MLQ [or 6.8 kDa mitochondrial proteolipid (Terzi et al., 1990)] and AGP [diabetes-associated protein in insulin-sensitive tissue (DAPIT); Paivarinne and Kainulainen, 2001]. The functional significance, if any, of the association of these two proteins with mtATPase remains to be demonstrated. Intriguingly, the AGP protein has 13% sequence identity (16% similarity) with yeast subunit k, however functional homology has yet to be demonstrated. It is noteworthy that the sequence identity between yeast subunit h and bovine subunit F6 is only 14.5% and these two proteins have been demonstrated to be functional homologues (Velours et al., 2001).

29

Structure and Function of mtATPases

3.4. The arrangement of mtATPase in mitochondrial membranes 3.1.1. Model A model based on investigations in yeast showing the relative arrangement of subunits at the interfaces between complexes was proposed (Paumard et al., 2002b) and more recently updated (Fronzes et al., 2006) taking into consideration the results of cross-linking studies (see Fig. 1.3). Both versions of the model focus on the role of subunit-subunit interactions within the F0 sector. Oligomeric forms of mtATPase imply two distinct interfaces. Crosslinking and other data suggested that subunit b participates at one interface; the second interface, as proposed by Paumard et al. (2002b), involves both subunits e and g. Detergents such as dodecylmaltoside disrupt both interfaces, whereas digitonin at relatively high concentrations preserves the eg/eg interface and at low digitonin-protein ratios (0.751 g  g1) also preserves the b/b interface. The results of FRET studies also suggest two interfaces (Gavin et al., 2005). Although the two distinct interfaces are proposed they are no longer thought to be independent as previously suggested. Subunits e and g stabilize the b/b dimer interface when it is exposed to detergents and are directly involved in the other interface between dimers that make up the oligomer. Rather the two monomers are viewed as interacting through their peripheral stalk structures particularly B

A Catalytic head

Su 9 digomer

Su g Su e

Su 6, 8 4, i, f

Dimer

Oligomer

Figure 1.3 A model for the supramolecular organization of the ATP synthase. (A) Organization of the dimerization interface in the membrane seen from the intermembrane space perpendicular to the main axis of the ATP synthase. The area in gray represents the cross section of the subunit c ring and of the F0 domain. The F1 sector is represented as a dashed line.The dimerization interface involves a large part of the Fo sector and may also extend to components localized in the matrix, such as subunit h (dotted line). Subunits e and g are localized at the periphery of the dimerization interface and stabilize it. In the dimer, subunits e and g are in close contact on each side of the dimer. (B) Oligomerization of ATP synthase may occur by interaction between the e þ g interfaces. [Reprinted with permission from Fronzes, R.,Weimann,T.,Vaillier, J.,Velours, J., Brethes, D.The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits. Biochemistry 45: 6715-6723. Copyright (2006), American Chemical Society.]

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Rodney J. Devenish et al.

subunits b and i at the membrane, and subunit h extrinsic to the membrane (Fronzes et al., 2006). As discussed above in mammalian mitochondria IF1 may also be involved in dimerization (Garcia et al., 2006). The adjacent dimers are tilted to prevent clashing of F1 headpieces, consistent with EM images (see Section 3.4.3). 3.1.2. A key role for subunit e? The available data could be taken to indicate that subunit e plays a central role in controlling oligomerization of mtATPase and mitochondrial function. It has been shown in yeast cells that regulated expression of subunit e can reversibly control the formation of mitochondrial cristae (Arselin et al., 2004). In mammalian cells and tissues, the expression level of subunit e has been linked to physiological stimuli such as diet and hypoxia (Levy and Kelly, 1997; Swartz et al., 1996), It has been suggested that subunit e could be involved in the Ca2þ-dependent regulation of mtATPase activity. Residues 34-65 of the subunit e are homologous with the Ca2þ-dependent tropomysin-binding region for troponin T (Arakaki et al., 2001). As discussed above the coiled-coil domains of subunit e contribute to the stability of d-mtATPase and oligomers as determined by BN-PAGE analysis (Bornhovd et al., 2006). However, the deletion of these domains did not result in the loss of mitochondrial cristae, but did result in a loss of mitochondrial membrane potential. Consequently, it was proposed that oligomerization is essential for maintenance of bioenergetically fully competent mitochondria; a drop in membrane potential would precede alterations in cristae morphology. 3.1.3. Visualisation of dimers As presented above, a considerable body of biochemical evidence exists in favor of a stable close interaction between individual mtATPase complexes. Remarkably, such interactions were first suggested to occur based on the evidence obtained from the pioneering rapid-freeze deep-etch EM of P. multimicronucleatum by Allen et al. (1989) conserving the close juxtaposition of F1 sectors. More recently, a number of single particle EM structures have been produced for d-mtATPase from a number of sources including bovine heart (Minauro-Sanmiguel et al., 2005), the colourless algae Polytomella (Dudkina et al., 2005) and yeast (Dudkina et al., 2006b). Rows of d-mtATPase dimers have been visualized using high-resolution atomic force microscopy in native mitochondrial inner-membranes isolated from the yeast Saccharomyces cerevisiae (Buzhynskyy et al., 2007). In each case, d-mtATPase was solubilized using digitonin and enriched using gradient ultracentrifugation. The structures visualized have revealed some important features in common. In each case the dimer interface was

Structure and Function of mtATPases

31

seen to be formed predominantly by the F0 domains in such way that the long axes of the two complexes are arranged at an angle. However, distinct differences exist in each of the published structures. Although the dimer in each case displayed a conic arrangement, the angle subtended between the individual complexes was estimated to be quite different in each case: bovine, 40
; Polytomella, 70
and yeast, 90
. The larger angles observed for yeast and Polytomella do not allow direct contact between the F1 headpieces as seen for the bovine structure. Since there was no evidence for the presence of the peripheral stalk, visualized in other EM representations of the bovine complex (Rubinstein et al., 2003), the dimeric interaction, based on some density in the images, was postulated to be mediated by IF1 (Minauro-Sanmiguel et al., 2005; Garcia et al., 2006). By contrast, EM images of the Polytomella complex clearly show the presence of the peripheral stalk sandwiched between each complex, but again interactions appear to be mediated by the F0 sector alone. MASP, a protein unique to the Polytomella complex and believed to be responsible for the high stability of the dimer is presumed to be located towards the top of the structure, but evidently not bridging the dimer (Dudkina et al., 2005). The apparent position of the of the peripheral stalk sandwiched between the individual complexes such that they interact through F0 suggests the these structures represent the dimer proposed on the basis of biochemical studies, in which membrane anchored subunits (b, together with e and g) are promoting or stabilising interactions (Fronzes et al., 2006; Paumard et al., 2002b). However, for yeast in addition to dimers subtending the large 90
angle, examples subtending a considerably smaller angle of 35
and having a smaller F0 contact area were also observed (Dudkina et al., 2006b). It was proposed that these ‘‘pseudo’’ dimers represent broken ‘‘true’’ dimers. Although they might represent nonspecific interactions that occur upon solubilisation of mitochondrial membranes, the intriguing suggestion has been made that these pseudo dimers represent complexes associated by interactions at the second interface present in the oligomeric form (Fig. 1.4). It should be noted that the working models based on biochemical data were developed by analysis using BN-PAGE or CN-PAGE and represent alternative structures to those visualized by EM. Although most likely representing a considerable technical challenge the recovery of stable oligomers (e.g., tetramers or hexamers) for analysis by EM would provide the possibility of visualizing both interfaces in the oligomeric form.

3.5. The role of mtATPase oligomerisation What purpose do mtATPase dimers/oligomers serve in mitochondria? Bacteria, apparently without having a requirement for ATP synthase dimers or oligomers, remain able to synthesise ATP in a highly regulated and

32

Rodney J. Devenish et al.

True-dimer

Pseudo-dimer

Figure 1.4 A model for the arrangement of ATP synthases dimers into multimers. Oligomers consist of dimeric ATP synthases. The oligomers can break down by detergent incubation into ‘‘true-dimers’’ or into ‘‘pseudo-dimers’’. The latter actually consist of two monomers from the neighboring dimers, symbolized by a blue and purple set of a3b3 subunits. Ochre and bright green densities symbolize dimer- and interdimer specific subunits, respectively. (Reprinted by permission of the Federation of the European Biochemical Societies from Characterization of dimeric ATP synthase and cristae membrane ultrastructure from Saccharomyces and Polytomella mitochondria, by Dudkina N. V., Sunderhaus, S., Braun, H. P., Boekema, E. J., FEBS Letters, 580: 3427^3432, Copyright 2006.)

efficient manner. It appears that the requirement to form oligomers may be specific to mitochondrial complexes. Investigations in yeast suggest that mitochondrial OXPHOS function is not greatly compromised in mitochondria unable to form dimers or oligomers. Thus, the specific activity of mtATPase was not affected in cells that do not have stable dimers or oligomers (Arnold et al., 1998; Boyle et al., 1999; Paumard et al., 2002b; Soubannier et al., 2002) and the uncoupled respiration rates and ATP/O ratios were not altered in these mutants (Boyle et al., 1999; Paumard et al., 2002b; Soubannier et al., 2002). Nevertheless, although capable of respiratory growth, such strains do display growth defects. The structure of the mitochondrion is now considered to be more complex than once thought. It is firmly established that the ‘‘infolds,’’ or cristae, of mitochondrial membrane are not random and are topologically complicated. The cristae are connected to the inner boundary membrane (IBM, that part of the inner membrane that runs parallel to the outer membrane) by narrow tubes called cristae junctions. Immunolabelling and transmission EM studies of bovine heart mitochondria (Gilkerson et al., 2003), together with application of more recent fluorescence imaging techniques in live yeast cells (Wurm and Jakobs, 2006), have led to the conclusion that nonuniform protein distributions exist within the IM indicating that the IBM and cristae are functionally distinct compartments.

Structure and Function of mtATPases

33

In excess of 90% of mtATPase and respiratory complexes (III, IV) were found enriched in cristal membranes, whereas proteins such as the TIM23 complex and presequence translocase motor were found enriched in the IBM (Wurm and Jakobs, 2006). Thus, the evidence supports cristae as being a regulated sub-mitochondrial compartment specialized for ATP production. The projections in the matrix space seen in EM images have long been known to represent the F1 sector of mtATPase (Racker et al., 1965). More recently, using freeze-fracture techniques, these projections were seen to be arranged in a highly ordered double-row in the tubular cristae of P. multimicronucleatum (Allen et al., 1989). It was these morphological observations that led to the first clear suggestion that the arrangement of ATP synthase complexes might have a role in promoting the tight curvature (50 nm diameter) of the membrane to form tubular cristae. Thus, the view was developed that ATP synthase, the molecular motor responsible for much of cellular energy generation, also appears to have an active role in the modelling of the membrane in which it resides. This concept gained further acceptance after the EM observations made of yeast lacking expression of subunits e and g and therefore presumably lacking oligomeric ATP synthase (Paumard et al., 2002b). In such cells normal cristae were absent and replaced by numerous digitations and onion-like structures containing many concentric membranes that was shown to have protein markers characteristic of the inner membrane. Given the apparent importance of cristae structure to mitochondrial metabolism it was remarkable to find that such yeast cells remained respiratory competent. The results of single-particle EM experiments indicate that interactions between the membrane regions of F0 subunits may be responsible for forming the tight curvature of the membrane. However, there is evidence to suggest that the F1 sector may also play role in cristae formation. Yeast strains lacking expression of proteins that mediate F1 assembly (e.g., Atp11p or Atp12p) lack F1 a3b3 hexamers and are devoid of mitochondrial cristae (Lefebvre-Legendre et al., 2005). It is not clear how this effect is mediated, but it may be related to the binding of other proteins to F1. In the case of mammalian cells IF1 may fulfil this role. However, EM projections of the ‘‘dimers’’ recovered from yeast mitochondria clearly show that the F0 sector is curved in those cases where one, or both of the F1 sectors was missing. The d-mtATPase isolated from bovine heart and visualized by single particle EM showed an interface formed by contacts on both the F0 and F1 domains (Minauro-Sanmiguel et al., 2005). A cross-bridging protein density was resolved which connects the two F0 domains on the inter-membrane space side of the membrane. On the matrix side of the complex, the two F1 moieties are connected by a protein bridge, which was attributed to the IF1 inhibitor protein. Close contact between neighboring mtATPase monomers is not sufficient to form and maintain cristae. It is clear from the results of in vivo

34

Rodney J. Devenish et al.

cross-linking experiments that a precise arrangement of ATP synthase complexes is required (Gavin et al., 2004). In this study enforced oligomerization of yeast mtATPase was achieved by expressing subunit g fused to DsRed an obligatory tetrameric fluorescent protein. The mitochondria of such cells resembled those found in cells lacking expression of subunit e in having numerous onion-like structures. Similar results also were obtained using the dimeric HcRed (Gong, Devenish and Prescott; unpublished results). These studies suggest that cross-linking of this type may disrupt the oligomeric array by causing localized distortion. It must be noted that other proteins are involved in cristae formation. The IM protein mitofilin, when deficient in heart muscle, leads to large membrane sworls. These structures were found to be composed of a complex, interconnected network of membranes totally lacking tubular connections to each other, or to the peripheral IM ( John et al., 2005). Reduced expression of the dynamin-like protein OPA1 in humans also causes disorganization of cristae (Griparic et al., 2004). The question remains as to why mitochondria form cristal subcompartments. Yeast cells expressing subunit e lacking one or more of its coils (from the C-terminal coiled-coil motif ) showed normal cristae, but had low membrane potential such that mitochondria could not accumulate membrane potential dyes (Bornhovd et al., 2006). It was concluded that membrane potential was dependent of the oligomeric state of mtATPase. It was postulated that increased plasticity of the mitochondrial membrane led to disruption of membrane micro-domains normally maintained by mtATPase oligomers. This in turn would result in altered cooperation between complexes such as mtATPase and ANC and consequently reduced flux through the respiratory chain arising from negative feedback effects and lower membrane potential. Although the relationship of ANC to mtATPase dimers and oligomers has yet to be established, ANC and PC have been reported to be part of the ATP synthasome (Chen et al., 2004; see Section 3.3.3).

3.6. Is oligomerization regulated in vivo? The mitochondrion is a highly dynamic structure. If dimerization and higher order structures are indeed involved in cristae formation and mitochondrial dynamics, we might expect both their formation and dissolution to be under tight control. However, such evidence is limited at present. Studies in yeast that manipulated membrane potential or cellular energy charge apparently did not alter the ratio of d-mATPase to m-ATPase observed using BN-PAGE (Arnold et al., 1998). On the other hand, cells isolated from patients with Leigh syndrome (T8993G/T8993C) mutation in the mitochondrially encoded ATP6 gene for subunit a) showed significant inhibition (60%) of ATP synthase

Structure and Function of mtATPases

35

activity, but increased amounts of oligomeric forms over the monomeric form compared with normal cells. This result suggests that the oligomeric form is regulated in vivo and apparently increased in pathological conditions presumably to overcome the cellular energetic defect (Cortes-Hernandez et al., 2007). Furthermore, mutations in ATP6 can cause a marked reduction in ATP production in neuronal mitochondria which have a very unusual inner membrane topology of rounded cristae compartments contiguous with flattened lamellar regions of the same membrane. A possible explanation for this highly unusual topological transition is that dimerization of ATP synthase is normally inhibited in flat, lamellar cristae and that the ATP6 mutation somehow weakens this inhibition, allowing mtATPase dimerization and, consequently, increased membrane curvature. The central role that subunit e appears to have in promoting dimer formation makes it a good candidate for a regulatory role in determining oligomerization. In support of this notion is the observation that by placing the expression of subunit e under control of the tetracycline promoter in yeast formation of mtATPase dimers could be reversibly depleted with characteristic alterations in cristae morphology (Arselin et al., 2004). Other evidence in support of a regulatory role for subunit e is that the level of its mRNA in C2C12 myotubes and myoblasts was found to be regulated in response to oxygen availability (Levy and Kelly, 1997). Although ATP synthase oligomeric structure can be correlated with cristae morphology, it is not clear that it alone is the key determinant for formation and maintenance of the mitochondrial cristae. Other factors clearly have a role to play. For example, expression of tBid caused a remodelling of cristal membranes (Scorrano et al., 2002), and reversal of curvature can occur in cardiolipin-containing membrane phases (Epand et al., 2002), suggesting that remodelling of the membranes is lipid mediated.

3.7. Supramolecular structures involving other respiratory complexes? In addition to ATP synthase discussed in detail above, the oxidative phosphorylation (OXPHOS) system comprises four oxidoreductases: NADH dehydrogenase: (complex I), succinate dehydrogenase (complex II), cytochrome c oxidase (complex III) and cytochrome oxidase (complex IV). High-resolution structures are available for complexes II, III and IV and in part for complexes I and V (ATP synthase). However, despite the detailed knowledge of the structures of the individual complexes comparatively little is known about their organization into supramolecular structures. Evidence exists to support two different views of the mitochondrial respiratory chain either as (i) a fluid state in which the complexes are free to diffuse in the plane of the manner membrane undergoing random

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Rodney J. Devenish et al.

collisions (Hackenbrock et al., 1986) or (ii) a solid state in which the complexes are more ‘‘rigidly’’ associated. The former view is supported by results of classical fractionation approaches developed some 40 years ago that led to the isolation individual complexes that are capable of carrying out their apparent physiological function. The latter view is supported by data showing increased complex activities when reconstituted in defined stoichiometry. Furthermore, one or more complexes can be isolated in association (Schagger and Pfeiffer, 2000). These two views represent different snapshots of the same system and, the final correct picture will have elements of both reflecting the dynamic nature of the mitochondrion. Interestingly, supercomplexes between mtATPase and other respiratory complexes do not appear to exist.

4. Extra-Mitochondrial Expression of F1F0-ATP Synthase 4.1. Introduction In recent years evidence for extra-mitochondrial expression of F1F0 ATP synthase has come from several laboratories. A number of reports provide evidence that complexes are located on the surface of various mammalian cells types, including endothelium, hepatocytes, adipocytes, keratinocytes and tumor cells. F1F0 ATP synthase components have been identified as cell-surface receptors for ligands in studies carried out on angiogenesis, lipoprotein metabolism, innate immunity, hypertension or regulation of food intake. The cell types, ligands and components of F1F0 ATP synthase identified have been comprehensively listed by Champagne et al. (2006) and Chi and Pizzo (2006a). However, as noted by Wahl et al. (2005) an exhaustive survey of cell types and tissues for evidence of cell surface ATP synthase components has not yet been made. Here we will refer to the complex as external ATP synthase (eAS). In some instances the function of eAS relates to a single report of a particular ligand interaction. However, in some instances a series of studies have contributed towards the development of the understanding of the specific ‘‘new’’ function of eAS, which is not the generation of cellular ATP so effectively carried out by the mitochondrial enzyme complex. In addition, one F0 component of F1F0 ATP synthase, F6 (usually designated CF6 in this context), again ectopically expressed, has been recognized as having function in its own right. In no case has an eAS complex been purified from the plasma membrane and all components of a fully assembled F1F0 ATP synthase shown to be present. The existence of fully assembled complexes generally has been inferred from the identification of an F1 component, most usually subunit b, and measurement of ATP hydrolysis and or ATP synthesis activity. If ATP

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synthesis is being correctly attributed to enzymatic activity of the complex under study then the expectation is that it must be fully assembled and membrane anchored, since only F1 functionally coupled to F0 can synthesize ATP. Also if F0 components were not present then F1, even if competent for ATP hydrolysis, would not be retained at cell surface since none of the F1 components are normally membrane anchored. Although the F0 subunits, b, d, e, F6 and O(SCP?), were shown to be protein components of detergent-insoluble lipid rafts isolated from rat liver (Bae et al., 2004), only recently have the F0 components, subunits d and OSCP, been directly shown to be present on the plasma membrane (PM) of an osteosarcoma cell line (Yonally and Capaldi, 2006). Definitive experiments remain to be performed to establish that cell surface eAS complexes and mtATPase have identical or closely similar arrangements of subunits. As pointed out by Champagne et al. (2006), the structure and subunit composition of eAS may differ between cell types and might possibly be, under different conditions, related to their different specific functions. How are eAS complexes distributed on membranes? In most instances this is not clear. There is no information as to whether eAS might be arrayed in dimeric or oligomeric forms. More recently, as discussed by Chi and Pizzo (2006a), it has emerged that eAS is found in caveolae/lipid rafts; see for example the studies of Ko and colleagues (Bae et al., 2004; Kim et al., 2004, 2006). In this context two recent reports are of interest. The first concerns regulation of HDL endocytosis where endothelial cells exposed to free cholesterol exhibited a subsequent increase in the concentration of caveolin-1 and the F1 subunit b within endothelial cell caveolae (Wang et al., 2006), together with an increase in the release of ATP from cells. The second, presents evidence suggesting that the localization of eAS to caveolae/lipid rafts is important for shear stress-induced ATP release by vascular endothelial cells (Yamamoto et al., 2007). An important implication of eAS localization to caveolae recognized by Chi and Pizzo (2006a) is the creation of microenvironments on the cell surface in which eAS, its binding partners or inhibitors plus substrates for enzymatic activity such protons, ADP and ATP can be concentrated. These authors give the example of a cell facing an acidic extracellular microenvironment where the eAS would be required to pump protons against the gradient in order to regulate intracellular pH. However, should the caveolar compartment have relatively lower proton concentration, then this would not be the case.

4.2. How might F1F0 ATP synthase get to the plasma membrane? The fully assembled multi-subunit mtATPase complex is anchored in, and assembled on, the inner mitochondrial membrane. The complex contains subunits made on cytosolic ribosomes and targeted to mitochondria, but

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also subunits encoded on mtDNA and made inside the mitochondrion (Table 1.1). As far as we are aware there is no evidence for the existence of additional copies of nuclear encoded mtATPase subunit genes that could specifically encode eAS components, or for mRNAs allowing for alternative translation products (i.e. having ER signal sequence rather than mitochondrial targeting signal). Moreover, such a scenario requires that the mitochondrial encoded subunits [in mammals subunits A6L (yeast subunit 8) and a (yeast subunit 6)] would also be nuclear encoded. [One of the reasons often suggested for the retention of these genes in the mitochondrial genome is to facilitate the synthesis and membrane insertion of the extremely hydrophobic subunits they encode (Claros et al., 1995).] If such genes did exist then the expectation would be for mRNAs to be translated by ER localized ribosomes, especially to facilitate the synthesis and membrane incorporation of hydrophobic membrane subunits such as subunits a and A6L. In such a scenario, the complex presumably would be assembled on the luminal surface of the ER and trafficked via vesicles through the Golgi into secretion vesicles that fuse with the PM thereby incorporating ATP synthase. Currently there is little evidence for the trafficking of complexes in such vesicles that are delivered to PM, although it has been recently reported that subunit a reaches the cell surface in an N-glycosylated form via the secretory pathway (Schmidt et al., 2007). Are there alternative pathways? Soltys and Gupta (1999, 2000) proposed specific export mechanisms by which proteins might exit directly mitochondria via channels, allowing them to reach specific extramitochondrial sites. The use of channels suggested by these authors for individual mitochondrial proteins (none of which were mtATPase components) would be unlikely to accommodate fully assembled mtATPase complexes. Transport of complexes assembled in mitochondria to the PM might occur by fusion events between the organelle and plasma membrane, facilitated by the dynamic fusion and fission of the mitochondrial network (Heath-Engel and Shore, 2006). There is no evidence for direct interaction of mitochondria and PM. In any case such interactions would be complicated by questions of membrane topology of the double membrane mitochondria. Fusion of the outer mitochondrial membrane with the PM would not result in complexes on the cell surface since most (94%) of the mtATPase complexes are located on cristae membranes (Gilkerson et al., 2003). If fusion occurred at contact sites (between inner and outer mitochondrial membranes) and the plasma membrane then it might be possible to have complexes on the external PM surface. Perhaps more feasible is direct interaction between the highly convoluted ER network with the highly dynamic mitochondrial network. Hypothetical pathways for export of matrix proteins to the ER have been presented by Soltys and Gupta (1999). If mitochondrial complexes could be ‘‘captured’’ within ER membranes, perhaps being sorted into specialized membrane regions (lipid rafts?), then subsequent delivery to the cell surface

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would require trafficking from the ER to the Golgi and presumably vesicle budding from the Golgi and finally vesicle fusion with the PM. A subdomain of the ER, the mitochondria-associated membrane (MAM) forms stable complexes with mitochondria, and in yeast is involved in phosphatidylserine traffic (see, for example, Choi et al., 2006). Furthermore sites of interaction between PM and ER in yeast (PAMs) have beeen characterized (Pichler et al., 2001). Thus mitochondria could associate with PAMs through MAMs. While MAMs could be used to capture and transport eAS, at present, the only available evidence supporting sequestration of mitochondria in membrane vesicles relates not to trafficking to the PM, but to turnover by autophagy that occurs following fusion with the vacuole (yeast) or lysosomes (mammals) (Mijaljica et al., 2007). Clearly, the mechanism by which complexes reach the plasma membrane remains elusive and is worthy of deliberate attention as a biological question in its own right. We now consider some aspects of the reported functions of eAS in the context of the structure and function.

4.3. The function of coupling factor 6 as a vasoconstrictor: Detachment and reattachment of an F0 component of eAS? A historical review of how we came to understand the cardiovascular, function of coupling factor 6 CF6, has been provided by Watts (2005). In summary, the current view is that CF6 circulates and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. In this context CF6, localized on the surface of the vascular endothelium, is released into the systemic circulation in response to pathological stimuli such as shear stress (Osanai et al., 2001a,b) and tumor necrosis factor alpha (TNF-a) (Sasaki et al., 2004). Other conditions related to cardiovascular dysfunction also result in increased circulating CF6 levels (Ding et al., 2004; Osanai et al., 2003a,b). Most recently the plasma CF6 level was shown to be markedly increased in patients with diabetes mellitus and that this increase could be positively correlated with disease severity (Li et al., 2007a). What is the source of the CF6 that is released into the circulation? It could be ‘‘free’’ on the PM or be ‘‘sequestered’’ by normal association as a component of eAS complexes. Within mtATPase CF6 is an important component of the stator stalk. Its functional homologue in yeast (subunit h) is essential for energy transduction (Velours et al., 2001; see section 2.4.2.4). As discussed above the N-terminal end of the predominantly a-helical CF6 subunit extends from near the top, to about the mid-point, of the F1 domain (Dickson, 2006). Significant contacts are made with subunits b, d and OSCP as well as the F1 domain surface. The available evidence suggests that CF6 stimulates cell surface ATPase activity of human umbilical vein endothelial cells (HUVECs) which can be

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blocked with efrapeptin, an ATP synthase inhibitor. On the basis of radioligand-binding studies Osanai et al. (2005) proposed the F1 subunit b as the receptor for CF6 on the PM of HUVECs. However, b-subunit antibody inhibited CF6 function by only 50%, suggesting other potential sites of action for CF6. It remains unclear how stimulation of ATPase by CF6 might occur through the b subunit. The questions that need to be addressed include the following. What is the binding site for CF6? Is CF6 binding to complexes lacking this subunit to ‘‘reconstitute’’ them? This seems unlikely as in yeast mtATPase complexes lacking subunit h the F1 sector is uncoupled from F0 and subunit 6 (a) is missing (Arselin et al., 1996). Reconstitution would thus require more than just CF6 fitting back into an ‘‘empty niche’’ on an otherwise fully assembled complex. In a similar vein it seems highly unlikely that CF6 released from eAS complexes by shear stress or other means just disassociates and then re-binds. Alternatively, a separate pool of free CF6, not associated with eAS, might exist on the cell surface and is it this CF6 that is released by shear stress or other stimuli. For reasons stated above it would seem unlikely that this CF6 would bind to eAS complexes lacking CF6 (i.e., reconstitution), rather than binding at a second site on fully assembled eAS complexes. However this remains an open question. The recent observation that the exposure of HUVECs to high glucose level results in a significant increase of CF6 expression and release, mediated by PKC and p38 MAPK activation, but antagonized by insulin treatment (Li et al., 2007b) starts to build a more detailed picture of signaling pathways involved CF6 activity, but does not resolve any of the questions concerning binding sites for CF6.

4.4. Multiple receptor functions of subunit b As well as being suggested as the binding site for CF6, subunit b is also identified as a putative receptor for at least three other ligands. These (and the proposed associated functional role) are: 

Enterostatin (Berger et al., 2002, 2004; Park et al., 2004)—regulation of fat intake.  HDL apolipoprotein A-I—contributes to HDL endocytosis (Martinez et al., 2003, 2004; Jacquet et al., 2005; Fabre et al., 2006; Zhang et al., 2006).  Angiostatin (an angiogenesis inhibitor derived from plasminogen)— regulates surface ATP levels, thereby modulating endothelial cell proliferation and differentiation (Moser et al., 1999, 2001). More recently, Veitonmaki et al. (2004) have shown that kringle 1-5, a different fragment of plasminogen with increased angiostatic activity compared to angiostatin, also binds to eAS (on bovine capillary cells) to trigger caspase-mediated apoptosis.

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At first it may seem improbable that these ligands all bind subunit b, but these ligands will function specifically in different cell and tissue types. There is little understanding of how ligand binding elicits the biological response in terms of eAS complex function. If these ligands all do bind subunit b, then they could influence ATP synthesis or hydrolysis by affecting a change in the ATP catalytic sites. The structure of subunit b is such that there is a hinge region whose conformation that could be influenced by ligand binding. These mechanistic questions remain to be addressed. For each of the ligands listed above (and for CF6), the reported biological effect of eAS relies on effects on enzymatic activity. In contrast there is another example involving cell surface subunit b which apparently does not involve enzymatic activity of the F1 moiety of eAS, but rather the recognition of a complex of F1 and apolipoprotein A-1 on tumor cells by a subset of gd T-lymphocytes that promotes innate tumor cell recognition and lysis (Scotet et al., 2005). Subunit b has also been proposed to serve as a ligand in NK cell-mediated cytotoxicity of tumor cells (Das et al., 1994). What is the biological effect in terms of eAS function elicited by binding of the ligand? Each of the ligands probably elicits different responses although this is not entirely clear as this question has not been explored in depth in all cases. In the case of enterostatin it is inhibition of ATP synthesis, for apoA-I it is inhibition of ATP hydrolysis, for angiostatin it is apparently inhibition of both ATP hydrolysis and synthesis leading to overall suppression of endothelial-surface ATP metabolism (Kenan and Wahl, 2005; Moser et al., 1999, 2001). The field would benefit from a more systematic and complete determination of enzymatic activities in response to ligand binding in the different cell systems. Even for a specific ligand there have been notable differences in determination of levels of enzymatic activity. For example, extracellular ATP synthesis on HUVECs was examined in detail by Arakaki et al. (2003), who measured a high ATP synthesis activity (50 nmol/min/106 cells) compared with that found by Moser et al. (2001) (about 40 pmol/min/106 cells). The reason for this significant difference remains unclear (Arakaki et al., 2003). The best-studied system is endothelial cell regulation by angiostatin (Chi and Pizzo, 2006a; Kenan and Wahl, 2005). As stated by Chi and Pizzo (2006a,b) the current model is that endothelial cells utilize eAS to generate extracellular ATP, and concomitantly for proton extrusion, as a means of regulating intracellular pH. They envisage such a scenario to be especially applicable to the tumor environment where the extracellular pH might be as low as, or even lower than, pH 6.7 and angiostatin is generated in vivo. These authors also present the evidence for angiostatin’s role as an antitumourigenic agent through a mechanism implicating eAS.

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4.5. The inhibitor action of IF1 can be demonstrated for eAS complexes The activity of IF1 on eAS activity has been assessed in two of the systems listed above. First Martinez et al. (2003) showed that exogeous IF1 inhibited ATP hydrolysis activity of eAS and HDL endocytosis. Burwick et al. (2005) assessed the effect of recombinant human IF1 on HUVEC eAS to determine whether it could serve as an angiostatin mimetic. Exogenous IF1 was found to inhibit ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. This finding agrees with the known function of IF1 in mitochondria (see Section 2.3.3). Thus IF1 was proposed to serve a protective function in the tumor microenvironment through the conservation of cellular ATP when pH is low (Burwick et al., 2005). Furthermore, it was demonstrated that angiostatin blocks IF1 binding to eAS, suggesting a relationship between the binding sites of IF1 and angiostatin on the complex. Such an observation is compatible both with angiostatin binding to subunit b and the IF1 binding site being largely to subunit b (Cabezon et al., 2000a,b). That angiostatin exerts its antiangiogenic effect, at least in part, by inhibiting IF1 binding to eAS has been proposed by Burwick et al. (2005) to explain why angiostatin has a stronger antiangiogenic effect at low pH than at physiological pH. Under the latter pH condition, conservation of ATP by IF1 inhibition of ATP hydrolysis would not be required, whereas in the low pH, low oxygen microenvironment of tumors, cells would have a greater need to conserve ATP through IF1 action (Burwick et al., 2005). These results raise the obvious question as to whether there is any evidence for IF1 on the cell surface. Burwick et al. (2005) demonstrated that endogenous IF1 is present on HUVEC surface, a finding confirmed by Cortes-Hernandez et al. (2005). The latter authors also showed that TNF-a decreased the level of subunit b and increased the amount of IF1. Further, because their studies showed that the ratio of IF1 to subunit b exhibited significant variation, they suggested that the function of IF1 in the PM of endothelial cells may not be limited to regulation of catalysis (in which case the ratio should be maintained at 1:1), although no suggestions of additional functions were made.

5. Concluding Remarks The recent structure of the peripheral stalk subunits reported by Walker and colleagues (Dickson et al., 2006) is a significant advance toward the ultimate goal of having a complete structure of the mtATPase complex. We still lack a high-resolution structure for subunits a and c assembled together, which potentially would enable us to fully dissect proton channel

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function. Furthermore, in view of our continually developing understanding of the dimeric and oligomeric forms of the complex in the inner mitochondrial membrane a high-resolution structure that includes the additional, nonproton channel, F0 membrane subunits and ‘‘dimer specific subunits’’ would be especially informative. Based on the present models, higher order supercomplexes of mtATPase may exist in the membrane. However, it may be difficult to study these in detail due to the difficulty in achieving their solubilization without disruption. It will be important to apply other techniques to investigate such supercomplexes. It may be easier and more informative to study these structures in vivo using fluorescence techniques as exemplified by the approaches utilised by Gavin et al. (2003, 2005). Such approaches also have the potential to provide spatial information and, more importantly, data on the assembly and possible dynamic nature of the supramolecular interactions. There is a weight of evidence supporting functional roles for eAS and cell surface CF6. However, there remain several questions related to biogenesis of the complexes on the cell surface that need to be addressed. It is noteworthy that ATP synthesis on the external surface of vascular endothelial cells attributed to eAS has been challenged. Yegutkin et al. (2001) presented evidence that ATP synthesis on HUVECs resulted from the activity of adenylate kinase and nucleoside diphosphokinase rather than eAS. More recently, Quillen et al. (2006) showed that a small amount of adenylate kinase activity alone could account for the observed extracellular ATP synthesis of HUVECs.

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Wilkens, S., and Capaldi, R. A. (1998b). Electron microscopic evidence of two stalks linking the F1 and F0 parts of the Escherichia coli ATP synthase. Biochim. Biophys. Acta 1365, 93–97. Wilkens, S., Borchardt, D., Weber, J., and Senior, A. E. (2005). Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy. Biochemistry 44, 11786–11794. Wilkens, S., Rodgers, A., Ogilvie, I., and Capaldi, R. A. (1997). Structure and arrangement of the delta subunit in the E. coli ATP synthase (ECF1F0). Biophys. Chem. 68, 95–102. Wilkens, S., Dahlquist, F. W., McIntosh, L. P., Donaldson, L. W., and Capaldi, R. A. (1995). Structural features of the epsilon subunit of the Escherichia coli ATP synthase determined by NMR spectroscopy. Nat. Struct. Biol. 2, 961–967. Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997). Solution structure of the N-terminal domain of the delta subunit of the E. coli ATP synthase. Nat. Struct. Biol. 4, 198–201. Wilkens, S., Zhou, J., Nakayama, R., Dunn, S. D., and Capaldi, R. A. (2000). Localization of the delta subunit in the Escherichia coli F(1)F(0)-ATP synthase by immuno electron microscopy: The delta subunit binds on top of the F(1). J. Mol. Biol. 295, 387–391. Wittig, I., and Schagger, H. (2005). Advantages and limitations of clear-native PAGE. Proteomics 5, 4338–4346. Wittig, I., and Schagger, H. (2007). Electrophoretic methods to isolate protein complexes from mitochondria. Methods Cell Biol. 80, 723–741. Wittig, I., Carrozzo, R., Santorelli, F. M., and Schagger, H. (2006). Supercomplexes and subcomplexes of mitochondrial oxidative phosphorylation. Biochim. Biophys. Acta 1757, 1066–1072. Wurm, C. A., and Jakobs, S. (2006). Differential protein distributions define two subcompartments of the mitochondrial inner membrane in yeast. FEBS Lett. 580, 5628–5634. Yamamoto, K., Shimizu, N., Obi, S., Kumagaya, S., Taketani, Y., Kamiya, A., and Ando, J. (2007). Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells. Am. J. Physiol. Heart Circ. Physiol. 293, H1646–H1653. Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., Jr., and Itoh, H. (2001). Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase. Nature 410, 898–904. Yegutkin, G. G., Henttinen, T., and Jalkanen, S. (2001). Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions. FASEB J. 15, 251–260. Yonally, S. K., and Capaldi, R. A. (2006). The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study. Mitochondrion 6, 305–314. Yoshida, Y., Sato, T., Hashimoto, T., Ichikawa, N., Nakai, S., Yoshikawa, H., Imamoto, F., and Tagawa, K. (1990). Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein. Eur. J. Biochem. 192, 49–53. Zhang, L., Meyers, C. D., Kamanna, V. S., and Kashyap, M. L. (2006). Niacin inhibits b chain ATP synthase cell surface expression in HepG2 cells: Mechanistic implications for raising HDL. Arterioscler. Thromb. Vasc. Biol. 26, e53.

C H A P T E R

T W O

Role of Proteasomes in Cellular Regulation Irina M. Konstantinova,* Anna S. Tsimokha,* and Alexey G. Mittenberg* Contents 1. Introduction 2. Proteasome Structure and Catalytic Activities 2.1. The core particle, 19S regulatory particle, alternative regulatory particles 2.2. Catalytic activities of proteasomes 3. Function of Proteasomes in Cell Proliferation, Differentiation, Apoptosis and Immune Response 3.1. Cell cycle control 3.2. Roles in differentiation and development 3.3. Proteasomes and apoptosis 3.4. Proteasomes and immune response 4. Modes of Regulation of Proteasome Activities in the Cell 4.1. Modulation of proteasome composition 4.2. Regulation of proteasome abundance in the cell and cellular compartments 5. Reprogramming of Proteasomes at Immune Response, Differentiation and Apoptosis 5.1. Changes of proteasome at immune response 5.2. Proteasome reprogramming at differentiation 5.3. Apoptosis-induced changes of proteasomes 6. Proteasomes in Regulation of Different Levels of Gene Expression 6.1. Action at multiple stages of transcription process 6.2. Participation in the regulation of posttranscriptional stages of gene expression 7. Concluding Remarks Acknowledgments References

*

60 61 61 63 65 65 66 68 74 76 76 92 96 97 98 98 100 100 103 105 106 106

Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00602-3

#

2008 Elsevier Inc. All rights reserved.

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Abstract The 26S proteasome is the key enzyme of the ubiquitin-dependent pathway of protein degradation. This energy-dependent nanomachine is composed of a 20S catalytic core and associated regulatory complexes. The eukaryotic 20S proteasomes demonstrate besides several kinds of peptidase activities, the endoribonuclease, protein-chaperone and DNA-helicase activities. Ubiquitinproteasome pathway controls the levels of the key regulatory proteins in the cell and thus is essential for life and is involved in regulation of crucial cellular processes. Proteasome population in the cell is structurally and functionally heterogeneous. These complexes are subjected to tightly organized regulation, particularly, to a variety of posttranslational modifications. In this review we will summarize the current state of knowledge regarding proteasome participation in the control of cell cycle, apoptosis, differentiation, modulation of immune responses, reprogramming of these particles during these processes, their heterogeneity and involvement in the main levels of gene expression. Key Words: Proteasome, Cell cycle, Apoptosis, Differentiation, Immune response, Different levels of gene expression. ß 2008 Elsevier Inc.

1. Introduction Degradation of cellular proteins is a tightly regulated process, carried out by the cascade of the proteasome-ubiquitin pathway. The 26S proteasome, an intensively studied protease, represents the sophisticated complex of subunits and is critical for life. This complex is implicated in the temporally controlled ATP-dependent degradation of key regulatory proteins controlling main cellular processes (cell cycle, apoptosis, differentiation, development, immune response, malignant transformation), as well as in regulation of different stages of gene expression (Collins and Tensey, 2006; Ferdous et al., 2007; Glickman and Ciechanover, 2002; Kloetzel, 2004; MaupinFurlow et al., 2006; Pajonk and McBride, 2001; Reed, 2006; Sikder et al., 2006; Wojcik et al., 2000). The participation of the proteasome-ubiquitin pathway in the control of degradation of regulatory proteins involves not only the regulation of the complicated enzyme system of conjugation of the ubiquitin molecules to the substrates but also the highly complex control of the composition and the activities of the proteasome particle itself. These particles are heterogeneous due to subtypes synthesized from duplicated genes and/or alternative splicing, to posttranslational modifications and to association of 20S core with alternative regulatory complexes (Glickman and Raveh, 2005; Maupin-Furlow et al., 2006). Here we will describe recent studies revealing the current knowledge about proteasomes involvement in the

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fundamental biological processes such as control of cell cycle, apoptosis, differentiation, modulation of immune responses, about their isoform complexity including subunits’ posttranslational modifications, structural and functional responsiveness to cell requirements and participation of these particles in the main stages of gene expression.

2. Proteasome Structure and Catalytic Activities 2.1. The core particle, 19S regulatory particle, alternative regulatory particles The 26S proteasome, further often named ‘‘the proteasome, ’’ is an ATPdependent multicatalytic enzyme complex found in the nucleus and cytoplasm not only of all eukaryotic cells, but also of Archebacteria and in some Eubacteria. Proteasomes are responsible for the degradation of most cellular proteins (Coux et al., 1994). The 26S proteasomes constitutes the central proteolytic machinery of the ubiquitin/proteasome system, and is composed of a core catalytic complex, called 20S proteasome, capped at both ends by a 19S regulatory complex (Baumeister et al., 1998; Dahlmann, 2005). 2.2.1. 20S core The 20S proteasome is a large, cylinder-shaped protease with the molecular weight of
700 kDa. This complex is formed by 28 subunits, which are arranged in four heptameric stacked rings in an a7b7b7a7 configuration (Fig. 2.1). The two outer rings are made up of a-type subunits whereas the inner two rings are composed of b-type subunits (Grziwa et al., 1991). In Archebacteria the a- and b-rings are composed of seven identical a and b subunits, respectively. The various a and b subunits of eukaryotic proteasome were shown to be homologous to archeabacterial ones (Heinemeyer et al., 2004). Three different b subunits have free N-terminal threonine (Thr) residues, which form the proteolytically active sites and, thus, are responsible for the proteolytic activity of proteasome (Arendt and Hochstrasser, 1997). The proteolytic active sites formed by the N-termini of b subunits make the innermost chamber which is flanked by the two outermost chambers (Lowe et al., 1995). Hence, there are two narrow (
1.3 nm) and gated pores at each end of the cylindrical complex (Groll et al., 2000). This structure and the different regulatory complexes protect the cell from unregulated protein degradation by the active sites of 20S proteasomes. Mammals have three additional proteasome subunits called LMP2, LMP7 and MECL-1 (also named b1i, b5i and b2i, respectively) (see Section 3.4).

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Ub Recognition Substrate

Deubiquitination a

Unfolding Translocation Cleavage

b2 b1 b1 a1

a1 b2

b b5 a5

a

ATPases

Base Non-ATPases Lid

Figure 2.1 Structure and function of the 26S proteasome.The 20S proteasome is composed of a stack of four rings composed of seven subunits each. The two outer rings are made up of seven different a-subunits (marked by blue, right caption), whereas the two central rings are composed of seven different b-subunits (marked by pink, right caption). The proteolytic active sites of the 20S proteasome are located in the central rings at subunits b1, b2 and b5 (marked by dark pink) and the endoribonuclease active sites of the 20S proteasome are located in the outer rings at subunits a1 and a5 (marked by light blue). 19S regulatory complexes are subdivided into two distinct base and lid subcomplexes (right caption) and composed of 11^12 non-ATPase (marked by yellow, right caption) and six AAA-type ATPase subunits (marked by green, right caption).The substrate and ubiquitin are marked by a black thread and violet circles respectively (right caption).The functions of the 26S proteasome necessary for proteolysis of ubiquitylated substrates are shown on the left. 19S regulatory complex of the 26S proteasome recognizes usually substrates via their polyubiquitin chain.The substrate is then unfolded via the ATPase ring. The unfolded polypeptide chain is translocated to the proteolytic chamber of the 20S proteasome where is subjected to the cleavage. The polyubiquitin chain is cleaved off by deubiquitinating enzymes of 19S regulator during the process of translocation.

They are induced by interferon-gamma (IFNg) and carry the catalytic N-terminal Thr residues. Under the action of IFNg, these subunits are incorporated into the newly assembled proteasomes instead of three constitutive catalytic subunits [designated traditionally X (b1), Y (b5) and Z (b2)] (Fruh et al., 1994). Proteasomes with IFNg-inducible subunits are termed immunoproteasomes and take part in immune response (Kloetzel et al., 1999). 2.1.2. Multiple roles for the 19S regulatory particle The 19S proteasome regulatory complex, or the PA700 activator, is composed of at least 17 protein subunits and subdivided into two distinct subcomplexes, called base and lid (DeMartino et al., 1996; Glickman et al., 1998). The lid subcomplex is formed by at least nine non-ATPase subunits (designated in yeast Rpn3, Rpn5–Rpn9, Rpn10, Rpn11 and Rpn12) (Glickman et al., 1998) and the base subcomplex contains six AAA-type

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ATPases (denoted Rpt1–Rpt6) and at least two non-ATPase components (designated Rpn1 and Rpn2) (Leggett et al., 2002). The base subcomplex has protein-chaperone activity (Lam et al., 2002) and is important for the regulatory activity of 26S proteasome (Lupas and Martin, 2002). The functions of the 19S regulatory complex consist in the recognition of the polyubiquitinated substrate, liberation of the polyubiquitin chain, unfolding of the substrate, opening of the central proteasome channel and translocation of the unfolded polypeptide chain toward the catalytic sites (Glickman et al., 1998; Wolf and Hilt, 2004). 2.1.3. Alternative regulatory particles Other regulatory complexes also associate with the 20S proteasome. One of them is the PA28 activator, or the 11S proteasome regulatory complex. It is a ring-shaped hexamer formed by two closely related subunits PA28a and PA28b, which are induced by IFNg (Dubiel et al., 1992). PA28 activator binds 20S proteasome and is implicated in the processing of MHC class I antigens (Rechsteiner et al., 2000). Subunits of the PA28g activator are different from PA28a and PA28b and PA28g is down-regulated by INFg (Tanahashi et al., 1997). Hence, the PA28g is not a participant of immune response but, possibly, plays a role in the cell division and carcinogenesis (Gao et al., 2004; Tanahashi et al., 1997). In a protozoan pathogen, Trypanosoma brucei, proteasomes an additional activator protein, PA26, has been identified (To and Wang, 1997). The protein sequence of PA26 is similar to sequence of mammalian activator proteins PA28a, PA28b or PA28g (Yao et al., 1999). The single-chain protein, called PA200 is present in the nuclei of mammalian cells. The association of PA200 activator with 20S core may facilitate release of digestion products or the entrance of substrates and it is thought to play a role in DNA repair (Ustrell et al., 2002, 2005).

2.2. Catalytic activities of proteasomes 2.2.1. Peptidase activity The proteasome belongs to the N-terminal nucleophile hydrolases, where the N-terminal Thr residue functions as the nucleophile (Orlowski and Wilk, 2000). The eukaryotic 20S proteasomes demonstrate at least five types of peptidase activity (Orlowski et al., 1993). The three classical catalytic activities of the proteasome are designated chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolyzing (i.e., caspase-like) (Adams, 2003b; Coux et al., 1996; Orlowski and Wilk, 2000). Moreover, the proteasomes are able to cleave bonds on the carboxyl side of branched chain amino acids and between the small neutral amino acids (Orlowski et al., 1993). The more detailed description of proteasomal peptidase

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activities is presented in the comprehensive reviews of Orlowski and Wilk (2000) and Groll et al. (2005). 2.2.2. RNase activity Despite high progress in the study of proteasomes, some questions still remain opened, and one of them is the RNase activity of these particles, mechanisms of its regulation and its possible physiological significance. A new view on the proteasome’s role in the regulation of gene expression is suggested by the recent observation that the proteasome harbors an endoribonuclease activity (Ballut et al., 2003; Evteeva et al., 2000; Evteeva et al., 2003; Jarrousse et al., 1999; Mittenberg et al., 2002; Petit et al., 1997a,b; Toktarova et al., 2004; Tsimokha et al., 2006, 2007a,b,c). RNase activity of 20S as well as 26S proteasomes has been studied, and the subunits possessing this activity (a-type subunits: a5/zeta and a1/iota) were identified. In contrast to 20S particles, 26S proteasomes can degrade cellular native messenger poly (A)-containing RNA molecules (Ballut et al., 2003; Evteeva et al., 2000). In addition, the endoribonuclease activity of 26S particles sharply differs from that of 20S proteasomes in the dependence on divalent cations: 20S proteasomes are active only in the presence of divalent cations, while 26S particles are active also in the absence of these cations as well (Evteeva et al., 2000; Mittenberg et al., 2002a,b). The different properties of the RNase activities of 20S and 26S particles could be due to the effects of the regulatory (19S) complexes on the activity. However, the RNase activity of proteasomes does not imply their involvement in mRNA stability regulation, and changes of this activity under the action of stimuli, which regulate the half time of mRNA molecules, favor this suggestion more strongly. Indeed, regulation of the 26S proteasome RNase activity has been demonstrated under several stimuli (Evteeva et al., 2000; 2003; Kulichkova et al., 2004a,b; Mittenberg et al., 2002a,b; Toktarova et al., 2004; Tsimokha et al., 2006; 2007a,b,c). In these works the above activity has been shown to be changed under the action of such agents as: the apoptosis inductors of proerythroleukemic K562 cells (doxorubicin and diethylmaleate), differentiating agent of K562 cells hemin and epidermal growth factor (EGF) in A431 cells. The RNase activity of 26S proteasomes is phosphorylation-dependent and specifically regulated by Ca and Mg ions. Phosphorylation of which of the subunits is essential for the activity as well as their phosphorylation status are under study at present. The studied agents specifically regulate phosphorylation of certain proteasomal subunits (Mittenberg et al., 2007; Tsimokha et al., 2006, 2007a,b,c). To assess the effect of the inductors on the RNase activity of 26S proteasomes, total cytoplasmic high-molecular-weight RNA (hmwRNA) was incubated with 26S proteasomes and the products of nucleolysis were separated by PAGE. Treatment of K562 cells with apoptosis and

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differentiation inductors decreased the proteasomal RNase activity toward hmw ribosomal RNAs. However, further data (Mittenberg et al., 2007; Tsimokha et al., 2007a,b,c) argue that different RNase centers of proteasomes are differentially regulated. To study the possible inductor’s effect on the specificity of the proteasomal RNase activity, the nucleolysis of individual messenger RNAs (mRNA) was checked. The obtained results have shown that the specificity of 26S proteasomes’ endoribonuclease activity is changed under the action of erythroid differentiation (hemin) and programmed cell death (diethylmaleate) inductors in K562 cells. Thus, treatment of K562 cells with apoptosis and differentiation inductors lead to the specific stimulation of RNase activity toward certain mRNAs (hemin: p53 and c-myc mRNAs), to unchanged activity toward other mRNA (DEM: c-myc mRNA) and to reduction of proteasome degradation of other specific mRNA (hemin and DEM: c-fos mRNA). Treatment of proteasomes with calf intestinal phosphatase revealed that the RNase activity specifically and selectively depends on phosphorylation of 26S proteasome subunits. Thus, the specificity of the proteasomes’ RNase activity is differentially and specifically regulated during differentiation and apoptosis.

3. Function of Proteasomes in Cell Proliferation, Differentiation, Apoptosis and Immune Response 3.1. Cell cycle control The cell cycle in eukaryotes is regulated by consecutive activation of cyclindependent kinases (CDKs) by various cyclins. Cyclins are synthesized during strongly defined moments of a cell cycle and, being the extremely unstable, exist and work only at the certain phases of the cell cycle and during the certain period of time. For example, cyclins D and E are active during phase G1; cyclins E and A, during phase S. Consecutive appearance and disappearance of the pairs cyclin-CDK at various stages of the cell cycle is defined by kinetics of cyclins synthesis and degradation. Therefore, the basic molecular mechanism of cell cycle regulation is periodic synthesis and destruction of major proteins during the cell cycle ( Johnson and Walker, 1999). Proteasome inhibitors were shown to block the cell cycle in the phases G1 (Kumeda et al., 1999; Rao et al., 1999), late S (Machiels et al., 1997) or G2/M (Wojcik et al., 1996). Ubiquitin- and proteasome-dependent proteolysis is one of the key mechanisms underlying cell cycle control. Thus, cyclins are degraded by this pathway. Furthermore, proteasomes are involved in the process of the regulation of CDK inhibitors stability (p27Kip1 and

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p21Cip1/WAF1) and of specific phosphatases of the family CDC25 activating the kinases CDK in mammals (Naujokat and Hoffmann, 2002; Reed, 2006). The ubiquitin-proteasome system realizes negative as well as positive control of cell cycle (Naujokat and Hoffmann, 2002). The following negative regulators of cell cycle progression, p21Cip1/WAF1, p27Kip1, p19INK4d, and Geminin (Table 2.1), are known to be substrates for the proteasomal proteolytic machine (Naujokat and Hoffmann, 2002). The proteasomal degradation of these proteins leads to ‘‘the release from brake’’ (Naujokat and Hoffmann, 2002) and the prolongation of cell cycle progression. Entrance into S-phase is determined by positive regulators of cell cycle progression, and both the timely proper S-phase beginning and the corresponding frequencies of entry into S-phase are supposed to be tightly controlled by proteasomal degradation of the above regulators (Naujokat and Hoffmann, 2002). The proteolysis during cell cycle can be characterized by two families of protein-ubiquitin ligases: APC/C and SCF (Reed, 2006). The anaphase promoting complex or cyclosome (APC/C) was initially described as a multisubunit protein complex (containing the ubiquitin-ligase E3) that ubiquitinates anaphase inhibitors thus targeting them for destruction by proteasomes (Clarke et al., 2005). Activation of this complex also occurs during mitosis and G1 phase. In this case, too, APC/C ubiquitinates the proteins inhibiting the mitotic progression (Reed, 2006). Thus, APC/C participates in ubiquitin-depending proteasomal degradation of the anaphase inhibitors during the transition from the metaphase to the anaphase and of the mitotic cyclins during the transition from mitosis (see review by Abramova et al., 2002). SCF (Skp1/Culin/F-box protein) protein-ubiquitin ligase is also a core component of the cell cycle machinery. Moreover, APC/C and SCF do not function in isolation from each other, and they are adjusted to coordinate the cell cycle events (Reed, 2006). SCF ligase ubiquitylates a large number of proteins involved in cell cycle control that are marked by phosphorylation at specific sequences known as phosphodegrons (see review by Reed, 2006). Therefore, the targeting of these proteins for destruction by phosphorylation provides the participation of signaling pathways via activation of specific kinases in the control of cell cycle. The comprehensive review has been published recently and the reader is referred to it for the full information on this subject (Reed, 2006).

3.2. Roles in differentiation and development Development and cellular differentiation as the other cellular processes are controlled by both gene expression and protein degradation. Hence, these processes are regulated, at least in part, by ubiquitin-proteasome pathway. For example, early studies in insects, including Drosophila and Manduca sexta, have demonstrated that early embryogenesis and metamorphosis depend

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Table 2.1 Cell cycle targets for ubiquitin-dependent proteasomal degradation

Organism

Substrate

Cell cycle targets of APC S. cerevisiae Securin Clb2 Clb5 Cdc5/Plk, Cdc20, Ase1, Hsl1

Dbf4 Cin8/Kip1 Metazoa

Cyclin B Cyclin A Geminin

Vertebrates Aurora A Nek2A Cdc6 Xkid Cdc25A Skp2 Cell cycle targets of SCF S. cerevisiae Sic1/Rum1, Far1 Cdc6/Cdc18 Cln1,2,3

Gic1,2 Swe1 Met4 Metazoa

Cyclin E

Cell cycle function

Anaphase inhibitor B-type cyclin (mitosis) B-type cyclin (S phase) Mitosis

Replication Mitotic spindle motor Mitosis S phase, mitosis Replication licensing Mitosis Centrosome development Replication Mitotic spindle motor S phase, mitosis SCF cofactor G1–S transition inhibitor DNA replication G1 cyclin

Budding Mitosis inhibitor G1-S transition inhibitor G1-S cyclin

References

Zur and Brandeis, 2001 Wasch and Cross, 2002 Shirayama et al., 1999 Cheng et al., 1998; Visintin et al., 1997; Juang et al., 1997; Martinez et al., 2006 Ferreira et al., 2000 Hildebrandt and Hoyt, 2001 Yamano et al., 2004 Geley et al., 2001 McGarry and Kirschner, 1998 Castro et al., 2002 Hames et al., 2001 Petersen et al., 2000 Castro et al., 2003 Donzelli et al., 2002 Wei et al., 2004 Feldman et al., 1997; Blondel et al., 2000 Perkins et al., 2001 King et al., 1996; Schweitzer et al., 2005 Reed, 2006 Kaiser et al., 1998 Patton et al., 2000 Ye et al., 2004 (continued)

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Table 2.1 (continued) Organism

Substrate

Vertebrates Wee1

Mammals

Cell cycle function

Mitosis inhibitor

Emi1

APC/C inhibitor

p27Kip1, p21Cip1, p130

G1-S transition inhibitor

Ctd1, Orc1 Cdc25A c-Myc

DNA replication S phase, mitosis S phase

References

Watanabe et al., 2004 Margottin-Goguet et al., 2003 Baldassarre et al., 2000; Bornstein et al., 2003; Tedesco et al., 2002 Reed, 2006 Jin et al., 2003 Gregory and Hann, 2000

on proteasome accumulation and proteasomal degradation of certain target proteins (Dawson et al., 1995; Jones et al., 1995; Klein et al., 1990; Low et al., 1997). Subsequent studies in the other organisms, such as sea urchin (Lytechinus pictus), frog (Xenopus laevis), mouse, and rat, have indicated that developmentally regulated expression of distinct subunits of the proteasomal 19S regulatory complex, as well as proteasomal degradation of cell cycle regulatory proteins, is necessary for the initiation of early embryonal mitosis and development ( Josefsberg et al., 2001; Kawahara et al., 2000a,b; Tokumoto et al., 1999a). Further, recent studies in vertebrates showed that the molecular assembly and the proteolytic activity of 26S proteasome undergo changes during the completion of meiosis (oocyte maturation) ( Josefsberg et al., 2000; Reverte et al., 2001; Sawada et al., 1999; Tokumoto et al., 1999a, 2000). The functions of the proteasome in cellular differentiation are very complex. The comprehensive reviews with more full information on this subject are accessible (Bowerman and Kurz, 2006; Naujokat and Hoffmann, 2002; Schwechheimer and Schwager, 2004).

3.3. Proteasomes and apoptosis Protease inhibitors inactivate enzymes reversibly or irreversibly by binding to the catalytic site of the enzyme. Proteasome inhibitors (Table 2.2) are attractive drug targets due to the importance of the ubiquitin-proteasome system in numerous biological processes including the apoptosis (Adams, 2003a; Ciechanover and Schwartz, 2004; Groll and Huber, 2004; Nandi et al., 2006; Rajkumar et al., 2005).

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Table 2.2 Different proteasome inhibitors Proteasome inhibitors

Chemical Peptide aldehydes inhibitors Peptide boronates

Peptide vinyl sulfones

Protein inhibitors

Examples

References

MG132, PSI (Z-IE (OtBu)AL-al), CEP1612 MG262 (Z-LLL-bor), PS341 (bortezomid), PS273 (MNLB) NLVS (Nip-LLL-vs), YLVS (YLLL-vs)

Lee and Goldberg, 1998 Adams et al., 1998; 1999

Peptide epoxyketones

Dihydroeponemycin, epoximicin, YU101 (Ac-hFLFL-ex)

Lactacystin and derivatives

Lactacystin, clastolactacystin-blactone, L-Lactone (omuralide) PI31

PR39 d- aminolevulinic acid dehydratase HIV encoded Tat Hepatitis B virus encoded X protein Protein aggregates

Lee and Goldberg, 1998; Adams et al., 2000 Kim et al., 1999; Meng et al., 1999; Glenn et al., 2004 Fenteany and Schreiber, 1998 McCutchenMaloney et al., 2000 Gao et al., 2000 Guo et al., 1994 Apcher et al., 2003 Hu et al., 1999 Grune et al., 2004

The materials of Nandi et al., 2006, were used for preparation of this table.

Apoptosis is regulated by two proteolytic systems: by the caspases, a family of specific cysteine proteases, and by the proteasomes. Interestingly, the proteasomes degrade specific proteins-regulators of apoptosis, but on the other hand some components of the proteasome system are degraded by caspases (Adrain et al., 2004). The proteasomal function in apoptosis appears

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to be very complex. Whereas proteasome inhibitors induce apoptosis in multiple cell types, while in other cells they are innocuous or even prevent apoptosis induced by other agents (An et al., 1998; Wojcik, 2002). The use of proteasome inhibitors has demonstrated that degradation or processing of proteins by the ubiquitin-proteasome system has a determinative influence on cell survival or cell death, depending on the cell type and/or the proliferative state of the cells (Drexler, 1998; Naujokat and Hoffmann, 2002). The proteasomes realize programmed proteolysis and processing of various groups of apoptosis-regulatory proteins possessing the pro- and anti-apoptotic functions in a cell. These proteins include transcription factors (c-Fos, c-Myc, NF-kB, AP-1), tumorous suppressor 53, IkBa (inhibitor of nuclear transcription factor NF-kB), regulators of cell cycle (p27Kip1, p21Cip1/WAF1), proteins of Bcl-2 family and regulators of caspase activity (inhibitors of apoptosis proteins [IAPs]) (Wojcik, 2002). Studies using the selective proteasome inhibitors have provided direct evidence for the proteasome functions both in promoting apoptosis and in protecting cells against apoptosis (Grimm et al., 1996; Lopes et al., 1997). These opposite roles of 26S proteasomes in regulation of apoptosis seem to depend on the proliferative state of the cell (Chen and Lin, 2004; Naujokat and Hoffmann, 2002; Wojcik and DeMartino, 2003). 3.3.1. Pro-apoptotic function of the proteasomes In some cell systems, proteasome inhibitors prevent apoptosis, indicating pro-apoptotic function of proteasomes (Grimm et al., 1996; Sadoul et al., 1996). The requirement of proteasomal activity for the progression of apoptosis has been shown in two nonproliferating mammalian cell types, namely in resting thymocytes and differentiated neurons (Grimm et al., 1996; Sadoul et al., 1996). Thus, the mouse thymocytes induced to undergo apoptosis by treatment with phorbol-12-myristate 13-acetate, dexamethasone or g-radiation were rescued from apoptosis when treated with proteasome inhibitors up to 1 h after the apoptotic stimulus (Grimm et al., 1996). It is important, that later addition (3-5 h) of proteasome inhibitors failed to rescue the cells from apoptosis, suggesting that proteasomal activity promotes apoptosis only at upstream points of apoptotic signal transduction pathways (Naujokat and Hoffmann, 2002). Similar findings were obtained in studies with differentiated neurons (Canu et al., 2000; Sadoul et al., 1996). Sympathetic neurons from rat superior cervical ganglia (Sadoul et al., 1996) and rat cerebellar neurons (Canu et al., 2000) induced to undergo apoptosis in response to deprivation of nerve growth factor and potassium, respectively, were rescued from apoptosis when treated with proteasome inhibitors early after the initiation of the apoptotic process. In some situations, proteasome inhibitors may have opposing effects even in the same cells, where they may either enhance or prevent apoptosis

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(Pleban et al., 2001; Sohn et al., 2006a; Wojcik, 2002; Yang et al., 2006). For example, pretreatment of non-small cell lung carcinoma cells in culture with proteasome inhibitors prevents the pro-apoptotic action of topoisomerase inhibitors, while when proteasome inhibitors are administered after the treatment with topoisomerase inhibitors, they enhance their pro-apoptotic effect (Tabata et al., 2001). Proteasome inhibitor PSI blocks T cell receptorinduced apoptosis in a T-cell hybridoma (Tanimoto and Kizaki, 2002). Proteasome activity is involved at an early step of glucocorticoidinduced apoptosis, preceding mitochondrial changes and caspase activation (Hirsch et al., 1998; Wallace and Cidlowski, 2001). There are many indications of the apoptosis induced by different proteasome inhibitors in various tumor cells (Adams, 2002; Nandi et al., 2006; Wojcik, 2002). However, the proteasome inhibitor MG132 rescued tumor cells (two HeLa cell lines: D98 and H21) from death receptor-induced apoptosis (Sohn et al., 2006a). One possible mechanism of proteasomal pro-apoptotic function has been uncovered recently in primary mouse thymocytes in the study of XIAP and c-IAP1, members of the family of inhibitors of apoptosis proteins IAPs (Duckett et al., 1996). These inhibitory proteins realize the anti-apoptotic activity, at least in part, by inhibiting the activation and enzymatic activity of caspases (Deveraux et al., 1997, 1998), and by ubiquitination and targeting of caspase 3 for proteasomal degradation (Suzuki et al., 2001). In response to various apoptotic stimuli, these inhibitory proteins are autoubiquitinated and subsequently degraded by the proteasomes (Yang et al., 2000). The proteasomal degradation of XIAP and c-IAP1 depends on an intact RING finger domain and appears to be highly operative in transducing apoptosis, because cells expressing XIAP and c-IAP1 with mutant RING finger domains display a lack of proteasomal degradation of the mutant proteins and fail to undergo apoptosis induced by several stimuli (Yang et al., 2000). Another possible mechanism of proteasomal pro-apoptotic function has been demonstrated in human umbilical vein endothelial cells (HUVECs) undergoing TNF-a-induced apoptosis (Naujokat and Hoffmann, 2002). Early after the initiation of TNF-a treatment of HUVECs, anti-apoptotic protein Bcl-2 is specifically degraded by the proteasome (Breitschopf et al., 2000a; Dimmeler et al., 1999). This event was indicated to be effective in inducing apoptosis, because pretreatment of HUVECs with specific proteasome inhibitors aborted both TNF-a–induced Bcl-2 degradation and induction of apoptosis (Breitschopf et al., 2000a; Dimmeler et al., 1999). 3.3.2. Anti-apoptotic functions of the proteasomes The proteasomes are abnormally highly expressed in rapidly growing metazoan embryonic and human neoplastic cells in contrast to differentiated and normally growing cells (Ichihara et al., 1993; Kanayama et al., 1991; Klein et al., 1990; Kumatori et al., 1990; Shimbara et al., 1992). Hence, in all

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probability, the proteasomes play a central role in maintaining survival and proliferation of rapidly and somehow abnormally growing cells (Ichihara and Tanaka, 1995). The effects of proteasome inhibitors on neoplastic and rapidly growing cells confirmed this hypothesis (Naujokat and Hoffmann, 2002). First indication of an anti-apoptotic function of the proteasomes came from studies focusing on the apoptosis induced by a proteasomal inhibitor lactacystin in human leukemia U937 cells in 1995 (Imajoh-Ohmi et al., 1995). After that, there were many other studies using different sets of proteasome inhibitors and several different cell lines: proteasome inhibitors have been found to induce apoptosis in neoplastic and rapidly growing mammalian cells of hematopoietic (Drexler, 1997; Naujokat et al., 2000; Shinohara et al., 1996; Tanimoto et al., 1997), neuronal (Kitagawa et al., 1999; Lopes et al., 1997; Qiu et al., 2000), mesenchymal (Drexler et al., 2000; Lopes et al., 1997) and epithelial origin (Adams et al., 1999; Herrmann et al., 1998). In several types of neoplastic cells, the proteasome inhibitors were found out to be active inducers of apoptosis, and they are able to induce apoptosis in cells resistant to other agents. Different proteasome inhibitors, including lactacystin and MG132, induce apoptosis in leukaemic B cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) at all stages of the disease, including those resistant to conventional chemotherapy (Wojcik, 2002). For example, the proteasome inhibitor MG132 is a potent deathinducing agent for PC3 prostate cancer cells and elicits activation of multiple signaling pathways in these cells (Yang et al., 2006). Proteasome inhibitors may induce apoptosis either by toxic effects or by inhibition of degradation and/or processing of specific regulatory proteins (Wojcik, 2002). One of the anti-apoptotic regulatory proteins which level is increased by proteasomes is NF-kB, a member of a large family of transcription factors found in the cytoplasm (Verma et al., 1995). Proteasome inhibitors reduce the activity of protein NF-kB through stabilization of protein IkBa, and start apoptosis in the number of transformed cells (Naujokat and Hoffmann, 2002; Wojcik, 2002). NF-kB forms an inactive complex with inhibitory protein IkBa in cytoplasm. In response to cellular stress (for example, the chemotherapy, irradiation, action of cytotoxic agents, viruses and oxidants), IkBa is phosphorylated, ubiquitinylated and degraded by the proteasomes. The active transcription factor NF-kB is transported from the cytoplasm into the nucleus, where it initiates the transcription of anti-apoptotic proteins (A1/Bfl1, IAP and bcl-2), growth factors (interleukins) and molecules of cellular adhesion, preventing the cells from the apoptosis. Proteasome inhibitors induce suppression of NF-kB activation in cancer cells, and these cells become more sensitive to chemotherapy and other stressful agents (Traenckner et al., 1994). The conjugation of the tumour necrosis factor TNF-a with the specific TNF-receptor leads to the activation of NF-kB and the induction of anti-apoptotic signal. The proteasomes are also believed to directly generate anti-apoptotic and

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survival signals in neoplastic cells by degrading pro-apoptotic proteins such as Bax and Bid (Breitschopf et al., 2000b; Chang et al., 1998; Li and Dou, 2000). One more mechanism of pro-apoptotic action of proteasome inhibitors involves the induction of a block in the cell cycle, which is regulated by cellular proteolysis of either cyclins or CDK inhibitors (Wojcik, 2002). Blocking of the cell cycle can actually prevent the cells from entering apoptosis in some situations and coexist or precedes the induction of apoptosis in other (Naujokat and Hoffmann, 2002). Another target for proteasome is the tumor suppressor p53, responsible for induction of apoptosis. In response to cellular stress (for example, damages of DNA and hypoxia), p53 causes the block in the cell cycle at the phases G1 or G2, DNA repair or induces apoptotic response (by activation of gene bax and repression of gene bcl-2). p53 acts as a transcriptional regulator inducing expression of several key genes mediating those effects (Burns and El-Deiry, 1999; Shen and White, 2001; Wang, 1999). p53 levels are normally very low in the cells, since it is constantly ubiquitinated by Mdm2 RING-finger ubiquitin ligase and then degraded by the proteasome (Fang et al., 2000). At cell damage, p53 degradation stops as the result of ubiquitin–proteasome proteolysis of Mdm2-ligase, and p53 level quickly increases in a cell. Furthermore, fast accumulation of p53 is promoted by the cleavage of Mdm2-ligase by caspase 3 (Cho et al., 2001). The proteasome inhibition also causes p53 accumulation and following induction of apoptosis in proliferating cells (MacLaren et al., 2001). Proteasome inhibitors can cause accumulation of the oncoprotein c-Myc. Transcription factor c-Myc controls the cell cycle, proliferation and apoptosis. c-Myc transactivates gene of Cdc25a-phosphatase, which removes inhibitory phosphorylation of Cdk2 and Cdk4 and also decreases expression of inhibitor p27Kip1. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt’s lymphoma (Gregory and Hann, 2000). The c-Myc protein is normally degraded very rapidly by the ubiquitin-proteasome pathway (Salghetti et al., 1999). c-Myc activation by proteasome inhibitors leads to stimulation of deregulated cell proliferation and to induction of the apoptosis (Wojcik, 2002). In human malignant glioma cells, the proteasome inhibitors cause an increase of c-Myc protein levels, which induces transiently FasL message to stimulate the Fas receptor-ligand apoptotic signaling pathway (Tani et al., 2001). Finally, the effects of proteasome’s anti-apoptotic action can include the regulation of the levels of specific secondary messenger molecules (for example, cAMP or nitric oxide), which in turn can induce apoptosis. So, for example, in human neutrophils, ubiquitin-proteasomal system of protein degradation regulates the balance of pro-apoptotic and anti-apoptotic proteins which plays a key role in the ability of cyclic AMP to delay neutrophil death (Lee et al., 2001; Martin et al., 2001).

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In all, both negative and positive regulators of apoptosis undergo proteasomal degradation in a tightly regulated and temporally controlled fashion. For example, proteasome inhibitors induce apoptosis in fastly proliferating cells (neoplastic cells). These cells show the raised level of proteasomal subunits expression, and, probably, proteasomes block the apoptosis and play an essential role in a survival and proliferation of only quickly growing and neoplastic cells (Naujokat and Hoffmann, 2002). Several of the specific proteasomal inhibitors have recently entered clinical trials due to their tremendous apoptosis-inducing capability. However, several recent studies provided substantial evidence that a combined treatment of tumors with apoptosis-inducing agents and proteasomal inhibitors might even cause adverse effects leading to a prolonged survival of tumor cells (Sohn et al., 2006a; Yang et al., 2006). Proteasomes provide balance between pro- and anti-apoptotic proteinsregulators in a cell and are the central figures in the balance between two opposite pathways: a survival and apoptosis of the cell (Sohn et al., 2006a,b; Yang et al., 2006). It has appeared that apoptotic action of proteasome inhibitors depends on a stage of apoptosis. Recently the model of biphasic role for the proteasome in apoptosis of tumor cells has been proposed (Sohn et al., 2006b). Thus, the proteasomes support a critical balance between the pools of pro- and anti-apoptotic proteins before the initiation of apoptosis (Sohn et al., 2006b). During the induction phase of apoptosis, however, this balance slowly moves toward cellular death due to an increase of the proapoptotic pool (e.g., active caspases), and it depends on proteasomal degradation of anti-apoptotic proteins (Sohn et al., 2006b). During the execution phase of apoptosis, the cell accumulates the pool of pro-apoptotic proteins in comparison with a decreasing pool of anti-apoptotic proteins. The inhibition of proteasomes before the induction phase of apoptosis leads to an increase of the pool of anti-apoptotic proteins as cellular biosynthesis constantly supplements this pool which is not decreased by proteolysis. Hence, in this case proteasomes cannot execute their pro-apoptotic role. However, inhibition of the proteasome after the induction phase of apoptosis leads to an increase of the pro-apoptotic proteins pool as the required proteasomal degradation of the anti-apoptotic proteins pool has been reached before (Sohn et al., 2006b). Similarly, both pro- and anti-apoptotic pathways can be regulated by proteasome inhibitor MG132 in prostate cancer PC3 cells (Yang et al., 2006).

3.4. Proteasomes and immune response Misfolded, foreign and other abnormal proteins are degraded through the ubiquitin- and proteasome-dependent pathway in the cells. This proteolytic system generates peptides from intracellular antigens, which are then presented to T cells, and thereby plays the central role in the cellular immune response.

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The IFNg controls the proteolytic properties of the proteasome to adapt them to the requirements of the immune system. The stimulation of cells with the cytokines IFNg or TNF-a induces the synthesis of three proteasomal subunits LMP2 (b1i), LMP7 (b5i), and MECL-1 (b2i). These subunits replace the three subunits Y(b1), X(b5), and Z(b2), which bear the proteasomal catalytically active sites (Fruh et al., 1994). The cytokine-induced exchange of three active site subunits of proteasome is unprecedented in molecular biology and one may expect a strong functional driving force for this system to evolve (Groettrup et al., 2001a). Furthermore, IFNg induces the synthesis of the proteasome activator PA28 and the formation of immunoproteasome. The PA28 synthesis and the immunoproteasomes formation, in tern, adapt the proteolytic properties of the proteasome and improve the proteasomal function in antigen presentation. Thus, a combination of several regulatory events tunes the proteasome system for maximal efficiency in the generation of MHC class I antigens (Kloetzel, 2004). Although the peptide production by constitutive proteasomes is able to maintain peptide-dependent MHC class I cell surface expression in the absence of LMP2 and LMP7, these subunits were recently shown to be central for the generation or destruction of several unique epitopes (Groettrup et al., 2001a). The proteasomal immune subunits exchanges have evolved not only to optimize class I peptide loading but also to generate LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites which are not generated in uninflamed tissues. This difference in epitope generation may serve to better stimulate T cells in the sites of an ongoing immune response and to avoid autoimmunity in uninflamed tissues (Groettrup et al., 2001b). Interestingly, the molecular interplay between the proteasome maturation protein (POMP) and the proteasomal LMP7 subunit has a key position in this immune adaptive program. IFNg-induced coincident biosynthesis of POMP and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of b5i (as well as b5) subunit into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression (Heink et al., 2005). The LMP2 and LMP7 are encoded in chromosome locus of MHC class I that is also an evidence of proteasomes participation in the immune response. Furthermore, proteasomal immune subunits are continually expressed in lymphoid organs (spleen, thymus and glands) cells (Rivett, 1998; Rock et al., 1994) as well as in other cells (Gomes et al., 2006). The expression of immunosubunits is cell tissue specific and is under high selective control (Kloetzel, 2004). It is significant, that the immunoproteasomes realize an additional function in antigen-presenting cells (APCs) of the immune system, including B-lymphocytes, macrophages, and dendritic cells. APCs absorb antigens

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entering the lymphoid organs with blood and lymph flows via endocytosis. Then immunoproteasomes transform antigens into antigenic epitopes, which are brought to the cell surface in a complex with molecules MHC class I, like in defective and virus-infected cells (Rock and Goldberg, 1999). APCs contain almost ten thousand molecules of MHC class I on their plasma membrane at the same time and an elevated number of immunoproteasomes ( Janeway and Travers, 1994). Proteasomes were shown to be indirectly involved in the development of both humoral and cell immune responses related to the cytotoxic activity of macrophages and T-killers (for review, see Sharova, 2006). Moreover, proteasomes were found to process antigenic polypeptides for their presentation in a complex with MHC class II to T-helper cells (Tewari et al., 2005). In conclusion, the proteasomes trigger a signal for destruction of defective cells and are involved in activation of T-killers and, thus, participate in the formation of the T-cell immune response. Moreover, proteasomes are involved in the activation of T-helper cells and, therefore, mediate both humoral (functioning of B-lymphocytes) and cellular (functioning of T-killers and macrophages) immunity.

4. Modes of Regulation of Proteasome Activities in the Cell 4.1. Modulation of proteasome composition 4.1.1. Heterogeneity of proteasomes in the cell The more in a complicated manner the organization of both the separate cells, and the entire organisms is, the more fancily becomes the main cellular machinery, aimed to non-lysosomal protein cleavage. The structure and sub-unit content of ancestral proteasome complex found in Prokaryota, both Archea (Maupin-Furlow and Ferry, 1995) and Eubacteria (Hu et al., 2006; Lupas et al., 1994; Tamura et al., 1995), is respectively simple: subunits are divided into only two types, a and b, forming four homoheptameric rings in abba order; the complex contains 14 identical proteolytic centers capable to cleave polypeptide chains after hydrophobic and basic amino acid residues (see also Section 2.1.1). However, in some Archea species, the first complication occurs consisting in appearance of two distinct kinds of a and/or b subunits (Humbard et al., 2006; Kaczowka and Maupin-Furlow, 2003; Madding et al., 2007; Zuehl et al., 1997). When rising up along the evolutionary staircase, we find eukaryotic proteasomes to be much more complex: parallel with appearance of at least 14 distinct subunits of the core particle (7 a and 710 b subunits), multiple forms of regulatory complexes bound to the core were observed,

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such as PA700 (19S), PA200, PA28 (11S) and PA26 (see Section 2.1.3). These complexes could be attached to 20S barrel either from one or from both sides. Combination of the listed above complexes attachment to core particle is one of the ways for appearance of multiple cellular forms of multicatalytic protease called 26S proteasome. How many other mysteries cover this surprising complex in itself ? Recent studies performed on different eukaryotic species have shown the widest variety of subunit forms, their different versions, appearing to be a result of gene duplication (Fu et al., 1998; Ma et al., 2002; Yang et al., 2004), alternative splicing (Gomes et al., 2006; Kawahara et al., 2000a,b) and/or of various post-translational modifications. Gene duplication was found in a wide range of organisms. Thus, in Arabidopsis, ten 20S proteasome subunits, encoded by two genes each, were found to be expressed in appropriate different isoforms (Fu et al., 1998; Yang et al., 2004), whereas in Drosophila, six genes encoding 20S proteasome subunits were shown to be represented by two or even three isoforms, encoded by separate genes (Ma et al., 2002; Yuan et al., 1996) and, like in previous case, additional isoforms were revealed to be expressed. Four of the 19S regulatory complex subunits were also found to have actively expressing gene duplications encoding male-specific isoforms (Ma et al., 2002). On the contrary, experiments performed on various mouse cells and tissues—cardiomyocytes (Gomes et al., 2006), testis and embryonic stem cells (Kawahara et al., 2000a,b)— have revealed several distinct mRNA forms for Rpn10 subunit of 19S regulatory complex generated by a single gene. All that is listed above speaks in favour of the fact that the proteasomal population in each separately taken cell is highly specialized, which was confirmed, in particular, via subpopulations fractionation by means of anionexchange chromatography on MonoQ resin (Dahlmann et al., 2000, 2001). Separate fractions (cytosolic and ER-membrane bound) were found to differ from each other in their enzymatic activities (Khan and Joseph, 2001). The detailed study of proteasomes from the mammalian tissues and their proteolytic activities has resulted in conclusion of heterogeneity of the proteasome population. The isolated proteasome samples revealed properties of the dominating subpopulation, while others remain unstudied (Dahlmann et al., 2000, 2001). The principal cause of existence of several proteasome subtypes is competition of 10 b subunit varieties for building in to form the sevenmember b-ring. The expression of these seven b subunits is constitutive, but three of them—b1, b2 and b5—can be replaced under influence of IFNg by b1i, b2i and b5i subunits, respectively, which leads to immunoproteasome formation (see Section 3.4). The above facts allow to consider the existence of the two main proteasome groups which can be separated chromatographically due to slight differences in their surface charges (Dahlmann et al., 2000, 2001).

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It currently is unknown whether the b subunit set is always similar in both halves of the proteasome cylinder. If unequal replacement is possible, then 36 different combinations of subunits may appear, and otherwise the number of combinations is limited to eight. Notwithstanding the fact that theoretically all configurations are possible, the more probable is simultaneous replacement of all three inducible subunits (Griffin et al., 1998; Groettrup et al., 1997). Thus, b1i and b2i are reciprocally necessary for inclusion into proteasome, but independently of b5i. However, presence of b1i and b2i favours inclusion b5i into the proteolytic particle. Thus, the most probable manner of immunoproteasome formation is that containing all three inducible b subunits, but also another variants seem to be possible— proteasomes containing either b1i and b2i without b5i or, in turn, only b5i, as the sole immunosubunit (Dahlmann et al., 2000, 2001). 4.1.2. Subunit modifications On a 2D map of proteasome subunits, a number of spots exceed 14 or even 17 corresponding to a number of constitutive or both constitutive and inducible subunits of 20S core particle (Claverol et al., 2002; Froment et al., 2005; Wang et al., 2007). This phenomenon becoming clear after any attempt of subunit identification, immunochemical or mass-spectrometric is based on several kinds of post-translational modifications undergone by subunits and some proteasome-interacting proteins co-purifying with proteasomes. Proteasomal subunits, like many other protein molecules, could be underwent to several post-translational modifications (see Table 2.3), such as phosphorylation (Benedict et al., 1995; Bose et al., 1999, 2001, 2004; Castano et al., 1996; Humbard et al., 2006; Mason et al., 1996; 1998; Wang et al., 2007; Wehren et al., 1996), N-acetylation and/or N-terminal propeptide processing (Arendt and Hochstrasser, 1999; Gomes et al., 2006; Huang et al., 2001; Humbard et al., 2006; Kimura et al., 2000, 2003; Tokunaga et al., 1990; Wang et al., 2007; Zong et al., 2006), 4-hydroxy-2-nonenal alkylation (Bulteau et al., 2001; Farout et al., 2006), N-myristoylation (Gomes et al., 2006; Kimura et al., 2000; Utsumi et al., 2001; Wang et al., 2007), O-glycosylation (Tomek et al., 1988; Schliephacke et al., 1991; Schmid et al., 1993; Su¨megi et al., 2003; Wells et al., 2002; Zhang et al., 2003), S-glutationylation (Demasi et al., 2001, 2003) and oxidation of sulphurcontaining amino acid residues (Humbard et al., 2006; Schmidt et al., 2006; Wang et al., 2007). Such modifications are found in a wide range of organisms, from archebacteria Haloferax volcanii (Humbard et al., 2006) to humans (Wang et al., 2007). The proteasome structures and functions have been thoroughly studied this last decade, and these complexes have been revealed to be as a mixture of several proteasome subpopulations possessing different enzymatic activities (Dahlmann et al., 2000, 2001).

Table 2.3 Post-translational modifications of 20S proteasome and proteasome regulatory particle proteins

Subunit

PTM

20S proteasome subunits a1 N-Acetyl; Met1-SO a1 N-Acetyl a1 Ser
P; Tyr
P; Thr
P a1 Met209-SO a2 N-Acetyl a2 N-Acetyl a2 N-Acetyl a2 N-Acetyl a2 Ser-acetyl a2 a2 a2 a2 a2 a2

Ser or Thr
P Tyr120
P Tyr
P; Thr
P Ser
P Tyr23
P; Tyr97
P O-GlcNAc

a2 a3 a3 a3

4-Hydroxy-2-nonenal N-Acetyl N-Acetyl Ser248
P

Organism

Haloferax volcanii S. cerevisiae Mus musculus S. cerevisiae Haloferax volcanii S. cerevisiae Rattus norvegicus Mus musculus Trypanosoma brucei S. cerevisiae Rattus norvegicus Rattus norvegicus Mus musculus Homo sapiens Drosophila melanogaster Rattus norvegicus S. cerevisiae Rattus norvegicus Candida albicans

PTM Catalyst

References

n.d. NatA/Nat1 n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d.

Humbard et al., 2006 Kimura et al., 2000 Zong et al., 2006 Iwafune et al., 2002 Humbard et al., 2006 Kimura et al., 2000 Tokunaga et al., 1990 Gomes et al., 2006 Huang et al., 2001

n.d. n.d. n.d. n.d. n.d. n.d.

Iwafune et al., 2002 Benedict et al., 1995 Wehren et al., 1996 Zong et al., 2006 Rush et al., 2005 Sumegi et al., 2003

n.d. NatA/Nat1 n.d. CK2

Bulteau et al., 2001 Kimura et al., 2000 Tokunaga et al., 1990 Fernandez-Murray et al., 2002 (continued)

Table 2.3 (continued) Subunit

PTM

Organism

PTM Catalyst

a3


P

Rattus norvegicus

CK2

a3 a3

Ser
P
P

Rattus norvegicus Homo sapiens

n.d. PLK

a3 a3 a3 a4 a4

Homo sapiens Mus musculus Drosophila melanogaster S. cerevisiae Homo sapiens

n.d. n.d. n.d. NatA/Nat1 n.d.

a4 a4

Ser75
P Ser
P O-GlcNAc N-Acetyl Processed, N-acetyl
P
P

Xenopus sp. Carassius auratus

n.d. CKI_

a4 a4 a4 a4 a4 a5 a5 a5 a5

Ser or Thr
P Met71-SO Cys63-SO 4-Hydroxy-2-nonenal O-GlcNAc N-Acetyl N-Acetyl N-Acetyl
P

S. cerevisiae Rattus norvegicus Rattus norvegicus Rattus norvegicus Drosophila melanogaster S. cerevisiae Mus musculus Homo sapiens Candida albicans

n.d. n.d. n.d. n.d. n.d. NatC/Mak3 n.d. n.d. CK2

References

Castano et al., 1996; Mason et al., 1996 Wehren et al., 1996 Mason et al., 1996; Bose et al., 2001; Feng et al., 2001 Wang et al., 2007 Zong et al., 2006 Sumegi et al., 2003 Kimura et al., 2000 Wang et al., 2007 Tokumoto et al., 1999b Tokumoto et al., 2000; Wakata et al., 2004; Horiguchi et al., 2005 Iwafune et al., 2002 Schmidt et al., 2006 Schmidt et al., 2006 Bulteau et al., 2001 Sumegi et al., 2003 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007 Fernandez-Murray et al., 2002

a5 a5 a5

Ser
P Ser56
P O-GlcNAc

a6 a6 a6 a6 a6

n.d. n.d. n.d.

Wehren et al., 1996 Beausoleil et al., 2004 Sumegi et al., 2003

N-Acetyl N-Acetyl O-GlcNAc
P
P

Rattus norvegicus Homo sapiens Drosophila melanogaster S. cerevisiae Homo sapiens Rattus norvegicus Oryza sativa Candida albicans

NatC/Mak3 n.d. OGT CK2 CK2

a6 a6 a6 a7

Ser
P; Tyr
P; Thr
P 4-Hydroxy-2-nonenal O-GlcNAc N-Acetyl

Mus musculus Rattus norvegicus Drosophila melanogaster Homo sapiens

PKA n.d. n.d. n.d.

a7 a7 a7 a7 a7 a7 a7 a7

N-Acetyl N-Acetyl N-Acetyl Processed, N-acetyl Ser-acetyl Ser
P Ser250
P Ser250
P

Rattus norvegicus S. cerevisiae Mus musculus Homo sapiens Trypanosoma brucei Rattus norvegicus Mus musculus Homo sapiens

n.d. NatA/Nat1 n.d. n.d. n.d. n.d. PKA n.d.

Kimura et al., 2000 Wang et al., 2007 Wells et al., 2002 Umeda et al., 1997 Fernandez-Murray et al., 2002 Zong et al., 2006 Bulteau et al., 2001 Sumegi et al., 2003 Claverol et al., 2002; Gillardon et al., 2007 Tokunaga et al., 1990 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007 Huang et al., 2001 Wehren et al., 1996 Zong et al., 2006 Mason et al., 1996; Bose et al., 2001; Wang et al., 2007; Gillardon et al., 2007 (continued)

Table 2.3 (continued) Subunit

PTM

Organism

PTM Catalyst

References

a7 a7 a7 a7 a7 a7 a7 a7 b b b1 b1 b2 b2 b2 b2 b3 b3 b3 b3 b4 b4 b4

Tyr160
P
P Tyr
P
P Ser243
P; Ser250
P Ser243
P; Ser250
P
P O-GlcNAc Ser129
P N-Acetyl; Met1-SO Processed Met4-SO, Met146-SO Ser
P; Thr
P Tyr154
P O-GlcNAc Processed N-Acetyl N-Acetyl Processed, N-acetyl Ser
P; Thr
P N-Acetyl N-Acetyl N-Acetyl

Homo sapiens Homo sapiens S. cerevisiae S. cerevisiae Rattus norvegicus Cercopithecus sp. Mus musculus Drosophila melanogaster Haloferax volcanii Haloferax volcanii Homo sapiens S. cerevisiae Mus musculus Homo sapiens Drosophila melanogaster Homo sapiens S. cerevisiae Mus musculus Homo sapiens Mus musculus S. cerevisiae Mus musculus Homo sapiens

n.d. PLK n.d. CK2 CK2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. PKA n.d. n.d. n.d. NatA/Nat1 n.d. n.d. PKA NatB/Nat3 n.d. n.d.

Rush et al., 2005 Feng et al., 2001 Iwafune et al., 2002 Pardo et al., 1998 Castano et al., 1996 Bose et al., 2004 Gomes et al., 2006 Sumegi et al., 2003 Humbard et al., 2006 Humbard et al., 2006 Wang et al., 2007 Iwafune et al., 2002 Zong et al., 2006 Rush et al., 2005 Sumegi et al., 2003 Wang et al., 2007 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007 Zong et al., 2006 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007

b4 Met1-SO b5 Processed b5 O-GlcNAc b6 Ser
P b7 Tyr102
P b7 Ser
P; Thr
P b7 Met14-SO, Met181-SO b7 O-GlcNAc 19S Regulatory particle ATPases Rpt1
P Rpt1 Processed Rpt2 Processed; N-myristoyl Rpt2 N-myristoyl Rpt2
P Rpt2 Processed; N-myristoyl Rpt2 Processed; N-myristoyl Rpt2 O-GlcNAc Rpt2 Rpt3 Rpt3 Rpt3 Rpt3 Rpt3 Rpt4 Rpt4

O-GlcNAc N-Acetyl
P N-Acetyl N-Acetyl O-GlcNAc Processed; N-acetyl Processed; N-acetyl

S. cerevisiae Homo sapiens Drosophila melanogaster Rattus norvegicus Homo sapiens Mus musculus S. cerevisiae Drosophila melanogaster

n.d. n.d. n.d. n.d. n.d. PKA n.d. n.d.

Iwafune et al., 2002 Wang et al., 2007 Sumegi et al., 2003 Wehren et al., 1996 Rush et al., 2005 Zong et al., 2006 Iwafune et al., 2002 Sumegi et al., 2003

Homo sapiens Homo sapiens S. cerevisiae Oryza sativa Homo sapiens Mus musculus Homo sapiens Homo sapiens; Oryctolagus sp. Drosophila melanogaster S. cerevisiae Homo sapiens Homo sapiens Mus musculus Drosophila melanogaster S. cerevisiae Homo sapiens

n.d. n.d. n.d. n.d. n.d. n.d. n.d. OGT

Mason et al., 1998 Wang et al., 2007 Kimura et al., 2003 Shibahara et al., 2002 Mason et al., 1998 Gomes et al., 2006 Wang et al., 2007 Zhang et al., 2003

n.d. NatB/Nat3 n.d. NatB/Nat3 n.d. n.d. NatA/Nat1 n.d.

Sumegi et al., 2003 Kimura et al., 2003 Mason et al., 1998 Wang et al., 2007 Gomes et al., 2006 Sumegi et al., 2003 Kimura et al., 2003 Wang et al., 2007 (continued)

Table 2.3 (continued) Subunit

PTM

Organism

Rpt4 O-GlcNAc Drosophila melanogaster Rpt5 Processed; N-acetyl S. cerevisiae Rpt5 N-acetyl Homo sapiens Rpt5 Ser9
P Homo sapiens Rpt6 Processed; N-acetyl S. cerevisiae Rpt6 N-Acetyl Oryza sativa Rpt6
P Sus scrofa Rpt6 N-Acetyl Mus musculus Rpt6 Processed; N-acetyl Homo sapiens 19S Regulatory particle non-ATPase subunits Rpn1 Processed S. cerevisiae Rpn1 N-Acetyl Homo sapiens Rpn1 Ser16
P Homo sapiens Rpn1 Ser361
P Homo sapiens Rpn1 N-Acetyl Mus musculus Rpn2 Processed; N-acetyl S. cerevisiae Rpn2 N-Acetyl Homo sapiens Rpn2 Met1-SO Homo sapiens Rpn2 Thr311
P Homo sapiens Rpn2 Thr311
P; Ser315
P Homo sapiens Rpn2 Thr273
P Homo sapiens Rpn3 Processed; N-acetyl S. cerevisiae Rpn3 Processed Oryza sativa Rpn3 Processed Daucus carota Rpn3 O-GlcNAc Drosophila melanogaster Rpn5 Processed; N-acetyl S. cerevisiae

PTM Catalyst

References

n.d. NatA/Nat1 n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d.

Sumegi et al., 2003 Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Shibahara et al., 2002 Satoh et al., 2001 Gomes et al., 2006 Wang et al., 2007

n.d. n.d. n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d. NatA/Nat1

Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Gomes et al., 2006 Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Shibahara et al., 2002 Smith et al., 1997 Sumegi et al., 2003 Kimura et al., 2003

Rpn5 Rpn5 Rpn6 Rpn6 Rpn6 Rpn6 Rpn6 Rpn6 Rpn7 Rpn7 Rpn8 Rpn8 Rpn8 Rpn8 Rpn9 Rpn10 Rpn10 Rpn10 Rpn10 Rpn10 Gankyrin Rpn11 Rpn11 Rpn12 Rpn12 Rpn13

N-Acetyl O-GlcNAc Processed; N-acetyl N-Acetyl Processed; N-acetyl Ser13
P Ser78
P O-GlcNAc Processed Processed Processed; N-acetyl Processed
P Thr186
P Ser106
P Processed Processed Ser266
P Ser358
P Ser358
P; Ser361
P N-Acetyl N-Acetyl Ser224
P Processed O-GlcNAc Processed; N-acetyl

Mus musculus Drosophila melanogaster S. cerevisiae Mus musculus Homo sapiens Homo sapiens Homo sapiens Drosophila melanogaster S. cerevisiae Homo sapiens S. cerevisiae Homo sapiens Homo sapiens Homo sapiens Homo sapiens S. cerevisiae Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens S. cerevisiae Homo sapiens S. cerevisiae Drosophila melanogaster Homo sapiens

The materials of Maupin-Furlow et al., 2006, were used for preparation of this table.

n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. NatB/Nat3 n.d. n.d. n.d. n.d.

Gomes et al., 2006 Sumegi et al., 2003 Kimura et al., 2003 Gomes et al., 2006 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Sumegi et al., 2003 Kimura et al., 2003 Wang et al., 2007 Kimura et al., 2003 Wang et al., 2007 Mason et al., 1998 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Wang et al., 2007 Kimura et al., 2003 Sumegi et al., 2003 Wang et al., 2007

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Multiple isoforms of proteasome subunits have been described, which further account for the structural complexity in 20S proteasome subunit composition (Claverol et al., 2002; Froment et al., 2005). The proteasome catalytic activity may be affected by various environmental factors such as oxidative stress, pathological states such as cancers or neurological disorders, aging, or pharmacological agents (Gillardon et al., 2007; Shah et al., 2001), and such fundamental cellular processes as immune response, programmed cell death and differentiation (Iwafune et al., 2002; Kulichkova et al., 2004b; Mason et al., 1996; Tsimokha et al., 2006). Structural (and more particularly, post-translational) modifications that affect either the protein to be degraded or the proteasome subunits may lead to altered or even inhibited proteolytic functions (Ermonval et al., 2001; Gillardon et al., 2007). Recently an evidence was obtained that changes in subunit modification of 20S proteasomes in humans suffering Alzheimer’s disease affect at least two of proteasome’s proteolytic activities (Gillardon et al., 2007). 4.1.2.1. Phosphorylation of proteasome subunits At the present time, several subunits of both 20S proteasomes and their regulatory complexes (19S and PA28) are known to be phosphorylated in vivo. For the first time, these data were obtained on 20S proteasome samples, isolated from the Drosophila larvae (Haass and Kloetzel, 1989), where four phosphorylated proteasome subunits were detected. Three unidentified mammalian 20S proteasome subunits hereafter were shown to be phosphorylated in vitro by cAMP-dependent protein-kinase, co-purifying with bovine proteasomes (Pereira, Wilk, 1990). As well a proteasome subunit with molecular mass 30 kDa was shown to be phosphorylated by casein kinase II, co-purifying with human erythrocyte proteasomes (Ludemann et al., 1993). Furthermore, it was shown that subunit a6 (C2, iota) isolated from rice cells is phosphorylated by the same casein kinase II (Umeda et al., 1997) despite the fact that the phosphorylation sites of this subunit are not conserved in different organisms. Fernandez-Murray and co-authors (2002) identified a6 (C2), a3 (C9), and a5 (Pup2) yeast proteasome subunits to be the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome components. The most intense isoform of the human erythrocyte a7 subunit was shown to be phosphorylated (Claverol et al., 2002). This modification has recently been described to play a role in 26S proteasome stability by interacting with the 19S regulatory complex rather than having a direct effect on proteolytic activity (Bose et al., 2004). Studies of the phosphorylated amino acids from rat liver and human placenta have shown what subunit a2 (C3) contains phosphotyrosine and phosphothreonine, whereas phosphoserine was detected in b6 (C5), a3 (C9), a7 (C8) and a5 (z) subunits (Bose et al., 1999; Castano et al., 1996; Mason et al., 1996; Wehren et al., 1996). Phosphorylation of serine residues of human a3 (C9) at Ser74 and a7 (C8) at Ser250 was described recently by

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Wang and co-authors (2007). Attempt to estimate the possible functional significance of phosphorylation of these subunits was undertaken. Phosphorylation of a2 (C3) subunit at Tyr120 demonstrated to be significant for proteasome’s nuclear localization (Benedict et al., 1995). A hypothesis was offered, that phosphorylation of proteasomal subunits could be involved in regulation of the enzymatic activity of the complex via conformational changes (Mason et al., 1996). Dephosphorylation of proteasome subunits a3 (C9) and a7 (C8) (which could be phosphorylated on serine residues by casein kinase II in vitro (Castano et al., 1996) led to a small, but significant, decrease in two peptidase activities (Mason et al., 1996). On the other hand, in vitro phosphorylation of murine cardiac 20S proteasome (subunits a1, a2, a3, b2, b3 and b7) by protein kinase A significantly increased the chymotrypsin- and caspase-like activities (Zong et al., 2006). Recent data of Wang and co-authors (2007) demonstrated approximately half of the phosphorylated amino acid residues identified to be serines or threonines followed by proline. The last is known to be a marker of phosphorylation by mitogen-activated or cyclin-dependent kinases, so regulation of proteasomal phosphorylation state might be proposed to be cell cycle dependent (Wang et al., 2007). Phosphorylation of 20S core complex subunits may influence the interaction of proteasome with other proteins. The potential phosphorylation sites of a3 (C9) and a7 (C8) subunits are located on the ends of the cylindershaped 20S proteasome, and therefore phosphorylation of these subunits can control binding of regulatory complexes to the core particle. Researchers from the same group (Bose et al., 2004) have recently found phosphorylation of subunit a7 (C8) to stabilize the association of 19S regulatory complexes with 20S proteasomes to form the 26S proteasome, while dephosphorylation facilitated the formation of 11S-containing proteasome complexes. For their turn, the proteasomal regulatory complexes can also contain phosphorylated subunits. For example, the phosphorylated subunits were detected in 11S regulators obtained from rabbit reticulocyte lysates (Li et al., 1996). The analysis of phosphorylated amino acids obtained from human erythrocytes has shown phosphorylation to occur at serine residues and antiphosphoserine antibodies to bind all three PA28 polypeptides which are recognizable by anti-PA28 antibodies. Thus, a, b, and nuclear g subunits of PA28 complex are subjected to phosphorylation (Bose et al., 1999). When 11S complex subunits were dephosphorylated, this regulator has lost the ability to stimulate peptidase activities of proteasomes, so one can conclude, that phosphorylation of the PA28 complex controls the activity of proteasome via conformational changes of this regulatory particle. Experiments performed on the HL-60 cell line also showed the phosphorylation of PA28 to be increased in the presence of IFNg (Bose et al., 1999, 2001).

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Besides the above-described complexes, the 19S (PA700) regulators could be phosphorylated too. Thus, several subunits of this complex, including at least, one of the ATPases (S4), were shown to be phosphorylated in vivo (Mason et al., 1998) although functional significance of ATPase subunits phosphorylation is not elucidated yet. Wang and co-authors (2007) in their recent investigation by means of tandem mass-spectrometry demonstrated 10 subunits of 26S proteasome complex from human 293 cell line to be phosphorylated at total of 16 sites and 12 of these sites have not been reported previously. For the most part, phosphorylated subunits reported (eight subunits: one ATPase and seven non-ATPases) were elements of 19S regulatory complex, and only two a subunits were parts of 20S core. It seems to be significant, that all recognized in the above work phosphorylation sites were either threonines or serines, but not tyrosines (Wang et al., 2007). Perhaps, phosphorylation of ATPase subunits, via conformational changes, can affect substrate recognition and opening of a channel inside the 20S core particle. The assembly of the 19S complex, consisting of ‘‘base’’ and ‘‘lid,’’ probably can be under control of protein subunits phosphorylation, too. Although the functional significance of phosphorylation on the 19S subunits remains to be established, identification of novel phosphorylation sites by Wang and co-authors (2007) is the first critical step toward an improved understanding of its role in regulating the function of the 26S proteasome complex. However, the phosphorylation state of proteasome subunits was shown to be not a constant value. In turn, it changed under the action of agents altering physiological state of the cell. The list of the above cellular models includes induction of erythroid differentiation by hemin in K562 proerythroleukemia cells (Mittenberg et al., 2007) and doxorubicin- or diethylmaleate-induced apoptosis of the same cell line (Toktarova et al., 2004; Tsimokha et al., 2006, 2007a,b,c). During the last decade, several works have appeared about the effect of the phosphorylation state of cytoplasmic 26S proteasome subunits on their proteolytic activity (Iwafune et al., 2002; Kulichkova et al., 2004b; Mason et al., 1996; Tsimokha et al., 2006). Moreover, a selective effect of dephosphorylation of cytoplasmic 26S proteasomes on their RNase activity regarding high molecular weight ribosomal RNA and c-fos, c-myc and p53 mRNAs was shown (Mittenberg et al., 2002a,b, 2007; Toktarova et al., 2004; Tsimokha et al., 2006). Earlier the efficiency of the 26S proteasomal RNase activity toward high molecular weight ribosomal RNA was shown to depend on phosphorylation state of the proteasomal subunits (Kulichkova et al., 2004b; Mittenberg et al., 2002a). To find out whether a change in the phosphorylation state affects nucleolysis efficiency regardless of substrate, or whether it can produce different effects on nucleolysis of different substrates, i.e., whether it can affect the specificity of the RNase activity, the influence of dephosphorylation of these

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particle subunits on the nucleolysis of various mRNAs were studied. Dephosphorylation of the proteasomal subunits from control K562 cells was found to lead to an increase in their nucleolysis efficiency for the p53 mRNA, while dephosphorylation of proteasomes from the cells induced to apoptosis or differentiation had no effect on nucleolysis of this RNA. This suggests that the mechanism responsible for an increase in the ability to cleave a particular mRNA is connected with dephosphorylation of proteasome subunits in the case of differentiation induction (Mittenberg et al., 2007). Earlier changes in the phosphorylation state of the proteasome subunits during apoptosis were revealed (Tsimokha et al., 2006); however, regulation of the phosphorylation state of the proteasome during cell differentiation was not studied. To estimate possible changes in the phosphorylation state of the proteasome subunits after induction of differentiation, the subunits were separated electrophoretically and analyzed by immunoblotting with monoclonal antibodies against phosphothreonine, phosphoserine, and phosphotyrosine. Hemin caused dephosphorylation of the proteasome subunits mainly at tyrosines, which argues in favor of the participation of specific phosphatases in this process. On the other hand, hemin action led to a more intensive phosphorylation of several proteasomal 20S core particle subunits, with molecular masses from 25 to 30 kDa, at threonine, as well as the stimulation of the phosphorylation of at least one subunit (with a molecular mass about 38 kDa) at serine residues. Thus, during induction of differentiation the phosphorylation state of proteasome subunits did, in fact, change. As well, during induction of apoptosis (Tsimokha et al., 2006, 2007a,b,c), changes in the composition of the proteasome subunits are observed, which also could be associated with the regulation of specificity of these particle RNase activities. The results indicate that the ‘‘switching’’ of proteasomes to the nucleolysis of other mRNA groups could occur via the phosphorylation-dephosphorylation of subunits of these particles. The identities of the particular subunits for which the phosphorylation state changes during regulation of the specificity of RNase activity, as well as the cellular pathways responsible for the regulation of proteasome phosphorylation, are as yet unknown. However, the data concerned with phosphorylation of a subunits responsible for possession of RNase activity (a1, a5) and for RNA binding (a6), obtained on various organisms, from yeast (Fernandez-Murray et al., 2002) to higher plants (Umeda et al., 1997) and mammals (Beausoleil et al., 2004; Wehren et al., 1996; Zong et al., 2006), favour a supposition that subunit phosphorylation state could be a mechanism of regulation of proteasome-associated RNase activity. 4.1.2.2. N-Acetylation and N-terminal propeptide processing of proteasome subunits The autocatalytic removal of the N-terminal propeptides is one of the detailedly described proteasomal post-trancriptional modifications. These propeptides were shown to promote 20S particle assembly and protect the Thr1 active sites from acetylation and inactivation

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(Arendt and Hochstrasser, 1999). Generally this processing occurs within b subunits and leads to exposure of active site N-terminal threonine residues (Seemueller et al., 1996). In eukaryotic proteasomes such exposure takes place only in three subunits shown to be catalytically active (b1, b2 and b5), whereas propeptides of other b-type subunits are either intermediately processed (b6 and b7, the last was proposed to possess peptidase activity consisting in cleavage of bonds after small neutral amino acids (Unno et al., 2002) or remaining unprocessed (b3 and b4) (Groll et al., 1997). Co-translational N-a-acetylation by N-acetyltransferases is one of the most common protein modifications in eukaryotes. It occurs in 5090% of cases (Kimura et al., 2000; 2003), thus proteasomal subunits were demonstrated not to be an exception from the above rule: there are at least three N-acetyltransferases which 20S proteasome subunits found to be modified by (NAT1, MAK3, and NAT3). The a1, a2, a3, a4, a7, and b3 subunits were acetylated with NAT1, the a5 and a6 subunits were acetylated with MAK3, and the b4 subunit was acetylated with NAT3. Moreover, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the a5 and a6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were N-a-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region (Kimura et al., 2000). Experiments performed on yeast mutants have shown significance of N-terminal propeptide of catalytic 20S proteasome b subunits for protection of N-terminal catalytic threonine against N-acetylation (Arendt and Hochstrasser, 1999). The yeast 20S proteasome purified from either N-a-acetyltransferase deletion mutants or normal strains exhibited similar hydrolytic activities suggesting that N-a-acetylation does not significantly affect proteasome function in yeast (Kimura et al., 2000). Huang and co-authors (2001) found two isoforms of a2 and a7 subunits from Trypanosoma brucei 20S proteasomes to be acetylated at serine residues. Recent data demonstrate evidence that 15 subunits of human 26S proteasomes are acetylated at their N-termini. Eight subunits, Rpt3, Rpt5, Rpn1, Rpn2, Gankyrin (a non-ATPase subunit), a5, a6, and b4, are acetylated at the first methionine residue, whereas seven subunits, Rpt4, Rpt6, Rpn6, Rpn13/ADRM1, a4, a7, and b3, underwent Met1 processing followed by N-acetylation of the next amino acid residue. The N-terminal modifications of 12 subunits (Rpt1, Rpt4, Rpt5, Rpn2, Rpn8, Rpn9, Gankyrin, Rpn13/ADRM1, a4, a6, b5, and b6) have been identified for the first time for mammalian proteasomes (Wang et al., 2007). 4.1.2.3. Other modifications of proteasomal subunits Mass-spectrometric studies of 26S proteasome subunit modifications revealed a glycine residue at the N-terminus of Rpt2 subunit to be modified by myristoylation, both in humans (Wang et al., 2007), mice (Gomes et al., 2006), yeast (Kimura et al., 2003) and rice (Shibahara et al., 2002; 2004). Although the function of

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N-myristoylation in Rpt2 is not clear, the conservation of this modification on Rpt2 from yeast to human suggests an important function for Rpt2 myristoylation. Since this modification promotes protein-protein and protein-membrane interactions, it is permissible to speculate about a role of Rpt2 in the interaction of the 26S proteasome with membranes or other proteins (Wang et al., 2007). Experiments performed on yeast proteasomes by Demasi and co-authors revealed another modification of sulphur-containing amino acids, namely glutationylation of cystein residues (Demasi et al., 2001, 2003). Chimotrypsinlike activity of 20S proteasomes was shown to be inhibited by S-glutationylation more significantly than trypsin-like, while caspase-like activity was not changed (Demasi et al., 2003). A number of cytoplasmic and nuclear proteins were shown to be modified by O-linked N-acetylglucosamine (Wells et al., 2002). Similar to phosphorylation, serine, threonine and tyrosine residues could be underwent to O-glycosylation. Furthermore, dynamism and reversibility of both these modifications have led to proposition about the role of glycosylation in reversible blocking of phosphorylation site, temporary terminating the last modification. While phosphorylation of many proteins is a ubiquitinylation signal following by further proteasome degradation, prevention of phosphorylation via N-acetylglucosamine binding can extend a protein’s half-life (Rechsteiner et al., 1993). Analysis of proteasomes purified from lenses of ageing humans revealed a number of subunits to be glycated, glyco-oxydated and carboxymethylated (Viteri et al., 2004). Such oxidative modifications were shown to inhibit proteolytic activities of proteasomes, and while trypsine- and chimotrypsinelike activities decreased partially, the caspase-like one fully vanished. Therefore, in aging eye lens accumulation of oxidized protein may occur, that could cause trouble to cell survival following oxidative stress (Viteri et al., 2004). Mass-spectrometric analysis of rat hepatocyte proteins interacting with N-acetylglucosamine-specific antibodies has revealed an a6 subunit of 20S proteasome among them (Wells et al., 2002). Immunoblotting with specific monoclonal antibodies allowed to recognize at least 8 of 19 subunits of PA700 regulatory complex and at least 8 of 14 components of proteasome 20S core complex purified from Drosophila cells to be O-glycosylated (Sumegi et al., 2003). Rpt2 ATPase subunit of 19S regulatory complex was found to be O-glycosylated both in vivo and in vitro, while appearance of this modification promoted a decrease of proteasome activity via inhibitory effect on the subunit’s ATPase activity (Zhang et al., 2003). Recent studies of Wang and co-authors (2007) have revealed that Rpn2 subunit of 19S regulator, when not N-acetylated, contains Met1 in oxidized form—methionine sulphoxide. However, Met1 of a1 subunit from archea Haloferax volcanii proteasome was shown to be oxidized when N-terminus is acetylated either. Moreover, both the same modification and methionine

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oxidation were found in b-type proteasome subunits of this organism (Humbard et al., 2006). Such modifications can lead to a shift of protein’s isoelectric point and to appearance of additional spots on 2D map. Detailed studies of yeast proteasomes allowed to detect methionine oxidation in such 20S core subunits as a1 (Met209), b1 (Met4, Met146), b4 (Met1), and b7 (Met14, Met181) (Iwafune, 2002). Recent proteomic studies of rat liver proteasomes have shown a4 subunit to consist of two distinct spots on a 2D map, one of which was proposed to correspond a protein containing oxidized Met71 (Schmidt et al., 2006). Moreover, the same authors have detected appearance of cysteine residues oxidation (cysteine sulphonic acid). The above modification is quite rare, and being not a product of electrophoretic sample preparation, it could be supposed to have a significant biological role in regulation of protein structure and function (Schmidt et al., 2006). The comprehensive overview on proteasome-associated proteins and their influence on stability, activities and other characterictics of the complexes is presented in the review by Glickman and Raveh (2005).

4.2. Regulation of proteasome abundance in the cell and cellular compartments 4.2.1. Regulation of proteasome subunit expression Unfortunately, current knowledge of regulation mechanisms of proteasome subunit expression, limited to several models described, seems to be insufficient and requires further studies. The Saccharomyces cerevisiae yeast were shown to have own unique system of coordinated control of proteasomal gene expression: the upstream activating cis-element called ‘‘proteasome-associated control element’’ being a target sequence for the transcription factor Rpn4 that activates genes encoding proteasomal subunits (Mannhaupt et al., 1999). The above transcription factor was found to be degraded by matured proteasomes (Xie and Varshavsky, 2001). Rpn4 was also demonstrated to participate in upregulation of all proteasomal subunits upon treatment with proteasome inhibitors (Fleming et al., 2002), so it is proposed to be a cellular tool responsible for the compensation of proteasome inhibition. Examples of regulation of proteasome expression were revealed also in mammals. An evidence was demonstrated that levels of proteasome expression depend on proliferative state of the cell. Thus, expression of proteasomes in actively proliferating hematopoietic malignancies was shown to be abnormally high (Kumatori et al., 1990), whereas in vitro terminal differentiation of immature leukemic cell lines led to significant decrease of proteasome expression independently of both cell type and differentiating agent (Shimbara et al., 1992). On the other hand, in normal cells up-regulation was demonstrated for mitogen-induced resting T-lymphocytes (Shimbara

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et al., 1992). Recent data showed proteasome expression to be up-regulated by action of proteasome inhibitors (MG132 and lactacystin) both in normal (primary culture of rat smooth muscle cells) and malignant (human HeLa, T2, HEK293 and green monkey COS7) cells (Meiners et al., 2003). The phenomenon observed is suggested to be general for all cells tested. The increase of proteasome subunit production was observed for both constitutive components of 20S core complex and proteins of 19S regulatory particles, whereas subunits of 11S proteasome activator and interferon-inducible subunits were not up-regulated at all. Authors showed that proteasomes are upregulated at both transcriptional and translational levels, so the data obtained suggest mammalian cells to have a compensatory mechanism lying in de novo proteasome formation in response to the action of proteasome inhibitors (Meiners et al., 2003). Experiment concerned with proteasomal subunit b5 overexpression in cultured human lens epithelial cells resulted in induction of expression of other 20S core catalytic subunits (b1 and b2), i.e., upregulation of proteasomes via transfection by a plasmid carrying b5 subunit sequence has been observed (Liu et al., 2007). The above model could be considered as a tool for prevention of age-related decline of proteasome activity in human lens epithelium, which can lead to cataract formation and is thought to contribute to the ubiquitinated and carboxymethylated proteins accumulation detected in elderly lenses (Viteri et al., 2004). However, the mechanism of proteasome expression regulation is not understood yet. The action of proteasome inhibitors did not affect expression of PA28 regulatory complex in a number of cell types (Meiners et al., 2003), whereas up-regulation of the above proteasome activator was observed during maturation of dendritic cells (Ossendorp et al., 2005). Moreover, in PA28b gene promoter, an NF-kB active site was found, so, therefore, expression of PA28b could be regulated by transcription factors of NF-kB family. Induction of PA28b subunit was shown to provide production of PA28 complex. In contrast to other cells, regulation PA28 expression in mature dendritic cells was demonstrated to be interferon-independent (Ossendorp et al., 2005). 4.2.2. Control of proteasome intracellular localization and assembly Proteasomes in eukaryotic cells are found both in nuclei and cytoplasm. Several proteasomal subunits were demonstrated to contain specific amino acid sequences called nuclear localization signals (NLSs) (Nederlof et al., 1995; Wang et al., 1997). In rat liver cells 16% of proteasomes revealed to be localized in nuclei, 14% detected to be associated with endoplasmic reticulum, whereas others were shown to be located in cytosol matrix (Rivett et al., 1992). However, studies of spermatozoa proteasomes detected 20S core and PA28g activator near the neck where centrosomes located,

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whereas no proteasomes were detected within nuclei (Wojcik et al., 2000). In contrast, in early stages of embryonal development and in malignant cells proteasomes were found to be accumulated in nuclei (Klein et al., 1990; Kumatori et al., 1990). In the cell nucleus, proteasomes are found not only in association with chromatin. During interphase, proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci (Rockel et al., 2005). Proteasomal activity has been detected in isolated nucleoplasmic cell fractions (Scharf et al., 2007), in isolated and purified nuclear proteasomes (Tsimokha et al., 2006) and nuclei of living cells (Scharf et al., 2007). Thus, microinjection of ectopic fluorogenic protein ovalbumin revealed that proteasomal protein degradation occurs in distinct nucleoplasmic foci (Rockel et al., 2005). Therefore, the proteasomes are proteolytically active in distinct nuclear domains. However, nuclear proteolysis remains mainly uninvestigated and little is known about the control of nuclear functions of proteasomes. In recent studies of Feist and co-authors (2007) performed on various cells, a suggestion was made that the distribution of proteasomes in the nucleus and cytoplasm is dependent on the cell type and cell cycle, where it may reflect changes in cell metabolism. Indeed, intracellular proteasome distribution is caused by requirements of certain cellular compartment. Thus, for example, respectively short cell cycle duration in yeast requires proteasomes to be abundant in those compartments where it is necessary to cleave a number of short-lived proteins known to control cell cycle progression and secretory and membrane protein biogenesis (Enenkel et al., 1999). When compared different immunochemical stainings of various cells, it turned out that the relative affinity with respect to nuclear and cytosolic proteasomes varies between different anti-proteasomal antibodies, probably due to different affinities regarding structural properties of proteasomal complexes or different interactions with regulatory proteins in examined compartments of the cell. The above observation shows a wide diversity of proteasome subsets within a single cell (Feist et al., 2007). The mechanisms controlling proteasome intracellular localization remain poorly studied at present time. However, the majority of the 20S core complex subunits was found to be phosphorylated (Gomes et al., 2006; Rush et al., 2005; Wang et al., 2007; Zong et al., 2006), while at least six of them underwent the above modification at tyrosine residues (Benedict et al., 1995; Iwafune et al., 2002; Rush et al., 2005; Wehren et al., 1996; Zong et al., 2006). Since tyrosine phosphorylation of proteasome subunits has been suggested to be involved in nucleo-cytoplasmic transfer of proteasomes (Tanaka et al., 1990; Wang et al., 1997), the specific kinases and phosphatases are considered to be candidates for the role of proteasome controller. Thus, experiments performed on fissing yeast discovered a

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complicated system of proteasomal nuclear localization control, containing heat-inducible regulatory protein Cut8, shown to be an upstream positive regulator of Cek1 kinase, which, in its turn, could subsequently control localization of proteasome via subunit phosphorylation. At the same time, Cut8, being very unstable, could be a substrate for nuclear proteasomes. Cut8 may be a feedback sensor for the amount of proteasome locating to the nucleus (Takeda and Yanagida, 2005; Tatebe and Yanagida, 2000). The authors (Takeda and Yanagida, 2005) suggest that regulation of nuclear proteasome abundance through ubiquitination and destruction of the sensor and anchor Cut8 is a conserved mechanism as a Cut8 homolog has been discovered in flies. Another localization controller, the internal one, was found in Schizosaccharomyces pombe: the Rpn5 subunit of 19S proteasome activator was shown to be involved in regulation of not only nuclear localization, but also proper assembly of proteasome complex ( Yen et al., 2003). The activity of purified nuclear proteasomes (isolated from control and apoptotic human K562 cells) is regulated by phosphorylation of proteasome subunits (Tsimokha et al., 2006, 2007a,b,c). The other enzyme, poly(ADPribose) polymerase (PARP), is also involved in the regulation of drug (adryamycin)-induced nuclear proteasome activation (Ciftci et al., 2001). Proteasome disassembly under severe stress is one of the mechanisms of proteasome’s abundance control (Glickman and Raveh, 2005). 4.2.3. Export from the cells Proteasomes were shown to be identified in various cellular compartments (see Section 4.2.2). Besides different intracellular populations, thoroughly studied by investigators all over the world, the extracellular one was detected in early 90th ( Wada et al., 1993). This proteasome population was found in the blood serum of both healthy subjects and patients with such hematologic malignancies as acute leukemia, chronic myelogenous leukemia, non-Hodgkin’s lymphoma, and multiple myeloma (Feist et al., 2007; Jakob et al., 2007; Lavabre-Bertrand et al., 2001; Wada et al., 1993). The estimated serum proteasome levels in control group were significantly lower than in patients. The data obtained suggest the elevated levels of serum proteasomes in patients to be derived from tumor cells ( Wada et al., 1993). The above results are in accordance with previously reported evidence of an abnormally high expression level of proteasomes in human leukemic cells first demonstrated by Kumatori and co-authors (1990). More recent studies recognized circulating proteasomes in blood serum of patients suffering various autoimmune disorders—systemic lupus erythematosus, dermatomyositis/polymyositis, primary Sjogren’s syndrome, undifferentiated connective tissue disease, etc. (Egerer et al., 2002; Feist et al., 1996, 1999, 2007; Zoeger et al., 2006), and authors consider extracellular proteasomes to be a suitable marker of numerous pathological processes listed below. Serum proteasome population was supposed to be a sensitive indicator for tissue injury and cellular turnover reflecting the chronic and destructive

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activity of rheumatic diseases (Egerer et al., 2002). Lavabre-Bertrand and co-authors (2001) proposed plasma proteasome level measurement to be an instrument for monitoring patients with neoplastic disease. They suppose that circulating proteasomes could be a kind of nonspecific warning signal which will allow detection of a still unknown neoplastic process: moreover, the abnormally high level may prompt a search for myeloid disorders or solid tumors. In contrast, a low level may suggest a lymphoid disorder. The plasma proteasome level also could be a kind of prognostic factor for disease burden detection ( Jakob et al., 2007). In patients with lymphoid disorders, an increase in the circulating proteasome level may lead to the disease transformation into an aggressive form of lymphoma (Lavabre-Bertrand et al., 2001). And, finally, plasma proteasome level determination may help to quantify patient response to chemotherapy. One also might hypothesize that plasma proteasome evaluation may help to monitor treatments based on the use of proteasome inhibitors (Lavabre-Bertrand et al., 2001). However, recent data of the same research group (Stoebner et al., 2005) suggest circulating proteasomes to represent themselves a marker more of nonspecific inflammation than of early cancer. Zoeger and co-authors (2006) have shown chromatographically that circulating proteasomes could be subdivided into at least six subpopulations, whereas subpopulations of intracellular ones purified from various blood cells encounter not more than five subtype peaks. Circulating proteasomes were shown to be intact both in healthy persons and patients with autoimmune disorders; they were enzymatically active, too (Zoeger et al., 2006). However, qualitative differences between cytoplasmic and extracellular proteasome populations and specificity of exported population were demonstrated by Kulichkova and colleagues (2004b). Thus, serum population of proteasomes retained enzymatic activities characteristic for these particles, whereas their both proteolytic and RNase activities differ from those obtained for intracellular proteasomes. Specific features of subunit content were also found in extracellular proteasomes. And finally, activities of these proteasome populations were shown to depend on physiological state of the cell. Thus, induction of apoptosis led to export of specific proteasome subpopulation with peptidase activities different from ones of extracellular proteasomes from control cells (Kulichkova et al., 2004b).

5. Reprogramming of Proteasomes at Immune Response, Differentiation and Apoptosis It was mentioned above that the proteasomes play important roles in most cellular processes. To perform their functions, proteasomes must be under a tight regulatory control and change their subunit composition and

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enzymatic activities to adapt them to the requirements of the each of the cellular processes during the cell life. Phosphorylation at serine, threonine, or tyrosine residues of regulatory proteins is the key event in signal transduction and cell cycle progression (Coux et al., 1996). Moreover, various protein kinases are involved in induction of apoptosis (Bandyopadhyay et al., 2004; Contri et al., 2005; Jin et al., 2005; Sabatini et al., 2004; Wang et al., 2005). Phosphorylation of substrates and enzymes also plays important roles in the ubiquitin-proteasome pathway (Glickman and Ciechanover, 2002; Wojcik and DeMartino, 2003). The 26S proteasome is posttranscriptionally modified by phosphorylation as well as by N-acetylation, glycosylation, 4-hydroxy-2-nonenal-alkylation in various species (Bose et al., 1999; Claverol et al., 2002; Farout et al., 2006; Fernandez Murray et al., 2002; Kimura et al., 2000; Schmid et al., 1993; Sumegi et al., 2003; see Chapter 4.1). These modifications of proteasomal subunits are intensively studied at present (see Section 4.1.2). Moreover, at present the changes of proteasomes proteolytic activity are widely investigated during various cell processes and under the action of different stimuli on a cell (Abramova et al., 2005; Ahn et al., 1991; Beyette et al., 1998; Ebisui et al., 1995; Hayashi and Goto, 1998; Kulichkova et al., 2004b; Low et al., 2001; Shibatani et al., 1996). Next, we consider in more detail changes in proteasomes during immune response, differentiation and apoptosis.

5.1. Changes of proteasome at immune response Under conditions of an intensified immune response, many eukaryotic cells adapt ubiquitin–proteasome system to the protein-breakdown process for optimized generation of antigenic peptide epitopes (see Section 3.4). Thus, as already mentioned above, during an immune response to pathogens, the pro-inflammatory cytokine INFg and tumor necrosis factor TNF-a are released and induce the replacement of constitutively expressed catalytic subunits of the proteasomes (b1, b2, b5) with subunits LMP2, LMP7, and MECL-1. Proteasome activity is further changed by the IFNg-mediated induction of the proteasome regulator PA28a/b (Groettrup et al., 2001b; see Section 3.4). IFNg greatly increases the levels of the mRNAs encoding LMP2 and LMP7, and the composition of the proteasome is changed in response to stimulation by IFNg, due to assembly of newly synthesized subunits (Aki et al., 1994). Interestingly, IFNg dramatically stimulates the trypsin-like and chymotrypsin-like proteasomal activities and inhibits the peptidyl-glutamyl-peptide hydrolyzing activity (Aki et al., 1994). After treatment of human embryonic lung L-132 cells with IFNg, the level of 26S proteasomes decreases and at once there is an increase in proteasome subcomplexes PA28. At that, free 19S regulatory complexes are not detected (Bose et al., 2001). It is significant that

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after IFNg treatment, the level of phosphorylation of proteasome subunits is decreased (Bose et al., 2001). Thus, the IFNg induces decrease of the phosphorylation of a3 (C9) and a7 (C8) proteasomal subunits (Bose et al., 2001). The decrease in phosphorylation of 19S component–ATPase subunit Rpt2 (S4) after IFNg treatment was also found (Rivett et al., 2001). Thus, modification of the proteasomes subunit composition and their functions under the action of IFNg suggests the reprogramming of the proteasomes, directed on specific function during immune response.

5.2. Proteasome reprogramming at differentiation Several studies have also shown the proteasome reprogramming during differentiation. For example, the molecular assembly and the proteolytic activity of certain 26S proteasome subunits undergo changes during starfish oocyte maturation induced by a maturation-inducing hormone, L-methyladenine (Sawada et al., 1999). Moreover, at least one component of 26S proteasomes changes during Xenopus oocyte maturation (Tokumoto et al., 1999a). Furthermore, the reprogramming of proteasomes at cellular differentiation is evidenced by changes in subcellular distribution and in the subunit composition of proteasomes during myogenic differentiation of satellite cells (Foucrier et al., 1999) and human lymphoblastic and monoblastic U937 cell line (Baz et al., 1997; Bureau et al., 1997; Henry et al., 1997). Other studies were focused on human proerythroleukaemic cell line K562 (Mittenberg et al., 2002a,b, 2007). The induction of erythroid differentiation of these cells by hemin is accompanied by changes in the phosphorylation state of several proteasome subunits at tyrosine, threonine, and serine residues (Mittenberg et al., 2007). Interestingly, treatment of K562 cells leads to redistribution of proteasomes and their migration mainly to the cytoplasm (Mittenberg et al., 2002b). The endoribonuclease activity of proteasomes also undergoes changes during differentiation of K562 cells (Mittenberg et al., 2002a,b, 2007).

5.3. Apoptosis-induced changes of proteasomes In thymocytes, changes in proteasomal proteolytic activity during glucocorticoid dexamethasone-induced apoptosis have been found (Beyette et al., 1998). So the dexamethasone treatment causes a decrease of the peptidylglutamyl peptide hydrolase, trypsin- and chymotrypsin-like activities of proteasomes. It is significant that the decreases of two proteasomes activities (peptidylglutamyl peptide hydrolase and trypsin-like) were canceled by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased

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further in the presence of this caspase inhibitor. The authors suggest that these changes of proteasomal proteolytic activity during the apoptosis in thymocytes may be responsible for the turnover of specific proteins, leading to apoptosis (Beyette et al., 1998). In tobacco hawkmoth, Manduca sexta after the appearance of the adult insect from its pupal cuticle, the intersegmental muscles undergo apoptotic regression in response to declining levels of the steroid hormone (Schwartz and Truman, 1983). At the same time, the proteasomal proteolytic activity increased about ninefold (Dawson et al., 1995; Jones et al., 1995). This increase of proteasomal proteolytic activity is correlated with the increase of the cellular amounts of the proteasomes and with the incorporation of new subunits into the 20S proteasomes (Dawson et al., 1995; Low et al., 1997). Furthermore, the proteasomes isolated from condemned muscles contained several new unidentified subunits that were not detected in the purified proteasomes from precommitment muscles (Dawson et al., 1995; Jones et al., 1995). Such changes in both proteasomal proteolytic activity and subunit composition during development of Manduca sexta show a reprogramming of the proteasomes that might result in an enhanced proteasomal degradation of certain ubiquitinated cellular anti-apoptotic proteins, leading to apoptosis of intersegmental muscle cells (Naujokat and Hoffmann, 2002). Interestingly, the accumulation of proteasomes in intersegmental muscle cells and thymocytes after induction of the apoptosis has been found (Beyette et al., 1998; Dawson et al., 1995; Jones et al., 1995). The increase of the proteasomes concentration together with regulatory reprogramming may facilitate the rapid proteolysis of proteins which function as anti-apoptotic regulators (Dawson et al., 1995; Low et al., 2001). Thus, the changes in proteasome subunit composition and proteolytic activity showing reprogramming of the proteasome have been observed during apoptosis of normally growing tissues. However, for a long time there have not been any reports on the changes of structure and function of the proteasomes in neoplastic tissues undergoing apoptosis. Recently, several works devoted to reprogramming of proteasomes during the apoptosis in leukemic cells have appeared. The proteasome proteolytic activity is changed under the action of the apoptotic inductors (doxorubicin [DR], diethylmaleate [DEM]) (Tsimokha et al., 2006, 2007a,b,c). At that, the apoptosis induction changes the specificity of the proteolytic activity of the proteasomes, isolated from both cytoplasm and nuclei of K562 cells. Thus, the trypsine- and chymotrypsine-like activity of proteasomes isolated from nuclei and cytoplasm of DR-induced cells is increased compared to the activity of these proteasomes from control cells K562 (Tsimokha et al., 2007a,c). Moreover, the endoribonuclease activity of proteasomes isolated from cytoplasm and nuclei of K562 cells undergoing apoptosis is also changed (Kulichkova et al., 2004b; Mittenberg et al., 2007; Toktarova et al., 2004;

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Tsimokha et al., 2006, 2007a,b,c). The characteristics of RNase activity of different proteasomal subpopulations differ. These changes of RNase activity of the proteasomes suggest the participation of proteasomal ribonuclease in the regulation of mRNA stability during the induction of apoptosis. For example, proteasomes can control the level of the apoptotic proteinsregulators both by ubiquitin-dependent proteasomal proteolysis and by the specific endonucleolysis of mRNAs encoding these proteins. And, thus, the cell can more quickly and effectively inactivate the anti-apoptotic genes and/or activate the pro-apoptotic genes during the realization of the death program. Treatment of K562 cells with the apoptotic inductor (DEM or DR) leads to yet unidentified modifications of proteasomal subunits (a1-a7 and b2, b3, b7) including catalytic subunits associated with proteolityc (b2) and RNase (a5/zeta, a1/iota/alpha type 6) activities (Tsimokha et al., 2006, 2007a,b,c). Moreover, the proteasomes isolated from control and apoptosisinduced K562 cells differ in the phosphorylation state of a number of subunits on threonine, serine and tyrosine residues (Tsimokha et al., 2006, 2007a,b,c). The specific changes of phosphorylation state of nuclear and cytoplasmic proteasomes and regulation of their phosphorylation state under the action of apoptotic inductors suggest the existence of specific regulatory cellular pathways, involving specific protein kinases and phosphatases. The observed changes in the phosphorylation state of specific subunits suggest that these modifications are necessary for the specific proteasomal functions in apoptosis in K562 cells. Thus, the subunit composition and enzymatic activities of proteasomes are changed in K562 cells undergoing apoptosis, and therefore the apoptosis of neoplastic cells also involves reprogramming of proteasomes.

6. Proteasomes in Regulation of Different Levels of Gene Expression 6.1. Action at multiple stages of transcription process A growing body of evidence reveals that proteasomes are involved in the control of different levels of gene expression: transcription process, messenger RNA stability and translation. The physical and functional association of subunits of the 20S core and 19S regulatory proteasome subparticles with approximately 6400 yeast genes has been studied (Sikder et al., 2006). The results revealed the crosslinking of the intact 26S proteasomes to most genes, while several hundred genes interacted with either the 20S or 19S subparticles. Correlation of many of these associations with gene expression levels and the presence of RNA polymerase II has been demonstrated. These data suggest

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independent functions for whole proteasome particles and for the proteasomal subparticles in transcription. Indeed, genetic and molecular biology evidence briefly summarized below, confirms that the intact 26S proteasome as well as their subparticles and subunits are involved in transcriptional regulation. The proteasomes participate in the regulation of multiple stages of gene transcription process through proteolytic and nonproteolytic activities; however, few targets of proteasomal regulation have been studied in detail. Proteasomes are implicated in transcription factor processing, proteolysis of co-activators, chromatin modifications, control of elongation, termination and other stages of transcription. Proteolytic roles of the proteasome in transcription involve the timely regulated stimulation of transcription factors via their processing or degradation of inhibitory proteins. The activity of a number of transcription factors and their regulation depends on this process, named ‘‘regulated ubiquitin/proteasome-dependent processing.’’ Proteins of the mammalian NF-kB family and the yeast proteins SPT23 and MGA2 are controlled by this pathway (see review of Rape and Jentsch, 2004). The proteolytic activity of the proteasome is also necessary for the stable recruitment of RNA pol II at promoters and re-initiation of transcription possibly through the proteolysis of the transcriptional activators by the proteasome. For example, repression of proteasome proteolytic activity blocks the ability of the yeast activator Gcn4 to recruit RNA pol II to promoters (Lipford et al., 2005). Accurate transcription termination depends on proteolytic activity of 26S proteasomes as indicated by the increased read-through of a transcription termination site in result of their activity inhibition (Gillette et al., 2004). Interestingly, the 20S proteasome catalytic b subunit LMP2 (low molecular mass polypeptide 2), associated with peptidylglutamyl peptidase activity, interacts directly with the steroid receptor coactivator (SRC)-interacting proteins. The recruitment of the 20S b subunit LMP2 by SRC coactivators is necessary for cyclic association of estrogen receptor (ER)-regulated transcription complexes on ER targets (Zhang et al., 2006). The proteasome proteolytic activity is required also for continued hormone response. This activity modulates glucocorticoid hormone receptordependent gene transcription by regulating turnover and recycling of receptor/transcriptional-DNA complexes (Kinyamu et al., 2007). The involvement of proteasomal proteolytic activity in transcriptioncoupled DNA repair is indicated by ubiquitin-mediated proteolysis of the elongating form of the RNA polymerase II in response to DNA damage (Kleiman et al., 2005; Krogan et al., 2004; Reid and Svejstrup, 2004; Somesh et al., 2005). The destruction of RNA pol II blocks transcription until DNA damage can be repaired.

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The 19S proteasome subunits, particularly the base ATPases, affect transcription of a number of genes through a nonproteolytic mechanism. The 19S complex is crucial for efficient elongation of RNA polymerase II in vitro and in vivo. Thus, yeast strains carrying mutant alleles of genes SUG1 (Rpt6) and SUG2 (Rpt4), encoding ATPases of 19S compplex, exhibit phenotypes indicative of elongation defects. Moreover, in vitro transcription was inhibited by immunodepletion of subunit Sug1, and the elongation was restored by addition of immunopurified 19S complex. The physical interaction of the elongation factor Cdc 68 with the 19S subparticle is suggested by the results of their coimmunoprecipitation. Inhibition of the proteasomal proteolytic activity did not affect elongation of transcription (Ferdous et al., 2001). Proteasome implication in dissociation of elongation complexes is indicated by the enrichment of proteasomes and RNA polymerase II binding at ribosomal protein genes in yeast proteasome mutant (Auld et al., 2006). The 19S subunits exhibit DNA-helicase and protein-chaperone activities. ATPase subunit SUG1 is a 30 -50 DNA-helicase whose activity depends on the intact ATP binding domain (Fraser et al., 1997). Therefore, 19S subcomplex can influence interactions of components of the transcription machinery with DNA via DNA-helicase activity, particularly, change protein-DNA interactions. The hypothesis has been offered that subunits of the 19S complex might affect transcription elongation and other stages of transcription process through their protein-chaperone activity, influencing protein folding/unfolding (Collins and Tansey, 2006). The 19S proteins can act at early steps in the transcription process: in the preinitiation interaction of co-activator (‘‘SAGA’’ complex containing 15 subunits, among them histone acetyltransferase) with a target promoter during activation of several genes. The authors (Lee et al., 2005) suggest that the 19S proteasome regulatory particle, possibly due to its chaperone activity, somehow alters SAGA conformation to stimulate its recruitment to transcriptional activator. Thus, the ATPase components of the 19S subparticle were shown to be nesessary to facilitate SAGA recruitment to promoters by transcriptional activators such as Gal4p (Lee et al., 2005). More recently (Ferdous et al., 2007), a new nonproteolytic activity of the proteasomal ATPases, i.e., the active destabilization of activatorpromoter complexes (between Gal4-VP16 and Gal4 binding sites) was discovered. This reaction depends on the presence of the activation domain and ATP. The obtained data suggest that proteasomal ATPase 19S subunits are required for active turnover of the activator-promoter complexes. Yeast regulatory proteasome 19S subparticle was found to be physically associated with many general transcription factors, including components of yeast FACT (Cdc68/Pob3), TFIID, TFIIH, and the RNA polymerase II holoenzyme; moreover, the whole 26S proteasome interacts with regions of the yeast inducible genes GAL1, GAL10, and HSP82, including the 30 ends, in a transcription-dependent fashion. These results demonstrate that

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proteasome subunits can interact both with components of the transcriptional machinery and gene regions (Sun et al., 2002). Chromatin structure is also affected by 19S subunits. For example, ATPases subunits of yeast 19S complex: Rpt4 and Rpt6 are necessary for methylation of histone H3 at lysine residues 4 and 79 that is a signal defining active sites of transcription (Ezhkova and Tansey, 2004). The suggestion has been put forward (Collins and Tansey, 2006) that bound 19S subunits affect local chromatin structure to facilitate recruitment of appropriate methyltransferases to their target sites on the histone molecules. Interestingly, binding of 19S subunits to sites of active transcription depends on ubiquitylation of histone H2B (Ezhkova and Tansey, 2004). Whether the subunits of 19S interact with DNA (due to their helicase activity) or histones (via chaperone activity) or both remains to be studied as well as the time of their appearance at gene regions. Chromatin immunoprecipitation assays have demonstrated contradictory results: subunits of the 19S complex were found at promoter sequences when the genes are already active (Gonzalez et al., 2002) as well as prior to activation (Ezhkova and Tansey, 2004). This recruited complex does not contain subunits of the 20S core particle. Furthermore, in reorganization of chromatin structure, proteolytic activity of proteasome is also involved. Thus, during glucocorticoid – induced transcription, inhibition of this activity increases tri-methyl histone H3K4 levels with a corresponding accumulation of this modification on glucocorticoid receptor-regulated promoters (Kinyamu et al., 2007). Probably, both proteolytic and nonproteolytic activities of the proteasome are involved in remodeling of chromatin structure. Moreover, proteasome association with chromatin, and transcription factor processing are interrelated. Thus, the transcription factor Gcn4 is destructed only when bound to the target promoter (Lipford et al., 2003). Finally, transcriptional regulation by the proteasome is highly complex, these particles are involved at multiple stages of the process, and, moreover, they act at the same stages through different mechanisms. The recent evidence also suggest that proteasomes and their subparticles might be independently involved in different steps of transcription of different genes. Although the main data were obtained with yeast cells, the evidence of the recent works with higher eukaryotic cells, cited above, and the homology between eukaryotic and yeast transcription factors allows assuming occurrence of similar mechanisms in higher eukaryotes.

6.2. Participation in the regulation of posttranscriptional stages of gene expression 6.2.1. Translation The data suggesting proteasome engagement in coordinated regulation of protein translation and degradation, although via two different mechanisms, have been obtained.

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Proteasomes have been shown to participate in selective alteration of different messenger RNA (mRNA) translation (Baugh and Pilipenko, 2004). The translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3, are degraded by ubiquitin-proteasome pathway. The cleavage of eIF4G or eIF3a selectively affects the assembly of ribosomal preinitiation complexes on different cellular and viral mRNAs in an in vitro system probably due to competition of different RNA molecules for translation factors. Inhibition of proteasomal proteolytic activity prevents degradation of both factors, restores assembly of ribosomal complexes in vitro, and differentially affects translation of different mRNAs in vivo. The results of these studies allow suggesting the involvement of the changes in proteolytic activity of proteasomes in coordination of selective protein synthesis and degradation. Proteasome implication in the regulation of transcription of ribosomal protein (RP) genes allowed assuming a direct feedback mechanism of maintaining homeostasis between protein synthesis and protein degradation (Auld et al., 2006). In conditions under which cellular proteins accumulate, including proteasome mutants (Auld et al., 2006) and during inhibition of proteasomal proteolytic activity (Dembla-Rajpal et al., 2004; Jang and Wek, 2005), the decrease of the expression of RP genes in yeast cells was found. This observation suggests that proteasomes are required to activate transcription of the RP genes. Auld and Silver (2006) has supposed that the presence and activity of the proteasome at ribosomal protein genes could provide a cellular mechanism of responding to defects in protein degradation by slowing down transcription of the protein biosynthesis machinery. The changes in the activity of proteasomes would influence coordinative degradation of cellular proteins, of ribosomal proteins’ transcription and the affectivity of protein synthesis in the cells. 6.2.2. Messenger RNA stability At present there is no information about the role of the RNase activity of proteasomes in the cell (see Section 2.2). However, regulation of this activity in response to extracellular stimuli argues for its possible involvement in controlling the life time of mRNA molecules by these stimuli or, in other words, for a new mechanism controlling RNA stability in the cell. The phosphorylation dependence of the RNase activity of proteasomes suggests that these particles can represent a link between signalling pathways and mRNA stability; however, the cellular signalling pathways controlling ribonuclease activity of proteasomes remain to be elucidated. One of the key levels of gene expression control is the regulation of mRNA degradation rates. It becomes increasingly clear that signal transduction pathways affect mRNA stability. Specific cis elements in mRNA molecules necessary for mRNA turnover are recognized by signal responsive

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trans-active factors that influence signal-regulated RNA decay (Garneau et al., 2007; Shim and Karin, 2002; Tourriere et al., 2002). The RNA-binding activity of a number of trans-acting factors is modulated by phosphorylation (Tourriere et al., 2001). The mRNA decay rate can be regulated not only by the activity of protective proteins, but also may be directly controlled by changing activity of ribonucleases (Tourriere et al., 2001). Phosphorylation could also modulate activity of specific endonucleases. For instance, G3BP protein harbors the endoribonuclease activity that cleaves mouse c-myc 30 UTR in the phosphorylation-dependent manner. Besides, the phosphorylation status of G3BP in the cell is modulated by RasGAP cascade (Tourriere et al., 2003). The RNase activity associated with 26S proteasomes represents the new example of regulated endoribonuclease activity. Moreover, Laroia and co-authors (1999, 2002) have obtained data favoring the other mechanism of proteasome involvement in the control of the messenger RNA molecules lifespan. These authors have shown that 26S proteasomes are involved in the control of stability of specific mRNAs through proteolysis of mRNA-binding trans-acting proteins: AUFs. Thus, it is possible that proteasomes could participate in the both processes (proteolysis and nucleolysis) determining mRNA stability regulation. Furthermore, the evidence obtained so far allows proposing the proteasomes involvement in the coordinated control of degradation of proteins and mRNAs encoding these proteins. However, much additional work is needed, in particular, in vivo studies, to elucidate function of the proteasome-associated RNase activity in cellular processes. In all, the growing body of evidence favours the proteasome involvement in coordinated regulation of multiple stages of gene expression, placing the mechanisms of the control of proteasomal activity in the nodes of converge of gene expression regulatory pathways.

7. Concluding Remarks Recent evidence show that proteasomes are involved in the control of the main cellular processes as well as in the main stages of gene expression and that these complexes themselves are subjected to tightly organized regulation. However, the cellular pathways of this regulation (enzymes responsible for modifications of subunits, control pathways of their activity, mechanisms of the regulation of subunit expression, of cellular localization and others) remain mainly uninvestigated. Furthermore, despite great progress in proteasome studies, many other questions remain unanswered, and among them is the problem of specificity of proteasome involvement in transcription and other levels of gene

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expression—whether or not there is selectivity in the action of specifically modified proteasome subpopulations in the expression of different genes. Studies of specialized subpopulations of these complexes participating in responses to different external and internal signals and in various cellular processes are at the initial stage at the moment. Moreover, at present there is no information about the role of the RNase activity of proteasomes in the cell and the functional significance of the proteasome export from the cells.

ACKNOWLEDGMENTS This work was supported by Russian Foundation for Basic Research (project No. 08-0400834) and St. Petersburg’s Scientific Center of Russian Academy of Sciences.

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Permissive and Repulsive Cues and Signalling Pathways of Axonal Outgrowth and Regeneration Sheng T. Hou,* Susan X. Jiang,* and Robert A. Smith† Contents 1. Introduction 2. Neuritogenesis and Developmental Patterning 2.1. Role of neurotrophins 2.2. Extracellular matrix cues 2.3. Establishing polarity and axonal specification 3. Repulsive Guidance Cues in Axonal Pathfinding 3.1. Repulsive ligands 3.2. Receptors for repulsive guidance cues 3.3. Intracellular signalling pathways for repulsive guidance cues 4. Guidance Cues in Axonal Damage and Neuronal Death 4.1. Ischemic neuronal death and axonal damage 4.2. Semaphorin/neuropilin in neuronal death 4.3. Netrin-1/UNC/DCC in neuronal death (the dependence receptor theory) 4.4. RGM/Neogenin dependence receptors in neuronal death 4.5. CRMP in neuronal death and survival 4.6. CRMP modulation by calpain and CaMK during neuronal death 5. Guidance Cues and Synaptic Plasticity in Stroke Brains 6. Evidence for Guidance Cues as Therapeutic Targets 7. Concluding Remarks and Future Perspectives Acknowledgment References

* {

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Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, G12 8QQ, Scotland

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00603-5

# 2008 Elsevier Inc. and Her Majesty the Queen in right of Canada All rights reserved.

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Abstract Successful axonal outgrowth in the adult central nervous system (CNS) is central to the process of nerve regeneration and brain repair. To date, much of the knowledge on axonal guidance and outgrowth comes from studies on neuritogenesis and patterning during development where distal growth cones constantly sample the local environment and respond to specific physical and trophic influences. Opposing permissive (e.g., growth factors) and hostile signals (e.g., repulsive cues) are processed, leading to growth cone remodelling, and a concomitant restructuring of the cytoskeleton, thereby permitting pioneering extension and a potential for establishing synaptic connections. Repulsive cues, such as semaphorins, ephrins and myelin-secreted inhibitory glycoproteins, act through their respective receptors to affect the collapsing or turning of growth cones via several pathways, such as the Rho GTPases signalling which precipitates the cytoskeletal changes. One of the direct modulators of microtubules is the family of brain-specific proteins, collapsin response mediator protein (CRMP). Exciting evidence emerged recently that cleavage of CRMPs in response to injury-activated proteases, such as calpain, signals axonal retraction and neuronal death in adult post-mitotic neurons, while blocking this signal transduction prevents axonal retraction and death following excitotoxic insult and cerebral ischemia. Regeneration is minimal in injured postnatal CNS, albeit the occurrence of some limited remodelling in areas where synaptic plasticity is prevalent. Frequently in the absence of axonal regeneration, there is not only an inevitable loss of functional connections, but also a loss of neurons, such as through the actions of dependence receptors. Deciphering the cues and signalling pathways of axonal guidance and outgrowth may hold the key to fully understanding nerve regeneration and brain repair, thereby opening the way for developing potential therapeutics. Key Words: Axonal development, Growth cone, Growth factors, ECM, Guidance cues, Apoptosis, Regeneration, Myelin-secreted inhibitory molecules. ß 2008 Elsevier Inc. and Her Majesty the Queen in right of Canada

1. Introduction The failure of axons of the adult mammalian central nervous system (CNS) to regenerate after lesion or damage does not represent an intrinsic inability of CNS axons to grow, but rather the non-permissive nature of the CNS environment. For a brief period, the CNS is able to support sprouting of axons at the lesion site, but the growth cones soon adopt a swollen dystrophic morphology typical of growth inhibition (Dickson, 2002; Schnorrer and Dickson, 2004; Liu et al., 2006). Evidence derived from genetic and in vitro studies has demonstrated that CNS environment may

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exert four kinds of actions on growth cones, namely, chemorepulsion, chemoattraction, contact independent repulsion and contact dependent attraction. For example, injured CNS axons are arrested in the adult injured brain partly due to the presence of growth inhibitory chemorepulsive and contact independent repulsive ligands secreted from oligodendrocytes/ myelin, reactive astrocytes and fibroblasts in the scar tissue. Neurons must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction and neuronal death. It is therefore not surprising that strategies to promote regenerative axonal growth in the CNS after brain injury are thwarted by the plethora of inhibitory ligands and the ligand promiscuity of some of their receptors (Carmeliet and Tessier-Lavigne, 2005; De Wit and Verhaagen, 2003; Pasterkamp and Kolodkin, 2003; Pasterkamp and Verhaagen, 2001). In the context of cerebral ischemia-induced brain damage, the molecular and biochemical mechanisms involved in the retraction and collapse of the axonal network remains unclear. One of the early morphological changes accompanying excitotoxicity-induced cell death in cultured neurons is the retraction/collapse of the neuritic network (S. T. Hou and S. X. Jiang, unpublished observations), which strongly argues that axonal damage occurs before the emergence of the typical morphological hallmarks of neuronal death (Deckwerth and Johnson, 1993, 1994). Typically, axonal degeneration is manifested by irregular blebbing of axons with thinning and fragmentation, followed by retraction and collapse of the axonal network. While axonal damage may be an outcome of the death process occurring within the cell body, more importantly, it may, in and of itself, be a trigger for death of the whole neuron. In studies of white matter damage, axonal injury in response to ischemia is associated with increased axonal membrane permeability with excess Naþ and/ or Ca2þ influx into the axon (Stys and Jiang, 2002; Stys, 2004). This imbalanced Ca2þ influx activates deleterious cascades of locally localized intracellular proteases and subsequent breakdown of cytoskeletons and disturbance of axon transport leading to degeneration and neuronal death (Aarts and Tymianski, 2004; Chan and Mattson, 1999; Hara and Snyder, 2006). Given that the expression of chemorepulsive signals, such as semaphorin-3A, Ephrins (Ephs) (Beck et al., 2002; Fujita et al., 2001; Goldshmit et al., 2006) and their respective receptors are elevated in the brain following cerebral ischemia, it is easy to envisage that the affected neurons may undergo repulsive guidance cue-mediated axonal retraction/ collapse and neuronal death. For example, class 3 semaphorins are inhibitory ligands which are secreted by the scar tissue and neurons in adult brains following traumatic injury and cerebral ischemia (Beck et al., 2002; Fujita et al., 2001; Zhang et al., 2001). However, the precise pathological significance of semaphorin-3A expression in vivo and its relationship with axonal damage, regeneration and neuronal death remain unclear.

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Stroke is the result of sudden onset of loss of neurological function because the blood flow to the brain has been cut off (Hou and MacManus, 2002). Functional outcome of brain ischemia is the result of a complex interplay between permanent damage and long-term plasticity, which may be beneficial or detrimental. Accordingly, limiting tissue damage and promoting useful plasticity are the two pillars of modern stroke management (Baron, 2005). However, no clinical effective neuroprotective or regenerative therapeutic compounds are currently available (Buchan and Kennedy, 2007; Papadakis and Buchan, 2006; Zivin, 2007). Investigation of repulsive guidance cues and their pathological role in neuronal regeneration failure may shed light on ways to design therapeutics for preserving axons, neurite networks and encouraging axonal outgrowth and regeneration. In this review, efforts are made to highlight the current understandings of inhibitory molecules and their roles in axonal degeneration and regeneration in the context of stroke-induced brain damage and neuronal death. We aim to present an overview of the literature on in vitro studies of the effects of the guidance cues that have elucidated many of the mechanisms underlying neuronal devlopment, and further highlight their important in vivo relevance in order to provide readers with insights into the basis of axonal guidance molecules and their potential prospects for modulation in brain regeneration and repair, particularly following stroke.

2. Neuritogenesis and Developmental Patterning The principal mechanisms involved in both neurite extension and axonal pathfinding in the developing nervous system rely upon the reorganization of their cytoskeletal elements, induced by a number of microtubule-associated proteins (MAPs) (Bouquet et al., 2004; Dehmelt and Halpain, 2004; Dent and Gertler, 2003) and Rho-GTPases (Govek et al., 2005; Li et al., 2006; Luo, 2000; Riederer, 2007; Thies and Davenport, 2003), and which result from the stimulation of an array of the surface receptors that bind ephrins, netrins, semaphorins and other major ligands (Gallard et al., 2005; Huber et al., 2003). Growth cones, the motile enlargements at the distal tips of the extending processes, first described by Ramon y Cahal over a century ago (1890), are instrumental in detecting these spatially and temporarily distributed (and frequently repulsive) molecular guidance cues (for review, see Gordon Weeks, 2004; Letourneau et al., 1991; Mueller et al., 2006), thereby ensuring the successful establishment of neuronal connections both in the embryo, and in early post-natal development in vertebrates (Tessier-Lavigne et al., 1996). Much of our knowledge concerning growth cone mobility and neurite extension has come from the

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study of cultured neurons (Smith and Jiang, 1994) since, in addition to monitoring fixed material (Lein et al., 1992), their dynamic properties can be visualized in living cells by use of sophisticated microscopy and automated time-lapse video imaging (Anderson et al., 2006; Keenan et al., 2006; Parpura et al., 1993), and by fluorescent dye techniques (Davenport et al., 1993; Taylor et al., 2005). Studies of living growth cones allow observations of the responses associated with changes in environmental cues (Kuhn et al., 1995), with developmental stage (Blackmore and Letourneau, 2006), and following CNS axonal injury and regeneration (Taylor et al., 2005). The growth cone consists of a central (C-) cytoplasmic domain, rich in microtubules and organelles, which is surrounded by a peripheral (P-) cortical domain from which protrude numerous fine finger-like projections, known as filopodia (abundantly rich in actin filaments), and flat, sheet-like lamellipodia (Gordon-Weeks, 2004). Unlike in the neurite shaft, where cytoskeletal microtubules exist in straight bundles, on entering the C-domain they mainly become de-fasciculated, and are present singly, either retaining a straight form or becoming curved and bent (Yamada et al., 1971). Microtubules exist as either short or long, hollow filaments composed of a- and b-tubulin heterodimers that bind GTP. They are intrinsically polarized (see Sections 2.3 and 3.3), with distally oriented ‘‘plus’’ (or fast-growing) and more centrally oriented ‘‘minus’’ (slow-growing) ends (Heidemann et al., 1981). The microtubules undergo cycles of slow, continuous growth due to a dynamic instability, and due to hydrolysis of GTP bound to the b-tubulin heterodimers, which permits restructuring and growth cone shape changes (Gordon-Weeks, 2004). A role for tubulin cofactor B (TBCB), localized in the transition zone between growth cones and the neurite shaft, in regulating the dynamics and stabilization of the microtubules has been shown recently by gene silencing studies, where enhanced axonal growth was observed and, conversely, where TBCB overexpression was seen to lead to neuronal degeneration (LopezFanarraga et al., 2007). A number of microtubule binding proteins are also important in ensuring that bending and turning movements of the growing neurites can occur (Dent and Gertler, 2003). Microtubules from the C-domain are also frequently aligned with the long axis of the filopodia where they become associated with the core of actin filaments (Letourneau, 1983), playing a role in permitting growth cones to turn in response to environmental cues (Zhou et al., 2004), and in regulating the cytoskeleton during neuronal outgrowth (Morii et al., 2006). The form of both the lamellipodia and filopodia are dependent on the polymerization and organization of F-actin filaments which, once formed, are retrogradely transported to the centre of the growth cone to be rapidly broken down into subunits for further recycling distally, thereby allowing consequent forward extension of the growth cone (Gallo and Letourneau, 2004). The filopodia therefore represent transient structures

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which undergo cycles of contractile activity forming, extending and retracting, and in consequence acting as antenna-like sensors as they explore and sample the local environment over a wide radius (Davenport et al., 1993). Recent studies, employing small interfering RNA silencing approaches, have demonstrated that the balance between the antagonistic forces of dynein (a microtubule transporter) and myosin-II (which retrogradely transports actin) is fundamental in controlling the interaction between long microtubules and actin, and in consequence underpinning growth cone turning and axonal retraction in the pursuit of pathfinding (Myers et al., 2006). Complex interactions exist between the growth cone and an array of guidance cues and inhibitory molecules, such as the ephrins and the semaphorins (Tamagnone et al., 2004; Wu et al., 2005) (see Section 3.1). The signalling cascades which are stimulated by these are in turn modulated by a variety of environmental signals including growth factors (and especially the neurotrophins (for review, see Huang and Reichardt, 2001; Lykissas et al., 2007; Markus et al., 2002), components of the extracellular matrix (ECM) (such as laminin and fibronectin; Kuhn et al., 1995), cell adhesion molecules (CAMs) (Skaper, 2005), neurotransmitters (van Kesteren and Spencer, 2003), electrical stimulation (McCaig et al., 2002; Ming et al., 2001; Rajnicek et al., 2006), calcium signalling (Bolsover, 2005; Gomez and Zheng, 2006; Henley and Poo, 2004), and a number of other influences that establish chemotactic gradients to guide axonal pathfinding (Ming et al., 2002). Intrinsic differences develop between young and old neurons during embryonic life, however, which can greatly restrict the ability of axons to regenerate, even in permissive environments. This was recently demonstrated by Blackmore and Letourneau (2006) in a study of organotypic cocultures of chick brainstem and spinal cord, where explants from E15 animals regenerated about 90% fewer axons, which also extended much more slowly on permissive substrates, than was the case in preparations from younger embryos (E9). A major goal of future research will be to elucidate how the interplay of the intrinsic and extrinsic factors can be manipulated successfully, and how the efficacy of positive signals early in embryonic life can be reactivated following insult to overcome the negative control by inhibitory cues, if regeneration in adult neurons is to become routine.

2.1. Role of neurotrophins Neurotrophins [the nerve growth factor (NGF) family] are important regulators with a multifunctional role, not only in neuronal survival in the developing nervous system but also acting in stimulating axonal growth and directing the establishment of functional patterning (Huang and Reichardt, 2001; Lykissas et al., 2007). They act via tyrosine kinase receptors (TrkA, TrkB, TrkC), and by a common low affinity receptor (p75NTR) (Barbacid, 1995). In the adult, neurotrophins are frequently responsible for maintaining a

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differentiated phenotype in both the central and peripheral nervous systems (Smith and Jiang, 1994; Markus et al., 2002). Neurotrophins, and other trophic factors, such as GDNF (glial cell line-derived neurotrophic factor) family members, are also of interest with regard to their therapeutic potential in stimulating axonal sprouting in response to injury or disease (Airaksinen and Saarma, 2002; Love et al., 2005). Neurite extension in sensory neuronal populations is particularly responsive to NGF ( Jiang et al., 1995; LeviMontalcini, 1987; Lindsay, 1988). The neurotrophins are thought to act by mediating rapid changes in growth cone responses to collapsin-1, although chronic exposures with NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) caused a differential sensitivity to the inhibitory guidance molecule if co-applications are given to neuronal cultures (Tuttle and O’Leary, 1998). Trophic factor interactions with components of the ECM are also of significance in regulating growth cone activity. In a study of axonal growth from embryonic sensory neurons, neurites were seen to extend towards each other in the presence of NT-3, but not NGF, which caused growth cone collapse and neurite repulsion when plated on laminin surfaces, whilst intermingled processes were observed when maintained on fibronectin or polylysine substrates (Hari et al., 2004). In the CNS, differential regulation has been demonstrated with NT-3 and BDNF affecting extension of axons in cultured embryonic rat hippocampal pyramidal neurons, whilst neurotrophin-4 (NT-4) stimulated growth only of undifferentiated minor neurites (Labelle and Leclerc, 2000). Cerebellar granule neurons from 2-day-old rats have also been shown to respond differently, with BDNF and NT-4 significantly increasing neurite length, and the speed of growth cone migration, whilst NT-3 was not effective (Tanaka et al., 2000), although NT-3 enhanced outgrowth and branching in 50% of neurites from embryonic hippocampal neurons (Morfini et al., 1994). In organotypic slice cultures, taken from the visual cortex of P14 ferret brains, BDNF and NT-3 exerted opposing effects on dendritic outgrowth in pyramidal neurons in layers 4 and 6, with BDNF having a positive effect in layer 4, but inhibiting outgrowth mediated by NT-3, whilst in layer 6 NT-3 inhibited the growth stimulated by BDNF (McAllister et al., 1997). One possible explanation to account for this opposing effect could be competitive binding at the Trk B receptor, although, since the inhibition occurs at very low doses, the authors speculated that subsequent stages of signal transduction were involved (McAllister et al., 1997). This certainly appeared to be the case in a study of BDNF signalling in adult pig retinal ganglion regeneration where treatments with either the PI3K (phosphatidylinositol 3-kinase) inhibitor Wortmannin, or the MAPK inhibitor, U0126, significantly reduced neurite extension after 5 days’ culture (Bonnet et al., 2004). Interestingly, Pfenninger and co-workers (2003) found that whilst BDNF enhanced neurite growth and elongation in embryonic rat hippocampal neurons, the actual initiation of membrane expansion within the distal growth cone was stimulated by insulin-like growth

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factor 1 (IGF-1). Further studies, incorporating siRNA silencing and antibody blocking strategies, have highlighted that it is indeed IGF-1, rather than BDNF, that activates PI3K (Sosa et al., 2006), in a manner that is quasi independent of the neuron’s perikaryon (Laurino et al., 2005). Synergistic responses to combinations of trophins are probably commonplace in embryonic development; the loss of such cooperative mechanisms in the adult may account for the reduced success in regenerative potential in the adult following neuronal trauma. Glial cell line-derived neurotrophic factor also signals via PI3-K, as demonstrated by Edstrom and Edstrom (2003), who applied the inhibitor LY294002 to cultures of adult mouse DRG explants, and who noted significant decreases in axonal outgrowth. Transient phosphorylation of mitogenassociated protein kinase kinase was also seen following GDNF treatments of cultured ventral mesencephalic (VM) neurons, whilst BDNF induced a longer acting signalling response (Feng et al., 1999). Total neurite length increased twofold in E18 rat VM dopaminergic and calretinin-expressing neurons following culture in medium supplemented with 10 ng/ml GDNF for 7 days (Schaller et al., 2005). Enhancement of sprouting also continues into adulthood for dopaminergic neurons in the substantia nigra, whilst neurturin (another closely related member of the GDNF family) failed to sustain fiber outgrowth both in vitro and in vivo lesioning, and in grafting experiments (Akerud et al., 1999). The effects of GDNF in neural transplantation strategies have been encouraging indeed, with enhanced fiber outgrowth into host striatum demonstrated in rodent models for Parkinson’s disease (McLeod et al., 2006); and in a clinical report where GDNF infusion into the brain of a 62-year-old man for a period of 43 months stimulated fiber sprouting (Love et al., 2005) and clearly warrants further investigation. A number of other growth factors are known to regulate axonal dynamics in the embryo, and include transforming growth factor b (TGFb) (Unsicker et al., 1991), epidermal growth factor (EGF) (Kenigsberg et al., 1992), and fibroblast growth factors (FGF) (Walicke et al., 1986), including FGF-2 (which increased extension and branching of neurites in cortical neurons by over 50% by increasing actin polymeristation, and the formation of microtubule loops in growth cones; Dent et al., 2004). Clearly a plethora of trophic factors influence both neurite extension and growth cone activity, acting either singly or in combination in embryonic neurons, and in addition with a role in maintaining the differentiated state in mature cells in health and potentially playing a role in regeneration.

2.2. Extracellular matrix cues The role of the surrounding permissive physical environment in orchestrating growth cone dynamics, resulting from adhesive interactions with cellular receptors (including Ig-cell adhesion molecules, integrins and cadherins;

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Huber et al., 2003) is well documented, and is vital in ensuring growth cone precision in pathfinding (Letourneau, 1975; Luckenbill-Edds, 1997; Smith and Jiang, 1994). During development, the growing neurite must contact, sample, and either accept or reject ECM cues in order to reach and establish functional connections that will lead to the complex circuitries present in the adult. Nanoscale fabrication technology has now provided the means to produce high-resolution patterned surfaces which can demonstrate in vitro that substrate geometry, including three-dimensional patterning (Li and Folch, 2005), is instrumental in regulating growth cone ‘‘choices’’ and ‘‘decisions’’ ( Johansson et al., 2006; Kleinfeld et al., 1988; Vogt et al., 2004; Withers et al., 2006). For instance, whether growth cones from hippocampal neurons pause or accelerate at specific space intersections, and whether they extend straight or branch over the surface, depends on the width of the grooves (Withers et al., 2006). The importance of the chemical composition of the ECM, in influencing neuronal behavior, particularly axonal outgrowth and guidance, has been well documented. Many studies of cultured neurons have benefited from adapting the patterned substrates and developing ‘‘stripe assays’’, where one or more ECM component (such as glycoproteins) is bound to restricted longitudinal lanes alongside uncoated regions of the substratum. The alternating striped patterns permit the observation of any differential axonal behavioral responses at borders with or without the component of interest (see Freire et al., 2002; LuckenbillEdds, 1997; Myers et al., 2006; Nguyen-Ba-Charvet et al., 2001; Snow et al., 2002; Turney and Bridgman, 2005). Early work focused on identifying the actual ECM molecules that promoted axonal sprouting. The glycoproteins, laminin and fibronectin, which bind to integrin receptors (Ivins et al., 2000; Luckenbill-Edds, 1997; Powell and Kleinman, 1997), were demonstrated to be two of the key molecules in in vitro and in vivo studies of both peripheral and central neurons (for example, see Baron-Van Evercooren et al., 1982; Manthorpe et al., 1983; Orr and Smith, 1988; Rogers et al., 1983, 1987; Smith and Orr, 1987), with laminin having the most significant effect on directional growth (Luckenbill-Edds, 1997; Sanes, 1989). Other glycoproteins, such as wnt protein, have also been shown to be of importance in morphogen signalling and in regulating axonal outgrowth in the embryonic development of CNS patterning (Sanchez-Camacho et al., 2005). More recent work has focused on elucidating the mechanisms involved. Kuhn and his colleagues (1995) showed, by using polystyrene beads coated with the glycoproteins, that laminin guideposts induced a preferential, rapid and sustained response in neurites extending from sensory neurons. Filopodial dynamics were altered causing the growth cones to change their direction and advance, with increased velocity, towards laminin sources, compared to fibronectin signals. Binding to b1-integrin sites, and

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subsequent activation of calcium-dependent protein kinase C (PKC) intracellular signalling, was implicated as shown by the addition of a number of selective inihibitors to the cultured cells (Kuhn et al., 1995). Stimulation of axonal growth by IGF-1 and laminin, via integrin receptor activation, in cortical neurons has been shown to involve phosphorylation of the transmembrane signal regulatory protein, SIRPa (Wang and Pfennigner, 2006). Responses to laminin also result with changes in growth cone myosin II activity, as shown in studies using the myosin II inhibitor, blebbistatin, and from culturing neurons from wild type and myosin II knockout mouse embryos (E13.5) (Turney and Bridgman, 2005). Loss of myosin II would affect the recycling of actin in growth cone remodelling (Myers et al., 2006). Others have shown that the level of neuritogenesis in cortical and retinal neurons is varied by the pH at which the laminin matrices are assembled, and moreover that in consequence distinct signalling pathways are activated (Freire et al., 2002). Neurite outgrowth was two to three times greater on acidic (pH 4) laminin, and with the growth cones exhibiting lamellipodia and filopodia extending for large areas, rather than on neutral (pH 7) laminin where the size of the growth cones was reduced and filopodia often absent. Interestingly, staurosporine (a wide-spectrum inhibitor of protein kinases, but with particular effects on PKC) reduced neurite lengths by 40% on neutral substrates, whilst neuritogenesis was not significantly affected on the acidic matrix by this inhibitor. However, the protein kinase A inhibitor H-89 significantly reduced outgrowth on acidic laminin, but not on the neutral substrate (Freire et al., 2002). Such subtle alterations in the organization of individual ECM components, and/or the downregulation of integrin receptors, or at least a reduction in their activation state (Ivins et al., 2000), could be important in the responsive switches which occur as development proceeds (Blackmore and Letourneau, 2006), thereby regulating neuritogenesis, and could also explain differences that contribute to the impairment of regeneration in the adult central nervous system following trauma. The interactions of the proteoglygans, and particularly the chondroitin sulfate proteoglycans (CSPGs; present in the ECM and also expressed on the surface of oligodenrocytes), have a bearing on the efficacy of the glycoproteins in regulating axonal pathfinding, since CSPGs are known to typically cause an inhibition of growth cone behavior and neurite outgrowth (Cole and McCabe, 1991; Niedero¨st et al., 1999; Wilson and Snow, 2000). Niedero¨st and co-workers (1999) demonstrated the CSPGs significantly inhibited fiber extension by both cerebellar granule cells and DRG neurons in the presence of laminin, and that oligodenrocytes maintained in the presence of proteoglycan inhibitors are less effective in blocking neurite growth. In many instances the extent of the opposing effects appears to depend upon the ratio of CSPGs to laminin, when the concentration of

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CSPCs (<50 mg/ml) exceeds that of laminin (25 mg/ml), by up to twofold, neurite outgrowth in a range of forebrain, dorsal root ganglion and retinal ganglion neurons is inhibited, and by 1000 mg/ml growth on the substratum is totally absent. With a higher ratio of laminin (25 mg/ml) to CSPGs (25 mg/ml), however, the proteoglycans are incapable of blocking outgrowth stimulated by laminin (Snow et al., 2002). In hippocampal neurons, CSPG type proved significant in how growth cone behavior was modified with respect to crossing, stalling or turning behavior on encountering different lanes of a stripe assay (Wilson and Snow, 2000), whilst earlier workers had reported that chondroitin sulfate type C specifically modified neurite extension patterns on laminin substrata from cultured embryonic thalamic, but not hippocampal, neurons (Fernaud-Espinosa et al., 1994). Others have shown that heparin chondroitin sulfate actually favored axonal growth, whilst dermatan sulfate stimulated both dendritc and axonal growth in embryonic rat mesencephalic neurons (Lafont et al., 1992). Such interplay between glycoprotein and proteoglycan components of the ECM therefore represents a potentially fundamental mechanism that can fine-tune what is permissive, and what is repulsive, for fiber generation as the embryo’s nervous system develops, and thereby precisely establishes complex neuronal circuitry. In a recent elegant study of embryonic murine cerebellar granule neurons, Manzini and co-workers (2006) have highlighted that the signal which arrests growth, and prevents invading pontine mossy fibers from contacting immature cells in the external germinal layer during development, is dependent on heparin-binding factors. More mature internal granule layer cells were not affected by heparin treatments and permitted pontine mossy fiber axonal growth to establish synaptic connections yielding correct patterning (Manzini et al., 2006). Laminin modulation, on interacting with a number of other cues which regulate axonal growth, appears to involve a variety of signalling mechanisms—for instance, DRG axons avoid the non-permissive glycoprotein, Slit2, in the presence of laminin, following stimulation of cGMP signalling pathways (Nguyen-Ba-Charvet et al., 2001). Activation of cGMP signalling is also sufficient to cause growth cone collapse when dentate granule neurons are exposed to nitric oxide releasing reagents, suggesting a regulatory role for the diffusible molecule that is also known to be involved in synaptic plasticity in these cells (Yamada et al., 2006). Growth cone pauses and collapse stimulated by Eph B proteins, in retinal ganglion cell axon pathfinding in laminin environments, on the other hand, were shown to result from a redistribution of filopodial microtubules, and a reduction in the levels of the microtubule destabilising protein, SCG10 (Suh et al., 2004), rather than specifically triggering changes in actin via Rho GTPases—a common occurrence during growth cone collapse (Luo, 2000)—although cross-talk between the longer microtubules which enter the outer actin-rich P-domain remains a probable scenario (Myers et al., 2006).

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2.3. Establishing polarity and axonal specification The means by which neurons establish polarity during differentiation, involving a coordinated control of cytoskeletal organization and membrane trafficking in the different regions of the cell, thereby generating functional axons and dendrites emanating from their perikarya, must be the most significant and fundamental event in neural development. Neuronal polarity produces axons in which a loose cytoskeletal meshwork of F-actin is associated with microtubules [with their (þ) ends directed away from the perikaryon; Sargent, 1989]. The stability and polymerization of the microtubules, together with associated local actin instability, within axonal processes depends upon the phosphorylative state of certain low molecular weight MAPs: tau proteins and collapsing response mediator protein 2 (CRMP-2) that preferentially enrich axons, and which are regulated by the Rho GTPases (Arimura et al., 2004; Dehmelt and Halpain, 2004; Govek et al., 2005; Wiggin et al., 2005). Dendritic processes on the other hand, have a preponderance of MAP-2 and lack tau protein (Dotti et al., 1988). Following polarization, synaptic connections are made, so that electrical activity generation and functional maturation are achieved. The underlying mechanisms have been extensively studied in vitro, particularly in cultures of hippocampal neurons (e.g., Da Silva and Dotti, 2002; Dotti et al., 1988; Jiang et al., 2005), such that our current understanding of the regulation of neuronal polarity is very good. Despite an increasing number of in vivo studies which on the whole corroborate those in vitro, much still remains to be verified in the whole brain, where external interactive cues from other cells and the local environment would be expected to add to the complexity as seen at the individual neuronal level (Wiggin et al., 2005). The five stages in the establishment of polarity have been documented in detail by Dotti and his co-workers (1988), and the sequence they described remains, with minor modifications, one of the most useful to date. Initially in stage 1 the neuronal sphere is broken as local buds, with characteristic lamellipodia and filopodia, form. During the next 24 hours (Stage 2) many such buds sprout neurites over the surface and these extend approximately 20 mm in length. The microtubule cytoskeleton of the developing neurites becomes stabilized by an up-regulation of MAPIB protein, and further extension is promoted distally (Bouquet et al., 2004; Li et al., 2006; Yu et al., 2001). In Stage 3, one of the neurites becomes dominant, extending rapidly as the future axon, whilst the others are arrested until several days later, when they resume growing and acquire a dendritic phenotype (Stage 4). Finally in Stage 5, both axon and dendrites mature with a full complement of marker proteins present, and by now they express full functional competence. Interestingly, even when differentiation has occurred, neurons retain a capacity for polarity re-programming in that transection of the axonal process of a Stage 3 neuron triggered one of the other neurites to transform

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into a new axon (Bradke and Dotti, 2000a). In this instance the former axon was seen to grow only 15 mm after axotomy and Tau-1 protein was lost whilst it became immuno-positive for MAP-2. Concomitantly, the ‘‘new’’ axon changed morphologically to resemble an axon and at the same time became Tau-1 positive (Bradke and Dotti, 2000a). The intracellular signalling pathways recruited to execute this ordered sequence in establishing polarity, and particularly the role of the Rho GTPase family members (Govek et al., 2005), are covered in more detail in Section 3.3, and so will only be considered briefly here. Much interest however, has focussed recently on a number of gene products, originally identified in Caenorhabditis elegans as conferring cellular asymmetry, known as PAR (partitioning-defective) proteins, since they play a crucial role in axonal specification (for review, see Wiggin et al., 2005), and may have relevance to neurodegeneration and regeneration. Indeed, Par-1/MARK phosphorylates tau protein in regulating normal axonal cytoskeletal dynamics, although hyperphosphorylation may lead to an aggregation of tau tangles in some human neurodegenerative diseases (Fortini, 2004). At Stage 2, prior to the appearance of the dominant axonal process, PAR-3 and PAR-6 have a global distribution within the neuron. By Stage 3 polarity is established, and the PAR proteins, which can bind the Rho family members, CDc42 and Rac1, become selectively restricted to the axon (Shi et al., 2003). This occurs downstream of PI3-kinase activation (Sosa et al., 2006), and appears to involve adenomatous polyposis coli (APC) and kinesin (KIF) 3A in protein-mediated transport which is regulated by the phosphorylation (the inactive form) of GSK-3b, glycogen synthase kinase-3b ( Jiang et al., 2005; Shi et al., 2004). In addition to regulating APC, GSK-3b also modulates CRMP-2, and in consequence the assembly of tubulin microtubules along with the actin network of the axonal cytoskeleton (Arimura et al., 2004; Yoshimura et al., 2005). The exact upstream signalling pathways involved in regulating GSK-3b are not fully determined. Indeed, others are also probably of significance, with some evidence that Wnt signalling at least has a vital part to play in establishing polarity (Sanchez-Camacho et al., 2005).

3. Repulsive Guidance Cues in Axonal Pathfinding 3.1. Repulsive ligands As seen, many guidance cues have bifunctional roles in growth cone guidance in that, dependent on changes in the level of intracellular cAMP/ cGMP concentrations; they can either attract or repel growth cones.

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For ease of the rest of this review, we will be focusing on the inhibitory aspects of these cues. 3.1.1. Semaphorins Semaphorins are secreted, transmembrane, and glycosyl-phosphatidylinositol (GPI)-anchored proteins, defined by cysteine-rich semaphorin protein domains, which have important roles in a variety of tissues (Table 3.1) (Fujisawa, 2004; Yazdani and Terman, 2006). Initially found to play an important role in axonal guidance during development of the nervous system, semaphorins are now known to be involved in the formation of cardiovascular, endocrine, immune, reproductive and respiratory systems. So far there are eight known classes of semaphorins with more than 28 members described by the Semaphorin Nomenclature Committee (1999), and the majority of the family members are found in mammalian cells (Yazdani and Terman, 2006) (Table 3.1). Semaphorins are, however, only found in animals, not in plants. During nervous system development, semaphorins are best known as repellents to growing axons, but semaphorin-3A can also function as a chemoattractant depending on the intracellular level of cyclic nucleotides (Nishiyama et al., 2003; Song et al., 1997). Semaphorin-3A, a prototypical class 3, is both a secreted and neuronal-expressed chemorepulsive molecule which consists of an N-terminal signal peptide followed by the Sema domain, and an IgG domain of 70 amino acids (Luo et al., 1993, 1995; Puschel et al., 1995, 1996; Steup et al., 1999). A basic domain is present at the carboxyl end of the molecule. A common theme of the mechanism of semaphorin function is through alteration of the cytoskeleton, such as the actin filaments and microtubule network (Morita et al., 2006; Rohm et al., 2000). These effects occur primarily through binding of semaphorin to their receptors, neuropilin and plexin family of transmembrane proteins. Semaphorin-3A plays a key role in axonal guidance during development through induction of growth cone collapse (Pasterkamp and Kolodkin, 2003). The process occurs at the tip of the growth cone and is manifested by depolymerization and loss of F-actin. The downstream pathways by which semaphorins exert their actions are still unclear, but are known to include the neuropilin and plexin receptors as well as intracellular collapsin response mediator proteins (CRMPs) and G-proteins (Kawasaki et al., 2002; Sahay et al., 2005). The biological activities of the repulsive axons guidance molecule semaphorin-3A are known to be responsible for the elimination of neurons during development where axons are still too far away from reaching the target (Beck et al., 2002; Pasterkamp and Kolodkin, 2003). The cellular target of semaphorin-3A also appears to be selective since it inhibits the outgrowth of a specific set of neurons such as spinal motor neurons and neurons in the embryonic dorsal root ganglion and sympathetic ganglion (Nakamura et al., 1998, 2000; Sandvig et al., 2004).

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Table 3.1 Semaphorins and neuropilin/plexins Ligands/Receptors

Organisms

Names

Characteristics

Ligands Semaphorins

Invertebrates

Sema-1a

Transmembrane proteins

Vertebrates

Virus Receptors Neuropilins and Plexins

Vertebrates

Sema-1b Sema-2a Sema-2b Sema-3A-G Sema-4A-G Sema-5A-C Sema-6A-D Sema-7A SemaVA and VB Neuropilin 1 Neuropilin 2

Plexin A1-4

Secreted proteins Secreted proteins Trans-membrane proteins

GPI-anchored

Short intracellular domain without tyrosine kinase domain. Forms homo- or hetero-dimers to interact with other receptors Long intracellular domain with tyrosine kinase domain

Plexin B1-3 Plexin C1 Plexin D1

3.1.2. Netrins Netrins are guidance cues for commissural neurons and they act as chemoattractants that guide axons to cross midline by binding to receptors of the DCC (deleted in colorectal carcinoma) family (Kennedy et al., 1994, 2006; Herincs et al., 2005). Netrins were purified from homogenates of embryonic chick brain using an in vitro assay designed to identify soluble cues that promote the outgrowth of commissural axons, mimicking the activity of the floor plate. Most of the components of the netrin family are secreted proteins, and surprisingly it is only recently that the diffusible gradient of netrin proteins has been directly visualized in the developing

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spinal cord confirming the widely accepted mechanism of axon guidance by diffusible chemoattractants (Kennedy et al., 2006). As a recurring theme, the netrin proteins are bi-functional signals that are chemoattractive for some neurons and chemorepellent for others, and can act as long or short range signals (Dickson, 2002; Hedgecock et al., 1990; Kennedy et al., 1994; Tessier-Lavigne et al., 1988; Tessier-Lavigne and Goodman, 1996). Midline cells express netrins and defects in the expression of netrin ligands and their receptors cause abnormalities in the reaching and crossing of the midline. Once crossing the midline, the response of axons to netrin must be silenced to prevent re-crossing the midline. This inactivation of netrin’s attraction is through binding to DCC by the slit/Robo complex (Giger and Kolodkin, 2001; Kaprielian et al., 2000, 2001; Plump et al., 2002). It has been shown that netrin-mediated axonal repulsion is through the Unc5 family proteins by forming DCC-Unc5 heterodimers (Hedgecock et al., 1990; Hong et al., 1999; Keleman et al., 2005). The secondary protein structure of the netrin family is highly conserved in all species. The N-terminal signal peptide is followed by domains VI, V-1, V-2 and V-3 which share sequence homologies with the globular domain VI of laminin and the EGF repetitions found in region V of the laminin chains, respectively (Barallobre et al., 2005; Brankatschk and Dickson, 2006). The C-terminal domain sequences are rich in basic amino acids, which act as binding sites for heparin, heparan sulfate proteoglycans or membrane glucolipids, thereby allowing interaction with components of the extracellular matrix or cell surface. Therefore, the diffusion of secreted netrins is determined by both their level of expression and by the concentration of binding sites in the surrounding tissue. The netrin family members and their receptors are summarized in Table 3.2. 3.1.3. Ephrins Ephrins (Ephs) represent a major class of short-range axon guidance molecules. First identified as contact repulsive guidance cues during development of the retino projections, Ephs were later found to also have attractant functions to axons (Davy and Soriano, 2005; Huot, 2004; Tessier-Lavigne, 1995). There are nine known mammalian Eph ligands and 16 Eph receptor tyrosine kinases (Eph RTK, Table 3.3). The nine ephrin ligands consist of ephrin A1-A6, which are GPI-anchored membrane proteins, and ephrin B1-B3, which are typical transmembrane proteins with a single cytoplasmic domain (Carmeliet and Tessier-Lavigne, 2005; Davy and Soriano, 2005; Dickson, 2002; Goldshmit et al., 2006; Huot, 2004; Price et al., 2006; Tessier-Lavigne, 1995; Zhang and Hughes, 2006). EphAs and EphBs appear to have opposing roles in neurite outgrowth in specific regions of the brain. For example, EphA4 knockout mice hippocampal neurons show morphologically disorganized dendritic spines which are long and overlapping each other, whereas triple knockout of EphB1/2/3 leads to failure for hippocampal neurons to

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Table 3.2 Netrins and DCC/UNC receptors Ligands/Receptors

Organisms

Names

Characteristics

Netrin 1

Midline cell secreted bifunctional proteins which attract or repel axons

Ligands

Netrins

Receptors DCC/UNC

Vertebrates

Netrin 4b

Netrin G1-2 NTN2L Vertebrates

DCC1-2

Dependence receptors (induce apoptosis)

UNC5 (C. elegance) UNC5H1-4 Table 3.3 Ephrins and Eph receptors Ligands/Receptors

Organisms

Names

Characteristics

Ligands Ephrins

Vertebrates

Receptors Eph RTK

Ephrin A1-6 Ephrin B1-3

GPI-anchored Transmembrane

Vertebrates

Eph A1-10

GPI-anchored proteins binds to A class Ephrins Transmembrane proteins binds to B class Ephrins

Eph B-1-6

produce spines, and the neurons appear to be long and immature (Dickson, 2002; Davy and Soriano, 2005; Goldshmit et al., 2006). The functions of Ephs are complex and intriguing in that Ephs can transmit not only a forward signal through interacting with Eph receptors, but this ligand-receptor interaction also causes transduction of a reverse signal into the Eph bearing cells. The Eph receptor and ligand interaction specificity is far from clear and some promiscuities within the same subclass of Eph family have

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been documented (Blits-Huizinga et al., 2004; Mendes et al., 2006). To add to the complexity, Ephs are found not only widely expressed in the developing and adult nervous system to participate in tissue patterning, axonal guidance and synaptic plasticity, but they are also involved in modulating angiogenesis and vascular remodelling (Zhang and Hughes, 2006). Readers are directed to the above excellent review articles for specific details. 3.1.4. Slits Slit protein was identified as a ligand for the Roundabout (Robo) receptor family (Brose and Tessier-Lavigne, 2000). Again as a recurring theme for axon guidance cues, the interaction of Slits with Robo can act as repellents or attractants for branching and elongation of axons. Three distinct Slit genes have been identified encoding slits1-3 in mammals (Table 3.4). Slit1 and Slit2 proteins have been found to control the development of the lateral olfactory tract (Nguyen-Ba-Charvet et al., 2002). All Slits are large extracellular matrix-associated glycolproteins of about 200 kD which are characterized by the presence of unusual tandem of four leucine-rich repeat domains in their N-terminal sequence. Slit2 is proteolytically processed into 140 kD N-terminal and 55–60 kD C-terminal fragments (Nguyen Ba-Charvet et al., 2001). Slit2 cleavage fragments appear to have different characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. Subsequent expression analysis has shown that the cleaved fragments have distinct axonal guidance properties (Plump et al., 2002; Nguyen-Ba-Charvet et al., 2002). Most importantly, Slits function to prevent midline crossing during nervous system development. At the midline, Slit is expressed by glia cells Table 3.4 Slits and Robos Ligands/Receptors

Organisms

Names

Characteristics

Ligands Slits

Vertebrates

Slits 1-3

Midline crossing repellent guidance cues

Vertebrates

Robo 1-3/Rig1 Robo4

Midline crossing (Magic Robo), vascular specific (binds to slit 2) Modulate slit presence

Receptors Roundabout (Robo)

Heparan Sulfate

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and is diffused away from the midline, thus setting up the gradient required for guidance (Hohenester et al., 2006). In addition to the well characterized role of Slits in the regulation of axon crossing at the midline, Slits are also found to specify the lateral and dorsoventral positioning of longitudinal axonal pathways, contribute to the formation of commissures by channelling axons into particular regions. As a recurring theme, in a similar fashion to Ephs and semaphorins, Slits also provide guidance for the vascular and immune system formation (Fouquet et al., 2007; Hohenester et al., 2006; Lopez-Bendito et al., 2007). 3.1.5. Repulsive guidance molecule The repulsive guidance molecule (RGM) cues represent another family of contact dependent repulsive factors comprising RGMa, RGMb, and RGMc (Table 3.5) (Matsunaga et al., 2004; Matsunaga and Chedotal, 2004; Monnier et al., 2002). RGMa and RGMb are abundantly expressed in the developing mouse nervous system, while RGMc expression is restricted to the muscles and blood cells. It has been discovered recently that RGMs play a key role in the guidance of optic nerve projection and crossing of the chiasma. The full-length RGMa is a GPI-anchored protein which is proteolytically processed into a 33 kD C-terminal fragment (C-RGMa) and an 11 kD N-terminal (N-RGMa) fragment (Matsunaga et al., 2004; Matsunaga and Chedotal, 2004; Monnier et al., 2002). Published studies demonstrated that C-RGMa inhibits axonal outgrowth through binding to its receptor, Neogenin. Noncleavable mutants of RGMa do not interact with Neogenin and do not have any function in axonal guidance unless it is proteolytically cleaved (Monnier et al., 2002; Brinks et al., 2004; Hata et al., 2006). Interestingly, most of the time, N-RGMa is linked with C-RGMa by a disulphide bond and it is still not

Table 3.5

RGM and Neogenin

Ligands/Receptors

Organisms

Names

Characteristics

Ligands RGM

Vertebrates

RGMa RGMb

Optic nerve projection and crossing of the chiasma Expressed in muscles and blood

RGMc Receptors Neogenin

Vertebrates

Neogenin

Dependence receptor (induce apoptosis)

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Table 3.6 Myelin-secreted inhibitory glycoproteins Ligands/ Receptors

Organisms

Names

Characteristics

Ligands Nogo

Vertebrates

Nogo-A Nogo-B Nogo-C Myelin-associated protein

GPI anchored proteins all contain Nogo-66 domain A sialic-dependent immunoglobulinlike protein GPI anchored protein

MAG

OMag

CSPG Receptors Nogo Vertebrates receptors

Oligodendrocytemyelin glycoprotein Myelin-associated chondroitin sulfate proteoglycans NRc and p75NTR

ECM

Nogo receptor is GPI anchored and has to function as a co-receptor for p75

yet known whether the N-RGMa is also an inhibitor to axonal outgrowth, and, if this is the case, what the receptor that N-RGMa interacts with is in mediating axon outgrowth inhibition. 3.1.6. Myelin, reactive glial and scar-derived axon growth inhibitors It has been demonstrated that scarring is a major impediment to successful repair of CNS connections after injury. Oligodendrocytes, myelin and reactive astrocytes within the scar tissue secrete inhibitory molecules which interact with growth cone receptors to arrest injured axons. Voluminous literature has been produced in the last few years describing factors such as Nogo, myelinassociated protein (MAG), oligodendrocyte myelin glycoprotein (OMag), and myelin-associated chondroitin sulfate proteoglycans (CSPGs) (Sandvig et al., 2004). Because of their apparent relevance to brain injury, extensive attempts have also been made to therapeutically modulate the expression and function of some of these inhibitors for brain repair, albeit with limited success (Domeniconi and Filbin, 2005; He and Koprivica, 2004; Johansson, 2007; Lenzlinger et al., 2005; Papadopoulos et al., 2002, 2006; Spencer et al., 2003). Nogo has three different spliced variants with the highest amino acid (aa) sequence homology towards the C-terminal ends. Nogo-A (1162 aa),

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Nogo-B (373 aa) and Nogo-C (199 aa) all share two hydrophobic domains close to the C-terminus (transmembrane domain) and a common 66 aa extracellular inhibitory domain (Nogo-66) in between the two hydrophobic motifs (He and Koprivica, 2004; Sandvig et al., 2004). MAG is secreted by myelin sheaths of oligodendrocytes and Schwann cells, and it is a sialic-dependent immunoglobulin-like protein with an extracellular domain, a transmembrane domain and a short intracellular domain. MAG is also known to be a bifunctional protein demonstrating a neurite outgrowth promotion function in developing young neurons, but causing neurite outgrowth inhibition in older neurons. The function of MAG appears to be regulated by the changes in the intracellular levels of cAMP (Mimura et al., 2006; Quarles, 2007). OMag is also a GPI-anchored inhibitor for neurite outgrowth and it signals neurite growth inhibition through Nogo receptors and p75 via Rho GTPases (Mikol and Stefansson, 1988; Wang et al., 2002a,b). Little is known yet about the functions of Omag, for although it is highly expressed on oligodendrocyte membranes and on neurons, the functions of neuronal expressed OMag are not clear. Although these molecules appear to be very diverse in structure, they share one common feature which is that they are all heavily glycosylated through either N- or O-linked glycosylation. It is not yet known whether modulation of glycosylation of these inhibitors would have any effects on their pathological roles in axonal regeneration. Detailed reviews of the structures and functions of these molecules have recently appeared, and readers are directed to the excellent treatises of others which are available (He and Koprivica, 2004; Sandvig et al., 2004; Quarles, 2007).

3.2. Receptors for repulsive guidance cues 3.2.1. Semaphorin receptors: Neuropilins Cellular receptors for semaphorins are neuropilins (He and Tessier-Lavigne, 1997; Kolodkin et al., 1997). Neuropilin contains two family members: neuropilin-1 and neuropilin-2. Both of them comprise an extracellular domain of two CUB motifs, next to two domains with homology to coagulation factors V and VIII, an MAM domain, a single transmembrane domain, and a short intracellular domain of 39 amino acids lacking any known signalling motifs. Neuropilins are non-tyrosine kinase transmembrane proteins. Their short intracellular segments lack cytoplasmic signal transduction domains. Therefore, neuropilins participate in signal transduction as co-receptors with plexins for axonal guidance and with vascular endothelial growth factor (VEGF) receptors for vascular guidance during development. Neuropilin-1 is a cell surface glycoprotein expressed on axons (Fujisawa, 2002; Kawasaki et al., 2002), and functions as a receptor for axon guidance

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factors such as semaphorin-3A during the process of axonal pathfinding. Peptides antagonizing the MAM domain have been found to inhibit neuropilin-1mediated inhibition of axonal outgrowth (Nakamura et al., 1998; Williams et al., 2005). The nature of the downstream effectors of neuropilin signal transduction remains elusive. One of the first cytosolic protein families identified to link neuropilin receptor complex to the cytoskeleton are the collapsin response mediator proteins (CRMPs), members of a small family of brain specific proteins (Liu and Strittmatter, 2001). There appears to be some selectivity in Neuropilin-1 and Neuropilin-2 interaction with their respective ligands. Neuropilin-1 binds to all classes of semaphorin 3, whereas neuropilin-2 binds selectively to the secreted semaphorins with the exception of semaphroin-3A. The specificity and selectivity of neuropilins are determined not only by their associated co-receptor proteins—plexins, but also by the way the neuropilin dimers are formed. For example, signalling via neuropilin-1 is mediated principally by plexin-A4, whereas signalling via neuropilin-2 is mediated principally by plexin-A3. Neuropilin-1 homodimers function as selective ligand-binding receptors for semaphorin-3A, while neuropilin-2 homodimers act as receptors for semaphorin-3F which is a promoter for axonal outgrowth (Pasterkamp and Kolodkin, 2003). In addition to functioning as guidance cue to axons, Neuropilins also guide developing blood vessels through interaction with VEGFs (Carmeliet and Tessier-Lavigne, 2005; Kim et al., 2006a; Schnorrer and Dickson, 2004). Furthermore, neuropilin interaction with VEGF165, but not Flt1 or KDR, enhances the survival of synviocytes from apoptosis by rapidly triggering phospho-AKT and phospho-ERK activities (Kim et al., 2006a). 3.2.2. Netrin receptors: DCC and UNC5 receptors DCC (Deleted in Colorectal Cancer) is a receptor for netrin-1 that mediates axonal growth cone turning dependent on the level of intracellular secondary messengers such as cAMP levels (Ming et al., 1997; Nishiyama et al., 2003). Elegant work from Dr. Poo’s lab has shown that Xenopus spinal neurons exhibited chemoattractive turning toward the source of netrin-1, but showed chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A, all of which are dependent on the Netrin receptor DCC (Ming et al., 1997). Recent studies also showed that DCC interacts with focal adhesion kinase in Netrin-1 signalling which revealed that a novel role of focal adhesion kinase in axonal guidance and also demonstrated the complexity in netrin downstream signalling (Ren et al., 2004) More importantly, the DCC and UNC5H receptors are also found to act as ligand dependent receptors in that etrin-1 is a survival factor through its receptors DCC and UNC5H (Furne et al., 2006; Herincs et al., 2005; Llambi et al., 2001, 2005; Mehlen and Llambi, 2005; Thiebault et al., 2003). Activation of DCC in the absence of netrin-1 induces apoptosis, while the presence

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of netrin-1 blocks DCC-mediated apoptosis (Arakawa, 2005; Tanikawa et al., 2003). It has therefore been proposed that DCC is a ‘‘conditional’’ tumor suppressor that is dependent on netrin-1. Similarly, UNC5H is also a regulator of p53-dependent apoptosis in the absence of netrin-1 (Arakawa, 2005). Further, netrin-1 itself is also a direct target of the p53 gene. Collectively, these findings indicate a close link between axon-guidance molecules and cellular death/survival machinery so that targeting this pathway may have an additional therapeutic effect in cancer treatment (Mehlen, 2005; Mehlen and Bredesen, 2004; Porter and Dhakshinamoorthy, 2004). 3.2.3. Ephrin receptor tyrosine kinases (Eph RTK) The vertebrate Eph RTK subfamily is by far the largest known subfamily of receptor tyrosine kinases and contains 16 receptor members EphA1–A10 and EphB1–B6 (Goldshmit et al., 2006). Although the molecular structures of the Eph RTK are well documented in recent reviews (Blits-Huizinga et al., 2004; Goldshmit et al., 2006; Huot, 2004; Pasquale, 2005), it is important to point out the key signalling motif which contains the two tyrosine residues near the juxtamembrane region that serves as the major autophosphorylation site involving receptor signalling. The tyrosine kinase domain functions as the binding site for activation of the GTPases which signal through the Rho kinase pathway to modulate cytoskeleton and axon guidance. Eph RTK activation is through conformational changes during Eph binding, autophosphorylation of the two tyrosine residues, and clustering of Eph receptorligand complexes. It has been shown that transcription regulation also plays a role in Eph RTK signalling. Mechanisms have been identified in that a sequence in the 30 -UTR of EhpA2 allows the mRNA to be translated only after it had crosses the midline, providing evidence to show another level of regulation of Eph (Brittis et al., 2002). 3.2.4. Slits receptors: Robos Advances in understanding the functions of Slits proteins come from the identification of Slits cellular receptors. Genetic studies have identified two types of cellular receptors for Slits: Robos (Roundabout)/Rig1 (Brose et al., 1999), and the heparan sulfate (HS) proteoglycan syndecan (Hohenester et al., 2006; Hussain et al., 2006). Several distinct Robos have been identified recently encoding Robo1, Robo2, Robo3 (also known as Rig1) and Robo4 (vascular specific) (Table 3.4). For the structures of these receptor proteins, refer to the excellent detailed recent review by Dickson and Gilestro (2006). The function of Robo receptor for Slits in midline crossing during development is relatively understood in that as the axon approaches the midline attracted by netrin, Robo is ‘‘silenced’’ by Slits, which keeps the Robo level low through an intracellular mechanism of preventing the Robo expression at the cellular membrane (Marillat et al., 2004; Sabatier et al., 2004).

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When the axon crosses the midline, this inhibition ceases and the reappearance of Robo on the cell surface enable repulsion to prevent re-entry of the growing axon. Remarkably, genetic disorder of Robo expression causes human diseases called ‘‘horizontal gaze palsy with progressive scoliosis’’ (HGPPS) ( Jen et al., 2004). Mutagenesis of Robo3 gene expression leads to abnormalities in reticular formation pathway to cross the midline which may explain the observed symptom, i.e., the lack of coordinated horizontal eye movement. The precise function of Slit interaction with HS is not absolutely clear, but their interactions may be important for shaping the presumed Slit gradient or presenting Slit at its target cell surface during midline crossing (Whitford et al., 2002). Much remains to be learned about this. 3.2.5. RGM receptors: Neogenin The receptor for RGMa is Neogenin. It is not yet clear how Neogenin signals growth cone collapse, and information on RGMa/Neogenin guidance of axon in vivo is also scant (Matsunaga et al., 2006; Wilson and Key, 2006a,b). During early embryonic development, RGM/Neogenin interaction repels axons. RGMa possesses activities similar to Ephrins which cause growth cone collapse and inhibits the outgrowth of temporal retinal ganglion cell axons (Matsunaga et al., 2006; Monnier et al., 2002). Recent studies showed that RGMa-induced growth cone collapse is also mediated by activation of the small GTPase RhoA and its downstream effector Rho kinase and PKC (Conrad et al., 2007). Neogenin is also a dependence receptor in that in the absence of its ligand, RGMa, Neogenein undergoes Caspase-3mediated cleavage to signal neuronal apoptosis (Matsunaga et al., 2004, 2006) (Table 3.2). From a mechanistic point of view, Neogenin induces death through formation of an RGMa-dependent complex that includes specific caspases such as caspase 3. In the absence of RGMa, activation of caspase 3 cleaves Neogenin thereby releases a pro-apoptotic peptide. Albeit the fact that it is still unclear how and in what order Neogenin cleavage and caspase activation occurs, it is believed that RGMa is a pro-survival factor and can be targeted for neuroprotection in stroke or traumatic brain injury (Doya et al., 2006; Matsunaga et al., 2004, 2006; Matsunaga and Chedotal, 2004; Rajagopalan et al., 2004; Schwab et al., 2005a,b). Caspase cleaves Neogenin at amino acid 1323 which leads to the production of two fragments, a 180 transmembrane fragment (N-terminal part) and an intracellular 23 kD fragment (C-terminal fragment) (Matsunaga and Chedotal, 2004). To add further to the complexity of RGM signaling, a hypothesis/ model is emerging that interaction of RGMa with Neogenin at the level of growth cone leads to growth cone collapse, while on the cell soma, their interaction promotes cell survival (Fitzgerald et al., 2006; Matsunaga et al., 2004; Matsunaga and Chedotal, 2004). Such an understanding of RGM

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function may have therapeutic implications in that it can be envisaged—for example, intravitreal injection delivering of RGMa to the retinal cell soma may promote retinal cell survival against optic nerve transection since the regenerating axons are not exposed to the negative effects of RGMa on the growth cone. 3.2.6. Receptors for myelin-secreted inhibitory glycoproteins The Nogo receptor is a 473 aa protein with a GPI anchor. Because it has no intracellular domain, it is understandable that Nogo receptor acts as a linker with another protein to transduce repulsive signals. It is now clear that Nogo receptor interacts with p75NTR which has transmembrane polypeptide sites. P75NTR is a member of the tumor necrosis factor superfamily which contains several important signal transduction domains such as the type II death domain which contains a GTPase-activating domain to activate the RhoA pathway (He and Koprivica, 2004; Wang et al., 2002a). Both MAG and OMag also bind to Nogo receptors with high affinity and require p75NTR as a co-receptor for signaling of MAG-mediated inhibition of axon growth and intracellular calcium elevation. Both MAG and Nogo bind to the same extracellular domain of the Nogo receptor (Wang et al., 2002b). Collectively, the complete trajectory of a given class of axons is likely to result from the effects of multiple positive and negative guidance cues that act at long and short ranges (Kaprielian et al., 2000, 2001; Plump et al., 2002). The growth cone must be able to integrate these signals and to modulate its responsiveness en route. Although the major mechanisms underlying this plasticity may be determined by the specific and selective intracellular signalling pathways (see the following section), it has come to light recently that even a single cyclic nucleotide, such as cAMP or cGMP, can modulate distinct responses of growth cones. For example, netrin-1 is sensitive to the levels of cAMP or protein kinase A (PKA) activity, while others including Sema 3A are modulated by cGMP and protein kinase G (PKG). It is generally believed that lowering of cAMP or cGMP levels, or inhibiting PKA or PKG activities, converts an attractive response to repulsive one, whereas elevating cAMP or cGMP, or activating PKA or PKG switches responses to attraction (Kao et al., 2002; Ming et al., 1997, 2002; Nishiyama et al., 2003; Song et al., 1997). It is therefore crucial to understand completely the relationship and interaction of these cues with their canonical receptors before the development of effective regenerative therapeutics can be advanced.

3.3. Intracellular signalling pathways for repulsive guidance cues The molecular signal transduction pathways for growth cone collapse and axonal guidance are hot topics of current research. The identification of the vast amount of guidance cues and the diverse range of molecules in response

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to the guidance cues towards their intended targets have also raised the question as just how all these proteins work together to cause growth cones to grow, turn and collapse in a very specific and precise manner. Although not completely understood, a large body of literature is out there suggesting that guidance of axonal growth is the result of activation of several distinct parallel signal transduction pathways and the cross-talk between these pathways. Three such intracellular pathways are highlighted in Figure 3.1, include the pathways of the ‘‘Rho GTPases’’, the ‘‘PI3K-GSK’’ and the ‘‘Fyn-Cdk5’’. 3.3.1. The ‘‘Rho GTPases’’ pathway The Rho family of GTPasaes includes Cdc42, Rac, and Rho, which are important regulators of the actin cytoskeleton affecting the shape and movement of the cells. Several interesting studies have shown that small GTPases of the Rho family are the major regulators of signalling pathways that link the extracellular cues (such as growth factors or repulsive factors mentioned above) to the assembly of focal adhesions and associated structures (Best et al., 1996; Kozma et al., 1996). Through a cascade of phosphorylation activation of downstream effectors, such as ROCK, PAK and coffilin, the Rho GTPases pathway ultimately affects the integrity of actins and tubulins. Rho GTPases play a pivotal role in many aspects of neuronal development, influencing neuritogenesis, axonal pathfinding and regulating dendritic spine formation (for review, see Govek et al., 2005). More recently, Rho family of GTPases has been shown be involved in excitotoxic neuronal death through directly modulating functions of the SAPKs (Semenova et al., 2007). For example, Cdc42 and Rac are activators of the c-Jun N-terminal kinase ( JNK) and p38 SAPKs and Rho A selectively mediates calcium-dependent activation of p38a to induce excitotoxic neuronal death (Semenova et al., 2007). Over-expression of dominant negative Cdc42 and Rac provides neuroprotection against neuronal death caused by the withdrawal of trophic support which strongly supports the idea that cdc42/Rac and their associated proteins contribute to the survival of neurons (Bazenet et al., 1998). These studies reinforce the idea that aberrant functions of the guidance signalling molecules may tip the balance to maintain growth and survival thereby causing neuronal death. 3.3.2. The ‘‘PI3K-GSK’’ pathway The phosphoinositide 3-kinase (PI3K)/AKT1 pathway is acknowledged as a key component of cell survival. More recently, PI3K is now viewed as an important player in many aspects of cell motility and adhesion (hence it contributes to metastatic/invasive phenotypes of various cancer cells). Inhibitors to PI3K affects both axon formation and elongation (Yoshimura et al., 2006). The PI3K/AKT activation leads to activation of glycogen synthase

151

Ligand

Guidance Cues of Axonal Outgrowth

Cdk5

Fyn

Intracellular

Receptor

Linker

Extracellular

Ligand: Sema, Eph, Nogo, MAG etc Linker: NRP1/2, Nogo receptor Receptor: Plexins, p75NTR

P Trk

PTEN PI3K

Rho

Rac

Par3/ Par6/ aPKC

Akt

Tau

PAK

SAPK p38a

GSK

LIMK

P CRMP

ROCK

CRMP

Numb Cofilin

? Actin dynamics

Endocytosis

Tubulin dynatmics

Reorganization of cytoskeleton

? Neuronal death

Axon guidance

Figure 3.1 A schematic diagram depicting several parallel signal transduction pathways and their potential cross-talks initiated in response to inhibitory guidance cues. The question marks indicate missing links and information.

kinase-3a and b (GSK-3a and b) through specific phosphorylation of Ser9 of GSK-3b by AKT, a kinase involved in many signalling pathways. PTEN, the lipid phophatase that breaks down that product of PI3K, prevents PI3K mediated activation of AKT therefore having an opposite effect on axonal outgrowth. PI3K/GSK in turn modulates the function of CRMP2 through

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specific phosporylation to affect the assembly of the cytoskeleton (Kim et al., 2006b; Yoshimura et al., 2005). Knockdown of both GSK-3a and b markedly reduced axon growth in dissociated cultures and slice preparations, led to the suggestion that GSK-3 is a downstream convergent point for many axon growth regulatory pathways (Kim et al., 2006b). Another group of PI3K downstream effectors of polarity setting molecules are the Par-3/Par-6/ aPKC polarity complex. However, major gaps exist in the picture as to whether Pars interact with AKT/GSKs/CRMPs (Fig. 3.1). Nevertheless, GSK has now been actively exploited as a drug target for brain therapeutics (Bhat et al., 2004). 3.3.3. The ‘‘Fyn-Cdk5’’ pathway Another pathway is emerging which involves Fyn (Morita et al., 2006) and Cdk5. Fyn is a member of the Src family of non-receptor tyrosine kinases and plays important roles in neuronal network building and behavior pattern determination. Cdk5 is a member of the serin/threonine kinase family which, due to the neuron-specific expression of the p35 regulatory subunit, has important roles in laminar formation of the cerebral cortex through regulation of neuronal migration, axon elongation and maintenance as well as stability and steering of their growth cones (Morfini et al., 2004; Nguyen and Bibb, 2003). In the cultured cortical neurons from fyn (-/-) and Cdk5, mutant (Tyr15 to Ala) mice, dendrites bear few spines and their response to Sema3A is also attenuated (Morita et al., 2006; Sasaki et al., 2002). Recent intense investigation of Cdk5 has also revealed a much larger role of this molecule in neuronal death and survival (Cheung and Ip, 2004; O’Hare et al., 2005). So how exactly do Fyn and Cdk5 regulate cytoskeleton reorganization to affect growth cone collapse? As illustrated in Figure 3.1, Sema3A activation of Plexin-As transduces signals to Cdk5 through activation of Fyn which is always associated with Plexins. Increased phosphoryaltion of Tyr15 of Cdk5 in the growth cone by Fyn Src kinase activates Cdk5 which leads to increased phosphorylation of the cytoskeleton system, such as Tau, thereby affecting the collapse of growth cone (Sasaki et al., 2002; Uchida et al., 2005). Alternatively, Cdk5 may modulate CRMPs and microtubules through GSKs to affect axonal guidance (Uchida et al., 2005). It is unclear at this time as to relatively how much weight each pathway contributes to the end result of growth cone guidance. One thing that is clear though is how these proximal pathways converge on several key points to impact on the integrity of the cytoskeleton system, and that one of the key converging molecules is the family of collapsin response mediator proteins (CRMPs). CRMPs are members of a small family of brain specific proteins consisting of five highly related members (CRMP-1 to -5) (Bretin et al., 2005; Wang and Strittmatter, 1996). They are homologues of Unc33 whose

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mutation in C. elegans causes an ubiquitous impairment in the formation of neural circuits and results in severely uncoordinated locomotion (Hedgecock et al., 1985, 1990; Li et al., 1992). CRMPs are also heavily involved in the regulation of axonal path-finding during development. During semaphorin 3A-mediated growth cone collapse, CRMPs act as cytosolic messengers through neuropilin receptors (Liu and Strittmatter, 2001). The most studied member of the family is CRMP-2 which is expressed in growth cones and distal parts of the growing axons (Arimura et al., 2000; Inagaki et al., 2001; Suzuki et al., 2003). It has be shown that CRMP-2 modulates axonal length by modifying F-actin filaments, microtubules and cytoplasmic flow (Bradke and Dotti, 1999, 2000b; Charrier et al., 2003; Franken et al., 2003). Although how CRMP-2 precisely modulates cytoskeleton to induce axonal collapse remains not well understood, it is known that CRMP-2 acts as a common intracellular target by integrating both positive and negative effects on axon extension possibly through distinct upstream activators. For example, CRMP-2 is phosphorylated in vitro and in vivo by the Rho family GTPase and a Rho effecter, Rhokinase (ROCK). ROCK-dependent and independent pathways exist for growth cone collapse through CRMP-2 phosphorylation (Arimura et al., 2000). Cdk5, as a result of association with Fyn, also targets CRMP2 through increasing phosphorylation of CRMP-2, thereby inactivating CRMP-2 to mediate growth cone changes (Uchida et al., 2005). Importantly, CRMPs have also recently been implicated in the death of neurons in a number of neurodegenerative diseases as described in the following section, which further support the notion that misbehave of the guidance signaling molecules may lead to neuronal death.

4. Guidance Cues in Axonal Damage and Neuronal Death 4.1. Ischemic neuronal death and axonal damage Glutamate receptor-mediated excitotoxicity is a major mechanism of neuronal death in various pathological conditions including cerebral ischemia. Cerebral ischemia-induced interruption of the supply of energy (glucose) to neurons leads to a reduction in ATP levels causing depolarization of the presynaptic membrane. Depolarization of the pre-synaptic membrane increases the release of glutamate with disturbed Ca2þ homeostasis, causing neuronal death. In many cases, glutamate toxicity can be attributed to excessive stimulation of the NMDA (N-methyl-D-aspartic acid) subtype glutamate receptors. NMDA receptors are important for both normal transmission and pathological damage, and it appears that the locations of the activated receptors determine the consequence of NMDA signalling (Aarts and

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Tymianski, 2004; Hou and MacManus, 2002; Kirino, 2000; Kobayashi and Mori, 1998; Love, 2003). Fascinating evidence emerged recently that growth and survival signals may in fact be derived from synaptic NMDA receptor complexes, whereas cell death signals are derived from extrasynaptic NMDA receptors (Hardingham, 2006; Hardingham et al., 2002; Hardingham and Bading, 2002, 2003; Riccio and Ginty, 2002). Distinct regulation of neuronal survival or death gene expression through synaptic versus extrasynaptic NMDA receptors, respectively, represents a fundamental and yet unexplored mechanism for neuronal response to excitotoxic insult including cerebral ischemia. Since NMDA receptors interact with many intracellular molecules, extrasynapse-to-nucleus signalling mechanisms are far from being clearly understood. A single second messenger, calcium, controls gene expression triggered by neuronal activity, and the spatial properties of calcium signalling determine the type of transcriptional response (Hardingham and Bading, 2003). In contrast to influx through synaptic NMDA receptors, calcium influx through extrasynaptic NMDA receptors is coupled to the cell death pathway. Identification of novel mediators responsible for death signal transduction in response to extrasynaptic NMDA receptor signalling will provide new insight into the mechanism of excitotoxicity-mediated neuronal death and provide the basis for designing novel drugs to achieve neuroprotection in diseases like cerebral ischemia. In this context, it is of significant interest to know if NMDA signalling requires the interaction with guidance pathways. Glutamate receptormediated Ca2þ influx modulation of cerebellar granule neuronal migration and EphB2 regulation of postnatal NMDA-dependent synaptic function are both especially highly suggestive of the existence of interactions of guidance pathway with mediators of excitotoxicity (Henderson et al., 2001; Yacubova and Komuro, 2003).

4.2. Semaphorin/neuropilin in neuronal death The role of semaphorins in the adult nervous system is far less clear. In addition to questions such as why are there so many related semaphorins, what are the underlying mechanisms of their complex regulation in expression patterns, and what of the poorly understood molecular mechanisms of semaphorin signalling, much remains to be learned about the importance of semaphorin in brain pathology and disease (De Winter et al., 2002a,b; De Wit and Verhaagen, 2003; Giger et al., 1998; Goshima et al., 2000; Pasterkamp et al., 1998). Because of these inhibitory roles of semaphorin-3A during development, it is not surprising to see researchers implicating an underlying role for semaphorins in a number of neurodegenerative diseases including Alzheimer’s, motor neuron degeneration and injuries caused by cerebral ischemia [as reviewed by De Winter and co-workers (2002b)].

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Semaphorin-3A expression is elevated in both neurons and components of the scar tissues such as glial cells in the injured adult brain (Beck et al., 2002; Fujita et al., 2001; Zhang et al., 2001). Our studies also demonstrated an increased expression of receptors for semaphorins, neuropilin1 and 2, during postnatal development and in the ischemic side of the brain (Fig. 3.2). The precise pathological significance of the induction of semaphorin-3A and neuropilins in the adult ischemic brain is unknown. In particular, little is known about the functions of the neuronal-expressed semaphorin-3A. However, it is highly possible that semaphorin-3A, both neuronal expressed and glial secreted from the scar tissue, form an inhibitory gradient to repel regenerating axons through interaction with its neuronal receptor neuropilin-1 resulting in the loss of connection and neuronal death (Fujisawa, 2002; He and Tessier-Lavigne, 1997; Kolodkin et al., 1997; Nakamura et al., 2000; B

A

1.2 13 d

16 d

28 d

NRP1 NRP2

1.0 NRP1 NRP2 b-actin

Ratio NRP1/actin

6d

0.8 0.6 0.5 0.4 0.3 0.2 0.1 0 6d

C

13 d

28 d

16 d

D Reperfusion time

Sham

2h

4h

6h

1.0

8h

*

NRP1

NRP2

b-actin

Ratio NRP1/actin

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Figure 3.2 Characterization of neuropilin expression. Brain mRNAs were extracted using the Trizol reagent and equal amount of the mRNA was reverse-transcribed into cDNA for PCR amplification. An internal control, b-actin, was used to indicate equal cDNA loading. As shown in panels A and B, the level of neuropilin 1 and 2 (NRP1/2) mRNA increased in the adult mice brains, and the level of NRP1/2 also increased in adult mouse brain after 1 h middle cerebral artery occlusion (MCAO) and followed by 2 and 4 h reperfusion (panels C and D). * in panel D, represents statistical significance with p <.01 (Student’s t-test).

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Pasterkamp et al., 1998). Interestingly, a recent in vitro study has demonstrated that neuronal-expressed semaphorin-3A causes the death of activated microglial thereby providing neuroprotection in the injured brain (Majed et al., 2006). Direct evidence showing semaphorin-3A’s involvement in neuronal degeneration comes from in vitro studies in that dopamine treatment of cultured sympathetic neurons up-regulates the expression of semaphorin-3A which eventually causes neuronal death (Shirvan et al., 1999, 2000, 2002), while blocking semaphorin-3A provides protection against neuronal death (Shirvan et al., 2002). These studies suggest that semaphorin-3A may transmit a death signal through neuropilins. Detailed investigation is urgently needed to unveil the role of semaphorin-3A in vivo following cerebral ischemia.

4.3. Netrin-1/UNC/DCC in neuronal death (the dependence receptor theory) It has been found that the netrin-1 receptor UNC5B is a molecular target of p53 (Arakawa, 2005; Tanikawa et al., 2003). Through bioinformatics searches and sequence comparison analysis, it has been shown that UNC5 contains the classical death domain sequence similar to that found in Fas (APO-1) and TNF receptors (Arakawa, 2005). Studies also found that Fas, the death receptor, can indeed mediate the pruning of neuronal branches during development (Zuliani et al., 2006), arguing that the death domain may be involved in microfilament re-organization and subsequently may lead to apoptosis. UNC5B is one of four related receptors for netrin-1 and all of which are type-1 transmembrane proteins. The proposed mechanism of p53 activating UNC5B in response to death stimuli is that membrane localized UNC5B is cleaved, possibly by activated caspases since the intracellular fragment of UNC5B contains a classical caspase-cleavage sequence DXXD, thereby releasing the peptides containing the death domain. This fragment then in turn interacts with DAPK and/or NRAGE which subsequently activates more capsases to initiate apoptosis. Therefore, UNC5B-induced apoptosis represents a pathway which is dependent on its interaction with netrin-1, but independent of mitochondrial and death receptor pathways (Arakawa, 2005c). These receptors have been dubbed dependence receptors (Mehlen, 2005; Mehlen and Bredesen, 2004; Mehlen and Goldschneider, 2005) because, in the absence of ligand availability, they induce programmed cell death, whereas in the presence of their trophic ligands, programmed cell death is inhibited as illustrated in Figure 3.3. So far, more than 10 such dependence receptors have been identified (Fig. 3.3). For example, p75NTR, the common neurotrophin low affinity receptor; the netrin-1 receptors DCC, UNC5H1, UNC5H2, and UNC5H3; the androgen receptor (AR); RET, the receptor for GDNF (glial cell line-derived

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Figure 3.3 The dependence receptor theory. In the presence of ligand, the dependence receptor mediates signal transductions to distal axonal guidance response. However, in the absence of appropriate guidance cues, apoptotic signals activate caspases or calpain to cleave the guidance receptor. The truncated membrane-anchored receptor and/or the cleavage product in turn activate apoptotic death pathways to induce neuronal death (A). The known possible ligands of dependence receptors are listed in B. (Modified and updated based on Mehlen and Bredesen, 2004; Mehlen, 2005.)

neurotrophic factor); integrins such as avb3 and a5b1, and the receptor for sonic hedgehog, patched (Ptc) (Mehlen and Bredesen, 2004).

4.4. RGM/Neogenin dependence receptors in neuronal death Works by Monnier and colleagues (Monnier et al., 2002; Schwab et al., 2001, 2005a,b) have shown that RGMa is highly expressed in the human adult nervous system and at the site of CNS injury. For example, following focal cerebral ischemia and traumatic brain injury, RGMa was found to

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increase in expression in the lesion site and in the penumbral area, both in neurons and in leucocytes infiltrating the lesion site (Schwab et al., 2005a,b). One week after insult, as the lesion matures, RGMa expression was observed in the glial scar on reactive astrocytes. The fact that RGMa is present in the lesion site has led to the suggestion that RGMa might exert inhibitory effects to the regenerating axons. However, questions remain as to what exactly causes the expression of RGMs, in particular, whether inflammation induces RGM expression in the injured brain is of significant interest. At least one report suggested that directly injected complete Freund’s adjuvant, although inducing spinal cord inflammation, did not elicit the up-regulation of RGMa (Doya et al., 2006; Hata et al., 2006). More experimental data are definitely required.

4.5. CRMP in neuronal death and survival Although the role of CRMPs in mediating growth cone collapse is well established, confounding evidence exists in the literature as to whether CRMPs modulate the death or survival of postmitotic neurons. The indication that CRMPs may be involved in neuronal death comes from the studies of dopamine-induced death of cerebellar granule neurons (CGNs) (Shirvan et al., 1999). Using the non-subjective differential display technique, it was found that CRMP-2 expression was up-regulated during the early stages of dopamine-induced neuronal apoptosis (Shirvan et al., 1999). Antibodies to Semaphorin 3A blocked CRMP-2 activities and neuronal death, whereas Semaphorin 3A-derived peptides induced apoptosis of cultured neurons (Gagliardini and Fankhauser, 1999; Shirvan et al., 2000). There is an increasing amount of evidence demonstrating that CRMPs participate in neuronal death both during development and in injured adult brains (Charrier et al., 2003; Franken et al., 2003; Hou et al., 2006; Zhang et al., 2007). In particular, our studies showed that a 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion caused a dramatic increase in the expression of CRMPs in injured neurons located in the infarct area on the ischemic side of the brain, but not in the contralateral side of the brain (see Fig. 3.4) (S. Jiang, J. Kappler, B. Zurakowski, A. Desbois, A. Alysworth and S. T. Hou, unpublished data; Hou et al., 2006). In response to glutamate toxicity in cultured CGNs, the expression of CRMP-3 increased dramatically in dying neurons (Hou et al., 2006). The full-length CRMP-3 (p63) was cleaved by calpain to produce a N-terminally truncated form of p54. MK801 and calpain inhibitors (calpastatin and ALLN) prevented CRMP-3 cleavage and neuronal death evoked by glutamate. Small interfering RNA to CRMP-3 (siRNA) significantly protected axons and neurons against glutamate toxicity. Over-expression of the full-length CRMP-3 (p63) in human HEK293 cells did not cause cell death, but over-expression of the N-terminally truncated p54 induced

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Figure 3.4 CRMP cleavage in response to MCAO reperfusion. Mice were subjected to 2 h MCAO and followed by 2^24 h reperfusion. Brains were collected at the time point as indicated in panel A.Western blotting was performed on these proteins using specific antibodies to CRMP 1^5. A clear cleavage band is visible in all MCAO brain samples. CRMP5 antibody to the C-terminus of the protein did not detect any cleavage suggesting that CRMP5 was cleaved towards the C-terminus of the protein. GAPDH was used as an internal control to show equal protein loading. The CRMP cleavage band was quantified using densitometry and normalized against that of GAPDH. As shown in panel B, the relative expression of the cleavage bands are plotted demonstrating that CRMP4 cleavage sharply increased after 6 h reperfusion.

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significant amounts of cell death. These studies demonstrated a potential role of CRMP-3 in modulating axonal damage and neuronal death (Hou et al., 2006). As shown in Figure 3.4, all CRMPs are targeted for cleavage during cerebral ischemia, and this cleavage can be inhibited by calpain inhibitors (S. Jiang and S. T. Hou, unpublished data), and ubiquitin proteasome system (UPS) inhibitor MG132, but not by protein translation inhibitor cychloheximide (Fig. 3.5), which strongly argues for the fact that posttranslational modification is very important for CRMP-mediated signal transduction in neuronal death. What is the evidence supporting the role of CRMPs role in neuronal survival? In normal adult brains, the expression of CRMPs is differentially regulated. Some members of CRMPs are significantly reduced in the level of expression (Bretin et al., 2005; Wang and Strittmatter, 1996) and are only found in areas undergoing neurogenesis and/or plasticity such as the hippocampus, the olfactory system and the cerebellum (Charrier et al., 2003; Kee et al., 2001). Over-expression of CRMP-2 has been found to accelerate nerve regeneration (Pasterkamp and Verhaagen, 2001; Suzuki et al., 2003), suggesting that CRMP-2 is associated with the survival and plays a maintenance role in postmitotic neurons. Following ischemic injury in the brain, induction of CRMP-4 expression was found in the ischemic brains and the level of CRMP-4 expression was associated with neurons having an

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Figure 3.5 Ubiqintin protease system inhibitor MG132 blocked the cleavage of CRMPs. Brain lysates were treated with Ca2þ at 0.5 mM which activates endogenous calpain to cleave CRMP (Hou et al., 2006). In the presence or absence of cycloheximide or MG132 at the concentration as indicated, MG132 completely prevented the cleavage of CRMPs, while CHX did not strongly support the argument that CRMP cleavage by UPS activity, but not by translation. Again, GAPDH was used as an internal control to show equal protein loading.

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intact morphology. These observations have led to the suggestion that CRMP-4 is a survival factor and may be involved in neuronal regeneration (Kee et al., 2001). Much remains to be learned as to the exact role of CRMPs in adult brain neurons during stress and injury.

4.6. CRMP modulation by calpain and CaMK during neuronal death The role of CRMPs in axonal guidance has been shown to be regulated by upstream kinases through increasing/decreasing phosphorylations. How CRMPs are modulated during neuronal death caused by excitotoxicity is very different. It appears that calcium activates calpain and CaM kinases are two very important candidates as summarized below. 4.6.1. Calpain targets CRMPs Excitotoxicity-mediated neuronal death involves the activation of Ca2þdependent proteases such as calpain. Calpains are a highly conserved family of calcium-dependent proteases. The ubiquitous -calpain and m-calpain are heterodimeric regulatory enzymes consisting of an 80 kD catalytic subunit and a 30 kD regulatory subunit. Whereas the two forms differ in their calcium requirements for in vitro activation, their substrate specificities are almost identical (Chan and Mattson, 1999; Lee et al., 1997). In almost all cases, the activation of calpain has been measured indirectly as increased proteolysis of endogenous -calpain substrates such as spectrin or MAP-2. It is important to note that calpain-mediated spectrin breakdown is specifically coupled to Ca2þ entry through the NMDA receptors. Nonspecific Ca2þ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. The importance of calpains is underscored by the fact that mice deficient in the 30 kD small regulatory subunit suffer from embryonic lethality (Carragher, 2007; Hara and Snyder, 2006; Kuchay and Chishti, 2007). In the CNS, calpains are widely expressed and their activities are modulated by an endogenously expressed inhibitory protein calpastatin. Calpain has been reported to cleave a large number of substrates including cytoskeletal proteins and important regulatory proteins such as cyclindependent protein kinase 5 (CDK -5) activator p39 producing a C-terminal truncated fragment at a molecular weight of 29 kD (Chan and Mattson, 1999; Hara and Snyder, 2006; Hou et al., 2006; Kulkarni et al., 2002). Expression of calpain inhibitor calpastatin is neuroprotective to many neurological diseases such as Parkinson’s and cerebral ischemia. Therefore identification of targets of calpain in response to NMDA receptor activation will shed light on the molecular mechanism of glutamate-induced neuronal death. Our recent studies showed that the activation of calpain through extrasynaptic NMDA receptors (by adding glutamate) induced proteolytic cleavage of CRMP-3 (Hou et al., 2006). Furthermore, the calpain inhibitor

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ALLN and calpastatin blocked the breakdown of CRMP-3 in response to glutamate toxicity. The CRMP-3 amino acid sequence indeed contained a putative calpain cleavage site between amino acid 73 and 85. Taken together, these data are highly suggestive that calpain activated in a calcium-dependent manner through NMDA receptors targets CRMP-3 during glutamatemediated neuronal death. Calpain also associates with focal adhesion proteins in platelets and regulates the attachment of modulatory proteins to the cytoskeleton and relaxes the retraction of fibrin clots. Because focal adhesion and stress-fiber formation are processes dependent on Rho GTPases, calpain has been postulated to regulate these processes (Kulkarni et al., 1999, 2002). Expression of Rho or Rac constructs overcomes the effect of calpain inhibition of adhesion and stress fiber formation; strongly suggesting that calpain regulates Rho and Rac. Moreover, calpain also cleaves RhoA to modulate actin filaments (Kulkarni et al., 1999, 2002). It is therefore highly possible that calpain modulation of guidance molecules causes neuronal death during excitotoxicity. 4.6.2. CaMK II targets CRMPs A potential alternative activator of CRMP-3 in response to calcium influx through NMDA receptors is the Ca2þ/calmodulin-dependent protein kinase II (CaMK II). Indeed, recent mass spectrometry studies have identified more than 30 proteins at the postsynaptic density (PSD) to be substrates of CaMK II (Fink et al., 2003; Fink and Meyer, 2002; Silva et al., 1992; Yoshimura et al., 2004) and, interestingly, CRMP-2 is one of them (Yoshimura et al., 2004). CRMPs are present in relatively high concentrations in the PSD fraction. However, whether CaMK II targets CRMPs to regulate plasticity and death of neurons in adult brains remains unknown. CaMK II functions as a link between Ca2þ stimuli and neuronal death caused by NMDA receptor activities. When calcium increases in the postsynaptic component, CaMK II is autophosphorylated and activated. Activated CaMK II is translocated to the PSD to target NR2B, a major component of the extrasynaptic NMDA receptors. The importance of CaMK II in neural functions is underscored by the fact that mice lacking CaMK II show numerous deficiencies in learning and neuronal plasticity (Silva et al., 1992). Loss of CaMK II activity also results in increased damage to neurons in response to both focal and global ischemia in mice (Hajimohammadreza et al., 1995; Takano et al., 2003; Waxham et al., 1996). However, numerous reports have shown that inhibitors to calmodulin and CaMK II are potently neuroprotective (Hajimohammadreza et al., 1995; Takano et al., 2003), which argue for a role of CaMK II in modulating neuronal death. Given that CaMK II has many intracellular targets, it is necessary to determine mechanistically whether CaMK II may target CRMP-3 to mediate neuronal death or survival.

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5. Guidance Cues and Synaptic Plasticity in Stroke Brains Glutamate over-activation of NMDA receptors increases intracellular Ca2þ concentrations which in turn activates Ca2þ-dependent proteases resulting in degradation of key structural and regulatory proteins and which ultimately leads to neuronal death. Even before the final demise of the injured neuron, distally located neurites undergo rapid physiological and structural alterations consisting of focal swelling and spine loss (Calabresi et al., 2003; Hasbani et al., 2001). It has also been shown that during cerebral ischemia reperfusion, the spine synapses can re-emerge in neurons indicating synaptic plasticity. Indeed, energy deprivation and anoxia evoke longterm potentiation (LTP), which also constitutes to synaptic plasticity (Calabresi et al., 2003; Smith and Jiang, 1994). Together, it has been postulated that ischemia-induced synaptic plasticity may be a crucial factor in determining the ensuing delayed neuronal death after ischemic injury to neurons. Although NMDA receptors (NMDAR) are known to cause ischemia-induced synaptic plasticity, the identities of mediators of NMDAR in ischemia-induced synaptic plasticity remain unknown. Nevertheless, it has been suggested that growth cone guidance molecules are intimately involved in synaptic plasticity as a result of local protein synthesis or in response to specific calcium channel activities. For example, local protein synthesis occurring in the mature axons has recently been confirmed and shown to be necessary for axonal guidance regulation and synaptic plasticity in neuronal physiology and pathology (Calabresi et al., 2003; Martin, 2004; Martin and Zukin, 2006; Otis et al., 2006; Thompson et al., 2004). Local translation of RhoA mRNA occurs in response to Sema3A-mediated growth cone collapse (Wu et al., 2005). With the design of novel growth cone selective expression vectors, many more growth cone guidance molecules are found to be expressed locally and in response to specific stimuli (Martin, 2004). EphA2 mRNA was found to be translated only after it has crossed the midline (Brittis et al., 2002). Moreover, BDNF potentiates neurotransmitter release from the developing synapse which requires local protein synthesis (Tyler et al., 2006; Zhang and Poo, 2002). More interestingly, protein degradation systems such as caspases (Williams et al., 2006), calpain and UPS are all found to play important roles in growth cone guidance and synapses during pruning of dendrites and neuronal death in nerve growth factor withdrawal (Zhai et al., 2003). Work from our own laboratory also showed that synaptosomal cleavage of growth cone guidance molecules such as CRMP also occurs during cerebral ischemia (Hou et al., 2006) (see the following section).

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There is also emerging evidence to suggest that transient receptor potential channels (TRPCs) may be an important target of NMDAR-mediated synaptic remodelling. TRPCs are members of a large family of TRP channels with conserved six-transmembrane domains forming nonselective cation-permeable channels (Ramsey et al., 2006). TRPCs mediate the transmembrane flux of cations down their electrochemical gradients, thereby raising intracellular Ca2þ and Naþ concentrations and depolarizing the cell. A striking number of biological functions have already been assigned to the various TRPC proteins, interestingly, with several members having similar functions in chemotropic axon guidance (TRPC1 and TRPC3) and axonal growth (TRPC5). TRPCs are widely expressed in the brain and are activated by the G protein–coupled receptors and receptor tyrosine kinases (RTKs). In the central nervous system, many trophic factors involved in modulating synaptic plasticity are RTKs, thereby implicating TRPCs in synaptic plasticity. Recent studies from Dr. Wang’s lab in Shanghai have shown that TRPCs are required in neuronal growth cone steering by the brain-derived neurotrophic factor (BDNF), confirming the function of TRPCs in axonal guidance and response to chemotaxis (Li et al., 2005). BNDF receptor TrkB contributes to dendritic spine development, synaptic strength and LTP induction (Lu, 2003; Tyler et al., 2002, 2006; Tyler and Pozzo-Miller, 2001) which further implicates TRPCs in modulating synaptic functions. Significantly, recent studies from Dr. Wang’s laboratory showed that TRPC3 and 6 are important in mediating neuronal survival during serum withdrawal. Although it is not clear if this protection involves synaptic plasticity, the fact TRPC may signal protection through CREB strongly argues for a role in synaptic and survival response.

6. Evidence for Guidance Cues as Therapeutic Targets The functional outcome of ischemic brain injury is the result of a complex interplay between permanent damage and long-term plasticity which can be beneficial or detrimental. Accordingly, limiting tissue damage and promoting useful plasticity are the two pillars of modern stroke management. It is therefore not surprising that efforts have been made to target the repulsive guidance cues and indeed in vivo evidence strongly support the hypothesis that blocking inhibitory guidance cues and their pathways have protective utilities in limiting brain damage. For example, our own work shows that specific polypeptides targeted against semaphorin-3A and neuropilin-1 (Williams et al., 2005) indeed protect neurons from oxygenglucose-deprivation induced neuronal death ( Jiang et al., 2007). A smallmolecule inhibitor of semaphorin-3A (SM-216289, isolated as a natural

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product from fungal fermentation) has also been shown to promote CNS repair in adult rats subjected to spinal cord transaction (Kaneko et al., 2006; Kikuchi et al., 2003). Treatment with SM-216289 increased regeneration of injured axons, Schwann cell-mediated myelination, enhanced angiogenesis and decreased apoptotic cell death. Delivery of Nogo antibody (IN-1) through direct injection into rat brains is also effective in improving neuroanatomical and functional recovery following middle cerebral artery occlusion (Papadopoulos et al., 2002, 2006). Inhibition of intracellular signalling pathways is also neuroprotective. Rho kinase inhibition has been shown to enhance axonal plasticity and confer neuroprotection (Lehmann et al., 1999; McKerracher and Higuchi, 2006; Ramer et al., 2004).

7. Concluding Remarks and Future Perspectives Significant progress has been made in understanding the role of growth promoting and inhibitory guidance molecules in both axonal initiation and specification during development, but also axonal degeneration and neuronal death in disease states and following trauma. A goal of future research which could have far-reaching consequences should focus on how the plethora of intrinsic and extrinsic factors, that act in promoting axonal growth, guidance and in establishing successful contacts in embryonic life, might be reactivated in the adult and thereby enhance regeneration. Degeneration of neurites occurring during neuronal death is controlled by events confined to the neurites and which occurs autonomously from the neuronal soma (Deckwerth and Johnson, 1994). Encouragingly, inhibitors blocking the inhibitory cues have been shown in proof-of-principle to be effective in facilitating axonal regeneration and providing neuroprotection. However, questions remain as to how these inhibitory molecules are activated in the injured brain. Recently, semaphorin 6A has been shown to be induced by interferon-g and defines an activation status of Langerhans cells (Gautier et al., 2006). Do cytokines regulate the guidance molecules in the injured CNS? Strong evidence exist that activated microglial causes neuronal toxicity by synthesizing and releasing neurotoxic compounds in response to neuronal injury thereby potentiating neuronal loss and synaptic dissolution, a typical response mediated by cytokines (Schwartz, 2003; Schwartz and Moalem, 2001). Emerging evidence also strongly supports the notion that immune response in the injured CNS may be beneficial to the repair and regrowth of the injured neurons (Banati et al., 1996; Kreutzberg, 1996; Schwartz, 2003) and it appears that the timing of the immune response is critical in determining the outcome. Future efforts will reveal the role of cytokines in the regulation of the production of guidance molecules.

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ACKNOWLEDGMENT We would like to thank Melissa Sheldrick and Angele Desbois for technical assistance. This work was supported by grants-in-aid from the Heart and Stroke Foundation of Ontario to S.T.H. (NA5393 and T5760). S.T.H. and R.A.S. thank the British Council for funding a number of Researcher Exchange Awards which enabled them to make reciprocal visits to each others laboratories whilst planning and writing this review.

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C H A P T E R

F O U R

New Insights into Mechanism and Regulation of Actin Capping Protein John A. Cooper* and David Sept† Contents 1. Introduction 2. Background 2.1. Physical and chemical properties 2.2. Biochemical activities 2.3. Cellular studies 2.4. Sequence conservation and isoforms 3. Mechanism of Binding Actin 3.1. Structural studies 3.2. Mobility of the C-terminal regions of subunits 4. Capping Protein Inhibitors and Uncapping 4.1. Contrasting results with CARMIL and V-1 4.2. Interaction of polyphosphoinositides with capping protein 4.3. CARMIL, CKIP-1 and CD2AP—A motif for inhibition of capping protein 4.4. Regulators that antagonize capping protein by indirect effects 4.5. Other interactors 5. Role of Capping Protein in Complex Cellular Processes 5.1. Actin-based motility at the plasma membrane 5.2. Z-line of the sarcomere in striated muscle 5.3. Drosophila development 5.4. Dynactin 6. Concluding Remarks and Future Directions Acknowledgments References

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Abstract The heterodimeric actin capping protein, referred to here as ‘‘CP,’’ is an essential element of the actin cytoskeleton, binding to the barbed ends of actin

* {

Department of Cell Biology, Washington University, St. Louis, MO, 63110 Department of Biomedical Engineering and Center for Computational Biology, Washington University, St. Louis, MO, 63130

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00604-7

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filaments and regulating their polymerization. In vitro, CP has a critical role in the dendritic nucleation process of actin assembly mediated by Arp2/3 complex, and in vivo, CP is important for actin assembly and actin-based process of morphogenesis and differentiation. Recent studies have provided new insight into the mechanism of CP binding the barbed end, which raises new possibilities for the dynamics of CP and actin in cells. In addition, a number of molecules that bind and regulate CP have been discovered, suggesting new ideas for how CP may integrate into diverse processes of cell physiology. Key words: Actin, Polymerization, Cell motility, Cell migration, Capping protein, Sarcomere. ß 2008 Elsevier Inc.

1. Introduction The actin capping protein (CP) was discovered, defined and named based on its ability to bind to the barbed ends of actin filaments, i.e., to ‘‘cap’’ them. The presence of CP at the barbed end inhibits the addition and loss of actin subunits at that end. In cells, CP is important for the dynamics of actin filament assembly, and this is important for the control of cell shape and movement. CP was called b-actinin when first characterized and purified from muscle by Maruyama and colleagues in the 1960s and 1970s in a remarkably prescient series of studies (Maruyama, 1966, 2002; Maruyama et al., 1977; Maruyama and Obinata, 1965). Nonmuscle CP was purified to homogeneity from Acanthamoeba in 1980 and shown to cap barbed ends (Isenberg et al., 1980). CP has continued to be an active subject of research, in part because it is found in essentially every eukaryotic organism and every metazoan cell type. Recent studies have produced new insights into the biochemistry of the interaction of CP with the actin filament, the mechanism of how this interaction can influence the architecture of actin filaments nucleated by Arp2/3 complex, the role of CP’s actin-binding activity in cells, and the identities and roles of molecules that bind and regulate CP. This review focuses on these recent discoveries. Other reviews of CP include the following: (Cooper et al., 1999; Schafer and Cooper, 1995; Wear et al., 2000; Wear and Cooper, 2004b).

2. Background 2.1. Physical and chemical properties CP is an a/b heterodimer with each subunit having a mass of 30 kDa. Individual subunits are unstable, but the heterodimer is very stable. The heterodimer remains folded in 0.6 M KI or 1% nonionic detergent (Wear and Cooper, 2004a), and it melts at 58  C in a single irreversible transition (Sizonenko et al., 1996). Individual subunits expressed in bacteria are largely insoluble, but they can be renaturated as heterodimers from

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urea (Remmert et al., 2000). Simultaneous expression of both subunits in bacteria produces large quantities of soluble active protein; the development of this expression system was a major technological advance in the field (Soeno et al., 1998). CP remains soluble, folded and active for capping actin under a variety of physiological conditions, including the presence or absence of divalent cation, and in a variety of salt concentrations, osmolality and pH. The CP molecule has the shape of a mushroom (Yamashita et al., 2003). The two subunits have very similar secondary structures, which is remarkable given their essentially complete lack of sequence similarity. The secondary structural elements of the subunits are arranged such that the molecule has a pseudo-two-fold axis of rotational symmetry down the center of the mushroom (Fig. 4.1A) (Yamashita et al., 2003). On the top surface of the mushroom, both subunits have C-terminal amphipathic a helixes, which appear to bind actin (Wear et al., 2003).

2.2. Biochemical activities CP was named for its ability to inhibit growth of the actin filament at the barbed end, i.e., to ‘‘cap’’ that end (Isenberg et al., 1980). CP binds to barbed ends with sub-nanomolar affinity (Wear et al., 2003). The presence of CP at the barbed end prevents the loss of the terminal actin subunit at the end of the filament, thus preventing depolymerization of the filament from that end. The critical concentration for actin polymerization is lower at the barbed end than at the pointed end, and the rate constants for actin elongation are higher at the barbed end than at the pointed end. These facts mean that capping of barbed ends by CP leads to an increase in the critical concentration, i.e., the actin monomer concentration at steady state. Cell cytoplasm has a high concentration of unpolymerized actin, for which capping of barbed ends is probably necessary. One molecule of CP appears to be sufficient to bind and attach a filament barbed end to an object, based on direct observation of single actin filaments by light microscopy (Bearer, 1991), including recent TIRF microscopy (Pavlov et al., 2007). TIRF microscopy confirms that the presence of CP at the barbed end abrogates the addition and loss of actin subunits (Kim et al., 2007). CP was one of the proteins found to be required for the reconstitution of motility based on actin assembly from pure proteins, in a landmark study (Loisel et al., 1999). Other molecules that cap barbed ends, not only CP, can serve this function (Revenu et al., 2007). One idea about the essential role of CP in the reconstitution system is that CP caps barbed ends that are older and thus located away from the surface of the object to be moved. By preventing actin subunits from adding in these undesired locations, the

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A

B

Figure 4.1 Illustrations of a model for the interaction of V-1 with CP and how their interaction inhibits actin capping activity. (A) The structure of the proposed molecular interaction between capping protein and V-1/myotrophin.The a subunit of capping protein is yellow, and its C-terminal actin-binding region is teal. The b subunit of capping protein is red, and its C-terminal actin-binding region is green. V-1/myotrophin is in pink. (B) The binding of V-1/myotrophin prevents capping protein from binding to actin filament barbed ends, i.e.,‘‘capping.’’ Growth of free barbed ends is an essential of the dendritic nucleation model for actin assembly, which involves Arp2/3 complex as the nucleating and branching agent. The color scheme is similar to the one in panel A, with Arp2/3 complex as the green oval at an end-to-side branch point for two actin filaments, whose subunits are teal.

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addition of actin subunits in the optimal locations is promoted, which can be considered as ‘‘funneling’’ of the subunits to these locations (Carlier and Pantaloni, 1997). Recent studies with synthetic systems show that CP can cause the shell of Arp2/3-nucleated actin filaments that assemble around a bead to break symmetry, which is necessary to produce polarity and movement (Orkun Akin and Dyche Mullins, personal communication). In reconstituted systems, high concentrations of CP lead to decreased actin assembly, making the plot of actin assembly or motility versus CP concentration a bell-shaped curve (Loisel et al., 1999). This biphasic nature of the effect of CP makes it difficult to interpret results in complex systems, such as mixtures of multiple actin regulators or even the cell cytoplasm. Mathematical modeling can help make predictions in such cases. For example, the concentration dependence of actin assembly on CP in the presence of Arp2/3 complex provided information about the end versus side nature of Arp2/3-mediated branching, based on predictions from mathematical modeling of the alternatives (Carlsson et al., 2004).

2.3. Cellular studies The abilities of CP to cap barbed ends and to tether barbed ends to objects appear to be a physiologically relevant in cells. The concentration of CP in cells is in the micromolar range, comparable to the number of actin filament barbed ends, and the binding affinity is in the sub-nanomolar range (Cooper et al., 1984; Wear et al., 2003). Analysis of a set of CP mutants in yeast showed a correlation of capping activity with the ability to rescue the null mutant phenotype (Kim et al., 2004). In cultured myotubes, injection of an anti-CP mAb that inhibited the actin-binding ability of CP caused a disruption in the early steps in myofibrillogenesis, as did expression of a mutant form of the CP b subunit that caps actin poorly (Schafer et al., 1995). In the mouse heart, expression of a capping-deficient CP b subunit during development caused disruption of myofibril architecture (Hart and Cooper, 1999). Other potential functions for CP are discussed below.

2.4. Sequence conservation and isoforms One of the most interesting and surprising features of the CP crystal structure was the two-fold rotational similarity between the tertiary structures of the two subunits (Yamashita et al., 2003). In vertebrates, the sequence similarity between the a and b subunits is very low, and given the lack of symmetry at the end of the actin filament, there was little reason to expect such structural symmetry in the CP heterodimer. When comparing the individual subunits in different organisms, sequence similarity is much higher. BLAST searches readily reveal apparent homologs of both subunits in vertebrates, invertebrates, plants, fungi, insects and protozoa (Fig. 4.2A, B). The sequences

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of the b subunits appear to be more strongly conserved than those of a subunits (Fig. 4.2C). The regions of conservation and variability are localized in a complementary manner on the two subunits. Within the b subunit, the actinbinding C-terminal region, the b tentacle, shows the highest sequence

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Figure 4.2 Phylogenetic analysis of CP subunits. Amino acid sequences were identified in a wide range of eukaryotes by BLAST.The sequences were aligned with CLUSTALW. (A) Phylogenetic trees for the a and b subunits are remarkably similar. Vertebrates have up to three isoforms of each subunit, while invertebrates and lower organisms have single isoforms of each subunit. Vertebrate isoforms 1 and 2 represent nearly all the CP outside of germ cells, they cluster into distinct groups. (B) Phylogenetic analysis of CP subunits compared with cofilin and profilin, other actin-binding proteins.The a and b subunits of CP are not more similar to each other than they are to the cofilin and profilin families, despite their similar secondary structures and interactions with actin. (C) For each organism, the similarity of its b subunit to the b subunits of other organisms is plotted versus the similarity of its a subunit to the a subunits of other organisms. For vertebrates, only a single isoform of each subunit was included per species. The results show that the b subunit sequences are more similar to each other than are those of the a subunits.

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variability. In contrast, the body of the a subunit is more weakly conserved than is the C-terminal region, giving rise to an inherent asymmetry in the molecule where the half of the molecule containing the b subunit and a tentacle is more conserved than the half with the a subunit and the b tentacle. This asymmetry may have implications for how CP interacts with the barbed end of the actin filament, and it appears to be consistent with structural studies discussed next. Organisms other than vertebrates have single genes encoding each of the CP subunits. Vertebrates, in contrast, have two somatically expressed isoforms of each subunit and one additional male germ-cell specific isoform (Hart et al., 1997b; Hurst et al., 1998; Schafer et al., 1994; von Bulow et al., 1997). For the a subunit, the somatic isoforms, termed a1 and a2, are encoded by different genes (Hart et al., 1997a), while the b subunit isoforms are produced from a single gene by alternative splicing (Schafer et al., 1994). The sequences of the a1 and a2 isoforms are conserved across vertebrates, as are those of the b1 and b2 isoforms, suggesting that they have distinct functions in vertebrates. Little evidence exists regarding specific functions of the a isoforms, but they are expressed at varying ratios in different cells and tissues (Hart et al., 1997b). The b1 isoform is located specifically at the Z-disc of the sarcomere of striated muscle; b2 is also present in the same cells, but it localizes elsewhere (Schafer et al., 1994). The b1 and b2 isoforms were not able to substitute for each other in muscle cells, supporting the hypothesis of distinct functions (Hart and Cooper, 1999). The biochemical nature of the functional difference has not been discovered. One would suspect that the b1 isoform interacts specifically with one or more components of the Z-disc. CP isoforms appear to bind equally well to actin and nebulin, as purified proteins in vitro (Pappas et al., 2008; Schafer et al., 1994), and other components remain to be tested.

3. Mechanism of Binding Actin 3.1. Structural studies An x-ray crystal structure of CP shows that the molecule has the shape of a mushroom and that the two subunits are arranged with a pseudo-two-fold axis of rotational symmetry (Yamashita et al., 2003). The N-termini of the subunits are located at the base of the stalk of the mushroom, and the subunits are extensively intertwined, with a large b sheet at the core of the mushroom cap structure. On the top surface of the mushroom, each subunit has an extended a helix oriented perpendicular to the strands of the

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b sheet on which it lies. Each subunit ends with a C-terminal amphipathic a helix on the top surface of the mushroom. Truncation and point mutations of the C-terminal regions reveal that both are important for high-affinity capping (Wear et al., 2003), with the C-terminal region of the a subunit being more important than that of the b. CP binds to the barbed end of the actin filament with high affinity, generally less than 1 nM. The second-order association rate constant is high, approaching the range of the diffusion limit, and the first-order dissociation rate constant is accordingly low. CP containing only one (either one) of the C-terminal regions is able to cap, and the C-terminal region of the b subunit alone is sufficient to cap, with decreased affinity. The binding affinity and rate constants have been inferred largely from actin polymerization experiments, and physical binding studies would provide a valuable confirmation of those results. Recent cryoEM work from the Mae´da lab has resulted in a low˚ ) structure of CP on the barbed end of an actin filament resolution (23 A (Narita et al., 2006; Narita and Maeda, 2007). CryoEM analysis of actin filament binding proteins that decorate the sides of the filament can benefit from helical averaging. Here, only one molecule of CP was present on the barbed end of each filament, so this analysis depended on the collection and averaging of single-particle images, a challenging task. A novel method for combining the images that were collected produced a new model for the structure of the CP-capped filament. This structure was able to unambiguously identify the a and b subunits, and their positions with respect to the actin protomers at the barbed end suggest that the body of the b subunit, along with the a subunit C-terminal region, make the primary contacts with the last two protomers of the filament. This finding is supported by the sequence conservation data discussed above. Mutational analysis confirmed the importance of residues in the C-terminal region of the a subunit and on the top surface. Computational modeling analysis showed that the b subunit C-terminus, a tentacle-like amphipathic helix, can bind to a hydrophobic cleft on the actin subunit, in a manner comparable to that of a WH2 domain (Dominguez, 2004; Hertzog et al., 2004). Based on these results, the authors proposed a model, shown in Figure 4.3, in which CP binds to the barbed end in two steps, first by the a subunit C-terminus and surrounding residues and second by the flexible b subunit C-terminus (Narita et al., 2006). This model raises the possibility that CP bound to the barbed end of the actin filament might dissociate from the a subunit site and thus be attached only by the b subunit tentacle. If this were to occur, the mobility of the b tentacle might allow the body of CP to move, or ‘‘wobble,’’ in place. If another molecule would bind to CP in this wobble state, then the presence of that molecule might inhibit rebinding and thus favor complete detachment, or ‘‘uncapping.’’

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Figure 4.3 A model for the binding of CP to the actin filament barbed-end proposed by Narita and colleagues (Narita et al., 2006) kindly provided by the authors and reproduced with their permission. First, basic residues on the CP a C-terminal region (blue) are attracted to acidic residues on the barbed end of the actin filament (red).These acidic residues include ones from the terminal and the penultimate protomers of the filament, labeled B and B-1. Next, the mobile b tentacle searches for its binding position on the filament. Finally, the hydrophobic surface (yellow) of the amphipathic b tentacle binds to the hydrophobic cleft (yellow) on the terminal protomer, B.

Uncapping will be an important subject for further study, because of its potential relevance in cells. In vitro, the dissociation rate for CP to leave the barbed end is quite long relative to the time scale on which actin filaments assemble and disassemble in cells and to the time scale on which CP appears to dissociate from the actin cytoskeleton in cells (Iwasa and Mullins, 2007; Miyoshi et al., 2006). Inducing uncapping might be a mechanism for cells to induce assembly or disassembly of the filament network, depending on other conditions in the local environment.

3.2. Mobility of the C-terminal regions of subunits As described above, the C-terminal region of the CP a subunit is an amphipathic helix that lies on the top surface of the protein in the crystal structure. Its hydrophobic side is oriented toward the body of the protein

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(i.e., the top of the mushroom) (Yamashita et al., 2003), and in molecular dynamics simulations, this region remains in that position on the mushroom surface (Bhattacharya et al., 2006). Residue Trp271 of the amphipathic helix occupies a hydrophobic pocket on the surface of the body (Yamashita et al., 2003). A short peptide corresponding to part of this same region of the CP a subunit was found to bind to the protein S100 (Ivanenkov et al., 1995, 1996), and an NMR structure of the S100 peptide complex showed that the Trp residue corresponding to position 271 occupies a hydrophobic pocket in S100 (Inman et al., 2002). This peptide from CP a can bind S100, and full-length unfolded CP a can bind S100, but native CP was found not to bind S100 (Schafer et al., 1996; Wear and Cooper, 2004a). Treatment of CP with high concentrations of nonionic detergent enabled S100 to bind weakly (Wear and Cooper, 2004a). Thus, the C-terminal region of the a subunit, which is necessary for binding actin, appears to be immobile in the native solution structure, as implied by the crystal structure and molecular dynamics results. Mutating the Trp271-analogous residue of yeast CP to Ala caused a large loss of capping activity, which may be due to alteration of the structure of the amphipathic helix (Kim et al., 2004). In the crystal structure, the C-terminal region of the CP b subunit is also an amphipathic helix, but the helix extends out from the body of the protein, surrounded by solvent ( Yamashita et al., 2003). In molecular dynamics simulations, this region is highly mobile, as expected (Bhattacharya et al., 2006). The mobilities of the C-terminal regions of the subunits are incorporated into the current model for CP binding to the barbed end of the actin filament proposed by Mae´da and colleagues (Narita et al., 2006; Narita and Maeda, 2007). In terms of the wobble hypothesis, the mobilities of the C-terminal actin-binding regions helps to predict that CP will not wobble when it is bound to a barbed end only by the C-terminal region of the a subunit, i.e., when the b subunit’s C-terminal tentacle is not bound to actin. In contrast, CP will wobble if the a subunit C-terminal region dissociates, leaving only the b tentacle attached (Bhattacharya et al., 2006).

4. Capping Protein Inhibitors and Uncapping 4.1. Contrasting results with CARMIL and V-1 CARMIL and V-1/myotrophin are two different proteins that can bind to CP and inhibit its ability to bind to the barbed end of the actin filament, i.e., to cap. When CP is already present on the barbed end, CARMIL appears to be able to remove it. This conclusion is based on physical and

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functional assays. First, CP is found in the supernatant after sedimentation of actin filaments following the addition of CARMIL (Uruno et al., 2006). Second, the concentration of free barbed ends increases rapidly on addition of CARMIL (Uruno et al., 2006; Yang et al., 2005). This increase occurs on the time scale of 10 s (Uruno et al., 2006; Yang et al., 2005), while the spontaneous dissociation rate of CP from the barbed end appears to be on the time scale of 10 min (Schafer et al., 1996). This difference suggests that CARMIL can bind to the CP/barbed-end complex in some manner, and we hypothesize that this interaction occurs in the wobble state. In support of this hypothesis, titration of CP with increasing concentrations of CARMIL in an actin-capping assay does not lead to complete inhibition of CP (Yang et al., 2005). Less than complete inhibition can be explained by the CARMIL/CP complex having a very low level of capping activity, suggesting that CARMIL and CP can co-exist in a ternary complex with the barbed end. In the future, identification of the CARMIL-binding site on CP should provide an important test of the wobble hypothesis, and imaging of single CP molecules on actin filaments should further our understanding of uncapping. V-1/myotrophin provides an important and interesting contrast with CARMIL. V-1 also binds to CP and inhibits its ability to cap the barbed end (see Fig. 4.1) (Bhattacharya et al., 2006; Taoka et al., 2003). However, V-1 has little or no uncapping activity in functional assays. That is, addition of a high concentration of V-1 to CP-capped actin filaments, at a level sufficient to inhibit all the CP in the reaction, produces little increase in the number of free barbed ends (Bhattacharya et al., 2006). Another difference between CARMIL and V-1 is that high concentrations of V-1 completely inhibit the capping activity of CP (Bhattacharya et al., 2006). Thus, both lines of evidence fail to indicate that V-1 can bind to CP that is bound to the barbed end. In the hypothetical wobble state, CP is attached to the barbed end only by the b subunit’s C-terminal region, i.e., the tentacle. V-1 requires the C-terminal region of the b subunit but not that of the a subunit for optimal binding to CP (Bhattacharya et al., 2006), suggesting that V-1 may interact with the b tentacle, among other parts of CP. Thus, V-1 would not be predicted to bind to the hypothetical wobble state, and this predicts that V-1 should not be able to uncap, which is the case. The binding site on CP for V-1 appears to include an area at the base of the b tentacle, as indicated in Figure 4.1A. This conclusion is based on several results. First, truncation of the CP a subunit C-terminal region was found to weaken the interaction of CP with V-1 by a small amount (Bhattacharya et al., 2006). The simplest interpretation of this observation alone would be a direct interaction between V-1 and the a subunit C-terminus. However, the dynamics of the a C-terminus are coupled to the rest of the protein, in contrast to the situation for the highly mobile ‘‘tentacle’’ of the b subunit. Molecular dynamics simulations of a CP

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molecule from which the C-terminal region of the a subunit was truncated revealed decreased dynamics of the a subunit at locations distant from the C-terminus, near the base of the b tentacle (Bhattacharya et al., 2006). The surface residues at the affected location were also part of a V-1 binding site identified on CP by computational docking analysis. In addition, these residues are among the relatively few that differ between the a1 and a2 isoforms, and V-1 binds slightly differently to the a isoforms. To account for all these observations, we suggest that V-1 binds to CP as depicted in Figure 4.1A. Structural data about the CP/V-1 complex should provide valuable information to test this model.

4.2. Interaction of polyphosphoinositides with capping protein Polyphosphoinositides, including PIP2, can bind to CP and inhibit its capping activity (Heiss and Cooper, 1991). PIP2 can also cause rapid uncapping, demonstrated recently by observations of the polymerization of single actin filaments by TIRF microscopy (Kim et al., 2007). In those studies, addition of PIP2 to a flow chamber with actin filaments that were capped by CP and thus not able to polymerize resulted in the rapid and complete conversion of ends from the nongrowing to the growing state. Computational docking analysis suggested that PIP2 binds to three conserved basic residues on the surface of CP near the a subunit C-terminus, and mutations of those residues weakened the affinity of PIP2 for CP, measured with functional and physical assays (Kim et al., 2007). Some of these residues were predicted to sit at the interface between CP and actin based on the cryoEM CP/actin filament structure (Narita et al., 2006), and mutations of these residues affected the ability of CP to cap actin (Kim et al., 2007; Narita et al., 2006). Thus, the PIP2 and actin binding sites on CP may overlap. In addition, these observations are consistent with the wobble model in that they suggest that the region of the a C-terminus is available for PIP2 binding when CP is on the barbed end, i.e., in the wobble state. Little recent work addresses the physiological significance of PIP2/CP interaction, but in older work, studies of the actin assembly that accompanies platelet activation suggested that an early step was uncapping of CP-capped actin filaments by polyphosphoinositides (Barkalow et al., 1996). In many other cells systems, CP appears to terminate actin assembly by capping free barbed ends that are created by other mechanisms. The actin assembly that results from treatment of Dictyostelium cells with chemoattractant appears to be such a case (Eddy et al., 1997). Since polymerization of free barbed ends at membranes appears to drive the movement of those membranes, one attractive hypothesis is that PIP2 generated in the membrane helps to inhibit capping by CP near the membrane.

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4.3. CARMIL, CKIP-1 and CD2AP—A motif for inhibition of capping protein 4.3.1. Motif for inhibition of capping protein The existence of a motif for binding and inhibiting CP has been suggested by comparative analysis of the sequences and biochemical properties of the proteins CARMIL, CKIP-1 and CD2AP (Bruck et al., 2006; Canton et al., 2005, 2006; Uruno et al., 2006). Each of the three proteins was found to bind directly to CP and inhibit the actin capping activity of CP. Structurefunction analysis of each protein revealed an essential region with a common set of essential amino-acid residues. The potential CP-binding motif appears to be LXHXTXXRPK(6X)P (Bruck et al., 2006). 4.3.2. CARMIL Acan125 was the original name for CARMIL when the protein was discovered in amoeba as a binding partner for the SH3 domain of certain class I myosins (Xu et al., 1995). Later, this protein was found to bind CP and Arp2/3 complex as well, leading to the acronym CARMIL ( Jung et al., 2001). The protein is relatively large, with a long leucine-rich repeat (LRR) region of unknown function (Xu et al., 1997). The LRR region may participate in autoinhibition of the CP-binding activity of CARMIL (Uruno et al., 2006). CARMIL binds tightly to CP, with a Kd in the nanomolar range (Yang et al., 2005). CARMIL purified from Acanthamoeba contains CP in near-stoichiometric amounts (Remmert et al., 2004), but the large majority of Acanthamoeba CP in cell extracts is free and able to cap actin (Cooper et al., 1984). CARMIL is important for actin-based motility, based on knockout and knockdown studies in Dictyostelium and cultured vertebrate cells ( Jung et al., 2001; Yang et al., 2005). The multiple biochemical functions associated with CARMIL raise many possibilities for its mechanism of action in cells. Loss of the CP-binding site, by internal deletion of 100 aa residues, produced a mutant form of CARMIL unable to rescue the knockdown phenotype in cultured vertebrate cells (Yang et al., 2005). 4.3.3. CKIP-1 CKIP-1 was discovered as an interaction partner for casein kinase 2, helping to recruit CK2 to the plasma membrane (Olsten et al., 2004). CKIP-1 was also found to interact biochemically with CP in cultured cells (Canton et al., 2005). The binding of CKIP-1 and CK2 to CP inhibits capping activity (Canton et al., 2005), and CKIP-1 expression in cultured cells causes changes in cell morphology and the actin cytoskeleton that depend on its interaction with CP (Canton et al., 2006). CK2 can phosphorylate CP by CK2 (Canton et al., 2005), which may be a novel regulatory mechanism.

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4.3.4. CD2AP CD2-associated protein (CD2AP) and its relative Cin85 (Cbl-interacting protein) have been found to bind to CP and inhibit its capping activity (Bruck et al., 2006; Hutchings et al., 2003). CD2AP and Cin85 appear to be adaptor proteins that provide signaling pathway connections from membrane receptors to the actin cytoskeleton (Dustin et al., 1998; Lynch et al., 2003; Shih et al., 1999). CD2AP and Cin85 also interact with cortactin (Lynch et al., 2003; Nam et al., 2007), which promotes actin assembly via Arp2/3 complex, so these adaptors have multiple potential connections to actin assembly.

4.4. Regulators that antagonize capping protein by indirect effects Formin proteins act as competitors of CP at the actin filament barbed end. Formins are a large family of proteins, with a diversity of structures and functions that have yet to be understood fully (Goode and Eck, 2007; Staiger and Blanchoin, 2006). A formin dimer can bind to an actin filament barbed end, which can inhibit the binding of CP (Fig. 4.4) (Zigmond et al., 2003). However, the presence of formin at the barbed end still allows an actin subunit to add to that barbed end. Most remarkably, one formin dimer can remain bound to the barbed end while more and more actin subunits add over time; the formin essentially ‘‘surfs’’ with the growing barbed end of the actin filament. Thus, formins are very effective anti-cappers, promoting the growth of barbed ends. A number of cellular studies of formins support the relevance of this mechanism in vivo (Goode and Eck, 2007; Staiger and Blanchoin, 2006). VASP also functions as an antagonist of CP, via interactions with the actin filament. VASP is a member of the Ena/VASP family of proteins, which have been implicated in actin-based motility and morphogenesis. VASP antagonizes the capping activity of CP in vitro (Barzik et al., 2005). The anti-capping effect of VASP is not specific for CP in that other barbed-end cappers, such as gelsolin, are also antagonized by VASP. Ena/VASP proteins are found at the tips of filopodia, where they may prevent capping, which would allow barbed ends of actin filaments to grow and filopodia to elongate (Fig. 4.4) (Applewhite et al., 2007; Mejillano et al., 2004).

4.5. Other interactors Twinfilin was characterized as a protein that binds and sequesters actin monomers, thus inhibiting actin polymerization (Goode et al., 1998; Lappalainen et al., 1998; Palmgren et al., 2002). Twinfilin binds directly to CP, and that interaction does not affect the interaction of either protein

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B Membrane Membrane protrusion

CP active CP inhibited

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+ F-actin (+; barbed / −, pointed) Arp2/3 complex-mediated

Arp2/3 complex CP

Filament nucleation

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Figure 4.4 Illustration of potential modes of actin assembly in cells with respect to CP, based on one created and provided by Dr. MartinWear (Wear and Cooper, 2004b). (A) When CP is active and actin nucleation is Arp2/3-mediated, lamellipodial assembly predominates. Newly created free barbed ends are near the membrane.They elongate to push the membrane forward and /or the actin filament network backward. After some time, CP caps those barbed ends, which would seem to be efficacious because the ends are no longer near the membrane and their further growth would not produce useful work. (B) In this setting, when CP is inactivated in one location, by any of several potential inhibitors, then the filaments in that small region may continue to grow, producing a thin protrusion that contains a bundle of actin filaments. (C) An alternative mechanism that may produce actin filament bundles, perhaps not associated with a plasma membrane, is the nucleation of actin polymerization by a formin. Formins allow actin subunits to add and do not allow CP to add.Thus, the filaments continue to grow.

with actin (Falck et al., 2004). In yeast, twinfilin’s ability to bind CP and its ability to bind actin are both necessary for its function in actin dynamics (Falck et al., 2004). Twinfilin alone can also cap barbed ends, with a preference for ADP-actin (Helfer et al., 2006; Paavilainen et al., 2007),

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raising important questions about the relative contributions of these various biochemical activities to cell physiology. Extracts of neutrophils were found to contain a low-molecular-weight inhibitor of CP (Huang et al., 2005), which has not yet been identified. This inhibitor was able to inhibit and reverse capping of barbed ends by purified CP in functional assays. The biochemical properties of the inhibitor indicated that it was not PIP2, V-1, CARMIL or VASP. In an earlier study with neutrophil extracts, Cdc42-induced actin polymerization was found to be insensitive to CP, relative to polymerization induced by actin seeds (Huang et al., 1999). In retrospect, this Cdc42-induced anti-capper may have been a formin.

5. Role of Capping Protein in Complex Cellular Processes 5.1. Actin-based motility at the plasma membrane In metazoan cells in culture, the ability to form lamellipodial type protrusions was found to depend on CP in siRNA knockdown studies (Iwasa and Mullins, 2007; Mejillano et al., 2004). In mouse melanoma cells, inhibition of lamellipodial assembly was accompanied by an increase in filopodia formation (Mejillano et al., 2004), consistent with older results in Dictyostelium with antisense (Hug et al., 1995). In contrast, filopodia were not increased on CP knockdown in Drosophila cultured S2 cells (Iwasa and Mullins, 2007). In the mouse melanoma cells, the increased filopodia formation depended on VASP (Mejillano et al., 2004), so the Drosophila S2 cells may have lacked sufficient activity of VASP or some other filopodial component (Iwasa and Mullins, 2007). The need for CP in lamellipodial assembly supports the relevance of a key element of the dendritic nucleation model proposed to account for the assembly of branched networks of actin filaments associated with membranes and Arp2/3 complex (Nicholson-Dykstra et al., 2005). In that model, the reason why capping of barbed ends by CP is important has been proposed to be to ‘‘funnel’’ actin assembly to the new filament ends at the membrane, as described above, or to keep the actin filaments short and highly branched, to strengthen the network (Fig. 4.4). Testing these ideas will likely require mathematical modeling and measurements of physical parameters on a microscopic time scale with high time resolution. Speckle and single-molecule fluorescence imaging of lamellipodial regions of cultured cells reveals that CP binds to the actin filament network very near the membrane and that it dissociates from the network after a short time and distance (Iwasa and Mullins, 2007; Miyoshi et al., 2006), also consistent with the proposed role for CP in the dendritic nucleation model.

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CP dissociation may result from severing-induced depolymerization of filaments or it may be the direct effect of an uncapper. Distinguishing these possibilities will require observing the behavior of CP in cells carrying a specific defect in severing or uncapping, which will require careful biochemical characterization of mutant proteins. A challenge for these studies will be identifying which of several potential uncappers or severing agents are the relevant actors in a given cell system. For CP, the ability to alter protein activity protein locally and rapidly should provide powerful information for testing predictions of models. Acute inactivation of CP has been achieved in fibroblasts by laser inactivation of GFP-CP. The result was a local increase in the concentration of free barbed ends, the polymerization of actin and the formation of actinbased protrusive structures (Vitriol et al., 2007). These results support the notion that the CP caps barbed ends and that barbed-end capping prevents actin polymerization. Note that intuitive reasoning from the dendritic nucleation model seems capable of explaining the increase in actin-based protrusions in this experiment but also the decrease in lamellipodial protrusions in the set of knockdown experiments discussed above. One can rationalize the opposing predictions for the effect of the loss of CP activity in these two experiments on the basis of the laser inactivation effect being local and acute, while the knockdown inhibition effect is global and chronic. The rigor and certainty of the conclusions would be greatly enhanced by the application of mathematical modeling so that predictions are based on more than intuition and rationalization. Other membrane movements, in addition to lamellipodial protrusions of the plasma membrane, appear to be based on actin assembly. Endocytosis is another good example, based on recent studies in yeast and vertebrate systems (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al., 2006). The endocytic process is composed of multiple steps of actin assembly and actin-based movement. Membrane receptors, endocytic adaptors, and actin-binding proteins, including Arp2/3 complex, are involved, with distinct roles at various steps in the process. Yeast CP null mutants showed a decrease in the initial movement of the cortical actin patch, the site of endocytosis, away from the plasma membrane, and the actin filaments of the patch still assembled (Kim et al., 2006), all of which appears to be consistent with the dendritic nucleation model. In contrast, other steps of endocytic traffic showed little to no effect from the loss of CP, so the model may not apply in these cases.

5.2. Z-line of the sarcomere in striated muscle CP purified from skeletal muscle was called ‘‘CapZ’’ because of its presence at the Z-disc of the sarcomere (Casella et al., 1987). The barbed ends of the actin-based thin filaments are also located at the Z-disc, and one molecule of

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CapZ appears to cap each barbed end (Schafer et al., 1993). The reason for capping the barbed end may be to help anchor the thin filament to the Z-disc, or it may be to prevent the growth of the thin filament into the adjacent sarcomere. CapZ and its actin-binding activity appear to be important for assembly of the sarcomere, as noted above. Recent studies have uncovered a biochemical interaction between nebulin, a giant protein of the sarcomere, and CapZ (Pappas et al., 2008; Witt et al., 2006). Nebulin knockdown in developing muscle cells leads to decreased accumulation of CapZ at the Z-disc and poor alignment of thin filament barbed ends (Pappas et al., 2008), consistent with a role for nebulin as a ‘‘ruler’’ specifying thin filament length by interacting with CapZ as the capper of the barbed end. The location of the CapZ binding site in nebulin suggests a model for the Z-disc in which nebulin connects one thin filament with an adjacent one, thus serving as a structural cross bridge to impart strength to the disc (Fig. 4.5).

5.3. Drosophila development In Drosophila, CP is essential for viability of the organism, and loss-offunction mutants die as embryos (Hopmann et al., 1996). In the bristles of the adult fly, actin bundles underlie and define the surface structure of the bristle, and the assembly of these actin bundles depends on CP and other actin regulators, including profilin and Arp2/3 complex (Frank et al., 2006; Hopmann and Miller, 2003). The effects of CP and the other proteins on the actin filament bundles appears to be an indirect one, mediated by their effects on a separate dynamic pool of actin filaments termed snarls (Frank et al., 2006). Studies of eye and wing development have also revealed an important role for CP, most likely through effects on actin assembly and morphogenesis (Iwasa and Mullins, 2007; Janody and Treisman, 2006). Nebulin

Z-disc Cap z a-actinin

∗M160M164

M177M181

SH3

Thin filament

Figure 4.5 An illustration of a new model for the structure of the Z-disc, provided by Dr. Carol Gregorio. Based on a structure-function analysis of the interaction of nebulin with CapZ (Pappas et al., 2008), the model proposes that the nebulin molecule crosses from one actin-based thin filament to another one, within the Z-disc.

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5.4. Dynactin CP is a biochemical component of dynactin, a multi-subunit complex necessary for the function of dynein (Schroer, 2004). Dynactin contains an actin-like filament composed largely of Arp1 (actin-related protein 1) and 1 mol per mol of CP. Single-particle EM image averaging of purified dynactin reveals lobes at the barbed end of the actin-like filament, which are likely to correspond to the subunits of CP (Hodgkinson et al., 2005; Imai et al., 2006). The presence of CP may be important to control the number of Arp1 subunits in the filament, which is remarkably constant among dynactin molecules. Whether the presence of CP affects the function of dynactin in cells has not been thoroughly tested. In yeast, CP null mutations produce no measurable effect on dynein function (Moore et al., 2008). Null mutations of some other dynactin subunits, including Arp1, produce a complete loss of dynein function. Biochemical approaches have not revealed CP to be a component of dynactin. Therefore, CP is either not an important component of dynactin in yeast or not a component at all. Yeast dynactin may lack other subunits of dynactin as well (Moore et al., 2008). To our knowledge, no tests of the functional role of CP have been done in other systems, which would be useful.

6. Concluding Remarks and Future Directions The emerging multiplicity of molecules that interact with CP and the diversity of their biochemical actions raise many new questions about how CP functions and is regulated in cells. The potential complexity is amplified by the cases where the interactors are proteins with multiple domains and may serve as adaptors with other molecules. Dissecting the individual roles of these interactions in biochemical and physiological terms will be an important challenge. New insight into the dynamic nature of the interaction of CP with the actin filament barbed end raises interesting possible mechanisms for the action of CP in the rapid assembly and disassembly of actin in cells. The potential existence of a wobble state needs to be established with direct physical methods, which will allow one to test whether the wobble state is part of the mechanism of uncapping. CP is present in essentially all cells and tissues of vertebrates, and actin filaments are proving to have multiple distinct roles in various cell settings. The roles that CP may play in these settings, especially the possibility of different roles for the conserved vertebrate isoforms of CP, will be an important avenue for the future.

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ACKNOWLEDGMENTS The authors are grateful to Drs. Carol Gregorio, Martin Wear, Yuichiro Mae´da, and Akihiro Narita for providing illustrations. Research in this area in the authors’ laboratories is supported by NIH GM 38542 to J.A.C. and GM 67426 to D.S.

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Eddy, R. J., Han, J., and Condeelis, J. S. (1997). Capping protein terminates but does not initiate chemoattractant-induced actin assembly in Dictyostelium. J. Cell Biol. 139, 1243–1253. Engqvist-Goldstein, A. E., and Drubin, D. G. (2003). Actin assembly and endocytosis: From yeast to mammals. Annu. Rev. Cell Dev. Biol. 19, 287–332. Falck, S., Paavilainen, V. O., Wear, M. A., Grossmann, J. G., Cooper, J. A., and Lappalainen, P. (2004). Biological role and structural mechanism of twinfilin-capping protein interaction. EMBO J. 15, 3010–3019. Frank, D. J., Hopmann, R., Lenartowska, M., and Miller, K. G. (2006). Capping protein and the Arp2/3 complex regulate nonbundle actin filament assembly to indirectly control actin bundle positioning during Drosophila melanogaster bristle development. Mol. Biol. Cell 17, 3930–3939. Goode, B. L., Drubin, D. G., and Lappalainen, P. (1998). Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein. J. Cell Biol. 3, 723–733. Goode, B. L., and Eck, M. J. (2007). Mechanism and function of formins in the control of actin assembly. Annu. Rev. Biochem. 76, 593–627. Hart, M. C., and Cooper, J. A. (1999). Vertebrate isoforms of actin capping protein beta have distinct functions in vivo. J. Cell Biol. 6, 1287–1298. Hart, M. C., Korshunova, Y. O., and Cooper, J. A. (1997a). Mapping of the mouse actin capping protein alpha subunit genes and pseudogenes. Genomics. 3, 264–270. Hart, M. C., Korshunova, Y. O., and Cooper, J. A. (1997b). Vertebrates have conserved capping protein alpha isoforms with specific expression patterns. Cell Motil. Cytoskeleton 2, 120–132. Heiss, S. G., and Cooper, J. A. (1991). Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids. Biochemistry 30, 8753–8758. Helfer, E., Nevalainen, E. M., Naumanen, P., Romero, S., Didry, D., Pantaloni, D., Lappalainen, P., and Carlier, M. F. (2006). Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: Implications in motility. EMBO J. 25, 1184–1195. Hertzog, M., van Heijenoort, C., Didry, D., Gaudier, M., Coutant, J., Gigant, B., Didelot, G., Preat, T., Knossow, M., Guittet, E., and Carlier, M. F. (2004). The beta-thymosin/ WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly. Cell 117, 611–623. Hodgkinson, J. L., Peters, C., Kuznetsov, S. A., and Steffen, W. (2005). Three-dimensional reconstruction of the dynactin complex by single-particle image analysis. Proc. Natl. Acad. Sci. USA 10, 3667–3672. Hopmann, R., Cooper, J. A., and Miller, K. G. (1996). Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila. J. Cell Biol. 133, 1293–1305. Hopmann, R., and Miller, K. G. (2003). A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle. Mol. Biol. Cell 14, 118–128. Huang, M., Pring, M., Yang, C., Taoka, M., and Zigmond, S. H. (2005). Presence of a novel inhibitor of capping protein in neutrophil extract. Cell Motil. Cytoskeleton 62, 232–243. Huang, M., Yang, C., Schafer, D. A., Cooper, J. A., Higgs, H. N., and Zigmond, S. H. (1999). Cdc42-induced actin filaments are protected from capping protein. Curr. Biol. 9, 979–982. Hug, C., Jay, P. Y., Reddy, I., McNally, J. G., Bridgman, P. C., Elson, E. L., and Cooper, J. A. (1995). Capping protein levels influence actin assembly and cell motility in Dictyostelium. Cell 81, 591–600. Hurst, S., Howes, E. A., Coadwell, J., and Jones, R. (1998). Expression of a testis-specific putative actin-capping protein associated with the developing acrosome during rat spermiogenesis. Mol. Reprod. Dev. 1, 81–91.

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Hutchings, N. J., Clarkson, N., Chalkley, R., Barclay, A. N., and Brown, M. H. (2003). Linking the T cell surface protein CD2 to the actin-capping protein CAPZ via CMS and CIN85. J. Biol. Chem. 25, 22396–22403. Imai, H., Narita, A., Schroer, T. A., and Maeda, Y. (2006). Two-dimensional averaged images of the dynactin complex revealed by single particle analysis. J. Mol. Biol. 359, 833–839. Inman, K. G., Yang, R., Rustandi, R. R., Miller, K. E., Baldisseri, D. M., and Weber, D. J. (2002). Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12. J. Mol. Biol. 5, 1003–1014. Isenberg, G., Aebi, U., and Pollard, T. D. (1980). A novel actin binding protein from Acanthamoeba which regulates actin filament polymerization and interactions. Nature 288, 455–459. Ivanenkov, V. V., Dimlich, R. V. W., and Jamieson, G. A. (1996). Interaction of S100a(0) protein with the actin capping protein, CapZ—Characterization of a putative S100a(0) binding site in CapZ-alpha-subunit. Biochem. Biophys. Res. Commun. 1, 46–50. Ivanenkov, V. V., Jamieson, G. A., Jr., Gruenstein, E., and Dimlich, R. V. (1995). Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ. J. Biol. Chem. 24, 14651–14658. Iwasa, J. H., and Mullins, R. D. (2007). Spatial and temporal relationships between actinfilament nucleation, capping, and disassembly. Curr. Biol. 17, 395–406. Janody, F., and Treisman, J. E. (2006). Actin capping protein alpha maintains vestigialexpressing cells within the Drosophila wing disc epithelium. Development 133, 3349–3357. Jung, G., Remmert, K., Wu, X., Volosky, J. M., and Hammer, J. A., 3rd. (2001). The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains. J. Cell Biol. 7, 1479–1497. Kaksonen, M., Toret, C. P., and Drubin, D. G. (2006). Harnessing actin dynamics for clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 7, 404–414. Kim, K., Galletta, B. J., Schmidt, K. O., Chang, F. S., Blumer, K. J., and Cooper, J. A. (2006). Actin-based motility during endocytosis in budding yeast. Mol. Biol. Cell 17, 1354–1363. Kim, K., McCully, M. E., Bhattacharya, N., Butler, B., Sept, D., and Cooper, J. A. (2007). Structure/function analysis of the interaction of phosphatidylinositol 4,5-bisphosphate with actin-capping protein: Implications for how capping protein binds the actin filament. J. Biol. Chem. 282, 5871–5879. Kim, K., Yamashita, A., Wear, M. A., Maeda, Y., and Cooper, J. A. (2004). Capping protein binding to actin in yeast: Biochemical mechanism and physiological relevance. J. Cell Biol. 4, 567–580. Lappalainen, P., Kessels, M. M., Cope, M. J., and Drubin, D. G. (1998). The ADF homology (ADF-H) domain: A highly exploited actin-binding module. Mol. Biol. Cell 9, 1951–1959. Loisel, T. P., Boujemaa, R., Pantaloni, D., and Carlier, M. F. (1999). Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 6753, 613–616. Lynch, D. K., Winata, S. C., Lyons, R. J., Hughes, W. E., Lehrbach, G. M., Wasinger, V., Corthals, G., Cordwell, S., and Daly, R. J. (2003). A cortactin-CD2-associated protein (CD2AP) complex provides a novel link between epidermal growth factor receptor endocytosis and the actin cytoskeleton. J. Biol. Chem. 24, 21805–21813. Maruyama, K. (1966). Effect of beta-actinin on the particle length of F-actin. Biochim. Biophys. Acta 126, 389–398. Maruyama, K. (2002). beta-Actinin, Cap Z, connectin and titin: What’s in a name? Trends Biochem. Sci. 27, 264–266. Maruyama, K., Kimura, S., Ishi, T., Kuroda, M., and Ohashi, K. (1977). Beta-actinin, a regulatory protein of muscle. Purification, characterization and function. J. Biochem. (Tokyo) 81, 215–232.

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Maruyama, K., and Obinata, T. (1965). Presence of beta-actinin in the soluble fraction of the muscle cells of the chick embryo. J. Biochem. (Tokyo) 57, 575–577. Mejillano, M. R., Kojima, S., Applewhite, D. A., Gertler, F. B., Svitkina, T. M., and Borisy, G. G. (2004). Lamellipodial versus filopodial mode of the actin nanomachinery; pivotal role of the filament barbed end. Cell 3, 363–373. Miyoshi, T., Tsuji, T., Higashida, C., Hertzog, M., Fujita, A., Narumiya, S., Scita, G., and Watanabe, N. (2006). Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: Evidence of frequent filament severing. J. Cell Biol. 175, 947–955. Moore, J. K., Li, J., and Cooper, J. A. (2008). Dynactin function in mitotic spindle positioning. Traffic 9, 510–527. Nam, J. M., Onodera, Y., Mazaki, Y., Miyoshi, H., Hashimoto, S., and Sabe, H. (2007). CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery. EMBO J. 26, 647–656. Narita, A., and Maeda, Y. (2007). Molecular determination by electron microscopy of the actin filament end structure. J. Mol. Biol. 365, 480–501. Narita, A., Takeda, S., Yamashita, A., and Maeda, Y. (2006). Structural basis of actin filament capping at the barbed-end: A cryo-electron microscopy study. EMBO J. 25, 5626–5633. Nicholson-Dykstra, S., Higgs, H. N., and Harris, E. S. (2005). Actin dynamics: Growth from dendritic branches. Curr. Biol. 15, R346–357. Olsten, M. E., Canton, D. A., Zhang, C., Walton, P. A., and Litchfield, D. W. (2004). The Pleckstrin homology domain of CK2 interacting protein-1 is required for interactions and recruitment of protein kinase CK2 to the plasma membrane. J. Biol. Chem. 40, 42114–42127. Paavilainen, V. O., Hellman, M., Helfer, E., Bovellan, M., Annila, A., Carlier, M. F., Permi, P., and Lappalainen, P. (2007). Structural basis and evolutionary origin of actin filament capping by twinfilin. Proc. Natl. Acad. Sci. USA 104, 3113–3118. Palmgren, S., Vartiainen, M., and Lappalainen, P. (2002). Twinfilin, a molecular mailman for actin monomers. J. Cell Sci. Pt 5, 881–886. Pappas, C. T., Bhattacharya, N., Cooper, J. A., and Gregorio, C. C. (2008). Nebulin interacts with CapZ and regulates thin filament architecture with in the Z-disc. Mol. Biol. Cell (in press). Pavlov, D., Muhlrad, A., Cooper, J., Wear, M., and Reisler, E. (2007). Actin filament severing by cofilin. J. Mol. Biol. 365, 1350–1358. Remmert, K., Olszewski, T. E., Bowers, M. B., Dimitrova, M., Ginsburg, A., and Hammer, J. A., 3rd. (2004). CARMIL is a bona fide capping protein interactant. J. Biol. Chem. 4, 3068–3077. Remmert, K., Vullhorst, D., and Hinssen, H. (2000). In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein. Prot. Exp. Purif. 1, 11–19. Revenu, C., Courtois, M., Michelot, A., Sykes, C., Louvard, D., and Robine, S. (2007). Villin severing activity enhances actin-based motility in vivo. Mol. Biol. Cell 18, 827–838. Schafer, D. A., and Cooper, J. A. (1995). Control of actin assembly at filament ends. Annu. Rev. Cell Dev. Biol. 11, 497–518. Schafer, D. A., Hug, C., and Cooper, J. A. (1995). Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments. J. Cell Biol. 1, 61–70. Schafer, D. A., Korshunova, Y. O., Schroer, T. A., and Cooper, J. A. (1994). Differential localization and sequence analysis of capping protein b-subunit isoforms of vertebrates. J. Cell Biol. 2, 453–465. Schafer, D. A., Waddle, J. A., and Cooper, J. A. (1993). Localization of CapZ during myofibrillogenesis in cultured chicken muscle. Cell Motil. Cytoskeleton 4, 317–335. Schafer, D. A., Jennings, P. B., and Cooper, J. A. (1996). Dynamics of capping protein and actin assembly in vitro: Uncapping barbed ends by polyphosphoinositides. J. Cell Biol. 135, 169–179.

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Schroer, T. A. (2004). Dynactin. Annu. Rev. Cell Dev. Biol. 20, 759–779. Shih, N. Y., Li, J., Karpitskii, V., Nguyen, A., Dustin, M. L., Kanagawa, O., Miner, J. H., and Shaw, A. S. (1999). Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science 5438, 312–315. Sizonenko, G. I., Karpova, T. S., Gattermeir, D. J., and Cooper, J. A. (1996). Mutational analysis of capping protein function in Saccharomyces cerevisiae. Mol. Biol. Cell 1, 1–15. Soeno, Y., Abe, H., Kimura, S., Maruyama, K., and Obinata, T. (1998). Generation of functional beta-actinin (CapZ) in an E. coli expression system. J. Muscle Res. Cell Motil. 6, 639–646. Staiger, C. J., and Blanchoin, L. (2006). Actin dynamics: Old friends with new stories. Curr. Opin. Plant Biol. 9, 554–562. Taoka, M., Ichimura, T., Wakamiya-Tsuruta, A., Kubota, Y., Araki, T., Obinata, T., and Isobe, T. (2003). V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein. J. Biol. Chem. 278, 5864–5870. Uruno, T., Remmert, K., and Hammer, J. A. (2006). CARMIL is a potent capping protein antagonist: Identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments. J. Biol. Chem. 281, 10635–10650. Vitriol, E. A., Uetrecht, A. C., Shen, F., Jacobson, K., and Bear, J. E. (2007). Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins. Proc. Natl. Acad. Sci. USA 104, 6702–6707. von Bulow, M., Rackwitz, H. R., Zimbelmann, R., and Franke, W. W. (1997). CP b-3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head. Exp. Cell Res. 1, 216–224. Wear, M. A., and Cooper, J. A. (2004a). Capping protein binding to S100B: Implications for the tentacle model for capping the actin filament barbed end. J. Biol. Chem. 279, 14382–14390. Wear, M. A., and Cooper, J. A. (2004b). Capping protein: New insights into mechanism and regulation. Trends Biochem. Sci. 8, 418–428. Wear, M. A., Schafer, D. A., and Cooper, J. A. (2000). Actin dynamics: Assembly and disassembly of actin networks. Curr. Biol. 24, R891–R895. Wear, M. A., Yamashita, A., Kim, K., Maeda, Y., and Cooper, J. A. (2003). How capping protein binds the barbed end of the actin filament. Curr. Biol. 13, 1531–1537. Witt, C. C., Burkart, C., Labeit, D., McNabb, M., Wu, Y., Granzier, H., and Labeit, S. (2006). Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo. EMBO J. 25, 3843–3855. Xu, P., Mitchelhill, K. I., Kobe, B., Kemp, B. E., and Zot, H. G. (1997). The myosin-Ibinding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins. Proc. Natl. Acad. Sci. USA 8, 3685–3690. Xu, P., Zot, A. S., and Zot, H. G. (1995). Identification of Acan125 as a myosin-I-binding protein present with myosin-I on cellular organelles of Acanthamoeba. J. Biol. Chem. 43, 25316–25319. Yamashita, A., Mae´da, K., and Mae´da, Y. (2003). Crystal structure of CapZ: Structural basis for actin filament barbed end capping. EMBO J. 7, 1529–1538. Yang, C., Pring, M., Wear, M. A., Huang, M., Cooper, J. A., Svitkina, T. M., and Zigmond, S. H. (2005). Mammalian CARMIL inhibits actin filament capping by capping protein. Dev. Cell 2, 209–221. Zigmond, S. H., Evangelista, M., Boone, C., Yang, C., Dar, A. C., Sicheri, F., Forkey, J., and Pring, M. (2003). Formin leaky cap allows elongation in the presence of tight capping proteins. Curr. Biol. 20, 1820–1823.

C H A P T E R

F I V E

Effects of Environmental Estrogens and Antiandrogens on Endocrine Function, Gene Regulation, and Health in Fish Mary Ann Rempel* and Daniel Schlenk* Contents 1. Introduction 2. Mechanisms of Estrogenic and Antiandrogenic Effects 2.1. Steroid receptor 2.2. Steroid synthesis 2.3. Steroid distribution 2.4. Metabolic clearance 2.5. Hypothalamus-pituitary-gonad axis 2.6. Indirect mechanisms of estrogenic and antiandrogenic effects 3. Consequences of Impaired Reproductive Endocrine Function 3.1. Impaired gene regulation 3.2. Effects of unscheduled protein synthesis 3.3. DNA damage 3.4. Intersex/sex reversal 3.5. Reproductive failure 4. Summary and Concluding Remarks References

208 209 209 219 223 226 230 233 236 236 237 238 239 241 243 243

Abstract A number of studies have indicated widespread reproductive endocrine disruption in wild fish populations. A number of laboratory studies have been conducted to determine the sources and to elucidate potential mechanisms of the disruption. This review explores the varied mechanisms of estrogenic and antiandrogenic effects in fish including effects at the steroid receptor level, effects on steroid synthesis, distribution, and excretion, actions up the hypothalamuspituitary-gonad axis, as well as indirect mechanisms including thyroid and growth hormone disruption. Consequences of reproductive endocrine disruption

*

Department of Environmental Sciences, University of California, Riverside, CA 92521

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00605-9

#

2008 Elsevier Inc. All rights reserved.

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will be touched on including non-reproductive responses such as impaired gene regulation, effects of unscheduled protein synthesis and DNA damage, and reproductive responses such as intersex, sex reversal and reproductive failure. Key words: Endocrine function, Endocrine disruption, Environmental, Estrogen, Antiandrogen, Fish. ß 2008 Elsevier Inc.

1. Introduction In the 1990s, local fishermen in the United Kingdom noted the presence of hermaphroditic fish in lagoons receiving wastewater-treatmentplant effluent. The presence of hermaphroditic fish was confirmed in a follow-up survey, leading to speculation that there could be a link between the state of the fish and exposure to the effluent from sewage-treatment works (STWs) (Purdom et al., 1994). In order to test this link, Purdom et al. (1994) conducted a study in which caged male trout were placed in the sewage effluent where the hermaphroditic fish were observed, as well as at other STWs and several control locations. Exposure to sewage-treatment effluent, but not water at control locations, led to the induction of the synthesis of a female-specific protein, the egg-yolk-precursor protein vitellogenin, in male fish. Production of this protein was previously shown to be under the control of estrogens (Clemens, 1978). This was the first published study to demonstrate that exposure to exogenous estrogens in the environment could lead to endocrine disruption in fish. Since that time a number of studies were conducted in the United Kingdom demonstrating that endocrine disruption was widespread in wild fish populations. Wild roach (Rutilus rutilus) were sampled from upstream (where possible) and downstream of eight STWs as well as five reference locations. The occurrence of intersex, in which developing eggs are present in the testes, and vitellogenin induction was highest at locations downstream of the STWs, lower at the upstream sites, and lowest at the reference locations ( Jobling et al., 1998). Similar results were demonstrated in another freshwater species, the gudgeon (Gobio gobio), demonstrating that the phenomenon was not species specific (van Aerle et al., 2001). Endocrine disruption extended even to saltwater species. Flounder (Platichthys flesus) in United Kingdom estuaries were found to have increased levels of vitellogenin in fish from contaminated estuaries over fish from reference estuaries (Allen et al., 1999). In the most severely contaminated estuary vitellogenin levels in males were found to be as high or higher than levels in reproducing females, more than six-fold higher than found in control males. In the same area 17% of the male fish sampled had intersex gonads, whereas none were present in the reference fish.

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Endocrine disruption in wild fish populations is not exclusive to the United Kingdom. Estrogenic effects, including high levels of vitellogenin, intersex, depressed levels of testosterone and low gonadosomatic index, were demonstrated in carp (Cyprinus carpio) collected near a sewage outfall in the Ebro River in Spain (Lavado et al., 2004). Coho salmon (Oncorhynchus kisutch) in Lake Erie, United States, had depressed testosterone, 11-ketotestosterone (the major androgen in fish), cortisol, and gonadotropin concentrations in plasma, demonstrated a high rate (91%) of precocious sexual maturation, yet lacked development of secondary sexual characteristics in males (Leatherland, 1993). Studies in Hong Kong have indicated that abiotic factors such as hypoxia can also lead to endocrine disruption in fish (Wu et al., 2003). There has even been some speculation that fish residing in the open ocean may be experiencing endocrine disruption, perhaps through exposure to toxicants from the food chain (Scott et al., 2006). In response to the numerous studies reporting reproductive endocrine disruption in fish, researchers have put forth an effort to determine which compounds are reproductive endocrine disruptors and how they act. This review will provide an overview of the various mechanisms of action of estrogenic and antiandrogenic compounds, as well as provide specific examples of compounds that act in each manner. It will also summarize the varying consequences of reproductive endocrine disruption.

2. Mechanisms of Estrogenic and Antiandrogenic Effects The most studied paradigm of estrogenic action is one in which compounds that have a similar structure as the endogenous estrogen 17b-estradiol (E2) bind to its receptor(s) and elicit downstream effects. Estrogen mimicking however is only one of many mechanisms of action. Inhibition of androgens can lead to estrogenic effects. Compounds can affect steroid synthesis, distribution, and excretion. Actions higher up the hypothalamus-pituitarygonad (HPG) axis can elicit downstream, estrogenic effects. Compounds can also affect the reproductive endocrine axis indirectly through other axes. Each one of these mechanisms is explored in further detail below.

2.1. Steroid receptor 2.1.1. Estrogen receptors in fish Nuclear estrogen receptors (ER) have six functional domains, as illustrated in Figure 5.1. The A and B domains have ligand-independent transactivation function AF-1. The C domain is the DNA binding-domain and is also involved in dimerization. The E domain is the ligand-binding domain and is

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NH2

A

B

C

D

E

F

COOH

Figure 5.1 Generic structural domains of nuclear estrogen receptors.

involved in dimerization as well (Menuet et al., 2005). The D domain acts as a hinge between the C and E domains (Urushitani et al., 2003). The function of the F domain remains unclear at this time. However, data suggest that it might have a role in modulation of ER activity (Menuet et al., 2005). Once a ligand, such as E2, binds to the ER, the receptor dimerizes with another receptor, translocates to the nucleus, and binds to estrogen-response elements (ERE) in the promoter region of estrogen-responsive genes to regulate transcription. Teleosts, in similarity to mammals, have two main subtypes of ER, ERa, and ERb. Rainbow trout (Oncorhynchus mykiss) have been shown to possess two subtypes of ERa derived from separate genes, ERa1 and ERa2 (Nagler et al., 2007), as well as two isoforms derived from alternative splicing, ERa-long and ERa-short. ERa-long has a structure similar to that found in mammals. ERa-short appears to be truncated by 45 residues at its N-terminus, in the A domain (Pakdel et al., 2000). The A domain in the absence of a ligand interacts with the C-terminal region, leading to an inhibition of the AF1 activity of the B domain (Metivier et al., 2000). The truncation of the A domain removes this interaction, increasing the activity of the receptor in the absence of a ligand. The short form has a ligandindependent transcriptional activity representing 30% of the E2-induced activity (Pakdel et al., 2000). ERa-short is expressed only in the liver, whereas ERa-long is expressed in a wide variety of tissues, demonstrating a tissue-specific distribution of the two isoforms (Menuet et al., 2001). It is conjectured that the short form is involved in the sustained production of vitellogenin necessary during reproduction (Menuet et al., 2001). There are also two subtypes of ERb, ERba (also known as ERg) and ERbb. Although the two subtypes of ERb are transcribed from separate genes, they share a higher degree of amino acid homology with each other than they do with ERa. They are believed to have arisen from the duplication of an ancestral ERb gene early in the teleost lineage (Hawkins and Thomas, 2004). Studies performed in largemouth bass (Micropterus salmoides), and sea bream (Sparus auratus) have indicated differential expression of the two isoforms, with ERbb being more prominently expressed in the liver than ERba, even though both are equally present in the gonads, pituitary and the male kidney (Pinto et al., 2006; Sabo-Attwood et al., 2004). Distribution of the two subtypes in the brain of the Atlantic croaker (Micropogonias undulates) and zebrafish (Danio rerio) is also tissue-specific (Hawkins et al., 2005; Menuet et al., 2002). Variability in tissue distribution between subtypes and isoforms of ER might be linked to the pleiotropic effects of estrogens in the body.

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While most of the amino acids in the binding pocket of ERs that are responsible for ER-estrogen interactions are conserved, there are some key differences between species and subtypes. For example, in human ERa there is a leucine in the binding pocket that interacts with the A ring of E2. This leucine is changed to a methionine in fish, and this change is found in fourteen fish ERas to date (Hawkins and Thomas, 2004). In the channel catfish there are three amino acid substitutions between ERa and ERbb in positions known to be important for ligand binding (Pinto et al., 2006). There are two amino acid changes between ERa and ERba (Y526 to H495 and C530 to M499) that are conserved in nine fish ERs designated as ERba. But Y526 is changed to a serine, not a histidine, and C530 is changed to an arginine, not a methionine, in all teleost ERbb identified to date. C530 is conserved, however, between tetrapod ERa and ERb (Hawkins and Thomas, 2004). The variability in the amino acid sequence in the ligand-binding domain between species is believed to lead to variations observed in ligand binding affinity. There also appears to be differences in inherent activity between the short and long forms of ERa. Recently a membrane-bound, G-protein
coupled estrogen receptor has been identified in the gonads of croaker. Similar to GPR30 in mammals, it is thought to lead to the rapid reactions to steroid hormones observed in the body (Thomas et al., 2006). Also, estrogen receptor-related receptors (ERRs) have been characterized in the killifish (Fundulus heteroclitus) (Tarrant et al., 2006). While ERRs do not bind E2, their expression appears to be regulated by E2 to some extent. In particular, expression of ERRa in the female heart was downregulated upon exposure to E2. Taking all of the variability in binding and response to estrogens into account, it may be important to take into consideration the type of estrogen receptor and the species in which it resides when determining whether a compound is ‘‘estrogenic’’ by its ability to bind to the ER. 2.1.2. Common biomarkers of estrogen receptor activation Researchers use several methods to demonstrate activation of the estrogen receptor. The most direct measurements are receptor-binding assays using either hepatic cytosolic/nuclear extractions of estrogen binding sites or receptor clones (Denny et al., 2005; Latonnelle et al., 2002; Nimrod and Benson, 1997; Segner et al., 2003; Tollefsen et al., 2002; Urushitani et al., 2003). MCF-7, an estrogen-sensitive breast cancer cell line, is used for both ER binding and cell proliferation assays (Souttou et al., 1993; Vanderburg et al., 1992). There are also assays using cell lines with reporter genes linked to estrogen receptors. The yeast estrogen screen (YES) assay utilizes a yeast cell into which the gene for human ER and ERE tied to the reporter gene lac-Z have been incorporated (Routledge and Sumpter, 1996). The estrogen receptor-mediated, chemical activated luciferase reporter gene expression (ER-CALUX) assay was developed in the T47D breast cancer

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cell line (Legler et al., 1999). A luciferase reporter gene was also transfected into the MCF-7 cell line (Kramer et al., 1997). More popular but also more indirect assays measure the activation of genes or quantify the products of genes known to be under the control of the ER. The most common female-specific genes targeted in fish are the vitellogenin and choriogenin (egg envelope) genes in the liver (Hansen et al., 1998; Lee et al., 2002). The difficulty with this type of assay is the inability to determine whether the compound of interest is directly binding to the ER (an ER mimic) or eliciting effects through more indirect routes. 2.1.3. Estrogen mimics Estrogen mimics are compounds that bind to the ER and elicit similar, downstream effects as the endogenous estrogen itself. There are a wide variety of compounds that have been shown to bind to the ER, including the more obvious pharmaceuticals and estradiol metabolites, to the less obvious phytochemicals, surfactants, pesticides, plasticizers and even sunscreen agents. Structures of some representative estrogenic compounds are presented in Figure 5.2. Many estrogenic compounds contain a phenol ring, though some, like o,p0 -DDT may have a halogen in place of the hydroxyl group on the aromatic ring and others, like dibutyl phthalate, may have an ester linkage. Even if a parent compound does not have the proper structure to bind to the ER, degradation and/or metabolism of the parent compound might lead to the formation of a ligand, as exemplified by the hydroxylated polychlorinated biphenyls (PCBs) OH-PCB 30 and OH-PCB 61 (Carlson and Williams, 2001). Although a compound may have a structure that enables it to bind to the ER, it does not necessarily mean that the compound will activate the ER. It may actually act as an inhibitor of ER in some tissues, such as tamoxifen and its metabolite, 4-hydroxytamoxifen (Sasson, 1991). Relative binding affinities for various compounds to fish estrogen receptors are presented in Table 5.1. The table includes endogenous estrogens and metabolites (E2, estrone, estriol, estradiol glucoronide, estradiol sulfate), pharmaceutical estrogens (diethylstilbesterol, ethinylestradiol, mestranol), estrogen antagonists (tamoxifen, 4-hydroxytamoxifen, ICI 182, 780), phytochemicals (genistein, zearalenone, coumesterol, daidzein, equol, formononetin), surfactants (nonylphenol, tert-octylphenol, bisphenol A), pesticides and degradation products of pesticides (methoxychlor, demethylated methoxychlor, o,p0 -DDT, o,p0 -DDE, chlordecone), placticizers (dibutyl phthalate, di[2-ethylhexyl]halite, dicyclohexyl phthalate), a sunscreen agent (benzophenone), industrial waste (octachlorostyrene), and an antifouling agent (tributyltin chloride). Many of these compounds are present in the environment, and given the proper dose and sufficient uptake could bind to the ER and elicit downstream, feminizing effects. Though the ER shows some binding capacity for all of the compounds listed, there is a wide variation in the affinity of the ER for each compound. The structure of the

OH OH

OH CH3

CH3

CH3

CH3

N

H

H

H

O

HO

H

H H

CH

OH

O

H3C

O

OH

HO

HO

17b -Estradiol Endogenous estrogen

Ethinylestradiol Pharmaceutical estrogen

Genistein Phytochemical

4-hydroxytamoxifen Pharmaceutical antiestrogen

Cl Cl

CH3

HO

CH3

Cl Cl

CH3

H3C O

O

OH

O

H3C H3C

OH

O O

OH

HO

CH3

Cl

o,p⬘-DDT p-tert-Octylphenol Surfactant degradation product Organochlorine pesticide

Dibutyl phthalate Plasticizer

Benzophenone-2 Sunscreen agent

Figure 5.2 Chemical structure of representative compounds that have been demonstrated to bind to the estrogen receptor.

Table 5.I estradiol

Relative binding affinities for estrogen receptor in various fish species. All binding affinities are expressed as a percentage relative to 17b-

Chemical

17b-Estradiol Estrone Estriol Estradiol glucoronide Estradiol sulfate Diethylestilbesterol Ethinylestradiol Mestranol Tamoxifen 4-Hydroxytamoxifen ICI 182, 780 Genistein Zearlenone Coumesterol Daidzein Equol Formononetin Nonylphenol tert-Octophenol Bisphenol A

Rainbow trouta (Denny et al., 2005)

100 5.0 4.0

Rainbow troutb (Laton nelle et al., 2002)

Rainbow trout a (Tollefse n et al., 2002)

Fathead minnowa (Denny et al., 2005)

Siberian sturgeonb (Laton nelle et al., 2002)

Atlantic salmon a (Tollefse n et al., 2002)

100

100

100

100

100 3.9

0.1 <0.07

0.039

0.07 0.05

0.012

Carpa (Seg ner et al., 2003)

Channel catfisha (Nim rod and Benson, 1997)

100

100

100

5.2

179 88.9 0.34

6.1

1.7

70 1.2

390 84

583 166

28.4 490

4.2

0.02

1.4

8.5 2.3

3.6 2.5

204 55

94

90 516 5.4 0.31

25

1.75 0.077 0.13 2.69 0.03 0.009

Mummichogc (Urushitani et al., 2003)

0.48 0.70

3.5 0.006 0.06 0.5 0.001 0.01

0.13 0.013

0.012 0.020

0.42 0.65

0.049 0.010 0.12

Methoxychlor Demethylated Methoxychlor o, p0 -DDT o, p0 -DDE Chlordecone Dibutyl phthalate Di(2-ethylhexyl)halate Dicyclohexyl phthalate Benzophenone Octachlorostyrene Tributyltin chloride a b c

0.028

ER derived from hepatic cytosol extraction. ER derived from hepatic nuclear extraction. ERa clone.

0.010

0.0038

0.015 0.10 0.0011

0.012 0.005 0.027 0.010 0.017 0.014 0.0075 0.019 0.17

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compound, in particular its similarity to E2, plays a large role in determining the affinity. Despite this variation the interspecies and intraspecies (interlab) ER affinity for each compound is surprisingly consistent, in most cases varying by an order of magnitude or less. Differences may be due in part to the source of the ER used by the authors. The study by Urushitani et al. (2003) used an ERa clone, whereas other studies used either hepatic cytosol or nuclear extraction to derive the ER used in the assay. The extraction method may be providing several ER subtypes and/or isoforms, not just one form of ERa, and as shown earlier, subtypes and isoforms may have differences in ligand-binding pocket structure and thus different affinity for estrogenic compounds. This may be why the ERa clone seems to show higher affinity for the surfactants nonylphenol and tert-octophenol than the extracted ER used in the other studies (Denny et al., 2005; Nimrod and Benson, 1997; Tollefsen, 2002; Urushitani et al., 2003). 2.1.4. Androgen receptors in fish Much less is known about androgen receptors (AR) than ER in fish. The functional regions of the nuclear AR are similar to other nuclear receptors, with regions A and B involved in transactivation, region C acting as the DNA binding domain, and region E as the ligand binding domain (Olsson et al., 2005). Most vertebrates are believed to have one active form of nuclear AR with a high specificity for the androgen 5a-dihydrotestosterone (DHT), whereas there appear to be two subtypes of the AR in some teleosts, ARa and ARb, differentially expressed in tissues, with high affinities for either testosterone (T) or DHT (Olsson et al., 2005). Two forms have been isolated in the Japanese eel (Anguilla japonica) testes, Nile tilapia (Oreochromis niloticus) testes, brain and ovary of Atlantic croaker and rainbow trout, which appears to have only one active form (Ikeuchi et al., 2001; Pasmanik and Callard, 1988; Sperry and Thomas, 1999; Takeo and Yamashita, 1999). One form of nuclear AR has been isolated from goldfish (Carassius auratus), red sea bream (Pagrus major), Japanese medaka (Oryzias latipes) and three-spined stickleback (Gasterosteus aculeatus). There are two splice variants, ARb1 and ARb2 in the stickleback (Olsson et al., 2005; Pasmanik and Callard, 1988; Touhata et al., 1999). In addition, a membrane AR was isolated and characterized recently in the ovary of the Atlantic croaker (Braun and Thomas, 2004). Analyses of the structure of teleost ARs in comparison to the human AR seem to indicate that the amino acids important for ligand binding are well conserved. An amino acid thought to be important for the architecture of the AR, R779 in humans, however, is not conserved. Mutational assays have indicated that the replacement of this arginine results in complete inactivation of the AR; however, in teleosts, where both R779S and R779T are found, the AR is still functional (Olsson et al., 2005). Therefore, even

Estrogenic and Antiandrogenic Actions in Fish

217

though the ligand-binding domains are similar between teleosts and mammals, they are not identical. Most of the ARs that have been assessed for binding affinity have a higher affinity for DHT or T than the major endogenous androgen in fish, 11-ketotestosterone (11-KT) (Olsson et al., 2005; Wells and Van Der Kraak, 2000). However, when the three-spined stickleback ARb2 was assessed in reporter-gene transfection experiments in two different cell lines, even though 11-KT had a lower binding affinity it was more effective than DHT in activating the luciferase gene, indicating that 11-KT preferentially activates ARb2. Human AR, however, was not preferentially activated by 11-KT (Olsson et al., 2005). 2.1.5. Common biomarkers of androgen receptor interaction/suppression Similar assays are used in measurement of AR activation as are used in ER activation analysis, including binding affinity assays and reporter-gene assays (Bauer et al., 1998; Olsson et al., 2005; Wells and Van Der Kraak, 2000). The reporter-gene assays have the added benefit of providing information not just on binding to the receptor but also activation of the receptor, important for determining whether the compound is acting as an inhibitor or an activator. One gene in some teleosts that has been shown to be under control of 11-KT is the gene for spiggin, a protein produced by the kidney of male fish that acts as glue in nest building once released from the body. An assay has recently been developed for the effects of antiandrogens on the production of this male-specific protein in three-spined sticklebacks (Katsiadaki et al., 2006). However, from this type of assay, it is unclear whether the compound is acting directly through the androgen receptor or through another, indirect route. The same is true for measuring other, higher order effects such as effects on gonadal development. 2.1.6. Antiandrogens Most antiandrogens are believed to exert their effects by occupying the AR without activating it (there are other mechanisms that will be described later). The result is an inactivation of the receptor because the endogenous androgens are unable to bind to it, and this leads to a demasculinization effect in the fish, or feminization by default. Environmental antiandrogens that have been identified include p,p0 -DDE, a DDT degradation product; dicarboximide fungicides such as procymidone and the M1 and M2 metabolites of vinclozolin; linuron, a herbicide; and interestingly methoxyclor, demethylated methoxychlor, and o,p0 -DDT, which have also been shown to bind to the ER (see earlier) (Katsiadaki et al., 2006; Kiparissis et al., 2003; Wells and Van Der Kraak, 2000). Structures of some antiandrogens are presented in Figure 5.3, including the model antiandrogen, flutamide, used to treat prostate cancer. There seems to be some variation in the species and

OH CH3

O CH3

Cl

H

O

CH3

Cl

Cl

F

O

H3C H

H Cl

O

O

11-ketotestosterone Endogenous androgen

Procymidone Dicarboximide fungicide

F

O

N

CH3

Cl

Cl

p,p⬘-DDE Organochlorine pesticide (DDT) metabolite

F

N H

+

N

CH3

Flutamide Pharmaceutical antiandrogen

Figure 5.3 Chemical structures of endogenous fish androgen,11-ketotestosterone, and representative antiandrogens.

O

–

Estrogenic and Antiandrogenic Actions in Fish

219

tissues in which AR binding of the earlier antiandrogens occur. Vinclozolin and its metabolites and procymidone have been shown to bind to mammalian AR, but failed to bind to ARs extracted from the cytosolic fractions of rainbow trout brains and goldfish brains, ovaries and testes (Wells and Van Der Kraak, 2000). o,p0 -DDT, p,p0 -DDE, methoxychlor and demethylated methoxychlor did bind to goldfish testes AR in addition to mammalian AR. As there are differences in sequence homology between AR in different teleost species (Olsson et al., 2005), data from receptor binding assays from more species is needed to determine whether chemicals identified as antiandrogens in mammals indeed pose a threat to fish exposed in the wild.

2.2. Steroid synthesis 2.2.1. Reproductive steroidogenesis Sex steroids are primarily, but not exclusively, produced in the gonads, specifically the thecal and granulosa cells of the ovary and the leydig cells of the testis. The chief estrogenic product produced is E2, and the chief androgenic products are T and 11-KT. In mammals T is converted to a more potent form for activity, DHT. While fish are capable of producing DHT as well, its role in reproduction is currently unknown (Thibaut and Porte, 2004). Another steroid important in the final maturation process of both male and female gametes is 17a,20b-dihydroxypregn-4-en-3-one, also known as maturation inducing hormone (MIH). As shown in Figure 5.4, steroidogenesis is initiated via a signal from a pituitary hormone, either follicle-stimulating hormone (also known as gonadotropin I) or luteinizing hormone (also known as gonadotropin II). Once the gonadotropins bind to their receptor, a signal is sent via an adenosine 30 ,50 -cyclicmonophospate (cAMP) and protein kinase A (PKA) pathway, which triggers the release of free cholesterol from a cholesterol ester. Free cholesterol is then transported to the mitochondria via the steroidogenic acute regulatory protein (StAR), where the enzyme P450 side-chain-cleavage (P450scc) produces pregnenolone from cholesterol. Pregnenolone then diffuses back into the cytosol where a host of P450 enzymes work to produce the final active reproductive steroids. While it is unclear whether the entire reproductive steroidogenic pathway is present in extragonadal tissues, some key enzymes are present in multiple regions of the body. P450 aromatase (CYP19), the enzyme responsible for the conversion of T to E2, is present in two forms in fish. The CYP19A1 is present mainly in the gonads, and the CYP19A2 is present mainly in the brain, although both are found in other tissues as well (Piferrer and Blazquez, 2005). The presence of 11b-hydroxysteroid dehydrogenase (11b-HSD) is relatively widespread in fish tissues, albeit levels are highest in gonadal steroidogenic cells and head kidney interrenal cells (Kusakabe et al., 2003). The enzyme is responsible for conversion of 11b-hydroxytestosterone to

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Mary Ann Rempel and Daniel Schlenk

FSH, LH Gonadotropin receptor cAMP

ATP

Cholesterol Ester

Cholesterol Ester Hydrolase

PKA

H3C CH3

StAR CH3

H

H

O CH3

Free cholesterol H 3C CH3

CH3

3b-HSD

CH3

H

HO

P450scc on inner mitochondrial CH3 membrane

P450C17

HO

3b-HSD

CH3

CH3

O

CH3 H HO

17b-HSD

H

H

5a -reductase

P45011b OH CH3

HO

O H

H

5a-dihydrotestosterone

O

H

17b-estradiol

HO

g -HCH

OH CH3

O

H

11b-hydroxytestosterone

H

CH3

11b-HSD

H

CH3 H

H

H

Testosterone

CH3

CH3

H

H

O

H

OH

EE

P450 aromatase

H

OH

CH3

OH

H

CH3

H

Dicofol, DBP, DBT

EtOH, EE, NP, BaP, atrazine? CH3

Androstenediol

HO

O

Androstenedione

17b-HSD 3b-HSD

17a,20bdihydroxypregn-4-en-3-one

H

O

OH CH3 H

CH3

Η

O

H

CH3

Dehydroepiandrosterone

CH3 OH

Η

CH3 Η

CH3

H

20b-HSD

17ahydroxyprogesterone

P450C17

3b-HSD

OH

H

O

H

HO CH3

O

H

H

17ahydroxypregnenolone

P450C17

NP, dicofol, p,p⬘-DDE H3C CH3

H

H

Dicofol

Progesterone

P450C17 CH3

H

H

Η

O

OH O CH3

CH3

O

H

H

Pregnenolone

HO

CH3 CH3

H

H

O

11-ketotestosterone

Figure 5.4 Reproductive steroidogenic pathway in teleosts. Enzymes are labeled in red. Potential sites of action of estrogenic and anti-androgenic compounds (labeled in blue) are represented by lightning rods. White lightning rods indicate stimulation. Black lightning rods indicate inhibition. FSH, follicle-stimulating hormone; LH, luteinizing hormone; NP, nonylphenol; EtOH, ethanol; EE, ethinylestradiol; BaP, benzo(a) pyrene; DBP, di-n-butyl phthalate; DBT, dibutyltin; g-HCH, g-hexachlorocyclohexane.

11-KT, as well as the conversion of cortisol to cortisone. In some fish species it appears that the testes secrete 11b-hydroxytestosterone, which is later converted to 11-KT outside of the gonad (Young et al., 2005). There has also been some speculation that cortisol can be metabolized to 11-KT, providing a second function for 11b-HSD in the head kidney (Ozaki et al., 2006).

Estrogenic and Antiandrogenic Actions in Fish

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Successful reproduction requires proper timing and level of expression of the range of enzymes responsible for steroid biosynthesis, perhaps in more than one tissue. Improper inhibition or stimulation of enzymes can lead to an imbalance of steroid hormones, resulting in reproductive impairment. Impacts on steroidogenesis primarily by estrogenic or antiandrogenic compounds will be discussed here. 2.2.2. Common biomarkers of steroidogenic impairment Measurement of impairment of steroidogenic enzymes can include quantifying the level of expression, or mRNA levels, for a specific enzyme in the tissue of interest (Hecker et al., 2005; Kazeto et al., 2004). One can also measure the activity of an enzyme of interest by measuring the amount of its substrate converted per unit time per amount of protein (Hecker et al., 2005; Kim et al., 2003; Thibaut and Porte, 2004). Reporter-gene assays have also been employed (Fan et al., 2007). Steroid levels in plasma or tissues and ratios of estrogens to androgens are also commonly measured to confirm in vivo correlation to the enzyme assays (Hecker et al., 2005; Kim et al., 2003). 2.2.3. Steroidogenic impairment linked to estrogenic/ antiandrogenic effects A sampling of enzymes demonstrated to be impacted by estrogenic or antiandrogenic compounds is shown in Figure 5.4 and described in more detail below. It is believed that E2 production is regulated at least in part by regulating CYP19 expression at the transcriptional level (Young et al., 2005). CYP19A2 expression has been linked to sexual differentiation, and suppression may be necessary for proper testicular differentiation (Piferrer and Blazquez, 2005). CYP19A1 mRNA is detected in ovaries during ovarian maturation, when both E2 and vitellogenin levels are high, and cannot be detected at other times. Stimulation of CYP19 by exogenous compounds could lead to varying results in fish depending on the isoform affected and the stage of development, from sex reversal and impaired sexual differentiation to production of vitellogenin in males. The pharmaceutical estrogen ethinylestradiol and the surfactant nonylphenol have been demonstrated to stimulate CYP19A2 expression in zebrafish (Kazeto et al., 2004). In contrast, ethinylestradiol was shown to inhibit CYP19A1, while nonylphenol had no effect (Kazeto et al., 2004). The endogenous estrogen E2 upregulates CYP19A2 (Young et al., 2005). Half and full sites for the ERE have been found in the regulatory region of the CYP19A1 and CYP19A2 genes, respectively, in several fish species. In addition, there has been some speculation that posttranscriptional regulation of the CYP19 genes may also be performed by estrogens (Piferrer and Blazquez, 2005). As ethinylestradiol and nonylphenol are known to bind

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to the ER (see earlier), it is possible that their modulation of CYP19 may be through activation of the ER. Interestingly, benzo(a)pyrene, a polycyclic aromatic hydrocarbon not generally thought of as a xenoestrogen, also simulated CYP19A2 expression in zebrafish (Kazeto et al., 2004). An aryl hydrocarbon receptor/aryl hydrocarbon nuclear translocator (AhR/ARNT) response element has been found in the regulatory region of the CYP19A1 gene in zebrafish, but not the CYP19A2 gene. Therefore, benzo(a)pyrene does not appear to be affecting CYP19A2 through binding of the AhR (Piferrer and Blazquez, 2005). However, at high concentrations, it is possible that benzo(a)pyrene and its metabolites may act through the ER (Kazeto et al., 2004). Ethanol has also been shown to increase expression of CYP19 as well as shift the ratio of estrogens
androgens toward estrogens in tilapia (Oreochromis mossambicus) (Kim et al., 2003). This has interesting implications in toxicity testing, as ethanol is commonly used as a solvent for estrogens in aquatic assays and therefore might affect estrogenic endpoints. Atrazine, a ubiquitous herbicide, has been implicated in the demasculinization of frogs, primarily through a decrease in T and an increase in E2 (Hayes et al., 2006; Hecker et al., 2005). Similar effects on steroid levels have been demonstrated in goldfish (Spano` et al., 2004). It has been postulated that stimulation of CYP19 by atrazine is the cause of the shift in steroid ratios (Hayes et al., 2006). However, support of this hypothesis has been spotty in fish and in other species (Fan et al., 2007; Hecker et al., 2005; Kazeto et al., 2004). Recently it was demonstrated that atrazine will bind to the orphan nuclear receptor, steroidogenic factor 1 (SF-1). Atrazine only seems to affect CYP19 expression in human cancer cell lines that express SF-1 and utilize an SF-1
dependent promoter, ArPII (Fan et al., 2007). Therefore it appears that atrazine may affect CYP19 indirectly, through SF-1. Response elements for SF-1 are present in the regulatory region of the genes for both isoforms of CYP19 in several fish species (Piferrer and Blazquez, 2005). However, it is unclear whether SF-1 is consistently coexpressed with CYP19. Therefore, the variability in response upon exposure to atrazine may be influenced by SF-1 levels in cells. 20b-Hydroxysteroid dehydrogenase (20b-HSD) is the enzyme responsible for producing MIH from 17a-hydroxyprogesterone. An increase in the activity of 20b-HSD is triggered by a luteinizing hormone surge during the final maturation stage of reproduction in both male and female fish. In females an increase in MIH is usually accompanied by a decrease in E2 synthesis. The shift toward MIH production causes a resumption of meiosis in the oocytes followed by ovulation. In males a similar decrease in T and 11-KT accompanies an increase in MIH. Upon this signal the gonads transition from spermatogenesis to spermiation. The reproductive repercussions of an unscheduled shift from the production of E2, 11-KT and testosterone to MIH are unclear and merit further research.

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The estrogenic compounds nonylphenol and dicofol (an organochlorine miticide) and the antiandrogenic compound p,p0 -DDE have been shown to stimulate the enzyme 20b-HSD and increase the production of MIH. In addition the androgenic compounds di-n-butyl phthalate (a placticizer) and fenarimol (a fungicide) also had a similar effect (Thibaut and Porte, 2004). Dicofol also had an inhibitory effect on the activity of 17b-HSD and the 5a-reductase (Thibaut and Porte, 2004). 17b-HSD converts dehydroepiandrosterone to androstenediol and also androstenedione to T. Inhibition of 17b-HSD could lead to decreased T, and perhaps demasculinization. 5aReductase is responsible for the production of DHT from T. DHT is generally more potent at the AR than T, therefore inhibition of the formation of DHT could lead to demasculinization. Although fish can produce DHT, it has not been shown that it is a major androgen in teleosts. Therefore, the implications of the inhibition of 5a-reductase in fish are unclear. Two known androgens, di-n-butyl phthalate and dibutyltin, also were shown to inhibit production of DHT by 5a-reductase, further clouding the issue (Thibaut and Porte, 2004). The organochlorine pesticide g-hexachlorocyclohexane (lindane) was shown to increase concentrations of T but decrease concentrations of 11-KT in goldfish testes (Kime and Singh, 1996). The pattern was seen regardless of whether endogenous or exogenous precursor was used, and led the authors to believe that the decreased level of 11-KT was likely due to a decrease in conversion from 11-hydroxytestosterone, indicating inhibition of 11b-HSD. 11-KT plays an important role in spermatogenesis and also perhaps sexual differentiation (Bhandari et al., 2006). Thus, decreases in 11-KT could lead to demasculinization of fish.

2.3. Steroid distribution 2.3.1. Sex steroid binding protein Sex steroid hormones, both estrogens and androgens, are transported in the plasma of many species bound to a carrier protein, or sex steroid binding protein (SBP). SBPs are thought to have several roles in fish, including protection of steroids from rapid metabolic clearance, modulation of steroid availability and uptake by target tissues, and control of rates of release and uptake across gills (Scott et al., 2005). In mammals unbound SBP binds to cell-surface receptors. Once bound SBP accepts a steroid ligand, which then activates a cellular response through cAMP pathways (Hryb et al., 1990; Nakhla et al., 1999). It is unknown, however, whether SBP serves the same function in fish. SBP is a glycoprotein that forms a homodimer, both units of which likely contain a binding site (Miguel-Queralt et al., 2004). In zebrafish, SBP could first be detected in 5- to 6-day-old larvae. It was detected in the liver and gut in larvae, and also in the testes in small amounts in the adults.

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Even though the liver is the main organ responsible for the synthesis of SBP, it was not readily detected in hepatocytes, most likely due to rapid secretion (Miguel-Queralt et al., 2004). There appears to be little difference in the levels of SBP in males and unreproductive females. However, in some species the binding capacity increases in vitellogenic females, purportedly to help maintain the high levels of estradiol necessary for vitellogenesis (Hobby et al., 2000). 2.3.2. Measurement of xenoestrogen binding to sex steroid binding protein To determine the potential for exongenous chemicals to interact with SBP, binding affinity assays are performed, similar to measurement of nuclear receptor interaction. The assays are usually performed on charcoal-treated plasma to remove endogenous steroids that may interfere with the assay. Both the disassociation constant, Kd, and the maximum binding capacity, Bmax, can be calculated. The disassociation constant gives a measurement of binding affinity, and the Bmax gives a measurement of effects on the concentration of SBG. It does not appear to date that exogenous chemicals have a marked impact on Bmax. Therefore that endpoint will not be discussed further in this paper. Relative binding affinities are calculated from the concentration of substrate required to displace 50% of bound estradiol, or the IC50 (Kloas et al., 2000; Tollefsen, 2002; Tollefsen et al., 2004). They can also be calculated by directly comparing inhibition constants (Ki) between estradiol and the compound of interest (Gale et al., 2004). 2.3.2.1. Steroid distribution and estrogenic effects The role of SBP in the endocrine system can have several impacts on the effects of endocrine disrupting compounds in fish. A study performed on Tinca tinca found that the rate of uptake of steroids from water was not only influenced by their concentration in the water versus the body, and their hydrophobicity, but also their binding affinities for SBP (Scott et al., 2005). SBP may also alter the diffusion gradient for endocrine disrupting compounds, increasing their bioconcentration. Once in the system, the amount of free versus bound compound will be a factor of both the relative binding affinity and the relative concentration compared to endogenous steroids. If the concentration or relative binding affinity is high for the compound, it could displace endogenous steroids, making them more available to exert their actions. If the compound is carried to a location or tissue in which the level of steroids is high (e.g., gonads), the steroids could displace the compound, leaving it free to exert its actions on the local tissue; a sort of shuttling effect (Tollefsen, 2002). Relative binding affinities for endogenous steroids and estrogenic compounds to SBP of four fish species are listed in Table 5.2. There is variation, sometimes marked, between the binding affinities of a given compound in

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Estrogenic and Antiandrogenic Actions in Fish

Table 5.2 Relative binding affinities (%) for endogenous steroids and estrogen mimics to steroid-binding proteins in plasma of various fish species. All binding affinities relative to 17b-estradiol

Compound

Common carp (Kloas et al., 2000)

Steroids 17b-Estradiol 100 Testosterone 104 11-Ketotestosterone 51 Progesterone 198 Pharmaceuticals Ethinylestradiol Diethylstilbestrol Phytochemicals Genistein Zearalenone Industrial Chemicals 4-Nonylphenol 0.6 4-Octylphenol 0.32 Bisphenol A 0.29 n-Butyl Benzyl Phthalate Pesticides o,p0 -DDT Endosulfan

Rainbow trout (Tollefsen, 2002)

Arctic charr Channel catfish (Gale (Tollefsen et al., 2004) et al., 2004)

100 86.6 13.3 0.22

100

100

0.77 0.014

307

0.25 0.035

0.018 0.017

0.0051 0.0018 0.00015

0.0012 0.018 0.03 0.1

0.0074 0.0091

0.00056 0.02

different fish (e.g., ethinylestradiol). As the Tollefsen studies with rainbow trout and Arctic charr have low variation in binding affinities, it is more likely that the differences are due to interlaboratory variability rather than species differences. Also noticeable is the discrepancy between the binding affinity of a compound to the ER versus SBP. The binding affinity of diethylstilbestrol to both the ER and SBP was measured in rainbow trout by the Tollefsen group. The binding affinity to the ER (relative to E2) was 390%, versus 0.014% for SBP (Tollefsen, 2002; Tollefsen et al., 2002). It appears that the binding properties between nuclear receptors and SBP are quite different, and binding affinities cannot be inferred between the two. In general, it appears that the exogenous compounds tested have much lower affinity for SBP than the endogenous steroids. Therefore, it may be unlikely, unless concentrations are very high, that the xenoestrogens exert their effects by displacing steroids from SBP and making them more

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bioavailable. It is more likely that if effects are occurring through SBP, it is by aiding in bioconcentration of estrogenic compounds from water, and by shuttling those compounds to areas of high steroid concentration, such as gonadal tissue (Kloas et al., 2000).

2.4. Metabolic clearance 2.4.1. Steroid metabolism Metabolism of steroids involves phase I and phase II metabolism. Phase I metabolism is catalyzed by P450 enzymes and most often results in the hydroxylation of the steroid, rendering it more polar. Phase II metabolism is catalyzed by transferases and results in the conjugation of the steroid with groups such as glucuronic acid and sulfates. Phase I and II reactions may occur separately or in conjunction and result in the efficient elimination of the steroid. Testosterone phase I reaction products in fish include 6a/b-, 16a/b-, 2b-, 15a/b-, and 12b-hydroxytestosterone (OHT) and androstenedione (Arukwe et al., 1997; Baldwin et al., 2005; Hasselberg et al., 2004; Kime, 1980; Kime and Saksena, 1980; Smeets et al., 2002). The major phase I reaction products are 6b-OHT, produced primarily by CYP3A and CYP2K, 16b-OHT, produced primarily by CYP3A, CYP2K and CYP1A, and androstenedione (Arukwe et al., 1997; Baldwin et al., 2005; Smeets et al., 2002). Phase II products are conjugations with glucuronic acid, either of the phase I metabolites or of unmetabolized T, the conjugation performed by the enzyme UDP-glucuronosyltransferase (Kime, 1980; Kime and Saksena, 1980; Thibaut and Porte, 2004). Free T may also be eliminated. 11-KT is eliminated in an unconjugated or conjugated (with glucuronic acid) form (Kime, 1980). Estradiol phase I reaction products in fish include 2-, 4-, 16b-, 7b-, and 6a/b-hydroxyestradiol (OHE2), and estrone (Butala et al., 2004; Hansson and Rafter, 1983; Willett et al., 2006). The major phase I reaction products are 2-OHE2, produced primarily by CYP3A and CYP1A, and estrone (Butala et al., 2004; Lin et al., 2002). 4-OHE2, a genotoxic metabolite of E2, is primarily produced by CYP1B (Willett et al., 2006). Phase II reaction products include conjugations of E2 with glucuronic acid and sulfates, utilizing UDP-glucuronosyltransferase or sulfotransferase (Thibaut and Porte, 2004). Catechol-O-methyltransferase, which adds a methyl group to 2-OHE2 and 4-OHE2, is present but its activity is low, at least in the ovary of the catfish (Heteropneustes fossilis) (Mishra and Joy, 2006). The clearance of steroids is dependent upon the level of expression and activity of phase I and II enzymes, and modulation of these enzymes by some xenoestrogens is thought to lead to some of the estrogenic effects observed in fish.

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2.4.2. Common biomarkers of steroid metabolism impairment Measurement of steroid metabolism impairment uses many of the same methods as measuring effects on steroidogenesis. Researchers have measured both mRNA and protein levels for enzymes in tissues (Arukwe et al., 1997; Baldwin et al., 2005; Mortensen et al., 2006; Navas and Segner, 2001; Willett et al., 2006). Researchers also measure the activity of enzymes in the presence of varying concentrations of inhibitor or activator by measuring the level of product produced when a given substrate is added. Common assays are T biotransformation and 7-ethoxy-O-deethylase (EROD) activity. These assays can either be run with tissue extracts (liver and gonad) or with enzymes expressed in bacteria (Arukwe et al., 1997; Hasselberg et al., 2004; Lin et al., 2002; Smeets et al., 2002; Thibaut and Porte, 2004). Exposure to the inhibitor or activator of enzyme activity can either be in vitro or in vivo. Analysis of inhibitor/enzyme complexes has also been carried out to determine the type of inhibition occurring (Lin et al., 2002). Levels of steroid hormones and products of metabolism in vivo have also been measured as confirmation of effect on enzymes under varying conditions (Arukwe et al., 1997; Kime, 1980; Kime and Saksena, 1980). 2.4.3. Impairment of steroid metabolism linked to estrogenic compounds Modulation of CYP1A expression and activity by estrogenic compounds has been demonstrated by several groups. Mortensen et al. (2006) examined effects on CYP1A1 expression in Atlantic salmon hepatocytes upon exposure to ethinylestradiol and hydroxylated PCBs. Ethinylestradiol decreased expression of CYP1A1 with doses of 0.01 to 0.1 mM, but at a concentration of 1 mM expression of CYP1A1 rose again, although not reaching control levels. A similar pattern was seen for the 4-hydroxy form of the PCB congener 187 (4-OH-PCB 187), where concentrations of 0.06 to 0.6 nM suppressed CYP1A1 but 60 nM significantly elevated CYP1A1 expression levels. The hydroxlated metabolites of congeners 107, 138 and 146 supressed CYP1A1 expression over all treatment levels. Navas and Senger (2001) noted a decrease in basal levels of CYP1A mRNA and CYP1A activity (measured by EROD) upon exposure to 0.01 to 1 mM E2 in rainbow trout hepatocytes. If the ER antagonist tamoxifen was added, it mitigated the effect of E2, indicating an ER-mediated effect of E2 on CYP1A. If the CYP1A was induced by the AhR agonist b-napthoflavone, the effect of E2 was also mitigated, which led the authors to believe that the effect of E2 was not through an ER-AhR interaction. It is possible that ethinylestradiol and the hydroxylated PCBs are acting through a similar mechanism as E2, at least over low doses. There is some evidence that ethinylestradiol also acts as a suicide inhibitor of CYP3A (see later) (Lin et al., 2002), if the same is occurring for CYP1A the result may be an increase in expression over high doses to compensate for the loss of active enzyme.

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Alkylphenols, surfactant degradates purported to be estrogenic, increased expression of CYP1A in male Atlantic cod, but had no effect on enzyme activity in vivo (Hasselberg et al., 2004). In females there was no change in expression of CYP1A, but there was a significant albeit transient increase in enzyme activity, indicating a potential difference in response between sexes. In vitro the alkylphenols inhibited enzyme activity, and the strength of inhibition increased with the length of the alkyl chain (Hasselberg et al., 2004). As CYP1A metabolizes both E2 and T, modulation of CYP1A by estrogenic chemicals may be thought to affect circulating levels of active hormones. Induction of CYP1A however had little effect on T metabolism in flounder (Platychthis flesus) or dab (Limanda limanda) hepatocyctes, which may indicate that CYP1A is a minor enzyme in T metabolism in fish and effects on CYP1A may not effect circulating T levels significantly (Smeets et al., 2002). CYPIA may have effects on E2 levels, however. Treatment of rainbow trout liver cells with CYP1A inducers decreased vitellogenin induction upon administration of E2, although whether the response was due to increased E2 clearance was in question (Anderson et al., 1996). Treatment of channel catfish (Ictalurus punctatus) microsomes with the CYP1A inducer benzo(a)pyrene did however increase the concentrations of 2- and 4-OHE2 (Butala et al., 2004). This may indicate that suppression of CYP1A expression and activity by the estrogenic chemicals described earlier decreases metabolism of E2, thereby increasing the amount of the active hormone in circulation, adding to any estrogenic effect. In addition to suppression, induction of CYP1A may also have estrogenic implications. Methoxychlor, an organochlorine pesticide, is believed to exert its estrogenic effects primarily through its demethylated metabolites. The metabolites are believed to be produced by CYP1 and CYP3 enzymes (Stuchal et al., 2006). Therefore induction of CYP1A may increase the estrogenicity of methoxychlor. In addition to CYP1A induction, benzo(a)pyrene exposure increased levels of CYP1B mRNA in the liver, gonad and blood of channel catfish (Willett et al., 2006). The co-induction of CYP1A and CYP1B altered the ratio of 2- and 4-OHE2 toward more 4-OHE2, the genotoxic metabolite, which could have implications on fish health (Butala et al., 2004). CYP3A and CYP2K are believed to be the most active P450 enzymes involved in steroid metabolism (Arukwe et al., 1997). Nonylphenol treatment of winter flounder (Pleuronectes americanus) produced an increase in 6b-OHT (nonsignificant) and 16b-OHT (significant) levels, both products of CYP3A and CYP2K (Baldwin et al., 2005). There was also a corresponding significant increase in hepatic CYP3A protein levels. CYP2K protein was not measured. Nonylphenol has been shown to bind to the pregnane-X-receptor (PXR) to induce CYP3A in rodents (Masuyama et al., 2000). CYP2 enzymes are activated by the constitutive

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androstane receptor (CAR) in mammals, but CAR has not been identified in fish. However, zebrafish PXR has similarities to CAR, and it was suggested that fish PXR is a precursor to both CAR and PXR in mammals (Moore et al., 2002). If this is the case, CYP2K may be activated by fish PXR, as is CYP3A. A dose-dependent increase in CYP3A protein levels in male Atlantic cod was also observed following exposure to alkylphenols (Hasselberg et al., 2004). However, Arukwe et al. (1997) found a different response in Atlantic salmon exposed to nonylphenol. At a dose of 1 mg/kg there was an increase in 6b-hydroxylation, as in the Baldwin study, and also a decrease in plasma E2 levels, indicating an increase in steroid clearance. However, there was no effect on CYP2K and CYP3A-like protein levels. At a dose of 125 mg/kg, there was a decrease in 6b-hydroxylation activity, no effect on plasma E2 levels, and a decrease in CYP2K and CYP3A-like protein levels, indicating inhibition on some level, not stimulation. The high dose in the Arukwe experiment was similar to the dose used in the Baldwin experiment (125 mg/kg body weight versus 100 mg/kg body weight) but the Arukwe experiment lasted 14 days, whereas the Baldwin experiment lasted 2 days. Therefore, the difference in response may be due to the duration of the exposure. It is possible that nonylphenol regulates CYP3A expression not only through PXR but also other receptor coactivators/ repressors, and the interaction between regulatory pathways is dependent on concentration and/or duration of exposure (Meucci and Arukwe, 2006). Ethinylestradiol, in contrast to nonylphenol, strongly decreased expression of CYP3A at 0.01 mM (Mortensen et al., 2006). The effect, while still significant, was weaker with increasing dose. The loss of effect at higher doses may be due to compensatory increases in transcription of the enzyme, because ethinylestradiol has also been shown to be a mechanism-based irreversible (suicide) inactivator of CYP3A, albeit in humans (Lin et al., 2002). The metabolite of ethinylestradiol covalently modifies the apoprotein, and also modifies and/or destroys the heme moiety. This demonstrates two very different effects by two xenoestrogens on phase I steroid metabolism enzymes. There appears to be a general inhibition of phase II enzymes by endocrine disrupting chemicals. Estrogenic chemicals including hydroxylated PCBs, ethinylestradiol, nonylphenol, and dicofol all decreased glucuronidation of T and/or E2 (Mortensen et al., 2006; Thibaut and Porte, 2004). In addition androgenic chemicals such as triphenyltin, tributyltin, and fenarimol had similar effects. Nonylphenol, triphenyltin, and tributyltin also decreased sulfation of estradiol (Thibaut and Porte, 2004). Temperature appears to increase the production of glucuronidated T and 11-KT, as well as the production of unconjugated 11-KT, in goldfish (Kime, 1980). This may have interesting implications on the role of temperature in sexual differentiation of fish.

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2.5. Hypothalamus-pituitary-gonad axis 2.5.1. Description of the hypothalamus-pituitary-gonad axis While reproduction is largely under the control of reproductive steroids, the production and release of those steroids is under the regulation of a suite of neurotransmitters and hormones that make up the hypothalamuspituitary-gonad (HPG) axis (Trudeau, 1997; Weltzein et al., 2004). In response to external and internal stimuli, neurotransmitters are released into the preoptic area of the hypothalamus. Dopamine, serotonin, GABA, and neuropeptide Y all appear to be involved in this signal transduction, dopamine as an inhibitor, and serotonin, GABA, and neuropeptide Y as stimulants (Corio et al., 1991; Khan and Thomas, 1993; Trudeau, 1997; Yu et al., 1991). Some of the neurotransmitters also act directly on the pituitary. Upon stimulation the decapeptide gonadotropin-releasing hormone (GnRH) is released from the hypothalamus. The neurons that produce GnRH directly innervate the proximal pars distalis region of the pituitary, releasing GnRH to bind to G-protein
coupled receptors on the surface of gonadotropic cells. The gonadotropin hormones subsequently released are either LH or FSH, each of which is produced in a distinct cell type. The gonadotropins consist of two subunits, an a subunit which they share, and a distinct b subunit. The released gonadotropins are carried through the bloodstream to the gonads. The hormones bind to G-protein
coupled receptors on the surface of gonadal cells. Both LH and FSH can bind to GTH-R1 (FSH) receptors located on Sertoli cells in the testes and thecal and granulosa cells in the ovaries. Only LH can bind to the GTH-R2 (LH) receptors that appear on Leydig cells of the testes and granulosa cells of the ovary during the final stages of gamete maturation ( Janz and Weber, 2000; Weltzein et al., 2004). FSH appears to play an active role in early vitellogenesis and spermatogenesis, most likely through stimulation of steroidogenesis and sertoli cell proliferation in the gonadal cells. LH is also involved in early steroidogenesis. Just prior to spawning there is a shift in the production of steroids, leading to a decrease in estrogens and androgens and an increase in MIH. This shift corresponds to an increase in LH and LH receptor levels, although the actual role of LH in the shift is unclear ( Janz and Weber, 2000). Steroid hormones can affect all levels of the HPG axis through feedback loops. The effects of neuropeptide Y on GnRH and the gonadotropins is potentiated by T and E2 (Trudeau, 1997). The effects of GABA are enhanced by T and inhibited by E2 in reproductively active fish. However the opposite is true in regressed fish. T, through aromatization to E2, increases GnRH responsiveness in the pituitary. 17a,20b-Dihydroxypregn-4-en-3-one, which not only acts as MIH but also as a potent pheromone in some fish species, stimulates GnRH neurons to cause a release of LH, and decreases

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pituitary dopamine turnover. Pituitary dopamine turnover is enhanced, however, by T and E2 in regressed fish (Trudeau, 1997). It appears that the feedback loops of steroids are likely mediated to some extent through nuclear receptors. ARa, ARb, ERba and ERbb have all been found in GnRH-producing preoptic area neurons in fish (Harbott et al., 2007; Hawkins et al., 2005). ARa, ARb, ERa, ERbb, and, to a lesser extent, ERba appear to be present in the pituitary as well (Choi and Habibi, 2003; Harbott et al., 2007). E2 increases ER mRNA in the preoptic area, and several putative hormone response elements have been found for GnRH. Therefore, it appears that steroids may cause feedback to some extent through effects on transcription (Harbott et al., 2007; Salbert et al., 1993). Steroids can also regulate pituitary hormone secretion though the stabilization of the mRNA of peptide hormones. Stabilization of mRNA increases the half-life of mRNA in cells, sometimes on the order of days, which in turn increases translation rates, and therefore protein levels. Progesterone stabilizes the mRNA of the b subunit of LH, and this stabilization is augmented by estrogen treatment (Ing, 2005). Androgens have been shown to stabilize the mRNA of the b subunit of FSH. Estradiol also stabilizes the mRNA of its own receptor. Thus, there could be multiple modes of regulation occurring in the same cell. 2.5.2. Estrogenic and antiandrogenic effects on the hypothalamus-pituitary-gonad axis Given the multiple feedback loops in steroid endocrinology, only a few of which were described here, the HPG axis appears more like a web, with multiple inducers and inhibitors acting on multiple levels to finely tune the reproductive process in fish. It is clear that any disturbance in one part of the axis is likely to cause effects on other parts, both upstream and downstream. Endocrine disruptors have been shown to cause effects on all levels of the HPG axis, and for some a clear path down the axis ultimately to reproductive effects has been demonstrated. The actions of a few endocrine disruptors are described below. The antidepressant mianaserin is a selective serotonin reuptake inhibitor (SSRI). It is an antagonist for serotonin receptors, and by blocking reuptake increases the effects of serotonin. Effects of mianaserin on zebrafish were evaluated using an oligo microarray. In the gonads of both sexes there was upregulation of genes associated with egg and embryo development, including genes encoding vitellogenin and zona pellucida glycoproteins, which are necessary for egg envelope formation. In the brain of males there was also upregulation of the same category of genes, at both 2 and 14 days. In the females at 2 days there were more genes down regulated than upregulated in this category, but at 14 days the opposite was true. The overall magnitude of response was greater in the males than in the females (Van der Ven et al., 2006a). This may indicate sex differences in the

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response of zebrafish to mianaserin, which could perhaps be brought about by differences in the feedback loops of each sex. LH was found to be downregulated in female brains in this study, which is in contrast with another study in which serotonin was shown to increase LH secretion, albeit in another species (Khan and Thomas, 1992). The effects of PCBs on the HPG axis have been well studied in Atlantic croaker, from the central nervous system neurotransmitters down through the hypothalamus and pituitary, with effects demonstrated in the gonad as well. In male Atlantic croaker, PCB 77, a coplanar congener present in the Aroclor 1254 mixture, was shown to decrease tryptophan hydroxylase activity, the rate-limiting enzyme in serotonin synthesis. Two non-coplanar congeners (PCB 47 and PCB 153) also present in Aroclor 1254 had no effect (Khan and Thomas, 2006). Aroclor 1254 decreased GnRH content in the preoptic area of the hypothalamus and also decreased GnRH receptors in the pituitary. The decrease was concomitant with a decrease in LH secretion (Khan et al., 2001). The male fish were exposed during their gonadal maturation stage, starting at the early-recrudescing stage and ending 30 days later when the controls were spermiating. The PCB-treated fish had hormone levels similar to those of early-recrudescing fish, indicating a possible inhibition of sexual maturation, which indeed appeared to be the case as the gonadosomatic index was significantly lower in the PCB-treated fish. It appears that the PCB mixture Aroclor 1254, through the actions of its coplanar congeners, inhibits serotonin synthesis, decreasing GnRH signaling, which in turn decreases LH secretion, which seems to ultimately have an effect on gonadal maturation in male Atlantic croaker. Nonylphenol has been mentioned to have effects in almost every category described thus far, from receptor interactions to steroid metabolism. Modulation of hormones up the HPG axis has been demonstrated as well. In juvenile Atlantic salmon both nonylphenol and estradiol increased mRNA levels of the b subunit of LH in females, but not in males, most likely because the basal levels were already high (Yadetie and Male, 2002). There were corresponding increases in zona radiata (an egg envelope protein) and vitellogenin mRNA. There was no measurable effect on FSHb mRNA levels, which could be because the background levels were highly variable. In a study with female rainbow trout going through gonadal maturation increases in plasma LH were demonstrated upon exposure to nonylphenol for 6 to 12 weeks; however, the difference between control and treatment disappeared at 18 weeks (Harris et al., 2001). Plasma FSH levels were suppressed over all time periods. Both LH and FSH pituitary protein concentrations and mRNA levels were depressed at 18 weeks. Plasma vitellogenin concentrations were elevated over all time periods as expected, but E2 levels were depressed, and gonadal development ceased. E2 appears to cause positive feedback on LHb and negative feedback on FSHb in salmonids undergoing gonadal maturation, supporting the idea that

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nonylphenol is acting through classic estrogen pathways on the HPG axis, at least in the short-term (Dickey and Swanson, 1998). However, the E2 exposure lasted a maximum of 7 days, whereas the nonylphenol exposure lasted for 18 weeks. It is possible that the effect of E2 on LH wanes over the duration of the maturational period, in a similar manner to the effects of nonylphenol.

2.6. Indirect mechanisms of estrogenic and antiandrogenic effects Reproduction in fish is also regulated by factors outside of the reproductive endocrine axis. Tangential endocrine, paracrine and autocrine systems can be involved in the signals leading to puberty in fish as well as potentiating processes involved in the cyclical reproductive cycle. Two examples are the growth hormone axis and the thyroid hormone axis. The effects of these axes on the reproductive axis are summarized in Figure 5.5, and presented in more detail below. Both axes can be influenced by exogenous chemicals, which may lead indirectly to reproductive dysfunction. 2.6.1. Growth hormone axis Growth hormone (GH) is produced primarily in the rostral pars distalis region of the pituitary. Its release is stimulated by GnRH, and inhibited by somatostatin and through negative feedback loops by itself and insulin-like growth factor (IGF) ( Janz and Weber, 2000). GH is also produced in the gonad, but its function there is likely autocrine or paracrine, and not GH +

GnRH

−

+ +

IGF-I +

LH

− +

+

T3

+

+

FSH

+

T

Arom

E2

Figure 5.5 Influence of growth hormone (GH), insulin-like growth factor I (IGF-1) and thyroid hormone (T3) on the reproductive endocrine axis in fish. GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone;T, testosterone; E2,17b-estradiol; Arom, P450 aromatase; þ, positive regulation;
, negative regulation.

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released into the general circulation (Filby and Tyler, 2007). GH release leads to an increase in transcription and release of two forms of IGF: IGF-1 and IGF-2. IGFs have a similar structure and function to insulin. IGF-1 has highest expression in the liver, but is also expressed in adipose tissue, brain, heart, kidney, muscle, spleen and the gonads ( Janz and Weber, 2000). GH and IGFs primarily affect somatic growth and osmoregulatory adaptation in fish. However, effects on the reproductive axis have also been demonstrated. GH potentiates LH-stimulated steroidogenesis and stimulates ovarian aromatase activity in seatrout. GH is high when the gonadosomatic index is increasing in goldfish (Trudeau, 1997). IGF-1 has been linked to increases in GnRH expression in the hypothalamus, and increases in LH and FSH content in the pituitary, as well as LH release (Baker et al., 2000; Hiney et al., 1996; Huang et al., 1998). Studies in red seabream and Coho salmon demonstrated that IGF-1 decreased the production of T in thecal cells, but increases the production of E2 in granulosa cells (Kagawa et al., 2003; Maestro et al., 1997). Aromatase activity and expression in granulosa cells were also increased upon exposure to IGF-1, although the effect was only present prior to final maturation of the oocytes. The decrease in T and increase in E2 seemed to balance each other, so that the increase in E2 production could only be demonstrated upon addition of exogenous T. This may indicate that endogenous IGF-1 does not act on steroidogenesis in the gonad in an endocrine manner, as the result on steroidogenesis would be null, but rather a paracrine or an autocrine manner, perhaps eliciting effects on each type of cell at a different time during reproductive development. In sexually maturing female sturgeon (Acipenser ruthenus), the levels of IGF-1 in the gonad increased to the same as in the liver, and IGF receptor levels were 20-fold higher in the gonad than in the liver (Wuertz et al., 2007). The increase appeared to be temporary, and the actions believed to be paracrine. The lowest expression of IGF-1 in the ovary was in arrested, pre-vitellogenic follicles and the highest in late vitellogenic follicles. In female trout, a peak in plasma IGF-1 a few months before the first spawn was observed, indicating IGF-1 signals the beginning of puberty in salmonids (Taylor et al., 2008). In fathead minnows (Pimephales promelas) there was apparent sexual dichotomy in the expression of GH, IGF-1, and GH receptors in gonads (Filby and Tyler, 2007). Expression of mRNA was in general higher in ovaries than in testes, and the levels of GH and IGF-1 mRNA increased progressively in ovaries but not in testes during gonadal development. However, despite the higher expression in ovaries than testes, IGFs still play a role in testes by acting directly on premeiotic germinal cells to induce proliferation (Le Gac et al., 2001). Prochloraz and nonylphenol ethoxylates have been shown to target IGF’s role in spermatogenesis of male trout. Both compounds increased the cellular binding capacity for IGF-1 in germ cells, and also noncompetitvely inhibited binding of IGF-1 to those sites (Le Gac et al., 2001). They also decreased IGF-stimulated DNA synthesis in germ cells. Similar results

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were demonstrated using Triton X-100 (a surfactant), but not E2, which the authors postulated might indicate that the inhibition of spermatogenesis is not caused by estrogen mimicking, but rather through the lipophilic or detergent properties of the compounds. Estrogenic compounds may act on other levels of the axis as well. E2, o,p0 -DDT and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) all increased GH expression in rainbow trout pituitary (Elango et al., 2006). The mechanism of action was believed to be ER-mediated for E2 and o,p0 -DDT, and through AhR/ER receptor cross-talk for TCDD. Bisphenol A has been shown to modulate somatostatin levels in the diencephalic regions of the brain of Coris julis and may affect GH levels by affecting somatostatin’s ability to inhibit GH release (Alo et al., 2005). 2.6.2. Thyroid hormone axis Release of thyroid-stimulating hormone (TSH) from the pars distalis region of the pituitary appears to be under hypothalamic control although the actual controlling factors in fish have not been well elucidated. Negative feedback from thyroid hormones also affects TSH synthesis and release. TSH has a similar structure as the gonadotropins, sharing the same a subunit but having a distinct b subunit. The function of TSH is to increase iodide uptake by thyroid follicles, and increase the synthesis and release of thyroxines. Thyroid follicles are not in glands as in mammals, but rather located diffusely in the ventral pharyngeal region of fish. There are two forms of thyroid hormone: L-thyroxine, T4, which is mostly inactive, and triiodothyronine, T3, which is the active form. Conversion of T4 to T3 mainly occurs in peripheral tissues by the action of deiodinases ( Janz and Weber, 2000). The primary functions of T3 are to increase lipid mobilization, protein synthesis, and metabolism. Thyroid hormone also increases GH mRNA expression, GH release from the pituitary and increases hepatic IGF-1 production. Thyroid hormone receptors are highly expressed in the brain and pituitary, and there is sexually dimorphic expression in the gonad and intestine, with higher expression in females (Filby and Tyler, 2007). The effects of thyroid hormones on reproduction are highly dependent on the species of fish, the sex of the fish, the reproductive cycle (e.g., daily spawner versus annual), and the stage of the given reproductive cycle. In medaka T3 promoted gonadotropin-stimulated E2 production in the ovary during vitellogenesis (Soyano et al., 1993). T3 and T4 levels peak 12 and 36 hours before spawning, followed by a peak in E2 4 hours later. T3 was also shown to increase gonadotropin sensitivity in ovaries of vendace (Coregonus albula) during final maturation (Tambets et al., 1997). The effects of compounds on the thyroid axis are as varied as the effects of the thyroid axis itself, and depend on exposure regimen, species, maturity stage, sex, etc. Different groups have seen different responses in fish with the

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same compound. A large variety of compounds have been shown to affect the thyroid axis, including PCBs, organophosphate pesticides, ammonia, perchlorate and pharmaceuticals as reviewed by Brown et al. (2004). There are fewer studies showing links between contaminant exposure, thyroid effects, and reproductive dysfunction. Propylthiouracil, while not necessarily an environmental contaminant, is a model antithyroid agent known to decrease production and conversion of T4 and T3. In a zebrafish partial life cycle test, the compound decreased the levels of circulating thyroid hormones, increased the total number of eggs, and decreased the size of mature eggs in exposed adults (Van der Ven et al., 2006b). Exposure of medaka starting at the egg stage to b-hexachlorocyclohexane (a byproduct of lindane production) was shown to influence both thyroid function and reproduction, though whether there was a cause-effect relationship is unclear (Wester and Canton, 1986). After 3 months’ exposure, medaka thyroid follicles decreased in size and content, indicative of activation. The number of TSH cells in the pituitary increased, and the cytoplasmic content was reduced, also indicative of activity. There was increased vitellogenin production in fish, and males treated with 0.18 to 1.0 mg/L presented ova-testes.

3. Consequences of Impaired Reproductive Endocrine Function Up to this point we have been focusing on the mechanisms through which compounds can cause estrogenic or antiandrogenic actions to occur. At this point, we focus on the consequences.

3.1. Impaired gene regulation The ER is involved in regulation of a large number of genes such as the P450 enzymes described earlier. The ER also regulates genes not directly involved in reproduction. Activation of the ER by estrogen mimics can lead to either upregulation or downregulation of genes which may in turn alter protein processing, homeostasis, and even enhance the toxicity of certain compounds. In addition antiandrogens appear to impair gene regulation in a sex specific manner different than estrogen mimics. Although there are many genes in mammals that have been determined to be under control of the ER, less is known in teleosts. Osteonectin is a glycoprotein in bone and fish scales that binds calcium and collagen, and its expression is markedly downregulated by E2, leading to calcium mobilization (Lehane et al., 1999). The fish orthologue to transforming growth factor beta
binding protein three is another protein whose gene has been determined to be under estrogenic control. The protein is upregulated by E2, which

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can modulate the effects that the growth factor has on early developmental processes (Andersson and Eggen, 2006). Flavin-containing monooxygenases (FMO) are a group of enzymes that are involved in the biotransformation of some exogenous compounds. FMOs sulfoxidate carbamate pesticides, such as aldicarb, rendering the pesticides more toxic. E2 has been shown to increase expression of FMO1-like and FMO3-like proteins in the gills of female medaka, enhancing the toxicity of aldicarb to the fish (El-Alfy and Schlenk, 2002). It is interesting to note the differences between gene expression profiles of E2, the estrogen mimic nonylphenol and the antiandrogen p,p0 -DDE in largemouth bass. E2 upregulated vitellogenin and choriogenin mRNA expression, as expected, and also mRNAs of aldose reductase and aspartic protease, genes involved in sugar metabolism and post-translational protein modification, respectively (Larkin et al., 2002). E2 downregulated transferrin, a protein involved in iron transport. Nonylphenol had a similar profile to E2, although it also upregulated expression of signal peptidase, an enzyme that cleaves signal sequences off secreted proteins, as well. Nonylphenol may be acting through an alternate pathway in addition to the ER, which has been demonstrated earlier. P,p0 -DDE upregulated expression of vitellogenin and choriogenin in males, but downregulated the genes in females. Several other genes were downregulated in females as well, including the androgen receptor. The dichotomy in expression between the sexes upon exposure to the same contaminant indicates that the responses to exposure will vary depending on the sex, and most likely the reproductive status, of the fish.

3.2. Effects of unscheduled protein synthesis Vitellogenin has served as an excellent biomarker for exposure to and uptake of estrogenic chemicals in oviparous animals such as fish. The production of the protein generally is not seen as a toxicological endpoint per se, but there has been some indication that production of large quantities of unusable vitellogenin can lead to pathological effects. In several fish species the unscheduled production of vitellogenin in the liver has been associated with moderate increases in hepatosomatic index as well as histopathology including thickening of nuclear and cell membranes, proliferation of rough endoplasmic reticulum, accumulation of clear eosinophilic material (most likely vitellogenin itself ), damaged cellular disarray, and reduction of glycogen deposits (Mills et al., 2001; Wester and Canton, 1986; Zha et al., 2007). Effects have been demonstrated in the kidney as well, with increased renal-somatic index and histopathology including lesions characterized by severe hemorrhaging in kidney tubules, the Bowman’s space and renal interstitium, and hypertrophy, degeneration and necrosis of tubular epithelia (Zha et al., 2007). Eosinophilic material accumulated in the head kidney, similar to in the liver. In general it appears that males are more affected pathologically by the accumulation of vitellogenin than females,

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most likely because the females can potentially incorporate some of the excess vitellogenin into oocytes if they are actively reproducing. There may be some effects on growth associated with production of vitellogenin (Wester and Canton, 1986; Zha et al., 2007), perhaps due to the shunting of energy and materials to the production of an unnecessary protein. This is supported by the depletion of glycogen stores in the liver.

3.3. DNA damage A metabolite of E2, 4-OHE2, is a genotoxicant. The mechanism of toxicity is discussed in detail in Cavalieri et al. (2000). Briefly, both 2-OHE2 and 4-OHE2 can be metabolized further to semiquinones by peroxidases or P450 enzymes. Further transformation to a quinone form can occur either by the same enzymatic process, or through redox cycling in the presence of oxygen. The quinone form can react directly with DNA, forming depurinating adducts. Redox cycling can also produce hydrogen peroxide, leading to formation of hydroxyl radicals that then react with DNA to produce oxidized DNA bases. In the presence of lipids, hydroxyl radicals can aid in the formation of lipid hydroperoxide-derived aldehyde-DNA adducts (Fig. 5.6). Generally, the 4-OHE2 form of catehol estrogen is more toxic than the 2-OHE2 form because the 2-OHE2 form is more readily conjugated and excreted before semiquinones and quinones can form. Also the quinone produced by 2-OHE2 produces stable adducts instead of Reductase CYP1A, peroxidases CYP1A, peroxidases 4-hydroxyestradiol Estradiol-3,4-semiquinone -OrEstradiol-3,4-quinone Non-enzymatic DNA CYP1B 17b -estradiol

O2

CYP3A, CYP1A

. Depurinating adducts O2 SOD

H2O2

2-hydroxyestradiol

Fe2+or Cu+ Lipid hydroperoxides DNA Aldehyde DNA adducts

Lipids

•OH DNA

Oxidized DNA bases

Figure 5.6 Activating metabolism pathway for 17b-estradiol metabolite 4-hydroxyestradiol. SOD, superoxide dismutase. Adapted from Cavalierei et al., 2000.

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depurinating adducts, which are less prone to cause mutation. The ratio of 4-OHE2 to 2-OHE2 formed and the level of detoxifying enzymes present in a given tissue can determine the tissue-specific genotoxicity of estrogens. While DNA damage caused by exogenous estrogens has been well studied in mammals, relatively little data is available on fish. Exposure to exogenous E2 has been shown to increase DNA damage in fish erythrocytes, albeit transiently and nonsignificantly (Teles et al., 2005). The estrogenic pesticide dieldrin was linked to oxidative DNA damage in gilthead seabream liver (Rodriguez-Ariza et al., 1999). The area deserves further research as a potential impact of estrogen exposure, especially in light of environmental contaminants like benzo(a)pyrene which shift E2 metabolism toward production of 4-OHE2 (see earlier).

3.4. Intersex/sex reversal Fish exhibit gender plasticity. Some fish, like the California sheephead (Semicossyphus pulcher), start life as one sex, in this case female, and switch to the other sex in adulthood under appropriate environmental stimuli. For other fish, the sex is determined at a very early age, soon after hatching, termed the sexual differentiation period. Medaka begin life with undifferentiated gonads which then differentiate into either male or female gonads ¨ rn et al., 2006). Zebrafish on within the first couple of months post-hatch (O the other hand all start out with ovary-like gonads which form at about 3 to 4 weeks post hatch, and then in males the ovaries morph into testes between ¨ rn et al., 2003). While many fish have genetic sex 4 to 5 weeks post-hatch (O determination, it can be overridden or partially reversed by exposure to steroids, endocrine disrupting compounds or even extremes in temperature, especially when the exposure occurs at a critical ‘‘window’’ in sexual differentiation. The critical time varies depending on the species of fish. Sticklebacks exposed to E2 and ethinylestradiol within 2 weeks after hatching presented both sex reversed and intersex males (Hahlbeck et al., 2004). However, exposure of the egg to E2 before hatching had no effect. Incidence of intersex was highest in medaka when exposure to octylphenol occurred at 3 days post hatch versus seven or 21 days (Gray et al., 1999). However, in a study exposing 5- to 8-day-old medaka to E2 for 28 days, all fish became phenotypic females, indicating that the fish were still sensitive at an older age (Nimrod and Benson, 1998). Genetically female Japanese flounder (Paralichthys olivaceus) exposed to elevated temperature between 30 and 100 days after hatching had suppressed expression of P450 aromatase and became phenotypic males (Kitano et al., 1999). Zebrafish exposed to the fungicide prochloraz (both a steroidogenesis inhibitor and an androgen receptor antagonist) from 1 to 60 days post-hatch had a higher ratio of males and the presence of intersex individuals (Kinnberg et al., 2007).

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However, exposure of zebrafish to 2 to 10 ng/L ethinylestradiol from 20 to 60 days post-hatch also produced sex reversal, this time to females, ¨ rn et al., 2003). indicating that the critical period may occur after 20 days (O Interestingly, the effect was reduced at 25 ng/L. No intersex individuals were observed at any concentration. Response to endocrine disruptor or steroid exposure during sexual differentiation and beyond also varies with the species. Both medaka and zebrafish were exposed to ethinylestradiol for 1 to 60 days post-hatch. In zebrafish, the sex ratio became 100% female at a concentration of ¨ rn et al., 2006). 10 ng/L, and complete mortality occurred at 100 ng/L (O No intersex occurred, similar to the experiment earlier. In medaka there was no change in sex ratio at 10 ng/L, but a small percentage of fish were intersex. At 100 ng/L the sex ratio became predominantly female, and a higher percentage of fish were intersex. Unlike sticklebacks, exposure of medaka to E2 in the egg, as little as 1 day beginning right after fertilization, can cause phenotypic sex reversal to females (Kobayashi and Iwamatsu, 2005). Medaka seem particularly sexually labile, as exposure even into adulthood can lead to intersex. Exposure to the herbicide oryzalin for 21 days led to intersex in 7-month-old, sexually mature medaka (Hall et al., 2007). Spontaneous intersex in untreated medaka has also been reported, and the incidence of intersex increased with age (Grim et al., 2007). While altered steroids levels, both androgens and estrogens, clearly have an effect on sexual differentiation, the trigger or mechanism of sexual differentiation still has not been elucidated. It was mentioned earlier that aromatase expression was suppressed during sexual differentiation in phenotypic Japanese flounder males. A study was performed on medaka exposing them for 14 days post-hatch to the estrogenic compound o,p0 -DDT with and without the aromatase inhibitors fadrozole (a pharmaceutical) and tributyltin (an antifouling agent). Even though aromatase activity was significantly suppressed in the presence of the inhibitors, 100% phenotypic sex reversal to females still occurred (Kuhl and Brouwer, 2006). A study with roach exposed to environmentally relevant concentrations of ethinylestradiol found a concentrationdependent increase in both ERa and ERb, with highest induction of ERa, alongside sex reversal (Katsu et al., 2007). If sexual differentiation is at least in part ER-mediated, then perhaps the presence of sufficient quantities of a compound that can interact with the ER are enough to elicit feminization and aromatase inhibition can be overcome. There seems to be some question whether intersex fish are still reproductively viable. Balch et al. (2004) indicated that intersex individuals were still able to reproduce successfully. However, Nash et al. (2004) believed that the intersex individuals were not sexually viable. The Nash intersex fish had other, extensive abnormalities including malformation of the sperm ducts, which could have had a greater impact on gonadal function than the presence of oocytes, however.

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While it is clear that if all males in a population were sex-reversed it would likely result in reproductive failure, the consequences of sex reversal in mixed populations is cloudier. Aquaculture studies with sex-reversed tilapia (Oreochromis aureus) demonstrated that phenotypic female/genetic male (pseudofemale) fish were still capable of reproducing, but the production of fry was lower due to reduced spawning frequency and a higher percentage of nonspawning pseudofemales versus control females (Desprez et al., 1995). The percentage of male offspring was also much higher (almost 100%) for pseudofemales (Desprez et al., 1995; Me´lard, 1995). Tilapia have ZZ (male) and ZW (female) genetic sex determination. Pairing of sex-reversed males with untreated males are likely to generate all ZZ, or male genotypes. Sex reversal in XX/XY sex-determined fish will likely provide different results. Pseudofemales paired with untreated males will likely result in XX, XY, and YY genotypes. Crossing YY males with females from various genetic backgrounds led to all male populations, as expected, but few of the offspring were sterile (Bongers et al., 1999). Therefore, even though pseudofemales are still capable of reproducing (albeit at a lower rate for tilapia), there may be an impact on the sex ratios of later generations.

3.5. Reproductive failure Estrogenic and antiandrogenic compounds have been shown in several species to have deleterious effects on reproduction. Treatment of rainbow trout with 280 ng/L nonylphenol for 60 days led to a decrease in semen quantity, a decrease in the percentage of eggs surviving to the eyed stage, and a decrease in the percentage of larvae that survived to the end of the yolk-sac stage (Lahnsteiner et al., 2005). Guppies (Poecilia reticulata) treated with at least 100 mg/L octylphenol and 1 mg/L E2 for 30 to 60 days had decreased testes growth but an increase in sperm count (Toft and Baatrup, 2001). There was a decrease in male secondary sexual characteristics upon treatment with both compounds. E2 decreased the birth rate, but the results were variable with octylphenol. Ninety days after the cessation of exposure there were still significant effects on sperm count and secondary sexual characteristics, indicating slow recovery. The antiandrogen flutamide decreased fecundity in fathead minnows treated for 21 days with 651 mg/L ( Jensen et al., 2004). The decrease in fecundity was caused by both a decrease in the number of spawns per female, and the number of eggs per spawn. There was a concomitant increase of atresia and a delay in egg development. Spermatocyte degeneration and necrosis was also present in males. At least two long-term reproductive studies have been conducted in the laboratory to try to determine the potential for population-level effects of exposure to environmentally relevant ethinylestradiol concentrations. Balch et al. (2004) exposed medaka from 2 to 5 days post-hatch until sexual maturity (about 4 to 6 months). Males had feminized secondary sexual

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characteristics, disorganized testicular tissue, and some intersex. There were no alterations to ovarian development in females, yet there was a trend toward a decrease in fecundity. It appeared that fish that copulated were able to produce fertilized eggs, but the number of copulations was decreased both in males paired with untreated females and females paired with untreated males; therefore, the impact on reproduction was more behavioral than physiological. The impact on behavior could be due to the feminized secondary sexual characteristics in males, and perhaps a decrease in pheromones produced by females. While cessation of exposure for six months did not alter gonadal deformities, effects on secondary sex characteristics decreased, indicating that a population may be able to recover somewhat and reproduce. Nash et al. (2004) performed a multigenerational study with zebrafish. Exposure of the F0 generation to 50 ng/L ethinylestradiol for up to 15 days led first to a decrease in egg production and viability, and eventually complete reproductive failure. Seven months’ exposure of the F1 generation to 5 ng/L led to the production of no viable eggs. There were no phenotypic males, no expressible sperm, and, therefore, no fertilization. Ethinylestradiol at 0.5 ng/L caused a decrease in the proportion of viable eggs, but the surviving larval integrity was not impaired. Therefore, it appears that long-term exposure to environmentally relevant concentrations of ethinylestradiol can impair or completely block reproduction. However, if there are surviving larvae there are no transgenerational effects. Partial recovery was also demonstrated upon removal of ethinylestradiol in this study. True population-level effects are extremely difficult to reproduce in the laboratory because it is difficult to unequivocally state that estrogenic and/or antiandrogenic compounds at environmentally-relevant concentrations will cause the demise of a fish population. However, a study was recently conducted in the field in which a lake was treated with environmentally realistic concentrations of ethinylestradiol over a period of 3 years (Kidd et al., 2007). The fathead minnow population in the lake was monitored. Increases in vitellogenin in both sexes were evident, along with testicular abnormalities in males (delayed spermatogenesis, fibrosis, malformed tubules, intersex, lowered gonadosomatic index) and females (delayed ovarian development, follicular atresia). Catch-per-unit-effort decreased consistently over time compared to two reference lakes. In the fall after the second season of ethinylestradiol additions, the fathead minnow population collapsed because of a loss of young-of-the-year animals. The reproductive failure continued through the third year of chemical additions, and an additional two years post-treatment. Another species in the lake, the pearl dace (Margariscus margarita), did not experience the same population crash perhaps because this species is longer lived. Therefore, it appears that populations of short-lived fish species may be at risk from chronic exposure to estrogenic compounds in the environment.

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4. Summary and Concluding Remarks There are many compounds found in the environment with estrogenic or antiandrogenic properties. There are several different types of ER and AR receptors within a species, as well as differences between species, and the binding properties and downstream responses vary. Therefore, the interaction of a given compound with a given receptor may not indicate how it will react with another. There are many other mechanisms for estrogenicity in addition to receptor interactions, including effects on steroid synthesis, distribution and excretion, effects on other parts of the HPG axis, and also indirect mechanisms. Some compounds can act through a myriad of different mechanisms to bring about an estrogenic response. And the response itself can vary, from non-reproductive effects on gene regulation, DNA damage, and liver and renal histopathology to reproductive effects such as gonadal histopathology, sex reversal and eventually reproductive and even population failure. While there are many in vitro tools available to assess the mechanisms of estrogenicity, relying on them to determine the magnitude of response in a whole animal may be narrow and misleading. In addition even whole animal responses will vary depending on species, sex, age, and reproductive status. Therefore, it is imperative to utilize endpoints throughout the biological hierarchy to better understand mechanisms of action, with the ultimate benefit being more accurate risk assessments for compounds of this nature.

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Roles of P21-Activated Kinases and Associated Proteins in Epithelial Wound Healing Mirjam Zegers* Contents 1. Introduction 2. Biology of Wound Healing in Different Model Systems 2.1. Developmental models 2.2. Scrape wound healing 2.3. Wound healing and cancer 2.4. Epithelial plasticity during wound healing 3. Rho GTPases and Epithelial Morphogenesis During Wound Healing 3.1. Steps in wound healing 3.2. Rho GTPases 4. P21-Activated Kinases 4.1. Background of P21-activated kinases 4.2. Structure 4.3. Mechanism of activation 4.4. Inactivation 5. Pak Activation During Wound Healing and Epithelial Sheet Migration 5.1. Background 5.2. Activation of Pak by wounding-associated signals 5.3. Kinase-independent functions and Pak-interacting proteins 5.4. The PIX-GIT complex 6. Regulation of Wound Healing Downstream of Pak 6.1. Cell motility and sheet migration 6.2. Regulation of cell proliferation by Pak, PIX and GIT 7. Concluding Remarks Acknowledgments References

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Department of Surgery, University of Chicago, Chicago, IL 60637

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00606-0

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Abstract The primary function of epithelia is to provide a barrier between the extracellular environment and the interior of the body. Efficient epithelial repair mechanisms are therefore crucial for homeostasis. The epithelial wound-healing process involves highly regulated morphogenetic changes of epithelial cells that are driven by dynamic changes of the cytoskeleton. P21-activated kinases are serine/threonine kinases that have emerged as important regulators of the cytoskeleton. These kinases, which are activated downsteam of the Rho GTPases Rac and cd42, were initially mostly implicated in the regulation of cell migration. More recently, however, these kinases were shown to have many additional functions that are relevant to the regulation of epithelial wound healing. Here, we provide an overview of the morphogenetic changes of epithelial cells during wound healing and the many functions of p21-activated kinases in these processes. Key words: Epithelial morphogenesis, Wound healing, Rho GTPases, p21-activated kinase, Cell adhesion, Cell migration. ß 2008 Elsevier Inc.

1. Introduction Epithelial cells are organized in sheets of adherent cells that are polarized, meaning that they have distinct apical and basolateral surfaces. An important function of polarized epithelial cells is to form barriers between distinct physiological environments (Nelson, 2003; O’Brien et al., 2002; Zegers et al., 2003b). Examples of such barriers are the skin and the luminal surfaces of internal organs such as the respiratory, gastrointestinal and uritogenitary tracts, as well as the mammary, prostate and other exocrine glands. The functions of epithelia rely entirely on the ability of epithelial cells to form a polarized monolayer. It is therefore essential that epithelia have efficient mechanisms to repair injuries induced by trauma, surgery, inflammation and toxic or ischemic insults. In general, epithelial repair can be divided into a start and a stop phase ( Jacinto et al., 2001). In the start phase, cells adjacent to injured areas partially dedifferentiate and migrate into the site of injury. These cells migrate by extending protrusions and lamellipodia into the wound, while pulling along cells located further back from the wound edge. In large wounds, cell proliferation is stimulated to replace lost or damaged cells. In the stop phase, newly formed cell-cell contacts block cell migration and proliferation. In this review I will discuss intracellular signaling pathways that control the diverse morphogenetic changes that epithelial cells undergo during the different stages of epithelial wound healing. In vivo wound healing is a complex and highly regulated process that involves a wide range of

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extracellular signals and epithelial cells, stromal and inflammatory cells. Interestingly, the signaling pathways that control epithelial behavior during injury repair appear to be highly conserved and bear strong resemblance with related behaviors that are observed during embryonic development. One of the conserved features for epithelial morphological changes is the crucial role of the cytoskeleton. Rho GTPases in particular, are ubiquitous intracellular signaling intermediates critical for cytoskeletal regulation and wound healing in a wide variety of models. In this review I will first discuss general aspects of epithelial morphogenesis during wound healing. Next, I will focus on the role of one particular effector family of Rho GTPases, the p21-activated kinases, and their role in epithelial cell behavior during wound healing.

2. Biology of Wound Healing in Different Model Systems Epithelial wound healing in vivo has been most widely studied in the context of the skin (Martin, 1997; Singer and Clark, 1999). Cutaneous wounds in adult tissue are temporarily repaired by the formation of a fibrinrich blood clot to which platelets bind. The clot acts as a reservoir for growth factors and cytokines secreted by platelets and damaged keratinocytes, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), the transforming growth factor a (TGF-a), and members of the transforming growth factor b (TGF-b) family. These factors attract neutrophils and fibroblasts, which also secrete growth factors and proteases such as matrix metalloproteases (MMPs). Upon secretion, MMPs degrade specific components of the extracellular matrix, thereby allowing matrix remodeling, while at the same time releasing additional growth factors that had been linked to the extracellular matrix. This complex mix of these growth factors generated in the stroma of wounded tissue will also induce angiogenesis, by stimulating endothelial cells to proliferate and form new capillaries into the wound stroma. A prime objective of wound healing is to restore epithelial function by inducing the epithelial cells to undergo sheet movements in which migration, proliferation and cell adhesion processes are highly coordinated. The released growth factors and proteases in the wound stroma profoundly affect the epithelial cells at the wound edge and induce phenotypical changes that resemble an epithelial-mesenchymal transition (EMT, see below). As a result, the interaction of epithelial cells with the underlying basement membrane and neighboring cells is reduced and cells migrate as a sheet over the provisional wound matrix. This provisional matrix forms under the clot and contains fibronectin, vitronectin and other matrix molecules.

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These morphological changes and rapid stimulation of epithelial cell migration is generally followed by sharp increase in cell proliferation to replace lost cells (Martin, 1997; Singer and Clark, 1999). The initial events at the ‘‘start phase’’ of wounding, which includes the inflammatory, angiogenic, migratory and mitogenic responses, need to be inhibited at the ‘‘stop phase’’ of wound closure ( Jacinto et al., 2001). Presently, the downregulation of these different responses remains poorly understood. In epithelial cells, ‘‘contact inhibition,’’ a mechanism that inhibits cell motility and proliferation upon reaching high density and/or establishment of cell-cell contacts (Abercrombie, 1979; Fagotto and Gumbiner, 1996; Middleton, 1972; Stoker and Rubin, 1967) is likely to be involved, but the molecular mechanisms underlying contact inhibition are still largely unknown. The basic wounding response in adult mammalian epithelia other then the skin appears to be generally similar to cutaneous wound healing. Some differences, however, exist dependent on the nature of the insult, the specific tissue involved, or the developmental stage of the organism. As has become clear from in vivo model systems, wound healing is a complex process that involves many different cell types and cellular behaviors. In addition, it generates a complex mixture of secreted growth factors, proteases and matrix molecules, which in turn will act on a wide array of membrane receptors and adhesion molecules. For this reason, the signaling pathways that underlie epithelial cell behavior at the cellular level have been difficult to decipher in mammalian in vivo models. For this reason, investigators have used a variety of alternative in vivo and in vitro model systems to study the mechanisms that control epithelial repair.

2.1. Developmental models The forward movement and fusion of epithelial sheets that occur during wound healing is not unique to wound repair, but is in fact a common phenomenon in many other morphogenetic processes, in particular during development. Examples are eyelid closure, in which fetal mouse eyelids move toward the center of the eye and tightly fuse which each other, only to open again 2 weeks after birth (Harris and McLeod, 1982). During late Drosophila embryogenesis, retraction of the germ band results in an epithelial hole, which is closed by lateral sheets of epithelia which move towards each other and fuse at the dorsal midline in a process called dorsal closure (Harden, 2002; Martin and Parkhurst, 2004). Embryonic tissue movements similar to Drosophila dorsal closure occur in the worm C. elegans, where the epidermis spreads from the dorsal surface of the C. elegans embryo, until the epithelial sheets encloses the embryo and seal at the ventral midline (Ding et al., 2004). As the molecular mechanism that drive these tissue movements appear to be largely conserved ( Jacinto et al., 2001; Martin and

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Parkhurst, 2004), aspects of epithelial sheet migration during embryonic development can serve as a model for epithelial wound healing. Not all aspects of wound healing are recapitulated during development, as these models for instance lack inflammatory responses. Nevertheless, the study of epithelial sheet movements during development has provided many insights into the regulation of sheet migration.

2.2. Scrape wound healing One of the simplest models systems for epithelial repair in vitro are scrape wound healing assays, in which closure of a monolayer is analyzed following the removal of a few rows of cells from a confluent monolayer of cells grown in culture dishes. Although this approach is obviously reductionistic, the advantages of these scrape wound models are the ease of experimental manipulation and the fact that such model systems only comprise epithelial cells. Thus, scrape wound healing assays allows investigators to study the intrinsic epithelial response to wounding in the absence of the complex mix of factors contributed by the stroma, and allows an analysis of the role of the individual components of this mix. In fact, most of our current knowledge on both the intracellular signaling pathways and the molecular machinery required for epithelial sheet migration during wound healing has been elucidated using scrape wound healing assays. Even though not all regulatory factors identified in scrape wound healing assays appear to be crucial in vivo (DiPersio, 2007), the mechanisms that drive wound healing in scrape wound healing assays have been found to be recapitulated to a remarkable extent in the different in vivo and developmental models (Van Aelst and Symons, 2002). This suggests that the epithelial wound healing response is driven by robust and conserved signaling pathways.

2.3. Wound healing and cancer Based on the similarities in both histology and signaling process that promote tumor progression, tumors have been proposed to behave as ‘‘wounds that never heal’’ (Dvorak, 1986). This notion has been further supported by recent genomic analyses comparing carcinoma cells and cells engaging in or mimicking a wounding response (Chang et al., 2004; Iyer et al., 1999; Pedersen et al., 2003). Data from those studies not only demonstrate significant overlap between transcriptional profiles of both cell types, but also indicate that increased similarities correlate with a tendency of tumor cells to metastasize (Chang et al., 2004). Thus, the extensive analysis of cellular behaviors of cancer cells, in particular those concerning regulation of motility and epithelial dedifferentiation, is highly relevant for understanding epithelial wound healing. Clearly, the opposite is

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equally true, in that understanding the epithelial wound healing response may yield novel insights in cancer progression.

2.4. Epithelial plasticity during wound healing As discussed above, epithelial wound healing is accompanied by dramatic cell shape changes of wound edge cells. In intact epithelia, epithelial cells have apical-basolateral polarization and their lateral membranes tightly interact through specialized structures such as tight junctions and E-cadherin-based adherens junctions. At the basal side, cells interact with basement membrane (a specialized form of the extracellular matrix) through adhesion receptors like integrins. Several of these epithelial characteristics will largely disappear at sites of injury. Cells will lose apical-basolateral polarization and tight cell-cell and cell-matrix interactions will be downregulated, weakened or altered. These morphological changes recapitulate aspects of epithelial-mesenchymal transition (EMT). EMT is a process mainly found during embryonic development in which epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype, allowing cells to migrate and invade the stroma. EMT is characterized by a down-regulation of epithelial-specific proteins, such as E-cadherin and the acquisition of mesenchymal-specific proteins like vimentin (Grunert et al., 2003; Hay and Zuk, 1995). The opposite process, mesenchymal-epithelial transition (MET), in which mesenchymal cells revert to cells with an epithelial phenotype, also exist and is instrumental for kidney development (Hay and Zuk, 1995). The term EMT has recently also been used to describe many types of epithelial plasticity. As a consequence, the initial stages of wound healing and sealing of epithelial sheets in the final stages of wound repair is sometimes suggested to represent EMT and MET, respectively. EMT in the strict sense however, is characterized by the ability of individual cells to leave the epithelial monolayer entirely, which does not occur during wound healing. Moreover, EMT is mainly regulated by transcriptional programs through transcriptional regulators such as Snail family proteins (Thiery and Sleeman, 2006) and Twist (Yang et al., 2004), and it is currently unclear to what extent these transcription factors play a role in normal epithelial wound healing. At least in in vitro scrape wound healing assays, wound healing can occur in the absence of protein synthesis (Altan and Fenteany, 2004), which would argue against a crucial role of transcriptional regulation, at least during some stages of epithelial wound healing. On the other hand, stromal growth factors such as TGF-b, which are released during wound healing and play important roles during the process, are well known to induce EMT. Furthermore, wounded epithelial cells are more susceptible to TGFb-induced EMT (Masszi et al., 2004), and EMT has been implicated in

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pathological wound healing processes such as kidney fibrosis in chronically injured kidney epithelia (Boutet et al., 2006; Zeisberg et al., 2003). Therefore, it is possible that wound healing shares the same signaling events with the initial stages of EMT, but that does normally not progress to a complete EMT.

3. Rho GTPases and Epithelial Morphogenesis During Wound Healing 3.1. Steps in wound healing Epithelial wound healing critically depends on the ability of cells to migrate. Cell migration can be regarded as a cyclical process in which the following distinct steps are distinguished (Ridley et al., 2003): I. Polarization. In response to migration-inducing factors, cells polarize and form protrusions towards the direction of migration. These protrusions can be lamellipodia, which are large and sheet-like and driven by formation of actin meshworks, or filopodia, which are spike-like and driven by actin bundles. II. Traction at the leading edge. The polarized protrusions at the leading edge are stabilized by adhesion to the ECM though transmembrane adhesion receptors which link to the actin cytoskeleton. The formation of such focal contacts allows the cell to generate traction force at the leading edge that the cell uses to move forward. III. Retraction at the trailing edge. Focal contacts at the trailing edge will disassemble and the tail of the cell will retract. These steps in cell migration are found in wide variety of cells and although they have been mainly characterized in single cells, epithelial sheet migration during wound healing appears to proceed in a similar fashion, with many cells acting in concert (Farooqui and Fenteany, 2005).

3.2. Rho GTPases Local rearrangements of the cytoskeleton drive the specific cell morphological changes that accompany cell migration. The small GTPases of the Rho family are crucial regulators of the actin cytoskeleton and it is therefore not surprising that these molecules are involved in the distinct steps of cell migration. With regard to epithelial wound healing, the role of Rho GTPase is not limited to regulation of migration, as these GTPases have also been implicated in many other aspects of this process, such as the regulation of cell-cell adhesion, apical-basolateral cell polarization and cell

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cycle control. Many excellent reviews on the role of Rho GTPases in these processes are available (Hall, 1998; Jaffe and Hall, 2005; Jaffer and Chernoff, 2004; Kaibuchi et al., 1999; Marshall, 1999; Schmidt and Hall, 2002; Schmitz et al., 2000; Settleman, 2000; Van Aelst and Symons, 2002). Rho GTPases act as switches between extracellular signals and intracellular effector molecules. Rho GTPases can be activated by activated growth factor receptors or in response to cell-cell or cell-matrix adhesion. With regards to the latter types of activation, they participate in bidirectional signaling with both cadherins (Kaibuchi et al., 1999) and cell-matrix receptors like integrins (Keely et al., 1998), meaning that they are not only activated through these adhesion receptors, but they also regulate their adhesive functions. Rho GTPases are activated via guanine nucleotide exchange factors (GEFs), which replace the GDP bound to the GTPase with GTP. Upon activation, Rho-GTPases activate different effector molecules, thereby stimulating signaling cascades that regulate a variety of cellular processes. Currently, over 20 Rho GTPase members have been identified in mammalian cells ( Jaffe and Hall, 2005). Most research however has focused on the prototypical family Rho GTPase family members Rac1, RhoA and cdc42. Rac, Rho and cdc42 all have been implicated in regulation of wound healing and sheet migration. Although their respective roles in these processes are to some extent cell type and tissue-specific, the roles of Rac1 appear the most widely conserved. In Drosophila, loss of function or inhibition of Rac leads to defects in dorsal closure, likely by an inhibition of lamellipodia and filopodia and inhibition of actin-myosin contractility (Hakeda-Suzuki et al., 2002; Harden et al., 1995, 1999; Woolner et al., 2005). In vitro scrape wound healing in the epithelial Madin-Darby canine kidney (MDCK) monolayers is blocked when dominant-negative Rac1 is microinjected in the first three rows of cells at the wound edge, whereas dominant-negative RhoA or cdc42 essentially have no effect (Fenteany et al., 2000). Studies in bronchial epithelial cells demonstrated similar requirement for Rac1, but in these cells wound healing also depends on RhoA (Desai et al., 2004). Together, these studies indicate a crucial role for Rac in wound healing. This was recently confirmed in vivo, as inhibition or deletion of Rac1 in mouse skin was shown to inhibit incisional epidermal wound healing (Tscharntke et al., 2007). The mechanism responsible for these wound healing defects likely involves the intrinsic inhibition of keratinocyte migration and proliferation (Castilho et al., 2007; Tscharntke et al., 2007), but may also involve the depletion of follicular stem cells (Benitah et al., 2005). Other in vivo studies however demonstrated that Rac1 deletion only inhibits hair follicle development by follicular stem cell depletion, but does not affect epidermal development and maintenance (Castilho et al., 2007; Chrostek et al., 2006).

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4. P21-Activated Kinases 4.1. Background of P21-activated kinases Rho GTPases, such as Rac, mediate their biological functions by activating effector molecules, which are mostly, but not always, kinases that initiate cellular behaviors by phosphorylating downstream substrates which may initiate signaling cascades. In order to understand the role of Rac in the regulation of wound healing and sheet migration, it is therefore crucial to identify and characterize the specific Rac effector molecules. P21-activated kinases (Paks) were the first identified binding partners of GTP-bound Rac and cdc42 (Manser et al., 1994) and are among the best characterized of the many Rho GTPase effector molecules currently known. Indeed, Paks were named after this characteristic, as the p21 in their name stands for Rac and cdc42, which, as all Rho GTPases, have a molecular weight around 21 kDa. Initial reports showed that Paks specifically interact with GTP-bound forms of Rac1 and cdc42, but not with the GDP-bound versions of these protein (Bagrodia et al., 1995; Knaus et al., 1995; Manser et al., 1994; Martin et al., 1995). More recently, several additional small GTPases of the Rac and cdc42 subfamilies (Bustelo et al., 2007) were found to activate Pak, including Rac2 (Knaus et al., 1998), Rac3 (Mira et al., 2000), Chp (Aronheim et al., 1998; Weisz Hubsman et al., 2007), TC10 (Neudauer et al., 1998) and Wrch-1 (Tao et al., 2001). Paks are not activated by Rho A-G or by Ras superfamily members (Bokoch, 2003). To date, six Pak family members have been identified. The human Pak1 (rat aPak), human Pak2 (rat gPak) and human Pak3 (rat bPak) are now classified as conventional, group I or group A Paks. In addition, there are the nonconventional, group II or group B Paks, which are named Pak4, Pak5 (sometimes described as Pak7) and Pak6 (Bokoch, 2003; Dan et al., 2001; Hofmann et al., 2004; Jaffer and Chernoff, 2002; Zhao and Manser, 2005). The structure and regulation of group II Paks differs significantly from the group I Paks and a detailed understanding of their roles is only beginning to emerge. For these reasons, this review will focus on Pak1-3.

4.2. Structure Pak1-3 share several conserved characteristic features. As shown in Figure 6.1, their general structure comprises a regulatory N-terminal domain and a catalytic C-terminal domain. The N-terminal domain contains a p21-binding domain (PBD), which interacts with the active, GTP-bound forms of Rac and cdc42. Partially overlapping with the PBD is an autoinhibitory domain (AID). A large part of the combined PBD and

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Grb2 Nck

Pak Rac/cdc42

PIX

PBD AID Regulatory domain

Catalytic domain

Figure 6.1 Structural features of Pak kinases. Image represents the domain structure of Pak1. Pak1-3 contain an N-terminal regulatory domain and a C-terminal serinethreonine kinase domain. Several domains within the regulatory domain are indicated: The dashed areas represent the canonical proline-rich, SH3-binding domains. The adaptor proteins Nck and Grb2 bind to the first and second proline-rich domain, respectively. In black, the non-canonical, proline-rich PIX-binding domain is indicated. In dark-grey, the p21-binding domain (PBD) is indicated, which binds GTP-bound forms of Rac and cdc42. Partially overlapping with the PBD is the autoinhibitory domain (AID) in light-grey, which inhibits Pak activity in trans by binding to the catalytic domain of another Pak molecule.

AID comprises the so-called ‘‘inhibitory switch domain’’ (Lei et al., 2000), which is crucial for the activation of Pak. The N-terminus furthermore contains several proline-rich domains with canonical PxxP SH3 binding domains (five in Pak1, two in Pak2 and four in Pak3) and a conserved nontypical proline-rich PxP SH3 domain which binds the GEFs of the PIX/Cool family (Bokoch, 2003; Jaffer and Chernoff, 2002). The serine/ threonine kinase domain of Pak is at the C-terminus of the protein and is at least 93% identical in Pak1, 2 and 3 ( Jaffer and Chernoff, 2002).

4.3. Mechanism of activation 4.3.1. Activation by Rho GTPases The mechanism of Pak activation has been analyzed at the molecular level in Pak1, for which a crystal structure of both the inactive and active kinase domain has been resolved (Lei et al., 2000; Lei et al., 2005). Based on the crystal structure, it was concluded that Pak1 exists as a dimer in a transautoinhibitory conformation in which the inhibitory switch domain of one Pak1 molecule inhibits the catalytic domain of the other. It is believed that Pak1 exist in this form both in solution and in unstimulated cells (Buchwald et al., 2001; Lei et al., 2000; Parrini et al., 2002). Binding of GTP-bound Rac or cdc42 induces a series of conformational changes, which results in a disruption of the dimer and ends with the kinase domain in a stable catalytically active conformation. Central to Pak1 activation is the phosphorylation of the Thr423 residue in the activation loop of Pak1. Thr423 is exposed upon Rac/cdc42 binding and its phosphorylation allows for kinase activation and stabilization of the active conformation. It furthermore allows for autophosphorylation of several other sites, which also contribute to kinase activation (Chong et al., 2001; Frost et al., 1998; Hoffman and Cerione, 2000; Lei et al., 2000, 2005; Tu and Wigler, 1999; Zenke et al., 1999; Zhao et al., 1998).

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Though Thr423 phosphorylation in solution can occur through autophosphorylation (Parrini et al., 2002), in cells it may be mediated by 3-phosphoinositide
dependent kinase 1 (PDK1), perhaps through a mechanism that depends on the membrane lipid sphingosine (Bokoch et al., 1998; King et al., 2000a,b). 4.3.2. Rho GTPase
independent activation Even though Paks are considered bona fide downstream targets of active Rac/cdc42, several Rac/cdc42-independent activation mechanisms have been reported as well. Initial studies that characterized Pak activity in vitro, had shown that proteolytic cleavage of Pak, which removes its N-terminus, yields a highly active Pak in solution (Benner et al., 1995; Roig and Traugh, 2001). Interestingly, proteolytic cleavage was demonstrated to be a physiological mechanism of Pak activation during apoptosis, when Pak2 is cleaved by caspase-3 (Rudel and Bokoch, 1997; Walter et al., 1998). Pak can be recruited to the plasma membrane by several different mechanisms, which activates the kinase by a process that is not fully understood. Membrane recruitment of Pak1, either through binding to the adaptor protein Nck (Lu et al., 1997), or experimentally induced by introduction of a C-terminal isoprenylation sequence (Daniels et al., 1998) activates Pak1 kinase activity, possibly through a sphingosine- and PDK1dependent phosphorylation of Pak1. Phosphorylation and activation of Paks by the kinase Akt, either downstream of Ras (Sun et al., 2000; Tang et al., 2000) or downstream of the heterotrimeric G-protein b/g subunits (Menard and Mattingly, 2004) has been reported as well. These Pak phosphorylations by PKD1 or Akt occur in the presence of dominant-negative mutants of Rac1 and cdc42, suggesting that they are independent of these GTPases. However, since the activating phosphorylations occur at sites at the catalytic domain that are masked by the inhibitory switch domains in inactive Pak1 dimers, the question remains how these residues are accessible to PKD1 or Akt. As will be discussed in detail later, Paks can also be recruited to the membrane by an interaction with the Pak-interacting exchange factor (PIX). Pak binds directly to PIX, which in turn tightly interacts with the G protein-coupled receptor kinase-interacting target (GIT1). This Pak-PIX-GIT complex accumulates at focal adhesions in migrating cells where the (indirect) interaction of GIT1 with Pak can activate Pak1. As this activation also occurs in the presence of dominantnegative Rac1 or cdc42 or in a Pak1 mutant that cannot bind active cdc42, this activation appears to be independent of Rho GTPases (Loo et al., 2004). In mitotic cells, GIT1 can target Pak1 to the centrosome, which results in Pak1 activation at this site (Zhao et al., 2005). Interestingly, when Pak1 is targeted to the centrosome by the addition of a centrosomal targeting domain, Pak1 is activated at the centrosome as well, suggesting that targeting to the centrosome is sufficient to drive Pak activation.

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The molecular mechanisms underlying GTPase-independent Pak activation is still unclear. At this point, it cannot be excluded that initial disruption of the Pak1 dimers in the studies mentioned above is mediated by a Rac/cdc42 subfamily GTPase other than Rac1 or cdc42 (Lu and Mayer, 1999) and that Pak phosphorylation by PKD1/Akt or other yet unidentified kinases cooperate in Pak1 activation. A role for sphingosine in making the activation loop accessible to phosphorylations by other kinases is possible as well (Zenke et al., 1999). Alternatively, ‘‘dimer breathing’’ has been proposed, in which the kinase domain is temporarily released from the inhibitory switch domain, thus allowing activation in a Rho-GTPase independent manner (Loo et al., 2004).

4.4. Inactivation As in all signaling pathways, it is important for cellular homeostasis that activating signals are counteracted by inactivating signals. Consistent with this notion are findings that the activity of Pak is tightly regulated: Pak activation in response to stimuli peaks within 15 seconds and returns to base levels after 3 minutes (Huang et al., 1998). The initial Pak-activating signals, i.e., GTP-bound Rho-GTPases are rapidly inactivated through the action of GTPase activating proteins (GAPs). The inactivation of Rho GTPases is however unlikely to deactivate Pak, since autophosphorylated Pak has a decreased affinity for the GTP-bound forms, and GTP-bound Rho GTPases are thought to be released from Pak upon its activation (Manser et al., 1994). Indeed, it was shown that separate signals activate and deactivate Pak (Huang et al., 1998). Several proteins have been implicated in the negative regulation of Pak. Two serine/threonine phosphatases of the PP2C family, POPX1 and POPX2, directly interact with PIX and form a heterotrimeric complex with PIX and Paks. POPX1 and POPX2 dephosphorylate and downregulate Pak activity, most likely by dephosphorylating the Thr423 residue (Koh et al., 2002). The phosphorylation and activation of Pak may also target it for degradation by the proteosome. Interestingly, this process is mediated by the small GTPases Chp or cdc42 (Weisz Hubsman et al., 2007). Since these GTPases also activate Pak1, these findings suggesting a dual role for Chp and cdc42 as both activators and as negative feedback regulators of Pak1. A third mechanism of negative regulation of Pak can be accomplished by blocking its activation, which can be mediated by a number of proteins. Caveolin (Kang et al., 2006), nischarin (Alahari et al., 2004), CRIPak (Talukder et al., 2006) and hPIP (Xia et al., 2001) all bind to Pak and prevent the activation of Pak by GTPbound Rac/cdc42. Also, even though one study implicated G protein b/g subunits upstream of Pak activation (Menard and Mattingly, 2004), G protein b/g subunits have also been implicated in Pak inhibition (Wang et al., 1999). Currently, it is unknown how the activity and expression of

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any of the Pak inhibitors is regulated. Clearly, this knowledge is required to fully understand the roles of Pak in regulation of wound healing and other processes.

5. Pak Activation During Wound Healing and Epithelial Sheet Migration 5.1. Background Since Rac is crucial for wound repair, it seems likely that Pak kinases are important regulators of this process. Indeed, several lines of evidence have implicated Pak1 in the regulation of developmental epithelial sheet movements or wound healing. During Drosophila dorsal closure, the Pak family member Dpak accumulates in the leading edge cells (Harden et al., 1996) where it is required for the integrity of the actin cytoskeleton and for epithelial sealing (Conder et al., 2004). Even though loss-of-function Dpak mutants survive, they are sterile (Hing et al., 1999) and have various defects in the follicular epithelium that covers the egg chamber, including a loss of apical-basolateral polarity (Conder et al., 2007). In C. elegans embryos, the Pak homologue CePak is highly expressed at hypodermal cell boundaries and regulates embryonic body elongation by controlling an actin-dependent process called hypodermal fusion (Chen et al., 1996). Pak1 may also have a role in epithelial morphogenesis during mammalian embryonic development since high levels of a phosphorylated form of Pak1 have been found in developing epithelial organs such as the lung, kidney, intestine and skin (Zhong et al., 2003). Furthermore, many in vitro studies in mammalian cells have demonstrated a role for Pak in cell migration during scrape wound healing or in cell migration of single fibroblasts or epithelial cells. Together, these studies support the hypothesis that Paks play important roles in the regulation of the cytoskeleton. (Bokoch, 2003). One of the clues that Pak1 is involved the regulation of the actin cytoskeleton and cell motility came from observations that a constitutively active form of Pak1, the phosphomimetic Pak1-T423E, induces lamellipodia and increases migration in 3T3 fibroblasts (Sells et al., 1997). Later studies showed that silencing Pak1 expression with small interference RNA (siRNA) inhibits fibroblast migration (Rhee and Grinnell, 2006). Consistent with a role in regulating actin polymerization at the leading edge, Pak1 distributes from the cytosol to the cortical actin in lamellipodia in v-Src
transformed fibroblasts or in normal cells during wound healing or after PDGF stimulation (Dharmawardhane et al., 1997). In breast cancer cells, Pak1 relocalizes to the leading edge of motile cells and promotes invasiveness in response to heregulin treatment (Adam et al., 2000). Finally, numerous other studies have demonstrated that Paks also localize to focal

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contacts, which stabilize the protrusions at the leading edge (Brown et al., 2002; Frost et al., 1998; Manser et al., 1997; Obermeier et al., 1998; Sells et al., 1997, 2000; Stofega et al., 2004; Zegers et al., 2003a). As will be discussed in a later section, this localization likely reflects their regulatory role in the formation and turnover of focal adhesions.

5.2. Activation of Pak by wounding-associated signals Growth factors and cytokines that are released during epithelial wound healing are able to activate Pak. Numerous studies have reported stimulation of Pak by PDGF in many different cell types, likely by a PDGF-induced activation of Rac (Dharmawardhane et al., 1997; Sells et al., 2000; Yoshii et al., 1999). At least one study reported that Pak1 activation by PDGF relies on bPIX (Lee et al., 2001). PDGF may also activate Pak1 through transactivation of the EGF receptor by the active PDGF receptor (He et al., 2001). Direct stimulation of EGF receptor family receptors with heregulin (Adam et al., 1998) or EGF (Galisteo et al., 1996) also activates Pak1. In addition, other growth factors, including hepatocyte growth factor (Royal et al., 2000) and VEGF (Stoletov et al., 2001) have been reported to activate Pak in epithelial and endothelial cells respectively. Even though several different growth factors can activate Pak, the downstream signaling response appears to some extent dependent on the specific growth factor. For instance, stimulation of HeLa and NIH-3T3 cells with either PDGF or EGF leads to activation of Pak1 and Pak2, but only PDGF stimulation links Pak kinases to extracellular-regulated kinase (ERK) activation (Beeser et al., 2005). The strong correlation of Pak signaling downstream of PDGF signaling is of particular importance for wound healing from a clinical standpoint. PDGFBB, a recombinant form of PDGF comprising PDGF b-chain homodimers, is currently the only FDA-approved growth factor in clinical use to accelerate wound healing (Harrison-Balestra et al., 2002; Papanas and Maltezos, 2007). Understanding the roles of Pak downstream of PDGF-signaling is therefore highly relevant for the development of future options for the treatment of wounds. Interestingly, activation of Paks upon in vitro scrape wounding does not rely on the addition of exogenous growth factors, and it currently unclear which wounding-induced signals are responsible for Pak activation. It is possible that factors released from damaged cells are involved. As an alternative, shear stress, which activates integrin signaling, or alterations or absence of cell-cell and cell-matrix contacts may be involved as well. Integrinmediated signaling is well known to activate Rho-GTPases and b1-integrin mediated attachment to extracellular matrix is required for GTP-bound Rac to interact with Pak1 and to activate the kinase (Chaudhary et al., 2000; del Pozo et al., 2000; Howe, 2001; Price et al., 1998). Activation of Pak1 is

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specific for some matrix-integrin interactions. For instance, Pak1 activity is induced by shear stress in endothelial cells plated on fibronectin, but not in cells plated on Matrigel basement membrane or on collagen I (Orr et al., 2007). Furthermore, cell attachment to laminin-332 through a3b1 integrin activates Pak1, whereas a2b1 integrin-mediated attachment to collagen I by did not influence Pak1 activation (Zhou and Kramer, 2005). With respect to these two latter studies, it is relevant to note that fibronectin is an important component of the wound provisional matrix (Martin, 1997; Singer and Clark, 1999), whereas laminin-332 is an important regulator of wound healing which synthesis is induced by both shear stress (Avvisato et al., 2007) and upon epithelial injury in vitro and in vivo (Mak et al., 2006; Schneider et al., 2007). Taken together, it seems likely that integrin signaling is an important factor in wounding-induced Pak1 activation.

5.3. Kinase-independent functions and Pak-interacting proteins Presently, over 40 different kinase substrates of activated Paks have been identified (Bokoch, 2003; Kumar et al., 2006). Pak substrates comprise a diverse group of proteins, many of which have been implicated in the regulation of the cytoskeleton. Pak effectors also include several transcriptional regulators and signaling proteins involved in regulation of cell proliferation and cell death. As will discussed below, many of the Paks’ functions in wound healing-related processes depend on its functional catalytic domain and involve phosphorylation of specific substrates. In addition, Pak has functions that do not rely on its catalytic activity. Numerous studies have demonstrated that at least some of the Paks’ morphological effects, such as stimulation of cell motility or the formation of actin-based structures like lamellipodia, invadapodia and podosomes are kinase-independent (Furmaniak-Kazmierczak et al., 2007; Manser et al., 1997; Sells et al., 1997, 1999; Webb et al., 2005; Zegers et al., 2003a). In addition, kinaseindependent transcriptional regulation by Pak has been reported (Hullinger et al., 2001). The kinase-independent effects of Pak depend on SH3-domain
containing proteins that bind to one of the several PxxP-containing motifs (in which x is any amino acid) within the Pak N-terminus (Frost et al., 1998). Two of such proteins are the SH3/SH2-domain
containing proteins adaptor proteins Nck and Grb2, which link activated receptor tyrosine kinases to intracellular signaling molecules through their SH2 and SH3 domains respectively. Several growth factor receptors that play important roles in in vivo wound healing interact with Pak1 through these adaptor proteins. Nck interacts with the first PxxP motif of Pak1 and links it to tyrosine-phosphorylated PDGF or EGF receptors or to activated integrins,

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thereby recruiting the kinase to the plasma membrane (Bokoch et al., 1996; Galisteo et al., 1996; Howe, 2001; Lu et al., 1997). In the keratinocyte cell line HaCaT, Grb2 recruits Pak1 to the plasma membrane by coupling it to the activated EGF receptor. When the interaction of Pak1 with Grb2 is inhibited with an inhibitory SH3-containing peptide, EGF-mediated lamellipodia extension is blocked, indicating a crucial role for Pak1 in this process (Puto et al., 2003). Using a similar approach, it was shown that the Pak-Nck interaction is important for endothelial cell migration and angiogenesis (Kiosses et al., 2002). Though membrane recruitment by adaptor proteins has generally been implicated in Pak activation (Galisteo et al., 1996; Lu et al., 1997), it is possible that the kinase-independent effects of Pak are mediated by Pak acting as a scaffold. As Nck and Grb2 bind exclusively to the first and second PxxP motif of Pak1 (Bokoch et al., 1996; Galisteo et al., 1996; Puto et al., 2003), respectively, and PIX binds to a third central proline-rich domain in Pak (Manser et al., 1998), it is possible that Pak integrates different signaling pathways during wound healing. Consistent with this notion are data from several studies that demonstrated the formation and membrane recruitment of Nck-PakPIX
containing protein complexes in response to either PDGF-stimulation (Yoshii et al., 1999), or as a result of cell-matrix adhesion and integrin signaling (Brown et al., 2005; Zhao et al., 2000a). Furthermore, Nck and Grb2 likely indirectly associate with PIX via GIT, as both the Nck and Grb2 SH2 domains directly bind to GIT when GIT is tyrosine-phosphorylated (Brown et al., 2005).

5.4. The PIX-GIT complex Of all the Pak-interacting proteins, PIX and its binding partner GIT have been studied most extensively and appear to be crucial for many of the Paks’ functions. aPIX and bPIX (Pak-interacting exchange factor) and the identical p85cool-1 and p85cool-2 were first identified as Pak-binding proteins and interact with a specific proline-rich domain in Paks through a SH3 domain (Bagrodia et al., 1998; Manser et al., 1998). Based on the presence of tandem DH/PH (Dbl homology/Pleckstin homology) domains, a conserved characteristic of Rho-GTPase GEF proteins, the PIX proteins were predicted to exhibit GEF activity. However, while GEF activity of aPIX towards cdc42 and Rac could be readily demonstrated (Feng et al., 2002), it is still uncertain to what extent bPIX exhibits GEF activity towards these GTPases. In vitro GEF assays have indicated that bPIX contains an autoinhibitory domain, and GEF activity towards cdc42 could only be demonstrated upon deletion of this domain (Feng et al., 2002). Data from some studies suggest that phosphorylation of bPIX by Pak2 or protein kinase A can relieve bPIX from its autoinhibitory state and allows it to act

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as a GEF in vitro (Chahdi et al., 2005; Shin et al., 2002, 2004), but evidence for such a mechanism at the molecular level is still lacking. Resolving this question has been further complicated by findings that aPIX and bPIX can both homo- and heterodimerize and that specificity of GEF activity of aPIX depends on its dimerization state (Feng et al., 2002, 2004). Currently, it is unclear whether its ability to activate Rac and/or cdc42 is the main role of PIX proteins. As many Pak functions depend on its ability of Pak to interact with PIX, it is possible that one of PIX functions is to recruit Pak to specific intracellular sites. Furthermore, PIX appears to facilitate the formation of large oligomeric complexes that function in the regulation of focal adhesions (see V.A-2). Indeed, PIX tightly binds to the highly homologuos family of G protein-coupled receptor interacting target (GIT) proteins, which comprises GIT1 (or Cat1/p95-APP1), GIT2 (or Cat2) and p95PKL (or p95APP2) (Bagrodia et al., 1999; Di Cesare et al., 2000; Paris et al., 2003; Premont et al., 1998, 2000; Turner et al., 1999). GITs are multidomain proteins. At their N-terminus, they contain an ARF-GAP domain, which exhibits activity towards several different small GTPases of the ARF family. They furthermore contain a Spa2-homology motif, which is required for its interaction with PIX proteins, a coiled-coil motif that mediates homo- and heterodimerization of GIT1 and GIT2 and a C-terminal paxillin-binding site, which binds the focal adhesion protein paxillin. In many migrating cells, Pak is recruited to focal contacts through a complex that forms through the sequential interactions with PIX, GIT and paxillin (Bagrodia et al., 1998, 1999; Manser et al., 1998; Turner et al., 1999; Zhao et al., 2000b). This complex plays important roles in cytoskeletal dynamics and cell motility. The dynamic assembly and disassembly of the complex, which we will call here Pak-PIX-GIT complexes, is highly regulated. The functions of Pak-PIX-GIT complexes are still not completely understood, but they appear to be involved in many aspects of epithelial wound healing, as will be discussed in the following sections.

6. Regulation of Wound Healing Downstream of Pak Although many studies have implicated Pak in the regulation of epithelial wound healing and sheet migration, knowledge about the molecular mechanisms by which Pak controls these processes is only beginning to emerge. As discussed earlier, wound healing occurs via distinct steps, and depends on many different interconnected signaling pathways. Here, an attempt is made to review the specific molecular mechanisms by which Pak regulates these distinct steps.

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6.1. Cell motility and sheet migration 6.1.1. Cell polarization One of the first processes during wound healing is the polarization of the actin cytoskeleton. In response to an extracellular migration signal, cells polarize and extend protrusions such as lamellipodia or filopodia in the direction of migration. Migratory cell polarization involves numerous interconnected signaling pathways and both positive and negative feedback loops that involve integrins, growth factor receptors, Rho GTPases and phosphoinositidemediated signaling. Pak kinases regulate cell polarity in many different organisms. In yeast, the Pak homologs Ste20 and Cla4p are required for polarized actin assembly during bud formation and cytokinesis (Eby et al., 1998; Holly and Blumer, 1999), while Pak induces actin polarization during directed cell migration of Dictyostelium and Entemoebe amoebas. In mammalian organisms, Paks regulates polarized actin rearrangements during many different cellular processes, including cell migration and polarized actin assembly that occur at the immunological synapse and during neurogenesis (Bokoch, 2003). Together, these findings suggest a rather direct role of Pak at the level of the cytoskeleton. 6.1.1.1. Lamellipodia extension Protrusion of lamellipodia involves the formation of a newly assembled actin meshwork. This is mediated by the Arp2/3 complex, which binds to the side or tip of an existing actin filament and nucleates and branches new filaments at the leading edge (Pollard and Borisy, 2003). Pak1 phosphorylates the p41-Arc subunit of the Arp2/3 complex, and phosphorylation of p41-Arc regulates its association with the Arp2/3 complex at actin nucleation sites at the leading edge of the cells (Vadlamudi et al., 2004). As a non-phosphorylatable mutant of p41-Arc slows cell migration in breast cancer epithelial cells, these data indicate a functional role for Pak1 in Arp2/3-regulated actin branching and lamellipodia extension (Vadlamudi et al., 2004). However, Pak1 may also inhibit the Arp2/3 complex by phosphorylating caldesmon, which increases the ability of caldesmon to compete with the Arp2/3 complex for actin binding (Morita et al., 2007). It remains to be determined whether these differences reflect cell type-dependent differences or that they may reflect different levels of regulation. Clearly, the activity of the Arp2/3 complex in actin branching needs to be spatially restricted to the leading edge of migrating cells, but it is currently unclear if the these apparent opposite roles of Pak are involved in the spatial restriction of the active Arp2/3 complex. In addition to its proposed role in actin nucleation by acting on the Arp2/3 complex, Pak may stabilize actin filaments. The actin depolymerizing factor/cofilin destabilizes actin filaments by severing actin filaments and by actin depolymerization. LIM kinase is activated by Pak1, and, upon activation, phosphorylates and inactivates cofilin, thus promoting actin

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filament stability (Edwards et al., 1999). The same study showed that a kinaseinactive LIM kinase abolishes many of Pak1-induced cytoskeletal changes and membrane ruffling. This suggests that Pak1 stabilizes lamellipodia by a mechanism that involves LIM kinase-mediated inactivation of cofilin. Caldesmon and tropomyosin are two actin-filament stabilizing proteins (Gunning et al., 2005; Hai and Gu, 2006) that have also been implicated in Pak-mediated stabilization of actin. In breast cancer epithelial cells, the kinase-dead Pak1K299R stabilizes F-actin filaments by causing an increased association of tropomyosin and caldesmon with actin stress fibers (Adam et al., 2000). Furthermore, Pak induces caldesmon phosphorylation in Rous sarcomatransformed fibroblasts (Morita et al., 2007) and in response to wounding in CHO cells (Eppinga et al., 2006). In the latter cells, wound healing is impaired in cells that express either the Pak-phosphomimetic or a nonphosphorylatable form of caldesmon (Eppinga et al., 2006). 6.1.1.2. Regulation of microtubules The polarization of the cortical actin at the leading edge during cell migration is accompanied by reorganization of the microtubule cytoskeleton. Though most attention has been focused on the dynamics of the actin cytoskeleton, recent work provided evidence that directional migration depends on microtubules as well, and that both components of the cytoskeleton are in fact tightly integrated during cell migration (Siegrist and Doe, 2007; Watanabe et al., 2005). Microtubules nucleate from their minus ends, which are generally located at the microtubule organizing center (MTOC). At their plus ends, they undergo phases of growth and shrinkage, known as dynamic instability. Plus ends can be captured at specific targets, often associated with the actin cytoskeleton, which prevents shrinkage and stabilizes the microtubules. In migrating cells, microtubule plus ends are selectively stabilized at the leading edge, where they can interact with the cortical actin. In addition, the MTOC usually reorients towards to direction of migration. Though reorganization of microtubules is likely not required for the protrusion of leading edge and migration per se, it is thought be essential for the positioning of the leading edge and persistent directional movement by stabilizing cell polarization of the migrating cell. Similar to the regulation of actin dynamics, Rho GTPases play important roles in the reorganization of microtubules during cell migration. Though a detailed understanding of the cross-talk between microtubules and Rho GTPases is only beginning to emerge and is reviewed in detail elsewhere (Fukata et al., 2003; Raftopoulou and Hall, 2004; Siegrist and Doe, 2007; Small et al., 2002; Small and Kaverina, 2003; Watanabe et al., 2005), several lines of evidence support a role for Pak family kinases in the regulation of microtubules. One of the mechanisms by which Pak regulates microtubules is by phosphorylating stathmin. Stathmin, also called oncoprotein 18 (Op18), binds a/b-tubulin dimers, thereby preventing tubulin polymerization and

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causing catastrophe, the rapid shrinkage of microtubule plus ends (Cassimeris, 2002). Microtubule destabilization by stathmin is inhibited by its phosphorylation on Ser16, which prevents binding of stathmin to tubulin. Several studies demonstrated that Pak1 is required for this phosphorylation (Daub et al., 2001; Wittmann et al., 2003, 2004). A later study showed that stathmin is a direct substrate of Pak1 in vitro, and that its phosphorylation on Ser16 by Pak1 results in a decreased ability of stathmin to inhibit tubulin polymerization in an in vitro assay (Wittmann et al., 2004). In vivo however, additional factors appear to be involved in stathmin phosphorylation (Wittmann et al., 2004), which would be consistent with reports that Pak1 is required but not sufficient for Rac1-mediated stimulation of microtubule growth at the leading edge of migrating cells (Wittmann et al., 2003). Both Pak1 (Zenke et al., 2004) and Pak4 (Callow et al., 2005) phosphorylate GEF-H1. GEF-H1 is a GEF for Rho whose activity is suppressed by binding to microtubules. As GEF-H1 can bind both actin and tubulin, it may locally integrate regulation of the actin and microtubule cytoskeleton by a spatial control of Rho activation (Krendel et al., 2002). Phosphorylation of GEF-H1 by Pak4 causes its release from microtubules in NIH 3T3 cells, which co-incided with a dissolution of stress fibers (Callow et al., 2005). In contrast, phosphorylation of GEF-H1 by Pak1 on an analogous Ser residue did not affect the association of GEF-H1 with microtubules in a study using HeLa cells. Rather, this study showed that Pak1-mediated GEF-H1 phosphorylation results in binding of the scaffold protein 14-3-3 to GEF-H1, thereby recruiting 14-3-3 to microtubules, which could potentially affect GEF-H1 function (Zenke et al., 2004). In addition to the potential roles of Pak in microtubule stabilization, Pak has also been implicated in the regulation of centrosomes and the centrosomal MTOC during mitosis. Pak1 is targeted to the MTOC of mitotic cells, which leads to its activation, as revealed by immunofluorescent staining of an antibody specific for rat Pak1 phosphorylated at Thr422 (Zhao et al., 2005). Furthermore, inducible overexpression of an active analogous human Pak1 phosphomimetic (Pak1-T423E) in epithelial breast cancer cells induces mitotic spindle abnormalities such as multiple spindles (Vadlamudi et al., 2000). The aberrant spindles may be due to phosphorylation of tubulin cofactor B by Pak1, as overexpression of tubulin co-factor B, but not expression of forms that cannot be phosphorylated by Pak1, gives rise to a similar phenotype (Vadlamudi et al., 2005). Alternatively, the phenotype may be mediated through Aurora A. Aurora A is a kinase that has been implicated in centrosome maturation and centrosomal microtubule assembly (Brittle and Ohkura, 2005), and was recently shown to be activated by Pak1 at the centrosome (Zhao et al., 2005). Finally, Pak1, but not Pak2 or Pak3 (Thiel et al., 2002), can be phosphorylated on T212 via p35/cdk5 kinase in neuronal cells (Nikolic et al., 1998), or by cyclinB1/cdc2 in mitotic fibroblasts and other cells (Banerjee et al., 2002; Thiel et al., 2002). This phosphorylation,

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which does not affects Pak1 activity (Thiel et al., 2002), targets Pak1 to the MTOC, where it has been implicated in microtubule destabilization during mitosis (Banerjee et al., 2002). Pak’s effect on microtubule stability and its association with the MTOC would suggest important roles of the kinase on microtubule organization during cell migration. However, to date stathmin is the only Pak substrate directly implicated in the regulation of directional motility (Wittmann et al., 2003). It is currently unclear to what extent other Pak substrates are involved in the control of microtubule polarization and MTOC reorientation during migration and wound healing. Recent studies in fibroblasts and astrocytes have indicated that polarized microtubule stabilization is initiated by localized activation of Rac and Cdc42 at the leading edge but does not involve Pak. Instead, upon activation, Cdc42 mediates the reorientation of the MTOC through a pathway that involves the Cdc42 effector PAR6, which forms a complex with PAR3 and the atypical PKC-zeta (Cau and Hall, 2005; Etienne-Manneville and Hall, 2001). Formation of polarized actin-based protrusions in response to active Cdc42 on the other hand, was reported to be regulated independently of PAR3/PAR6/PKC-zeta, through a pathway that did depend on a Pak1-dependent recruitment of bPIX at the leading edge (Cau and Hall, 2005). The Pak-bPIX complex may then facilitate downstream activation of Rac and/or cdc42, which in turn may control spatial actin reorganization through downstream effectors, which may include Pak. It is possible that these data are cell type-dependent, considering the diverse functions of Pak in regulating microtubule stabilization in other cells. Also, as will discussed below, Pak may be involved in the polarization of microtubules through its interaction with bPIX. Furthermore, Pak can directly regulate atypical PKC-zeta in prostate carcinoma cells where PKC-zeta constitutively associates with Pak1 and is phosphorylated in a Pak1-dependent manner (Even-Faitelson and Ravid, 2006). Thus, these findings may suggest that PKC-zeta and Pak can integrate Cdc42 signaling to microtubules and filaments respectively. Finally, it is relevant to note that all studies discussed above were done in either non-epithelial cells or in epithelial cells that lacked apico-basolateral polarity. In such cells microtubules radiate out from a perinuclear MTOC, which is often, but not always, oriented towards the leading edge (Salaycik et al., 2005). In contrast, in polarized epithelial cells (i.e., cells with apicalbasolateral polarization), microtubules do not radiate from a centrosomal MTOC. Rather, they are organized in parallel arrays, in which the minus ends are associated with the apical membrane, and the plus ends extend towards the basal surface (Fukata et al., 2003; Luders and Stearns, 2007). This organization must undergo significant changes upon wounding since epithelial cells at wound edges exhibit a radial organization similar to that observed in non-polarized cells. How the transitions in microtubule organization during wound healing are regulated is unclear, and it would be

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important to know if the downstream effectors of Pak are involved in this process. In that respect, it is interesting to note that MARK2/Par-1 induces a change from a parallel organization to an organization in which microtubules nucleate from a single MTOC in the MDCK cell line (Cohen et al., 2004). In neuronal cells, MARK2/Par-1 destabilizes microtubules by phosphorylating tau, causing its dissociation from microtubules (Nishimura et al., 2004). Pak5 binds MARK2/Par-1 and when both molecules are overexpressed in CHO cells, Pak5 counteracts the function of MARK2/Par-1, thereby stabilizing microtubules. Though it is tempting to speculate that Pak may be involved in regulating parallel or radial microtubule organizations, the kidney-derived MDCK are unlikely to express Pak5 (Dan et al., 2002), and it remains to be established if other Pak forms are involved in this process. 6.1.1.3. The Pak-PIX-GIT complex in cell polarization PIX-GIT
containing complexes may regulate cell polarization by recruiting other cell polarity protein complexes. Three major protein complexes that localize at apical cell junctions and control epithelial polarization were initially identified in Drosophila and C. elegans. The general function and key components of these complexes are highly conserved in different vertebrate and invertebrate organisms. The Par3/Par6/aPKC complex is recruited to cadherin-based junctions and appears to initiate formation of the apical membrane. Maintenance of ‘‘apical identity’’ of the apical membrane is mediated by the Crb/Stardust complex, which antagonizes the function of the Lgl/Dlg/Scrib complex. This Lgl/Dlg/Scrib complex is proposed to generate and maintain basolateral identity by counteracting Par3/Par6/ aPKC function (Nelson, 2003). Although these three complexes have been mostly implicated in apical-basolateral polarization, it has become increasingly clear that they also function in other types of cell polarization. For instance, the Par3/Par6/aPKC and the Lgl/Dlg/Scrib complexes engage in bidirectional signaling with Rho GTPases and have been implicated in regulation of cell polarization during migration (Humbert et al., 2006). Mass spectrometry analysis of proteins that co-immunoprecipitate with Scrib in mammary epithelial cells identified bPIX (and associated GIT1) as a main binding partner of Scrib (Audebert et al., 2004). Recent studies indicate that Scrib plays a crucial role in directional motility and epithelial wound healing by a mechanism that depends on bPIX. Previously it was found that loss of the Drosophila forms of Scrib and Dlg results in defects of dorsal closure (Bilder et al., 2000), while mice that carry Scrib mutations exhibit defects in embryonic fusion events such as eyelid- and neural tube closure (Murdoch et al., 2003; Zarbalis et al., 2004). These observed effects on sheet migration suggest a potential role of these proteins in wound healing. Indeed, expression of mutant mammalian Scrib was recently shown to inhibit epidermal wound healing in an in vivo mouse model (Dow et al., 2007). It appears that deregulation of migratory polarity

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underlies these defects. In scrape wound healing assays, bPIX and Scrib are recruited to the leading edge in mammary epithelial cells (Dow et al., 2007) and astrocytes (Osmani et al., 2006). Knockdown of Scrib expression significantly interferes with migratory polarization of these cells; it blocks recruitment of bPIX, cdc42 (Osmani et al., 2006) and Rac (Dow et al., 2007) to lamellipodia and results in a loss of polarized actin and microtubule organization and directional motility. In astrocytes, it also inhibits wounding-induced activation of cdc42 (Osmani et al., 2006). Interestingly, whereas knockdown of Scrib abolishes sheet migration in mammary epithelial cells, it does not affect general rates of cell motility when the cells are subconfluent (Dow et al., 2007), suggesting functional cross-talk with cell-cell adhesions. Subsequent experiments suggested that the phenotype of Scrib knockdown cells depends on the interaction of Scrib with bPIX. This conclusion was based on findings that knockdown of bPIX or expression of bPIX mutants that lack the Scrib binding motif or the DH domain (required for GEF function) phenocopied the Scrib knockdown phenotype (Osmani et al., 2006). Taken together, these data are consistent with a model in which Scrib and bPIX-GIT1 complexes recruit and regulate the activation of Rac and cdc42 at the leading edge of migrating cells. The active Rac and cdc42 in turn, may then induce cytoskeletal rearrangements and lamellipodia formation by activating effector proteins, including, quite likely, Pak kinases. It must be noted that some of Scrib’s effects on cell migration may be context or cell-type dependent and/or appear to mediated by alternative mechanisms. For instance, while knockdown of Scrib results in a loss of directional migration in MDCK cells, it increases overall motility in these cells (Qin et al., 2005). In these cells, however, increased motility appeared to be caused by a destabilization of adherens junctions, which occurs independently of bPIX function. Thus, it is possible that Scrib-containing complexes with different compositions and/or distinct intracellular localizations have different and perhaps even opposite functions in directional migration. Such distinct functions have already been demonstrated for GIT1-containing complexes, which, depending on intracellular localization and molecular composition either promote or inhibit lamellipodia formation. Thus, while Pak1-PIX-GIT1
containing complexes, in association with paxillin, stimulate motility and protrusion of lamellipodia, likely by promoting activation of Rac at the leading edge of the cell, GIT1 inhibits Rac activation at the trailing edge when it is associated with a4 integrin through paxillin (Nishiya et al., 2005). As a consequence, GIT1 promotes cell polarization by mediating opposite effects at the leading and trailing edge of the cell. On a related note, even though the different GIT family members appear to interact equally well with paxillin and PIX, they may have distinct roles, as it was recently shown that GIT2, but not GIT1 represses motility in nontransformed mammary epithelial cells (Frank et al., 2006).

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6.1.2. Stabilization of cell protrusions and the dynamic regulation of focal contacts To promote cell migration, protrusions at the leading edge must be stabilized and anchored to the underlying extracellular matrix. The main proteins that mediate this process are integrins; heterodimeric matrix receptors that bind to different components of the extracellular matrix. At the inside of the cell, integrins link to the cytoskeleton. The connection of the extracellular matrix to the actin cytoskeleton allows the cells to exert traction forces, which are required to pull the cell forward. In addition, integrins are important signaling molecules that transmit intracellular signals upon binding to the extracellular matrix (‘‘outside in signaling’’), while their function is also being regulated by intracellular signals (‘‘inside out signaling’’). Key regulators of bidirectional integrin signaling are Rho GTPases (Schmitz et al., 2000; Wozniak et al., 2004; Yu et al., 2005). The formation and regulation of integrin-based adhesion sites is not completely understood. Upon adhesion, integrins are activated and cluster in focal complexes, in which many different multidomain proteins, including paxillin, interact and ultimately link to the actin cytoskeleton. Different types of integrin clusters exist: Focal complexes are relatively small, are found at the cell periphery, form by a mechanism that depends on Rac activity and exhibit high turnover rates (Ballestrem et al., 2001; Hall, 1998; Zaidel-Bar et al., 2003). Focal complexes are also thought to be the precursors of focal adhesions (Hall, 1998), which are larger, more stationary complexes that generally localize more distally and form in Rho-dependent manner. As differences between the two different integrin-based contact sites are not always obvious, I will use the term focal contact to refer to either complex. Focal contacts need to turn over to allow the cell to move forward and highly migratory cells tend to have many smaller focal contacts that turn over rapidly. Numerous studies have shown that Pak1-PIX-GIT
containing complexes are targeted to focal contacts and have implicated the complex in the dynamic regulation of these adhesion sites. The precise targeting mechanisms and functions of these proteins at focal contacts is still not entirely clear as apparently conflicting evidence have been reported. As mentioned earlier, Paks are activated in response to integrin-mediated adhesion (Chaudhary et al., 2000; del Pozo et al., 2000; Howe, 2001; Orr et al., 2007; Price et al., 1998; Zhou and Kramer, 2005). Activated Pak1 mutants localize to focal contacts (Kiosses et al., 1999; Sells et al., 1997; Stofega et al., 2004) and endogenous Pak1 is recruited to focal contacts upon its activation by PDGF or VEGF (Dharmawardhane et al., 1997; Sells et al., 2000; Stoletov et al., 2001) or following expression of active Rac1 or cdc42 mutants (Manser et al., 1997), although this latter study did not find that active Pak mutants localized to focal contacts (Manser et al., 1997). Conversely, many studies demonstrated that inactive Pak accumulates in focal adhesions. Thus, inhibition of Pak function by expression of the

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Pak-autoinhibitory domain or by expression of kinase-dead Pak results in recruitment of Pak in focal adhesions (Kiosses et al., 1999; Royal et al., 2000; Zegers et al., 2003a; Zhao et al., 2000a). How Pak is initially targeted to focal contacts is also matter of some debate. Pak can be recruited to focal contacts by both Nck (Kiosses et al., 1999; Zhao et al., 2000a) and PIX (Manser et al., 1998; Zegers et al., 2003a). PIX-dependent recruitment is likely mediated by sequential interactions of paxillin, GIT family proteins and PIX, in which GIT serves as a linker between paxillin and PIX (Brown et al., 2002, 2005; Manabe Ri et al., 2002; Turner et al., 1999; Zhao et al., 2000b). The precise function of the complex at focal contacts is still under investigation, but appears to be multifaceted. Overexpression of active Pak mutants leads to disassembly of focal contacts in some systems (Manser et al., 1997; Sells et al., 1997), but was not observed in endothelial or epithelial cells (Kiosses et al., 1999; Zegers et al., 2003a). In fact, Pak activity and recruitment to focal contacts is required for formation of these structures in VEGF-stimulated endothelial cells (Stoletov et al., 2001). Conversely, an increase of the number of large focal adhesions upon inhibition of Pak function has been widely reported in many cell types (Kiosses et al., 1999; Royal et al., 2000; Zegers et al., 2003a; Zhao et al., 2000a) (Fig. 6.2). In summary, although most studies are consistent with the hypothesis that active Pak promotes focal contact turnover, there is no straightforward correlation between the recruitment of Pak and its binding partners PIX and GIT to focal contacts, the formation of these structures, and the effect on cell motility. This may not be surprising, as motility depends on a tightly regulated balance of focal contact formation and breakdown. The functional effects of a disruption of this balance will likely depend on the spatial and molecular context of the complex. In that respect, it is relevant to note that the Pak-PIX-GIT complex is subject to different intermolecular interactions and posttranslational modifications. For instance, phosphorylation of Pak1 on Ser21 by Akt decreases the interaction of Pak1 with Nck, which leads to the release of Pak1 from focal contacts and an increase in cell motility (Zhou et al., 2003). Autophosphorylation of Pak1 also decreases its affinity for PIX and Nck binding (Manser et al., 1997; Zhao et al., 2000a), and induces disassembly of focal contacts and retraction of peripheral membrane, which suggests a potential inhibitory effect on migration. Finally, Pak1 phosphorylates paxillin on Ser273 (Nayal et al., 2006) and Ser709 (Webb et al., 2006), which increases the affinity of paxillin for GIT and promotes cell protrusion (Webb et al., 2006). Pak-mediated phosphorylation of paxillin also induces formation of small highly dynamic focal contacts that promote cell motility by a mechanism that depends on Pak-PIX and PIX-GIT interactions (Nayal et al., 2006). Taken together, the data appear to be consistent with the hypothesis that Pak-PIX-GIT complexes may promote formation of focal complexes by a

Pak / Vinculin

Pak

Wild type Pak1

Vinculin

Pak / Vinculin

Pak

Vinculin

Kinase-dead Pak1

Figure 6.2 Dominant-negative Pak1 accumulates in focal contacts in scrape-wounded epithelial cells. Image represents MDCK cells, which express wild type or a dominant-negative (Pak1-K299R, kinase-dead) under control of a tetracycline-regulatable promoter (Zegers et al., 2003a). Monolayers of MDCK cells expressing these HA-tagged forms of Pak1were scrape wounded.The next day, cells were fixed and stained using antibodies against the HA-tag and the focal contact marker vinculin. Note that wild type Pak1 localized to focal contacts to a limited extent, and localization is restricted to very peripheral focal contacts. In contrast, kinase-dead Pak1 accumulates at focal contacts and is found both peripheral and more distally within lamellipodia.

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mechanism that relies on the local, PIX-mediated activation of Rac at the leading edge. This mechanism may not require Pak activation, or perhaps relies on a partial activation, but could be mainly mediated by recruiting PIX to the leading edge. Full activation, mediated by the resulting local Rac activation, may subsequently lead to full Pak activation, which results in a release of Pak from focal contacts through its diminished affinity for PIX and Nck. Furthermore, activation of Pak will induce the degradation of focal contacts. How this latter process is regulated is still an open question. It is possible that degradation occurs through proteolytic cleavage by calpain, which is recruited to focal contacts by interacting with aPIX (Rosenberger et al., 2005). Also, as will discussed below, Pak can regulate myosin activity, which may be involved in adhesion disassembly (Crowley and Horwitz, 1995). Finally, microtubules have recently also emerged as regulators of focal adhesions (Palazzo and Gundersen, 2002). Hence, Pakmediated focal adhesion disassembly may also be regulated indirectly by through Pak’s diverse effects on microtubules. 6.1.3. Generation of traction forces The traction forces required to move cells forward are generated by the interaction of the non-muscle myosin II with actin filaments. Crucial to actin-myosin contractility is the phosphorylation of myosin II, which regulates both its association with actin and its motor activity. The phosphorylation of the regulatory myosin light chain (MLC) is controlled by myosin light chain kinase (MLCK). This kinase needs to be non-phosphorylated to be active, and phosphorylation of MLCK negatively inhibits the activity of the kinase. The role of Pak in the regulation of actin-myosin contractility has been somewhat controversial. One study provided evidence that Pak phosphorylates MLCK, thereby promoting dephosphorylation of MLC, thus potentially decreasing actin-myosin contractility (Sanders et al., 1999). Others however, showed that active Pak1 mutants lead to phosphorylation of MLC (Kiosses et al., 1999; Sells et al., 1999), and that MLC is a direct substrate of Pak1 (Bokoch, 2003). In addition to MLC phosphorylation, the myosin heavy chain (MHC) can be phosphorylated as well. The function of MHC phosphorylation in actinmyosin contractility is somewhat unclear, but at least for non-muscle myosin II-B, it may promote myosin filament assembly (Even-Faitelson and Ravid, 2006; van Leeuwen et al., 1999). Bradykinin-induced Rac activation results in MHC phosphorylation in PC12 cells, which is inhibited by dominantnegative Pak1. However, as active Pak1 mutants does not increase MHC phosphorylation, MHC may not be a direct substrate of Pak1 (van Leeuwen et al., 1999). In that respect, it was recently shown that Pak can mediate MHC phosphorylation through atypical PKC-zeta in the metastatic prostate carcinoma cell line TSU-pr1. In these cells, EGF stimulation drives the formation of protein complex containing Pak1, the atypical PKC-zeta, and the MHC of

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myosin II-B. Pak1 induces phosphorylation of PKC-zeta, and, upon stimulation with EGF, PKC-zeta phosphorylates myosin II-B directly, leading to slower filament assembly of myosin II-B (Even-Faitelson and Ravid, 2006). Since PKC-zeta has a clear role in directional cell migration (Cau and Hall, 2005; Etienne-Manneville and Hall, 2001), it would be of considerable interest to know how this interaction is regulated.

6.2. Regulation of cell proliferation by Pak, PIX and GIT 6.2.1. Positive regulation of mitogenic signaling Although the repair of minor wounds and other epithelial injuries relies on epithelial sheet migration and can occur independently of cell proliferation, healing of larger wounds is accompanied and critically depends on proliferation to replace lost cells (Mammen and Matthews, 2003; Martin, 1997; Singer and Clark, 1999). There is increasing evidence that Paks, in addition to their well-established roles in migration, play significant roles in the regulation of cell proliferation. As discussed previously, Paks are activated downstream of several mitogenic growth factors and interact with the EGF and PDGF receptors through adaptor proteins. Furthermore, Paks play important roles in growth factor-induced effects on cell migration. The canonical Raf!MEK!ERK pathway is well known for regulating cell proliferation in response to adhesion or growth factors, and appears to be regulated by Pak on several different levels. Pak is required for ERK activation and transformation by Ras (Tang et al., 1997), and both Raf-1 and MEK1 (King et al., 1998; Li et al., 2001; Slack-Davis et al., 2003; Sun et al., 2000) are believed to be direct substrates of Pak. ERK is activated upon scrape wounding in many cell types, and at least in some epithelial cells, the wounding-induced activation depends on an upstream activation of Src (Matsubayashi et al., 2004). Several groups showed that activated ERK localizes to focal contacts in fibroblasts and poorly differentiated epithelial cells (Fincham et al., 2000; Slack-Davis et al., 2003; Yin et al., 2005). Recently, it has become evident that Pak-PIX-GIT
containing complexes play a crucial role in recruiting and activating ERK at these sites. For instance, cell matrix-adhesion sequentially activates FAK, Src and Pak1. Active Pak1, in turn, phosphorylates MEK1 at S298, which primes MEK for its activation and allows for subsequent MEK and ERK activation (summary: adhesion!FAK!Src!Pak1!p-S298-MEK1!p-MEK1(S218/ S222, active)!p-ERK (T202,Y204, active) (Eblen et al., 2004; Slack-Davis et al., 2003). Moreover, GIT1, when phosphorylated by Src, is required for recruitment of ERK to focal adhesions and can bind both MEK1 and ERK2. Then, acting as a scaffold for MEK and ERK, GIT1 mediates sustained ERK activation at focal adhesions (Yin et al., 2004, 2005). Finally, Pak1 interacts with the MEK-ERK scaffold MP1, and this interaction is required for Pak1-mediated ERK activation (Pullikuth et al., 2005). The signaling pathway at focal contacts mentioned above and summarized

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here for simplicity as Src! GIT1/Pak1!MEK!ERK, is under control of several negative feedback steps. Specifically, ERK phosphorylates MEK on T292, which blocks the ability of Pak to activate MEK, and thus subsequent ERK activation. ERK also phosphorylates Pak1 on T212, which attenuates ERK signaling as well (Sundberg-Smith et al., 2005). On the other hand, ERK-dependent phosphorylation of bPIX and activation of Pak2 was also reported (Shin et al., 2002). Interestingly, available studies suggest that this type of Pak1-mediated ERK activation is particularly important in signaling downstream of cell-matrix adhesion. Although the same pathway has been reported to be activated in response to growth factors, the response seems specific for some, but not all growth factors that are known to activate ERK and may therefore be of lesser importance. Also, even though several groups have shown that Pak1 phosphorylates Raf-1 on S338, the most recent studies show that Raf is, at least in some systems, not required for Pak-mediated ERK activation (Beeser et al., 2005 and discussion therein). Mitogenic signaling by growth factors is under tight control. One of the negative regulators of EGF signaling is ubiquitin ligase Cbl, which binds and mono-ubiquitinates the activated EGF receptor (EGFR), thereby targeting it for endocytosis and degradation in the lysosome (Dikic, 2003). Recently, a series of studies showed that bPIX inhibits Cbl-mediated EGFR downregulation, thereby prolonging EGF signaling. EGF stimulation of cells induces a Src and Fak-dependent phosphorylation of bPIX on Tyr442, which stimulates its GEF activity toward Cdc42 (Feng et al., 2006), likely by releasing autoinhibitory constraints (Feng et al., 2002; Peterson and Chernoff, 2006). Upon phosphorylation, bPIX forms a complex with both activated Cdc42 and Cbl, thereby sequestering Cbl away from the EGFR, leading to an inhibition of EGFR endocytosis and degradation. As a result, EGFR-coupled signaling, such as the activation of ERK is sustained (Schmidt et al., 2006). Furthermore, these studies have provided evidence for an essential role of bPIX for cellular transformation and deregulated cell growth induced by either v-Src or Cdc42 (Feng et al., 2006; Wu et al., 2003). Though the effects on epithelial wound healing was not specifically addressed in these studies, others showed that either silencing of Cdc42 or overexpression of a Cbl mutant that cannot bind bPIX inhibits cell proliferation and wound closure in scrape wound healing assays in EGFR-overexpressing breast cancer cells (Hirsch et al., 2006). Based on these observations, a role for bPIX in EGF-stimulated wound healing seems likely. 6.2.2. Regulation of the cell cycle Many of the extracellular signals that regulate cell division are interpreted during the G1 phase of the cell cycle, which precedes DNA replication (S phase). Cyclin-dependent kinases are the major kinases that drive the cell cycle and are activated by cyclins. D-type cyclins, such as cyclin D1, control cyclin-dependent kinase 4 and are major regulators of cell cycle progression

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through the G1 phase. The expression of cyclin D1 is controlled by both mitogens and signaling pathways downstream of integrins. Studies with constitutive and dominant-negative Rho GTPases have shown that Rho GTPases are essential for progression through G1 (Olson et al., 1995). Rho GTPases appear to regulate the cell cycle through several different mechanism, which are not yet completely understood (Coleman et al., 2004). Several studies have indicated that Paks can induce cyclin D1. Pak is required for Ras-induced cyclin D1 expression (Nheu et al., 2004) and in normal and transformed epithelial cell lines, overexpression of wild-type and constitutively active Pak1 stimulates cyclin D1 promoter activity as measured by in vitro reporter assays. Furthermore, overexpression of active Pak induces an increase of cyclin D1 protein levels and accumulation of cyclin D1 in the nucleus (Balasenthil et al., 2004), whereas inhibition of Pak by siRNA-mediated knockdown, expression of the Pak1 autoinhibitory domain, or by a pharmacological inhibitor decreased cyclin D1 expression. Interestingly, cyclin D1 expression is also inhibited by a peptide that inhibits Pak-PIX interaction (Nheu et al., 2004), which may suggest a role for PIX in this process. On the other hand, another study demonstrated an inhibitory effect of Pak1 on cyclin D1 expression and cell cycle progression. The mechanism underlying this inhibition, which can be induced by overexpression of either wild-type or kinase-dead Pak1, is still unclear but is mediated by the domain that comprises the Pak-autoinhibitory domain (Thullberg et al., 2007). Surprisingly, the mechanism does not involve an inhibition of Pak kinase activity, since the same study showed that an inactive autoinhibitory domain elicits the same effect and that active Pak cannot rescue the defect. This suggests that inhibition of proliferation by the Pak autoinhibitory domain is mediated by a yet unidentified function of this domain in the control of cell proliferation. Pak phosphorylation on Thr212 is regulated in a cell cycle-dependent manner and markedly increases in mitotic cells (Banerjee et al., 2002; Li et al., 2002; Thiel et al., 2002). While it is unclear how this phosphorylation affects Pak activity, it is suggested to promote its association with centrosomes, as a Thr212-phosphomimic Pak peptide is targeted to centrosomes (Banerjee et al., 2002). Active endogenous Pak also localizes to centrosomes during metaphase where it phosphorylates the centrosomal Aurora A kinase (Li et al., 2002; Zhao et al., 2005). Finally, overexpression of an active Pak1 mutant interferes with normal spindle formation (Vadlamudi et al., 2000). Taken together, these data point to a regulatory role of Pak in spindle formation in mitotic cells. 6.2.3. Contact inhibition and Pak signaling at cell-cell contacts The roles of Pak in wound healing I have discussed in this review suggest that Pak functions primarily in the ‘‘start phase’’ of wound healing, i.e., the promotion of cell migration and proliferation upon initial wounding.

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However, in order to properly close a wound, this initial phase is temporary, and is followed by a ‘‘stop phase’’ ( Jacinto et al., 2001), in which cell migration and proliferation is inhibited and the epithelial cells regain their apico-basolateral polarization. Contact inhibition, the ability of cells to cease cell migration and proliferation upon the establishment of cell-cell contacts, is thought to be in important factor in the final stage of wound healing ( Jacinto et al., 2001). However, even though it has been known for over 50 years that cells can undergo contact inhibition, the mechanism underlying this process are still poorly understood (Abercrombie, 1979; Middleton, 1972; Stoker and Rubin, 1967). Most likely, signaling pathways that induce contact inhibition in epithelial cells are initiated by the E-cadherin-based adherens junctions, which form when adjacent cells come into contact with each other (Fagotto and Gumbiner, 1996). Rho GTPases are well known regulators of cell-cell junctional integrity and involved in many signaling pathways that are activated in response to cell-cell adhesion ( Jaffer and Chernoff, 2004). Thus, Rho GTPases may be important signaling intermediates in the regulation of contact inhibition. Several studies have indicated that Pak-PIX-GIT complexes are involved in adherens junction-related signaling. In epithelial and endothelial cell, Pak, PIX and GIT can be recruited to cell-cell contacts (Audebert et al., 2004; Orr et al., 2007; Stockton et al., 2007; Zegers et al., 2003a) but the precise functions of the complex at cell-cell contacts is still unclear and may be celltype dependent. In endothelial cells, shear stress induces recruitment of endogenous active Pak to cell junctions, where it promotes vascular permeability (Orr et al., 2007; Stockton et al., 2007). Others however proposed that activation of Pak and junctional recruitment of Pak, PIX and GIT in response to oxidized phospholipids enhances the barrier function of endothelial cells (Birukova et al., 2007a,b). Pak may negatively regulate contact inhibition by inhibiting the tumor suppressor protein Merlin. Merlin has high homology to members of the ERM (Ezrin-radixin-moesin) family of actin linker proteins, which link the cytoskeleton to the plasma membrane. When active, Merlin is in a closed conformation and acts as a growth suppressor by inducing contact inhibition through mechanisms that are not well understood (Okada et al., 2007). Pak phosphorylates Merlin on Ser518, which inactivates the protein and enables cell proliferation (Kissil et al., 2002; Xiao et al., 2002, 2005). Interestingly, Pak and Merlin engage in bidirectional signaling, as Merlin also inhibit Pak function and inhibits the recruitment and activation of Rac and Pak at the plasma membrane, which may be part of the mechanism by which Merlin regulates contact inhibition (Kissil et al., 2003; Okada et al., 2005; Shaw et al., 2001). Indeed, in endothelial cells, Pak1 activity reduces when cells reach confluency, and expression of an active membrane-targeted form of Pak1 is sufficient to release cells from contact inhibition of growth (Okada et al., 2005).

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On the other hand, Pak may also promote contact inhibition, since localization of Pak-PIX-GIT complexes at cell-cell contacts is required for the establishment of contact inhibition upon wound closure in epithelial cells (Zegers et al., 2003a). Using a model system of scrape-wounded MDCK cells, it was shown that the complex localizes to focal contacts in cells at wound edges, but is dramatically retargeted to areas of cell-cell contacts upon establishment of cell-cell contacts and wound closure. Inhibition of endogenous Pak1 blocks the ability of cells to undergo contact inhibition of proliferation by causing an accumulation of Pak1 and bPIX at focal contacts, which results in an inhibition of their recruitment to lateral membranes. Interestingly, although these cells are unable to undergo contact inhibition of proliferation, they still form adherens junctions and are able to polarize (Zegers et al., 2003a). This suggests that Pak-PIX-GIT complex may act as a sensor of extracellular environment, and acts as a signaling intermediate downstream of integrin in wounded epithelial cells, but downstream of E-cadherin upon wound closure. Such a dual role would be consistent with the apparent opposite roles of Rac, which is both necessary for cell migration (see Section 3.2) and for the establishment of adherens junctions (Van Aelst and Symons, 2002).

7. Concluding Remarks While it is obvious that Paks play important roles in the control of cellular behaviors that accompany and drive epithelial wound healing and sheet migration, many questions remain. An important outstanding issue is the spatiotemporal regulation of Pak function. This type of regulation likely entails both its spatiotemporal activation as well as the formation of distinct Pak-containing protein complexes. Clearly, many processes during wound healing need to be locally controlled, and a process of cell motility and cell polarization often depends on opposite behaviors at the leading versus the trailing edge of cells. As is obvious from the reviewed literature, a common theme of wounding-associated roles of Pak that are reviewed here are the often opposing effects that Pak appears to have in different cell types or under slightly different experimental conditions. One possible explanation of such findings is that most studies rely on approaches that modify Pak functions uniformly within the cell, and, it that light it may not be surprising that, depending on the context, opposite results can be obtained. A related question is how Pak is inactivated. While our knowledge of signals upstream of Pak is fairly extensive, our understanding of Pak inhibitors is scarce. Although several Pak inhibitory proteins, such as for instance the Pak phosphatase POPX, have been identified, insight into the regulation of these inhibitors is almost entirely lacking. Studies that specifically address

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Pak’s activation and deactivation in space and time, will undoubtedly lead to significant new insights in Pak biology and our understanding of epithelial wound healing.

ACKNOWLEDGMENTS I thank Martin ter Beest for critical comments on the manuscript. The work in my laboratory is funded by the NIH (GM076363) and the Concern Foundation.

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Tu, H., and Wigler, M. (1999). Genetic evidence for Pak1 autoinhibition and its release by Cdc42. Mol. Cell Biol. 19, 602–611. Turner, C. E., Brown, M. C., Perrotta, J. A., Riedy, M. C., Nikolopoulos, S. N., McDonald, A. R., Bagrodia, S., Thomas, S., and Leventhal, P. S. (1999). Paxillin LD4 motif binds Pak and PIX through a novel 95-kDa ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling. J. Cell Biol. 145, 851–863. Vadlamudi, R. K., Adam, L., Wang, R. A., Mandal, M., Nguyen, D., Sahin, A., Chernoff, J., Hung, M. C., and Kumar, R. (2000). Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells. J. Biol. Chem. 275, 36238–36244. Vadlamudi, R. K., Barnes, C. J., Rayala, S., Li, F., Balasenthil, S., Marcus, S., Goodson, H. V., Sahin, A. A., and Kumar, R. (2005). p21-activated kinase 1 regulates microtubule dynamics by phosphorylating tubulin cofactor B. Mol. Cell Biol. 25, 3726–3736. Vadlamudi, R. K., Li, F., Barnes, C. J., Bagheri-Yarmand, R., and Kumar, R. (2004). p41Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate. EMBO Rep. 5, 154–160. Van Aelst, L., and Symons, M. (2002). Role of Rho family GTPases in epithelial morphogenesis. Genes. Dev. 16, 1032–1054. van Leeuwen, F. N., van Delft, S., Kain, H. E., van der Kammen, R. A., and Collard, J. G. (1999). Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading. Nat. Cell Biol. 1, 242–248. Walter, B. N., Huang, Z., Jakobi, R., Tuazon, P. T., Alnemri, E. S., Litwack, G., and Traugh, J. A. (1998). Cleavage and activation of p21-activated protein kinase gamma-Pak by CPP32 (caspase 3). Effects of autophosphorylation on activity. J. Biol. Chem. 273, 28733–28739. Wang, J., Frost, J. A., Cobb, M. H., and Ross, E. M. (1999). Reciprocal signaling between heterotrimeric G proteins and the p21-stimulated protein kinase. J. Biol. Chem. 274, 31641–31647. Watanabe, T., Noritake, J., and Kaibuchi, K. (2005). Regulation of microtubules in cell migration. Trends Cell Biol. 15, 76–83. Webb, B. A., Eves, R., Crawley, S. W., Zhou, S., Cote, G. P., and Mak, A. S. (2005). PAK1 induces podosome formation in A7r5 vascular smooth muscle cells in a Pak-interacting exchange factor-dependent manner. Am. J. Physiol. Cell Physiol. 289, C898–C907. Webb, D. J., Kovalenko, M., Whitmore, L., and Horwitz, A. F. (2006). Phosphorylation of serine 709 in GIT1 regulates protrusive activity in cells. Biochem. Biophys. Res. Commun. 346, 1284–1288. Weisz Hubsman, M., Volinsky, N., Manser, E., Yablonski, D., and Aronheim, A. (2007). Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp. Biochem. J. 404, 487–497. Wittmann, T., Bokoch, G. M., and Waterman-Storer, C. M. (2003). Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J. Cell Biol. 161, 845–851. Wittmann, T., Bokoch, G. M., and Waterman-Storer, C. M. (2004). Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1. J. Biol. Chem. 279, 6196–6203. Woolner, S., Jacinto, A., and Martin, P. (2005). The small GTPase Rac plays multiple roles in epithelial sheet fusion–dynamic studies of Drosophila dorsal closure. Dev. Biol. 282, 163–173. Wozniak, M. A., Modzelewska, K., Kwong, L., and Keely, P. J. (2004). Focal adhesion regulation of cell behavior. Biochim. Biophys. Acta 1692, 103–119. Wu, W. J., Tu, S., and Cerione, R. A. (2003). Activated Cdc42 sequesters c-Cbl and prevents EGF receptor degradation. Cell 114, 715–725.

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Xia, C., Ma, W., Stafford, L. J., Marcus, S., Xiong, W. C., and Liu, M. (2001). Regulation of the p21-activated kinase (Pak) by a human Gbeta-like WD-repeat protein, hPIP1. Proc Natl. Acad. Sci. USA 98, 6174–6179. Xiao, G. H., Beeser, A., Chernoff, J., and Testa, J. R. (2002). p21-activated kinase links Rac/Cdc42 signaling to merlin. J. Biol. Chem. 277, 883–886. Xiao, G. H., Gallagher, R., Shetler, J., Skele, K., Altomare, D. A., Pestell, R. G., Jhanwar, S., and Testa, J. R. (2005). The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression. Mol. Cell Biol. 25, 2384–2394. Yang, J., Mani, S. A., Donaher, J. L., Ramaswamy, S., Itzykson, R. A., Come, C., Savagner, P., Gitelman, I., Richardson, A., and Weinberg, R. A. (2004). Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927–939. Yin, G., Haendeler, J., Yan, C., and Berk, B. C. (2004). GIT1 functions as a scaffold for MEK1-extracellular signal-regulated kinase 1 and 2 activation by angiotensin II and epidermal growth factor. Mol. Cell Biol. 24, 875–885. Yin, G., Zheng, Q., Yan, C., and Berk, B. C. (2005). GIT1 is a scaffold for ERK1/ 2 activation in focal adhesions. J. Biol. Chem. 280, 27705–27712. Yoshii, S., Tanaka, M., Otsuki, Y., Wang, D. Y., Guo, R. J., Zhu, Y., Takeda, R., Hanai, H., Kaneko, E., and Sugimura, H. (1999). alphaPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase. Oncogene 18, 5680–5690. Yu, W., Datta, A., Leroy, P., O’Brien L, E., Mak, G., Jou, T. S., Matlin, K. S., Mostov, K. E., and Zegers, M. M. (2005). b1-Integrin orients epithelial polarity via rac1 and laminin. Mol. Biol. Cell 16, 433–445. Zaidel-Bar, R., Ballestrem, C., Kam, Z., and Geiger, B. (2003). Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. J. Cell. Sci. 116, 4605–4613. Zarbalis, K., May, S. R., Shen, Y., Ekker, M., Rubenstein, J. L., and Peterson, A. S. (2004). A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development. PLoS Biol. 2, E219. Zegers, M. M., Forget, M. A., Chernoff, J., Mostov, K. E., ter Beest, M. B., and Hansen, S. H. (2003a). Pak1 and PIX regulate contact inhibition during epithelial wound healing. EMBO J. 22, 4155–4165. Zegers, M. M., O’Brien, L. E., Yu, W., Datta, A., and Mostov, K. E. (2003b). Epithelial polarity and tubulogenesis in vitro. Trends Cell Biol. 13, 169–176. Zeisberg, M., Hanai, J., Sugimoto, H., Mammoto, T., Charytan, D., Strutz, F., and Kalluri, R. (2003). BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat. Med. 9, 964–968. Zenke, F. T., King, C. C., Bohl, B. P., and Bokoch, G. M. (1999). Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity. J. Biol. Chem. 274, 32565–32573. Zenke, F. T., Krendel, M., DerMardirossian, C., King, C. C., Bohl, B. P., and Bokoch, G. M. (2004). p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor. J. Biol. Chem. 279, 18392–183400. Zhao, Z. S., Lim, J. P., Ng, Y. W., Lim, L., and Manser, E. (2005). The GIT-associated kinase Pak targets to the centrosome and regulates Aurora-A. Mol. Cell 20, 237–249. Zhao, Z. S., and Manser, E. (2005). Pak and other Rho-associated kinases—Effectors with surprisingly diverse mechanisms of regulation. Biochem. J. 386, 201–214. Zhao, Z. S., Manser, E., Chen, X. Q., Chong, C., Leung, T., and Lim, L. (1998). A conserved negative regulatory region in alphaPAK: inhibition of Pak kinases reveals their morphological roles downstream of Cdc42 and Rac1. Mol. Cell Biol. 18, 2153–2163.

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C H A P T E R

S E V E N

Biology and Biophysics of the Nuclear Pore Complex and Its Components Roderick Y. H. Lim,* Katharine S. Ullman,† and Birthe Fahrenkrog* Contents 1. Introduction 2. Nuclear Pore Complex Structure 2.1. Overall nuclear pore complex architecture 2.2. The nuclear pore complex at atomic level 2.3. Nuclear pore complex density and distribution 3. Nucleoporin Function(s) 3.1. FG-nucleoporins and nucleocytoplasmic transport 3.2. Nucleoporins and kinetochores 3.3. Nucleoporins and transcription 3.4. Nucleoporins, the immune system and Parkinson’s disease 4. Selective Cargo Translocation Across the Nuclear Pore Complex 4.1. The NPC as a selective gate 4.2. Current models of selective gating 4.3. In vitro studies of FG-domain function 4.4. In silico studies of the FG-domains and barrier function 4.5. Kinetic aspects of nucleocytoplasmic transport 4.6. Toward an understanding of FG-domain behavior in the NPC 5. Nuclear Pore Complex Assembly and Disassembly 5.1. Building a nuclear pore: Who’s on first? 5.2. Nuclear pore building blocks: The transmembrane proteins 5.3. Collaboration between nucleoporins in NPC assembly 5.4. Peripheral pore structures 5.5. Regulation of NPC assembly 5.6. Clues from a second site for NPC assembly 5.7. Nuclear pore assembly is never-ending 5.8. Deconstructing the NPC 6. Concluding Remarks

* {

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M.E. Mu¨ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00632-1

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2008 Elsevier Inc. All rights reserved.

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Acknowledgments References

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Abstract Nucleocytoplasmic exchange of proteins and ribonucleoprotein particles occurs via nuclear pore complexes (NPCs) that reside in the double membrane of the nuclear envelope (NE). Significant progress has been made during the past few years in obtaining better structural resolution of the three-dimensional architecture of NPC with the help of cryo-electron tomography and atomic structures of domains from nuclear pore proteins (nucleoporins). Biophysical and imaging approaches have helped elucidate how nucleoporins act as a selective barrier in nucleocytoplasmic transport. Nucleoporins act not only in trafficking of macromolecules but also in proper microtubule attachment to kinetochores, in the regulation of gene expression and signaling events associated with, for example, innate and adaptive immunity, development and neurodegenerative disorders. Recent research has also been focused on the dynamic processes of NPC assembly and disassembly that occur with each cell cycle. Here we review emerging results aimed at understanding the molecular arrangement of the NPC and how it is achieved, defining the roles of individual nucleoporins both at the NPC and at other sites within the cell, and finally deciphering how the NPC serves as both a barrier and a conduit of active transport. Key words: Nuclear pore complex, Nuclear envelope, Nucleoporins, Nucleocytoplasmic transport, Transmembrane proteins. ß 2008 Elsevier Inc.

1. Introduction In interphase eukaryotic cells, transcription takes place in the cell nucleus while proteins are synthesized in the cytoplasm. Exchange of material between these two cellular compartments occurs via nuclear pore complexes (NPCs) located in the double membrane of the nuclear envelope (NE). NPCs support passive diffusion of small molecules and ions and facilitate receptor-mediated translocation of proteins and ribonucleoprotein complexes. Overall, the vertebrate NPC is a
120 MDa protein complex made up
30 different proteins called nucleoporins (or Nups) that are repetitively arranged as distinct subcomplexes to form the NPC (Cronshaw et al., 2002; Lim and Fahrenkrog, 2006; Rout et al., 2000; Schwartz, 2005; Tran and Wente, 2006). In the plane of the NE, the eightfold symmetric central framework of the NPC embraces a central pore that is
50 nm long and is narrowest (
40 nm) at the NE midplane (Beck et al., 2004, 2007; Stoffler et al., 2003). Attached to the central framework are cytoplasmic filaments and a nuclear basket (Fig. 7.1). We begin, here, by reviewing recent advances towards the elucidation of NPC

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architecture at the ultrastructural and atomic level by electron tomography and X-ray crystallography. These inroads into NPC structure lay important groundwork for understanding the function of the nuclear pore and we will overview progress that has been made in our understanding how the NPC acts as a selective barrier for macromolecular cargo. We will also discuss recent insights into the function of individual nucleoporins in nuclear organization that go beyond their well-characterized role in nucleocytoplasmic transport. Last but not least, we will review recent progress in addressing how the NPC disassembles and assembles at the beginning and end of mitosis, respectively.

2. Nuclear Pore Complex Structure 2.1. Overall nuclear pore complex architecture The NPC is a highly complex structure and electron microscopy (EM) and, more recently, cryo-electron tomography (CET) have proven to be the methods of choice to study intact NPCs at high resolution. The NPC consists of an approximately cylindrical central framework, eight cytoplasmic filaments and a nuclear basket composed of eight filaments that join into

Cytoplasmic filaments

Nuclear envelope

Nuclear basket

Central framework

100 nm

Figure 7.1 Electron micrograph with partially overlaid schematic representation of a cross-sectioned nuclear pore complex. The major structural components include the central framework, the cytoplasmic filaments and a nuclear basket.

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a distal ring (Fig. 7.1). Early EM studies provided 3D reconstructions of the central framework using negatively stained and frozen-hydrated NPCs from Xenopus laevis oocyte NEs (Akey and Radermacher, 1993; Hinshaw et al., 1992) or frozen-hydrated yeast cells (Yang et al., 1998). The central framework of the NPC (also called the spoke complex) resides between the inner and outer nuclear membranes, anchored where these parallel membrane bilayers curve to meet each other. Early structural studies showed that cytoplasmic and nuclear ring moieties are integral to the central framework. Recent CET studies in Xenopus oocyte isolated nuclei (Stoffler et al., 2003) and in intact, transport-competent nuclei isolated from Dictyostelium discoideum (Beck et al., 2004, 2007) have improved the resolution of the central framework to 89 nm and revealed the first reconstructions of peripheral, flexible components of the NPC, i.e., the cytoplasmic filaments and the nuclear basket. In Dictyostelium, the cytoplasmic filaments have a length of
35 nm and the nuclear basket is about 60 nm long. Together with the
50 nm central framework, the NPC therefore has a total length of
150 nm with the outer diameter of the structure being 125 nm (Beck et al., 2004, 2007). The overall linear dimensions of the NPC varies between species, whereas the overall 3D architecture appears to be evolutionarily conserved (Fahrenkrog et al., 1998; Kiseleva et al., 2004; Yang et al., 1998). Enclosed by the central framework is the hourglass-shaped central pore of the NPC with a diameter of 6070 nm at its cytoplasmic and nuclear periphery and
45 nm in the midplane of the NPC/NE (Beck et al., 2004, 2007; Pante´ and Kann, 2002; Stoffler et al., 2003). This central pore mediates all exchange between the cytoplasm and the nucleus and enables transport of macromolecules with diameters of up to 39 nm (Pante´ and Kann, 2002). Increasing concentrations of signal-carrying cargoes selectively interferes with the passage of other molecules that utilize a facilitated pathway, but not with the diffusion of inert molecules and vice versa, suggesting that passive and facilitated transport across the NPC proceed via routes that are sterically nonoverlapping (Naim et al., 2007). Whether these two routes exist in the central pore, i.e., facilitated transport along the walls of the central pore and passive diffusion through a hollow diffusion tube located at the pore center (Peters, 2005), or whether passive diffusion might additionally utilize peripheral channels (Akey and Radermacher, 1993; Beck et al., 2004; Hinshaw et al., 1992; Stoffler et al., 2003) remains to be seen. Peripheral channels of the NPC have a diameter of
8 nm and have been implicated in the diffusion of small molecules and ions (Feldherr and Akin, 1997; Hinshaw et al., 1992) and/or in trafficking of integral membrane proteins to the inner nuclear membrane (Soullam and Worman, 1995). However, the more recent observation that the cytoplasmic openings of the peripheral channels are not topologically continuous with the nuclear openings (Stoffler et al., 2003) challenges the view that they act as transport channels. Other potential roles have been proposed, such as in

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maintenance the NE electrical conductance (Danker et al., 1999; Enss et al., 2003; Mazzanti et al., 2001; Shahin et al., 2001) or as buffer zones that accommodate deformations of the central framework upon translocation of large cargoes (Fahrenkrog and Aebi, 2003).

2.2. The nuclear pore complex at atomic level Based on secondary structure prediction, nucleoporins can be grouped into three classes (Devos et al., 2006). The transmembrane group, which contains transmembrane a-helices and a cadherin-fold, comprises the outermost features of the NPC central framework and at least some members of this group are thought to help anchor the NPC in the NE. The second group of nucleoporins contain b-propeller and a-solenoid folds and these nucleoporins localize towards the inside of the NPC, whereas the third class harbors the conserved sequence motif of phenylalanine-glycine (FG)repeats (see Sections 3 and 4) in combination with a coiled-coil fold and may contribute to the formation of the NPC’s inner central framework and the peripheral structures (Devos et al., 2006; Schwartz, 2005; Tran and Wente, 2006). Other less frequent structural motifs found in nucleoporins are zinc-finger domains as in Nup153 and RanBP2/Nup358 (Higa et al., 2007) or RNA-recognition motifs as in Nup35 (Handa et al., 2006). b-Propellers are predicted in a third of the nucleoporins, and in fact sevenbladed b-propellers have been resolved from the N-terminal domains (NTD) of the human nucleoporins Nup133 and Nup214 and its yeast homologue Nup159p by X-ray crystallography (Berke et al., 2004; Napetschnig et al., 2007; Weirich et al., 2004). Proteins with b-propeller folds participate in diverse cellular functions and serve as platforms for multiple dynamic protein– protein interactions. Along this line, yeast Nup133p and Nup159p both play roles in mRNA export from the nucleus, and deletion or mutations in their NTDs impair their functions in mRNA export, probably by preventing the association of multiple mRNA export factors with the NPC (Berke et al., 2004; Weirich et al., 2004). The NTD of human Nup133 furthermore contains an amphipathic a-helical motif capable of sensing membrane curvature (Drin et al., 2007). This motif corresponds to an exposed loop, which connects two blades of the b-propeller and folds into an a-helix upon interacting with small liposomes. Whether the membrane curvature sensor in Nup133 serves to recognize the topology of the nuclear pore membrane to anchor the NPC during interphase or to recognize vesicles or tubules containing NE fragments critical for NE reassembly after mitosis, or both, remains to be seen (Drin et al., 2007). The NTD of human Nup214, in comparison to its yeast homologue Nup159p, consists of two distinct structural elements: the b-propeller and a 30-residue C-terminal extended peptide segment (Napetschnig et al., 2007).

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This extension binds to the bottom of the b-propeller with low affinity and has been suggested to play an ‘‘autoinhibitory’’ role in NPC assembly. The first crystal structure obtained for a nucleoporin was the NPC targeting domain of human Nup98 (Hodel et al., 2002). This domain, similar to the nuclear pore targeting domain of its yeast homologue Nup116p, consists of a six-stranded b-sheet sandwiched against a two-stranded b-sheet and flanked by two a-helical regions (Hodel et al., 2002; Robinson et al., 2005). This domain exhibits multiple conformations and is stabilized only when bound to a ligand, i.e., Nup96 and Nup145p-C in the case of Nup98 and Nup116p, respectively (Robinson et al., 2005). Conformational diversity might allow Nup98 and Nup116p to bind to multiple targets within the NPC or to associate and dissociate fast from the NPC to increase the mobility of the nucleoporins, as described for Nup98, which shuttles in an transcriptiondependent manner (Griffis et al., 2002, 2004). The attempt to crystallize the first subcomplex of the NPC, the Nup62 complex, yielded the structure of the a-helical coiled-coil domain of one of its components, rat Nup58/45 (Melcak et al., 2007). Nup58/45 forms tetramers in the crystal structure consisting of two antiparallel dimers. Each dimer consists of two a-helices that are connected by a short loop. The intradimer interactions are of hydrophobic nature, whereas two dimers associate through hydrophilic residues. The tetramer can adopt various conformations leading to a lateral displacement between tetramers suggesting an intermolecular sliding mechanism (Melcak et al., 2007). The Nup62 complex has recently been mapped to the cytoplasmic periphery of the NPC’s central pore (Schwarz-Herion et al., 2007), so that sliding of Nup58/45, and most likely of Nup62 and Nup54 as well, could contribute to modulating the diameter of the central pore in response to transport activity (Melcak et al., 2007).

2.3. Nuclear pore complex density and distribution The number of NPCs per cell varies greatly with cell size and activity. Yeast cells have
200 NPCs, proliferating human cells
30005000 and a mature Xenopus oocyte
5  107 (Gorlich and Kutay, 1999). A comprehensive ultrastructural study using freeze-fracture EM of yeast cells in combination with 3D reconstruction has shown that the distribution of yeast NPCs in the NE is not equidistant, but rather clustered into regions of higher density (Winey et al., 1997). The number of NPCs was found to increase steadily, beginning in G1- and peaking in S-phase of the cell cycle, suggesting that NPC assembly occurs continuously throughout the cell cycle (see Section 5) (Winey et al., 1997). Similarly, the density of NPCs increases through-out the cell cycle in HeLa S3 cells (Maeshima et al., 2006). Interestingly, these HeLa S3 cells exhibit large subdomains in the NE devoid of NPCs. These ‘‘pore-free islands’’ are present in telophase and

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G1 nuclei and are enriched in the inner nuclear membrane proteins emerin and lamin A/C, but not lamin B (Maeshima et al., 2006). Knock-down of lamin A/C by RNAi resulted in the disappearance of the pore-free islands, whereas upregulation of lamin A/C facilitated the formation of pore-free islands. Although the physiological relevance of pore-free islands remains to be elucidated, in HeLa cells the presence of such regions correlates with lower proliferative activity. Consistent with this, embryonic cells lack lamin A/C and have a high density of NPC along with high proliferative activity (Maeshima et al., 2006; Maul et al., 1980). Further indication of a relationship between lamin expression and pore density was obtained using Xenopus oocytes, whose giant nuclei lack lamin A/C and exhibit a high density of NPCs. Overexpression of human lamin A in these oocyte nuclei leads to the appearance of stretches in the NE that are devoid of NPCs (B. Fahrenkrog and B. Maco, unpublished results; Fig. 7.2). Recent studies using mouse embryonic stem (ES) cells addressed the adaptation of NPC structure and density during cardiac differentiation (Perez-Terzic et al., 2003, 2007). Accordingly, NPC density increases somewhat when ES cells differentiate into proliferative cardiomyocytes.

Figure 7.2 Electron micrographs of cross sections along a nuclear envelope of isolated Xenopus oocyte nuclei. (A) The nuclear envelope of a stage 6 nucleus is characterized by a high density of nuclear pore complexes (black arrows). (B) Overexpression of human lamin A in these Xenopus oocyte nuclei causes a decrease in nuclear pore complex (black arrows) density and a thickened nuclear lamina (gray arrowheads). c, cytoplasm; n, nucleus. Scale bar,100 nm.

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With a few significant exceptions, genes encoding components of the nucleocytoplasmic transport machinery, i.e., nuclear transport receptors, nucleoporins and Ran-related factors, were found to be broadly down regulated in cardiomyocytes derived from ES cells compared to the undifferentiated cells, supporting the notion that changes in transport occur concomitantly (and maybe help to drive) differentiation. Further differences have been observed between stem cellderived cardiomyocytes and adult heart-isolated cardiomyocytes, which have a density of
15 NPCs/mm2 and
28 NPCs/mm2, respectively. While the overall diameter and height of the NPC are similar in both cell types, there is greater central density in the NPCs of stem cellderived cardiomyocytes, indicative of greater transport activity (Perez-Terzic et al., 2003). Drosophila Nup154, the homologue of rat Nup155, is essential for gametogenesis (Gigliotti et al., 1998) and regulated expression of a testisspecific isoform of RanBP2/Nup358, BS-63, and Nup50/Npap60 may also influence gamete/testis maturation (Hogarth et al., 2005). Another case in which the nuclear transport machinery appears to be involved in cellular fate is found in malignant cells resistant to chemotherapy (Lewin et al., 2007). Multidrug resistance commonly limits efficiency in treating malignant cells with chemotherapy and is classically described as a plasma membrane phenomenon. However, multidrug-resistant cells specifically exclude chemotherapeutic drugs from the nucleus and have now been shown to exhibit an increased number of NPCs compared to drug-sensitive cells (Lewin et al., 2007). The increase in NPC number somehow correlates with the exclusion of chemotherapeutic drugs from the nucleus, suggesting that nuclear export is selectively enhanced in the resistant cells. The mechanism by which NPCs export chemotherapeutic drugs remains elusive, but inhibition of nucleocytoplasmic transport with injection of wheat germ agglutinin can reverse multidrug resistance in these cells (Lewin et al., 2007). All together, these data indicate that regulation of NPC number, composition and nucleocytoplasmic transport may drive and influence more cellular processes than previously assumed.

3. Nucleoporin Function(s) 3.1. FG-nucleoporins and nucleocytoplasmic transport FG-repeat domains are found in about one third of the nucleoporins and mediate the interaction between soluble transport receptors loaded with signal-bearing cargo and the NPC. These FG-repeat domains also likely contribute to the selective barrier that limits diffusion through the NPC (see section 4). Atomic structures of FG-repeat peptides in complex with, for

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example importin b, NTF2 or the mRNA export factor TAP/NXF1, have consistently shown that the interaction between FG-repeats and the different transport receptors involves primarily the phenylalanine ring of the FG-repeat core and hydrophobic residues on the surface of the receptor. Hydrophilic linker between individual FG-motifs, which constitute the majority of amino acid mass in the overall FG-domain, appear to influence the strength of the binding and allow simultaneous binding of several FGcores to the receptor (Liu and Stewart, 2005). Based on biophysical measurements, the FG-repeat domains of yeast nucleoporins were found to be natively unfolded, i.e., having no or only little secondary structure. Similarly, FG-repeat domains of human, fly, worm and other yeast species are most likely disordered based on their amino acid composition (Denning and Rexach, 2007). This notion is further supported by immuno-EM studies on two vertebrate FG-repeat nucleoporins, Nup153 and Nup214, which suggested that FG-repeat domains are flexible and mobile within the NPC (Paulillo et al., 2005, 2006). Atomic force microscopy (AFM) studies on recombinantly expressed FG-repeat domain of human Nup153 further revealed that this
700 residue domain in fact is an unfolded molecule with a length of
180 nm, resembling an unfolded polypeptide chain (Lim et al., 2006b). Nup153 and Nup214 are both known to play roles in distinct nucleocytoplasmic transport pathways and to interact with a number of nuclear transport receptors via their FG-repeats (Ball and Ullman, 2005; Bernad et al., 2006; Hutten and Kehlenbach, 2006; Sabri et al., 2007; van Deursen et al., 1996). The location of the FG-repeat domains of Nup153 and Nup214 in the NPC shifts in a transport-dependent manner, further supporting their role in nucleocytoplasmic transport (Paulillo et al., 2005). Systematic deletion of FG-repeat regions in yeast nucleoporins revealed, however, that yeast NPCs are able to compensate the loss of
50% of their FG-repeats with only little effect on distinct nuclear transport pathways, indicating that FG-repeats are highly redundant within the NPC, that individual FG-nucleoporins appear critical for specific nuclear transport pathways but not for bulk nucleocytoplasmic transport and/or that other interaction sites for transport receptors exist within the NPC. Besides playing important roles in nucleocytoplasmic transport, FG-repeat domains may have other functions as well. The crystal structure of the RRM domain of mouse Nup35 revealed that all three FG-sequences of this nucleoporin are in ordered secondary structure elements and consistent with these FG-sequences do not interact with transport receptors, such as importin b, but rather with, for example, the integral membrane protein Ndc1. Thus, the FG-sequences of Nup35 may contribute to the formation of the NPC’s central framework (Handa et al., 2006).

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3.2. Nucleoporins and kinetochores A well-studied and conserved subcomplex of the NPC is the vertebrate Nup107-160 complex and its yeast homologue the Nup84p complex. The Nup107-160 complex is composed of nine nucleoporins and resides on both sides of the central framework of the NPC (Belgareh et al., 2001; Krull et al., 2004; Loiodice et al., 2004; Orjalo et al., 2006). The Nup107-160 complex seems to represent the core element of the central framework, since depletion of any member of this NPC subcomplex in nuclear reconstitution assays or by RNAi led to the assembly of NPC-free nuclei or nuclei with severe deficiencies in NPC formation (Boehmer et al., 2003; Harel et al., 2003b; Loiodice et al., 2004; Walther et al., 2003a) (see Section 5). A fraction of the Nup107-160 complex is targeted to kinetochores from prophase to late anaphase (Belgareh et al., 2001; Loiodice et al., 2004), to spindle poles and proximal spindle fibers in prometaphase mammalian cells and throughout reconstituted spindles in Xenopus egg extracts (Orjalo et al., 2006). Anchoring of the human Nup107-160 complex to kinetochores is mediated by the Ndc80 complex, which is part of the outer kinetochore and involved in formation and maintenance of stable kinetochoremicrotubule (MT) interaction, and CENP-F, which is also involved in MT attachment (Zuccolo et al., 2007). Kinetochores depleted of the Nup107-160 complex fail to establish proper MT attachment, which leads to a checkpoint-dependent mitotic delay (Zuccolo et al., 2007). Another nucleoporin recruited to kinetochores and the spindle in mitosis is RanBP2/Nup358 in complex with RanGAP1 ( Joseph et al., 2004, 2002; Matunis et al., 1998). RNAi approaches revealed that the RanBP2/ RanGAP1 complex is involved in chromosome congression and segregation, stable kinetochore-MT association, and kinetochore assembly (Askjaer et al., 2002; Joseph et al., 2004; Salina et al., 2003). The nuclear export receptor CRM1 provides the anchoring site for RanBP2 and RanGAP1 at the kinetochores (Arnaoutov et al., 2005), and the Nup107-160 complex in turn is required for the recruitment of CRM1, RanBP2 and RanGAP1 to the kinetochores (Zuccolo et al., 2007). Therefore, the Nup107-160 complex helps to recruit distinct kinetochore subcomplexes required for stable kinetochore-MT interaction.

3.3. Nucleoporins and transcription In the past few years it became evident that the NPC plays a role in chromatin organization in the nucleus. In this context, the nuclear periphery and the NPCs have been considered a zone of gene repression caused by the presence of heterochromatin and silencing factors (Brown and Silver, 2007). Consistently, in S. cerevisiae, two nucleoporins, Nup60p and Nup145p-C, are required for repression of the silent mating type loci

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HML and HMR and for the proper silencing of telomeres (Brown and Silver, 2007; Feuerbach et al., 2002; Galy et al., 2000). However, yeast NPCs can also positively regulate gene expression by preventing the spread of heterochromatin regions and by recruiting actively transcribed genes to the nuclear periphery (Cabal et al., 2006; Casolari et al., 2004, 2005; Ishii et al., 2002; Luthra et al., 2007; Menon et al., 2005; Schmid et al., 2006), indicating a function for nucleoporins in transcription activation as well as gene silencing. Most important, the same set of nucleoporins, namely Nup2p and the Nup84p complex, can have repressive and activating functions (Dilworth et al., 2005; Ishii et al., 2002; Menon et al., 2005; Schmid et al., 2006; Therizols et al., 2006), and it will be interesting to see how their dual functions in gene expression are regulated at the molecular level. In higher eukaryotes, the first clues to function of nucleoporins in transcription came from studies with the chimeric NUP98-HOXA9 protein, a chromosomal translocation product that occurs in myelodysplastic syndromes and acute myeloid leukemia. These studies consistently showed that NUP98-HOXA9 acts as an aberrant transcription factor, with the N-terminal FG-repeat domain of Nup98 enhancing the transcriptional activity of the DNA binding domain derived from the transcription factor HoxA9 (Ghannam et al., 2004; Kasper et al., 1999). The NUP98 gene has been found to fuse with 19 different fusion partners causing different forms of acute and myeloid leukemia. Recently, it became evident that NUP98 fusions can also act as trans-repressors of transcription (Bai et al., 2006). The intranuclear localization of these fusion proteins (Kasper et al., 1999) suggests that transcription regulation by the NUP98 fusion may not occur at the NPC or the NE. Additionally, it is not yet clear whether transcriptional regulation is the sole role of Nup98 sequences in oncogenic fusions or how this ability to modulate transcription relates to the role of endogenous Nup98. It is notable, however, that in yeast, human Nup98 was found capable of stimulating the transcription of a reporter gene at the nuclear periphery (Menon et al., 2005). Nup153 and Nup98 both dynamically interact with the NPC and their mobility within the cell appears transcription-dependent (Griffis et al., 2002, 2004). The transcription factor PU.1, which is expressed in several hematopoietic cell lineages and plays a pivotal role in the differentiation of myeloid cells and lymphocytes, is proposed to be imported into the nucleus via direct interaction with Nup153 (Zhong et al., 2005). Binding of PU.1 to Nup153 is stimulated by RanGTP but is independent of any nuclear transport receptor of the karyopherin family. In the presence of a source of energy (and presumably elevated RanGTP levels), PU.1 associates with the nuclear side of the NPC (Zhong et al., 2005), suggesting that PU.1dependent active genes might be targeted to the NPC, at least in part. More direct evidence that Nup153 in fact targets genes directly to the NPC came from a recent study on dosage compensation in Drosophila, a phenomenon

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that is distinguished by the hypertranscription of the male X chromosome (Mendjan et al., 2006). Proteins of the dosage compensation complex (DCC) were found to associate with NPC components, in particular Nup153 and Mtor, the Drosophila homologue of the mammalian nucleoporin Tpr. Nup153 and Mtor/Tpr are part of the NPC’s nuclear basket, and knock-down of Nup153 or Mtor resulted in a shift of DCC localization away from the nuclear rim, coinciding with a loss of dosage compensation in male cells (Mendjan et al., 2006). The putative transcription factor ELYS was identified as binding partner of the Nup107-160 complex and found to localize to NPCs during interphase and to kinetochores in mitosis (Rasala et al., 2006). ELYS, and its homologue in C. elegans named Mel-28, is required for NPC assembly (see Section 5; Fernandez and Piano, 2006; Franz et al., 2007; Galy et al., 2006; Rasala et al., 2006), and as a DNA binding protein it potentially targets active genes to the NPC. Taken together, recent results lend support to the notion that NPCs act in gene gating (Blobel, 1985) and that locating genes directly to the NPC is indeed a ubiquitous, evolutionary conserved mechanism for regulating gene expression.

3.4. Nucleoporins, the immune system and Parkinson’s disease The nucleoporin Nup96 is autocatalytically cleaved from a Nup98/Nup96 precursor protein, which results in the two nucleoporins Nup96 and Nup98 (Enninga et al., 2003). Nup96, like Nup98, localizes to both sides of the NPC, and is a component of Nup107-160 complex (Enninga et al., 2002). Both Nup96 and Nup98 are induced by interferons (Enninga et al., 2002). Heterozygous Nup96þ/ mice show downregulation of interferon-regulated genes and defects in the mRNA export of major players of immune response, MHCI and MHCII gene products, coinciding with alterations in MHCrelated T cell function. Additionally, B cell function is impaired in Nup96þ/ mice, resulting in Nup96þ/ cells and mice highly susceptible to viral infection. Therefore, Nup96 appears to function in antiviral response and in innate and adaptive immunity (Faria et al., 2006). A homologue of Nup96 has recently been identified in Arabidopsis thaliana, but in contrast to vertebrates the AtNup98 and AtNup96 genes locate to different chromosomal regions (Mans et al., 2004; Zhang and Li, 2005). AtNup96 is required for basal defense and constitutive resistance response to pathogens (Li et al., 2001; Zhang et al., 2003), indicating a conserved function of Nup96 in immune response. Similarly conserved is the function of Nup96 in mRNA export: plants depleted for Nup96 accumulate polyadenylated RNA within their nuclei (Parry et al., 2006). Moreover, plants depleted for Nup96 and Nup160, another component of the Nup107-160 complex, exhibit pleiotropic growth defects implicating

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these nucleoporins in hormone signaling (Parry et al., 2006). In different contexts, other roles for nucleoporins in signaling and development have been documented: a genome-wide RNA interference screen in Drosophila identified Nup153 and Nup98 as positive regulators of the Hedgehog signaling pathway (Nybakken et al., 2005). Additionally, Drosophila Nup154 was found to play a critical role in oogenesis due to its interaction with the germline specific protein Cup, which is implicated in multiple aspects of female gametogenesis (Grimaldi et al., 2007). A pleiotropic role in cell function has also been suggested for the nucleoporin RanBP2/Nup358. RanBP2 is a large modular protein and several molecular partners with distinct functions interacting with specific domains of RanBP2 have been identified. Several roles of RanBP2 have emerged that implicate RanBP2 in nucleocytoplasmic transport (Bernad et al., 2004; Yokoyama et al., 1995), protein biogenesis (Ferreira et al., 1996, 1997), the formation of the mitotic spindle and NE assembly (Askjaer et al., 2002), and the integration of NE breakdown with kinetochore formation and maturation during early mitotic progression (Salina et al., 2003). Some protein partners interact with RanBP2 in a tissue-specific manner, such as a subset of G proteincoupled receptors, the red/green opsin, in photosensory neurons (Ferreira et al., 1996, 1997) or the kinesins KIF5B and KIF5C selectively in the central nervous system (CNS) (Cai et al., 2001). CNS-selective effects of RanBP2 may underlie the pathogenesis of certain neuropathies, in particular Parkinson’s disease (PD). The Parkin protein, which has E3 ubiquitin ligase activity, has been implicated in autosomal recessive juvenile Parkinsonism, and RanBP2 has been identified as target for Parkin leading to the ubiquitination of RanBP2 and its subsequent proteosomal degradation (Um et al., 2006). Abnormal processing of RanBP2 by Parkin might therefore play a role in PD pathogenesis. RanBP2 itself possesses SUMO-E3 ligase activity (Pichler et al., 2002), and it will therefore be interesting to see if RanBP2-mediated sumoylation or the loss of it contributes to PD progression, in particular since NPC-regulated sumoylation appears to also play a role in other cellular processes, such as DNA repair and cytokinesis (Makhnevych et al., 2007; Palancade et al., 2007). Haploinsufficient RanBP2þ/ mice show a selective reduction of hexokinase type I (HKI) in the CNS, whereas skeletal muscle, spleen and liver levels of HKI remained largely unaffected. HKI is a key player in glucose metabolism and ATP production, and RanBP2 appears to prevent the inhibition of HKI and its degradation by binding the HKI antagonist COX 11 (Aslanukov et al., 2006). Haploinsufficiency in RanBP2 consequently promotes the destabilization and degradation of HKI, decreases ATP production and, hence, reduces the responsiveness of neurons. These observations may help to explain how targeting of RanBP2 for degradation by Parkin (Um et al., 2006) contributes to the pathophsyiological mechanisms underlying Parkinsonism and other neurodegenerative disorders.

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Together, mouse models as for Nup96 and RanBP2 present a powerful tool to link cellular pathways, as well as pathophysiological states, to the NPC—and, in doing so, expose roles for nucleoporins that had not been anticipated.

4. Selective Cargo Translocation Across the Nuclear Pore Complex Whereas small molecules (e.g. H2O and ions) can diffuse freely through the NPC, large cargoes (>40 kDa) require the assistance of soluble transport receptor molecules, known collectively as karyopherins (Kaps; also called importins, exportins and transportins), to be effectively chaperoned through the NPC (Stewart, 2007). Appropriate macromolecules (i.e., cargo) are identified through a short sequence of residues known as nuclear localization/export signals (i.e., NLS/NES), which exhibit binding interactions with the Kaps. Import of NLS-cargo into the nucleus usually entails the use of importin a, which acts as an adaptor to importin b through an importin bbinding (IBB) domain (Gorlich et al., 1996). However, there are some proteins that can bind directly with importin b. The directionality of nucleocytoplasmic transport is driven by an asymmetric distribution of the two nucleotide states of Ran (GTP/GDP) (reviewed in Gorlich and Kutay, 1999; Macara, 2001; Weis, 2002). RanGTP is found predominantly in the nucleus and functions to release NLS-cargo from its import receptor by binding to the import receptor itself (Gorlich et al., 1996). The importin-RanGTP complex is then recycled back into the cytoplasm. Similarly, trimeric complexes formed by an export receptor, its cargo and RanGTP, are ferried to the cytoplasm. Once in the cytoplasm, RanGAP1 (together with RanBP1 and RanBP2/Nup358) catalyzes GTP hydrolysis, which drives the disassembly of the complexes (reviewed in Gorlich and Kutay, 1999; Macara, 2001; Weis, 2002). In this manner, the receptors are recycled while a large pool of RanGDP in the cytoplasm is constantly replenished (Stewart, 2007; Weis, 2002). A current controversy remains as to how passage through the NPC is obstructed for non-NLS/NES harboring molecules that do not bind to the karyopherins or have a noncanonical means of transport (Paine et al., 1975). Hence, the selection criterion for transport through the NPC is not simply based on size exclusion per se and alludes to the presence of a selective gating mechanism within the NPC that simultaneously prevents the passive passage of molecules while promoting the translocation of receptor-mediated cargo. In this section, we will review the various concepts and supporting evidence that have led to the current understanding of selective gating, as

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well as highlight outstanding aspects of the NPC which need to be addressed in order to provide for a more refined description of NPC function.

4.1. The NPC as a selective gate Initial EM-based structural studies linked the biophysical origin of the selective gate to the presence of a ‘‘central plug’’ or ‘‘transporter’’ module located within the NPC (Akey, 1990; Feldherr and Akin, 1997). With its nanometerresolution imaging capability and its ability to be used in physiologically relevant environments, AFM was subsequently used to resolve the basis of the central plug, but these studies resulted in controversy ( Jaggi et al., 2003a,b; Mooren et al., 2004; Stoffler et al., 1999; Wang and Clapham, 1999). Today, by using state-of-the-art CET, it has been shown that the central plug most likely represents cargo caught in transit (Beck et al., 2004, 2007; Stoffler et al., 2003). Several lines of evidence now indicate that the key constituents of the NPC selective gate consist of FG-repeat nucleoporins and reside at both the cytoplasmic and nuclear peripheries surrounding the central pore (Rout et al., 2000). Cargo selection relies on binding interactions that occur between karyopherins and the FG-motifs (Bayliss et al., 2000, 2002; Bednenko et al., 2003; Liu and Stewart, 2005). Instead of possessing any well-defined structure, the FG-repeat domains exhibit large Stokes radii and are natively unfolded (Denning et al., 2003). Accordingly, AFM-based stretching experiments (i.e., single molecule force spectroscopy [SMFS]) show that the FG-repeat domains exhibit a highly flexible entropic elasticity (Lim et al., 2006a, 2007). Interestingly, studies reveal a high level of functional redundancy between the various FG-repeat domains in the NPC: 1) the asymmetric FG-domains have been shown to be dispensable for nucleocytoplasmic transport (Strawn et al., 2004; Zeitler and Weis, 2004); 2) the direction of transport through the NPC can be inverted by reversing the gradient of RanGTP (Nachury and Weis, 1999); 3) active transport is able to proceed in NPCs lacking cytoplasmic filaments (i.e., FGrich RanBP2/Nup358) (Walther et al., 2002); and perhaps most tellingly, 4) the selective gating mechanism has been found to remain functional even after 50% of the FG-repeats have been depleted (Strawn et al., 2004).

4.2. Current models of selective gating The manner in which the FG-repeat domains contribute to the selective gating of the NPC is widely speculated and has been the subject of several reviews (Fahrenkrog and Aebi, 2003; Lim et al., 2006b; Stewart, 2007; Suntharalingam and Wente, 2003; Weis, 2003). As illustrated in Figure 7.3, it is generally agreed that the FG-repeat domains form the physical

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Figure 7.3 Main models of selective gating in the NPC. (A) The Brownian/virtual gating model (Rout et al., 2000, 2003) predicts that the entropic fluctuations of the unfolded FG-domains form an effective barrier to passive cargo. Although the central pore appears unobstructed, the highly stochastic motion of the elongated FG-domains (shaded area) generates a high-density FG-domain entropic barrier or ‘‘cloud’’ that surrounds and extends beyond the immediate peripheries of the NPC (dark)(Lim et al., 2006a). (B) The selective phase model predicts that hydrophobic interactions between the FG-repeats drive the FG-domains to form an randomly interconnected gel-like meshwork within the central pore that acts as a sieve to passive, hydrophilic cargo (Ribbeck and Gorlich, 2002). Receptor-cargo complexes can dissolve through and negotiate the meshwork by breaking the‘‘links’’ between the FG-domains via receptorFG interactions. The gray area denotes the ‘‘range’’ of the meshwork while three FGdomains are drawn in red to emphasize that the FG-domains have to be elongated in order to cross-link with each other. (C) By combining aspects of Brownian gating and the selective phase, the two-gate model suggests that the more central GLFG-domains form a cohesive meshwork in the central pore while the peripheral FxFG-domains give rise to an entropic barrier (Patel et al., 2007).The shaded areas represent the locations of the two gates.

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constituents of the underlying barrier (Ben-Efraim and Gerace, 2001; Macara, 2001; Ribbeck and Gorlich, 2002; Rout et al., 2000). The Brownian affinity gating model (Rout et al., 2000) or virtual gating (Rout et al., 2003) proposes that the entropic behavior of peripheral FG-repeat domains acts as a substantial barrier to inert cargo. Translocation is anticipated for receptor-mediated cargoes due to interactions between the FG-repeats and the transport receptors (Bayliss et al., 2000, 2002) which increases the residence time and probability of entry into the NPC. In a similar manner, the ‘‘oily-spaghetti’’ model (Macara, 2001) postulates that noninteracting FG-repeat domains are pushed aside by cargo complexes but otherwise obstruct the passage of passive cargo. The selective phase model (Ribbeck and Gorlich, 2002) predicts that FG-repeat domains attract each other via hydrophobic inter-FG-repeat interactions to form a hydrophobic gel or meshwork. This interpretation is based on experiments which show that the addition of hydrophobic solvents disrupts the meshwork and triggers a nonselective opening of the central pore (Ribbeck and Gorlich, 2002; Shulga and Goldfarb, 2003). Hence, it is predicted that passive, more hydrophilic material is obstructed while hydrophobic cargo complexes are able to ‘‘dissolve’’ through the sieve-like meshwork. Most recently, Patel et al. (2007) have proposed a two-gate model that combines elements of both Brownian gating and the selective phase. Based on the observation that the centrally located yeast FG-repeat domains (i.e., GLFG) exhibited cohesion as opposed to the peripheral yeast FG-repeat domains (i.e., FxFG) that did not, the authors deduced that the more centralized FG-repeat domains formed a cohesive meshwork while the peripheral FG-repeat domains functioned as an entropic barrier.

4.3. In vitro studies of FG-domain function Despite progress in characterization of the NPC and its individual components, an accurate picture of how selective gating is achieved by the FGrepeat domains remains unclear due to a general lack of information about FG-domain behavior in the context of the NPC. The source of this ambiguity stems in part from the difficulty in trying to visualize the FG-repeat domains in vivo, which is evident given the lack of resolution, even when using state-ofthe-art structural techniques such as CET, to detect the FG-repeat domains (Beck et al., 2004, 2007; Stoffler et al., 2003). Direct imaging of the NPC with AFM is also limited in resolution and chemical sensitivity due to the complexity of the NPC and its cellular environment ( Jaggi et al., 2003a,b; Mooren et al., 2004; Stoffler et al., 1999; Wang and Clapham, 1999). Presently, only immunogold-EM has been able to provide positional information of the FG-repeat domains and has been used to show that the FG-repeat domains of Nup153 and Nup214 (Paulillo et al., 2005) appear diffuse and mobile within the nuclear and cytoplasmic peripheries of the NPC, respectively.

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The FG-repeat domains themselves have so far been directly visualized only in isolation as individual biopolymers by AFM (Lim et al., 2006b). To reproduce the contextual dimensions of the NPC (i.e., the FGrepeat domains are anchored to the NPC surface and not free-floating in solution), Lim et al. (2006a) developed an experimental platform that allowed for the collective, biophysical behavior of surface tethered FGrepeat domains to be probed at the nanoscopic level. In support of the Brownian affinity model (Rout et al., 2000, 2003), they found that FGdomain clusters of Nup153 (termed cNup153) are entropically dominated and resemble a polymer brush (Halperin et al., 1992; Milner, 1991; Zhao and Brittain, 2000). Being surface-anchored, the molecular chains exhibit a predisposed net directionality normal to the surface because of lateral packing constraints, which causes them to stretch away from the surface, i.e., forming a brush. This provided an explanation as to how FG-repeat domains could give rise to an effective repulsive entropic barrier in and around the NPC. The observation that the extended brush-like conformation of the FG-repeat domains collapses in hexanediol provides an explanation as to why NPCs appear to reversibly open and close when the same reagent is added/removed (Patel et al., 2007; Ribbeck and Gorlich, 2002; Shulga and Goldfarb, 2003). This was substantiated with SMFS-AFM analysis, which showed that individual Nup153 FG-domain molecules could be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, resembling a worm-like chain (Bustamante et al., 1994; Marko and Siggia, 1995). These measurements indicate a lack of intra-FG interactions within each individual FG-repeat domain and provide a nanomechanical verification of the natively unfolded conformation of this domain. In comparison, SMFS analysis detected an interaction between importin b and cNup153 when importin bmodified AFM tips were used (Lim et al., 2007) and, further, provided evidence for multiple points of contact of importin b with the cNup153 region. This is in agreement with the fact that importin b consists of five hydrophobic FG-binding sites (Bayliss et al., 2000, 2002; Bednenko et al., 2003; Liu, and Stewart, 2005) (with an additional five binding sites predicted by molecular dynamics (MD) simulations (Isgro and Schulten, 2005)) that can be simultaneously occupied (Isgro and Schulten, 2005). This led to the suggestion that 1) cooperativity between FG-repeat domains arises from FG-receptor interactions instead of FG-FG interactions, and 2) binding promiscuity allows for a ‘‘capture’’ mechanism that involves the coiling or wrapping of the FG-domain(s) around receptor molecules (Lim et al., 2007). At the macroscopic level Frey et al. (2006) showed that the yeast FGnucleoporin, Nsp1p, can be cast in the form of a macroscopic hydrogel to lend support to the ‘‘selective phase’’ model (Ribbeck and Gorlich, 2002). Remarkably, the authors showed that a saturated hydrogel made of Nsp1p FG-repeats can reproduce the permeability properties of NPC (Frey and

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Gorlich, 2007). In order to investigate the cohesiveness of different FGnucleoporins, Patel et al. devised a low affinity assay, which could detect the binding of CFP-nucleoporins to GST-nucleoporins immobilized on Sepharose beads (Patel et al., 2007). Interestingly, they found that only GLFG-domains showed weak cohesive interactions whereas FxFGdomains (such as in Nsp1p) did not. By overexpressing the FG-repeat domains in yeast cells, the authors observed a similar pattern of interactions in vivo, albeit not visualized in the context of the NPC. A systematic depletion of FG-repeat domains in yeast showed, however, that the NPCs displayed similar qualitative ‘‘leakiness’’ in all the cases studied, which indicated that the FG-repeat domains in both peripherally and centrally anchored nucleoporins play an important role in maintaining the selective gating mechanism. These findings led to the conclusion that FxFG domains on both faces of the NPC act as an entropic repulsive barrier while the GLFG-domains form a cohesive meshwork in the NPC’s central pore (Patel et al., 2007).

4.4. In silico studies of the FG-domains and barrier function Computational studies have also been useful in providing additional insight into the possible aspects of FG-repeat domain behavior. By modelling a cross-linked network, Bickel and Bruinsma (2002) showed that a receptor molecule would have a lower, and not higher, mobility than a passive molecule due to its attachments to the FG-repeat domains. In agreement with the cohesive properties observed between GLFG-domains (Patel et al., 2007), Kustanovich and Rabin (2004) predicted that FG-repeat domains would exhibit low equilibrium affinities for each other. In support of brushlike behavior for FG-domains (Lim et al., 2006a), Nielsen et al. (2006) showed that the conformational entropy of non-interacting FG-repeat domains was enough to provide for a robust barrier around the NPC by modelling the FG-repeat domains as surface grafted, polymeric random coils. By solving a rigorous mathematical model of transport through the NPC, Zilman et al. (2007) showed that selectivity, efficiency, directionality, and robustness of nucleocytoplasmic transport could be explained by combining the interaction strengths of binding to the flexible FG-repeat domains with the physics of diffusion inside a channel. Besides finding that NPC selectivity arises from a balance between the probability of forming receptor-mediated cargo complexes and their speed, they propose that the competition between specific receptor-mediated versus nonspecific interaction for FG-binding also contributes to the selectivity of the NPC mechanism. In contrast to predictions of the selective phase model, the authors find the inherent flexibility of the FG-repeat domains to play a key

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role in maintaining the high throughput and relative robustness (i.e., insensitive to FG-repeat deletions) of the NPC. Using molecular dynamics simulations, Isgro and Schulten predicted that additional hydrophobic binding spots could exist on the transport receptors importin b (Isgro and Schulten, 2005), NTF2 (Isgro and Schulten, 2007a) and the Cse1p:Kap60p:RanGTP complex (Isgro and Schulten, 2007b) that could enhance receptor interactions with the FG-repeats. Besides requiring several binding spots, the authors predict that close physical proximity between binding spots on each receptor molecule is likely to be an important criterion for transport selection. Furthermore, by using identical FG-repeat peptides derived from both FxFG domains (i.e., Nsp1p) and GLFG-domains (i.e., Nup116p) in all three studies, these simulations show that both classes of FG-domains interact with overlapping binding spots on importin b, NTF2 and Cse1p, respectively. Indeed, such overlap has been experimentally observed previously for importin b (Bayliss et al., 2002).

4.5. Kinetic aspects of nucleocytoplasmic transport Selective gating appears to be a rapid process given the relatively short residence times of receptor-cargo complexes at the NPC as shown by single molecule fluorescence microscopy (Kubitscheck et al., 2005; Yang et al., 2004). By directly monitoring the transport of a model protein substrate (i.e., NLS-2xGFP) through individual NPCs in permeabilized HeLa cells, Yang et al showed that movement through the NPC is bidirectional, resembling a random walk whereby the import substrate spends the majority of its
10 ms interaction time within the central pore (Yang et al., 2004). Kubitscheck et al. (2005) obtained kinetic data regarding the dwell times of the nuclear transport receptors NTF2 (5.8 ms) and transportin (7.2 ms) at their respective NPC binding sites. They observed that the dwell times decreased from 5.8 ms to 5.2 ms for NTF2 and 7.2 ms to 5.6 ms for transportin when each respective transport receptor was bound to specific transport substrates, indicating that translocation is accelerated for receptorcargo complexes. By comparing their data with known bulk transport rates, they suggested that nucleocytoplasmic transport proceeds via multiple parallel pathways within each NPC. More recently, Yang and Musser (2006) showed that the transport efficiency and import time of cargo was modulated by importin b concentration and suggested that in vivo mechanisms that altered the expression levels of the receptor could dramatically affect transport rates. In addition, the recent findings of Paradise et al. (2007) and Timney et al. (2006) revealed that karyopherins compete nonspecifically with other cytosolic structures and proteins. By colliding with a large number of nonspecific partners in the crowded cytosolic environment, karyopherins could be ‘‘shielded’’ from the FG-repeat domains. Indeed, Timney et al. (2006) reported that nonspecific competition

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resulted in each karyopherin having to take
10 s to search for and successfully import the appropriate cargo—100 times longer than previous estimates, which ignored such effects. Based on the 510 ms residence/dwell time at the NPC (Kubitscheck et al., 2005; Yang et al., 2004), this led the authors to suggest that the limiting factor in cargo transport arises from the receptor having to seek out specific partners (i.e., cargo) in the milieu of nonspecific interactions instead of the actual process of translocation through the NPC. To obtain a direct thermodynamic perspective of nuclear transport, Kopito and Elbaum (2007) conducted quantitative transport measurements in reconstituted nuclei and showed that nuclear accumulation follows Michaelis-Menten first-order kinetics as a function of the cytoplasmic cargo concentration. Importantly, this suggests that 1) the fate of a protein population led by receptor-mediated transport is dictated by the NLS, and 2) individual molecules are free to shuttle back and forth through the NPC.

4.6. Toward an understanding of FG-domain behavior in the NPC While the biophysical behavior of the FG-repeat domains in vivo remains unsubstantiated, the effect of nonphysiological reagents (e.g., hexanediol) to abolish the NPC barrier as observed in transport assays (Patel et al., 2007; Ribbeck and Gorlich, 2002; Shulga and Goldfarb, 2003) provides an important clue to their physiologically relevant conformations. By observing that the FG-repeat domains ‘‘collapse’’ in hexanediol (Lim et al., 2006a), it can be inferred that the FG-repeat domains are predominantly extended to a degree in the NPC in the midst of ongoing receptor-FG interactions. How then does the movement of karyopherins (and cargo) through the NPC occur? To achieve a rational mechanistic picture of how the NPC selectively gates nuclear transport, it will be essential to understand how the different FGdomain conformations (i.e., gel vs. brush) simultaneously prevent the passage of passive molecules while promoting the translocation of receptor-cargo complexes through the NPC (i.e., definition of selective gating) at the observed transport rates (
5 ms). Thus, newer structural/biophysical techniques will be required to elucidate even finer dynamic, molecular details of FG-repeat domain behavior within the NPC and how they respond to the biochemical interactions that govern nucleocytoplasmic transport.

5. Nuclear Pore Complex Assembly and Disassembly Consideration of the massive, ornate structure of the NPC and its central role in creating distinct nuclear and cytoplasmic environments leads to the question of how this macromolecular machine is assembled with each

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cell division. In a proliferating human cell, thousands of NPCs are formed de novo during each cell cycle. NPCs are assembled both concomitantly with membrane recruitment to the newly forming nuclei, as well as after the chromatin is fully enclosed by the two lipid bilayers that comprise the NE. Whether these are truly distinct modes of assembly remains to be determined. If so, in post-mitotic cells or in organisms with a ‘‘closed mitosis’’ only the latter path is relevant. The process of NPC assembly is extremely rapid; in the Xenopus egg extract system, NPCs are estimated to form at the rate of
140 per minute (D’Angelo et al., 2006). Given the observation that during an ‘‘open mitosis’’ NPC components disperse into subunits and individual components, as well as evidence that formally rules out NPC splitting to create new NPCs (D’Angelo et al., 2006), a pathway of selfassembly clearly exists. Many key players and steps in this process are now known, although significant gaps remain to be elucidated.

5.1. Building a nuclear pore: Who’s on first? One strategy to set the stage for understanding NPC formation has been to delineate the order of nucleoporin recruitment during post-mitotic nuclear assembly (Bodoor et al., 1999; Haraguchi et al., 2000). Certain nucleoporins are thought to be present, albeit initially on a restricted region of the chromatin surface, from the very beginning since they reside at the kinetochore during mitosis (see Section 3.2). These include the Nup107-Nup160 complex (Belgareh et al., 2001) and a newly identified associated protein, ELYS/MEL28, as well as RanBP2/Nup358 ( Joseph et al., 2002). Broader recruitment of Nup107 and Nup133 has been reported to also occur very early—during anaphase, similar to Nup153 and before Nup62 (Belgareh et al., 2001). Membrane recruitment naturally brings with it integral membrane proteins of the NPC, although notably the recruitment of transmembrane proteins (or at least their stable association with the nuclear rim) does not occur simultaneously. Recruitment of POM121 and likely Ndc1, which appears to localize to the same vesicle population in Xenopus egg extracts (Mansfeld et al., 2006), is an early event, whereas gp210 does not accumulate until later in the nuclear assembly process (Bodoor et al., 1999). mAb414 reactivity is detected relatively late during nuclear reconstitution in egg extracts (Antonin et al., 2005); although this antibody recognizes at least four nucleoporins, the bulk of its reactivity usually reflects Nup62 levels. Nup214 was noted to arrive after Nup62 when these nucleoporins were tracked individually in NRK cells (Bodoor et al., 1999). Nup155 arrives relatively late in the assembly process as well, although in its absence nucleoporins that get recruited earlier do not accumulate at the rim, suggesting that Nup155 plays a critical role in stabilizing interactions that lead to NPC formation (Franz et al., 2005). Tpr is also a late-arriving

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nucleoporin (Bodoor et al., 1999; Haraguchi et al., 2000; Hase and Cordes, 2003), but in this case its presence is dispensable for the core structure of the NPC (Frosst et al., 2002; Hase and Cordes, 2003; Shibata et al., 2002). Another approach to understanding the early steps of nuclear pore formation has been to observe this process at high resolution, using electron microscopy. Analysis of nuclear assembly using transmission EM led to the notion that an intermediate structure, termed a ‘‘pre-pore’’, forms on the surface of chromosomes independent of membranes (Sheehan et al., 1988). Further visual analysis in both Xenopus and Drosophila systems, using scanning EM, has provided a more detailed conceptual framework of the structural stages of pore assembly (Goldberg et al., 1997; Kiseleva et al., 2001). The molecular composition of NPC assembly intermediates is not yet known, although there is speculation that the Nup107-160 complex is a good candidate for forming a pre-pore-type structure (Walther et al., 2003a). Revisiting these structures in combination with immuno-detection techniques will result in a more integrated picture of nucleoporin recruitment and the step-wise assembly of the NPC (Drummond et al., 2006).

5.2. Nuclear pore building blocks: The transmembrane proteins Integral membrane proteins of the nuclear pore are predicted to play unique and essential roles in NPC formation, as such proteins seem likely to be involved in facilitating creation of the pore itself (or, described from a different perspective, in joining the inner and outer nuclear membranes) and in anchoring the soluble NPC building blocks to this site of the NE. In the Xenopus egg extract system, depletion of POM121-containing vesicles results in an early block to NE assembly: vesicles appear to bind the chromatin, but do not fuse (Antonin et al., 2005). This phenotype precludes direct analysis of the role for POM121 in nuclear pore formation. Nonetheless, an interesting layer of regulatory cross-talk between POM121 and the Nup107-160 subcomplex was observed (see below). Knockdown approaches in mammalian cells have not led to a unified view on the role of POM121; the degree of impairment in NE/NPC assembly may depend on the extent of depletion (Antonin et al., 2005; Imreh et al., 2003; Stavru et al., 2006b). In any case, the observation that POM121 appears to be restricted to vertebrates indicated a priori that another transmembrane protein would likely play a pivotal role. Indeed, the integral membrane protein Ndc1 (Chial et al., 1998) has emerged in several studies as an important player in NPC assembly (Lau et al., 2004; Madrid et al., 2006; Mansfeld et al., 2006; Stavru et al., 2006a). Even depletion of Ndc1, however, does not lead to an absolute defect in NPC assembly; this was best illustrated by a C. elegans strain bearing a deletion that disrupts the ORF of NDC1. This mutant strain has high embryonic and larval mortality and dramatically

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reduced mAb414 reactivity at the nuclear rim, but rare survivors can be propagated (Stavru et al., 2006a). RNAi-directed depletion of the third metazoan transmembrane protein, gp210, in mammalian cells did not have a significant effect on NPCs in some studies (Eriksson et al., 2004; Mansfeld et al., 2006; Stavru et al., 2006b), but did alter NPC and NE phenotype in another case (Cohen et al., 2003). In one study, simultaneous knockdown of gp210 and Ndc1 was found to have a synergistic effect (Mansfeld et al., 2006). Likewise, in yeast, Ndc1p was found to be partially redundant with POM152p (Madrid et al., 2006). Results in Xenopus egg extracts have been complicated by initial mis-identification of the C-terminus of this orthologue (Antonin et al., 2005; Drummond and Wilson, 2002). In C. elegans, gp210 is required for viability, but the NPC phenotype associated with its depletion is primarily a problem in NPC positioning rather than formation (Cohen et al., 2003). Tissue-specific expression patterns for gp210 (Olsson et al., 2004) indicate that it may be more likely to modulate rather than to dictate NPC structure. One overriding theme of these studies is that redundancy is built into the NPC assembly pathway, ensuring that NPC formation is a robust biological process (Kitano, 2004; Stavru et al., 2006a).

5.3. Collaboration between nucleoporins in NPC assembly Understanding how the transmembrane components of the nuclear pore are linked to the soluble pore building blocks is key to understanding NPC assembly. One such connection exists between Ndc1 and Nup35 (sometimes referred to as Nup53 as it is the homologue of yeast Nup53p), which associate in a manner independent from the Nup35-Nup93 interaction (Mansfeld et al., 2006). Nup35 itself has been implicated as an important player in NPC assembly (Hawryluk-Gara et al., 2005). Although this nucleoporin is not a transmembrane protein, it appears to be in close apposition to the nuclear membrane and associates with lamin B. Indeed, overexpression of the yeast homologue, Nup53p, causes accumulation of extramembrane structures within the nucleus, in which Nup53p is found (Marelli et al., 2001). Ndc1p is also targeted to these extra-membrane structures and the C-terminal region of Nup53p that is functionally implicated in membrane recruitment is also important for the interaction with Ndc1p. A complex relationship appears to exist between the Nup107-160 complex and POM121 during NE formation. The observations are 1) when the Nup107-160 complex is depleted, POM121 is no longer recruited to the NE during assembly (Harel et al., 2003b) and 2) the arrest in NE assembly seen when membrane vesicles are depleted of POM121 is dependent on the Nup107-160 complex (Antonin et al., 2005). Although there is no evidence for a direct interaction between the Nup107-160 complex and POM121

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itself, these interesting interdependent phenotypes have been proposed to suggest that chromatin-associated Nup107-160 complex exerts negative feedback on NE formation, which is relieved by the presence of POM121. Whatever the exact nature of this regulatory relationship, the Nup107160 complex is clearly playing an important collaborative role in creating the NPC. When this NPC subunit is depleted, the membranes that enclose chromatin in an in vitro nuclear assembly system completely lack NPCs (Antonin et al., 2005; Harel et al., 2003b; Walther et al., 2003a) and knockdown of Nup107 in mammalian cells leads to severe NPC defects as well (Boehmer et al., 2003; Walther et al., 2003a). This NPC subunit is thought to eventually create an important core aspect of the nuclear pore. Interestingly, this complex, which shares two members with COPII and possesses additional general features similar to coatomer complexes, is hypothesized to form a coat-like structure at the pore membrane (Devos et al., 2004; see also Antonin and Mattaj, 2005).

5.4. Peripheral pore structures Tpr is proposed to be the critical building block of the nuclear pore basket in vertebrates, with its recruitment to the NPC dependent on Nup153 (Frosst et al., 2002; Hase and Cordes, 2003; Krull et al., 2004). The role of Nup153, however, may be complicated by the presence of more than one population of this nucleoporin at the NPC. One possibility is that a stably associated population of Nup153 at the nuclear ring moiety is dedicated to tethering Tpr to form the core basket, while another population dynamically associates with the NPC (Ball and Ullman, 2005; Fahrenkrog and Aebi, 2003). The basket structure itself is conserved in yeast (Fahrenkrog et al., 1998; Kiseleva et al., 2004), although its composition is not welldefined in this organism. Tpr homologues in S. cerevisae, Mlp1p/Mlp2p, have been described as being on intranuclear filaments connected to the nuclear pore (Strambio-de-Castillia et al., 1999), leaving open the question of how the basket structure itself is formed in this case. These proteins may have the potential for dual (multiple) localization, as Tpr has been reported to be on intranuclear filaments/channels in certain metazoan cells (Cordes et al., 1997; Fontoura et al., 2001; Zimowska et al., 1997) and Mlp2p binds to the spindle pole body in yeast (Niepel et al., 2005). Based on knockdown analysis in cultured human cells, the partner proteins Nup214 and Nup88 are proposed to play a key role in recruiting RanBP2/Nup358 to create the cytoplasmic filaments of the NPC (Bernad et al., 2004). This does not seem to be the case in Drosophila cells, where knocking down Nup214 leaves functions ascribed to RanBP2/Nup358 intact (Forler et al., 2004). Likewise in nuclei assembled in Xenopus egg extract, depletion of Nup214 does not interfere with RanBP2/Nup358 targeting to the NPC (Walther et al., 2002). Corresponding ultrastructural

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analysis suggested that Nup214 and RanBP2/Nup358 localize to distinct structures on the cytoplasmic face of the NPC (Walther et al., 2002). The apparent discrepancy in the interdependence of Nup214 and RanBP2/ Nup358 targeting may be due to the role of Nup88, which is proposed to anchor each of these nucleoporins. Nup88 levels are closely linked to those of Nup214, but in certain cases, Nup88 may be present in enough excess to recruit RanBP2/Nup358 to the NPC when Nup214 (and certain amounts of Nup88) are depleted. A central role for Nup88 in organizing the features of the cytoplasmic face of the NPC is underscored by its role in anchoring Nup98 to this site as well; in contrast, interactions with Nup96, a member of the Nup107-160 complex, target Nup98 to the nuclear face of the nuclear pore (Griffis et al., 2003). Only a fraction of known interactions between nucleoporins have been highlighted here, but almost all of such contacts ultimately contribute to NPC assembly. Gaining an even more complete map of this interaction network is an important step in understanding the process of NPC assembly and its overall structure.

5.5. Regulation of NPC assembly NPC assembly is regulated at several levels. One particularly striking observation is that the calcium chelator BAPTA completely prevents NPC assembly in the Xenopus nuclear reconstitution assays (Macaulay and Forbes, 1996). The early observation that EGTA does not phenocopy BAPTA (Macaulay and Forbes, 1996) suggests that it is not absolute calcium levels, but rather a burst of calcium, which may not be quickly enough quenched by EGTA, that is required during NPC assembly. Whether this reflects a calcium flux requirement for inner and outer membrane fusion or some earlier step has not been formally addressed. Among many possibilities, calcium could be involved in a SNARE-related event (Baur et al., 2007) or in modulating the calciumbinding protein Cdc31p/centrin, which was identified as a component of yeast nuclear pores (Rout et al., 2000) but not found, as yet, at the vertebrate NPC. A role for calcium, though intriguing, is ill-defined and there is evidence against a requirement for lumenal calcium stores during the process of NE/ NPC assembly (Marshall et al., 1997). Indeed, the dearth of information on the molecular events that underlie BAPTA inhibition leave open the possibility that this small molecule in fact exerts its effects on nuclear pore assembly via a mechanism distinct from calcium chelation. Whatever the mechanism, an important clue may lie in the observation that depletion of the Nup107-160 complex gives rise to a similar morphological phenotype as BAPTA inhibition. In addition to post-translational modification, which will be discussed below, association with transport receptors has proven to provide another important layer of regulation for nucleoporins. Considered in this context, the transport receptors serve as chaperones and, in doing so, guide the spatial

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and temporal order of nuclear pore protein recruitment. This chaperone role of transport receptors has also been proposed to be important to formation of an FG-domain based hydrogel within the confines of the NPC (Frey and Gorlich, 2007). Just as in the case of nucleocytoplasmic trafficking, the small GTPase Ran works as a switch to regulate nucleoporin association with importin b, the transport receptor best studied in the context of nuclear pore assembly. In fact, this role of Ran is intimately linked to the role of chromosomal DNA as the surface on which the NE and NPCs are assembled. This is because the guanine nucleotide exchange factor (GEF) for Ran, RCC1, is targeted to chromatin and its activity is stimulated by histones (Nemergut et al., 2001). Thus, RanGEF activity is high in the vicinity of chromosomes, in turn creating a gradient of RanGTP even in the absence of a nuclear membrane (Kalab et al., 2002, 2006; see also Gorlich et al., 2003). RanGTP modulates the binding activity of importin b, promoting the release of associated proteins, such as Nup107, Nup153, and RanBP2/Nup358 (Walther et al., 2003b), as well as (presumably) other factors required for fusion of the NE and assembly of nuclear pores. Yet, additional regulation of importin b appears to be at play: RanQ69L, which reverses the inhibitory effects of excess importin b on nuclear membrane fusion, does not reverse its ability to inhibit NPC insertion in a preassembled NE (Harel et al., 2003a). A role for importin b, and for Ran, in NPC assembly is also found in yeast (Ryan et al., 2003, 2007), suggesting similarity in fundamental regulatory mechanisms despite certain differences in NPC assembly due to the open vs. closed configuration of mitosis. A chaperone-like role is not restricted to importin b: in yeast, Kap121p helps to target Nup53p and is involved in NPC remodeling at mitosis (Lusk et al., 2002; Makhnevych et al., 2003); transportin may aid in escorting Nup153 to the NPC (Nakielny et al., 1999). Other components of the reforming nucleus impinge on nuclear pores as well. This was recently illustrated by the observation that patches of newlyformed NEs are initially pore-free and correspond to regions that are enriched in underlying lamin A/C and have lower levels of lamin B (Maeshima et al., 2006) (see Section 2.3). Whether this is a case of inhibiting NPC assembly in particular regions or of preventing NPC anchorage in certain domains of the lamina network remains to be addressed. And, in either case, the molecular mechanism and mediating proteins have yet to be explored. Nonetheless, this report underscores the many levels of nuclear assembly that are simultaneously orchestrated and cross-regulated.

5.6. Clues from a second site for NPC assembly Although chromatin is typically the favored scaffold on which to build NPCs, an alternate site for NPC assembly exists in the annulate lamellae (AL). These cytoplasmic membrane cisternae house tightly arrayed NPCs.

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AL formation is more pronounced in rapidly proliferating cells and has been proposed to be a storage site for NPC components. These NPC-like complexes might seem poised to contribute to NPC formation at the NE itself during membrane expansion, but at least under certain circumstances, this does not appear to be the case. Specifically, in Drosophila embryos the excess nucleoporins, presumably the stores for new NPC formation, were found to be largely soluble rather than AL associated (Onischenko et al., 2004). There was no decrease in AL-associated pore complexes concomitant with increases in NPC numbers at the NE. Although NPC formation at the AL may be a separable event, rather than a prelude to the appearance of NPCs at the NE, understanding what initiates this chromatin-independent assembly process and how it differs at this site is a way of gaining insight into the pathways that converge to create NPC structure. Experimental manipulations that lead to increased AL formation include increasing levels of RanGTP (Harel et al., 2003a; Walther et al., 2003b). Similarly, Ran-coated beads are sufficient to direct formation of double membrane bilayer replete with nuclear pores (Zhang et al., 2002), consistent with the notion that local levels of RanGTP direct NE/NPC formation to the chromatin surface and are capable of driving this process at other sites as well. Another interesting observation is that inhibition of microtubule formation and/or kinesin function prevents NPC assembly at the NE, but does not perturb formation of a nuclear envelope or NPC-containing AL (Ewald et al., 2001). This suggests that delivery of certain components, perhaps a vesicle population, to the chromatin surface is facilitated by microtubules whereas this same component is either not needed at the AL or is incorporated independently of microtubules.

5.7. Nuclear pore assembly is never-ending Beyond the fact that new NPCs are assembled throughout much of the cell cycle, the dynamic nature of NPC components reveals that this structure is not assembled to a static end-point but rather is continuously remodeled. Dynamic association with the NPC was first observed for Nup153 (Daigle et al., 2001) and for Nup98 (Griffis et al., 2002). An extensive survey of this property with respect to nucleoporins later revealed that several nucleoporins move on and off the NPC structure (Rabut et al., 2004; Tran and Wente, 2006). Interestingly, this movement has been shown in certain cases to be dependent on ongoing transcription (Griffis et al., 2002, 2004). NPC structure has additional layers of dynamics as well. For instance, largescale conformational rearrangements have been observed by scanning EM (Goldberg et al., 2000; Kiseleva et al., 1996, 1998), by AFM (Shahin et al., 2001, 2005), and by CTE (Beck et al., 2004). In addition, domains within individual pore proteins have been found to be (or have the potential to be) flexibly arranged within the NPC (Fahrenkrog et al., 2002; Paulillo et al.,

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2005, 2006; Schwarz-Herion et al., 2007), indeed, the dynamic arrangement of these domains is likely central to trafficking and the selectivity of the nuclear pore (see Section 4). Functional alterations of the NPC, such as the greater upper limit of cargo diameter in proliferating vs. quiescent cells, may also reflect specific reconfiguration of NPC structure (Feldherr and Akin, 1990, 1991).

5.8. Deconstructing the NPC Several pore proteins are targets of phosphorylation at mitosis. These include both integral membrane proteins of the NPC, such as Ndc1 (Mansfeld et al., 2006; Stavru et al., 2006a) and gp210 (Favreau et al., 1996), as well as nonmembrane anchored, such as Nup35, Nup153, Nup98, Nup62, Nup214, and Nup107-160 complex members (Belgareh et al., 2001; Glavy et al., 2007; Lusk et al., 2007; Macaulay et al., 1995; Walther et al., 2003b). A phosphorylation-dephosphorylation switch has long been thought to aide in driving NPC disassembly and re-assembly. Indeed, when isolated nuclei from Drosophila embryos are incubated with cdc2-cyclin, several pore proteins are released (Onischenko et al., 2005). It is difficult to formally prove that this is due to nucleoporins being direct targets of cdc2-cyclin, but clearly they are responsive to a mitotic signaling cascade driven by phosphorylation events. Hallberg and colleagues also recently demonstrated that a phosphomimetic mutation in gp210, at a serine (1880) known to be phosphorylated at mitosis, interfered with its incorporation into the NPC (Onischenko et al., 2007). Consistent with these observations, phosphatase activity is implicated in the process of post-mitotic nuclear pore assembly. In Drosophila embryos, okadaic acid inhibits NPC assembly, suggesting that PP1 and/or PP2A are involved (Onischenko et al., 2005). In certain organisms, nuclear pore remodeling at mitosis is limited to discrete, but still significant, rearrangements. In Aspergillus nidulans these changes lead to an increase in the diffusion cut-off of the NPC, whereas in S. cerevisae mitotic changes at the nuclear pore appear more subtle and selectively alter particular transport paths (De Souza et al., 2004; Marelli et al., 1998). In organisms that undergo open mitosis, changes in the diffusion cut-off of the NPC herald the prophase-to-prometaphase transition (Lenart and Ellenberg, 2006; Lenart et al., 2003) and correspond to an early wave of nucleoporin exit from the NPC (Lenart et al., 2003), perhaps stimulated by phosphorylation. Extensive dispersal of the NPC components ensues and, concomitantly, the membranes that enclose the nucleus are also remodeled, allowing complete intermixing between cytoplasmic and the nuclear space. Although Ran is not implicated in the initial steps of NPC remodeling, it has recently been shown to have a regulatory role in mitotic nuclear membrane remodeling (Muhlhausser and Kutay, 2007).

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Mitotic membrane remodeling brings up the question of the fate of integral membrane proteins of the nuclear pore at mitosis. Direct observation of a GFP fusion with the inner nuclear membrane protein LBR in COS-7 cells indicated that this protein is ultimately intermixed with endoplasmic reticulum (ER) at mitosis (Ellenberg et al., 1997), as was also the case for two other residents of the inner nuclear membrane (LAP1 and LAP2) as well as for the nucleoporin gp210 (Yang et al., 1997). This intermixing could be explained by a passive process in which interactions that normally serve to keep the nuclear membranes distinct from the ER are lost at mitosis, allowing lateral diffusion between these membrane domains. In another study, however, LAP1 and LAP2 were found to have distinct localizations at mitosis (Maison et al., 1997). Whether differing results are due to the timing of detection within mitosis or to the specific isoforms detected or to some technical issue is not clear. There is independent evidence from HeLa cells for distinct vesicle populations (Chaudhary and Courvalin, 1993); however, an alternate interpretation is that such vesicles are derived artificially from microdomains within a contiguous ER-like network (Collas and Courvalin, 2000). Studies using nuclei reconstituted in Xenopus egg extract have implicated the coatomer complex, COPI, in nuclear disassembly (Cotter et al., 2007; Liu et al., 2003; Prunuske et al., 2006). Given the role of COPI in forming vesicles at the Golgi (Bethune et al., 2006), this brings up the possibility of a more active mechanism for dismantling the nuclear membrane and, at least in some cases, delivering it to the ER. It is possible that COPI-mediated vesicle formation occurs only local to the nuclear pores and represents one of two distinct routes of membrane disassembly (the second being passive lateral diffusion). Indeed, Allen and colleagues saw evidence of both vesicles and ER-like tubules when they examined the mitotic breakdown of reconstituted nuclei by field emission scanning EM (Cotter et al., 2007). Vesicles enriched in certain proteins such as POM121 and Ndc1, but not necessarily the highly mobile pore membrane protein gp210, may have escaped observation in earlier studies, but could account for the presence of distinct vesicle populations present in Xenopus egg extract. Alternatively, COPI components may play a noncanonical role, perhaps participating in the establishment or maintainance of microdomains within the ER network. Interestingly, impairing the function of many nucleoporins results in NE abnormalities, and likewise alterations in particular membrane proteins that reside in the NE/ER results in phenotypic differences at the NPC (Franz et al., 2005; Lewis et al., 2007; Liu et al., 2007; Miao et al., 2006; Stavru et al., 2006a). A recent example of the latter is the protein Apq12p; when this gene is deleted in yeast, NPCs appear to be embedded in only the inner nuclear membrane (Scarcelli et al., 2007). This advance in identification of important players in proper NPC assembly in fact exposes the general gap in our understanding of how fusion between the inner and outer nuclear

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membranes is coordinated as the core framework of the NPC coalesces. Future study of membrane dynamics at the nuclear envelope and how this is integrated with NPC assembly and disassembly will yield new insight into this important central question.

6. Concluding Remarks Although it is not yet possible to build a complete molecular picture of the nuclear pore complex, the structures of individual nucleoporins and NPC subcomplexes are indispensable to our growing understanding of NPC assembly and nucleocytoplasmic transport. Being able to merge this information with CET on intact nuclei will allow for detection of distinct functional states of the NPC and may reveal changes in the configuration of individual NPC subcomplexes during nucleocytoplasmic transport. Single molecule approaches in combination with immunogold-labelling will provide additional insight into the molecular composition of individual NPCs and variation among tissues or even from NPC to NPC. Newer structural and biophysical techniques in combination with molecular simulations will be required to elucidate even finer molecular details of the NPC, such as the identity of the anchoring sites and the exact numbers of FG-repeat domains per NPC and FG-repeat domain behavior in a cellular context. Recent data also points to intriguing roles for nucleoporins that are distinct from, but perhaps coordinated with, their roles at the NPC. As nucleoporins are studied in further detail, it is likely that the scope of their roles will continue to hold surprises. Finally, as more detailed knowledge of how the NPC is put together emerges, this will go hand-in-hand with a deeper understanding of the molecular architecture of the NPC and in turn will aide in building a comprehensive model of trafficking through the NPC.

ACKNOWLEDGMENTS This work was supported by a grant from the Swiss National Science Foundation (to B.F.), the M.E. Mu¨ller Foundation and the Kanton Basel Stadt, and from the N.I.H. (GM61275) and the Leukemia and Lymphoma Society (to K.U.).

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Endocytosis and the Actin Cytoskeleton in Dictyostelium discoideum Francisco Rivero* Contents 1. Introduction 2. Tools to Study Endocytosis 3. Role of D. discoideum Actin and ABPs in Endocytosis 3.1. Actin 3.2. The F-actin nucleation machinery 3.3. Monomeric actin binding proteins 3.4. Severing and capping proteins 3.5. Actin crosslinking proteins 3.6. Lateral ABPs 3.7. Membrane-associated ABPs 3.8. Actin-based molecular motors 4. Molecular Events During the Uptake Phase: A Simplified Model 5. Concluding Remarks Acknowledgments References

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Abstract Endocytosis, an essential process of all eukaryotic cells, requires the actin cytoskeleton for proper functioning. The soil amoeba Dictyostelium discoideum is well known for its contribution to the actin cytoskeleton field. The genetic tractability and the availability of appropriate tools have made of Dictyostelium an attractive model for studies of endocytosis and vesicle trafficking as well. These tools include a large palette of fluorescent protein fusions and the combination of improved fractionation methods with high throughput techniques along with the recently propagated use of the amoeba a host for microbial pathogens. In this review I discuss in a comprehensive manner the evidence accumulated in the literature towards a participation of components of the

*

The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, United Kingdom

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00633-3

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2008 Elsevier Inc. All rights reserved.

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microfilament system of D. discoideum in endocytic trafficking and conclude with a model that describes the sequence of events and the components involved during the well-investigated uptake phase of the endocytic process in the soil amoeba. Key Words: Dictyostelium, Actin, Actin-binding protein, Phagocytosis, Pinocytosis, Cytoskeleton. ß 2008 Elsevier Inc.

1. Introduction Endocytosis, the uptake of fluid or solid phase material, is an essential process of all eukaryotic cells. In single cell organisms, such as Dictyostelium discoideum, endocytosis serves primarily a nutritive function in vegetative cells, whereas in the multicellular stage a small population of sentinel cells serves a defensive purpose (Chen et al., 2007). Dictyostelium is a notorious professional phagocyte: cells are able to engulf and internalize particles of various sorts and sizes, ranging from bacteria to yeast and apoptotic cells of the same species, as well as synthetic beads. Commonly used laboratory strains are also able to grow in synthetic liquid media. In Dictyostelium the bulk of fluid is taken up by macropinocytosis, which takes place at crownlike protrusions apparent at the dorsal and lateral cell surface of adherent cells (Hacker et al., 1997). Additional pathways of fluid uptake also exist in Dictyostelium, like clathrin-dependent or independent micropinocytosis, but they are less well understood and will not be considered further (Neuhaus et al., 2002; O’Halloran and Anderson, 1992). The endocytic pathway of Dictyostelium has been operationally divided into three major steps: uptake at the plasma membrane, transit through endosomal compartments and finally release of indigestible components by exocytosis (Maniak, 2002, 2003). The first step consists in the formation of a cell surface protrusion that engulfs a particle or an aliquot of surrounding medium, involves a considerable amount of signaling and is driven by actin remodeling. This step is accomplished very rapidly: within approximately 1 min after internalization the actin coat that surrounds the nascent endosome begins to dissociate and the endosome is captured by peripheral microtubules. During its transit through the endosomal pathway the endosome matures, a process that requires numerous fusion and fission events. Concomitantly with the dissociation of the actin coat, fusion with vesicles that carry vacuolar HþATPases results in acidification of the endosomal lumen, lasting for approximately 30 min. Several sets of lysosomal enzymes are then delivered, allowing digestion of the endosomal contents. The vacuolar HþATPase and the lysosomal enzymes are retrieved and recycled. The pH of the endosome returns to a neutral value, which enables homotypic fusion. Large neutral endosomes form that acquire a coat of filamentous actin. The release of the late endosomal content by exocytosis occurs

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then within seconds. Judging from population studies, the whole process takes about one hour, but recent studies based on tracking of individual cells are beginning to challenge this view, indicating that at least early steps of endosome maturation occur more rapidly than inferred from population studies (Clarke and Maddera, 2006). In what follows I will present in a comprehensive manner the evidence that has accumulated in the literature towards a participation of components of the microfilament system of D. discoideum in endocytic trafficking (Tables 8.1 and 8.2). Details on structure, biochemical activities and other functions of these proteins not given here can be found in (Rivero and Eichinger, 2005). Due to space constrains, aspects like signaling to the cytoskeleton (in particular by small GTPases) and other signaling pathways as well as the role of calcium and phospholipids, which are important regulators of numerous ABPs, are not treated here in detail. This aspects have been covered elsewhere (Cardelli, 2001; Maniak, 2002; Vlahou and Rivero, 2006). More recently Dictyostelium has begun to be used as a model system for the study of pathogenic bacteria that manipulate the endocytic pathway to create a favorable environment for replication and dissemination (Farbrother et al., 2006; Steinert and Heuner, 2005). The relevant information has been incorporated into Tables 8.1 and 8.2, but this aspect is not treated in depth here. To close this review I will make an attempt to integrate most of the information into a model that describes the sequence of events and the components involved during the uptake phase, which is the best investigated phase of the endocytic process in Dictyostelium.

2. Tools to Study Endocytosis One fundamental advantage of Dictyostelium discoideum as a model organisms for studies of endocytosis and vesicle trafficking, apart from the genetic tractability and the availability of a fully sequenced and assembled genome, is the availability of tools that allow the investigation of almost every step of the endocytic pathway. Laboratory strains of Dictyostelium relay on an efficient uptake of both particles and fluid as the predominant way to bring into the cell nutrients needed for growth and multiplication, therefore measuring the growth rate in a bacterial suspension or in axenic medium or the expansion of a plaque on a bacterial lawn are first approximations to whether a strain is capable of normal endocytosis or not. The major drawback of these determinations is that the rate of cell multiplication also depends on the efficiency of cell division, which may be controlled independently of endocytic performance. For a precise analysis, it is therefore instrumental to quantify the uptake of cargo directly. Phagocytosis is commonly quantitated using fluorescent particles (latex beads, yeasts or bacteria), whereas pinocytosis, exocytosis and changes in endosomal pH are most frequently quantitated

Table 8.1 Cytoskeleton components reportedly or putatively involved in endocytosis in Dictyostelium Protein Class

Gene

Monomeric actin-binding Profilin proA, (I and II) proB

CAP

cap

Capping and/or severing Capping protein acpA, (Cap32/34, acpB aginactin)

Cofilin 1

cofA

Severin

sevA

GRP125

gnrA

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Phagocytic cup (IF, beads). Isolated phagosomes.

Normal (double KO; latex beads)/ increased (double KO; latex beads, bacteria)

Decreased (double KO)

Decreased (double KO)

Decreased (double KO)

Cortex (IF, GFP).

Normal (KO; bacteria, yeast)

Decreased (KO)

ND

ND

Cortex.

ND

ND

ND

ND

Crowns, phagocytic cup (IF, GFP, beads). Isolated phagosomes. Phagosomes (IF, bacteria). Isolated phagosomes. Vesicles (IF).

Normal (OE; latex beads)

ND

ND

ND

ND

ND

ND

ND

KO described but not assayed.

ND

ND

ND

ND

KO described but not assayed.

Notes

Single KOs unimpaired. KO cells are more susceptible to infection with Legionella and Legionella-like bacteria. proA gene downregulated at 3 and 6 hours upon infection with Legionella.

Localization inferred from studies in A. castellani. No KO of any of the subunits described; OE and UE strains described but not assayed. KO is lethal.

vilA

Golgi, ER vesicles (GFP, IF).

Normal (KO; yeast)

Normal

ND

ND

KO showed reduced uptake and intracellular growth of pathogenic Legionella.

arpB, arpC, arcA, arcB, arcC, arcD, arcE

Cortex, crowns, macropinosomes, phagocytic cup (yeast), postlysosomal endosomes (IF, fluorescent proteins). Isolated phagosomes (Arp3)

ND

ND

ND

ND

Scar

scrA

Cytosolic, weakly cortical (IF, GFP).

Decreased (KO; beads, bacteria)

Decreased (KO)

Decreased (KO)

Decreased (KO)

No KO of any of the subunits described. An Arp2 hypomorphic strain described but not assayed. GFP fusions of Arp3 and p41-Arc, mRFP fusion of p41-Arc, and antiArp3 and anti-p21-Arc antibodies used to monitor the complex. Additive defects in triple mutants of Scar and profilins I and II.

CARMIL VASP

carmil vasP

Crowns (IF). Tips of filopods (GFP).

Decreased (KO) ND

ND ND

ND ND

Formin C

forC

Crowns (GFP).

ND Normal (KO; beads) ND

ND

ND

ND

Formin H

forH

Tips of filopods (GFP).

ND

ND

ND

ND

Cortex, phagocytic cup (IF; bacteria, beads). Isolated phagosomes.

Increased (KO; yeast)

Normal (KO)

ND

ND

Villidin

Nucleation Arp2/3 complex

Cross-linking ABP34 abpB

Reduced particle (beads) adhesion in KO. KO strain described but not assayed. KO strain described but not assayed. Effector for Rac1a. Reduced pinocytosis in ABP34/a-actinin double KO. Normal pinocytosis in ABP34/filamin double KO. Increased phagocytosis (yeast), normal pinocytosis and exocytosis in ABP34/ fimbrin double KO.

(continued)

Table 8.1

(continued)

Protein Class

Gene

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Fimbrin

fimA

Cortex, phagocytic cup (IF, GFP; yeast), macropinosomes (IF, GFP).

Normal (KO)

Normal (KO)

Normal (KO)

ND

Filamin (gelation factor, ABP120)

abpC

Cortex. Phagocytic cup (IF, beads).

Decreased (KO, beads, bacteria)/ normal (KO) (depending on parental strain)

Normal (KO)

ND

ND

a-actinin

abpA

Late phagosomes (IF). Isolated phagosomes (weakly or absent).

Normal (KO)

Normal (KO)

ND

ND

Cortexillin 1 and 2

ctxA, ctxB

Cortex, crowns (IF, GFP). Isolated phagosomes.

ND

ND

ND

ND

Notes

Increased phagocytosis (yeast), normal pinocytosis and exocytosis in ABP34/ fimbrin double KO. Reduced phagocytosis (bacteria) and pinocytosis in filamin/ a-actinin double KO. Normal pinocytosis in ABP34/filamin double KO. Reduced uptake and intracellular growth of L. pneumophila in filamin/a-actinin double KO. Reduced phagocytosis (bacteria) and pinocytosis in filamin/ a-actinin double KO. Reduced pinocytosis in ABP34/a-actinin double KO. Reduced uptake and intracellular growth of L. pneumophila in filamin/a-actinin double KO. KO (singles and double) reported but not assayed.

Dynacortin

dct

eEF1A (ABP50) efaAI eEF1B

efa1B

Lateral actin-binding Coronin corA

Aip

wdpA

Coactosin

coaA

Abp1 LimC

abpE limC

LimD1 (LimD) limD1

Cortex, crowns (IF, GFP).

ND

ND

ND

ND

Cortex (IF). Isolated phagosomes. Cortex (IF). Isolated phagosomes.

ND

ND

ND

ND

Both genes upregulated after 3 hours upon infection with L. pneumophila. Strain expressing almost no dynacortin (gene silencing) described but not assayed. No KO described.

ND

ND

ND

ND

No KO described.

Phagocytic cup (IF, GFP; yeast, bacteria), crowns, macropinosomes (IF, GFP) Isolated phagosomes. Post-lysosomal vesicles. Phagocytic cup (IF, GFP), macropinosomes (IF, GFP). Isolated phagosomes.

Decreased (KO; yeast, bacteria)

Decreased (KO)

ND

ND

Reduced uptake of L. pneumophila in KO, but more permissive for intracellular growth of L. pneumophila and Mycobacterium marinum.

Decreased (KO; yeast) Increased (OE, yeast) ND

Decreased (KO) Normal (OE)

ND

ND

ND

ND

ND

Cortex (IF). Cortex, macropinosomes, phagocytic cup (GFP; yeast).

Normal (KO) Normal (yeast; KO)

Normal (KO) Normal (KO)

ND ND

ND ND

Cortex, macropinosomes, phagocytic cup (GFP; yeast). Isolated phagosomes.

Normal (KO; yeast)

Normal (KO)

ND

ND

No KO, no localization studies. Double KO limC/limD also unimpaired. Reduced uptake and growth of L. pneumophila in double KO. Double KO limC/limD also unimpaired. Reduced uptake and growth of L. pneumophila in double KO.

(continued)

Table 8.1 Protein Class

(continued)

Gene

LimE (DdLim) limE

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Cortex, macropinosomes (IF, GFP), Phagocytic cup (GFP; yeast). Vesicles, phagocytic cups (YFP, yeast).

ND

ND

ND

ND

KO and OE strains described but not assayed.

Decreased (KO; yeast) Increased (OE yeast) Increased (KO; yeast) Decreased (OE; yeast)

Normal (KO, OE)

ND

ND

Double KO limF/CH-lim behaves like limF null.

Normal (KO, OE)

ND

ND

Double KO limF/CH-lim behaves like limF null.

KO and OE strains described but not assayed. MhkA gene downregulated at 6 hours upon infection with L. pneumophila. Effector for Rac1a, not RacC, RacE.

LimF

limF

CH-Lim

ChLim

Cortex, vesicles, phagocytic cups, phagosomes (GFP, yeast).

MHCK A

mhkA

Macropinosomes (GFP), phagocytic cup (IF; yeast).

ND

ND

ND

ND

DGAP1

rgaA

Cortex (IF, GFP).

ND

ND

ND

RacGEF1

gxcA

Cortex (GFP).

Normal (KO; yeast) Decreased (OE; yeast) ND

ND

ND

ND

Notes

KO strain and diverse overexpressors described but not assayed. Exchange factor for RacB, weakly for Rac1b, not for RacC, RacE, RacG.

Trix

gxcB

Cortex (GFP; yeast).

Normal (KO)

ND

Decreased (KO)

ND

GxcDD

gxcDD

ND

Normal (KO, yeast)

Normal (KO)

ND

ND

Cortex (GFP), tips of filopods.

Decreased (KO; yeast, bacteria, beads)

Normal (KO)

ND

ND

Membrane-associated Talin A talA (filopodin)

Comitin

comA

Golgi, vesicles (IF). Isolated phagosomes.

Decreased (yeast, bacteria); normal (latex beads) (KO)

Normal (KO)

Normal (KO)

ND

Ponticulin A

ponA

Normal (KO; bacteria, beads)

Normal (KO)

ND

ND

Ponticulin B Annexin C1

ponB nxnA

ND Normal (KO)

ND Normal (KO)

ND ND

Annexin C2

nxnB

PM, Golgi vesicles, phagocytic cup (yeast) (IF). PM, vesicles (IF). PM, nucleus, vesicles (IF, GFP). Isolated phagosomes. PM, Golgi, vesicles (GFP).

ND

ND

ND

ND Normal (KO) Reduced on low Ca ND

N-terminal fragment (CH domains) accumulates at (presumably) late endosomes. No exchange factor for Rac1a, RacC, RacE. Interacts with Rac1a, RacA, RacC, RacE, RacH, RacI; not with RacB, RacD. Reduced particle adhesion in KO. TalA gene upregulated at 24 hours upon infection with L. pneumophila. KO more permisive to infections with L. pneumophila and Legionella-like bacteria; delayed degradation of S. enterica.

No KO described.

No KO described.

(continued)

Table 8.1

(continued)

Protein Class

Gene

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Hisactophilins

hatA, hatB

ND

ND

ND

ND

KO and OE strains described but not assayed.

Interaptin

abpD

PM (IF, GFP), phagocytic cup (IF; yeast). Isolated phagosomes. Nuclear envelope, ER, Golgi (IF, GFP).

ND

ND

ND

ND

Motors Conventional myosin (Myosin II)

KO and OE strains described but not assayed.

mhcA

Cortex, vesicles (IF). Phagocytic cup (GFP; yeast). Isolated phagosomes.

Normal (KO, beads)

Normal (KO)

ND

ND

Crowns (MyoB, MyoC, MyoK, IF; MyoE, YFP). Phagocytic cup (MyoB, IF, bacteria and GFP, yeast; MyoE, YFP, yeast; MyoK, IF, yeast). Purified early pinosomes (MyoB).

See Table 9.2

See Table 9.2

See Table 9.2

See Table 9.2

Strong inhibition of phagocytosis and pinocytosis upon inactivation with blebbistatin. MhcA gene upregulated at1 and 3 hours upon infection with L. pneumophila. See Table 9.2

Class I myosins myoA, myoB, myoC, myoD, myoE, myoF, myoK

Notes

Myosin-I

myoI

Cortex, tips of filopods, phagocytic cup (yeast; GFP).

Decreased (beads, yeast, bacteria; KO)

Normal (KO)

Normal (KO)

ND

Myosin-J Myosin-M

myoJ myoM

ND Cortex, crowns, macropinosomes (GFP).

ND Normal (KO)

Normal (KO) ND

ND ND

ND ND

Reduced particle adhesion in KO KO permissive to infection by acapsular strains of the pathogen fungus Cryptococcus neoformans.

The table is a compilation of ABPs reportedly involved in endocytic trafficking based on functional studies or likely to be involved in this process by virtue of their subcellular localization or by analogy with homologs in other species. Note that some Dictyostelium proteins have been described but not analyzed in terms of localization or role in endocytosis, like protovillin, WASP, WIPa, enlazin and most formins, and have therefore been excluded from the table. See main text for references on localization and functional studies. For details on domain architecture and biochemical properties, see Rivero and Eichinger (2005). Additional information can also be retrieved from dictybase (www.dictybase.org) using the gene name. Data on infection studies are compiled from Steinert and Heuner (2005) and Farbrother et al. (2006). Where the protein has been localized at phagosomes and macropinosomes, this is indicated. In many cases this has not specifically been investigated, therefore missing information in the table should not be taken as to imply that the protein is not localized at relevant structures. It should also be considered that, although not indicated in the table, many proteins are (sometimes predominantly) cytosolic but show some enrichment at relevant places. Only localization data determined for the full-length protein has been considered. Data on phagocytosis and pinocytosis is based exclusively on quantitative analyses of particle or fluid phase uptake, not on defects in growth on bacterial lawn, growth in suspension with bacteria or growth in nutrient medium. Where reported, the particle (yeasts, bacteria, latex beads) used for localization and phagocytosis studies is indicated. IF, immunofluorescence; GFP, green fluorescent protein fusion; RFP, red fluorescent protein fusion; YFP, yellow fluorescent protein fusion; PM, plasma membrane; ER, endoplasmic reticulum; KO, knockout strain; OE, overexpressor strain; ND, not determined.

Table 8.2 Mutant strains of class I myosins and endocytosis in Dictyostelium Strai n

Phagocytosis

Pinocytosis

Exocytosis

Othe r phenotypes

References

ND

Normal

ND

(Neuhaus and Soldati, 2000; Novak et al., 1995; Peterson et al., 1995)

myoB

# 30% (bacteria) # 37% (beads)

Normal

Normal/#

myoBþ

ND

# 66%

Normal clearing (5h) of bacterial suspension. Oversecretion of lysosomal enzymes. Normal membrane recycling from early endosomes. Slow growth on bacterial lawn. Normal clearing (5h) of bacterial suspension. Normal intravesicular pH. Normal intracellular retention time. Impaired membrane recycling from early endosomes. Oversecretion of lysosomal enzymes. Deficient pressureinduced rocketing of phagosomes.

ND

myoA



(Clarke et al., 2006; Jung and Hammer III, 1990; Jung et al., 1996; Neuhaus and Soldati, 2000; Novak et al., 1995; Temesvari et al., 1996)

(Novak and Titus, 1997)

myoBþSH3

ND

Normal

ND

myoBþS332A ND

Normal

ND

myoC

# 36% (beads)

Normal

Normal

myoCþ myoD myoEþ

ND Normal " 40% (yeast) ND # 30% (yeast) (adherent cells)

# 60% Normal # 30%

ND ND ND

Normal Normal

ND ND

myoF myoK

Fails to complement the pinocytosis defect of myoA/ myoB. Localization not altered. Fails to complement the pinocytosis defect of myoA/ myoB. Localization not altered. Normal clearing (5h) of bacterial suspension. Normal intravesicular pH. Normal intracellular retention time. Normal secretion of lysosomal enzymes

(Novak and Titus, 1997; Novak and Titus, 1998)

(Novak and Titus, 1997; Novak and Titus, 1998)

( Jung et al., 1996; Novak et al., 1995; Peterson et al., 1995; Temesvari et al., 1996)

(Dai et al., 1999) ( Jung et al., 1996) (Du¨rrwang et al., 2005)

Normal steady state levels of particle uptake

(Titus et al., 1995) (Schwarz et al., 2000)

(continued)

(continued )

Table 8.2 Strain

myoK

þ

myoA/ myoB

myoB/ myoC

Phagocytosis

Pinocytosis

Exocytosis

Other phenotypes

References

# 30% (yeast) (adherent cells) ND

ND

ND

Normal steady state levels of particle uptake

(Schwarz et al., 2000)

# 60%

ND

(Clarke et al., 2006; Neuhaus and Soldati, 2000; Novak et al., 1995; Solomon et al., 2000; Temesvari et al., 1996)

ND

# 60%

Normal

Normal clearing (5h) of bacterial suspension. Slow growth in suspension, low saturation density. Delayed and decreased membrane internalization. Impaired membrane recycling from early endosomes. Oversecretion of lysosomal enzymes. Deficient pressureinduced rocketing of phagosomes. Increased intracellular growth of L. pneumophila. Normal clearing (5h) of bacterial suspension.

(Novak et al., 1995; Temesvari et al., 1996)

myoB/ myoDAS myoA/ myoF myoB/ myoCAS/ myoD

# 37% (beads) ND

# 39%

##

ND

ND

# 37% (beads)

# 60%

###

Slow growth in suspension, low saturation density. Delayed and decreased membrane internalization. Normal intravesicular pH. Normal intracellular retention time. Oversecretion of lysosomal enzymes. Slow growth in suspension. Only motility analyzed. Slow growth in suspension. Increased intracellular retention time.

( Jung et al., 1996) (Falk et al., 2003) ( Jung et al., 1996)

Data on phagocytosis and pinocytosis is based exclusively on quantitative analyses of particle or fluid phase uptake. Where reported, the particle (yeasts, bacteria, latex beads) used for phagocytosis studies is indicated. Unless otherwise indicated, initial rates of phagocytosis in suspension are given. In two strains the levels of one myosin have been reduced by antisense techniques (indicated with AS); in all other cases homologous recombination has been employed. A myoE knockout has not been described; ND, not determined.

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Francisco Rivero

using fluorescently labeled dextran. A detailed methodological discussion can be found in Rivero and Maniak (2006). Biochemical, immunological and microscopy tools allow monitoring the localization and dynamics of molecules and organelles along the endocytic pathway. In vivo studies using fusions of fluorescent proteins have played and will continue to play a major part in elucidating the role of the cytoskeleton in endocytic trafficking. Such studies were pioneered in Dictyostelium, with coronin being one of the first proteins whose dynamics during particle uptake was monitored in vivo (Maniak et al., 1995), soon followed by actin (Westphal et al., 1997) and many other proteins. In fact, the use of fluorescent protein fusions can be considered as de rigueur in the functional characterization of any novel protein. Early attempts to identify Dictyostelium proteins associated with endosomes were performed more than ten years ago and yielded a reduced amount of information due to technical limitations (Adessi et al., 1995; Rezabek et al., 1997). More recently the combination of improved fractionation methods with high throughput techniques is allowing dissection of the phagosome maturation pathway at increased resolution in several organisms (Griffiths and Mayorga, 2007). In these studies actin, actin-binding proteins (ABPs) and numerous signaling components directly associated with the actin cytoskeleton are consistently found. An important milestone has been the publication of proteomes over the course of maturation of Dictyostelium latex beads phagosomes (Gotthardt et al., 2006). Such studies are revealing a hitherto unexpected complexity of the phagosome maturation process. While diverse approaches have been employed to address the role of a particular protein in the endocytic pathway, the limitations of each method should always be taken into account. In many cases localization studies suggest a role in endocytosis, yet disruption of the corresponding gene results in no overt quantitative alteration of this process, frequently due to functional compensation by one or more other genes. Conversely, disruption of a particular gene may result in impaired endocytosis in the absence of biochemical or immunohistochemical association of the corresponding protein with endosomal compartments. In general it can be said that only a combination of several approaches will provide a complete picture on the role of a protein in endocytosis.

3. Role of D. discoideum Actin and ABPs in Endocytosis 3.1. Actin Because filamentous actin can be easily stained with fluorescent derivatives of phalloidin, the accumulation of actin and its colocalization with numerous ABPs during formation of phagosomes and macropinosomes is reported

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359

almost routinarily. Indeed, actin is a major protein of isolated endocytic vesicles and phagosomes (Adessi et al., 1995; Nolta et al., 1994; Rezabek et al., 1997; Rodriguez-Paris et al., 1993; Yuan and Chia, 1999). Formation of an actin coat is essential for endocytosis, as shown in studies using the actin polymerization inhibitor drug cytochalasin A. Cytochalasin A has a dosisdependent and reversible effect on phagocytosis of yeast particles (Maniak et al., 1995) and on fluid-phase uptake (Hacker et al., 1997). Nevertheless, the requirements for actin in pinocytosis and phagocytosis seem to differ, because the 50% inhibitory concentration of cytochalasin A for pinocytosis is one order of magnitude lower than for phagocytosis. Also the effects of latrunculin A, a drug that sequesters monomeric actin, distinguish phagocytosis from pinocytosis: latrunculin A exerts a stimulatory effect on phagocytosis whereas pinocytosis becomes inhibited (Konzok et al., 1999). Because introduction of fluorescent probes in Dictyostelium cells, either by microinjection or electroporation, is notoriously cumbersome, monitoring the in vivo behavior of actin had to await the advent of GFP technology. As already mentioned (Section 2), coronin-GFP was the first probe introduced (Maniak et al., 1995), followed by GFP-actin (Westphal et al., 1997) and fluorescent protein fusions that allow specific visualization of actin filaments, like the actin-binding domain of filamin (Lee and Knecht, 2002; Pang et al., 1998), the N-terminus of LimE (LimE△coil) and the C-terminal 63 kDa fragment of talin A (TalC63). LimE△coil in particular is a sensitive probe for rapid assembly of new filaments because it labels newly polymerized filaments more strongly that older filaments (Diez et al., 2005). TalC63 has been used as a trap for actin filaments because it forms complexes with F-actin that dissociate slowly enough to be carried with the flow of actin, allowing visualization of transient actin flows (Weber et al., 2002). Actin accumulates at the cell cortex during formation of the phagocytic cup around solid particles. Once the particle is engulfed, the actin coat disassembles within 1 min. (Konzok et al., 1999; Maniak et al., 1995; Peracino et al., 1998). A similar behavior has been reported during macropinosome formation, where actin accumulates at crowns, remains transiently associated with the nascent macropinocytic vesicle and finally dissociates within 1 min after internalization (Lee and Knecht, 2002). In many cases disassembly of actin is visibly more rapid from the side of the phagosome facing the cytosol. Studies with the TalC63 probe showed that during formation of macropinosomes or phagosomes an actin flow is induced at the border of the cup and filaments disassemble at the base of the cup (Weber et al., 2002). Recent in vivo studies using bacteria instead of yeasts and multiple fluorescent probes have allowed a more accurate description of actin’s behavior during particle uptake and the role of the Arp2/3 complex in assembly and of coronin in disassembly of actin filaments (Sections 3.2.1 and 3.6.2) (Clarke and Maddera, 2006; Lu and Clarke, 2005). Such studies have

360

Francisco Rivero

revealed the formation, shortly after detachment of actin from the phagosome, of an actin comet tail between the phagosome and the plasma membrane that propels the phagosome away from the site of uptake. Actin-powered movement lasts for few seconds to up to 30 sec before actin again dissipates and the phagosome is captured by microtubules. A comet-like burst of actin accumulation after engulfment of yeast particles has went unnoticed probably due to the large size of the phagosome, but has also been reported recently, overlapping with accumulation of vacuolar proton pumps (Pikzack et al., 2005). Actin-mediated rocketing of phagosomes can also be induced mechanically by compressing the cells so as to bring the phagosome into contact with the plasma membrane, but the mechanism that triggers actin polymerization and the functional implications of this phenomenon are not clear (Clarke et al., 2006). Actin is present at vesicles along the entire postlysosomal pathway (Rauchenberger et al., 1997). Treatment of cells with latrunculin A or expression of a post-lysome targeted cofilin disrupts the actin coat of postlysosomal vesicles and results in vesicle clustering, indicating that one function of the actin coat is keeping the late endosomes in a disperse state throughout the cytoplasm (Drengk et al., 2003). Because cytochalasin A has an inhibitory effect on exocytosis, the actin coat may facilitate the association of the exocytotic vesicle with the cell cortex (Rauchenberger et al., 1997). The in vivo actin dynamics during exocytosis has been seldom reported, in great part because, contrary to uptake, release of endocytosed material cannot be readily synchronized and is therefore difficult to capture. Using GFP-ABD as a probe, Lee and Knecht (2002) have shown that postlysosomal vesicles with a weak actin coat persist within the cell for long periods of time, an indication that actin coating does not by itself trigger the exocytotic process. Rather, vesicle docking is followed by release of contents and, apparently, a concomitant increase of actin polymerization while the vesicle collapses. Accumulation of actin (as monitored by the actin-binding protein LimC) during exocytosis of yeast particles has also been reported (Khurana et al., 2002a).

3.2. The F-actin nucleation machinery Local generation of actin filaments in response to signals is tightly regulated. Two major protein complexes are employed by the cell to initiate new actin filaments, the Arp2/3 complex and formins (Pollard, 2007). The Arp2/3 complex produces branched filaments and remains attached to the pointed end; it is a stable equimolar assembly of seven subunits consisting of two actin related proteins (Arp2 and Arp3) and five more proteins (ARPCs). The complex is intrinsically inactive and requires so-called nucleation promoting factors, like WASP/Scar family proteins and CARMIL and in yeast (but not in Dictyostelium) myoI and Abp1. Formins, on the contrary,

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361

form ring-shaped flexible dimers that associate to barbed ends, where they antagonize capping proteins and allow processive elongation of unbranched actin filaments. 3.2.1. The Arp2/3 complex The Arp2/3 complex plays a primary role in the nucleation of actin filaments in many cellular processes. It is required for phagosome formation in macrophages (May et al., 2000) and for clathrin-mediated endocytosis in yeast and mammalian cells (Kaksonen et al., 2006). In Dictyostelium the localization of the complex has been investigated using Arp3 and p21-Arc specific antibodies and fluorescent protein fusions of Arp3 and ARPC1 (p41). The complex is localized diffusely in the cytoplasm, occasionally with a punctate pattern, and accumulates at the cell cortex, crowns and, in a discontinuous pattern, at large vesicles (Insall et al., 2001; Jung et al., 2001). In vivo studies revealed that the complex accumulates at sites of particle attachment and later on at the border of the extending phagocytic cup until the membrane seals around the particle. A similar behavior was observed during formation of macropinosomes. The complex is stripped off the endosome shortly after closure, coincident with acidification of its lumen, and re-associates with postlysosomal endosomes, suggesting that the Arp2/3 complex participates in reorganization of the actin system both during uptake and exocytosis (Insall et al., 2001). In isolated phagosomes Arp3 can be detected predominantly during phagosome formation (Gotthardt et al., 2006). The Arp2/3 complex accumulates also at comet tails that propel phagosomes around ingested bacteria after the phase of actin assembly and disassembly that follows uptake (Clarke and Maddera, 2006). In an in vivo study in which actin-mediated rocketing of yeast-containing phagosomes was induced by compression, mRFP-p41 and GFP-Arp3 were found at places where the phagosome contacts the plasma membrane, almost completely overlapping with an actin probe. Interestingly, when the phagosome moved away a ring or track of GFP-Arp3 was left behind and dissipated (Clarke et al., 2006). There are no molecular genetics studies on the Arp2/3 complex of Dictyostelium to date. Because each component of the complex is encoded by a single gene, it is very likely that targeting of any of them results in a lethal phenotype. 3.2.2. WASP/Scar family In Dictyostelium this family consists of Scar, WASP (Wiskott-Aldrich syndrome protein) and two uncharacterized WASP-related proteins. These proteins share a central proline-rich region and a C-terminal region composed of one WH2 (WASP-homology 2) domain that binds actin monomers followed by one acidic region that interacts with the Arp2/3 complex. The proline-rich region binds profilin as well as SH3 domains from a variety of proteins (Takenawa and Suetsugu, 2007). Both Scar and WASP are positive regulators of actin polymerization in Dictyostelium (Myers et al., 2005;

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Seastone et al., 2001). WASP plays important roles in motility and chemotaxis, however its subcellular localization and participation in endocytic processes have not been addressed. By contrast, a requirement for WASP is well established in other organisms. For example, it is recruited to nascent FcgR-mediated phagosomes in macrophages, as well as to Golgi vesicles and to clathrin coated vesicles, where it induces actin polymerization (Takenawa and Suetsugu, 2007). It is conceivable that WASP, similar to other cytoskeleton components like class I myosins (Section 3.8.2), plays dual roles in cell motility and endocytosis both in aggregation competent and vegetative cells, respectively. Although it remains to be verified experimentally, WASP might be targeted to sites of particle or fluid phase uptake through binding to phosphoinositides. WASP binds in vitro to phosphatidylinositol (4,5) bisphosphate (PIP2) and phosphatidylinositol (3,4,5) trisphosphate (Myers et al., 2005), the latter accumulating transiently at phagocytic cups and macropinocytic crowns (Dormann et al., 2004). Interaction with phosphoinositides and Rho GTPases present at the plasma membrane would then result in a conformational change that renders WASP active. WASP is able to interact with several Rho GTPases, and RacC, a Rho GTPase involved in phagocytosis, appears to be the major regulator (Han et al., 2006), providing a link of WASP to endocytic processes that needs further analysis. One more regulator of WASP is the recently described WIPa (WASP-interacting protein a), a member of the verprolin family of proline-rich ABPs. Verprolins act as scaffolds that interact with many SH3 domain-containing proteins as well as with profilin and, through its C-terminal region, with WASP. In Dictyostelium WIPa is important for actin remodeling during chemotaxis, but a role in vegetative cells has not been explored (Myers et al., 2006). Unlike WASP, the role of Scar in endocytosis has been extensively investigated in a knockout cell line (Seastone et al., 2001). These cells have reduced rates of phagocytosis (80%), pinocytosis (40%) and exocytosis, delayed transit from the lysosomal to the post-lysosomal compartment and defective secretion of lysosomal enzymes. The levels of F-actin are reduced by 50% and macropinocytic crowns are absent. Scar is a predominantly cytosolic protein only weakly enriched at the cell cortex at actin-rich protrusions. It does not associate stably with endolysosomes, however the association might be transient and might be needed to trigger actin polymerization, because endolysosomes of Scar null cells lack an F-actin coat. Consistent with this role, inhibition of actin polymerization with cytochalasin A results in similar phenotypes as those described above for the Scar null mutant. Disruption of Scar in a profilin I and II null background results in additive effects on fluid phase uptake and release as well as on secretion of lysosomal enzymes, consistent with profilin participating in the F-actin nucleation process through binding to the proline-rich region of Scar. Like its mammalian homolog WAVE (WASP family verprolin homology protein), Scar exist as a multimolecular complex with PIR121, Nap1,

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Abi2 and HSPC300 (Blagg et al., 2003; Vlahou and Rivero, 2006). Strains deficient in PIR121 or Nap1 have been described, but only their roles in motility and filopod formation, respectively, have been investigated (Blagg et al., 2003; Steffen et al., 2006). Although lacking a Rho GTPase binding domain WAVE is subject to regulation by Rac, but the exact mechanism and the role of other components of the complex in targeting and activation of Scar/WAVE remain controversial. 3.2.3. CARMIL CARMIL (p116; capping protein, Arp2/3 and myosin I linker) was identified in a search for proteins interacting with the SH3 domain of class I myosins MyoB and MyoC ( Jung et al., 2001). It turned out to be homologous to Acanthamoeba p125, and similar proteins have been subsequently found in metazoa. Members of the CARMIL family have a C-terminal half that harbors a region functionally related to the WH2 and acidic domains of WASP family proteins. The C-terminus binds all subunits of the Arp2/3 complex and displays Arp2/3 dependent actin nucleation activity, although weaker than WASP. This region is followed by a proline-rich region that does not contain runs of five or six consecutive proline residues, and therefore is not expected to bind profilin; by contrast, it contains PXXP motifs that bind SH3 domains. This proline-rich region is immediately followed by a CAH3 domain (CARMIL homology domain 3), a region responsible for binding of both subunits of the capping protein (CP). In fact, CARMIL proteins are potent antagonists of CP (Section 3.4.2) (Remmert et al., 2004; Yang et al., 2005). CARMIL localizes in dynamic actin-rich extensions, like macropinocytic crowns, where components of the complex like MyoC and Arp3 also localize. Consistent with a role in pinocytosis, CARMIL deficient cells show a 45% reduction of the rate of fluid phase uptake. Mutant cells display defective formation of macropinocytic crowns and a smaller intracellular endocytic compartment ( Jung et al., 2001). It has been proposed that the predominant role of CARMIL would be as a regulator of the functional levels of CP. The interaction with SH3-bearing class I myosins would be used to translocate and concentrate the complex at the vicinity of the plasma membrane, facilitating the extension of actin filaments and the formation of protrusions. Studies on the Acanthamoeba ortholog suggest that CARMIL forms homodimers that might exist in an autoinhibited state (Remmert et al., 2004). In this respect, CARMIL would behave like the other nucleators described in this section, and even activation by Rho GTPases has been postulated (Uruno et al., 2006). 3.2.4. Formins, VASP and IQGAP Evidence is accumulating, particularly in mammalian cells, towards a participation of formins in endocytosis. Some formins localize on endosomes and are apparently required for their motility, although there are still many

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open questions regarding targeting and regulation of these proteins (Faix et al., 1996). Only four of the 10 formins of Dictyostelium (ForA to ForJ) have been characterized to some extent (Rivero et al., 2005). ForH localizes predominantly at the tips of filopods, along with VASP (vasodilator-stimulated phosphoprotein), with which it interacts, and both are required for formation of filopods. In addition to this role, VASP is required for particle and substrate adhesion, although cells lacking VASP were able to take up latex beads at a normal rate (Han et al., 2002). Mutants lacking ForA, ForB or both have been described as showing no detectable growth phenotype. Finally, cells lacking ForC, a formin that plays roles at late developmental stages, grew normally in nutrient medium and in bacterial lawns, indicating that this formin might be dispensable for endocytosis (Kitayama and Uyeda, 2003). However, in vivo experiments with a GFP fusion showed that ForC accumulates at macropinocytic crowns and that the N-terminal region is sufficient for targeting the protein to places of active actin reorganization, like macropinosomes, phagocytic cups and cell-to-cell contacts (Kitayama and Uyeda, 2003). Several formins might conceivably play redundant roles, which would explain the absence of detectable phenotypes in some mutants. Clearly, much work is still needed to delineate the roles of formins in endocytosis and other processes in Dictyostelium and other organisms, and how localization and activity are regulated, in particular by Rho GTPases, for which formins function as effectors. More recently IQGAP1 has been identified as interaction partner of the formin Dia1, and this interaction appears to be required for phagocytic cup formation in macrophages (Brandt et al., 2007). IQGAPs are scaffolding proteins that interact with cytoskeletal and signal transduction proteins and are primarily involved in cell adhesion, binding to microtubules and cytokinesis (Vlahou and Rivero, 2006). A possible implication in endocytic processes has been investigated only in one of the four IQGAPs of Dictyostelium, DGAP1, which accumulates at the cell cortex and is an effector of Rac1a. Cells lacking DGAP1 display unaffected uptake of yeasts, and overexpression of the protein results in a moderately (25%) reduced rate of particle uptake (Faix et al., 1998). This alteration might be consequence of the reduced levels of F-actin and the absence of F-actin at cortical protrusions found in the overexpressor cells, which in turn might result from misregulated formin activity.

3.3. Monomeric actin binding proteins ABPs that bind G-actin are essential both to maintain and regenerate the pool of unpolymerized actin and to make the actin monomers available for filament elongation by constituting part of the nucleation machinery discussed above (Section 3.2). Profilin and the cyclase-associated protein are

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the best characterized ABPs of this class, which also includes the unexplored actobindins and twinfilin. 3.3.1. Profilin The abundant profilins catalyze the exchange of ADP bound to G-actin for ATP. In complex with ATP-actin profilins then efficiently promote elongation of F-actin barbed ends, particularly in association with proteins that contain poly-L-proline-rich motifs, like WASP, Scar, VASP and formins. In addition, profilins bind with high affinity phosphoinositides (in particular PIP2), causing an inhibition of their hydrolysis by phospholipase C. The Dictyostelium genome encodes three profilins of which two have been studied in detail (Haugwitz et al., 1991). Profilin I and II display a uniform cytoplasmic localization and concentrate and colocalize with actin at nascent phagosomes (Lee et al., 2000; Yuan and Chia, 1999). This localization at early phagosomes seems independent of the ability to bind poly-L-proline motifs, as shown with a profilin II mutant (W3N) with abolished poly-L-proline binding capacity (Lee et al., 2000). Profilin I and II have been identified as constituents of isolated early phagosomes, from where they detached together with actin and cofilin during phagosome processing (Yuan and Chia, 1999), although more sophisticated profiles of phagosome proteins show a more persistent localization of profilin along the endocytic pathway (Gotthardt et al., 2006). Although profilins I and II differ in their biochemical properties (Haugwitz et al., 1991), they appear to be functionally equivalent. Single mutants lacking either of them show an unaltered phenotype, which to some extent can be explained by compensatory increased expression of the remaining isoform. Double mutants, by contrast, displayed a severe phenotype. They have increased levels of F-actin with notable defects in uptake and transit of fluid phase: low rate of pinocytosis, less numerous macropinosomes of small size, delayed progress during the acidification phase, defective secretion of lysosomal enzymes and delayed exocytosis (Haugwitz et al., 1994; Seastone et al., 2001; Temesvari et al., 2000). Phagocytosis, by contrast, has been reported as normal (Haugwitz et al., 1994) or increased (Temesvari et al., 2000). The phenotypes elicited by loss of profilin may result not only from an altered actin polymerization, but also from an altered phosphoinositide turnover. An increased availability of PIP2 in the double knockout mutant has been invoked to explain the normal or increased rate of phagocytosis versus the profoundly reduced rate of fluid uptake. PIP2 is a substrate of phospholipase C, which is specifically required for phagocytosis (Duhon and Cardelli, 2002). Moreover, it is interesting that most of the vesicle transport defects of the profilin deficient mutant are partially rescued upon disruption of the gene encoding DdLIMP, a glycoprotein of the

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endolysosomal pathway (Karakesisoglou et al., 1999; Temesvari et al., 2000). DdLIMP belongs to the CD36/LIMP-II (lysosomal integral membrane protein) family, which includes plasma membrane receptors and lysosomal membrane proteins of unclear function in the endolysosomal pathway (Kuronita et al., 2002). DdLIMP does not bind profilin or actin, but it binds PIP2, and therefore DdLIMP and profilin might compete for PIP2 binding on endolysosomal membranes. 3.3.2. Cyclase-associated protein The cyclase-associated protein (Srv2/CAP) is a multifunctional protein composed of two domains separated by a central proline-rich stretch and a conserved WH2 domain of unclear role. The C-terminal domain is responsible for binding to monomeric actin, whereas the N-terminal domain inhibits this activity in a PIP2-dependent manner (Gottwald et al., 1996). The function of CAP appears to be more complex than simple monomer sequestering. CAP forms high molecular weight complexes with actin that function as monomer processing intermediates that accelerate cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhance nucleotide exchange by profilin (Balcer et al., 2003; Paavilainen et al., 2004). CAP accumulates at the cell cortex (Gottwald et al., 1996). Using GFP fusions of CAP and several domain combinations it was shown that targeting to the cell cortex is mediated by the N-terminal domain and that the proline-rich central region is dispensable for targeting (Noegel et al., 1999). In homologs from other organisms the proline-rich region binds to SH3 domains. The yeast protein (Srv2), for example, is targeted to cortical actin patches through binding of the SH3-domain containing protein Abp1p (Hubberstey and Mottillo, 2002). The proline-rich region of Dictyostelium CAP is less prominent than in yeast or mammalian homologs: there is only one motif that could potentially bind SH3 domains, but this has not been analyzed. For the same reason, a direct interaction with profilin reported in Srv2 is unlikely in Dictyostelium CAP. Compatible with a role at the cell cortex, a mutant strain that expresses less than 5% of the endogenous protein showed a pinocytosis defect but phagocytosis was not affected (Noegel et al., 1999). Additionally, CAP plays roles at the interface between the actin cytoskeleton and the endolysosomal system. CAP displays partial colocalization with components of the endocytic pathway, like V-ATPase, N-ramp1 and vacuolin. It binds to subunits of the V-ATPase and the CAP mutant has reduced levels and altered distribution of this proton pump. The function of the V-ATPase is also altered, resulting in a slightly higher basal endolysosomal pH, but unaffected time course of acidification of endosomes and relocalization of the V-ATPase during phagocytosis (Sultana et al., 2005).

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3.4. Severing and capping proteins Severing and capping of actin filaments are essential activities during remodeling of actin-based structures. Capping of barbed ends prevents addition of actin monomers and growth of filaments, whereas severing facilitates disassembly of filaments and subsequent release of actin monomers. 3.4.1. Cofilin and Aip Two of the five functional Dictyostelium cofilins have been investigated. Cofilin-1 is expressed in vegetative cells and during early development, and is apparently essential (Aizawa et al., 2001, 1995). Cofilin-2 predominates in the aggregation stage and is absent in vegetative cells; disruption of the corresponding gene does not cause any apparent phenotype (Aizawa et al., 2001). I will focus therefore on cofilin-1. It binds to and depolymerizes actin filaments in a pH-dependent and phosphoinositide-regulated manner and has a relatively weak monomer sequestering activity. Phosphorylation of a serine residue close to the N-terminus appears to be a major inhibitor of cofilin function in mammals and A. castellani, but such a regulatory mechanism is apparently absent in Dictyostelium cofilin-1 (Aizawa et al., 1997; Yuan and Chia, 1999). Cofilin-1 distributes uniformly in the cytoplasm and at sites of active remodeling of actin networks, such as crown-like protrusions, phagocytic cups and leading edges of migrating cells, where the protein rapid and transiently accumulates (Aizawa et al., 1995, 1997; Konzok et al., 1999; Yuan and Chia, 1999). Cofilin-1 was also identified as a constituent of early phagosomes, from which it detached together with actin and profilins during phagosome processing (Yuan and Chia, 1999). Cells that overexpress cofilin-1 have increased levels of F-actin, organized in actin bundles, but otherwise grow normally in nutrient medium and perform normal phagocytosis (Aizawa et al., 1996). Nevertheless, the fact that cofilin-1 appears to be essential is consistent with an absolute requirement for actin remodeling during food uptake. Cofilin activity is also regulated by interaction with other components of the actin cytoskeleton, like actin interacting protein (Aip) and CAP. Aip1 is a nine WD40-repeat protein that enhances the severing activity of cofilin (Aizawa et al., 1999) with which it associates to form a barbed end cap that prevents reannealing of severed filaments (Balcer et al., 2003). Aip1 localizes to regions of active microfilament remodeling like phagocytic cups and macropinosomes, where it colocalizes with actin (Aizawa et al., 1999; Konzok et al., 1999). The enrichment of Aip at these structures is transient; at phagosomes in particular, Aip detaches within 1 min after internalization. Consistent with a role in the rapid remodeling of the cortical actin meshwork, Aip1 null cells were strongly impaired in phagocytosis and fluid-phase uptake, and engulfment of yeast particles was prolonged, as monitored using GFPactin. The localization of cofilin during phagocytosis was not altered in Aip

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null cells. Overexpression of the protein did not affect fluid phase uptake, but increased the rate of yeast particle uptake (Konzok et al., 1999). 3.4.2. Capping protein Capping protein (CP; aginactin, Cap32/34) caps but does not sever or nucleate actin filaments and thereby prevents the addition or loss of actin subunits at the barbed filament end. It is composed of two subunits, each encoded by a distinct gene (Schleicher et al., 1984). The subcellular localization of CP in Dictyostelium has not been reported, but since the A. castellani homolog localizes in the cytoplasm and accumulates in dynamic actin-rich regions like the cell cortex and filopods (Cooper et al., 1984), a similar localization in Dictyostelium can be inferred. Strains deficient in one or both subunits of the CP have not been described, but the function of the heterodimer has been investigated using strains where simultaneous expression of both subunits is upregulated or downregulated. These studies have shown that CP is the major regulator of the number of free barbed ends and contributes to terminating the actin polymerization response upon chemoattractant stimulation (Hug et al., 1995). One would expect endocytosis to be severely affected in those strains, and although uptake of fluid or particles has not been investigated, underexpressors grew slowly both in nutrient medium and in bacterial suspension, whereas overexpressors displayed no defects. The capping activity of CP is inhibited by PIP2 (Haus et al., 1991) and by proteins harboring a CAH3 domain (Section 3.2.3). 3.4.3. Gelsolin family Members of this family are characterized by the presence of several copies (three to seven) of the conserved gelsolin repeat. Of the eight members of this family, four have been characterized to some extent: severin, protovillin (Cap100), villidin and GRP125. The most extensively studied is severin, an abundant protein that displays nucleating, severing and capping activity in addition to G-actin binding and is regulated by Ca2þ, phosphoinositides and phosphorylation (Eichinger et al., 1998, 1991; Eichinger and Schleicher, 1992). Severin appears diffusely distributed in the cytosol, with some enrichment at pseudopods and phagosomes (Andre et al., 1989; Brock and Pardee, 1988), and it has also been reported at isolated phagosomes (Gotthardt et al., 2006). Cells deficient in severin grow normally both in nutrient medium and on bacterial lawns, indicating that endocytosis is not severily impaired in this mutant (Andre et al., 1989). Protovillin displays a strong capping activity and binds G-actin. Its main function might be in regulating the G- and F-actin pools, but there are no data on the subcellular localization of this protein and functional studies are also lacking (Hofmann et al., 1992). Villidin and GRP125 share many features (Gloss et al., 2003; Stocker et al., 1999). Both lack the first gelsolin repeat and may not display nucleating, severing and capping activities, and

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both localize at vesicular compartments. Villidin however has a coroninrelated module in the N-terminus that seems to be the major actin-binding region of this protein. Villidin and GRP125 have been proposed as links between membranes and the cytoskeleton, but appear dispensable for endocytosis (Gloss et al., 2003; Stocker et al., 1999).

3.5. Actin crosslinking proteins F-actin crosslinking proteins stabilize three-dimensional networks or densely packed bundles of actin filaments, depending on the spatial arrangement of their actin-binding sites and the length and flexibility of spacer elements that separate the actin-binding sites. This class of proteins contains some of the most extensively characterized ABPs, which will be discussed first. 3.5.1. Filamin, a-actinin, ABP34 and fimbrin Filamin (ddFLN, formerly gelation factor or ABP-120), a-actinin, the 34 kDa actin-bundling protein (ABP34) and fimbrin are abundant regulators of the organization of cortical microfilaments. Whereas ABP34 is apparently unique to Dictyostelium and related species, fimbrin, filamin and a-actinin are widely distributed among the eukaryotes. ABP34 and fimbrin are the only calcium inhibited ABPs that function primarily to induce the formation of F-actin bundles (Fechheimer and Taylor, 1984; Prassler et al., 1997). Filamin crosslinks actin filaments to form branched networks, whereas a-actinin, also a calcium inhibited ABP, gives rise to lateral arrays (Condeelis et al., 1984). ABP34, fimbrin and filamin have been found enriched in the cell cortex, including protrusions like pseudopods and filopods (Carboni and Condeelis, 1985; Johns et al., 1988; Ogihara et al., 1988; Prassler et al., 1997). By contrast, a-actinin displays a diffuse and patchy distribution throughout the cytoplasm and is also enriched in pseudopods and at the leading edge (Brier et al., 1983). ABP34 colocalizes with actin during formation of phagocytic cups and dissociates as the phagosome matures (Furukawa et al., 1992; Furukawa and Fechheimer, 1994). The protein has also been identified in isolated phagosomes (Rezabek et al., 1997). Fimbrin localizes at phagocytic cups and macropinosomes, where it also colocalizes with actin (Pikzack et al., 2005). The enrichment of fimbrin at these structures is transient, and detachment takes place within 1 min after internalization, as revealed in in vivo studies with GFP-fimbrin and deletion mutants (Pikzack et al., 2005). Filamin colocalized with actin at phagocytic cups (Cox et al., 1996) whereas a-actinin did not consistently accumulate at the phagocytic cup (Maniak et al., 1995), rather it was found to associate with phagosomes at later stages (after ABP34 has been recruited) and to remain associated with the maturing phagosome after ABP34 has dissociated (Furukawa and Fechheimer, 1994). In agreement with a role at late stages of phagocytosis, other studies

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show only very low amounts (Schreiner et al., 2003) or complete absence (Maniak et al., 1995) of the protein in phagosomal fractions. From the localization and the presumed role of actin crosslinkers in stabilizing the actin coat a prominent role during the uptake phase would be anticipated. Early experiments in which a specific monoclonal antibody that blocks the interaction of ABP34 with actin in vitro were loaded into cells specifically inhibited, but did not abolish phagocytosis, supporting a specific implication of ABP34 in phagocytosis (Furukawa et al., 1992). However, single knockout mutants of a-actinin, ABP34 and fimbrin did not present obvious defects in the rates of phagocytosis or fluid phase uptake, ABP34 null cells even displayed increased phagocytosis in quantitative tests (Pikzack et al., 2005; Rivero et al., 1996a, 1996b, 1999; Wallraff et al., 1986). Mutants deficient in filamin have been generated in different genetic backgrounds with conflicting outcomes. Whereas those generated in the AX2 strain (either by chemical mutagenesis or by homologous recombination) lacked any obvious phenotype (Brink et al., 1990; Rivero et al., 1996b, 1999; Witke et al., 1992), those generated in AX3 displayed abnormal formation of phagocytic cups and 50% reduced uptake of bacteria or latex beads, but normal rates of uptake of a fluid phase marker (Cox et al., 1996). The mild phenotype of the fimbrin null mutant is in contrast to the situation in yeast, where fimbrin is essential for endocytosis (Kubler and Riezman, 1993) and illustrates very nicely the caveats of extracting universal conclusions from studies on a particular organism. Precisely yeast is equipped with a rather limited repertoire of ABPs, and gene disruptions lead more frequently to overt phenotypes. The absent or mild phenotypes of single mutants has prompted several studies on strains deficient in combinations of two proteins aimed at verifying the hypothesis that some extent of functional redundancy exists among actin crosslinkers. Some combinations of double knockouts, like a-actinin/ filamin or a-actinin/ABP34, were found more deleterious than others, like ABP34/filamin or ABP34/fimbrin (Pikzack et al., 2005; Rivero et al., 1996b, 1999). These studies have revealed a complex network of unique and shared roles among actin crosslinkers. Nevertheless, the roles of additional yet uncharacterized crosslinkers uncovered after sequencing of the Dictyostelium genome need to be investigated in order to have a better picture of the contribution of this class of ABPs to endocytosis. 3.5.2. Other actin crosslinkers Cortexillins and dynacortin are best known for their roles in maintaining cortical viscoelasticity both in interphase and during cytokinesis (Girard et al., 2004; Simson et al., 1998). In interphase cells both cortexillin isoforms and dynacortin are enriched at the cell cortex and macropinocytic crowns (Faix et al., 1996; Robinson et al., 2002), and cortexillins have been identified in isolated phagosomes both in Dictyostelium (Gotthardt et al., 2006),

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and in E. histolytica (Marion et al., 2005). However, the behavior of these proteins during endocytosis has not been addressed, and endocytosis studies in knockout or overexpression mutants have not been reported. Some transcriptional elongation factors constitute important physical and functional links between the translational machinery and the actin cytoskeleton. The best studied is elongation factor 1A (ABP50, eEF1A) an abundant protein that binds G-actin and bundles F-actin, but also eEF1B displays actin-binding properties. Both proteins distribute diffusely throughout the cytosol and are enriched at actin-rich regions of the cell cortex (Dharmawardhane et al., 1991; Furukawa et al., 2001). Noncanonical roles of eEF1A have been established for example in studies with yeast eEF1A, where mutants with reduced actin bundling activity cause alterations in actin cytoskeleton organization (Gross and Kinzy, 2005). It remains speculative, albeit not unlikely, that elongation factors are implicated in actin remodeling during endocytic traffic, but this aspect has not been addressed specifically and mutants are not available. In fact these proteins have been repeatedly identified in isolated phagosomes in several organisms, including Dictyostelium (Gotthardt et al., 2006) and E. histolytica (Okada et al., 2005).

3.6. Lateral ABPs In general the ABPs included in this section function as scaffolds for the recruitment of multiprotein complexes that frequently include signaling molecules, like small GTPases. These proteins therefore constitute important elements at the interface between signal transduction, remodeling of the actin cytoskeleton and vesicle trafficking. 3.6.1. ADF/cofilin family The ADF (actin depolymerization factor) is found in cofilin and in several proteins that bind to filamentous actin without displaying severing activity. Two of them, coactosin and Abp1, have been reported. For coactosin only in vitro data is available: the protein binds but does not bundle actin and appears to counteract the capping activity of CP and severin (Rohrig et al., 1995). Coactosin was identified as a protein associated to pre-spore vesicles, along with profilin in a proteomics study (Srinivasan et al., 2001) and as a component of isolated phagosomes (Gotthardt et al., 2006) but the subcellular localization of this protein has not been addressed in detail and there are no functional studies, therefore the cellular function of coactosin remains unclear. Abp1 is a conserved protein and has homologs in metazoa (drebrin F) and yeast (Abp1p). In Abp1p the ADF domain is followed by an acidic region, a proline-rich domain and a SH3 domain. In yeast and mammals Abp1 constitutes an important functional link between the endocytic machinery and actin filament dynamics: the SH3 domain interacts with

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numerous proteins all involved in endocytosis, including dynamin, synaptojanin, CAP and the class I myosin Myo5 (Qualmann and Kessels, 2002). While binding to Dictyostelium CAP is unlikely (Section 3.3.2), class I myosins appear as relevant binding partners of Abp1, and indeed an interaction with MyoK has been reported (Soldati, 2003). It was also shown that Abp1p binds and activates the Arp2/3 complex presumably via its acidic region (Higgs and Pollard, 2001), but in the Dictyostelium homolog the acidic region cannot be recognized and it remains to be seen whether it has a similar function as the yeast Abp1p. In Dictyostelium Abp1 localizes at the cell cortex of vegetative cells where it co-localizes with actin to a large extent, but in depth studies addressing the localization of Abp1 during endocytosis are missing. Work on strains lacking or overexpressing Abp1 has revealed a role during early aggregation, where the protein regulates the number of pseudopods, but the protein seems to be dispensable for pinocytosis and phagocytosis (Wang and O’Halloran, 2006). 3.6.2. Coronins Coronins are proteins of the WD repeat family that apart from binding F-actin are also able to bind to and inhibit the nucleation activity of the Arp2/3 complex, although this last aspect has been addressed biochemically only in yeast and mammalian coronin (Uetrecht and Bear, 2006). Coronin strongly accumulates at crown-like surface extensions (hence the name of the protein) as well as at phagocytic cups and other regions of active actin remodeling (de Hostos et al., 1991). GFP-coronin accumulates at the phagocytic cup and at crowns, and is gradually released within 1 min after closure of the phagosome or macropinosome (Hacker et al., 1997; Lu and Clarke, 2005; Maniak et al., 1995). Coronin is also present at actin-coated vesicles of the post-lysosomal compartment prior to accumulation of vacuolin (Rauchenberger et al., 1997). More accurate observations using GFPcoronin and mRFP-LimE△coil show binding of coronin to actin filaments a few seconds after they have formed. At comet tails that form after ingestion of bacteria or when actin-driven rocketing of phagosomes is induced by compression of the cell to bring the phagosome into contact with the plasma membrane, coronin is recruited to the end of the actin tails, where actin filaments disassemble. All this is consistent with an inhibitory role of coronin on the Arp2/3 complex (Clarke and Maddera, 2006; Clarke et al., 2006). Mutants lacking coronin display markedly decreased rates of phagocytosis and macropinocytosis (de Hostos et al., 1993; Hacker et al., 1997; Maniak et al., 1995). Interestingly, coronin deficient cells, contrary to many cytoskeleton mutants, are more permissive for intracellular growth of Legionella pneumophila and Mycobacterium marinum (Fajardo et al., 2004; Solomon et al., 2000, 2003). These studies might point towards roles of coronin at later steps of the endocytic pathway. For example, in macrophages coronin 1A associates with the p40phox subunit of the NADPH oxidase complex

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(Grogan et al., 1997). If Dictyostelium coronin functions in a similar way and is required for the assembly of the NADPH oxidase complex, its absence would favor survival of the ingested pathogens. Besides a typical coronin, two more members of the coronin family are encoded by the Dictyostelium genome, villidin (Section 3.4.3) and a coronin 7/POD-1 homolog. Because roles in intracellular trafficking have been proposed for members of the coronin 7 family (Uetrecht and Bear, 2006), it will be of great interest to study the Dictyostelium homolog. 3.6.3. LIM proteins Many LIM proteins associate directly or indirectly with the actin cytoskeleton (Khurana et al., 2002b), and several of the LIM proteins of Dictyostelium participate in vesicle trafficking processes. LimC, LimD and LimE (DdLim) have a similar pattern of subcellular localization: they localize preferentially at the cell cortex, where they colocalize with actin, and are transiently recruited to macropinosomes and phagocytic cups (Khurana et al., 2002a; Prassler et al., 1998; Schneider et al., 2003). Recruitment of LimC to the cell cortex during exocytosis has also been reported, and LimD has been identified in isolated phagosomes (Khurana et al., 2002a). Fluid phase and particle uptake of single and double mutants of LimC and LimD are unimpaired under standard conditions. Rather, these proteins appear more important for maintenance of cortical strength, establishment of cell polarity and chemotaxis (Khurana et al., 2002a). LimE appears to exist in a complex with activated Rho GTPases, but the implications of this association are unknown (Prassler et al., 1998). Although knockout and overexpressor strains of LimE have been described, these studies address mainly the role of the protein in cytokinesis. LimD is more closely related to LimE than to LimC, therefore it is likely that they play redundant roles in endocytosis. It would be interesting to study the phenotype of a LimD/LimE double mutant. A clear role in phagocytosis has been assigned to two more LIM proteins, LimF and CH-Lim, that interact with each other and with Rab21 (Khurana et al., 2005). Both proteins appear enriched at intracellular vesicles, but the identity of these vesicles has not been investigated. In addition, CH-Lim accumulates at the cell cortex as well as at phagocytic cups, and remains attached as the yeast particle traffics within the cell. The CH domain of this protein is probably responsible for association with actin at the cell cortex and during particle uptake, but the LIM domain region, which alone is purely cytosolic, seems to be required for prolonged association of the whole protein to the phagosome. LimF appears also enriched at phagocytic cups. Interestingly, Rab21 also displays a cytosolic and vesicular localization, but it was not determined whether all three components of the complex localize at the same vesicles. Extensive quantitative analyses performed on knockout and overexpressor strains of both LIM proteins in connection with Rab21 have revealed that all three components act

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cooperatively. Knockout of LimF and CH-Lim have opposite effects. Rab21 has a positive effect on phagocytosis, but both LIM proteins are required for Rab21 to function. Because LimF and CH-Lim null cells are not defective in adhesion, it is probable that the complex is not needed for particle attachment and uptake, but rather operates at the interface of the actin cytoskeleton and vesicle fusion during phagosome formation, in agreement with the localization of the components at vesicles. The interaction of these proteins with Rab21 is independent of the activation state of the GTPase, indicating that these LIM proteins are not effectors but might regulate availability or targeting of Rab21. It would be interesting to investigate the nature of these vesicles and their behavior in mutants of the complex. 3.6.4. ABPs containing a Rho GTPase exchange factor domain Three ABPs harboring one or more calponin homology (CH) domains and a Rho GTPase exchange factor (RhoGEF) domain have been reported very recently: RacGEF1, Trix and GxcDD. RacGEF1 associates partially with the cell cortex through a CH domain and plays roles in chemotaxis; a participation in endocytosis has not been addressed (Park et al., 2004). Trix contains an N-terminal region with three CH domains that binds and bundles actin filaments. Its localization has been studied with the help of GFP fusions of the full length protein or the N-terminal region. Both fusions accumulate at the cell cortex. Dynamics studies of yeast uptake with the N-terminal fusion have revealed that the protein does not enrich at the phagocytic cup, but progressively and very intensely accumulates after 45 min of incubation with yeast, presumably at late endosomes. It has not been determined, however, whether this pattern of distribution reflects the behavior of the full length protein. Further in support of a role in the regulation of late steps of the endocytic pathway, presumably exocytosis, cells deficient in Trix displayed normal rates of yeast uptake, but 30% reduction in exocytosis (Strehle et al., 2006). In the multidomain protein GxcDD the single CH domain functions as a membrane association domain, whereas an ArfGAP (GTPase activating protein for Arf )-PH (plekstrin homology) tandem colocalizes with actin at the cell cortex and accumulates at phagocytic cups. The targeting of the ArfGAP-PH tandem may be mediated by binding to phosphoinositides. As for Trix, however, the behavior of the full length protein has not been reported. A GxcDD deficient strain displayed normal pinocytosis and phagocytosis rates (Mondal et al., 2007).

3.7. Membrane-associated ABPs The establishment of links between membranes and the actin cytoskeleton is essential to endocytosis, and many of the ABPs discussed above are targeted to membranes and become regulated by phospholipids. In this

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section I will discuss several families of proteins for which the association to membranes is the most salient feature. Many unconventional myosins are able to establish links between actin filaments and membrane lipids, however myosins are discussed separately below. 3.7.1. Talin Talins exist as homodimers and are components of focal adhesions in metazoan cells where they act as links between integrins and the actin cytoskeleton (Critchley, 2005). Two talins have been characterized in Dictyostelium, talin A (TalA, originally called filopodin) and talin B (TalB). They are 50% similar to each other, but apparently functionally distinct, because neither talin compensates the defects of the other. This indicates that each isoform plays unique roles, TalA at the vegetative and TalB at the multicellular stage (Tsujioka et al., 1999). TalA accumulates at the tips of filopods, in the cortex and in dot-like structures on the ventral surface but is apparently excluded from podosome-like structures known as eupodia (Fukui and Inoue, 1997; Hibi et al., 2004; Kreitmeier et al., 1995). Although talin is enriched in phagocytic cups during Fc and complement receptor mediated particle uptake in macrophages (Lim et al., 2007), an enrichment of TalA during phagocytosis has not been reported in Dictyostelium (Weber et al., 2002). Cells lacking TalA showed defects in adhesion to the substrate, Ca2þdependent cell-to-cell adhesion and phagocytosis (Gebbie et al., 2004; Niewohner et al., 1997). The phagocytosis defect was traced down to the initial phases of uptake that require adhesion, because uptake of yeast particles improved with shaking at low frequency and only bacterial species devoid of carbohydrate moieties of the cell surface lipopolysaccharides are efficiently taken up by the TalA mutant. In addition, macropinocytosis, which does not depend on adhesion, was unaffected, and actin distribution to macropinosomes was not altered in TalA null mutants (Gebbie et al., 2004; Weber et al., 2002). There is genetic and biochemical evidence that TalA and MyoI function in the same pathway. Both null mutants display the same phenotype and both co-immunoprecipitate (Tuxworth et al., 2005). However, TalA is not needed for targeting of MyoI and vice versa, therefore a model has been put forward in which the interaction induces a conformational change in each protein; in the absence of one partner the other can no longer promote formation or stabilization of adhesion complexes. The membrane anchor of these complexes might be provided by Sib proteins, members of a family of type I transmembrane proteins with features found in integrin b. The cytosolic domain of these proteins interacts with TalA and a mutant defective in one of the Sib proteins (SibA) behaves in many respects like the TalA mutant (Cornillon et al., 2006). This mechanism of action resembles in many respects that of mammalian talin, that is specifically required for uptake through complement receptor 3

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(which is identical to integrin aMb2) through direct binding of the cytosolic domain of the b2 subunit and activation of the receptor (Lim et al., 2007). 3.7.2. Comitin Comitin is an unusually basic homodimeric protein with some relationship to mannose-specific plant lectins. By virtue of its mannose binding activity comitin might bind to vesicle membranes exhibiting mannosylated glycans ( Jung et al., 1996a). Comitin is present on Golgi membranes as well as on vesicles of unclear affiliation, probably of the ER and endosomes, although not lysosomes (Weiner et al., 1993). An enrichment in isolated early phagosomes has been reported, although immunolocalization studies have failed to reveal a specific enrichment around phagosomes (Gotthardt et al., 2006; Schreiner et al., 2003). Comitin null cells are impaired in the early steps of phagocytosis of yeast particles or bacteria, but not of latex beads, and accumulation of actin at phagosomes and subsequent dissociation was found unaffected. Other processes like pinocytosis, exocytosis and maturation of glycoproteins, were also found unaltered (Schreiner et al., 2003). The interpretation has been put forward that comitin associates with (and is needed for proper targeting) of a particular class of receptors. In support of this, comitin is enriched in the Triton-insoluble floating fraction (TIFF), where it co-caps, among others, with the cell adhesion molecule gp80 (contact site A) (Harris et al., 2003). In addition, comitin null cells are more pemissive to infections with Legionella pneumophila and Legionellalike bacteria; the mutation also delays degradation of Salmonella enterica (Skriwan et al., 2002). All this indicates a reduced efficiency of phagosome processing. This is in agreement with the proposal that comitin, by virtue of its biochemical activities and its localization, acts as a linker between intracellular membrane vesicles and the actin cytoskeleton ( Jung et al., 1996a). Nevertheless, the genome sequence has uncovered an uncharacterized comitin-related gene, therefore it is expected that the available data do not reveal the full spectrum of comitin’s roles in the cell. 3.7.3. Ponticulin Ponticulins constitute a large family of small atypical integral membrane proteins with a C-terminal glycosylated lipid anchor. Two ponticulins have been reported, ponticulin A and B, and only ponticulin A has been characterized extensively. Ponticulin A is abundant in axenically growing cells and during early development, where it accounts for most of the actinbinding activity of the plasma membrane (Hitt et al., 1994). Ponticulin A was found throughout the plasma membrane and in vesicles of the Golgi apparatus and, like comitin, it is enriched in the TIFF fraction (Harris et al., 2003). During phagocytosis the protein is present, although not enriched, in phagocytic cups and also associates with intracellular vesicles around the engulfed particle (Wuestehube et al., 1989). However, although membranes

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of ponticulin A null cells displayed only 10% of the high affinity actinbinding of wild type cells, the cells displayed unaffected pinocytosis and phagocytosis (Hitt et al., 1994), suggesting that the activities of ponticulin A are dispensable for endocytosis. Interestingly, ponticulin B, which exhibits 50% sequence similarity to ponticulin A and also localizes at the plasma membrane and in vesicles, is only expressed in cells grown on bacterial lawns and may play a clearer role in phagocytosis (Hitt et al., 2003). 3.7.4. Other membrane-associated ABPs Dictyostelium expresses two members of the annexin family of calcium and phospholipid binding proteins, annexin C1 (originally described as annexin VII or synexin) and annexin C2 (Marko et al., 2006). Annexins have been proposed to participate in the regulation of membrane organization, membrane trafficking and Ca2þ homeostasis. A number of annexins have been described as F-actin-binding proteins, and it has been suggested that they could participate in regulating membrane-cytoskeleton dynamics (Gerke et al., 2005). However, a direct binding of actin to any of the two annexins of Dictyostelium has not been demonstrated yet, therefore these proteins will not be discussed further. Hisactophilins by contrast are well documented ABPs targeted to the plasma membrane by virtue of myristoylation and positive charges of their numerous histidine residues (Hanakam et al., 1996). It has been proposed that hisactophilin acts as a pH sensor at the plasma membrane and reversibly connects the cortical actin network to the membrane in response to local changes of the proton concentration (Hanakam et al., 1996; Stoeckelhuber et al., 1996). During attachment and uptake of particles hisactophilin remains associated to the plasma membrane, where it re-shuffles after internalization (Maniak et al., 1995). Not surprisingly, histactophilin has been identified in isolated phagosomes (Gotthardt et al., 2006). Further evidence linking hisactophilins to endocytosis processes is missing: although a hisactophilin1/2 double mutant and an overexpressor strain have been generated, those processes have not been specifically addressed in these strains, and the Dictyostelium genome harbors a third hisactophilin gene that awaits characterization. Interaptin is an ABP of the a-actinin superfamily specifically targeted to the nuclear envelope, endoplasmic reticulum and Golgi apparatus (Rivero et al., 1998). It constitutes the Dictyostelium member of the metazoan nesprin family of proteins functioning as bridges that connect the nuclear matrix with the actin cytoskeleton (Ma¨a¨tta¨ et al., 2004). Despite its ER and Golgi localization, there is no evidence for interaptin playing roles in vesicle trafficking processes. Dictyostelium also has an uncharacterized member of the Sla2p/ Hip1 family of talin-related proteins. Proteins of this family bind to actin and clathrin at endocytic sites in yeast and mammals, functioning as adaptors that link the actin cytoskeleton to vesicle transport (Hyun and Ross, 2004).

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3.8. Actin-based molecular motors Myosins are actin-based motor proteins that convert the energy of ATP hydrolysis into movement. Dictyostelium expresses one conventional and twelve unconventional myosin heavy chains, along with up to eight myosin light chains (Kollmar, 2006). The roles of these myosins in diverse cellular processes and the pathways involved in their regulation have been the subject of several excellent reviews (Bosgraaf and van Haastert, 2006; de la Roche and Cote, 2001; Ma et al., 2001; Soldati, 2003). Here I will focus on the evidence linking Dictyostelium myosins (referring to the heavy chains only) to endocytosis and membrane traffic. 3.8.1. Myosin II and the myosin heavy chain kinase A The single conventional myosin of Dictyostelium has the characteristic long coiled-coil tail and assembles into bipolar filaments in a phosphorylationdependent manner. Myosin II accumulates at the cleavage furrow of dividing cells and at the rear end of migrating cells, and plays well established roles in cytokinesis, cell motility, chemotaxis and multicellular development. The evidence in support of a role of Dictyostelium myosin II in endocytosis and vesicle trafficking is scarce. The need for such studies was not appreciated because first, myosin II was not found to localize at phagosomes in immunofluorescence studies (Yumura et al., 1984) and second, it was soon realized that cells in which the myosin II heavy chain gene was disrupted were able to phagocytose bacteria but displayed more severe and attractive phenotypes (De Lozanne and Spudich, 1987; Manstein et al., 1989). By contrast, the participation of myosin II in phagocytosis is well established in other organisms. Myosin II is present in isolated phagosomes in E. histolytica, where it is recruited around the nascent phagosome during erythrophagocytosis. A strain that expresses a dominant negative myosin II displays a reduced erythrophagocytosis activity (Marion et al., 2005). It must be noted however that E. histolytica has a very limited repertoire of myosins (only a class I myosin, apart from myosin II), leaving little room for functional compensation. Myosin II has been also identified in phagosomes isolated from mouse macrophages (Garin et al., 2001). In these cells myosin II plays roles during CR3 and Fcg receptor-mediated phagocytosis, being required for phagosome closure (Araki et al., 2003; Olazabal et al., 2002). Is there any evidence linking Dictyostelium myosin II to any step of endocytosis? Electron microscopy studies on isolated cortices revealed an association of myosin II with unidentified cytoplasmic vesicles (Ogihara et al., 1988). Myosin II accumulates below ConA patches and moves with the patches into the cap (Carboni and Condeelis, 1985), a process that takes place less efficiently in cells lacking myosin II (Pasternak et al., 1989). This led to propose that this myosin participates in maintaining the resting-state cortical stiffness. Myosin II has been found in isolated early phagosomes,

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from which it is lost upon maturation (Rezabek et al., 1997), and in isolated early pinosomes, probably associated to actin filaments via the head domain (Neuhaus and Soldati, 2000). In one study with a GFP fusion myosin II was found at the base of phagocytic cups (but not at the protruding rim or at the entire phagosome), where it co-localized with PAKa (Mu¨llerTaubenberger et al., 2002). A shorter myosin II (HMM-140) unable to assemble into thick filaments does not localize at phagocytic cups (Fukui et al., 1990). Interestingly, accumulation of enzymatically inactive myosin II (either by treatment of wild type cells with the specific inhibitor blebbistatin, or by introducing the E476K mutant in myosin II deficient cells) results in strong inhibition of pinocytosis and phagocytosis, but this is probably due to formation of cytoplasmic aggregates, without altering the distribution of actin (Shu et al., 2005). In summary, the evidence gathered so far assigns a secondary part to myosin II in the endocytosis play, and the reason can be suspected at least in one of the proteins involved in its regulation, the myosin heavy chain kinase (MHCK) A. MHCK A is one of four closely related atypical kinases that phosphorylate the tail of Dictyostelium myosin heavy chain on specific threonine residues, driving disassembly of myosin filaments. These kinases have in common a catalytic domain followed by a WD repeat domain that binds directly to myosin II. Only MHCK A, which is unique to Dictyostelium, has an N-terminal coiled-coil extension responsible for homooligomerization and for binding and bundling of actin filaments (Bosgraaf and van Haastert, 2006). This coiled-coil region is necessary and sufficient for translocation of the protein to F-actin-rich structures (Steimle et al., 2001) and binding to F-actin leads to a 40-fold increase in MHCK activity. This might prevent myosin II filament accumulation at sites of actin-based protrusive activity, like the leading edge of migrating cells, but also the phagocytic and macropinocytic cup, where MHCK A accumulates transiently (Steimle et al., 2001). Knockout and overexpressor strains of MHCK A have been reported, but only the effects on myosin assembly have been studied (Kolman et al., 1996). It would be interesting to investigate how myosin II behaves during phagocytosis in the knockout mutant, where an increased accumulation of myosin II at the phagocytic cup is to be expected. 3.8.2. Class I myosins A large number of unconventional myosins belong to class I, probably reflecting their important roles in processes involving membrane dynamics. In fact, roles for these myosins in several steps of the endocytic pathway are well established in several fungal and mammalian species (Soldati, 2003). Three of them (MyoB, C and D) carry tails with the characteristic three regions TH1, TH2 and TH3, whereas three more (MyoA, E and F) have only the TH1 region. The TH1 region is a basic phospholipid-binding domain involved in attachment to membranes (Senda et al., 2001), the

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TH2 region constitutes an ATP-independent F-actin binding site and the TH3 region harbors an SH3 domain. One more class I myosin, MyoK has virtually no tail, but ends in a prenylation motif that targets the protein to the plasma membrane. This myosin has a TH2-like insertion in the motor domain that represents an additional F-actin-binding site, and contains poly-L-proline motifs that might bind profilin and SH3-domain containing proteins. Several class I myosins of Dictyostelium have been extensively studied, and single and multiple mutants have been characterized, in some cases extensively (Table 8.2 and references therein). From these studies a role in maintaining cortical tension, rather than in facilitating vesicle movement, has emerged (Dai et al., 1999; Schwarz et al., 2000). This role as ‘‘cortical managers’’ (Soldati, 2003) has been invoked to explain the motility and endocytosis defects observed in several myosin 1 mutants. Class I myosins are predominantly cytosolic, but a significant fraction (less than 15%) associates with the plasma membrane, as reported for MyoA, B, C and D in a detailed fractionation study (Senda et al., 2001). Several class I myosins accumulate at macropinocytic crowns (Du¨rrwang et al., 2005; Jung et al., 2001; Novak et al., 1995; Schwarz et al., 2000) and some have been also described at phagocytic cups (Du¨rrwang et al., 2005; Fukui et al., 1989) and in purified early pinosomes (Neuhaus and Soldati, 2000). Class I myosins have also been found to accumulate at phagocytic cups in Acanthamoeba, Entamoeba and macrophages (Allen and Aderem, 1995; Baines et al., 1992; Voigt et al., 1999). Extensive studies on single, double and triple mutants of class I myosins in several combinations have revealed a complex network of redundant, shared and non-redundant roles in endocytosis (Table 8.2 and Titus, 2000). For example, elimination of one class I myosin does not result in fluid phase uptake or exocytosis defects, whereas elimination of two or three leads to substantial (and in cases additive) pinocytosis and exocytosis defects, and consequently reduced growth rates in suspension. Phagocytosis is in general less affected: 30–40% reduction of the initial rate of particle uptake (but otherwise normal steady state levels) has been reported for MyoB and MyoC null mutants (but not for MyoA or MyoD null), and this defect does not become more intense in double or triple mutants. MyoA and MyoB null mutants display increased secretion of a-mannosidase and acid phosphatase, suggesting that these myosins play some role in recycling or retention of lysosomal enzymes. Some short tailed mammalian class I myosins associate to lysosomes and are important for delivery to internalized molecules (Raposo et al., 1999), but there is no evidence of association of Dictyostelium myosins with lysosomes (Senda et al., 2001). MyoB, but not MyoA, seems to be important for efficient recycling of membrane components from early endosomes to the plasma membrane (Neuhaus and Soldati, 2000). Perhaps all these defects explain why the MyoA/B double mutant is more permissive for intracellular growth of L. pneumophila (Solomon et al., 2000).

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Detailed analyses of endosomal trafficking performed in some myosin I mutants allowed also to conclude that these myosins are probably not involved in intracellular movement of vesicles, but rather at events that take place at the cell cortex. Here class I myosins are needed for achieving a balanced cortical tension; changes in one or the other direction result in a similar phenotype of deficient pinocytosis or phagocytosis, as demonstrated with Dictyostelium strains that lack or overexpress MyoB, C or K, or with an E. histolytica strain that overexpresses MyoIB, the unique unconventional myosin of this organism (Voigt et al., 1999). One aspect that has not been addressed in depth in the studies summarized above is the contribution of each class I myosin to endocytosis and other processes in terms of intracellular concentration and functional significance. MyoB and MyoC are more abundant than MyoD, which would explain in part why MyoD null mutants phagocytose normally compared to MyoB and MyoC null mutants ( Jung et al., 1996b). Another aspect to be considered is the ratio of phosphorylated (active) versus unphosphorylated (inactive) myosin. Class I myosins require phosphorylation of the so-called TEDS site of the motor domain for actin-activated ATPase activity (de la Roche and Cote, 2001). The functional relevance of phosphorylation has been addressed with a S332A mutant of the TEDS site of MyoB. This mutant failed to complement the pinocytosis and growth defects of a MyoA/B double mutant. Phosphorylation of class I myosins is performed by kinases of the PAK family, which are in turn regulated, among others, by GTPases of the Rho family. This is just one line of evidence that links unconventional myosins with the actin nucleation and elongation machinery (Soldati, 2003). Studies on class I myosins from several organisms highlight the functional relevance of the SH3 domain. SH3 domains interact specifically with proline-rich motifs and both SH3 domains and proline-rich motifs are found in numerous proteins involved in actin dynamics and endocytic trafficking. In Dictyostelium a truncated MyoB lacking the SH3 domain is unable to revert the growth and endocytosis defects of a MyoA/B double mutant, and when overexpressed it did not provoke the reduced rate of pinocytosis of the wild type myosin (Novak and Titus, 1997; Novak and Titus, 1998). As already mentioned (Section 3.2.3), the SH3 domains of MyoB and MyoC interact with the C-terminus of the scaffolding protein CARMIL, which in turn is an activator of the Arp2/3 complex ( Jung et al., 2001). On the other hand, the TH2-like insertion of MyoK harbors proline-rich motifs able to recruit profilin-actin and to interact with the SH3 domain of Abp1 (Soldati, 2003). Some class I myosins would be therefore needed for translocating multiprotein complexes involved in actin dynamics towards the barbed end of existing filaments, concentrating the complex in the vicinity of the plasma membrane. In support of such a role, MyoB is enriched at the plasma membrane close to phagosomes before the onset of actin-driven rocketing

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induced by compression of the cell. In MyoB null and MyoA/B double knockout cells rocketing is inefficient (Clarke et al., 2006). 3.8.3. MyoI and other unconventional myosins MyoI was initially described as belonging to class VII myosins, but recent sequence analyses seem to indicate that this myosin might constitute a common ancestor of several classes of myosins whose tail consists of a tandem repeat of TH4-FERM domains separated by an SH3 domain (Kollmar, 2006). The same architecture is found in the as yet uncharacterized MyoG. A GFP fusion of MyoI localizes diffusely in the cytosol and accumulates at the tip of filopods and some areas of the cell cortex. During particle uptake MyoI is enriched at the phagocytic cup, but only during the initial engulfment stages (Tuxworth et al., 2001). As already mentioned (Section 3.7.1), MyoI deficient cells exhibit a phenotype similar to that of TalA deficient cells: defective Ca2þ-dependent cell-to-cell adhesion, adhesion to the substrate and phagocytosis, but normal rates of fluid phase uptake and exocytosis (Titus, 1999; Tuxworth et al., 2001). The phagocytosis defect is severe, has been tracked down to the initial step of uptake and is caused by reduced adhesion of particles. In contrast to the TalA defect, the phagocytosis defect persisted when the assay was performed on cells attached to a surface or when a more adherent bacterial strain was used (Titus, 1999). When successful, extension of the phagocytic cup proceeded normally (Tuxworth et al., 2001). MyoI might promote the formation of links between the cytoskeleton and receptors at the plasma membrane needed for adhesion to the substrate and for engulfment of particles (Tuxworth et al., 2001). MyoM defines a novel class of myosins involved in Rho signaling. Its tail harbors a short coiled-coil stretch followed by a proline-rich region and a RhoGEF-PH domain combination. Such architecture has not been identified thus far in myosins of other organisms, while in higher eukaryotes myosins with a RhoGAP domain (class IX) exist (Geissler et al., 2000; Oishi et al., 2000). The motor domain of MyoM contains a phosphorylatable serine residue at the TEDS rule site, suggesting a mechanism of activation in common with class I myosins. In fixed cells a GFP fusion of MyoM appeared enriched at the cell cortex, crowns and large vesicles, probably macropinosomes. MyoM deficient cells grow normally (indicating that pinocytosis is not severely impaired) and perform phagocytosis normally. Two myosins, MyoJ and MyoH, are closely related to class V myosins (Kollmar, 2006). These myosins possess a tail with a coiled-coil dimerization domain and a large globular domain. Class V myosins are processive mechanoenzymes, which fits with their well established activity as vesicle motors in yeast and mammals, involved in transport of a broad repertoire of cargo organelles (Wu et al., 2000). MyoJ deficient cells did not present altered pinocytosis and apparently also phagocytosis, indicating that its

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function might be compensated by the uncharacterized MyoH (Peterson et al., 1996), but clearly more work is needed to establish the role of these interesting myosins.

4. Molecular Events During the Uptake Phase: A Simplified Model I will now make an attempt to incorporate the information summarized above into a simplified model that describes the sequence of molecular events that take place during formation and closure of the phagosome (Fig. 8.1). A similar sequence of events is likely to occur during formation of macropinosomes, with the difference that macropinocytosis does not need the local trigger of particle attachment. 1. Attachment of the particle (Fig. 8.1A). Phagocytosis frequently begins with the contact of filopods to the particle. Components of a complex formed by TalA and MyoI are required for this step. TalA is linked to the plasma membrane by interaction with integrin-related Sib proteins. MyoI probably also interacts with receptors at the plasma membrane. Proteins like VASP and formins are needed for the formation of filopods, but at least VASP is dispensable for particle uptake. Although some more membrane proteins essential for substrate adhesion and particle uptake have been described, it remains unclear how these proteins elicit the signaling changes required for recruitment and activation of the actin polymerization machinery. 2. Activation of the actin polymerization machinery (Fig. 8.1B). Although many details are still unclear, a hierarchy of signaling events involving heterotrimeric G proteins, GTPases of the Ras family and activation of phosphatidylinositol 3-kinases leads to de novo F-actin formation at the site of particle contact. Phosphoinositides play important roles for the recruitment of proteins bearing PH and other domains, like RhoGEFs. Recruitment and activation of small GTPases of the Rho family are instrumental for the activation of the actin polymerization machinery. Several multimolecular assemblies may be involved in positioning and activating the Arp2/3 complex in the vicinity of the plasma membrane. MyoB, MyoC and presumably also MyoD recruit CARMIL, whose main role is to inhibit CP and therefore enable elongation of actin filaments. CARMIL also activates, albeit weakly, the Arp2/3 complex. A more potent activator of the complex is WASP, in turn dependent on Rho GTPases and phosphoinositides for its activation. The role of WASP has not been addressed in Dictyostelium but is very likely, and the participation of the other major Arp2/3 complex activator, Scar, is

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A

?

Sib

Myol

Talin A

VASP P

P

Formin F-actin Rac B WIPa

CAP

Rac

P

WASP P P

Scar

MyoB,C,D

PAK Arp2/3 complex

IQGAP

CARMIL

Formin P

MyoA,E,F MyoM Abp1

MyoK P

CP

F-actin

ABP34 Fimbrin Filamin

MHCKA

LimC LimD LimE

LimF

CH-Lim Ponticulin Hisactophilin

Rab21

Other crosslinkers

Myosin II C

Severin Arp2/3 complex

F-actin

Cofilin Coronin

CP

Aip

ADP

ATP

C

P CAP

V-ATPase

Figure 8.1 Model of the role of actin cytoskeleton components during the uptake phase. See Section 4 of the main text for a detailed description. Although Rac occupies a central place in this model, this is not intended as to underplay the role of other signaling pathways. To avoid unnecessary complexity or because of incompleteness of the available information, some interactions are oversimplified. For example, it is not specified which Racs, formins or PAKs are involved and which specific myosins are targeted. Note that many more interactions are possible, particularly between SH3 domainbearing proteins and proteins with proline-rich regions, but only those interactions are depicted here that have been demonstrated in Dictyostelium or appear very likely by analogy to what we know from other organisms. While the role of WASP, WIPa, formins and IQGAP during uptake has not been specifically addressed in Dictyostelium, these components have been included based on data from other organisms. (A) Components

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unclear. In any case, there must be some overlapping in nucleation activity because none of the single knockouts of Scar, WASP or CARMIL is lethal nor results in abolished F-actin polymerization and actindependent functions. Additional unconventional myosins may also be required to position multimolecular complexes like MyoK (that binds profilin and Abp1) and MyoM (an activator of Rho GTPases). Class I myosins and MyoM would become activated by phosphorylation through kinases of the PAK family, which are in turn effectors of Rac. The exact role of one or more formins at this stage, eventually in complex with proteins of the IQGAP family, has not been defined yet. Profilin plays an important role in this phase, supplying actin monomers freshly charged with ATP to the nucleators. Profilin acts synergistically with cofilin and CAP to increase actin filament turnover at the phagocytic cup. 3. Progression and closure of the phagocytic cup. De novo assembly of actin filaments pulls the borders of the rim of the phagocytic cup following the contour of the engulfed particle. The forces exerted by de novo actin polymerization are probably sufficient for closure of the phagocytic cup. Myosin II is not required for phagosome closure in Dictyostelium, probably an effect of MHCKA, which is recruited to the actin meshwork and ensures that myosin II remains in a phosphorylated, inactive state. New actin filaments are crosslinked by fimbrin, ABP34, filamin and other crosslinkers, thus contributing to the stabilization of the meshwork. Numerous other ABPs with largely unknown roles also interact with F-actin at the phagocytic cup. 4. Detachment of the actin coat (Fig. 8.1C). While the polymerization machinery progresses from the base to the rim of the phagocytic cup, coronin begins to inactivate the Arp2/3 complex from the base of the phagosome. Removal or inactivation of CARMIL would enable repositioning of CP at barbed ends, preventing actin filament elongation. In this region of the phagocytic cup the activity of cofilin and severin dominates, leading to severing and depolymerization of filaments. CAP and Aip regulate the activity of cofilin. Detachment of actin and ABPs from the phagosome is completed usually within one minute after phagosome closure. In many cases residual actin polymerization at the point of phagosome closure is visible as a comet tail that pushes the phagosome away from the involved in the formation of filopods and attachment to the particle. (B) Components involved in activation of the actin polymerization machinery, subsequent elongation of actin filaments and stabilization of F-actin networks. (C) Components involved in disassembly of the actin coat. Black arrows indicate interactions (a discontinuous line means that the interaction is indirect or has not been proved in Dictyostelium). Green arrows indicate interaction and/or activation. Red arrows indicate interaction and/or inhibition. Gray arrows indicate interaction and/or activation. Open arrows indicate interaction and/or inhibition. P, profilin; C, cofilin.

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plasma membrane. As occurs during formation of clathrin-coated vesicles, actin polymerization takes place at the plasma membrane and not at the membrane that surrounds the vesicle. Away from the plasma membrane, the phagosome is captured by microtubules. Concomitant with the detachment of the actin coat, delivery of proton pumps and acidification of the phagosome starts.

5. Concluding Remarks One observation that emerges after examining the literature on actin cytoskeleton components is that a large number of those play dual roles, participating in endocytosis during the vegetative phase and in cell migration during the aggregation phase. In fact, the actin remodeling machinery used during the uptake phase is probably the same as needed for motility during chemotaxis (Gerisch et al., 1999), therefore many components whose role in chemotaxis is well established may be anticipated to play roles during uptake as well and vice versa. Although phagocytosis and macropinocytosis proceed in similar ways, either process has different requirements for particular sets of proteins as becomes apparent from inspection of Table 8.1, with macropinocytosis depending more strongly on G-actin binding proteins (Maniak, 2002). If a comparison could be made, it is as if phagocytosis and micropinocytosis (and cell motility too) were variations on the same musical theme played with the same set of instruments but with different arrangements. Dictyostelium is traditionally well known for its contribution to the actin cytoskeleton field. It is therefore not surprising that a large body of literature covering diverse aspects of the actin cytoskeleton, endocytosis among them, has accumulated over the years. In that way the roles of many components have been defined, although clearly, the roles of many others remain to be addressed. With the advent of the genome era we can now easily see whether homologs of components already studied in other organisms are also present in Dictyostelium and play similar roles. Nevertheless, a key task that needs to be accomplished in the future is to define networks of interactions, elucidate the timing with which events occur along the endocytic pathway and determine how these events are coordinated by regulatory elements. Further refinements of the imaging and high throughput technologies will help to overcome the current imbalance in our knowledge of the endocytosis process, in which a very well studied uptake phase contrasts with the poorly understood transit and exocytosis phases. These approaches along with the recently propagated use of Dictyostelium as a genetically tractable model for infectious disease promise to yield substantial contributions to a better general understanding of the endocytosis process.

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ACKNOWLEDGMENTS This review was elaborated in part while at the Institute for Biochemistry, Medical Faculty, University of Cologne. Support by the Deutsche Forschungsgemeinschaft and the Ko¨ln Fortune Programme of the Medical Faculty, University of Cologne is acknowledged.

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Index

A Acan125. See CARMIL proteins Acanthamoeba, 184, 195, 363, 380 Acipenser ruthenus, 234 Actin based molecular motors, 378–383 crosslinking proteins, 369–371 cytoskeleton components, model of, 383–386 in endocytosis, 358–360 related proteins, 360 Actin-bundling protein (ABP), 369 lateral ADF/cofilin family, 371–372 coronins, 372–373 LIM proteins, 373–374 RhoGEF, 374 membrane-associated, 374–375 comitin, 376 ponticulin, 376–377 talin, 375–376 Actin capping protein (CP) biochemical activies inhibiting molecular interaction, 186 motility reconstitution, role in, 185, 187 cellular studies, 187 CKIP-1, 196 isoforms, 189 physical and chemical properties, 184–185 sequence conservation BLAST, 187, 189 phylogenetic analysis, 188 in vertebrates, 187 a-Actinin, 369–371 b-Actinin, 184 Actin interacting protein (Aip), 367 Active transcription factor NF-kB, 72 Adenine nucleotide carrier (ANC) and phosphate carrier (PC) ratio, 28 Adenomatous polyposis coli (APC), 137 Adryamycin-induced nuclear proteasome activation, 95 AhR/ER receptor cross-talk for TCDD, 235 Aip protein, 367–368 Aldicarb, sulfoxidate carbamate pesticides, 237 Alkylphenols, surfactant, 228 Androgen receptors (AR) biomarkers of, 217 in fish, 216–217

Angiostatin, 41 Anguilla japonica, 216 Annexin protein, 377 Annulate lamellae (AL), 325 Antiandrogenic effects, mechanisms of metabolic clearance of biomarkers of, 227 steroid metabolism and, 226 steroid distribution sex steroid binding protein, 223–224 xenoestrogen binding measurement of, 224–226 steroid receptor androgen receptors, 216–217 antiandrogens, 217–219 biomarkers of estrogen receptor activation, 211–212 estrogen mimics, 212–216 estrogen receptors in, 209–211 steroid synthesis biomarkers of, 221 estrogenic/antiandrogenic effects, 221–223 reproductive steroidogenesis, 219–221 Antiandrogens, 217–219 Anti-apoptotic regulatory proteins, 72 Antigen-presenting cells (APCs) of immune system, 75–76 apoA-I and inhibition of ATP hydrolysis, 41 Apoptosis, 141, 143, 146–148, 156–158 regulatory proteins, 70 Arabidopsis thaliana, 310 Arp2/3 complex, in endocytosis, 361 Aryl hydrocarbon receptor/aryl hydrocarbon nuclear translocator (AhR/ARNT), 222 Aspergillus nidulans, 327 Atomic force microscopy (AFM), 20, 30, 307, 313, 315–316, 326 ATP synthase supramolecular organization, model for, 29 ATP synthesis by F1F0-ATPase, 5 Axonal development extracellular matrix in adhesive interactions, 132 glycoproteins and laminin, 133–135 nanoscale fabrication technology, 133 proteoglygans, 134–135 neuronal polarity in, 136–137 neurotrophins in differential regulation, 131–132

399

400

Index

Axonal development (cont.) glial cell line-derived neurotrophic factor, 132 receptors support, 130 with trophic factors, 131 Axon regeneration, 130, 132, 134, 145, 165 B Bacterial e subunit, ratchet mechanism, 10 BAPTA inhibition, 324 B-Cell chronic lymphocytic leukaemia (B-CLL), 72 BDNF. See Brain-derived neurotrophic factor 20b-Hydroxysteroid dehydrogenase (20b-HSD), 222 Binding actin mechanism C-terminal regions, mobility, 192 structural studies complete detachment, 191, 193 cryoEM analysis, 190–191 crystal structure, 189 truncation and point mutations, 190 Binding-change mechanism for steady-state catalysis, 6 Bovine ATP synthase from peripheral stalk, crystal structure of, 13–14 Bovine F1 bound with inhibitor dicyclohexylcarbodiimide (DCCD), 9 Brain-derived neurotrophic factor (BDNF), 131 Burkitt’s lymphoma, 73 C 2þ

Ca /calmodulin-dependent protein kinase II (CaMK II), 162 Caenorhabditis elegans, 137 Calpain protein, 158, 161–162, 279 Calponin homology (CH), 374 CaMK II. See Ca2þ/calmodulin-dependent protein kinase II CAP. See Cyclase-associated protein Capping protein (CP), 363, 368 actin polymerization inhibition, 198 antagonist regulators formin protein, 196 VASP protein, 196–197 complex cellular processes Cap Z, 200 dendritic nucleation model, 198–199 drosophila development, 201 dynactin, 201 lamellipodial regions, cultured cells, 199 Z-disc, sarcomere, 200 inhibition motif CARMIL proteins, 195 CD2AP and CKIP-1, 196 polyphosphoinositides interaction

computational docking analysis, 194 PIP2 capping activity, 194–195 protein inhibitors CARMIL and V-1 myotrophin, 193 molecular dynamics simulations, 194 Carassius auratus, 216 CARMIL protein, in endocytosis, 363 Caspase inhibitor benzyloxycarbonyl-Val-AlaAsp(OMe)-fluoromethylketone, 98 Caveolae/lipid rafts and eAS localisation, 37 Cell adhesion, 255, 259, 275, 283 Cell cycle regulation, 282 Cell cycle targets for ubiquitin-dependent proteasomal degradation, 67 Cell migration endothelial cells, VEGF-stimulated, 277 focal complex formation, 277, 279 focal contacts, regulation of, 276 traction force generation, 279 Cell motility based on actin assembly, 185 CARMIL, 195 vs. CP concentration, 187 Cell proliferation EGF signaling, negative regulators, 281 Merlin on Ser518 role in, 283 Pak-PIX-GIT–containing complexes and Raf-MEK-ERK pathway, 280 Cerebral ischemia, 156–157, 160 calpain inhibitor calpastatin in, 161 induced brain damage, 127 neuron death, 153–154 reperfusion, 163 CET. See Cryo-electron tomography CF6 expression in HUVECs, 40 Chlamydomonas reinhardtii, 24, 27 Chondroitin sulphate proteoglycans (CSPGs), 134–135, 144 Chromatin structure reorganization, 103 CKIP-1 protein, 196 Cofilin protein, 367–368 Collapsin response mediator protein (CRMP) in neuron death, 158–159 in neuron survival, 160–161 Comitin protein, 376 Computational docking analysis, 194 Coronin-GFP, 359 Coronins protein, 372–373 Cortexillins protein, 370–371 CRMP. See Collapsin response mediator protein Cryo-electron tomography, 301 CryoEM analysis, 190 Cyclase-associated protein, 366 Cyclin dependent kinases (CDKs), 65 CYP1A1 expression in Atlantic salmon hepatocytes, 227 Cyprinus carpio, 209 Cytochalasin A drug, 359–360

401

Index D Danio rerio, 210 DCC. See Dosage compensation complex DCC receptor, 146–147 Deleted in colorectal cancer receptor. See DCC receptor DHT. See 5a-Dihydrotestosterone Dicofol, estrogenic chemicals, 229 Dictyostelium discoideum, actin and endocytosis, 344 ABPs lateral, 371–374 membrane-associated, 374–377 actin, 358–360 F-actin nucleation machinery, 360–364 molecular motors, 378–383 monomeric actin binding proteins, 364–366 proteins actin crosslinking, 369–371 severing and capping, 367–369 Diethylmaleate (DEM), apoptotic inductors, 99 5a-Dihydrotestosterone (DHT), 216 Dosage compensation complex (DCC), 310 Doxorubicin/diethylmaleate-induced apoptosis of cell line, 88 Doxorubicin (DR), apoptotic inductors, 99 Dynacortin, 370–371 Dynactin, 201 Dynein, 130, 201 E ECM. See Extracellular matrix E. coli F1, central stalk protein complex from, 9 E. histolytica, 371, 378, 381 eIF4G translation initiation factors and ubiquitin-proteasome pathway, 104 ELYS/MEL28 protein, 320 Endocrine disruption, in fish, 208–209 Endocrine function consequences of, 236–237 DNA damage and, 238–239 intersex/sex reversal, 239–241 reproductive failure and, 241–242 unscheduled protein synthesis effects of, 237–238 Endocytosis analysis of cytoskeleton components, 346–353 myosins, mutant strains of, 354–357 Dictyostelium discoideum actin, role of ABPs lateral, 371–374 membrane-associated, 374–377 actin, 358–360 F-actin nucleation machinery, 360–364 molecular motors, 378–383 monomeric actin binding proteins, 364–366 proteins

actin crosslinking, 369–371 severing and capping, 367–369 Endocytosis, analysis of, 345 Endothelial cell proliferation and differentiation, 40 Enterostatin and fat intake regulation, 40 and inhibition of ATP synthesis, 41 Ephrin receptor tyrosine kinases (Eph RTK), 147 Ephrins (Ephs) molecules, 140 receptors, 141 Eph RTK. See Ephrin receptor tyrosine kinases Epidermal growth factor (EGF), 255 Epithelial cells barrier formation, 254 repair process outline, 254–255 Epithelial-mesenchymal transition (EMT), 258 Epithelialmorphogenesis, 259–260 ER-CALUX. See Estrogen receptor-mediated, chemical activated luciferase reporter gene expression ERE. See Estrogen-response elements ERM (Ezrin-radixin-moesin), 283 17b-Estradiol metabolite 4-hydroxyestradiol activating metabolism pathway, 238 Estradiol phase I reaction products in fish, 226 Estrogenic effects, mechanisms of steroid receptor androgen receptors, 216–217 antiandrogens, 217–219 biomarkers of estrogen receptor activation, 211–212 estrogen mimics, 212–216 estrogen receptors in, 209–211 Estrogen mimics, 212–216 Estrogen receptor, 209 activation, biomarkers of, 211–212 mediated, chemical activated luciferase reporter gene expression, 211 regulated transcription complexes on ER targets, 101 related receptors, 211 Estrogen receptor-mediated, chemical activated luciferase reporter gene expression (ER-CALUX), 211 Estrogen-response elements, 210 Ethinylestradiol, estrogenic chemicals, 229 Eukaryotes cell cycle and cyclindependent kinases (CDKs) activation, 65 Eukaryotes protein modifications and Co-translational N-a-acetylation by N-acetyltransferases, 90 Excitotoxicity-induced cell death, 127 External ATP synthase (eAS), 36 Extracellular ATP synthesis on HUVECs, 41–42 Extracellular matrix (ECM) role in axonal growth

402

Index

Extracellular matrix (ECM) (cont.) adhesive interactions, 132 glycoproteins in, 133, 135 laminin in, 133–135 nanoscale fabrication technology, 133 proteoglygans in, 134–135 F F-actin filaments, 129 nucleation machinery, 360–364 Fadrozole, aromatase inhibitors, 240 F1 and apolipoprotein A-1 on tumour cells complex, 41 Fas receptor-ligand apoptotic signaling pathway, 73 Fenarimol, androgenic chemicals, 229 F1F0-ATP synthases enzyme, 2 extra-mitochondrial expression of, 36 FG-FG interactions, 316 FG-nucleoporins, 306–307 Fibroblast growth factors (FGF), 132 Filamin protein, 369–371 Filopodia, 129 Fimbrin protein, 369–371 Flavin-containing monooxygenases (FMO), 237 Formin, 196 in endocytosis, 360–361, 363–364 Fundulus heteroclitus, 211 Fyn-Cdk5 pathway, in guidance cue, 152–153 G Gasterosteus aculeatus, 216 Gcn4 yeast activator and proteasome proteolytic activity, 101 GDNF. See Glial cell line-derived neurotrophic factor GEF. See Guanine nucleotide exchange factor Gelsolin protein, 368–369 Glial cell line-derived neurotrophic factor (GDNF), 131–132 GnRH expression in hypothalamus, 234 Gobio gobio, 208 Gonadotropin-releasing hormone (GnRH), 230 G-Proteins, 138 Growth cones composition of, 129 in guidance cue detection, 128 interaction with guidance cues, 130 Growth factors, 130, 132 GTH-R1 (FSH) receptors, 230 Guanine nucleotide exchange factors (GEFs), 260, 325 Guidance cues, repulsive axonal damage and neuronal death calpain in, 161–162 CaMK effects and CRMP modulation, 161

cerebral ischemia mediated, 153–154 collapsin response mediator protein, 158–159 CRMP in neuronal death and survival, 158 dependence receptor theory, 156 netrin mediated, 156–157 repulsive guidance molecule mediated, 157–158 RGM/Neogenin dependence receptors, 157 semaphorins and neuropilin mediated, 154–156 axonal pathfinding ephrins, 140–142 Fyn-Cdk5 pathway, 152–153 intracellular signalling pathways, 149 myelin, reactive glial and scar-derived inhibitors, 144–145 netrins, 139–141 PI3K-GSK pathway, 150–152 receptors for, 146–149 Rho GTPases pathway, 150 semaphorins, 138–139 slit protein, 142–143 future research potential, 165 and synaptic plasticity, 163 as therapeutic agents, 164 H HDL apolipoprotein A-I, 40 HeLa S3 cells, 304 Hemin and proteasome subunits dephosphorylation, 89 Heparan sulphate (HS), 147 Heparin-binding factors, 135 Hermaphrodism, in fishes, 208 Hexokinase type I (HK1), 311 Hisactophilins protein, 377 HKI. See Hexokinase type I Horizontal gaze palsy with progressive scoliosis (HGPPS), 148 HPG. See Hypothalamus-pituitary gonad Human umbilical vein endothelial cells (HUVECs), 39–40 Hydroperoxide-derived aldehyde-DNA adducts, 238 Hypodermal fusion, 265 Hypothalamus-pituitary-gonad axis, 209 description of, 230–231 estrogenic and antiandrogenic effects on, 231–233 indirect mechanisms of, 233–236 I IBB. See Importin b-binding IF1 inhibited ATP hydrolysis activity of eAS and HDL endocytosis, 42

403

Index

Importin b-binding, 312 Inhibitor protein (IF1), 5 Insulin-like growth factor I (IGF-1), 131–132, 233–234 Integrin receptors, 133 Interaptin protein, 377 Intracellular signaling pathways, in guidance cue Fyn-Cdk5 pathway, 152–153 P13K-GSK pathway, 150–152 Rho GTPase pathway, 150 IQGAP, in endocytosis, 363–364 Ischemia-induced synaptic plasticity, 163 Ischemic neuronal death, 157. See also Neuronal death K Karyopherins, 312 11-Ketotestosterone (11-KT), 217 Kringle 1–5, plasminogen fragments, 40–41 L Laminin, 131, 133–135, 140 Latrunculin A drug, 359–360 Leber hereditary optic neuropathy (LHON), 20 Legionella pneumophila, 372, 376, 380 Leigh syndrome, 20, 34 Limanda limanda, 228 LimEDcoil, 359 LIM proteins, 373–374 LMP7-dependent degradation of POMP, 75 LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites, 75 M mAb414 protein, 320 Madin-Darby canine kidney (MDCK), 260 MAG. See Myelin associated glycoprotein Malignancies, hematologic, 95 MAM. See Mitochondria-associated membrane Mammalian cells mtATPase, subunit composition, genetic specification and stoichiometry, 4 Manduca sexta, 99 MAPIB protein, 136 MAPs. See Microtubule-assosiated proteins Maternally inherited Leigh syndrome (MILS), 20 Mdm2 RING-finger ubiquitin ligase, 73 Merlin, 283 Mesenchymal-epithelial transition (MET), 258 MG132 proteasome inhibitor, 72 Micropogonias undulates, 210 Micropterus salmoides, 210 Microtubule-assosiated proteins (MAPs), 128, 136 Microtubule organizing center (MTOC), 271–274

Microtubules, 129, 271 Mitochondria-associated membrane (MAM), 39, 145–146 Mitochondrial ATP synthase (mtATPase) F0 sector, function and structure of, 18–22 F1 sector, 7 central stalk, 9–10 inhibitor protein (IF1), 10–12 function and structure of, 3 mitochondrial membranes, arrangement in, 29–31 mtATPase oligomerisation, role of, 31–34 peripheral/Stator stalk, 12–13 bacterial and bovine OSCP, 13–18 rotary catalysis of, 3–7 Monomeric actin binding proteins, 364–365 cyclase-associated protein, 366 profilin, 365–366 mtATPase oligomerisation, role of, 29–34 MTOC. See Microtubule organizing center Multi-subunit mtATPase complex, 37 Mutant strains of class I myosins and endocytosis in, 354–357 Mycobacterium marinum, 372 c-Myc oncoprotein accumulation, 73 Myelin associated glycoprotein (MAG), 144–145 Myelin-secreted inhibitory molecule, 144, 149 Myosin class I, 379–382 II, 378–379 MyoM, 382 Myosin heavy chain kinase (MHCK) A, 378–379 Myosin heavy chain (MHC), 279 Myosin-II, 130, 378, 385 Myosin light chain kinase (MLCK), 279 Myosin light chain (MLC), 279 N þ

2þ

Na and Ca influx, 127 Nanoscale fabrication technology, 133 Neogenin receptors, 148–149 Netrin proteins in axon pathfinding, 139–140 and DCC/UNC receptors, 141 in neuron death, 156–157 Netrin receptors, 146–147 Neuritogenesis, 128, 134, 150 Neurogenic ataxia and retinitis pigmentosa (NARP), 20 Neuronal death, 127–128, 150 CRMP in, 158–161 CRMP modulation by calpain and CaMK, 161–162 Netrin-1/UNC/DCC in, 156–157

404 Neuronal death (cont.) RGM/Neogenin dependence receptors in, 157–158 semaphorin/neuropilin in, 154–156 Neuronal regeneration, 128, 161 Neuron polarity, in axonal growth, 136–137 Neuropilin-1 glycoprotein, 145 Neuropilin receptor in axon pathfinding, 145–146 in neuron death, 154–156 Neurotrophin-4 (NT-4), 131 Neurotrophins, axonal growth differential regulation, 131–132 glial cell line-derived neurotrophic factor, 132 receptors support, 130 with trophic factors, 131 NK cell-mediated cytotoxicity of tumour cells, 41 N-methyl-D-aspartic acid (NMDA), 153–154 Nogo receptor, 149 Nonylphenol, estrogenic chemicals, 229 NPCs. See Nuclear pore complexes Nsp1p nucleoporin, 316 NTD. See N-terminal domains N-terminal domains, 12–15, 22, 261, 303, 366 Nuclear envelope (NE), 300 Nuclear estrogen receptors, domains of, 209–211 Nuclear pore complexes (NPC), 300 agreement and disagreement, 319–320 interpretation of, 327–329 nuclear pore, 320–321 nuclear pore building blocks, 321–322 nucleoporins, 322–323 peripheral pore structures, 323–324 regulation of, 324–325 cargo translocation in, 312–313 FG-domain behavior, 319 FG-domain function, 315–317 FG-domains and barrier function, 317–318 nucleocytoplasmic transport, 318–319 selective gating, 313–315 structure of architecture of, 301–303 atomic level, 303–304 density and distribution, 304–306 Nucleation promoting factors, 360 Nucleoporins (Nups) function nucleocytoplasmic transport, 306–307 nucleoporins and kinetochores, 308 nucleoporins and transcription, 308–310 Parkinson’s disease, 310–312 Nup107–160, 323 Nup133, 303 Nup214, 303–304 Nup107–160 complex, 308 NUP98 gene, 309 Nup35-Nup93, 322 Nup153, role of, 323

Index O 4-OHE2, genotoxicant, 238 Oncoprotein 18 (Op18). See Stathmin Oncorhynchus kisutch, 209 Oncorhynchus mykiss, 210 Oreochromis aureus, 241 Oreochromis niloticus, 216 Oryzias latipes, 216 Osteosarcoma cell line plasma membrane (PM), 37 P PA28a/b proteasome regulator, 97 PA700 activator protein, 62 P21-activated kinases activation, mechanism of Rho GTPase–independent activation, 263–264 by Rho GTPases, 262–263 background functions, 261 inactivation, 264–265 structure of, 261–262 PA28 expression in mature dendritic cells, 93 Pagrus major, 216 Pak activation wound healing and epithelial sheet migration background functions, 265–266 kinase-independent functions, 267–268 PIX-GIT complex, 268–269 wounding-associated signals, 266–267 Paralichthys olivaceus, 239 Paramecium multimicronucleatum, 23 Parkin protein, 311 Parkinson’s disease, 310–312 Partitioning-defective proteins (PAR), 137 Phosphatidylinositol (4,5) bisphosphate, 362 Phosphoinositide 3-kinase (PI3K), 150 Phosphorylation, in class I myosins, 381 Platelet-derived growth factor (PDGF), 255 Platichthys flesus, 208, 228 Plexin-A4 and A3, 146 Poecilia reticulata, 241 Poly(ADPribose) polymerase (PARP), 95 Polymerization, 194 actin inhibiting, 198–199 in binding actin mechanism, 190 in biochemical activities, 185 PIP2 uncapping in, 194 Ponticulin protein, 376–377 Pore-free islands’’, 305 Pregnane-X-receptor (PXR), 228 Prochloraz and spermatogenesis, 234 Profilin protein, 365–366 b-Propellers, 303–304 Proteasome enzyme antiapoptotic functions of, 71–74 and apoptosis, 68–70

405

Index

apoptosis-induced changes of, 98–100 catalytic activities of peptidase activity and, 63 RNase activity and, 64 in cell regulation of and proteasome subunit expression, 92–93 composition modulation of heterogeneity in cell, 76–78 N-terminus of Rpt2 subunit by myristoylation, 90–91 post-translational, 78–86 gene expression and posttranslational stages, 103–105 transcription process, 100–103 immune response and, 74–76 N-acetylation and N-terminal propeptide processing of, 89–90 proapoptotic function of, 70–71 reprogramming at differentiation, 98 19S regulatory complex, 62–63 structure of, 61 subunits phosphorylation of, 86–89 Proteasome maturation protein (POMP), 75 Proteasome proteolytic activity, 103 Proteasome-ubiquitin pathway, 60 Protein cross-bridge at F1-F1 interface of dimeric structure, 11 Protein kinase C (PKC), 40, 134, 148, 273, 279–280 Proton-driven ATP synthesis by F1F0-ATPase, 6 Protovillin, role in G-actin, 368 p53 tumor suppressor and apoptosis induction, 73 R Rac/cdc42, 262–264 RanBP2 protein, 303, 308, 311–312, 324 RanGTP, 309, 312, 318, 325–326 Repulsive guidance molecule (RGM) in axon pathfinding, 143–144 and Neogenin, 143 in neuron death, 157–158 RGM. See Repulsive guidance molecule Rho GTPase exchange factor (RhoGEF), 374 Rho GTPase pathway, in guidance cue, 150 Rho-GTPases protein, 128, 135, 259 RNA silencing, 130 Robo receptor, 142, 147–148 Roundabout receptor. See Robo receptor Rpn5 subunit of 19S proteasome activator and proteasome complex, 95 Rutilus rutilus, 208 S Salmonella cerevisiae, 308, 323, 327 Salmonella enterica, 376 Sarcomere

in striated muscle, 200 Z-disc, located at, 189 Semaphorin protein in axon pathfinding, 138–139 in neuron death, 154–156 and neuropilin/plexins, 139 semaphorin-3A, 138, 155–156 Semicossyphus pulcher, 239 Serotonin reuptake inhibitor (SSRI), 233–234 Sewage-treatment works, 208 Sex steroid binding protein (SBP), 223–226 SH3/SH2-domain, 267 Single-molecule experiments and rotational catalysis in F1F0-ATP synthase, 6 Single molecule force spectroscopy, 313 siRNA silencing, 132 Sjogren’s syndrome, 95 Skp1/Culin/F-box protein (SCF) protein-ubiquitin ligase, 66 Slit protein, 142–143 SMFS. See Single molecule force spectroscopy SMFS-AFM analysis, 316 Snail family proteins, 258 Sparus auratus, 210 Stathmin, 271–272 Stator stalk, subunit i/j forms, 22 Steroid receptor coactivator (SRC)-interacting proteins, 101 Stroke, 128 STWs. See Sewage-treatment works Supramolecular ATP synthase dimers and oligomers, 23–25 e, g and k, dimer specific subunits, 25–28 Supramolecular structures and respiratory complexes, 35–36 Synaptic plasticity, 163–164 T Talin protein, 375–376 Tau protein, 136–137, 152, 274 TBCB. See Tubulin cofactor B T Cell receptor-induced apoptosis, 71 Testosterone phase I reaction products in fish, 226 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 235 Thr423 residue, 262–264 Thyroid-stimulating hormone (TSH), 235 TIFF. See Triton-insoluble floating fraction TIRF microscopy, 185, 194 TM2 swivelling, protonation/deprotonation cycle, 20 TNF-a-induced apoptosis, 71 Tpr nucleoporin, 320–321 Transforming growth factor a (TGF-a), 255 Transforming growth factor b (TGF-b), 132, 255 Transient receptor potential channels (TRPC), 164

406

Index

Tributyltin, androgenic chemicals, 229 Trigger caspase-mediated apoptosis, 40–41 Triphenyltin, androgenic chemicals, 229 Triton-insoluble floating fraction, 376 Triton X-100, surfactant, 235 TRPC. See Transient receptor potential channels Trp271 yeast residue, 192 a-and b-Tubulin, 129 Tubulin cofactor B (TBCB), 129 Twinfilin protein, 198, 365 Two serine/threonine phosphatases, 264 U Ubiquitin-dependent proteasomal proteolysis, 99 Ubiquitin-proteasome system, 66 UDP-Glucuronosyltransferase, 226 UNC5 receptor, 146–147 V Vascular endothelial cells and stress-induced ATP release, 37 Vascular endothelial growth factor (VEGF), 145, 255 VASP protein, in endocytosis, 363–364 Vitellogenin, biomarker, 237 V-1, myotrophin protein, 193 W WASP/Scar family, in endocytosis, 361–363 Wobble hypothesis, 192–193 Wound healing actin-myosin, regulation of MHC phosphorylation, function of, 279 role of Pak in, 279 cell-cell contacts contact inhibition by Pak, 284 scrape-wounded MDCK cells, 284 stop phase, apico-basolateral polarization, 283 tumor suppressor protein (Merlin) inhibition, 283 cell migration cell protrusion stabilization, 276 endothelial cells, VEGF-stimulated, 277

focal complex formation, 277, 279 focal contacts, regulation of, 276 traction force generation, 279 cell polarization cell protrusion stabilization, 276 lamellipodia protrusion, 270–271 microtubule cytoskeleton reorganisation, 271 microtubule stabilization, 272–273 Pak-PIX-GIT complex, 274–275 polarized vs. nonpolarized cells, 273–274 Scrib complex proteins, 274–275 traction forces, generation of, 279–280 developmental models, 256–257 epithelial plasticity apical-basolateral polarization, 258 future research directions Pak inhibitory proteins, 284–285 spatiotemporal regulation of Pak function, 284 mitogenic signaling, regulation of, 280–281 regulation of cell motility and sheet migration, 270–275 cell proliferation by Pak, PIX and GIT, 280–284 mitogenic signaling, 280–281 Rho GTPases and epithelial morphogenesis, 259–260 scrape wound healing, 257 steps involved in, 259 wound healing and cancer, 257–258 X Xenopus laevis, 68, 302 Y Yeast estrogen screen (YES), 211 Yeast F1 structure, 7 Yeast mtATPase, 4 regulation, 12 Yeast regulatory proteasome 19S subparticle, 101–102

CONTRIBUTORS

John A. Cooper Department of Cell Biology, Washington University, St. Louis, MO, 63110 Rodney J. Devenish Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Microbial Structural and Functional Genomics, Monash University, Clayton Campus, Victoria, 3800, Australia Birthe Fahrenkrog M.E. Mu¨ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Sheng T. Hou Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada Susan X. Jiang Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada Irina M. Konstantinova Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia Roderick Y. H. Lim M.E. Mu¨ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Alexey G. Mittenberg Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia Mark Prescott Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Microbial Structural and Functional Genomics, Monash University, Clayton Campus, Victoria, 3800, Australia Mary Ann Rempel Department of Environmental Sciences, University of California, Riverside, CA 92521

ix

x

Contributors

Francisco Rivero The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, United Kingdom Andrew J. W. Rodgers Industrial Biotechnology Group, CSIRO Division of Molecular and Health Technologies, Clayton, Victoria, 3168, Australia Daniel Schlenk Department of Environmental Sciences, University of California, Riverside, CA 92521 David Sept Department of Biomedical Engineering and Center for Computational Biology, Washington University, St. Louis, MO, 63130 Robert A. Smith Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, G12 8QQ, Scotland Anna S. Tsimokha Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia Katharine S. Ullman Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112 Mirjam Zegers Department of Surgery, University of Chicago, Chicago, IL 60637

C H A P T E R

O N E

The Structure and Function of Mitochondrial F1F0-ATP Synthases Rodney J. Devenish,* Mark Prescott,* and Andrew J. W. Rodgers† Contents 1. Introduction 2. Mitochondrial ATP Synthase 2.1. Overview of the structure and subunit composition 2.2. Rotary catalysis 2.3. The F1 sector 2.4. The peripheral/‘‘Stator’’ stalk 2.5. The F0 sector 3. Supramolecular ATP Synthase 3.1. Introduction 3.2. Dimers and oligomers 3.3. Subunits relevant to dimer formation 3.4. The arrangement of mtATPase in mitochondrial membranes 3.5. The role of mtATPase oligomerisation 3.6. Is oligomerization regulated in vivo? 3.7. Supramolecular structures involving other respiratory complexes? 4. Extra-Mitochondrial Expression of F1F0-ATP Synthase 4.1. Introduction 4.2. How might F1F0 ATP synthase get to the plasma membrane? 4.3. The function of coupling factor 6 as a vasoconstrictor: Detachment and reattachment of an F0 component of eAS? 4.4. Multiple receptor functions of subunit b 4.5. The inhibitor action of IF1 can be demonstrated for eAS complexes 5. Concluding Remarks References

* {

2 3 3 5 7 12 18 23 23 23 25 29 31 34 35 36 36 37 39 40 42 42 43

Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Microbial Structural and Functional Genomics, Monash University, Clayton Campus, Victoria, 3800, Australia Industrial Biotechnology Group, CSIRO Division of Molecular and Health Technologies, Clayton, Victoria, 3168, Australia

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00601-1

#

2008 Elsevier Inc. All rights reserved.

1

2

Rodney J. Devenish et al.

Abstract We review recent advances in understanding of the structure of the F1F0-ATP synthase of the mitochondrial inner membrane (mtATPase). A significant achievement has been the determination of the structure of the principal peripheral or stator stalk components bringing us closer to achieving the Holy Grail of a complete 3D structure for the complex. A major focus of the field in recent years has been to understand the physiological significance of dimers or other oligomer forms of mtATPase recoverable from membranes and their relationship to the structure of the cristae of the inner mitochondrial membrane. In addition, the association of mtATPase with other membrane proteins has been described and suggests that further levels of functional organization need to be considered. Many reports in recent years have concerned the location and function of ATP synthase complexes or its component subunits on the external surface of the plasma membrane. We consider whether the evidence supports complete complexes being located on the cell surface, the biogenesis of such complexes, and aspects of function especially related to the structure of mtATPase. Key Words: Cristae, Dimers, External ATP synthase (eAS), Mitochondrial ATP synthase (mtATPase), Mitochondrial inner membrane, Peripheral or ‘‘stator’’ stalk. ß 2008 Elsevier Inc.

1. Introduction F1F0-ATP synthases are enzyme complexes found in eubacterial plasma membranes, chloroplast thylakoid membranes and the inner membranes of mitochondria. Their function is to harness energy from a gradient of protons (or sodium ions, in some bacteria) across the membrane to synthesise ATP from ADP and Pi. This chapter will review recent advances in the structure and function of mitochondrial ATP synthase (mtATPase). We focus on three areas: (i) the emerging understanding of the structure of the peripheral, or stator, stalk and the subunits comprising it; (ii) the relationship between oligomeric ATP synthase complexes and mitochondrial cristae; and (iii) the extra-mitochondrial location and function of enzyme complexes apparently equivalent to ATP synthase. We draw principally on studies in yeast and mammalian cells (bovine and rat), but refer to relevant studies of the bacterial enzyme particularly in relation to structure and function of the complex. The assembly of mtATPase is not considered here as it has been a major focus of a recent review (Ackerman and Tzagoloff, 2005).

3

Structure and Function of mtATPases

2. Mitochondrial ATP Synthase 2.1. Overview of the structure and subunit composition F1F0-ATP synthases are traditionally viewed as consisting of a soluble portion (the F1 sector), where the sites catalysing the formation and hydrolysis of ATP are located, and a membrane-bound portion (the F0 sector), which functions as a proton channel (Fig. 1.1). Table 1.1 shows the subunit a

OSCP

b

b

d

F6 g e d

C10

b

a +e,f,g, A6L

Figure 1.1 The subunit organization in mtATPase. Subunits are labelled. F1 is the globular domain made of subunits a, b and the three central stalk subunits, g, d and e.The F0 domain is comprised of the subunit c ring (10 copies in yeast), subunit a, and the peripheral stalk subunits b, d, F6(h) and OSCP. The so-called minor subunits [e, f, g, and A6L(8)] are not shown individually, but they all span the membrane and are probably present in a 1:1:1:1 stoichiometry.The rotor is made up of the central stalk and the c-ring. The remainder of the subunits make up the stator. F1 is shown with one a subunit removed for clarity.The inhibitor protein (IF1) is also not shown; it binds in a catalytic a/b interface near the bottom of (ab)3. [This article was published in Biochimica et Biophysica Acta,Vol. 757,Walker, J. E. and Dickson,V. K.,The peripheral stalk of the mitochondrial ATP synthase, 286^296, Copyright Elsevier (2006).]

4

Rodney J. Devenish et al.

Table 1.1 Subunit composition, genetic specification and stoichiometry of mtATPase from yeast and mammalian cells Mitochondria

Bacteria (E. coli) Subunit

Genea

Subunitb

ATP1 ATP2 ATP3

a

a b g (see OSCP) d e Su6

a b g (see OSCP) d e Su6m

1 1 1

b c

B Su9

b Su9

1 10

A6Lm

1

OSCP d e f g F6

1 1 1 (2d ) 1 1 1 1 ? 1 1

Sector Subunit

F1

a b g d e

F0

Mammalian Stoichiometryc

Yeast

Su8 OSCP D E F G H i/j K INH STF1 (9 kDa) STF2 (15 kDa) STF3

ATP16 ATP15 ATP6 m (oli2) ATP4 ATP9m (olil) ATP8m (aap1) ATP5 ATP7 ATP21 ATP17 ATP20 ATP14 ATP18 ATP19 INH1 STF1

IF1

3 3 1

STF2

1

STF3

?

Subunits are aligned horizontally based on sequence or functional homology. The bacterial (E. coli) subunits are shown for comparison with mtATPase subunits. a Genes are in nuclear DNA of Saccharomyces cerevisiae, except those marked with m, which are in mitochondrial DNA. References for individual subunits are as follows: ATP1, Takeda et al., 1986; ATP2, Takeda et al., 1985; ATP3, Paul et al., 1994; ATP16, Giraud and Velours, 1994; ATP15, Guelin et al., 1993; ATP6 (oli2), Macino and Tzaqgoloff, 1980; ATP4, Velours et al., 1988; ATP9 (oli1), Hensgens et al., 1979; Macino and Tagoloff, 1979; ATP8 (aap1), Macreadie et al., 1983; ATP5, Uh et al., 1990; ATP7, Norais et al., 1991; ATP21, Arnold et al., 1997; ATP17, Spannagel et al., 1997; ATP20, Boyle et al., 1999; ATP14, Arselin et al., 1996; ATP18, Arnold et al., 1999 and Vaillier et al., 1999; ATP19, Arnold et al., 1998; INH1, Ichikawa et al., 1990; STF1, Akashi et al., 1988; STF2, Yoshida et al., 1990; STF3, Hong and Pedersen, 2002.

Structure and Function of mtATPases

5

composition of mtATPase. In the mitochondrial enzyme, F1 is composed of three copies of each of subunits a and b, and one each of subunits g, d and e. The inhibitor protein (IF1) is not traditionally considered as an F1 subunit, but we will consider it here in this context because the available evidence suggests that when bound to mtATPase it associates principally with F1 subunits. F1 subunits g, d and e constitute the ‘‘central’’ stalk of mtATPase. F0 consists of a subunit c ring (comprising 10 copies in the case of the yeast enzyme, but varying to up to 14 copies in other organisms) and one copy each of subunits a, b, d, h (F6) and OSCP. Subunits b, d, F6 (h) and OSCP form the peripheral stalk which lies to one side of the complex. A number of additional subunits (e, f, g, i/j, k and A6L) are associated with F0, although their precise locations within the complex remain uncertain (see discussion below). The F0 and F1 sectors of the enzyme, and the two stalks connecting them, are clearly visible in electron cryomicroscopy images of mtATPase (Rubinstein et al., 2003).

2.2. Rotary catalysis Synthesis of ATP by F1F0-ATPase is achieved by coupling the activities of two rotary motors; one in F0, for which a rotational mechanism was first proposed by Cox et al. (1984), and the other in F1 (Boyer and Kohlbrenner, 1981). The presence of a proton motive force drives protons through a channel in F0 at the interface between subunit a and the subunit c ring. In the case of the mitochondrial enzyme, protons pass from the intermembrane space into the matrix. This releases energy which causes rotation of the ring (relative to subunit a), along with subunits g, d, and e, to which it is attached. In turn, rotation of subunit g within the F1 a3b3 hexamer provides energy for ATP synthesis at the catalytic sites (located in each of the three b subunits, at the interface with an adjacent a subunit). The rotary mechanism of F1-ATPase was proved in remarkable singlemolecule experiments carried out by Noji et al. (1997). The a3b3 hexamer of bacterial F1 was immobilised on a flat surface, and ATP-dependent rotation of a fluorescently labelled actin filament attached to the g subunit was directly observed under the fluorescence microscope. A similar technique was used to show that the hydrolysis of one molecule of ATP causes a

b a, b, g, d, e, Walker et al., 1985; Su6, A6L, Fearnley and Walker, 1986; Su9, Sebald and Hoppe, 1981; b, d, Walker et al., 1987b; OSCP, F6, INH, Walker et al., 1987a; e, Walker et al., 1991; f,g, Collinson et al., 1994a. c Compilation of data for both yeast and bovine systems presented in Arnold et al., 1998; Fronzes et al., 2003; Gregory and Hess, 1981; Hekman et al., 1991; Muraguchi et al., 1990; Okada et al., 1986; Paumard et al., 2000; Stephens et al., 2003; Stutterheim et al., 1981; Todd et al., 1980; and Walker et al., 1985. A question mark indicates subunits for which no reliable stoichiometric data are available. d Arakaki et al., 2001.

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rotation of 120
(Yasuda et al., 2001), as would be expected from the existence of three catalytic sites. Binding of ATP causes a rapid (<0.1 ms) 80
rotation, which is followed by hydrolysis (during the 2 ms ‘‘interim dwell’’), and another rapid 40
movement (Shimabukuro et al., 2003). Recent experiments have revealed the sequence of catalytic and rotational steps in even more detail (Adachi et al., 2007). The interim dwell is divided into a 1 ms ‘‘catalytic’’ dwell, in which cleavage of ATP occurs, and a second phase, the length of which is dependent on Pi concentration. The second of the 1 ms dwells at the 80
point marks Pi release, and energy from the Pi release drives the last 40
substep (Adachi et al., 2007). Single-molecule experiments have also shown that rotational catalysis occurs in F1F0-ATP synthase (Sambongi et al., 1999), and that ATP synthesis by F1 can be driven by externally applied rotational force (Itoh et al., 2004; Rondelez et al., 2005). A framework for understanding rotary catalysis in F1 is provided by the ‘‘binding-change’’ mechanism, first proposed by Boyer (1975). According to this mechanism, the three catalytic sites undergo cyclical interconversion between three states with markedly different nucleotide-binding affinities. When the enzyme is operating in the direction of ATP synthesis, each site switches cooperatively through conformations in which ADP and phosphate bind, ATP is formed, and then released. It is now accepted that these transitions are brought about by rotation of the g subunit. The catalytic mechanism of the enzyme when operating in the opposite direction (ATP hydrolysis) appears likely to use essentially the same pathway, but in reverse (Adachi et al., 2007). The most detailed picture yet of the binding and catalytic events taking place in each of the b subunits over the full 360
rotation of the g subunit has emerged from recent experiments by Ariga et al. (2007). These authors studied ATP hydrolysis and rotation by a hybrid F1 containing one or two mutant b subunits with altered catalytic kinetics, and showed that all three b subunits participate in driving each 120
rotation of the g subunit, with a 120
phase difference. In any particular b subunit, ATP bound when g is positioned at 0
is cleaved when g has rotated 200
(Ariga et al., 2007). For proton-driven ATP synthesis by F1F0-ATPase to occur, the a3b3 hexamer must remain fixed relative to subunit a during catalysis; this occurs by virtue of a physical bridge (the peripheral stalk) formed by subunits b, d, F6 (h) and OSCP. From a mechanistic point of view, the enzyme can therefore be divided into ‘‘rotor’’ (c10-14, g, d, e) and ‘‘stator’’ [a3b3, a, b, d, h (F6), OSCP] components (Fig. 1.1). The binding-change mechanism as presented by Boyer (1993) predicted that during steady-state catalysis, only two of the three catalytic sites would be occupied (on time average) with nucleotide (‘‘bi-site’’ catalysis). This reaction scheme appears unlikely, however, as it would require (when the reaction proceeds in the direction of ATP hydrolysis) that ATP bind to the lowest affinity site in preference to the medium affinity site (Weber and

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Senior, 2000). In addition, Weber and Senior (1997) have used a sensitive assay based on quenching of fluorescence from tryptophan residues introduced into the active sites to show that in bacterial (E. coli) F1, the Kd for binding of MgATP to the lowest-affinity site corresponds to Km (MgATP), and that all three catalytic sites must be filled to obtain Vmax (and physiologically relevant) rates of ATPase activity (‘‘tri-site’’ catalysis). Similar techniques have been employed to show that this is also the case with F1 from the thermophilic bacterium Bacillus PS3 (Ren et al., 2006) and yeast mitochondrial F1 (Corvest et al., 2005). Experiments carried out with mitochondrial F1 using indirect methods of measuring nucleotide binding appear to favor ‘‘bi-site’’ catalysis (Milgrom and Cross, 2005). However, the enzyme preparation used in these studies hydrolyzed ATP at a rate which was about 50% of that reported in other work ( Jault and Allison, 1993; Milgrom et al., 1998), suggesting that it had defective catalytic properties (Ren et al., 2006). To accommodate the wealth of data supporting ‘‘tri-site’’ catalysis, alternative reaction schemes for ATP synthesis and hydrolysis have been proposed in which three catalytic sites are filled, on time average, over the course of the reaction cycle (Weber and Senior, 2003). A recent structure of yeast F1 (Kabaleeswaran et al., 2006) showed two molecules of AMP-PNP and a phosphate bound to the three catalytic sites. This structure appears to support ‘‘tri-site’’ catalysis as this arrangement is predicted to be an intermediate state during the tri-site (Weber and Senior, 2003), but not bi-site (Milgrom and Cross, 2005) reaction schemes. Singlemolecule experiments in which both rotation and nucleotide binding/ release were monitored simultaneously in real time (Adachi et al., 2007) have provided the most detailed picture yet of the relationship between the rotational angle of the g subunit and nucleotide occupancy of the catalytic sites, and strongly support a ‘‘tri-site’’ mechanism similar to that proposed by Weber and Senior (2003).

2.3. The F1 sector 2.3.1. Structure and subunit composition The landmark crystal structure of bovine mitochondrial F1 (Abrahams et al., ˚ , provided many valuable insights into 1994), solved at a resolution of 2.4 A the function of the enzyme. The protein was crystallized in the presence of Mg2þ, ADP, azide and an ATP analogue, AMP-PNP. The three a subunits and three b subunits were shown to pack alternately, like segments of an orange, around a cental spindle formed by the N- and C-terminal helices of subunit g. The presence of six nucleotide-binding sites was confirmed; three catalytic sites, located in the three b subunits at the interface with an adjacent a subunit, and three noncatalytic sites in the three a subunits. Each of the a and b subunits features an N-terminal b-barrel, a central mixed a/b domain (where the nucleotide-binding sites are located) and a

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C-terminal helical bundle. The structures of the three a subunits were very similar, with AMP-PNP bound to each of the three noncatalytic nucleotide binding sites. By contrast, an important feature of the structure was the asymmetry of the three b subunits. They were reported to contain ADP (bDP), AMP-PNP (bTP), or no nucleotide (bE) (Abrahams et al., 1994). The structure of bDP and bTP were very similar to each other with the bound nucleotide being the only major difference. However, the structure of bE was significantly different, being referred to as the open conformation. Here a large displacement of the C-terminal domain was observed (some portions ˚ ), that caused a disruption in a small b-sheet of the displaced by 20 A nucleotide binding domain. The existence of three conformations provided clear support for binding-change models of ATP synthesis/hydrolysis, which predict that each b subunit cycles through conformations with markedly different nucleotide-binding affinities (Boyer, 1993; Weber and Senior, 2003). In addition, both the tip of the C-terminal helix of subunit g, and the sleeve around it formed by regions of subunits a and b immediately below the b-barrel domains, are predominantly composed of hydrophobic residues. This assembly was suggested to act as a molecular bearing, facilitating rotation of subunit g within the F1 a3b3 hexamer during catalysis (Abrahams et al., 1994). Several other high-resolution structures of F1 have been solved since 1994 with an array of different inhibitors bound, and with various states of nucleotide occupancy (Abrahams et al., 1996; Bianchet et al., 1998; Bowler et al., 2006, 2007; Braig et al., 2000; Chen et al., 2006; Gibbons et al., 2000; Kabaleeswaran et al., 2006; Menz et al., 2001; Orriss et al., 1998; van Raaij et al., 1996). However, a high-resolution structure with all three catalytic sites filled with either MgATP or MgAMP-PNP has yet to be solved, despite their presence in crystallization buffers at concentrations greater than their respective Kd values for the lowest affinity site. This would indicate that, in these cases at least, the process of crystal formation tends to dissuade nucleotide occupancy of the bE site, in a manner not yet understood. The sole example of a structure in which all three sites are filled with nucleotide (Menz et al., 2001) is of F1 crystallized in the presence of ADP and fluoroaluminate. The presence of the very tight-binding inhibitor ADP-fluoroaluminate in two of the catalytic sites allows binding of ADP and a sulfate ion (probably mimicking phosphate) in the third site (bE). This structure is proposed to represent a post-hydrolysis, pre-product release step on the catalytic pathway (Menz et al., 2001). In a more recent structure of yeast F1 (Kabaleeswaran et al., 2006), a phosphate ion is bound to the bE site. Comparison of the position of this phosphate ion with the position of the g-phosphate of AMP-PNP in previous structures provides a description of the path followed by the phosphate as it binds to the enzyme, is moved into position ready for reaction with MgADP, and subsequent catalysis. Two recent structures of bovine F1 (Bowler et al., 2006, 2007)

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reveal the binding site for azide and explain how it inhibits ATPase activity. In the structure from crystals grown in the presence of ADP, AMP-PNP and azide (Bowler et al., 2006), the azide anion occupies a position in the ADP-binding catalytic subunit, bDP, very similar to the site occupied by the g-phosphate in the AMP-PNP (ATP) binding subunit, bTP. The structure from crystals grown in the absence of azide (Bowler et al., 2007) has AMPPNP bound in both bDP and bTP. Azide therefore appears to inhibit the enzyme by sterically blocking the pocket where the g-phosphate of ATP would bind. The presence of azide in bDP also tightens the binding of several amino acid side chains to the ADP, enhancing its affinity and thereby stabilizing the ‘‘ADP-inhibited’’ state of the enzyme (Bowler et al., 2006). 2.3.2. The ‘‘Central’’ stalk Although the F1 structures published prior to 2000 provided a nearly complete picture of the architecture of the a3b3 hexamer, and of the Nand C-terminal a-helices of the g subunit, crystal disorder in the central stalk proteins of F1 (g, d and e) resulted in a lack of detailed structural information on this portion of the enzyme. NMR (Wilkens et al., 1995; Wilkens and Capaldi, 1998a) and crystal (Uhlin et al., 1997) structures of the isolated bacterial e subunit (equivalent to the d subunit in mtATPase) showed that this protein consists of a 10-stranded b-sandwich at the N-terminus, followed by a pair of antiparallel coiled-coil a-helices which fold back to make contact with the b-sandwich. The crystal structure of bovine F1 bound with the inhibitor dicyclohexylcarbodiimide (DCCD) (Gibbons et al., 2000) revealed the arrangement of central stalk proteins in the mitochondrial enzyme for the first time. The long C-terminal helix of subunit g extends to the bottom of F1. A short loop connects it to an a/b domain featuring a Rossman fold, and this domain is connected to the N-terminal a-helix. The d subunit folds in a way very similar to that seen in the isolated bacterial subunit e, with a b-sandwich at the N-terminus and a helical hairpin at the C-terminus. The protein is packed against a face formed by both the N- and C-terminal helices of subunit g. The third component of the central stalk in mitochondrial F1, subunit e, has no equivalent in bacterial and chloroplast ATP (Table 1.1). This protein is wedged into a cleft between the two domains of the d subunit, thereby forming a connection between them. The crystal structure of the central stalk protein complex from bacterial (E. coli) F1 (comprising the central domain of subunit g plus subunit e) (Rodgers and Wilce, 2000) highlighted differences in subunit organization and in the mechanism of regulation between the bacterial and mitochondrial complexes. Although the folds of subunit g and the N-terminal b-sandwich domain of bacterial subunit e are very similar to their mitochondrial equivalents, the arrangement of the C-terminal helices of bacterial subunit e is markedly different. In the bacterial structure, the helices are separated from one another and wrap around subunit g. Cross-linking studies carried

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out on the bacterial complex show that the subunit e-helices can adopt both this conformation and the contracted arrangement seen in the mitochondrial structure (Tsunoda et al., 2001), as well as an even more extended conformation in which the e-helices point directly up and deeply penetrate the F1 a3b3 hexamer (Suzuki et al., 2003). Recent chemical labelling experiments (Ganti and Vik, 2007) confirm that the C-terminal helices of bacterial subunit e have a high degree of flexibility in the intact enzyme. Nucleotide occupancy of the catalytic sites and the presence of a proton motive force across the membrane influence movement of the helices between these conformations (Feniouk et al., 2007; Suzuki et al., 2003; Tsunoda et al., 2001). Importantly, ATP synthesis and hydrolysis activities are affected differently by the conformation of the subunit e-helices (Tsunoda et al., 2001). When they lie close to F0, the enzyme is active in both ATP hydrolysis and synthesis; when they point toward F1, ATP hydrolysis is inhibited, yet the enzyme is fully functional in ATP synthesis. The bacterial e subunit therefore acts via a ‘‘ratchet’’ mechanism to switch the enzyme to different gears depending on whether rotation of the subunit ge complex is in the direction of synthesis or hydrolysis (Tsunoda et al., 2001). This mechanism, which involves interaction between the C-terminal helix of subunit e and the ‘‘DELSEED’’ region of subunit b (Hara et al., 2001) is believed to be important for survival in bacteria, by preventing depletion of intracellular ATP under starving conditions (Feniouk et al., 2007; Suzuki et al., 2003; Tsunoda et al., 2001). Conformational changes in subunit e have also been implicated in regulation of chloroplast ATP synthase ( Johnson and McCarty, 2002) and may involve a ‘‘ratchet’’ mechanism similar to that seen in bacteria (Richter et al., 2005). 2.3.3. Inhibitor protein (IF1) In contrast to the bacterial enzyme, mtATPase is regulated to carry out ATP synthesis exclusively. Wasteful hydrolysis of ATP is not desirable, and must be prevented when oxygen supply to the electron transport chain is limited (such as in the case of ischaemia). The involvement of a ‘‘ratchet’’ mechanism similar to that seen in the bacterial enzyme is very unlikely, given that the C-terminal helices of the mitochondrial subunit d are packed securely at the foot of the central stalk (Gibbons et al., 2000) in such a way that would tightly constrict their movement. Rather, this regulation is carried out by the inhibitor protein IF1, which potently inhibits mitochondrial F1-ATPase activity (Pullman and Monroy, 1963) in a pH-dependent manner (Van Heeke et al., 1993). IF1 can exist in at least two conformations (active and inactive), with the active form predominating at pH values <6.5 (Cabezon et al., 2000b). In its active form, bovine IF1 exists as a dimer, stabilized by formation of an antiparallel coiled-coil by a-helices in the C-terminal portion of the

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monomers (Cabezon et al., 2001). In the presence of ATP, the N-terminal portion of each of the monomers is able to bind to F1 (Milgrom, 1991), causing formation of F1 dimers in solution (Cabezon et al., 2000a), and stabilization of mtATPase dimers which occur in the mitochondrial inner membrane (Garcia et al., 2006). IF1 probably contributes to dimerization by forming part of a protein cross-bridge at the F1-F1 interface of the dimeric structure, observed for the first time in transmission EM images of the bovine heart mtATPase (Minauro-Sanmiguel et al., 2005). At higher pH values, a-helices in the N-terminal portion of IF1 associate with one another, causing formation of tetramers (Cabezon et al., 2001); the masking of the inhibitory N-terminal portions prevents binding to F1 and thus renders IF1 inactive as an inhibitor. In the event of a collapse in the proton gradient across the inner membrane, the mitochondrial matrix becomes more acidic, favoring formation of the active form of IF1. The inhibitor protein therefore appears to play two crucial roles in the cell. Firstly, it acts as a pH-dependent sensor that prevents mtATPase from wastefully hydrolysing ATP in the mitochondrial matrix under anoxic conditions. Secondly, IF1 stabilizes the dimer structure, which plays a part in determining the morphology of mitochondrial cristae of the inner membrane (see Section 3.3.2 for detailed discussion). Cross-linking experiments showed that IF1 makes contact with both the F1 a3b3 hexamer and the rotor subunits of the central stalk (MinauroSanmiguel et al., 2002). These results were confirmed by crystal structures of dimeric IF1 associated with two F1-ATPase moieties [(F1-IF1)2] (Cabezon et al., 2003) and of F1 bound to a truncated (residues 1-60), monomeric form of IF1 (Gledhill et al., 2007). These structures showed that IF1 binds mainly at the interface between subunits aDP and bDP (using the notation of Abrahams et al., 1994), and also makes contact with subunit g. Bound IF1 therefore appears to inhibit enzyme activity by interfering with the conformational changes in the nucleotide-binding sites (at the a/b interface) required for catalysis, and possibly by blocking rotation of the central stalk subunits (Cabezon et al., 2003; Garcia et al., 2006; Gledhill et al., 2007; Minauro-Sanmiguel et al., 2002). The binding of IF1 to F1 requires ATP to be bound first (de GomezPuyou et al., 1980; Milgrom, 1991). Interestingly, the (F1-IF1)2 crystal structure (Cabezon et al., 2003) features a molecule of ATP (or its analogue AMP-PNP) in the bDP catalytic site, rather than the ADP molecule present in the F1 structure with no IF1 bound (Abrahams et al., 1994). This property is shared with the ‘‘ground-state’’ F1 structure derived from crystals grown in the absence of azide (Bowler et al., 2007). This would suggest that binding of IF1 traps ATP in this site, and that the (F1-IF1)2 structure represents a prehydrolysis state in the catalytic cycle (Cabezon et al., 2003; Gledhill et al., 2007). Corvest et al. (2005) investigated the effect of nucleotide occupancy of the catalytic sites on the rate of inhibition by IF1, and

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found that at least two of the sites must be filled to allow binding of IF1. Subsequent hydrolysis of ATP was proposed to lock IF1 in place (Corvest et al., 2005). The yeast complex has an inhibitory protein associated with it, Inh1p, homologous to IF1. Additionally, in yeast other small proteins are involved. STF1 is reported as having similar functional properties and sensitivity to the same effectors as Inh1p, but different affinity for mtATPase. Despite much detailed investigation the role of STF1 in vivo remains unclear (Venard et al., 2003). While both yeast proteins can form dimers there is no evidence that they cannot function as monomers. Furthermore, the role of dimeric yeast Inh1p has been questioned (Ichikawa et al., 2002). Two other small proteins, STF2 and STF2, share some 65% homology and are proposed to contribute to regulation of yeast mtATPase through modulation of Inh1p and STF1 function. How this modulation is effected remains unclear (Hong and Pederson, 2002).

2.4. The peripheral/‘‘Stator’’ stalk 2.4.1. Overall structure and subunit composition Although early EM images of bacterial (E. coli ) ATP synthase reconstituted into phospholipid vesicles (Gogol et al., 1987) clearly showed the presence of a central stalk linking F1 to F0, these pictures did not reveal the presence of proteins linking the two sectors at the periphery of the complex. Rather, information on the location and structure of the peripheral stalk was initially built up from cross-linking and reconstitution experiments. Collinson et al. (1994b) combined purified bovine subunits d, OSCP, F6 and the membraneextrinsic portion of subunit b and found that these proteins formed a 1:1:1:1 complex. Moreover, this sub-complex was able to bind with purified F1 in a 1:1 ratio (Collinson et al., 1994b, 1996). The crystal structure of most of the peripheral stalk from bovine mtATPase has recently been solved (Dickson et al., 2006; see below). Proving that the peripheral stalk acts as a ‘‘stator’’ in ATP synthase required functional studies on catalytically active enzyme. This has largely involved disulfide cross-linking experiments carried out with the bacterial (E. coli) enzyme, which offers the advantage that site-directed mutant enzymes with introduced cysteine residues can be readily constructed and purified, and the effect of the zero-length cross-link on enzyme activity easily measured. Cross-links from either subunit g or e to either subunit a or b abolish activity completely (Aggeler et al., 1995; Aggeler and Capaldi, 1996), as expected if rotation of subunits g and e relative to subunits a and b is required for catalysis. The bacterial d subunit (Ogilvie et al., 1997) and the C-terminus of subunit b (Rodgers and Capaldi, 1998) can be cross-linked to the N-terminal domain of an a subunit, subunit d can be cross-linked to subunit b (McLachlin and Dunn, 2000) and subunit g can be cross-linked

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to the subunit c ring (Watts et al., 1995). Crucially, these cross-links have little effect on enzyme activity. After it had been established experimentally that the a-b, a-d and g-c cross-links had no effect on rotational catalysis, the peripheral stalk could be properly designated as a ‘‘stator’’ that holds subunit a and the F1 a3b3 hexamer in position as the rotor component of the complex rotates. Once the composition and function of the stator stalk of the bacterial enzyme had emerged, it became clear that the b:d:OSCP:F6 complex (Collinson et al., 1994b) was very likely to play an equivalent role in bovine mtATPase. The peripheral stalk may also contribute to transient accumulation of elastic energy during catalysis (Cherepanov et al., 1999) that would arise from a mismatch in symmetry between the rotary motors of F1 (containing 3 catalytic sites) and F0 (with 1014 copies of the proton-translocating c subunit). In the late 1990s, a number of EM studies were carried out on single particles of negatively stained ATP synthase complexes, and were successful in visualizing the peripheral stalk. This technique, which relies on averaging of many images, revealed the presence of protein mass at the periphery of the enzyme purified from bovine mitochondria (Karrasch and Walker, 1999), chloroplast (Bottcher et al., 1998) and E. coli (Wilkens and Capaldi, 1998b). More recently, EM images with higher resolution have been obtained from single unstained bovine mitochondrial F1F0 particles embedded in vitreous ice (Rubinstein et al., 2003). In these pictures, electron density outside the central F1/c-ring complex is more clearly defined, and appears large enough to accommodate the subunits predicted to make up the peripheral stalk [b (extramembrane domain), d, OSCP and F6] and F0 components outside the subunit c ring [subunits a and A6L (8), along with the membrane spanning regions of subunits b, e, f and g] (Dickson et al., 2006; Rubinstein et al., 2003). EM of negatively stained bacterial (E. coli) F1F0 complexes that had been decorated with monoclonal antibodies against the bacterial subunit d (equivalent to mitochondrial OSCP) showed that this protein is located at the top of F1 (Wilkens et al., 2000). EM has also been used to probe the positions of subunits h and OSCP within the yeast mitochondrial stator stalk. By tagging these subunits with biotin and avidin and examining the position of the avidin molecule in negatively stained images, subunit h appeared to reside close to the membrane surface (Rubinstein et al., 2005), while OSCP was located toward the top of F1 (Rubinstein and Walker, 2002). This result is consistent with cross-linking studies in the bacterial (E. coli) (Ogilvie et al., 1997) and chloroplast (Lill et al., 1996) enzymes, which place subunit d in close proximity to the N-terminal domain of subunit a. The crystal structure of most of the peripheral stalk from bovine ATP synthase has recently been solved at 2.8 A˚ resolution (Dickson et al., 2006). The crystallized complex contains most of the extramembranous

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portion of subunit b [residues 79184 (missing residues 185214)], most of subunit d [residues 1124 (missing 125160)] and all of subunit F6 (h). All of the subunits are predominantly a-helical (structures of the individual proteins are discussed below). Importantly, the structure fits well into a region of electron density at the periphery of the low-resolution structure of the whole complex derived from EM studies (Rubinstein et al., 2003), see Figure 1.2. This density in the structure is that which is unaccounted for once the F1 (Abrahams et al., 1994; Gibbons et al., 2000) and the subunit c ring (Stock et al., 1999) structures are docked into it and is very likely to be occupied by the peripheral stalk subunits [OSCP, b, d, and F6 (h) plus membrane-bound subunits outside the subunit c ring (a, e, f, g and A6L] (Dickson et al., 2006; Rubinstein et al., 2003). 2.4.2. Structures of individual subunits 2.4.2.1. OSCP The NMR solution structure of the N-terminal domain for both bacterial (E. coli) subunit d (Wilkens et al., 1997) and bovine OSCP (Carbajo et al., 2005) have been determined. The C-terminal regions (residues 112190 in OSCP and 134177 in subunit d) are highly susceptible to proteolysis, and appear from NMR studies to be mostly disordered (Carbajo et al., 2005; Wilkens et al., 1997). The N-terminal domains of both proteins consist of a bundle of six a-helices, and their folds are extremely similar. The N-terminal domain of OSCP appears to fit well into a region of density in the EM-derived low-resolution structure which lies directly above the top of F1 (Rubinstein et al., 2003), while the C-terminus appears to be located in a region of the peripheral stalk further away from the central axis of the F1 a3b3 hexamer (Dickson et al., 2006); see Figure 1.2. The F1-binding surface on bacterial subunit d has been studied by mutagenesis and tryptophan fluorescence (Weber et al., 2003b), and found to be localized to helices 1 and 5 of the N-terminal domain. A synthetic peptide consisting of the first 22 residues of bacterial subunit a was shown to bind to the N-terminal domain of subunit d specifically and with high affinity (Weber et al., 2003a). The binding interface within this complex was analyzed using NMR (Wilkens et al., 2005). Similar techniques were used by Carbajo et al. (2005) to demonstrate binding of the N-terminal residues of bovine subunit a to the N-terminal domain of bovine OSCP, and also to rule out any involvement of N-terminal residues of subunit b in binding OSCP. These and later studies indicate that a stretch of residues at the N-terminus of subunit a is likely to form an a-helix which binds to OSCP/d by packing into a groove between helices 1 and 5 (Carbajo et al., 2005, 2007; Wilkens et al., 2005). Weber et al. (2002) used a fluorimetric assay to determine a Kd value of 1.4 nM for the binding affinity of bacterial subunit d to F1, suggesting a standard free energy of binding of 50.2 kJ/mol. This would be roughly equivalent with the strain demands placed upon the

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Figure 1.2 The composite structure of mtATPase. Detailed structures of the F1c10 subcomplex (gray), the N-terminal domain of the OSCP (cyan) and the peripheral stalk subcomplex (magenta, orange and green) were introduced by eye into an electron density map determined by averaging single particles of the intact bovine complex observed by electron cryomicroscopy. (A) Side view and (B) residual density corresponding to the peripheral stalk and the second domain of F0 (Rubinstein et al., 2003). Dotted lines represent the lipid bilayer. (C) View looking down onto the ‘‘crown’’ of the F1 catalytic ˚ . [Adapted by permission from Macmillan Publishers Ltd. domain. The scale bar is 50 A EMBO Journal (Dickson, V. K., Silvester, J. A., Fearnley, I. M., Leslie, A. G., Walker, J. E. On the structure of the stator of the mitochondrial ATP synthase. EMBO J. 25: 2911^2918) Copyright 2006.]

stator by the torque energy generated by the rotor during catalysis (Hasler et al., 1999), although the direct binding of subunit b to F1 may also contribute to the stability of the stalk/F1 connection in the bacterial enzyme (Weber et al., 2004).

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2.4.2.2. Subunit b of the bacterial, chloroplast and mitochondrial ATP synthases are predicted to play similar roles in their respective enzymes (Velours et al., 1988; Walker et al., 1987b), in that they constitute a continuous link from the membrane-integral F0 components to near the top of F1, and provide a scaffold for assembly of other subunits of the peripheral stalk. However, there are marked differences in stoichiometry and topology between the subunits from the different sources. The bacterial enzyme features a homodimer of two b subunits, while in some other bacteria and chloroplasts, there is a heterodimer of two versions of b subunits (referred to as b and b’ in bacteria, or CF0I and CF0II in chloroplasts). However, only a single copy of subunit b is present in mtATPase (Bateson et al., 1999; Collinson et al., 1994a, 1996). This subunit features two membrane-spanning domains at the N-terminus, presumed to be a-helices, with the N-terminal residue on the matrix side of the inner membrane (Velours et al., 1989; Soubannier et al., 2002). The hydrophilic C-terminal portion extends into the matrix, and binds subunits d, F6 (h) and OSCP in 1:1:1:1 ratio (Collinson et al., 1994b, 1996). Given that OSCP is located near the top of F1 (Carbajo et al., 2005) the b subunit must extend to this region. In yeast, two-hybrid and cross-linking (Soubannier et al., 1999; Velours et al., 1998) as well as FRET (Gavin et al., 2003) studies provide evidence for interaction between the C-terminus of OSCP with the C-terminus of subunit b. These findings are consistent with disulfide cross-linking experiments in bacterial (E. coli) ATP synthase, which place the C-terminus of subunit b close to the N-terminus of subunit a (Rodgers and Capaldi, 1998) and to bacterial subunit d (McLachlin and Dunn, 2000; Rodgers et al., 1997). The crystal structure of the peripheral stalk from bovine mtATPase (Dickson et al., 2006) contains residues 79184 of the b subunit, which ˚ long. The N-terminal amino acid of form a continuous a-helix about 160 A the fragment (residue 79 in the full-length protein) is predicted from hydropathy profiles to lie at the interface between the mitochondrial inner membrane and the matrix (Walker et al., 1987b). It would therefore be expected that in the intact mtATPase, the helix in the b subunit would extend away from the membrane surface towards the top of F1 in the N- to C-terminal direction (Dickson et al., 2006). If the peripheral stalk crystal structure is docked into the electron density map of the F1F0 EM structure (Rubinstein et al., 2003), with residue 79 of the b subunit positioned at the predicted membrane surface, the length of the b subunit helix fits very well with the distance from the membrane to the top of F1 (Dickson et al., 2006). Part of the density unaccounted for by the peripheral stalk proteins may be due to a segment of subunit b which is missing from the crystallized complex (residues 185214). The position of this density suggests that there is a turn in subunit b around residues 185187, followed by another a-helix (residues 188204) which lies antiparallel to the long a-helix (Dickson et al., 2006; see Fig. 1.2).

Structure and Function of mtATPases

17

The structure of the membrane-bound segment of subunit b (residues 179) is presently unknown. Secondary structure and hydropathy analyses of the sequence (Walker and Dickson, 2006) predict that the long a-helix continues uninterrupted across the inner mitochondrial membrane to the intermembrane space. A turn, followed by a second transmembrane a-helix (residues 3347), results in the N-terminal region (residues 130) being exposed to the mitochondrial matrix (Walker and Dickson, 2006). 2.4.2.3. Subunit d is unique to mtATPases being characterized first in mammals by Walker et al. (1987b), and in yeast by Norais et al. (1991). The protein is predominantly hydrophilic, and has been shown to be essential for enzyme function (Norais et al., 1991). Reconstitution experiments showed that subunit d makes contact with all of the other three components of the peripheral stalk (b, F6 and OSCP) and is present in one copy per ATP synthase complex (Collinson et al., 1994b, 1996). The portion of subunit d (residues 3123) which is resolved in the crystal structure of the bovine peripheral stalk (Dickson et al., 2006) is composed of 5 a-helices separated by extended linker regions. The protein has an extensive interface with subunit b, interacting predominantly via a parallel and two antiparallel coiled-coil interactions in the region of residues 99162 of subunit b, placing the protein approximately halfway along the extramembranous portion of the peripheral stalk (Dickson et al., 2006; see Fig. 1.2). 2.4.2.4. Subunit h (F6 ) Subunit h of yeast mtATPase is an acidic watersoluble protein, present in one copy per complex (Fronzes et al., 2003). Yeast mutants lacking this protein are deficient in oxidative phosphorylation, showing that subunit h plays an essential role in assembly and/or catalysis (Velours et al., 2001). The bovine subunit F6 can replace subunit h in such mutants, indicating that the two proteins are functionally homologous, despite sharing just 14.5% sequence identity (Velours et al., 2001). Reconstitution experiments with purified bovine subunits demonstrated that F6 binds directly with the b subunit, and interacts with subunit d either directly or through subunit b (Collinson et al., 1994b). Disulfide and chemical crosslinking experiments with yeast ATP synthase place subunit h in close proximity to subunits b and d, and also to the membrane bound subunit f (Fronzes et al., 2003). This would imply that the location of subunit h within the peripheral stalk is in a region close to the mitochondrial inner membrane. EM images of yeast F1F0 particles containing subunit h labelled with biotin/avidin also suggested that the protein is close to the F0 sector of the enzyme (Rubinstein et al., 2005). In contrast, the crystal structure of the bovine peripheral stalk (Dickson et al., 2006) shows that F6 is likely to be located in a region close to OSCP and the N-terminal portions of the a and b subunits (at the top of F1) and that the C-terminus of the protein would lie about 70 A˚ from the membrane surface (Dickson et al., 2006; see Fig 1.2).

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Rodney J. Devenish et al.

Even when the slightly greater length of the yeast subunit h is taken into account (16 amino acids) it appears unlikely that this protein would extend as far as the membrane surface. The solution structure of isolated bovine subunit F6 has been determined by NMR, and shows two loosely-packed a-helices separated by an unstructured linker (Carbajo et al., 2004). In this form, the protein is highly flexible and shows a high degree of structural heterogeneity. However, the crystal structure of the bovine peripheral stalk (Dickson et al., 2006) revealed that this was due to the subunit being studied in isolation. When bound to other components of the peripheral stalk, F6 adopts a more elongated conformation, and the observed interactions between the helices within isolated F6 are replaced by other inter-subunit contacts. In the peripheral stalk structure, the helices (residues 824 and 3451) are linked by an extended region; extended regions are also present at the N- and C-termini of the protein. Hydrophobic patches on the helices and portions of the extended regions make specific interactions with the b subunit, and a segment of the C-terminal helix of F6 makes a parallel interaction with a helix from subunit d (Dickson et al., 2006).

2.5. The F0 sector 2.5.1. Overall structure, function and subunit composition The F0 sector of mtATPase consists of three subunits with homologues in the bacterial enzyme (subunits a, b and c), whose roles are clear, along with at least six others (e, f, g, i/j, k and A6L) the functions of which are less wellcharacterized. All complexes contain a single copy of subunit a, while the stoichiometry of subunit c appears to vary between organisms. Yeast mtATPase contains 10 copies (Stock et al., 1999), as does the bacterium E. coli ( Jiang et al., 2001). By contrast, the bacterium Ilyobacter tartaricus (Meier et al., 2005) and spinach chloroplast (Seelert et al., 2003) complexes possess 11 and 14 copies, respectively. The function of F0 is to couple proton translocation across the membrane with rotation of the subunit c ring relative to subunit a. An acidic amino acid residue present in subunit c from all known sources (Glu58 in the human mitochondrial enzyme), which lies in the middle of the membrane (Meier et al., 2005; Stock et al., 1999), plays a critical role in proton movement. Alternative models have been proposed to explain how protons access this residue. Dimroth et al. (2000) suggested that the subunit c ring contains an aqueous channel through which protons (or Naþ ions in the case of the I. tartaricus enzyme) would gain access to the conserved glutamic acid side-chain. However, the crystal structure of the I. tartaricus subunit c ring (Meier et al., 2005) did not reveal the presence of an aqueous access channel. More likely, residues in subunit a enable access of protons to the

Structure and Function of mtATPases

19

conserved glutamic acid residue from both sides of the membrane, via two spatially distinct ‘‘half-channels’’ (Angevine et al., 2007; Junge et al., 1997). F0 is thought to operate as a ‘‘Brownian ratchet’’ in which directional motion is generated based on stochastic thermal fluctuations of the relative positions of subunit a and the subunit c ring, with rotation biased to proceed in a particular direction based on the polarity of the prevailing proton electrochemical gradient ( Junge and Nelson, 2005). Fluctuations are constrained by the need for the essential acidic acid side-chain of subunit c to be negatively charged when facing a positively charged residue in subunit a, but neutralized by protonation when facing hydrophobic membrane lipids. The presence of a proton gradient across the inner mitochondrial membrane would favor a proton entering the lower ‘‘half-channel’’ (from the mitochondrial intermembrane space) and binding to the essential acidic residue in subunit c at the interface with subunit a, disrupting electrostatic interactions between it and residues in subunit a. This would allow the subunit c ring (and the attached subunit g) to rotate one step counterclockwise. Simultaneously, the acidic residue of the adjacent subunit c would move into the subunit a interface and release a proton into the matrix through the upper ‘‘half-channel’’ ( Junge and Nelson, 2005). Due to the lack of high-resolution structures of F0 subunits from mtATPases, our present understanding of the architecture of this portion of the enzyme has come mainly from studies on their bacterial equivalents. In the case of subunit a, genetic and biochemical studies in E. coli have defined the likely topology and packing arrangement of the transmembrane helices. With regards to subunit c, an electron density map of modest resolution, obtained from crystals of a subcomplex of yeast mtATPase, was sufficiently detailed to show the presence of a ring of 10 subunits (Stock et al., 1999). However, a clearer picture of how the subunit ring is ˚ ) crystal structure of assembled was provided by the high-resolution (2.4 A þ the unadecameric ring of the Na -translocating ATP synthase from I. tartaricus (Meier et al., 2005). The structure and arrangement of the individual proteins present in F0 are discussed below. 2.5.1.1. Subunit a (Subunit 6) Cox et al. (1984) first suggested that proton translocation through F0 is accompanied by rotation of subunit a relative to the subunit c ring. It was also proposed that amino acid side chains from both of these proteins contribute to the proton channel across the membrane (Cox et al., 1986). Based on hydropathy profiles and secondary structure predictions, these authors suggested a model for folding of the bacterial a subunit in which there are five transmembrane a-helices, with the N-terminus on the periplasmic side of the membrane (equivalent to the intermembrane space in the case of the mitochondrial subunit a) and the C-terminus on the cytoplasmic side (mitochondrial matrix). Subsequent work involving site-directed mutagenesis, analysis of second-site revertants,

20

Rodney J. Devenish et al.

cross-linking and labelling has confirmed this topology (Fillingame et al., 2003; Hatch et al., 1995; Vik and Ishmukhametov, 2005). Cox et al. (1986) proposed that conserved residues on the fourth transmembrane helix, including Arg210 (Arg159 in human mtATPase) could form a proton translocation pathway, along with the conserved acidic residue on the second transmembrane helix of subunit c (Asp61 in the bacterial protein, Glu59 in yeast mtATPase). Site-directed mutagenesis showed that Arg210 is required absolutely for proton translocation (Cain and Simoni, 1989; Lightowlers et al., 1987). Recent cross-linking experiments with the bacterial (E. coli) enzyme, in which Cys residues were introduced into subunit a, indicated that the packing arrangement for helices 2, 3, 4 and 5 is a four-helix bundle (Schwem and Fillingame, 2006). Chemical modification studies indicated that protons may access the critical residues (subunit a Arg210 and subunit c Asp61) from the periplasmic (intermembrane space) side of the membrane via an aqueous channel in the middle of the helical bundle, and that access to the cytoplasm (matrix) is provided by residues on the external faces of helices 4 and 5, which contact subunit c (Angevine et al., 2007). These authors propose that gating of the two half-channels would be achieved as follows. During proton-transport driven rotation of the subunit c ring relative to subunit a, a protonated Asp61 of subunit c would release its proton to the cytoplasmic half-channel due to its interaction with Arg210 of subunit a; the acidic side-chain would be neutralized by protons sourced from the periplasmic half channel and moved from the centre of the helical bundle to the outside by means of swivelling of one or both of helices 4 and 5 (Angevine et al., 2007). Several debilitating human mitochondrial diseases are caused by point mutations in subunit a of mtATPase (Schon et al., 2001). These include maternally inherited Leigh syndrome (MILS) (Tatuch et al., 1992), neurogenic ataxia and retinitis pigmentosa (NARP) (Holt et al., 1990), and some cases of Leber hereditary optic neuropathy (LHON) (Majander et al., 1997). 2.5.1.2. Subunit c (Subunit 9) Structural information on subunit c has come from NMR experiments on the isolated subunit in organic solvent (Girvin et al., 1998; Nakano et al., 2006; Rastogi and Girvin, 1999) along with studies on the oligomeric subunit c ring involving cryo-electron microscopy (Vonck et al., 2002), atomic force microscopy (Seelert et al., 2000; Stahlberg et al., 2001) and x-ray crystallography (Meier et al., 2005; Stock et al., 1999). The protein folds as a hairpin comprised of two transmembrane helices, with the critical acidic residue (Asp61 in E. coli, Glu58 in human mitochondria) situated approximately halfway between the two sides of the membrane. The NMR-derived structures of the isolated bacterial (E. coli) subunit c, determined at pH 5 (Girvin et al., 1998) and pH 8 (Rastogi and Girvin

Structure and Function of mtATPases

21

et al., 1999) revealed different conformations of the protein, with Asp61 in protonated and deprotonated states, respectively. The deprotonated form (likely to represent subunit c at the interface with subunit a) differed from the protonated form (likely to represent subunit c facing the lipid bilayer) in that the C-terminal helix was rotated by 140
(Girvin et al., 1999). Fillingame and Dmitriev (2002) suggested a model for a 10-mer subunit c ring in which the individual subunits pack in a ‘‘front-to-back’’ manner with the first transmembrane helix (TM1) facing inside the ring, and the second helix (TM2) on the outside. On the basis of the protonated and deprotonated conformations seen in the NMR structures (Girvin et al., 1998, 1999), it was proposed that access of protons to Asp61 during the protonation/deprotonation cycle would require TM2 to rotate, such that Asp61 moves from an occluded position (protonated state) to a position on the outside of the ring (deprotonated state, in the interface with the a subunit). This swivelling motion was proposed to drive the rotation of the subunit c ring relative to subunit a (Fillingame and Dmitriev, 2002). The high-resolution structure of the 11-mer subunit c ring from I. tartaricus (Meier et al., 2005) confirmed that the subunits pack with TM1 on the inside and TM2 on the outside, but did not support an ion-translocating mechanism involving swivelling of TM2. In this structure, the side-chains of all of the critical acidic residues (each with bound cation) face toward the outside surface of the ring, eliminating the need for swivelling of TM2 during the protonation/deprotonation cycle. Meier et al. (2005) also noted that the highly compact structure of the subunit c ring would impose steric hindrance which would be likely to severely restrict the proposed swivelling. A more detailed understanding of the conformational changes undergone by subunit c during ion translocation, and of the access pathways to the conserved acidic residues await a high-resolution structure of F0 in which both subunit a and the subunit c ring are packed together in their native arrangement. 2.5.1.3. Subunits e, g and k The ability of mtATPase to form a homodimer was first observed by native gel electrophoresis of solubilized yeast mitochondria (Arnold et al. 1998). Three proteins, e, g and k, were found associated with the dimeric form of the enzyme, but not with the monomer, and were not essential for enzyme function as an ATP synthase (see Section 3). 2.5.1.4. Subunit 8 (A6L) This small hydrophobic subunit is unique to the mtATPase complex (Table 1.1), having a stoichiometry of one per complex (Stephens et al., 2003b). It is a membrane-embedded protein oriented with the C-terminus facing into the mitochondrial matrix (Stephens et al., 2000). The protein has no clear function within the complex, but yeast mutants failing to express subunit 8 are unable to assemble subunit 6 into the enzyme

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complex (Hadikusumo et al., 1988) and therefore have nonfunctional mtATPase. The yeast subunit has been shown by cross-linking to be proximal to several other subunits of the peripheral stalk. The N-terminus of subunit 8 forms chemical cross-links with subunits b, d, f and subunit 6 in the intermembrane space, and the C-terminus interacts with subunits b, d and f within the mitochondrial matrix (Stephens et al., 2000, 2003a). Based on its proximity to the other component protein of the peripheral stalk as well as its interaction with subunit 6, it was postulated that subunit 8, in combination with subunit d, may complement the single copy of subunit b as a component of the stator stalk (Bateson et al., 1999). 2.5.1.5. Subunit f Another yeast subunit lacking a bacterial homologue, its presence is required for the stable assembly of the three mitochondrially encoded subunits, 6 (a), 8, and 9 (c) into mtATPase (Spannagel et al., 1997). The C-terminus domain of this subunit possesses a hydrophobic transmembrane domain which is not essential for enzyme function, but does seem to play a role in functional coupling and structural stabilization of the enzyme (Roudeau et al., 1999). The N-terminal domain of the subunit resides within the mitochondrial matrix (Belogrudov et al., 1996; Roudeau et al., 1999). The 66 N-terminal residues within the matrix (Roudeau et al., 1999) would extend two-thirds of the way up the side of F1 if in a-helical confirmation. Interactions of subunit f with subunits b, i, h and 8 have been demonstrated (Fronzes et al., 2003; Spannagel et al., 1998b; Stephens et al., 2003a; Velours et al., 2000). 2.5.1.6. Subunit i/j This small membrane integral protein was identified concurrently by two research groups, who named this protein subunit i (Vaillier et al., 1999) and j (Arnold et al., 1999). It has a single hydrophobic transmembrane domain orientated with its N-terminus inside the mitochondrial matrix (Paumard et al., 2000). Functional assessments of the protein differed between the two groups. One group found that a null mutant strain, while having reduced ATP catalytic activity and retarded proton pumping, was still able to grow on a nonfermentable carbon source (Vaillier et al., 1999). The null strain of the other group, however, displayed no oligomycin-sensitive ATPase activity, was unable to grow in medium containing a nonfermentable carbon source, and adopted a petite phenotype (Arnold et al., 1999). Subunit i/j forms cross-links with subunits d and f of the stator stalk, as well as subunit 6 and subunit g (Paumard et al., 2000). Its presence is required also for the stable expression of subunit 6 and subunit f (Arnold et al., 1999). Interactions between proximal mtATPase complexes can occur through this subunit, indicating it has a peripheral location within the complex (Paumard et al., 2002a), and may be involved in mtATPase dimerization (Fronzes et al., 2006).

Structure and Function of mtATPases

23

3. Supramolecular ATP Synthase 3.1. Introduction The mitochondrial inner-membrane (IM) contains one of the highest protein concentrations of cellular membranes. Thus, one might expect these proteins would be highly organised and, possibly arranged into large supramolecular complexes. Such a view of the mitochondrial IM is now generally accepted. In this section we will focus on supramolecular organization of mtATPase. In the last decade it has become clear that strong, stable interactions exist between individual mtATPase complexes that have important ramifications for cristae structure/function and mitochondrial bioenergetics. The significant advances in our knowledge of protein complex organization in mitochondrial membranes can be attributed, in-part, to the development and application of new high-performance analytical approaches. Gel based electrophoretic approaches, in particular blue-native PAGE (BNPAGE) and clear native PAGE (CN-PAGE), have been important for the high-resolution separations and visualising the complexes. These techniques and their application to protein complexes in different membrane systems are the subject of recent comprehensive reviews (Krause, 2006; Wittig and Schagger, 2007). BN-PAGE is a charge-shift method relying on the binding of an anionic dye to membrane proteins and complexes which allows separation according to size with superior resolution compared to more traditional techniques such as gel filtration or density gradients. Although constrained to separating proteins with a native negative charge, CN-PAGE performed without the addition of dye, allows weaker interactions to be preserved and enzyme activity to be more efficiently monitored in situ. Of equal importance to the success of such studies has been the selection of the best detergent that upon solubilization of membranes preserves both enzymatic activity and supramolecular organization. In the case of mitochondrial membranes the use of nonanionic detergents, in particular digitonin, has been found to be very effective in preserving many supramolecular structures from mitochondrial membranes.

3.2. Dimers and oligomers First identified in mitochondrial membranes isolated from yeast cells (Arnold et al., 1998), the existence of dimers of mtATPase (d-mtATPase) was initially met with some scepticism even though such an arrangement had been hinted by the remarkable EM images of Paramecium multimicronucleatum mitochondria (Allen, 1989; Allen et al., 1995) (see below). The existence of such dimers and indeed other oligomeric forms is now well accepted.

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Indeed, d-mtATPase can be recovered from a number of sources including mammals, fungi and plants (Table 1.2). Oligomeric forms of mtATPase have to date not been identified in bacteria presumably due to the absence of those F0 subunits believed to be involved in promoting oligomer formation (Schagger, 2002). In contrast to the rather unstable nature of d-mtATPase from most mitochondrial sources requiring careful use of the gentle detergents such as digitonin or Triton X-100, Chlamydomonas reinhardtii mitochondria contained a particularly stable d-mtATPase that was readily recovered using dodecylmaltoside (van Lis et al., 2003), a detergent that disrupts d-mtATPase from other sources. Indeed m-mtATPase could not be recovered under these conditions. The initial reports of d-mtATPase were soon followed by those showing that tetramers (Arselin et al., 2004; Paumard et al., 2002b) could be recovered in small amounts from yeast mitochondria through the careful selection of alternative detergent/protein ratios. More recently hexamers and even octamers of mtATPase, in addition to dimers, have been recovered from rat Table 1.2 Dimers and oligomers of ATP synthase identified by BN-PAGE or CN-PAGE Organism and/or cell type a

Human fibroblast and osteosarcoma Bovine heart

Liver, skeletal muscle, brain kidney, brain Rat liver Higher plants (Arabidopsis) Yeast Saccharomyces cerevisiae

Filamentous fungus Podospora anserina Chlamydomonad Algae C. reinhardtii chloroplasts Polytomella sp. a

Dimer

Oligomers

References

X

X

X

X

X

X

Cortes-Hernandez et al., 2007 Schagger and Pfeiffer 2000; Pfeiffer et al., 2003 Krause et al., 2005

X

X

Garcia et al., 2006; Krause et al., 2005 Eubel et al., 2003, 2004

X

Arnold et al., 1998; Gavin et al., 2005; Paumard et al., 2002b

X

X

X

Krause et al., 2004

X X X

van Lis et al., 2003 Rexroth et al., 2004 Dudkina et al., 2006a,b

Unless otherwise indicated, listings relate to mitochondria.

Structure and Function of mtATPases

25

heart mitochondrial membranes through careful titration with digitonin combined with analysis by CN-PAGE (Wittig et al., 2005). These oligomeric forms of mtATPase are evidently more labile, but readily observed by CN-PAGE rather than BN-PAGE where the binding of the blue dye to proteins presumably weakens protein–protein interactions (Wittig et al., 2006). Interestingly in this study only even numbered oligomers were recovered, a finding that has relevance to the organization of mtATPase complexes within the membrane (see Section 3.4). The evidence for the existence of oligomeric forms of mtATPase is largely based on their analysis after detergent extraction from mitochondrial membranes leaving the possibility that they might arise from nonspecific aggregation of mtATPase monomers under the high protein concentrations used in BN-PAGE or CN-PAGE analysis. However, this possibility now seems unlikely. Recovery of d-mtATPase is dependent on the presence of specific subunits in mitochondrial membranes (see below). Moreover, d-mtATPase can be stabilized upon cross-linking in membranes of isolated yeast mitochondria (Paumard et al., 2002a). The close proximity of mtATPase monomers in intact yeast cells could be inferred using a noninvasive fluorescent protein cross-linking approach (Gavin et al., 2004).

3.3. Subunits relevant to dimer formation 3.3.1. The dimer specific subunits e, g and k Use of BN-PAGE resulted in the identification of proteins now considered subunits of mtATPase. Two of these subunits, e and g, first identified as being associated with bovine heart mtATPase (Collinson et al., 1994a; Walker et al., 1991) and subsequently found to exist in yeast (Arnold et al., 1997, 1998; Boyle et al., 1999) are often referred to as the dimer specific subunits. An analysis of the subunit composition of mtATPase complexes solubilized from yeast with different detergents revealed these subunits to be associated with d-mtATPase but not m-mtATPase. The importance of these subunits was underscored by the finding that d-mtATPase could only be recovered only in very small amounts, or not at all, from yeast mitochondria lacking expression of subunit g or e, respectively (Arnold et al., 1998; Schagger, 2002). A third protein, subunit k, was also found to be associated with d-mtATPase, but is not considered important to the formation of dimers as d-mtATPase was readily recovered from cells lacking expression of this subunit. Although both subunits e and g are involved in the formation of d-mtATPase, it is subunit e that appears to play a more central role as its presence is required for the stability of subunit g, whereas subunit e is stable, albeit present in lower amounts, in the absence subunit g. Furthermore, in mitochondria from the strain lacking subunit e small amounts (5%) of dimer could be isolated, and subunit g can in some cases be found bound to m-mtATPase complexes (Schagger, 2002). However, a recent report indicates that phosphorylation of

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a serine residue located in the matrix domain of subunit g regulates formation of d-mtATPase (Reinders et al., 2007). Both subunit g and e are small transmembrane hydrophobic proteins with orientation Nin- Cout relative to the mitochondrial matrix. In rat liver mitochondria the stoichiometry of subunit e was found to be two per mmtATPase (Arakaki et al., 2001). In yeast two copies of the protein are in close proximity as homodimers were observed upon oxidation of its unique cysteine (Everard-Gigot et al., 2005). However, cross-linking studies suggest that there are two populations of subunit e that exist in different environments (see model below). Homodimerization and heterodimerization of subunits e and g has been shown through the cross-linking of both endogenous and introduced cysteines (Brunner et al., 2002). The subunit e/g heterodimer is specific to d-mtATPase; the g/g and e/e homodimers are found in oligomeric forms (Arselin et al., 2003; Bustos and Velours, 2005). The 95-amino acid subunit e has a unique transmembrane span containing a conserved GXXXG dimerization motif essential for the stability of the supramolecular structure (Arselin et al., 2003). Substitutions within this motif abolish the recovery of d-mtATPase from yeast mitochondria. In such mitochondria subunit e associated only weakly with mtATPase and subunit g was absent indicating the mutual dependence of these two subunits. Subunit g also contains a GXXXG motif in its single transmembrane segment that has been demonstrated to be important for function and stability of the subunit within mtATPase (Saddar and Stuart, 2005). However, these authors also showed that an intact GXXXG motif in subunit g was not essential for its interaction with subunit e. Subunit e has a C-terminal coiled-coil motif projecting into the intermembrane space that is also involved increasing the stability of d-mtATPase (Everard-Gigot et al., 2005). Removal of one or more of the coils from yeast subunit e resulted in decreased stability of d-mtATPase and oligomers as determined by detergent solubilization and BN-PAGE analysis (Bornhovd et al., 2006). Under these conditions subunit g was subject to increased degradation. Clearly subunit e has an important role in the formation or stability of d-mtATPase and oligomers. However, it is not clear if the absence of subunit e alone is sufficient to abolish interactions between monomers. This issue was addressed in intact yeast cells in which the single copy subunit b was expressed fused in equal amounts to blue fluorescent protein and green fluorescent protein (Gavin et al., 2005). Analysis of Fo¨rster resonance energy transfer (FRET) between the subunit b fusion proteins in cells lacking expression of subunit e indicated that in vivo interactions between the subunits b were maintained suggesting that subunit e was not involved in the interface stabilized by the b–b interactions. This result was in keeping with the presence of two distinct interfaces between the monomers in d-ATPase, as previously suggested by Paumard et al. (2002b). Subsequently it has been found that subunit e

Structure and Function of mtATPases

27

is not involved in driving formation of the b–b interface, but rather helps stabilise it on solubilization of membranes in detergents (Fronzes et al., 2006). 3.3.2. Other proteins that promote/stabilise dimerization and oligomerization The finding that chloroplast d-mtATPase can be extracted from Chlamydomonas reinhardtii where there is, as yet, no evidence for subunits e and g (or homologous proteins) suggests other proteins are involved in dimer formation in chloroplasts (van Lis et al., 2003). Despite the interactions mediated through subunits e and g, data exist to suggest other proteins play an important role in dimerization. Although d-mtATPase cannot be isolated by the use of detergents from yeast mitochondria lacking subunit e or g there is convincing evidence to indicate that d-mtATPase exists in the membranes of such mutant mitochondria. Thus, it was still possible to recover d-mtATPase from mitochondrial membranes of cells expressing subunit i/j modified by introduced cysteines in place of the native subunit and under conditions which facilitate formation of cross-links (Fronzes et al., 2006). The stator stalk appears to play an important role in formation or stabilization of d-mtATPase. Homodimers of subunit b can be isolated by SDS-PAGE after cross-linking of introduced cysteines in the hydrophilic loop of this protein (Spannagel et al., 1998a). However, in this case crosslinked d-mtATPase could not be solubilized from the membranes using detergents suggesting that the mtATPase had in some way been altered. In a different study, d-mtATPase or oligomers could not be recovered from mitochondria expressing subunit b with a truncation of the N-terminal portion of the loop traversing the inner membrane (Soubannier et al., 2002). Such a truncation did not affect the function or assembly of monomeric mtATPase (m-mtATPase). By contrast, subunit h, in cross-linking studies was able to form homo-dimers, and in this case d-mtATPase could be solubilized with digitonin and visualized by BN-PAGE (Fronzes et al., 2006). Importantly these same cross-linked dimers could be isolated in the absence in mitochondria lacking subunit e or g indicating the importance of stator stalk interactions for mtATPase dimerization. There have been suggestions that IF1 may play a role in dimer formation. It has been demonstrated that bovine IF1 self-associates as an inhibitory dimer (Cabezon et al., 2000b) that can induce the dimerization of F1 particles (Cabezon et al., 2000a). A crystal structure F1 reconstituted with IF1 shows a dimeric structure (Cabezon et al., 2003). However, dimeric complexes could be isolated from the mitochondria of yeast cells lacking expression of Inh1p, the homologue of IF1 (Dienhart et al., 2002), or bovine heart submitochondrial particles in which were depleted of IF1 (Tomasetig et al., 2002). Although the role of IF1 in this respect remains controversial more recently depletion of IF1 from rat liver and bovine heart

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sub-mitochondrial particles was found to decrease the dimer to monomer (D/M) ratio of mtATPase whereas reconstitution with recombinant IF1 restored partially the D/M ratio as determined using BN-PAGE (Garcia et al., 2006). Furthermore, 2-fold increased expression of IF1 in AS30D hepatoma mitochondria led to a 1.4-fold increase in D/M ratio, compared to mitochondria from normal liver cells having a sub-stoichiometric amount of IF1 (Garcia et al., 2006). A working model for IF1 in dimerization has been proposed accommodating data accumulated from crytallographic, cross-linking and EM studies. In this model, depicting the arrangement of IF1 in rat liver d-mtATPase, the C-termini of the two IF1 molecules are proposed to bend and cross at the dimer interface to interact with OSCP (Garcia et al., 2006). 3.3.3. Associations with non-mtATPase proteins We have discussed the homodimerization of mtATPase. Are there other proteins associated with the complex? This certainly appears to be the case based on the isolation of the ATP synthasome from rat liver mitochondria, a monomeric ATPase complex together with the adenine nucleotide carrier (ANC) and the phosphate carrier (PC) in a ratio of 1:1:1 (Ko et al., 2003). Subsequent EM analysis of the ATP synthasome indicated an increased dimension of F0 in the plane of the membrane sufficient to allow the docking of a putative heterodimer of ANC and PC. It is intriguing that the components of the ATP synthasome have not been recognized in the EM analyses of d-ATPase (see discussion above). The reasons for this discrepancy are not clear but may be related to differences in the properties of the detergents used in each study. CHAPS, and not digitonin, was used to solubilise the ATP synthasome. Nevertheless, it will be important to ultimately define the relationship of the components of the ATP synthasome to oligomeric forms of mtATPase. If the d-mtATPase interface involves the stator stalk, perhaps components of the ATP synthasome are located on an alternative interface. The stator stalk was not visualized in the ATP synthsome structure determined by EM (Chen et al., 2004). In a recent report BN-PAGE was used to identify two small proteins, denoted MLQ and AGP, associated with bovine mtATPase (Meyer et al., 2007). Interestingly, both proteins have been identified in different contexts: MLQ [or 6.8 kDa mitochondrial proteolipid (Terzi et al., 1990)] and AGP [diabetes-associated protein in insulin-sensitive tissue (DAPIT); Paivarinne and Kainulainen, 2001]. The functional significance, if any, of the association of these two proteins with mtATPase remains to be demonstrated. Intriguingly, the AGP protein has 13% sequence identity (16% similarity) with yeast subunit k, however functional homology has yet to be demonstrated. It is noteworthy that the sequence identity between yeast subunit h and bovine subunit F6 is only 14.5% and these two proteins have been demonstrated to be functional homologues (Velours et al., 2001).

29

Structure and Function of mtATPases

3.4. The arrangement of mtATPase in mitochondrial membranes 3.1.1. Model A model based on investigations in yeast showing the relative arrangement of subunits at the interfaces between complexes was proposed (Paumard et al., 2002b) and more recently updated (Fronzes et al., 2006) taking into consideration the results of cross-linking studies (see Fig. 1.3). Both versions of the model focus on the role of subunit-subunit interactions within the F0 sector. Oligomeric forms of mtATPase imply two distinct interfaces. Crosslinking and other data suggested that subunit b participates at one interface; the second interface, as proposed by Paumard et al. (2002b), involves both subunits e and g. Detergents such as dodecylmaltoside disrupt both interfaces, whereas digitonin at relatively high concentrations preserves the eg/eg interface and at low digitonin-protein ratios (0.751 g  g1) also preserves the b/b interface. The results of FRET studies also suggest two interfaces (Gavin et al., 2005). Although the two distinct interfaces are proposed they are no longer thought to be independent as previously suggested. Subunits e and g stabilize the b/b dimer interface when it is exposed to detergents and are directly involved in the other interface between dimers that make up the oligomer. Rather the two monomers are viewed as interacting through their peripheral stalk structures particularly B

A Catalytic head

Su 9 digomer

Su g Su e

Su 6, 8 4, i, f

Dimer

Oligomer

Figure 1.3 A model for the supramolecular organization of the ATP synthase. (A) Organization of the dimerization interface in the membrane seen from the intermembrane space perpendicular to the main axis of the ATP synthase. The area in gray represents the cross section of the subunit c ring and of the F0 domain. The F1 sector is represented as a dashed line.The dimerization interface involves a large part of the Fo sector and may also extend to components localized in the matrix, such as subunit h (dotted line). Subunits e and g are localized at the periphery of the dimerization interface and stabilize it. In the dimer, subunits e and g are in close contact on each side of the dimer. (B) Oligomerization of ATP synthase may occur by interaction between the e þ g interfaces. [Reprinted with permission from Fronzes, R.,Weimann,T.,Vaillier, J.,Velours, J., Brethes, D.The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits. Biochemistry 45: 6715-6723. Copyright (2006), American Chemical Society.]

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subunits b and i at the membrane, and subunit h extrinsic to the membrane (Fronzes et al., 2006). As discussed above in mammalian mitochondria IF1 may also be involved in dimerization (Garcia et al., 2006). The adjacent dimers are tilted to prevent clashing of F1 headpieces, consistent with EM images (see Section 3.4.3). 3.1.2. A key role for subunit e? The available data could be taken to indicate that subunit e plays a central role in controlling oligomerization of mtATPase and mitochondrial function. It has been shown in yeast cells that regulated expression of subunit e can reversibly control the formation of mitochondrial cristae (Arselin et al., 2004). In mammalian cells and tissues, the expression level of subunit e has been linked to physiological stimuli such as diet and hypoxia (Levy and Kelly, 1997; Swartz et al., 1996), It has been suggested that subunit e could be involved in the Ca2þ-dependent regulation of mtATPase activity. Residues 34-65 of the subunit e are homologous with the Ca2þ-dependent tropomysin-binding region for troponin T (Arakaki et al., 2001). As discussed above the coiled-coil domains of subunit e contribute to the stability of d-mtATPase and oligomers as determined by BN-PAGE analysis (Bornhovd et al., 2006). However, the deletion of these domains did not result in the loss of mitochondrial cristae, but did result in a loss of mitochondrial membrane potential. Consequently, it was proposed that oligomerization is essential for maintenance of bioenergetically fully competent mitochondria; a drop in membrane potential would precede alterations in cristae morphology. 3.1.3. Visualisation of dimers As presented above, a considerable body of biochemical evidence exists in favor of a stable close interaction between individual mtATPase complexes. Remarkably, such interactions were first suggested to occur based on the evidence obtained from the pioneering rapid-freeze deep-etch EM of P. multimicronucleatum by Allen et al. (1989) conserving the close juxtaposition of F1 sectors. More recently, a number of single particle EM structures have been produced for d-mtATPase from a number of sources including bovine heart (Minauro-Sanmiguel et al., 2005), the colourless algae Polytomella (Dudkina et al., 2005) and yeast (Dudkina et al., 2006b). Rows of d-mtATPase dimers have been visualized using high-resolution atomic force microscopy in native mitochondrial inner-membranes isolated from the yeast Saccharomyces cerevisiae (Buzhynskyy et al., 2007). In each case, d-mtATPase was solubilized using digitonin and enriched using gradient ultracentrifugation. The structures visualized have revealed some important features in common. In each case the dimer interface was

Structure and Function of mtATPases

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seen to be formed predominantly by the F0 domains in such way that the long axes of the two complexes are arranged at an angle. However, distinct differences exist in each of the published structures. Although the dimer in each case displayed a conic arrangement, the angle subtended between the individual complexes was estimated to be quite different in each case: bovine, 40
; Polytomella, 70
and yeast, 90
. The larger angles observed for yeast and Polytomella do not allow direct contact between the F1 headpieces as seen for the bovine structure. Since there was no evidence for the presence of the peripheral stalk, visualized in other EM representations of the bovine complex (Rubinstein et al., 2003), the dimeric interaction, based on some density in the images, was postulated to be mediated by IF1 (Minauro-Sanmiguel et al., 2005; Garcia et al., 2006). By contrast, EM images of the Polytomella complex clearly show the presence of the peripheral stalk sandwiched between each complex, but again interactions appear to be mediated by the F0 sector alone. MASP, a protein unique to the Polytomella complex and believed to be responsible for the high stability of the dimer is presumed to be located towards the top of the structure, but evidently not bridging the dimer (Dudkina et al., 2005). The apparent position of the of the peripheral stalk sandwiched between the individual complexes such that they interact through F0 suggests the these structures represent the dimer proposed on the basis of biochemical studies, in which membrane anchored subunits (b, together with e and g) are promoting or stabilising interactions (Fronzes et al., 2006; Paumard et al., 2002b). However, for yeast in addition to dimers subtending the large 90
angle, examples subtending a considerably smaller angle of 35
and having a smaller F0 contact area were also observed (Dudkina et al., 2006b). It was proposed that these ‘‘pseudo’’ dimers represent broken ‘‘true’’ dimers. Although they might represent nonspecific interactions that occur upon solubilisation of mitochondrial membranes, the intriguing suggestion has been made that these pseudo dimers represent complexes associated by interactions at the second interface present in the oligomeric form (Fig. 1.4). It should be noted that the working models based on biochemical data were developed by analysis using BN-PAGE or CN-PAGE and represent alternative structures to those visualized by EM. Although most likely representing a considerable technical challenge the recovery of stable oligomers (e.g., tetramers or hexamers) for analysis by EM would provide the possibility of visualizing both interfaces in the oligomeric form.

3.5. The role of mtATPase oligomerisation What purpose do mtATPase dimers/oligomers serve in mitochondria? Bacteria, apparently without having a requirement for ATP synthase dimers or oligomers, remain able to synthesise ATP in a highly regulated and

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True-dimer

Pseudo-dimer

Figure 1.4 A model for the arrangement of ATP synthases dimers into multimers. Oligomers consist of dimeric ATP synthases. The oligomers can break down by detergent incubation into ‘‘true-dimers’’ or into ‘‘pseudo-dimers’’. The latter actually consist of two monomers from the neighboring dimers, symbolized by a blue and purple set of a3b3 subunits. Ochre and bright green densities symbolize dimer- and interdimer specific subunits, respectively. (Reprinted by permission of the Federation of the European Biochemical Societies from Characterization of dimeric ATP synthase and cristae membrane ultrastructure from Saccharomyces and Polytomella mitochondria, by Dudkina N. V., Sunderhaus, S., Braun, H. P., Boekema, E. J., FEBS Letters, 580: 3427^3432, Copyright 2006.)

efficient manner. It appears that the requirement to form oligomers may be specific to mitochondrial complexes. Investigations in yeast suggest that mitochondrial OXPHOS function is not greatly compromised in mitochondria unable to form dimers or oligomers. Thus, the specific activity of mtATPase was not affected in cells that do not have stable dimers or oligomers (Arnold et al., 1998; Boyle et al., 1999; Paumard et al., 2002b; Soubannier et al., 2002) and the uncoupled respiration rates and ATP/O ratios were not altered in these mutants (Boyle et al., 1999; Paumard et al., 2002b; Soubannier et al., 2002). Nevertheless, although capable of respiratory growth, such strains do display growth defects. The structure of the mitochondrion is now considered to be more complex than once thought. It is firmly established that the ‘‘infolds,’’ or cristae, of mitochondrial membrane are not random and are topologically complicated. The cristae are connected to the inner boundary membrane (IBM, that part of the inner membrane that runs parallel to the outer membrane) by narrow tubes called cristae junctions. Immunolabelling and transmission EM studies of bovine heart mitochondria (Gilkerson et al., 2003), together with application of more recent fluorescence imaging techniques in live yeast cells (Wurm and Jakobs, 2006), have led to the conclusion that nonuniform protein distributions exist within the IM indicating that the IBM and cristae are functionally distinct compartments.

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In excess of 90% of mtATPase and respiratory complexes (III, IV) were found enriched in cristal membranes, whereas proteins such as the TIM23 complex and presequence translocase motor were found enriched in the IBM (Wurm and Jakobs, 2006). Thus, the evidence supports cristae as being a regulated sub-mitochondrial compartment specialized for ATP production. The projections in the matrix space seen in EM images have long been known to represent the F1 sector of mtATPase (Racker et al., 1965). More recently, using freeze-fracture techniques, these projections were seen to be arranged in a highly ordered double-row in the tubular cristae of P. multimicronucleatum (Allen et al., 1989). It was these morphological observations that led to the first clear suggestion that the arrangement of ATP synthase complexes might have a role in promoting the tight curvature (50 nm diameter) of the membrane to form tubular cristae. Thus, the view was developed that ATP synthase, the molecular motor responsible for much of cellular energy generation, also appears to have an active role in the modelling of the membrane in which it resides. This concept gained further acceptance after the EM observations made of yeast lacking expression of subunits e and g and therefore presumably lacking oligomeric ATP synthase (Paumard et al., 2002b). In such cells normal cristae were absent and replaced by numerous digitations and onion-like structures containing many concentric membranes that was shown to have protein markers characteristic of the inner membrane. Given the apparent importance of cristae structure to mitochondrial metabolism it was remarkable to find that such yeast cells remained respiratory competent. The results of single-particle EM experiments indicate that interactions between the membrane regions of F0 subunits may be responsible for forming the tight curvature of the membrane. However, there is evidence to suggest that the F1 sector may also play role in cristae formation. Yeast strains lacking expression of proteins that mediate F1 assembly (e.g., Atp11p or Atp12p) lack F1 a3b3 hexamers and are devoid of mitochondrial cristae (Lefebvre-Legendre et al., 2005). It is not clear how this effect is mediated, but it may be related to the binding of other proteins to F1. In the case of mammalian cells IF1 may fulfil this role. However, EM projections of the ‘‘dimers’’ recovered from yeast mitochondria clearly show that the F0 sector is curved in those cases where one, or both of the F1 sectors was missing. The d-mtATPase isolated from bovine heart and visualized by single particle EM showed an interface formed by contacts on both the F0 and F1 domains (Minauro-Sanmiguel et al., 2005). A cross-bridging protein density was resolved which connects the two F0 domains on the inter-membrane space side of the membrane. On the matrix side of the complex, the two F1 moieties are connected by a protein bridge, which was attributed to the IF1 inhibitor protein. Close contact between neighboring mtATPase monomers is not sufficient to form and maintain cristae. It is clear from the results of in vivo

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cross-linking experiments that a precise arrangement of ATP synthase complexes is required (Gavin et al., 2004). In this study enforced oligomerization of yeast mtATPase was achieved by expressing subunit g fused to DsRed an obligatory tetrameric fluorescent protein. The mitochondria of such cells resembled those found in cells lacking expression of subunit e in having numerous onion-like structures. Similar results also were obtained using the dimeric HcRed (Gong, Devenish and Prescott; unpublished results). These studies suggest that cross-linking of this type may disrupt the oligomeric array by causing localized distortion. It must be noted that other proteins are involved in cristae formation. The IM protein mitofilin, when deficient in heart muscle, leads to large membrane sworls. These structures were found to be composed of a complex, interconnected network of membranes totally lacking tubular connections to each other, or to the peripheral IM ( John et al., 2005). Reduced expression of the dynamin-like protein OPA1 in humans also causes disorganization of cristae (Griparic et al., 2004). The question remains as to why mitochondria form cristal subcompartments. Yeast cells expressing subunit e lacking one or more of its coils (from the C-terminal coiled-coil motif ) showed normal cristae, but had low membrane potential such that mitochondria could not accumulate membrane potential dyes (Bornhovd et al., 2006). It was concluded that membrane potential was dependent of the oligomeric state of mtATPase. It was postulated that increased plasticity of the mitochondrial membrane led to disruption of membrane micro-domains normally maintained by mtATPase oligomers. This in turn would result in altered cooperation between complexes such as mtATPase and ANC and consequently reduced flux through the respiratory chain arising from negative feedback effects and lower membrane potential. Although the relationship of ANC to mtATPase dimers and oligomers has yet to be established, ANC and PC have been reported to be part of the ATP synthasome (Chen et al., 2004; see Section 3.3.3).

3.6. Is oligomerization regulated in vivo? The mitochondrion is a highly dynamic structure. If dimerization and higher order structures are indeed involved in cristae formation and mitochondrial dynamics, we might expect both their formation and dissolution to be under tight control. However, such evidence is limited at present. Studies in yeast that manipulated membrane potential or cellular energy charge apparently did not alter the ratio of d-mATPase to m-ATPase observed using BN-PAGE (Arnold et al., 1998). On the other hand, cells isolated from patients with Leigh syndrome (T8993G/T8993C) mutation in the mitochondrially encoded ATP6 gene for subunit a) showed significant inhibition (60%) of ATP synthase

Structure and Function of mtATPases

35

activity, but increased amounts of oligomeric forms over the monomeric form compared with normal cells. This result suggests that the oligomeric form is regulated in vivo and apparently increased in pathological conditions presumably to overcome the cellular energetic defect (Cortes-Hernandez et al., 2007). Furthermore, mutations in ATP6 can cause a marked reduction in ATP production in neuronal mitochondria which have a very unusual inner membrane topology of rounded cristae compartments contiguous with flattened lamellar regions of the same membrane. A possible explanation for this highly unusual topological transition is that dimerization of ATP synthase is normally inhibited in flat, lamellar cristae and that the ATP6 mutation somehow weakens this inhibition, allowing mtATPase dimerization and, consequently, increased membrane curvature. The central role that subunit e appears to have in promoting dimer formation makes it a good candidate for a regulatory role in determining oligomerization. In support of this notion is the observation that by placing the expression of subunit e under control of the tetracycline promoter in yeast formation of mtATPase dimers could be reversibly depleted with characteristic alterations in cristae morphology (Arselin et al., 2004). Other evidence in support of a regulatory role for subunit e is that the level of its mRNA in C2C12 myotubes and myoblasts was found to be regulated in response to oxygen availability (Levy and Kelly, 1997). Although ATP synthase oligomeric structure can be correlated with cristae morphology, it is not clear that it alone is the key determinant for formation and maintenance of the mitochondrial cristae. Other factors clearly have a role to play. For example, expression of tBid caused a remodelling of cristal membranes (Scorrano et al., 2002), and reversal of curvature can occur in cardiolipin-containing membrane phases (Epand et al., 2002), suggesting that remodelling of the membranes is lipid mediated.

3.7. Supramolecular structures involving other respiratory complexes? In addition to ATP synthase discussed in detail above, the oxidative phosphorylation (OXPHOS) system comprises four oxidoreductases: NADH dehydrogenase: (complex I), succinate dehydrogenase (complex II), cytochrome c oxidase (complex III) and cytochrome oxidase (complex IV). High-resolution structures are available for complexes II, III and IV and in part for complexes I and V (ATP synthase). However, despite the detailed knowledge of the structures of the individual complexes comparatively little is known about their organization into supramolecular structures. Evidence exists to support two different views of the mitochondrial respiratory chain either as (i) a fluid state in which the complexes are free to diffuse in the plane of the manner membrane undergoing random

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collisions (Hackenbrock et al., 1986) or (ii) a solid state in which the complexes are more ‘‘rigidly’’ associated. The former view is supported by results of classical fractionation approaches developed some 40 years ago that led to the isolation individual complexes that are capable of carrying out their apparent physiological function. The latter view is supported by data showing increased complex activities when reconstituted in defined stoichiometry. Furthermore, one or more complexes can be isolated in association (Schagger and Pfeiffer, 2000). These two views represent different snapshots of the same system and, the final correct picture will have elements of both reflecting the dynamic nature of the mitochondrion. Interestingly, supercomplexes between mtATPase and other respiratory complexes do not appear to exist.

4. Extra-Mitochondrial Expression of F1F0-ATP Synthase 4.1. Introduction In recent years evidence for extra-mitochondrial expression of F1F0 ATP synthase has come from several laboratories. A number of reports provide evidence that complexes are located on the surface of various mammalian cells types, including endothelium, hepatocytes, adipocytes, keratinocytes and tumor cells. F1F0 ATP synthase components have been identified as cell-surface receptors for ligands in studies carried out on angiogenesis, lipoprotein metabolism, innate immunity, hypertension or regulation of food intake. The cell types, ligands and components of F1F0 ATP synthase identified have been comprehensively listed by Champagne et al. (2006) and Chi and Pizzo (2006a). However, as noted by Wahl et al. (2005) an exhaustive survey of cell types and tissues for evidence of cell surface ATP synthase components has not yet been made. Here we will refer to the complex as external ATP synthase (eAS). In some instances the function of eAS relates to a single report of a particular ligand interaction. However, in some instances a series of studies have contributed towards the development of the understanding of the specific ‘‘new’’ function of eAS, which is not the generation of cellular ATP so effectively carried out by the mitochondrial enzyme complex. In addition, one F0 component of F1F0 ATP synthase, F6 (usually designated CF6 in this context), again ectopically expressed, has been recognized as having function in its own right. In no case has an eAS complex been purified from the plasma membrane and all components of a fully assembled F1F0 ATP synthase shown to be present. The existence of fully assembled complexes generally has been inferred from the identification of an F1 component, most usually subunit b, and measurement of ATP hydrolysis and or ATP synthesis activity. If ATP

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synthesis is being correctly attributed to enzymatic activity of the complex under study then the expectation is that it must be fully assembled and membrane anchored, since only F1 functionally coupled to F0 can synthesize ATP. Also if F0 components were not present then F1, even if competent for ATP hydrolysis, would not be retained at cell surface since none of the F1 components are normally membrane anchored. Although the F0 subunits, b, d, e, F6 and O(SCP?), were shown to be protein components of detergent-insoluble lipid rafts isolated from rat liver (Bae et al., 2004), only recently have the F0 components, subunits d and OSCP, been directly shown to be present on the plasma membrane (PM) of an osteosarcoma cell line (Yonally and Capaldi, 2006). Definitive experiments remain to be performed to establish that cell surface eAS complexes and mtATPase have identical or closely similar arrangements of subunits. As pointed out by Champagne et al. (2006), the structure and subunit composition of eAS may differ between cell types and might possibly be, under different conditions, related to their different specific functions. How are eAS complexes distributed on membranes? In most instances this is not clear. There is no information as to whether eAS might be arrayed in dimeric or oligomeric forms. More recently, as discussed by Chi and Pizzo (2006a), it has emerged that eAS is found in caveolae/lipid rafts; see for example the studies of Ko and colleagues (Bae et al., 2004; Kim et al., 2004, 2006). In this context two recent reports are of interest. The first concerns regulation of HDL endocytosis where endothelial cells exposed to free cholesterol exhibited a subsequent increase in the concentration of caveolin-1 and the F1 subunit b within endothelial cell caveolae (Wang et al., 2006), together with an increase in the release of ATP from cells. The second, presents evidence suggesting that the localization of eAS to caveolae/lipid rafts is important for shear stress-induced ATP release by vascular endothelial cells (Yamamoto et al., 2007). An important implication of eAS localization to caveolae recognized by Chi and Pizzo (2006a) is the creation of microenvironments on the cell surface in which eAS, its binding partners or inhibitors plus substrates for enzymatic activity such protons, ADP and ATP can be concentrated. These authors give the example of a cell facing an acidic extracellular microenvironment where the eAS would be required to pump protons against the gradient in order to regulate intracellular pH. However, should the caveolar compartment have relatively lower proton concentration, then this would not be the case.

4.2. How might F1F0 ATP synthase get to the plasma membrane? The fully assembled multi-subunit mtATPase complex is anchored in, and assembled on, the inner mitochondrial membrane. The complex contains subunits made on cytosolic ribosomes and targeted to mitochondria, but

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also subunits encoded on mtDNA and made inside the mitochondrion (Table 1.1). As far as we are aware there is no evidence for the existence of additional copies of nuclear encoded mtATPase subunit genes that could specifically encode eAS components, or for mRNAs allowing for alternative translation products (i.e. having ER signal sequence rather than mitochondrial targeting signal). Moreover, such a scenario requires that the mitochondrial encoded subunits [in mammals subunits A6L (yeast subunit 8) and a (yeast subunit 6)] would also be nuclear encoded. [One of the reasons often suggested for the retention of these genes in the mitochondrial genome is to facilitate the synthesis and membrane insertion of the extremely hydrophobic subunits they encode (Claros et al., 1995).] If such genes did exist then the expectation would be for mRNAs to be translated by ER localized ribosomes, especially to facilitate the synthesis and membrane incorporation of hydrophobic membrane subunits such as subunits a and A6L. In such a scenario, the complex presumably would be assembled on the luminal surface of the ER and trafficked via vesicles through the Golgi into secretion vesicles that fuse with the PM thereby incorporating ATP synthase. Currently there is little evidence for the trafficking of complexes in such vesicles that are delivered to PM, although it has been recently reported that subunit a reaches the cell surface in an N-glycosylated form via the secretory pathway (Schmidt et al., 2007). Are there alternative pathways? Soltys and Gupta (1999, 2000) proposed specific export mechanisms by which proteins might exit directly mitochondria via channels, allowing them to reach specific extramitochondrial sites. The use of channels suggested by these authors for individual mitochondrial proteins (none of which were mtATPase components) would be unlikely to accommodate fully assembled mtATPase complexes. Transport of complexes assembled in mitochondria to the PM might occur by fusion events between the organelle and plasma membrane, facilitated by the dynamic fusion and fission of the mitochondrial network (Heath-Engel and Shore, 2006). There is no evidence for direct interaction of mitochondria and PM. In any case such interactions would be complicated by questions of membrane topology of the double membrane mitochondria. Fusion of the outer mitochondrial membrane with the PM would not result in complexes on the cell surface since most (94%) of the mtATPase complexes are located on cristae membranes (Gilkerson et al., 2003). If fusion occurred at contact sites (between inner and outer mitochondrial membranes) and the plasma membrane then it might be possible to have complexes on the external PM surface. Perhaps more feasible is direct interaction between the highly convoluted ER network with the highly dynamic mitochondrial network. Hypothetical pathways for export of matrix proteins to the ER have been presented by Soltys and Gupta (1999). If mitochondrial complexes could be ‘‘captured’’ within ER membranes, perhaps being sorted into specialized membrane regions (lipid rafts?), then subsequent delivery to the cell surface

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would require trafficking from the ER to the Golgi and presumably vesicle budding from the Golgi and finally vesicle fusion with the PM. A subdomain of the ER, the mitochondria-associated membrane (MAM) forms stable complexes with mitochondria, and in yeast is involved in phosphatidylserine traffic (see, for example, Choi et al., 2006). Furthermore sites of interaction between PM and ER in yeast (PAMs) have beeen characterized (Pichler et al., 2001). Thus mitochondria could associate with PAMs through MAMs. While MAMs could be used to capture and transport eAS, at present, the only available evidence supporting sequestration of mitochondria in membrane vesicles relates not to trafficking to the PM, but to turnover by autophagy that occurs following fusion with the vacuole (yeast) or lysosomes (mammals) (Mijaljica et al., 2007). Clearly, the mechanism by which complexes reach the plasma membrane remains elusive and is worthy of deliberate attention as a biological question in its own right. We now consider some aspects of the reported functions of eAS in the context of the structure and function.

4.3. The function of coupling factor 6 as a vasoconstrictor: Detachment and reattachment of an F0 component of eAS? A historical review of how we came to understand the cardiovascular, function of coupling factor 6 CF6, has been provided by Watts (2005). In summary, the current view is that CF6 circulates and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. In this context CF6, localized on the surface of the vascular endothelium, is released into the systemic circulation in response to pathological stimuli such as shear stress (Osanai et al., 2001a,b) and tumor necrosis factor alpha (TNF-a) (Sasaki et al., 2004). Other conditions related to cardiovascular dysfunction also result in increased circulating CF6 levels (Ding et al., 2004; Osanai et al., 2003a,b). Most recently the plasma CF6 level was shown to be markedly increased in patients with diabetes mellitus and that this increase could be positively correlated with disease severity (Li et al., 2007a). What is the source of the CF6 that is released into the circulation? It could be ‘‘free’’ on the PM or be ‘‘sequestered’’ by normal association as a component of eAS complexes. Within mtATPase CF6 is an important component of the stator stalk. Its functional homologue in yeast (subunit h) is essential for energy transduction (Velours et al., 2001; see section 2.4.2.4). As discussed above the N-terminal end of the predominantly a-helical CF6 subunit extends from near the top, to about the mid-point, of the F1 domain (Dickson, 2006). Significant contacts are made with subunits b, d and OSCP as well as the F1 domain surface. The available evidence suggests that CF6 stimulates cell surface ATPase activity of human umbilical vein endothelial cells (HUVECs) which can be

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blocked with efrapeptin, an ATP synthase inhibitor. On the basis of radioligand-binding studies Osanai et al. (2005) proposed the F1 subunit b as the receptor for CF6 on the PM of HUVECs. However, b-subunit antibody inhibited CF6 function by only 50%, suggesting other potential sites of action for CF6. It remains unclear how stimulation of ATPase by CF6 might occur through the b subunit. The questions that need to be addressed include the following. What is the binding site for CF6? Is CF6 binding to complexes lacking this subunit to ‘‘reconstitute’’ them? This seems unlikely as in yeast mtATPase complexes lacking subunit h the F1 sector is uncoupled from F0 and subunit 6 (a) is missing (Arselin et al., 1996). Reconstitution would thus require more than just CF6 fitting back into an ‘‘empty niche’’ on an otherwise fully assembled complex. In a similar vein it seems highly unlikely that CF6 released from eAS complexes by shear stress or other means just disassociates and then re-binds. Alternatively, a separate pool of free CF6, not associated with eAS, might exist on the cell surface and is it this CF6 that is released by shear stress or other stimuli. For reasons stated above it would seem unlikely that this CF6 would bind to eAS complexes lacking CF6 (i.e., reconstitution), rather than binding at a second site on fully assembled eAS complexes. However this remains an open question. The recent observation that the exposure of HUVECs to high glucose level results in a significant increase of CF6 expression and release, mediated by PKC and p38 MAPK activation, but antagonized by insulin treatment (Li et al., 2007b) starts to build a more detailed picture of signaling pathways involved CF6 activity, but does not resolve any of the questions concerning binding sites for CF6.

4.4. Multiple receptor functions of subunit b As well as being suggested as the binding site for CF6, subunit b is also identified as a putative receptor for at least three other ligands. These (and the proposed associated functional role) are: 

Enterostatin (Berger et al., 2002, 2004; Park et al., 2004)—regulation of fat intake.  HDL apolipoprotein A-I—contributes to HDL endocytosis (Martinez et al., 2003, 2004; Jacquet et al., 2005; Fabre et al., 2006; Zhang et al., 2006).  Angiostatin (an angiogenesis inhibitor derived from plasminogen)— regulates surface ATP levels, thereby modulating endothelial cell proliferation and differentiation (Moser et al., 1999, 2001). More recently, Veitonmaki et al. (2004) have shown that kringle 1-5, a different fragment of plasminogen with increased angiostatic activity compared to angiostatin, also binds to eAS (on bovine capillary cells) to trigger caspase-mediated apoptosis.

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At first it may seem improbable that these ligands all bind subunit b, but these ligands will function specifically in different cell and tissue types. There is little understanding of how ligand binding elicits the biological response in terms of eAS complex function. If these ligands all do bind subunit b, then they could influence ATP synthesis or hydrolysis by affecting a change in the ATP catalytic sites. The structure of subunit b is such that there is a hinge region whose conformation that could be influenced by ligand binding. These mechanistic questions remain to be addressed. For each of the ligands listed above (and for CF6), the reported biological effect of eAS relies on effects on enzymatic activity. In contrast there is another example involving cell surface subunit b which apparently does not involve enzymatic activity of the F1 moiety of eAS, but rather the recognition of a complex of F1 and apolipoprotein A-1 on tumor cells by a subset of gd T-lymphocytes that promotes innate tumor cell recognition and lysis (Scotet et al., 2005). Subunit b has also been proposed to serve as a ligand in NK cell-mediated cytotoxicity of tumor cells (Das et al., 1994). What is the biological effect in terms of eAS function elicited by binding of the ligand? Each of the ligands probably elicits different responses although this is not entirely clear as this question has not been explored in depth in all cases. In the case of enterostatin it is inhibition of ATP synthesis, for apoA-I it is inhibition of ATP hydrolysis, for angiostatin it is apparently inhibition of both ATP hydrolysis and synthesis leading to overall suppression of endothelial-surface ATP metabolism (Kenan and Wahl, 2005; Moser et al., 1999, 2001). The field would benefit from a more systematic and complete determination of enzymatic activities in response to ligand binding in the different cell systems. Even for a specific ligand there have been notable differences in determination of levels of enzymatic activity. For example, extracellular ATP synthesis on HUVECs was examined in detail by Arakaki et al. (2003), who measured a high ATP synthesis activity (50 nmol/min/106 cells) compared with that found by Moser et al. (2001) (about 40 pmol/min/106 cells). The reason for this significant difference remains unclear (Arakaki et al., 2003). The best-studied system is endothelial cell regulation by angiostatin (Chi and Pizzo, 2006a; Kenan and Wahl, 2005). As stated by Chi and Pizzo (2006a,b) the current model is that endothelial cells utilize eAS to generate extracellular ATP, and concomitantly for proton extrusion, as a means of regulating intracellular pH. They envisage such a scenario to be especially applicable to the tumor environment where the extracellular pH might be as low as, or even lower than, pH 6.7 and angiostatin is generated in vivo. These authors also present the evidence for angiostatin’s role as an antitumourigenic agent through a mechanism implicating eAS.

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4.5. The inhibitor action of IF1 can be demonstrated for eAS complexes The activity of IF1 on eAS activity has been assessed in two of the systems listed above. First Martinez et al. (2003) showed that exogeous IF1 inhibited ATP hydrolysis activity of eAS and HDL endocytosis. Burwick et al. (2005) assessed the effect of recombinant human IF1 on HUVEC eAS to determine whether it could serve as an angiostatin mimetic. Exogenous IF1 was found to inhibit ATP hydrolysis but not ATP synthesis, in contrast to angiostatin, which inhibited both. This finding agrees with the known function of IF1 in mitochondria (see Section 2.3.3). Thus IF1 was proposed to serve a protective function in the tumor microenvironment through the conservation of cellular ATP when pH is low (Burwick et al., 2005). Furthermore, it was demonstrated that angiostatin blocks IF1 binding to eAS, suggesting a relationship between the binding sites of IF1 and angiostatin on the complex. Such an observation is compatible both with angiostatin binding to subunit b and the IF1 binding site being largely to subunit b (Cabezon et al., 2000a,b). That angiostatin exerts its antiangiogenic effect, at least in part, by inhibiting IF1 binding to eAS has been proposed by Burwick et al. (2005) to explain why angiostatin has a stronger antiangiogenic effect at low pH than at physiological pH. Under the latter pH condition, conservation of ATP by IF1 inhibition of ATP hydrolysis would not be required, whereas in the low pH, low oxygen microenvironment of tumors, cells would have a greater need to conserve ATP through IF1 action (Burwick et al., 2005). These results raise the obvious question as to whether there is any evidence for IF1 on the cell surface. Burwick et al. (2005) demonstrated that endogenous IF1 is present on HUVEC surface, a finding confirmed by Cortes-Hernandez et al. (2005). The latter authors also showed that TNF-a decreased the level of subunit b and increased the amount of IF1. Further, because their studies showed that the ratio of IF1 to subunit b exhibited significant variation, they suggested that the function of IF1 in the PM of endothelial cells may not be limited to regulation of catalysis (in which case the ratio should be maintained at 1:1), although no suggestions of additional functions were made.

5. Concluding Remarks The recent structure of the peripheral stalk subunits reported by Walker and colleagues (Dickson et al., 2006) is a significant advance toward the ultimate goal of having a complete structure of the mtATPase complex. We still lack a high-resolution structure for subunits a and c assembled together, which potentially would enable us to fully dissect proton channel

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function. Furthermore, in view of our continually developing understanding of the dimeric and oligomeric forms of the complex in the inner mitochondrial membrane a high-resolution structure that includes the additional, nonproton channel, F0 membrane subunits and ‘‘dimer specific subunits’’ would be especially informative. Based on the present models, higher order supercomplexes of mtATPase may exist in the membrane. However, it may be difficult to study these in detail due to the difficulty in achieving their solubilization without disruption. It will be important to apply other techniques to investigate such supercomplexes. It may be easier and more informative to study these structures in vivo using fluorescence techniques as exemplified by the approaches utilised by Gavin et al. (2003, 2005). Such approaches also have the potential to provide spatial information and, more importantly, data on the assembly and possible dynamic nature of the supramolecular interactions. There is a weight of evidence supporting functional roles for eAS and cell surface CF6. However, there remain several questions related to biogenesis of the complexes on the cell surface that need to be addressed. It is noteworthy that ATP synthesis on the external surface of vascular endothelial cells attributed to eAS has been challenged. Yegutkin et al. (2001) presented evidence that ATP synthesis on HUVECs resulted from the activity of adenylate kinase and nucleoside diphosphokinase rather than eAS. More recently, Quillen et al. (2006) showed that a small amount of adenylate kinase activity alone could account for the observed extracellular ATP synthesis of HUVECs.

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Soltys, B. J., and Gupta, R. S. (1999). Mitochondrial-matrix proteins at unexpected locations: Are they exported? Trends Biochem. Sci. 24, 174–177. Soltys, B. J., and Gupta, R. S. (2000). Mitochondrial proteins at unexpected cellular locations: Export of proteins from mitochondria from an evolutionary perspective. Int. Rev. Cytol. 194, 133–196. Soubannier, V., Rusconi, F., Vaillier, J., Arselin, G., Chaignepain, S., Graves, P. V., Schmitter, J. M., Zhang, J. L., Mueller, D., and Velours, J. (1999). The second stalk of the yeast ATP synthase complex: Identification of subunits showing cross-links with known positions of subunit 4 (subunit b). Biochemistry 38, 15017–15024. Soubannier, V., Vaillier, J., Paumard, P., Coulary, B., Schaeffer, J., and Velours, J. (2002). In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize. J. Biol. Chem. 277, 10739–10745. Spannagel, C., Vaillier, J., Arselin, G., Graves, P. V., Grandier-Vazeille, X., and Velours, J. (1998a). Evidence of a subunit 4 (subunit b) dimer in favor of the proximity of ATP synthase complexes in yeast inner mitochondrial membrane. Biochim. Biophys. Acta 1414, 260–264. Spannagel, C., Vaillier, J., Arselin, G., Graves, P. V., and Velours, J. (1997). The subunit f of mitochondrial yeast ATP synthase—Characterization of the protein and disruption of the structural gene ATP17. Eur. J. Biochem. 247, 1111–1117. Spannagel, C., Vaillier, J., Chaignepain, S., and Velours, J. (1998b). Topography of the yeast ATP synthase F0 sector by using cysteine substitution mutants. Cross-linkings between subunits 4, 6, and f. Biochemistry 37, 615–621. Stahlberg, H., Muller, D. J., Suda, K., Fotiadis, D., Engel, A., Meier, T., Matthey, U., and Dimroth, P. (2001). Bacterial Na(þ)-ATP synthase has an undecameric rotor. EMBO Rep. 2, 229–233. Stephens, A. N., Khan, M. A., Roucou, X., Nagley, P., and Devenish, R. J. (2003a). The molecular neighborhood of subunit 8 of yeast mitochondrial F1F0-ATP synthase probed by cysteine scanning mutagenesis and chemical modification. J. Biol. Chem. 278, 17867–17875. Stephens, A. N., Nagley, P., and Devenish, R. J. (2003b). Each yeast mitochondrial F1F0-ATP synthase complex contains a single copy of subunit 8. Biochim. Biophys. Acta 1607, 181–189. Stephens, A. N., Roucou, X., Artika, I.M, Devenish, R. J., and Nagley, P. (2000). Topology and proximity relationships of yeast mitochondrial ATP synthase subunit 8 determined by unique introduced cysteine residues. Eur. J. Biochem. 267, 6443–6451. Stock, D., Leslie, A. G., and Walker, J. E. (1999). Molecular architecture of the rotary motor in ATP synthase. Science 286, 1700–1705. Stutterheim, E., Henneke, M. A., and Berden, J. A. (1981). Subunit composition of mitochondrial F1-ATPase isolated from Saccharomyces carlsbergensis. Biochim. Biophys. Acta 634, 271–278. Suzuki, T., Murakami, T., Iino, R., Suzuki, J., Ono, S., Shirakihara, Y., and Yoshida, M. (2003). F0F1-ATPase/synthase is geared to the synthesis mode by conformational rearrangement of epsilon subunit in response to proton motive force and ADP/ATP balance. J. Biol. Chem. 278, 46840–46846. Swartz, D. A., Park, E. I., Visek, W. J., and Kaput, J. (1996). The e subunit gene of murine F1F0-ATP synthase. Genomic sequence, chromosomal mapping, and diet regulation. J. Biol. Chem. 271, 20942–20948. Takeda, M., Vassarotti, A., and Douglas, M. G. (1985). Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor. J. Biol. Chem. 260, 15458–15465. Takeda, M., Chen, W. J., Saltzgaber, J., and Douglas, M. G. (1986). Nuclear genes encoding the yeast mitochondrial ATPase complex. Analysis of ATP1 coding the F1-ATPase alpha-subunit and its assembly. J. Biol. Chem. 261, 15126–15133.

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C H A P T E R

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Role of Proteasomes in Cellular Regulation Irina M. Konstantinova,* Anna S. Tsimokha,* and Alexey G. Mittenberg* Contents 1. Introduction 2. Proteasome Structure and Catalytic Activities 2.1. The core particle, 19S regulatory particle, alternative regulatory particles 2.2. Catalytic activities of proteasomes 3. Function of Proteasomes in Cell Proliferation, Differentiation, Apoptosis and Immune Response 3.1. Cell cycle control 3.2. Roles in differentiation and development 3.3. Proteasomes and apoptosis 3.4. Proteasomes and immune response 4. Modes of Regulation of Proteasome Activities in the Cell 4.1. Modulation of proteasome composition 4.2. Regulation of proteasome abundance in the cell and cellular compartments 5. Reprogramming of Proteasomes at Immune Response, Differentiation and Apoptosis 5.1. Changes of proteasome at immune response 5.2. Proteasome reprogramming at differentiation 5.3. Apoptosis-induced changes of proteasomes 6. Proteasomes in Regulation of Different Levels of Gene Expression 6.1. Action at multiple stages of transcription process 6.2. Participation in the regulation of posttranscriptional stages of gene expression 7. Concluding Remarks Acknowledgments References

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Institute of Cytology, Russian Academy of Sciences, St. Petersburg, 194064, Russia

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00602-3

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2008 Elsevier Inc. All rights reserved.

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Abstract The 26S proteasome is the key enzyme of the ubiquitin-dependent pathway of protein degradation. This energy-dependent nanomachine is composed of a 20S catalytic core and associated regulatory complexes. The eukaryotic 20S proteasomes demonstrate besides several kinds of peptidase activities, the endoribonuclease, protein-chaperone and DNA-helicase activities. Ubiquitinproteasome pathway controls the levels of the key regulatory proteins in the cell and thus is essential for life and is involved in regulation of crucial cellular processes. Proteasome population in the cell is structurally and functionally heterogeneous. These complexes are subjected to tightly organized regulation, particularly, to a variety of posttranslational modifications. In this review we will summarize the current state of knowledge regarding proteasome participation in the control of cell cycle, apoptosis, differentiation, modulation of immune responses, reprogramming of these particles during these processes, their heterogeneity and involvement in the main levels of gene expression. Key Words: Proteasome, Cell cycle, Apoptosis, Differentiation, Immune response, Different levels of gene expression. ß 2008 Elsevier Inc.

1. Introduction Degradation of cellular proteins is a tightly regulated process, carried out by the cascade of the proteasome-ubiquitin pathway. The 26S proteasome, an intensively studied protease, represents the sophisticated complex of subunits and is critical for life. This complex is implicated in the temporally controlled ATP-dependent degradation of key regulatory proteins controlling main cellular processes (cell cycle, apoptosis, differentiation, development, immune response, malignant transformation), as well as in regulation of different stages of gene expression (Collins and Tensey, 2006; Ferdous et al., 2007; Glickman and Ciechanover, 2002; Kloetzel, 2004; MaupinFurlow et al., 2006; Pajonk and McBride, 2001; Reed, 2006; Sikder et al., 2006; Wojcik et al., 2000). The participation of the proteasome-ubiquitin pathway in the control of degradation of regulatory proteins involves not only the regulation of the complicated enzyme system of conjugation of the ubiquitin molecules to the substrates but also the highly complex control of the composition and the activities of the proteasome particle itself. These particles are heterogeneous due to subtypes synthesized from duplicated genes and/or alternative splicing, to posttranslational modifications and to association of 20S core with alternative regulatory complexes (Glickman and Raveh, 2005; Maupin-Furlow et al., 2006). Here we will describe recent studies revealing the current knowledge about proteasomes involvement in the

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fundamental biological processes such as control of cell cycle, apoptosis, differentiation, modulation of immune responses, about their isoform complexity including subunits’ posttranslational modifications, structural and functional responsiveness to cell requirements and participation of these particles in the main stages of gene expression.

2. Proteasome Structure and Catalytic Activities 2.1. The core particle, 19S regulatory particle, alternative regulatory particles The 26S proteasome, further often named ‘‘the proteasome, ’’ is an ATPdependent multicatalytic enzyme complex found in the nucleus and cytoplasm not only of all eukaryotic cells, but also of Archebacteria and in some Eubacteria. Proteasomes are responsible for the degradation of most cellular proteins (Coux et al., 1994). The 26S proteasomes constitutes the central proteolytic machinery of the ubiquitin/proteasome system, and is composed of a core catalytic complex, called 20S proteasome, capped at both ends by a 19S regulatory complex (Baumeister et al., 1998; Dahlmann, 2005). 2.2.1. 20S core The 20S proteasome is a large, cylinder-shaped protease with the molecular weight of
700 kDa. This complex is formed by 28 subunits, which are arranged in four heptameric stacked rings in an a7b7b7a7 configuration (Fig. 2.1). The two outer rings are made up of a-type subunits whereas the inner two rings are composed of b-type subunits (Grziwa et al., 1991). In Archebacteria the a- and b-rings are composed of seven identical a and b subunits, respectively. The various a and b subunits of eukaryotic proteasome were shown to be homologous to archeabacterial ones (Heinemeyer et al., 2004). Three different b subunits have free N-terminal threonine (Thr) residues, which form the proteolytically active sites and, thus, are responsible for the proteolytic activity of proteasome (Arendt and Hochstrasser, 1997). The proteolytic active sites formed by the N-termini of b subunits make the innermost chamber which is flanked by the two outermost chambers (Lowe et al., 1995). Hence, there are two narrow (
1.3 nm) and gated pores at each end of the cylindrical complex (Groll et al., 2000). This structure and the different regulatory complexes protect the cell from unregulated protein degradation by the active sites of 20S proteasomes. Mammals have three additional proteasome subunits called LMP2, LMP7 and MECL-1 (also named b1i, b5i and b2i, respectively) (see Section 3.4).

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Ub Recognition Substrate

Deubiquitination a

Unfolding Translocation Cleavage

b2 b1 b1 a1

a1 b2

b b5 a5

a

ATPases

Base Non-ATPases Lid

Figure 2.1 Structure and function of the 26S proteasome.The 20S proteasome is composed of a stack of four rings composed of seven subunits each. The two outer rings are made up of seven different a-subunits (marked by blue, right caption), whereas the two central rings are composed of seven different b-subunits (marked by pink, right caption). The proteolytic active sites of the 20S proteasome are located in the central rings at subunits b1, b2 and b5 (marked by dark pink) and the endoribonuclease active sites of the 20S proteasome are located in the outer rings at subunits a1 and a5 (marked by light blue). 19S regulatory complexes are subdivided into two distinct base and lid subcomplexes (right caption) and composed of 11^12 non-ATPase (marked by yellow, right caption) and six AAA-type ATPase subunits (marked by green, right caption).The substrate and ubiquitin are marked by a black thread and violet circles respectively (right caption).The functions of the 26S proteasome necessary for proteolysis of ubiquitylated substrates are shown on the left. 19S regulatory complex of the 26S proteasome recognizes usually substrates via their polyubiquitin chain.The substrate is then unfolded via the ATPase ring. The unfolded polypeptide chain is translocated to the proteolytic chamber of the 20S proteasome where is subjected to the cleavage. The polyubiquitin chain is cleaved off by deubiquitinating enzymes of 19S regulator during the process of translocation.

They are induced by interferon-gamma (IFNg) and carry the catalytic N-terminal Thr residues. Under the action of IFNg, these subunits are incorporated into the newly assembled proteasomes instead of three constitutive catalytic subunits [designated traditionally X (b1), Y (b5) and Z (b2)] (Fruh et al., 1994). Proteasomes with IFNg-inducible subunits are termed immunoproteasomes and take part in immune response (Kloetzel et al., 1999). 2.1.2. Multiple roles for the 19S regulatory particle The 19S proteasome regulatory complex, or the PA700 activator, is composed of at least 17 protein subunits and subdivided into two distinct subcomplexes, called base and lid (DeMartino et al., 1996; Glickman et al., 1998). The lid subcomplex is formed by at least nine non-ATPase subunits (designated in yeast Rpn3, Rpn5–Rpn9, Rpn10, Rpn11 and Rpn12) (Glickman et al., 1998) and the base subcomplex contains six AAA-type

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ATPases (denoted Rpt1–Rpt6) and at least two non-ATPase components (designated Rpn1 and Rpn2) (Leggett et al., 2002). The base subcomplex has protein-chaperone activity (Lam et al., 2002) and is important for the regulatory activity of 26S proteasome (Lupas and Martin, 2002). The functions of the 19S regulatory complex consist in the recognition of the polyubiquitinated substrate, liberation of the polyubiquitin chain, unfolding of the substrate, opening of the central proteasome channel and translocation of the unfolded polypeptide chain toward the catalytic sites (Glickman et al., 1998; Wolf and Hilt, 2004). 2.1.3. Alternative regulatory particles Other regulatory complexes also associate with the 20S proteasome. One of them is the PA28 activator, or the 11S proteasome regulatory complex. It is a ring-shaped hexamer formed by two closely related subunits PA28a and PA28b, which are induced by IFNg (Dubiel et al., 1992). PA28 activator binds 20S proteasome and is implicated in the processing of MHC class I antigens (Rechsteiner et al., 2000). Subunits of the PA28g activator are different from PA28a and PA28b and PA28g is down-regulated by INFg (Tanahashi et al., 1997). Hence, the PA28g is not a participant of immune response but, possibly, plays a role in the cell division and carcinogenesis (Gao et al., 2004; Tanahashi et al., 1997). In a protozoan pathogen, Trypanosoma brucei, proteasomes an additional activator protein, PA26, has been identified (To and Wang, 1997). The protein sequence of PA26 is similar to sequence of mammalian activator proteins PA28a, PA28b or PA28g (Yao et al., 1999). The single-chain protein, called PA200 is present in the nuclei of mammalian cells. The association of PA200 activator with 20S core may facilitate release of digestion products or the entrance of substrates and it is thought to play a role in DNA repair (Ustrell et al., 2002, 2005).

2.2. Catalytic activities of proteasomes 2.2.1. Peptidase activity The proteasome belongs to the N-terminal nucleophile hydrolases, where the N-terminal Thr residue functions as the nucleophile (Orlowski and Wilk, 2000). The eukaryotic 20S proteasomes demonstrate at least five types of peptidase activity (Orlowski et al., 1993). The three classical catalytic activities of the proteasome are designated chymotrypsin-like, trypsin-like, and peptidyl-glutamyl-peptide hydrolyzing (i.e., caspase-like) (Adams, 2003b; Coux et al., 1996; Orlowski and Wilk, 2000). Moreover, the proteasomes are able to cleave bonds on the carboxyl side of branched chain amino acids and between the small neutral amino acids (Orlowski et al., 1993). The more detailed description of proteasomal peptidase

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activities is presented in the comprehensive reviews of Orlowski and Wilk (2000) and Groll et al. (2005). 2.2.2. RNase activity Despite high progress in the study of proteasomes, some questions still remain opened, and one of them is the RNase activity of these particles, mechanisms of its regulation and its possible physiological significance. A new view on the proteasome’s role in the regulation of gene expression is suggested by the recent observation that the proteasome harbors an endoribonuclease activity (Ballut et al., 2003; Evteeva et al., 2000; Evteeva et al., 2003; Jarrousse et al., 1999; Mittenberg et al., 2002; Petit et al., 1997a,b; Toktarova et al., 2004; Tsimokha et al., 2006, 2007a,b,c). RNase activity of 20S as well as 26S proteasomes has been studied, and the subunits possessing this activity (a-type subunits: a5/zeta and a1/iota) were identified. In contrast to 20S particles, 26S proteasomes can degrade cellular native messenger poly (A)-containing RNA molecules (Ballut et al., 2003; Evteeva et al., 2000). In addition, the endoribonuclease activity of 26S particles sharply differs from that of 20S proteasomes in the dependence on divalent cations: 20S proteasomes are active only in the presence of divalent cations, while 26S particles are active also in the absence of these cations as well (Evteeva et al., 2000; Mittenberg et al., 2002a,b). The different properties of the RNase activities of 20S and 26S particles could be due to the effects of the regulatory (19S) complexes on the activity. However, the RNase activity of proteasomes does not imply their involvement in mRNA stability regulation, and changes of this activity under the action of stimuli, which regulate the half time of mRNA molecules, favor this suggestion more strongly. Indeed, regulation of the 26S proteasome RNase activity has been demonstrated under several stimuli (Evteeva et al., 2000; 2003; Kulichkova et al., 2004a,b; Mittenberg et al., 2002a,b; Toktarova et al., 2004; Tsimokha et al., 2006; 2007a,b,c). In these works the above activity has been shown to be changed under the action of such agents as: the apoptosis inductors of proerythroleukemic K562 cells (doxorubicin and diethylmaleate), differentiating agent of K562 cells hemin and epidermal growth factor (EGF) in A431 cells. The RNase activity of 26S proteasomes is phosphorylation-dependent and specifically regulated by Ca and Mg ions. Phosphorylation of which of the subunits is essential for the activity as well as their phosphorylation status are under study at present. The studied agents specifically regulate phosphorylation of certain proteasomal subunits (Mittenberg et al., 2007; Tsimokha et al., 2006, 2007a,b,c). To assess the effect of the inductors on the RNase activity of 26S proteasomes, total cytoplasmic high-molecular-weight RNA (hmwRNA) was incubated with 26S proteasomes and the products of nucleolysis were separated by PAGE. Treatment of K562 cells with apoptosis and

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differentiation inductors decreased the proteasomal RNase activity toward hmw ribosomal RNAs. However, further data (Mittenberg et al., 2007; Tsimokha et al., 2007a,b,c) argue that different RNase centers of proteasomes are differentially regulated. To study the possible inductor’s effect on the specificity of the proteasomal RNase activity, the nucleolysis of individual messenger RNAs (mRNA) was checked. The obtained results have shown that the specificity of 26S proteasomes’ endoribonuclease activity is changed under the action of erythroid differentiation (hemin) and programmed cell death (diethylmaleate) inductors in K562 cells. Thus, treatment of K562 cells with apoptosis and differentiation inductors lead to the specific stimulation of RNase activity toward certain mRNAs (hemin: p53 and c-myc mRNAs), to unchanged activity toward other mRNA (DEM: c-myc mRNA) and to reduction of proteasome degradation of other specific mRNA (hemin and DEM: c-fos mRNA). Treatment of proteasomes with calf intestinal phosphatase revealed that the RNase activity specifically and selectively depends on phosphorylation of 26S proteasome subunits. Thus, the specificity of the proteasomes’ RNase activity is differentially and specifically regulated during differentiation and apoptosis.

3. Function of Proteasomes in Cell Proliferation, Differentiation, Apoptosis and Immune Response 3.1. Cell cycle control The cell cycle in eukaryotes is regulated by consecutive activation of cyclindependent kinases (CDKs) by various cyclins. Cyclins are synthesized during strongly defined moments of a cell cycle and, being the extremely unstable, exist and work only at the certain phases of the cell cycle and during the certain period of time. For example, cyclins D and E are active during phase G1; cyclins E and A, during phase S. Consecutive appearance and disappearance of the pairs cyclin-CDK at various stages of the cell cycle is defined by kinetics of cyclins synthesis and degradation. Therefore, the basic molecular mechanism of cell cycle regulation is periodic synthesis and destruction of major proteins during the cell cycle ( Johnson and Walker, 1999). Proteasome inhibitors were shown to block the cell cycle in the phases G1 (Kumeda et al., 1999; Rao et al., 1999), late S (Machiels et al., 1997) or G2/M (Wojcik et al., 1996). Ubiquitin- and proteasome-dependent proteolysis is one of the key mechanisms underlying cell cycle control. Thus, cyclins are degraded by this pathway. Furthermore, proteasomes are involved in the process of the regulation of CDK inhibitors stability (p27Kip1 and

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p21Cip1/WAF1) and of specific phosphatases of the family CDC25 activating the kinases CDK in mammals (Naujokat and Hoffmann, 2002; Reed, 2006). The ubiquitin-proteasome system realizes negative as well as positive control of cell cycle (Naujokat and Hoffmann, 2002). The following negative regulators of cell cycle progression, p21Cip1/WAF1, p27Kip1, p19INK4d, and Geminin (Table 2.1), are known to be substrates for the proteasomal proteolytic machine (Naujokat and Hoffmann, 2002). The proteasomal degradation of these proteins leads to ‘‘the release from brake’’ (Naujokat and Hoffmann, 2002) and the prolongation of cell cycle progression. Entrance into S-phase is determined by positive regulators of cell cycle progression, and both the timely proper S-phase beginning and the corresponding frequencies of entry into S-phase are supposed to be tightly controlled by proteasomal degradation of the above regulators (Naujokat and Hoffmann, 2002). The proteolysis during cell cycle can be characterized by two families of protein-ubiquitin ligases: APC/C and SCF (Reed, 2006). The anaphase promoting complex or cyclosome (APC/C) was initially described as a multisubunit protein complex (containing the ubiquitin-ligase E3) that ubiquitinates anaphase inhibitors thus targeting them for destruction by proteasomes (Clarke et al., 2005). Activation of this complex also occurs during mitosis and G1 phase. In this case, too, APC/C ubiquitinates the proteins inhibiting the mitotic progression (Reed, 2006). Thus, APC/C participates in ubiquitin-depending proteasomal degradation of the anaphase inhibitors during the transition from the metaphase to the anaphase and of the mitotic cyclins during the transition from mitosis (see review by Abramova et al., 2002). SCF (Skp1/Culin/F-box protein) protein-ubiquitin ligase is also a core component of the cell cycle machinery. Moreover, APC/C and SCF do not function in isolation from each other, and they are adjusted to coordinate the cell cycle events (Reed, 2006). SCF ligase ubiquitylates a large number of proteins involved in cell cycle control that are marked by phosphorylation at specific sequences known as phosphodegrons (see review by Reed, 2006). Therefore, the targeting of these proteins for destruction by phosphorylation provides the participation of signaling pathways via activation of specific kinases in the control of cell cycle. The comprehensive review has been published recently and the reader is referred to it for the full information on this subject (Reed, 2006).

3.2. Roles in differentiation and development Development and cellular differentiation as the other cellular processes are controlled by both gene expression and protein degradation. Hence, these processes are regulated, at least in part, by ubiquitin-proteasome pathway. For example, early studies in insects, including Drosophila and Manduca sexta, have demonstrated that early embryogenesis and metamorphosis depend

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Table 2.1 Cell cycle targets for ubiquitin-dependent proteasomal degradation

Organism

Substrate

Cell cycle targets of APC S. cerevisiae Securin Clb2 Clb5 Cdc5/Plk, Cdc20, Ase1, Hsl1

Dbf4 Cin8/Kip1 Metazoa

Cyclin B Cyclin A Geminin

Vertebrates Aurora A Nek2A Cdc6 Xkid Cdc25A Skp2 Cell cycle targets of SCF S. cerevisiae Sic1/Rum1, Far1 Cdc6/Cdc18 Cln1,2,3

Gic1,2 Swe1 Met4 Metazoa

Cyclin E

Cell cycle function

Anaphase inhibitor B-type cyclin (mitosis) B-type cyclin (S phase) Mitosis

Replication Mitotic spindle motor Mitosis S phase, mitosis Replication licensing Mitosis Centrosome development Replication Mitotic spindle motor S phase, mitosis SCF cofactor G1–S transition inhibitor DNA replication G1 cyclin

Budding Mitosis inhibitor G1-S transition inhibitor G1-S cyclin

References

Zur and Brandeis, 2001 Wasch and Cross, 2002 Shirayama et al., 1999 Cheng et al., 1998; Visintin et al., 1997; Juang et al., 1997; Martinez et al., 2006 Ferreira et al., 2000 Hildebrandt and Hoyt, 2001 Yamano et al., 2004 Geley et al., 2001 McGarry and Kirschner, 1998 Castro et al., 2002 Hames et al., 2001 Petersen et al., 2000 Castro et al., 2003 Donzelli et al., 2002 Wei et al., 2004 Feldman et al., 1997; Blondel et al., 2000 Perkins et al., 2001 King et al., 1996; Schweitzer et al., 2005 Reed, 2006 Kaiser et al., 1998 Patton et al., 2000 Ye et al., 2004 (continued)

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Table 2.1 (continued) Organism

Substrate

Vertebrates Wee1

Mammals

Cell cycle function

Mitosis inhibitor

Emi1

APC/C inhibitor

p27Kip1, p21Cip1, p130

G1-S transition inhibitor

Ctd1, Orc1 Cdc25A c-Myc

DNA replication S phase, mitosis S phase

References

Watanabe et al., 2004 Margottin-Goguet et al., 2003 Baldassarre et al., 2000; Bornstein et al., 2003; Tedesco et al., 2002 Reed, 2006 Jin et al., 2003 Gregory and Hann, 2000

on proteasome accumulation and proteasomal degradation of certain target proteins (Dawson et al., 1995; Jones et al., 1995; Klein et al., 1990; Low et al., 1997). Subsequent studies in the other organisms, such as sea urchin (Lytechinus pictus), frog (Xenopus laevis), mouse, and rat, have indicated that developmentally regulated expression of distinct subunits of the proteasomal 19S regulatory complex, as well as proteasomal degradation of cell cycle regulatory proteins, is necessary for the initiation of early embryonal mitosis and development ( Josefsberg et al., 2001; Kawahara et al., 2000a,b; Tokumoto et al., 1999a). Further, recent studies in vertebrates showed that the molecular assembly and the proteolytic activity of 26S proteasome undergo changes during the completion of meiosis (oocyte maturation) ( Josefsberg et al., 2000; Reverte et al., 2001; Sawada et al., 1999; Tokumoto et al., 1999a, 2000). The functions of the proteasome in cellular differentiation are very complex. The comprehensive reviews with more full information on this subject are accessible (Bowerman and Kurz, 2006; Naujokat and Hoffmann, 2002; Schwechheimer and Schwager, 2004).

3.3. Proteasomes and apoptosis Protease inhibitors inactivate enzymes reversibly or irreversibly by binding to the catalytic site of the enzyme. Proteasome inhibitors (Table 2.2) are attractive drug targets due to the importance of the ubiquitin-proteasome system in numerous biological processes including the apoptosis (Adams, 2003a; Ciechanover and Schwartz, 2004; Groll and Huber, 2004; Nandi et al., 2006; Rajkumar et al., 2005).

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Table 2.2 Different proteasome inhibitors Proteasome inhibitors

Chemical Peptide aldehydes inhibitors Peptide boronates

Peptide vinyl sulfones

Protein inhibitors

Examples

References

MG132, PSI (Z-IE (OtBu)AL-al), CEP1612 MG262 (Z-LLL-bor), PS341 (bortezomid), PS273 (MNLB) NLVS (Nip-LLL-vs), YLVS (YLLL-vs)

Lee and Goldberg, 1998 Adams et al., 1998; 1999

Peptide epoxyketones

Dihydroeponemycin, epoximicin, YU101 (Ac-hFLFL-ex)

Lactacystin and derivatives

Lactacystin, clastolactacystin-blactone, L-Lactone (omuralide) PI31

PR39 d- aminolevulinic acid dehydratase HIV encoded Tat Hepatitis B virus encoded X protein Protein aggregates

Lee and Goldberg, 1998; Adams et al., 2000 Kim et al., 1999; Meng et al., 1999; Glenn et al., 2004 Fenteany and Schreiber, 1998 McCutchenMaloney et al., 2000 Gao et al., 2000 Guo et al., 1994 Apcher et al., 2003 Hu et al., 1999 Grune et al., 2004

The materials of Nandi et al., 2006, were used for preparation of this table.

Apoptosis is regulated by two proteolytic systems: by the caspases, a family of specific cysteine proteases, and by the proteasomes. Interestingly, the proteasomes degrade specific proteins-regulators of apoptosis, but on the other hand some components of the proteasome system are degraded by caspases (Adrain et al., 2004). The proteasomal function in apoptosis appears

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to be very complex. Whereas proteasome inhibitors induce apoptosis in multiple cell types, while in other cells they are innocuous or even prevent apoptosis induced by other agents (An et al., 1998; Wojcik, 2002). The use of proteasome inhibitors has demonstrated that degradation or processing of proteins by the ubiquitin-proteasome system has a determinative influence on cell survival or cell death, depending on the cell type and/or the proliferative state of the cells (Drexler, 1998; Naujokat and Hoffmann, 2002). The proteasomes realize programmed proteolysis and processing of various groups of apoptosis-regulatory proteins possessing the pro- and anti-apoptotic functions in a cell. These proteins include transcription factors (c-Fos, c-Myc, NF-kB, AP-1), tumorous suppressor 53, IkBa (inhibitor of nuclear transcription factor NF-kB), regulators of cell cycle (p27Kip1, p21Cip1/WAF1), proteins of Bcl-2 family and regulators of caspase activity (inhibitors of apoptosis proteins [IAPs]) (Wojcik, 2002). Studies using the selective proteasome inhibitors have provided direct evidence for the proteasome functions both in promoting apoptosis and in protecting cells against apoptosis (Grimm et al., 1996; Lopes et al., 1997). These opposite roles of 26S proteasomes in regulation of apoptosis seem to depend on the proliferative state of the cell (Chen and Lin, 2004; Naujokat and Hoffmann, 2002; Wojcik and DeMartino, 2003). 3.3.1. Pro-apoptotic function of the proteasomes In some cell systems, proteasome inhibitors prevent apoptosis, indicating pro-apoptotic function of proteasomes (Grimm et al., 1996; Sadoul et al., 1996). The requirement of proteasomal activity for the progression of apoptosis has been shown in two nonproliferating mammalian cell types, namely in resting thymocytes and differentiated neurons (Grimm et al., 1996; Sadoul et al., 1996). Thus, the mouse thymocytes induced to undergo apoptosis by treatment with phorbol-12-myristate 13-acetate, dexamethasone or g-radiation were rescued from apoptosis when treated with proteasome inhibitors up to 1 h after the apoptotic stimulus (Grimm et al., 1996). It is important, that later addition (3-5 h) of proteasome inhibitors failed to rescue the cells from apoptosis, suggesting that proteasomal activity promotes apoptosis only at upstream points of apoptotic signal transduction pathways (Naujokat and Hoffmann, 2002). Similar findings were obtained in studies with differentiated neurons (Canu et al., 2000; Sadoul et al., 1996). Sympathetic neurons from rat superior cervical ganglia (Sadoul et al., 1996) and rat cerebellar neurons (Canu et al., 2000) induced to undergo apoptosis in response to deprivation of nerve growth factor and potassium, respectively, were rescued from apoptosis when treated with proteasome inhibitors early after the initiation of the apoptotic process. In some situations, proteasome inhibitors may have opposing effects even in the same cells, where they may either enhance or prevent apoptosis

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(Pleban et al., 2001; Sohn et al., 2006a; Wojcik, 2002; Yang et al., 2006). For example, pretreatment of non-small cell lung carcinoma cells in culture with proteasome inhibitors prevents the pro-apoptotic action of topoisomerase inhibitors, while when proteasome inhibitors are administered after the treatment with topoisomerase inhibitors, they enhance their pro-apoptotic effect (Tabata et al., 2001). Proteasome inhibitor PSI blocks T cell receptorinduced apoptosis in a T-cell hybridoma (Tanimoto and Kizaki, 2002). Proteasome activity is involved at an early step of glucocorticoidinduced apoptosis, preceding mitochondrial changes and caspase activation (Hirsch et al., 1998; Wallace and Cidlowski, 2001). There are many indications of the apoptosis induced by different proteasome inhibitors in various tumor cells (Adams, 2002; Nandi et al., 2006; Wojcik, 2002). However, the proteasome inhibitor MG132 rescued tumor cells (two HeLa cell lines: D98 and H21) from death receptor-induced apoptosis (Sohn et al., 2006a). One possible mechanism of proteasomal pro-apoptotic function has been uncovered recently in primary mouse thymocytes in the study of XIAP and c-IAP1, members of the family of inhibitors of apoptosis proteins IAPs (Duckett et al., 1996). These inhibitory proteins realize the anti-apoptotic activity, at least in part, by inhibiting the activation and enzymatic activity of caspases (Deveraux et al., 1997, 1998), and by ubiquitination and targeting of caspase 3 for proteasomal degradation (Suzuki et al., 2001). In response to various apoptotic stimuli, these inhibitory proteins are autoubiquitinated and subsequently degraded by the proteasomes (Yang et al., 2000). The proteasomal degradation of XIAP and c-IAP1 depends on an intact RING finger domain and appears to be highly operative in transducing apoptosis, because cells expressing XIAP and c-IAP1 with mutant RING finger domains display a lack of proteasomal degradation of the mutant proteins and fail to undergo apoptosis induced by several stimuli (Yang et al., 2000). Another possible mechanism of proteasomal pro-apoptotic function has been demonstrated in human umbilical vein endothelial cells (HUVECs) undergoing TNF-a-induced apoptosis (Naujokat and Hoffmann, 2002). Early after the initiation of TNF-a treatment of HUVECs, anti-apoptotic protein Bcl-2 is specifically degraded by the proteasome (Breitschopf et al., 2000a; Dimmeler et al., 1999). This event was indicated to be effective in inducing apoptosis, because pretreatment of HUVECs with specific proteasome inhibitors aborted both TNF-a–induced Bcl-2 degradation and induction of apoptosis (Breitschopf et al., 2000a; Dimmeler et al., 1999). 3.3.2. Anti-apoptotic functions of the proteasomes The proteasomes are abnormally highly expressed in rapidly growing metazoan embryonic and human neoplastic cells in contrast to differentiated and normally growing cells (Ichihara et al., 1993; Kanayama et al., 1991; Klein et al., 1990; Kumatori et al., 1990; Shimbara et al., 1992). Hence, in all

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probability, the proteasomes play a central role in maintaining survival and proliferation of rapidly and somehow abnormally growing cells (Ichihara and Tanaka, 1995). The effects of proteasome inhibitors on neoplastic and rapidly growing cells confirmed this hypothesis (Naujokat and Hoffmann, 2002). First indication of an anti-apoptotic function of the proteasomes came from studies focusing on the apoptosis induced by a proteasomal inhibitor lactacystin in human leukemia U937 cells in 1995 (Imajoh-Ohmi et al., 1995). After that, there were many other studies using different sets of proteasome inhibitors and several different cell lines: proteasome inhibitors have been found to induce apoptosis in neoplastic and rapidly growing mammalian cells of hematopoietic (Drexler, 1997; Naujokat et al., 2000; Shinohara et al., 1996; Tanimoto et al., 1997), neuronal (Kitagawa et al., 1999; Lopes et al., 1997; Qiu et al., 2000), mesenchymal (Drexler et al., 2000; Lopes et al., 1997) and epithelial origin (Adams et al., 1999; Herrmann et al., 1998). In several types of neoplastic cells, the proteasome inhibitors were found out to be active inducers of apoptosis, and they are able to induce apoptosis in cells resistant to other agents. Different proteasome inhibitors, including lactacystin and MG132, induce apoptosis in leukaemic B cells from patients with B-cell chronic lymphocytic leukaemia (B-CLL) at all stages of the disease, including those resistant to conventional chemotherapy (Wojcik, 2002). For example, the proteasome inhibitor MG132 is a potent deathinducing agent for PC3 prostate cancer cells and elicits activation of multiple signaling pathways in these cells (Yang et al., 2006). Proteasome inhibitors may induce apoptosis either by toxic effects or by inhibition of degradation and/or processing of specific regulatory proteins (Wojcik, 2002). One of the anti-apoptotic regulatory proteins which level is increased by proteasomes is NF-kB, a member of a large family of transcription factors found in the cytoplasm (Verma et al., 1995). Proteasome inhibitors reduce the activity of protein NF-kB through stabilization of protein IkBa, and start apoptosis in the number of transformed cells (Naujokat and Hoffmann, 2002; Wojcik, 2002). NF-kB forms an inactive complex with inhibitory protein IkBa in cytoplasm. In response to cellular stress (for example, the chemotherapy, irradiation, action of cytotoxic agents, viruses and oxidants), IkBa is phosphorylated, ubiquitinylated and degraded by the proteasomes. The active transcription factor NF-kB is transported from the cytoplasm into the nucleus, where it initiates the transcription of anti-apoptotic proteins (A1/Bfl1, IAP and bcl-2), growth factors (interleukins) and molecules of cellular adhesion, preventing the cells from the apoptosis. Proteasome inhibitors induce suppression of NF-kB activation in cancer cells, and these cells become more sensitive to chemotherapy and other stressful agents (Traenckner et al., 1994). The conjugation of the tumour necrosis factor TNF-a with the specific TNF-receptor leads to the activation of NF-kB and the induction of anti-apoptotic signal. The proteasomes are also believed to directly generate anti-apoptotic and

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survival signals in neoplastic cells by degrading pro-apoptotic proteins such as Bax and Bid (Breitschopf et al., 2000b; Chang et al., 1998; Li and Dou, 2000). One more mechanism of pro-apoptotic action of proteasome inhibitors involves the induction of a block in the cell cycle, which is regulated by cellular proteolysis of either cyclins or CDK inhibitors (Wojcik, 2002). Blocking of the cell cycle can actually prevent the cells from entering apoptosis in some situations and coexist or precedes the induction of apoptosis in other (Naujokat and Hoffmann, 2002). Another target for proteasome is the tumor suppressor p53, responsible for induction of apoptosis. In response to cellular stress (for example, damages of DNA and hypoxia), p53 causes the block in the cell cycle at the phases G1 or G2, DNA repair or induces apoptotic response (by activation of gene bax and repression of gene bcl-2). p53 acts as a transcriptional regulator inducing expression of several key genes mediating those effects (Burns and El-Deiry, 1999; Shen and White, 2001; Wang, 1999). p53 levels are normally very low in the cells, since it is constantly ubiquitinated by Mdm2 RING-finger ubiquitin ligase and then degraded by the proteasome (Fang et al., 2000). At cell damage, p53 degradation stops as the result of ubiquitin–proteasome proteolysis of Mdm2-ligase, and p53 level quickly increases in a cell. Furthermore, fast accumulation of p53 is promoted by the cleavage of Mdm2-ligase by caspase 3 (Cho et al., 2001). The proteasome inhibition also causes p53 accumulation and following induction of apoptosis in proliferating cells (MacLaren et al., 2001). Proteasome inhibitors can cause accumulation of the oncoprotein c-Myc. Transcription factor c-Myc controls the cell cycle, proliferation and apoptosis. c-Myc transactivates gene of Cdc25a-phosphatase, which removes inhibitory phosphorylation of Cdk2 and Cdk4 and also decreases expression of inhibitor p27Kip1. Deregulated expression of c-Myc is associated with many human cancers, including Burkitt’s lymphoma (Gregory and Hann, 2000). The c-Myc protein is normally degraded very rapidly by the ubiquitin-proteasome pathway (Salghetti et al., 1999). c-Myc activation by proteasome inhibitors leads to stimulation of deregulated cell proliferation and to induction of the apoptosis (Wojcik, 2002). In human malignant glioma cells, the proteasome inhibitors cause an increase of c-Myc protein levels, which induces transiently FasL message to stimulate the Fas receptor-ligand apoptotic signaling pathway (Tani et al., 2001). Finally, the effects of proteasome’s anti-apoptotic action can include the regulation of the levels of specific secondary messenger molecules (for example, cAMP or nitric oxide), which in turn can induce apoptosis. So, for example, in human neutrophils, ubiquitin-proteasomal system of protein degradation regulates the balance of pro-apoptotic and anti-apoptotic proteins which plays a key role in the ability of cyclic AMP to delay neutrophil death (Lee et al., 2001; Martin et al., 2001).

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In all, both negative and positive regulators of apoptosis undergo proteasomal degradation in a tightly regulated and temporally controlled fashion. For example, proteasome inhibitors induce apoptosis in fastly proliferating cells (neoplastic cells). These cells show the raised level of proteasomal subunits expression, and, probably, proteasomes block the apoptosis and play an essential role in a survival and proliferation of only quickly growing and neoplastic cells (Naujokat and Hoffmann, 2002). Several of the specific proteasomal inhibitors have recently entered clinical trials due to their tremendous apoptosis-inducing capability. However, several recent studies provided substantial evidence that a combined treatment of tumors with apoptosis-inducing agents and proteasomal inhibitors might even cause adverse effects leading to a prolonged survival of tumor cells (Sohn et al., 2006a; Yang et al., 2006). Proteasomes provide balance between pro- and anti-apoptotic proteinsregulators in a cell and are the central figures in the balance between two opposite pathways: a survival and apoptosis of the cell (Sohn et al., 2006a,b; Yang et al., 2006). It has appeared that apoptotic action of proteasome inhibitors depends on a stage of apoptosis. Recently the model of biphasic role for the proteasome in apoptosis of tumor cells has been proposed (Sohn et al., 2006b). Thus, the proteasomes support a critical balance between the pools of pro- and anti-apoptotic proteins before the initiation of apoptosis (Sohn et al., 2006b). During the induction phase of apoptosis, however, this balance slowly moves toward cellular death due to an increase of the proapoptotic pool (e.g., active caspases), and it depends on proteasomal degradation of anti-apoptotic proteins (Sohn et al., 2006b). During the execution phase of apoptosis, the cell accumulates the pool of pro-apoptotic proteins in comparison with a decreasing pool of anti-apoptotic proteins. The inhibition of proteasomes before the induction phase of apoptosis leads to an increase of the pool of anti-apoptotic proteins as cellular biosynthesis constantly supplements this pool which is not decreased by proteolysis. Hence, in this case proteasomes cannot execute their pro-apoptotic role. However, inhibition of the proteasome after the induction phase of apoptosis leads to an increase of the pro-apoptotic proteins pool as the required proteasomal degradation of the anti-apoptotic proteins pool has been reached before (Sohn et al., 2006b). Similarly, both pro- and anti-apoptotic pathways can be regulated by proteasome inhibitor MG132 in prostate cancer PC3 cells (Yang et al., 2006).

3.4. Proteasomes and immune response Misfolded, foreign and other abnormal proteins are degraded through the ubiquitin- and proteasome-dependent pathway in the cells. This proteolytic system generates peptides from intracellular antigens, which are then presented to T cells, and thereby plays the central role in the cellular immune response.

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The IFNg controls the proteolytic properties of the proteasome to adapt them to the requirements of the immune system. The stimulation of cells with the cytokines IFNg or TNF-a induces the synthesis of three proteasomal subunits LMP2 (b1i), LMP7 (b5i), and MECL-1 (b2i). These subunits replace the three subunits Y(b1), X(b5), and Z(b2), which bear the proteasomal catalytically active sites (Fruh et al., 1994). The cytokine-induced exchange of three active site subunits of proteasome is unprecedented in molecular biology and one may expect a strong functional driving force for this system to evolve (Groettrup et al., 2001a). Furthermore, IFNg induces the synthesis of the proteasome activator PA28 and the formation of immunoproteasome. The PA28 synthesis and the immunoproteasomes formation, in tern, adapt the proteolytic properties of the proteasome and improve the proteasomal function in antigen presentation. Thus, a combination of several regulatory events tunes the proteasome system for maximal efficiency in the generation of MHC class I antigens (Kloetzel, 2004). Although the peptide production by constitutive proteasomes is able to maintain peptide-dependent MHC class I cell surface expression in the absence of LMP2 and LMP7, these subunits were recently shown to be central for the generation or destruction of several unique epitopes (Groettrup et al., 2001a). The proteasomal immune subunits exchanges have evolved not only to optimize class I peptide loading but also to generate LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites which are not generated in uninflamed tissues. This difference in epitope generation may serve to better stimulate T cells in the sites of an ongoing immune response and to avoid autoimmunity in uninflamed tissues (Groettrup et al., 2001b). Interestingly, the molecular interplay between the proteasome maturation protein (POMP) and the proteasomal LMP7 subunit has a key position in this immune adaptive program. IFNg-induced coincident biosynthesis of POMP and LMP7 and their direct interaction essentially accelerate immunoproteasome biogenesis compared with constitutive 20S proteasome assembly. The dynamics of this process is determined by rapid LMP7 activation and the immediate LMP7-dependent degradation of POMP. Silencing of POMP expression impairs recruitment of b5i (as well as b5) subunit into the proteasome complex, resulting in decreased proteasome activity, reduced MHC class I surface expression (Heink et al., 2005). The LMP2 and LMP7 are encoded in chromosome locus of MHC class I that is also an evidence of proteasomes participation in the immune response. Furthermore, proteasomal immune subunits are continually expressed in lymphoid organs (spleen, thymus and glands) cells (Rivett, 1998; Rock et al., 1994) as well as in other cells (Gomes et al., 2006). The expression of immunosubunits is cell tissue specific and is under high selective control (Kloetzel, 2004). It is significant, that the immunoproteasomes realize an additional function in antigen-presenting cells (APCs) of the immune system, including B-lymphocytes, macrophages, and dendritic cells. APCs absorb antigens

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entering the lymphoid organs with blood and lymph flows via endocytosis. Then immunoproteasomes transform antigens into antigenic epitopes, which are brought to the cell surface in a complex with molecules MHC class I, like in defective and virus-infected cells (Rock and Goldberg, 1999). APCs contain almost ten thousand molecules of MHC class I on their plasma membrane at the same time and an elevated number of immunoproteasomes ( Janeway and Travers, 1994). Proteasomes were shown to be indirectly involved in the development of both humoral and cell immune responses related to the cytotoxic activity of macrophages and T-killers (for review, see Sharova, 2006). Moreover, proteasomes were found to process antigenic polypeptides for their presentation in a complex with MHC class II to T-helper cells (Tewari et al., 2005). In conclusion, the proteasomes trigger a signal for destruction of defective cells and are involved in activation of T-killers and, thus, participate in the formation of the T-cell immune response. Moreover, proteasomes are involved in the activation of T-helper cells and, therefore, mediate both humoral (functioning of B-lymphocytes) and cellular (functioning of T-killers and macrophages) immunity.

4. Modes of Regulation of Proteasome Activities in the Cell 4.1. Modulation of proteasome composition 4.1.1. Heterogeneity of proteasomes in the cell The more in a complicated manner the organization of both the separate cells, and the entire organisms is, the more fancily becomes the main cellular machinery, aimed to non-lysosomal protein cleavage. The structure and sub-unit content of ancestral proteasome complex found in Prokaryota, both Archea (Maupin-Furlow and Ferry, 1995) and Eubacteria (Hu et al., 2006; Lupas et al., 1994; Tamura et al., 1995), is respectively simple: subunits are divided into only two types, a and b, forming four homoheptameric rings in abba order; the complex contains 14 identical proteolytic centers capable to cleave polypeptide chains after hydrophobic and basic amino acid residues (see also Section 2.1.1). However, in some Archea species, the first complication occurs consisting in appearance of two distinct kinds of a and/or b subunits (Humbard et al., 2006; Kaczowka and Maupin-Furlow, 2003; Madding et al., 2007; Zuehl et al., 1997). When rising up along the evolutionary staircase, we find eukaryotic proteasomes to be much more complex: parallel with appearance of at least 14 distinct subunits of the core particle (7 a and 710 b subunits), multiple forms of regulatory complexes bound to the core were observed,

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such as PA700 (19S), PA200, PA28 (11S) and PA26 (see Section 2.1.3). These complexes could be attached to 20S barrel either from one or from both sides. Combination of the listed above complexes attachment to core particle is one of the ways for appearance of multiple cellular forms of multicatalytic protease called 26S proteasome. How many other mysteries cover this surprising complex in itself ? Recent studies performed on different eukaryotic species have shown the widest variety of subunit forms, their different versions, appearing to be a result of gene duplication (Fu et al., 1998; Ma et al., 2002; Yang et al., 2004), alternative splicing (Gomes et al., 2006; Kawahara et al., 2000a,b) and/or of various post-translational modifications. Gene duplication was found in a wide range of organisms. Thus, in Arabidopsis, ten 20S proteasome subunits, encoded by two genes each, were found to be expressed in appropriate different isoforms (Fu et al., 1998; Yang et al., 2004), whereas in Drosophila, six genes encoding 20S proteasome subunits were shown to be represented by two or even three isoforms, encoded by separate genes (Ma et al., 2002; Yuan et al., 1996) and, like in previous case, additional isoforms were revealed to be expressed. Four of the 19S regulatory complex subunits were also found to have actively expressing gene duplications encoding male-specific isoforms (Ma et al., 2002). On the contrary, experiments performed on various mouse cells and tissues—cardiomyocytes (Gomes et al., 2006), testis and embryonic stem cells (Kawahara et al., 2000a,b)— have revealed several distinct mRNA forms for Rpn10 subunit of 19S regulatory complex generated by a single gene. All that is listed above speaks in favour of the fact that the proteasomal population in each separately taken cell is highly specialized, which was confirmed, in particular, via subpopulations fractionation by means of anionexchange chromatography on MonoQ resin (Dahlmann et al., 2000, 2001). Separate fractions (cytosolic and ER-membrane bound) were found to differ from each other in their enzymatic activities (Khan and Joseph, 2001). The detailed study of proteasomes from the mammalian tissues and their proteolytic activities has resulted in conclusion of heterogeneity of the proteasome population. The isolated proteasome samples revealed properties of the dominating subpopulation, while others remain unstudied (Dahlmann et al., 2000, 2001). The principal cause of existence of several proteasome subtypes is competition of 10 b subunit varieties for building in to form the sevenmember b-ring. The expression of these seven b subunits is constitutive, but three of them—b1, b2 and b5—can be replaced under influence of IFNg by b1i, b2i and b5i subunits, respectively, which leads to immunoproteasome formation (see Section 3.4). The above facts allow to consider the existence of the two main proteasome groups which can be separated chromatographically due to slight differences in their surface charges (Dahlmann et al., 2000, 2001).

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It currently is unknown whether the b subunit set is always similar in both halves of the proteasome cylinder. If unequal replacement is possible, then 36 different combinations of subunits may appear, and otherwise the number of combinations is limited to eight. Notwithstanding the fact that theoretically all configurations are possible, the more probable is simultaneous replacement of all three inducible subunits (Griffin et al., 1998; Groettrup et al., 1997). Thus, b1i and b2i are reciprocally necessary for inclusion into proteasome, but independently of b5i. However, presence of b1i and b2i favours inclusion b5i into the proteolytic particle. Thus, the most probable manner of immunoproteasome formation is that containing all three inducible b subunits, but also another variants seem to be possible— proteasomes containing either b1i and b2i without b5i or, in turn, only b5i, as the sole immunosubunit (Dahlmann et al., 2000, 2001). 4.1.2. Subunit modifications On a 2D map of proteasome subunits, a number of spots exceed 14 or even 17 corresponding to a number of constitutive or both constitutive and inducible subunits of 20S core particle (Claverol et al., 2002; Froment et al., 2005; Wang et al., 2007). This phenomenon becoming clear after any attempt of subunit identification, immunochemical or mass-spectrometric is based on several kinds of post-translational modifications undergone by subunits and some proteasome-interacting proteins co-purifying with proteasomes. Proteasomal subunits, like many other protein molecules, could be underwent to several post-translational modifications (see Table 2.3), such as phosphorylation (Benedict et al., 1995; Bose et al., 1999, 2001, 2004; Castano et al., 1996; Humbard et al., 2006; Mason et al., 1996; 1998; Wang et al., 2007; Wehren et al., 1996), N-acetylation and/or N-terminal propeptide processing (Arendt and Hochstrasser, 1999; Gomes et al., 2006; Huang et al., 2001; Humbard et al., 2006; Kimura et al., 2000, 2003; Tokunaga et al., 1990; Wang et al., 2007; Zong et al., 2006), 4-hydroxy-2-nonenal alkylation (Bulteau et al., 2001; Farout et al., 2006), N-myristoylation (Gomes et al., 2006; Kimura et al., 2000; Utsumi et al., 2001; Wang et al., 2007), O-glycosylation (Tomek et al., 1988; Schliephacke et al., 1991; Schmid et al., 1993; Su¨megi et al., 2003; Wells et al., 2002; Zhang et al., 2003), S-glutationylation (Demasi et al., 2001, 2003) and oxidation of sulphurcontaining amino acid residues (Humbard et al., 2006; Schmidt et al., 2006; Wang et al., 2007). Such modifications are found in a wide range of organisms, from archebacteria Haloferax volcanii (Humbard et al., 2006) to humans (Wang et al., 2007). The proteasome structures and functions have been thoroughly studied this last decade, and these complexes have been revealed to be as a mixture of several proteasome subpopulations possessing different enzymatic activities (Dahlmann et al., 2000, 2001).

Table 2.3 Post-translational modifications of 20S proteasome and proteasome regulatory particle proteins

Subunit

PTM

20S proteasome subunits a1 N-Acetyl; Met1-SO a1 N-Acetyl a1 Ser
P; Tyr
P; Thr
P a1 Met209-SO a2 N-Acetyl a2 N-Acetyl a2 N-Acetyl a2 N-Acetyl a2 Ser-acetyl a2 a2 a2 a2 a2 a2

Ser or Thr
P Tyr120
P Tyr
P; Thr
P Ser
P Tyr23
P; Tyr97
P O-GlcNAc

a2 a3 a3 a3

4-Hydroxy-2-nonenal N-Acetyl N-Acetyl Ser248
P

Organism

Haloferax volcanii S. cerevisiae Mus musculus S. cerevisiae Haloferax volcanii S. cerevisiae Rattus norvegicus Mus musculus Trypanosoma brucei S. cerevisiae Rattus norvegicus Rattus norvegicus Mus musculus Homo sapiens Drosophila melanogaster Rattus norvegicus S. cerevisiae Rattus norvegicus Candida albicans

PTM Catalyst

References

n.d. NatA/Nat1 n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d.

Humbard et al., 2006 Kimura et al., 2000 Zong et al., 2006 Iwafune et al., 2002 Humbard et al., 2006 Kimura et al., 2000 Tokunaga et al., 1990 Gomes et al., 2006 Huang et al., 2001

n.d. n.d. n.d. n.d. n.d. n.d.

Iwafune et al., 2002 Benedict et al., 1995 Wehren et al., 1996 Zong et al., 2006 Rush et al., 2005 Sumegi et al., 2003

n.d. NatA/Nat1 n.d. CK2

Bulteau et al., 2001 Kimura et al., 2000 Tokunaga et al., 1990 Fernandez-Murray et al., 2002 (continued)

Table 2.3 (continued) Subunit

PTM

Organism

PTM Catalyst

a3


P

Rattus norvegicus

CK2

a3 a3

Ser
P
P

Rattus norvegicus Homo sapiens

n.d. PLK

a3 a3 a3 a4 a4

Homo sapiens Mus musculus Drosophila melanogaster S. cerevisiae Homo sapiens

n.d. n.d. n.d. NatA/Nat1 n.d.

a4 a4

Ser75
P Ser
P O-GlcNAc N-Acetyl Processed, N-acetyl
P
P

Xenopus sp. Carassius auratus

n.d. CKI_

a4 a4 a4 a4 a4 a5 a5 a5 a5

Ser or Thr
P Met71-SO Cys63-SO 4-Hydroxy-2-nonenal O-GlcNAc N-Acetyl N-Acetyl N-Acetyl
P

S. cerevisiae Rattus norvegicus Rattus norvegicus Rattus norvegicus Drosophila melanogaster S. cerevisiae Mus musculus Homo sapiens Candida albicans

n.d. n.d. n.d. n.d. n.d. NatC/Mak3 n.d. n.d. CK2

References

Castano et al., 1996; Mason et al., 1996 Wehren et al., 1996 Mason et al., 1996; Bose et al., 2001; Feng et al., 2001 Wang et al., 2007 Zong et al., 2006 Sumegi et al., 2003 Kimura et al., 2000 Wang et al., 2007 Tokumoto et al., 1999b Tokumoto et al., 2000; Wakata et al., 2004; Horiguchi et al., 2005 Iwafune et al., 2002 Schmidt et al., 2006 Schmidt et al., 2006 Bulteau et al., 2001 Sumegi et al., 2003 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007 Fernandez-Murray et al., 2002

a5 a5 a5

Ser
P Ser56
P O-GlcNAc

a6 a6 a6 a6 a6

n.d. n.d. n.d.

Wehren et al., 1996 Beausoleil et al., 2004 Sumegi et al., 2003

N-Acetyl N-Acetyl O-GlcNAc
P
P

Rattus norvegicus Homo sapiens Drosophila melanogaster S. cerevisiae Homo sapiens Rattus norvegicus Oryza sativa Candida albicans

NatC/Mak3 n.d. OGT CK2 CK2

a6 a6 a6 a7

Ser
P; Tyr
P; Thr
P 4-Hydroxy-2-nonenal O-GlcNAc N-Acetyl

Mus musculus Rattus norvegicus Drosophila melanogaster Homo sapiens

PKA n.d. n.d. n.d.

a7 a7 a7 a7 a7 a7 a7 a7

N-Acetyl N-Acetyl N-Acetyl Processed, N-acetyl Ser-acetyl Ser
P Ser250
P Ser250
P

Rattus norvegicus S. cerevisiae Mus musculus Homo sapiens Trypanosoma brucei Rattus norvegicus Mus musculus Homo sapiens

n.d. NatA/Nat1 n.d. n.d. n.d. n.d. PKA n.d.

Kimura et al., 2000 Wang et al., 2007 Wells et al., 2002 Umeda et al., 1997 Fernandez-Murray et al., 2002 Zong et al., 2006 Bulteau et al., 2001 Sumegi et al., 2003 Claverol et al., 2002; Gillardon et al., 2007 Tokunaga et al., 1990 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007 Huang et al., 2001 Wehren et al., 1996 Zong et al., 2006 Mason et al., 1996; Bose et al., 2001; Wang et al., 2007; Gillardon et al., 2007 (continued)

Table 2.3 (continued) Subunit

PTM

Organism

PTM Catalyst

References

a7 a7 a7 a7 a7 a7 a7 a7 b b b1 b1 b2 b2 b2 b2 b3 b3 b3 b3 b4 b4 b4

Tyr160
P
P Tyr
P
P Ser243
P; Ser250
P Ser243
P; Ser250
P
P O-GlcNAc Ser129
P N-Acetyl; Met1-SO Processed Met4-SO, Met146-SO Ser
P; Thr
P Tyr154
P O-GlcNAc Processed N-Acetyl N-Acetyl Processed, N-acetyl Ser
P; Thr
P N-Acetyl N-Acetyl N-Acetyl

Homo sapiens Homo sapiens S. cerevisiae S. cerevisiae Rattus norvegicus Cercopithecus sp. Mus musculus Drosophila melanogaster Haloferax volcanii Haloferax volcanii Homo sapiens S. cerevisiae Mus musculus Homo sapiens Drosophila melanogaster Homo sapiens S. cerevisiae Mus musculus Homo sapiens Mus musculus S. cerevisiae Mus musculus Homo sapiens

n.d. PLK n.d. CK2 CK2 n.d. n.d. n.d. n.d. n.d. n.d. n.d. PKA n.d. n.d. n.d. NatA/Nat1 n.d. n.d. PKA NatB/Nat3 n.d. n.d.

Rush et al., 2005 Feng et al., 2001 Iwafune et al., 2002 Pardo et al., 1998 Castano et al., 1996 Bose et al., 2004 Gomes et al., 2006 Sumegi et al., 2003 Humbard et al., 2006 Humbard et al., 2006 Wang et al., 2007 Iwafune et al., 2002 Zong et al., 2006 Rush et al., 2005 Sumegi et al., 2003 Wang et al., 2007 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007 Zong et al., 2006 Kimura et al., 2000 Gomes et al., 2006 Wang et al., 2007

b4 Met1-SO b5 Processed b5 O-GlcNAc b6 Ser
P b7 Tyr102
P b7 Ser
P; Thr
P b7 Met14-SO, Met181-SO b7 O-GlcNAc 19S Regulatory particle ATPases Rpt1
P Rpt1 Processed Rpt2 Processed; N-myristoyl Rpt2 N-myristoyl Rpt2
P Rpt2 Processed; N-myristoyl Rpt2 Processed; N-myristoyl Rpt2 O-GlcNAc Rpt2 Rpt3 Rpt3 Rpt3 Rpt3 Rpt3 Rpt4 Rpt4

O-GlcNAc N-Acetyl
P N-Acetyl N-Acetyl O-GlcNAc Processed; N-acetyl Processed; N-acetyl

S. cerevisiae Homo sapiens Drosophila melanogaster Rattus norvegicus Homo sapiens Mus musculus S. cerevisiae Drosophila melanogaster

n.d. n.d. n.d. n.d. n.d. PKA n.d. n.d.

Iwafune et al., 2002 Wang et al., 2007 Sumegi et al., 2003 Wehren et al., 1996 Rush et al., 2005 Zong et al., 2006 Iwafune et al., 2002 Sumegi et al., 2003

Homo sapiens Homo sapiens S. cerevisiae Oryza sativa Homo sapiens Mus musculus Homo sapiens Homo sapiens; Oryctolagus sp. Drosophila melanogaster S. cerevisiae Homo sapiens Homo sapiens Mus musculus Drosophila melanogaster S. cerevisiae Homo sapiens

n.d. n.d. n.d. n.d. n.d. n.d. n.d. OGT

Mason et al., 1998 Wang et al., 2007 Kimura et al., 2003 Shibahara et al., 2002 Mason et al., 1998 Gomes et al., 2006 Wang et al., 2007 Zhang et al., 2003

n.d. NatB/Nat3 n.d. NatB/Nat3 n.d. n.d. NatA/Nat1 n.d.

Sumegi et al., 2003 Kimura et al., 2003 Mason et al., 1998 Wang et al., 2007 Gomes et al., 2006 Sumegi et al., 2003 Kimura et al., 2003 Wang et al., 2007 (continued)

Table 2.3 (continued) Subunit

PTM

Organism

Rpt4 O-GlcNAc Drosophila melanogaster Rpt5 Processed; N-acetyl S. cerevisiae Rpt5 N-acetyl Homo sapiens Rpt5 Ser9
P Homo sapiens Rpt6 Processed; N-acetyl S. cerevisiae Rpt6 N-Acetyl Oryza sativa Rpt6
P Sus scrofa Rpt6 N-Acetyl Mus musculus Rpt6 Processed; N-acetyl Homo sapiens 19S Regulatory particle non-ATPase subunits Rpn1 Processed S. cerevisiae Rpn1 N-Acetyl Homo sapiens Rpn1 Ser16
P Homo sapiens Rpn1 Ser361
P Homo sapiens Rpn1 N-Acetyl Mus musculus Rpn2 Processed; N-acetyl S. cerevisiae Rpn2 N-Acetyl Homo sapiens Rpn2 Met1-SO Homo sapiens Rpn2 Thr311
P Homo sapiens Rpn2 Thr311
P; Ser315
P Homo sapiens Rpn2 Thr273
P Homo sapiens Rpn3 Processed; N-acetyl S. cerevisiae Rpn3 Processed Oryza sativa Rpn3 Processed Daucus carota Rpn3 O-GlcNAc Drosophila melanogaster Rpn5 Processed; N-acetyl S. cerevisiae

PTM Catalyst

References

n.d. NatA/Nat1 n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d.

Sumegi et al., 2003 Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Shibahara et al., 2002 Satoh et al., 2001 Gomes et al., 2006 Wang et al., 2007

n.d. n.d. n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d. NatA/Nat1

Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Gomes et al., 2006 Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Shibahara et al., 2002 Smith et al., 1997 Sumegi et al., 2003 Kimura et al., 2003

Rpn5 Rpn5 Rpn6 Rpn6 Rpn6 Rpn6 Rpn6 Rpn6 Rpn7 Rpn7 Rpn8 Rpn8 Rpn8 Rpn8 Rpn9 Rpn10 Rpn10 Rpn10 Rpn10 Rpn10 Gankyrin Rpn11 Rpn11 Rpn12 Rpn12 Rpn13

N-Acetyl O-GlcNAc Processed; N-acetyl N-Acetyl Processed; N-acetyl Ser13
P Ser78
P O-GlcNAc Processed Processed Processed; N-acetyl Processed
P Thr186
P Ser106
P Processed Processed Ser266
P Ser358
P Ser358
P; Ser361
P N-Acetyl N-Acetyl Ser224
P Processed O-GlcNAc Processed; N-acetyl

Mus musculus Drosophila melanogaster S. cerevisiae Mus musculus Homo sapiens Homo sapiens Homo sapiens Drosophila melanogaster S. cerevisiae Homo sapiens S. cerevisiae Homo sapiens Homo sapiens Homo sapiens Homo sapiens S. cerevisiae Homo sapiens Homo sapiens Homo sapiens Homo sapiens Homo sapiens S. cerevisiae Homo sapiens S. cerevisiae Drosophila melanogaster Homo sapiens

The materials of Maupin-Furlow et al., 2006, were used for preparation of this table.

n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. NatA/Nat1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. NatB/Nat3 n.d. n.d. n.d. n.d.

Gomes et al., 2006 Sumegi et al., 2003 Kimura et al., 2003 Gomes et al., 2006 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Sumegi et al., 2003 Kimura et al., 2003 Wang et al., 2007 Kimura et al., 2003 Wang et al., 2007 Mason et al., 1998 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Wang et al., 2007 Kimura et al., 2003 Wang et al., 2007 Kimura et al., 2003 Sumegi et al., 2003 Wang et al., 2007

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Multiple isoforms of proteasome subunits have been described, which further account for the structural complexity in 20S proteasome subunit composition (Claverol et al., 2002; Froment et al., 2005). The proteasome catalytic activity may be affected by various environmental factors such as oxidative stress, pathological states such as cancers or neurological disorders, aging, or pharmacological agents (Gillardon et al., 2007; Shah et al., 2001), and such fundamental cellular processes as immune response, programmed cell death and differentiation (Iwafune et al., 2002; Kulichkova et al., 2004b; Mason et al., 1996; Tsimokha et al., 2006). Structural (and more particularly, post-translational) modifications that affect either the protein to be degraded or the proteasome subunits may lead to altered or even inhibited proteolytic functions (Ermonval et al., 2001; Gillardon et al., 2007). Recently an evidence was obtained that changes in subunit modification of 20S proteasomes in humans suffering Alzheimer’s disease affect at least two of proteasome’s proteolytic activities (Gillardon et al., 2007). 4.1.2.1. Phosphorylation of proteasome subunits At the present time, several subunits of both 20S proteasomes and their regulatory complexes (19S and PA28) are known to be phosphorylated in vivo. For the first time, these data were obtained on 20S proteasome samples, isolated from the Drosophila larvae (Haass and Kloetzel, 1989), where four phosphorylated proteasome subunits were detected. Three unidentified mammalian 20S proteasome subunits hereafter were shown to be phosphorylated in vitro by cAMP-dependent protein-kinase, co-purifying with bovine proteasomes (Pereira, Wilk, 1990). As well a proteasome subunit with molecular mass 30 kDa was shown to be phosphorylated by casein kinase II, co-purifying with human erythrocyte proteasomes (Ludemann et al., 1993). Furthermore, it was shown that subunit a6 (C2, iota) isolated from rice cells is phosphorylated by the same casein kinase II (Umeda et al., 1997) despite the fact that the phosphorylation sites of this subunit are not conserved in different organisms. Fernandez-Murray and co-authors (2002) identified a6 (C2), a3 (C9), and a5 (Pup2) yeast proteasome subunits to be the main in vivo phosphorylated and in vitro CK2-phosphorylatable proteasome components. The most intense isoform of the human erythrocyte a7 subunit was shown to be phosphorylated (Claverol et al., 2002). This modification has recently been described to play a role in 26S proteasome stability by interacting with the 19S regulatory complex rather than having a direct effect on proteolytic activity (Bose et al., 2004). Studies of the phosphorylated amino acids from rat liver and human placenta have shown what subunit a2 (C3) contains phosphotyrosine and phosphothreonine, whereas phosphoserine was detected in b6 (C5), a3 (C9), a7 (C8) and a5 (z) subunits (Bose et al., 1999; Castano et al., 1996; Mason et al., 1996; Wehren et al., 1996). Phosphorylation of serine residues of human a3 (C9) at Ser74 and a7 (C8) at Ser250 was described recently by

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Wang and co-authors (2007). Attempt to estimate the possible functional significance of phosphorylation of these subunits was undertaken. Phosphorylation of a2 (C3) subunit at Tyr120 demonstrated to be significant for proteasome’s nuclear localization (Benedict et al., 1995). A hypothesis was offered, that phosphorylation of proteasomal subunits could be involved in regulation of the enzymatic activity of the complex via conformational changes (Mason et al., 1996). Dephosphorylation of proteasome subunits a3 (C9) and a7 (C8) (which could be phosphorylated on serine residues by casein kinase II in vitro (Castano et al., 1996) led to a small, but significant, decrease in two peptidase activities (Mason et al., 1996). On the other hand, in vitro phosphorylation of murine cardiac 20S proteasome (subunits a1, a2, a3, b2, b3 and b7) by protein kinase A significantly increased the chymotrypsin- and caspase-like activities (Zong et al., 2006). Recent data of Wang and co-authors (2007) demonstrated approximately half of the phosphorylated amino acid residues identified to be serines or threonines followed by proline. The last is known to be a marker of phosphorylation by mitogen-activated or cyclin-dependent kinases, so regulation of proteasomal phosphorylation state might be proposed to be cell cycle dependent (Wang et al., 2007). Phosphorylation of 20S core complex subunits may influence the interaction of proteasome with other proteins. The potential phosphorylation sites of a3 (C9) and a7 (C8) subunits are located on the ends of the cylindershaped 20S proteasome, and therefore phosphorylation of these subunits can control binding of regulatory complexes to the core particle. Researchers from the same group (Bose et al., 2004) have recently found phosphorylation of subunit a7 (C8) to stabilize the association of 19S regulatory complexes with 20S proteasomes to form the 26S proteasome, while dephosphorylation facilitated the formation of 11S-containing proteasome complexes. For their turn, the proteasomal regulatory complexes can also contain phosphorylated subunits. For example, the phosphorylated subunits were detected in 11S regulators obtained from rabbit reticulocyte lysates (Li et al., 1996). The analysis of phosphorylated amino acids obtained from human erythrocytes has shown phosphorylation to occur at serine residues and antiphosphoserine antibodies to bind all three PA28 polypeptides which are recognizable by anti-PA28 antibodies. Thus, a, b, and nuclear g subunits of PA28 complex are subjected to phosphorylation (Bose et al., 1999). When 11S complex subunits were dephosphorylated, this regulator has lost the ability to stimulate peptidase activities of proteasomes, so one can conclude, that phosphorylation of the PA28 complex controls the activity of proteasome via conformational changes of this regulatory particle. Experiments performed on the HL-60 cell line also showed the phosphorylation of PA28 to be increased in the presence of IFNg (Bose et al., 1999, 2001).

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Besides the above-described complexes, the 19S (PA700) regulators could be phosphorylated too. Thus, several subunits of this complex, including at least, one of the ATPases (S4), were shown to be phosphorylated in vivo (Mason et al., 1998) although functional significance of ATPase subunits phosphorylation is not elucidated yet. Wang and co-authors (2007) in their recent investigation by means of tandem mass-spectrometry demonstrated 10 subunits of 26S proteasome complex from human 293 cell line to be phosphorylated at total of 16 sites and 12 of these sites have not been reported previously. For the most part, phosphorylated subunits reported (eight subunits: one ATPase and seven non-ATPases) were elements of 19S regulatory complex, and only two a subunits were parts of 20S core. It seems to be significant, that all recognized in the above work phosphorylation sites were either threonines or serines, but not tyrosines (Wang et al., 2007). Perhaps, phosphorylation of ATPase subunits, via conformational changes, can affect substrate recognition and opening of a channel inside the 20S core particle. The assembly of the 19S complex, consisting of ‘‘base’’ and ‘‘lid,’’ probably can be under control of protein subunits phosphorylation, too. Although the functional significance of phosphorylation on the 19S subunits remains to be established, identification of novel phosphorylation sites by Wang and co-authors (2007) is the first critical step toward an improved understanding of its role in regulating the function of the 26S proteasome complex. However, the phosphorylation state of proteasome subunits was shown to be not a constant value. In turn, it changed under the action of agents altering physiological state of the cell. The list of the above cellular models includes induction of erythroid differentiation by hemin in K562 proerythroleukemia cells (Mittenberg et al., 2007) and doxorubicin- or diethylmaleate-induced apoptosis of the same cell line (Toktarova et al., 2004; Tsimokha et al., 2006, 2007a,b,c). During the last decade, several works have appeared about the effect of the phosphorylation state of cytoplasmic 26S proteasome subunits on their proteolytic activity (Iwafune et al., 2002; Kulichkova et al., 2004b; Mason et al., 1996; Tsimokha et al., 2006). Moreover, a selective effect of dephosphorylation of cytoplasmic 26S proteasomes on their RNase activity regarding high molecular weight ribosomal RNA and c-fos, c-myc and p53 mRNAs was shown (Mittenberg et al., 2002a,b, 2007; Toktarova et al., 2004; Tsimokha et al., 2006). Earlier the efficiency of the 26S proteasomal RNase activity toward high molecular weight ribosomal RNA was shown to depend on phosphorylation state of the proteasomal subunits (Kulichkova et al., 2004b; Mittenberg et al., 2002a). To find out whether a change in the phosphorylation state affects nucleolysis efficiency regardless of substrate, or whether it can produce different effects on nucleolysis of different substrates, i.e., whether it can affect the specificity of the RNase activity, the influence of dephosphorylation of these

Role of Proteasomes in Cellular Regulation

89

particle subunits on the nucleolysis of various mRNAs were studied. Dephosphorylation of the proteasomal subunits from control K562 cells was found to lead to an increase in their nucleolysis efficiency for the p53 mRNA, while dephosphorylation of proteasomes from the cells induced to apoptosis or differentiation had no effect on nucleolysis of this RNA. This suggests that the mechanism responsible for an increase in the ability to cleave a particular mRNA is connected with dephosphorylation of proteasome subunits in the case of differentiation induction (Mittenberg et al., 2007). Earlier changes in the phosphorylation state of the proteasome subunits during apoptosis were revealed (Tsimokha et al., 2006); however, regulation of the phosphorylation state of the proteasome during cell differentiation was not studied. To estimate possible changes in the phosphorylation state of the proteasome subunits after induction of differentiation, the subunits were separated electrophoretically and analyzed by immunoblotting with monoclonal antibodies against phosphothreonine, phosphoserine, and phosphotyrosine. Hemin caused dephosphorylation of the proteasome subunits mainly at tyrosines, which argues in favor of the participation of specific phosphatases in this process. On the other hand, hemin action led to a more intensive phosphorylation of several proteasomal 20S core particle subunits, with molecular masses from 25 to 30 kDa, at threonine, as well as the stimulation of the phosphorylation of at least one subunit (with a molecular mass about 38 kDa) at serine residues. Thus, during induction of differentiation the phosphorylation state of proteasome subunits did, in fact, change. As well, during induction of apoptosis (Tsimokha et al., 2006, 2007a,b,c), changes in the composition of the proteasome subunits are observed, which also could be associated with the regulation of specificity of these particle RNase activities. The results indicate that the ‘‘switching’’ of proteasomes to the nucleolysis of other mRNA groups could occur via the phosphorylation-dephosphorylation of subunits of these particles. The identities of the particular subunits for which the phosphorylation state changes during regulation of the specificity of RNase activity, as well as the cellular pathways responsible for the regulation of proteasome phosphorylation, are as yet unknown. However, the data concerned with phosphorylation of a subunits responsible for possession of RNase activity (a1, a5) and for RNA binding (a6), obtained on various organisms, from yeast (Fernandez-Murray et al., 2002) to higher plants (Umeda et al., 1997) and mammals (Beausoleil et al., 2004; Wehren et al., 1996; Zong et al., 2006), favour a supposition that subunit phosphorylation state could be a mechanism of regulation of proteasome-associated RNase activity. 4.1.2.2. N-Acetylation and N-terminal propeptide processing of proteasome subunits The autocatalytic removal of the N-terminal propeptides is one of the detailedly described proteasomal post-trancriptional modifications. These propeptides were shown to promote 20S particle assembly and protect the Thr1 active sites from acetylation and inactivation

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(Arendt and Hochstrasser, 1999). Generally this processing occurs within b subunits and leads to exposure of active site N-terminal threonine residues (Seemueller et al., 1996). In eukaryotic proteasomes such exposure takes place only in three subunits shown to be catalytically active (b1, b2 and b5), whereas propeptides of other b-type subunits are either intermediately processed (b6 and b7, the last was proposed to possess peptidase activity consisting in cleavage of bonds after small neutral amino acids (Unno et al., 2002) or remaining unprocessed (b3 and b4) (Groll et al., 1997). Co-translational N-a-acetylation by N-acetyltransferases is one of the most common protein modifications in eukaryotes. It occurs in 5090% of cases (Kimura et al., 2000; 2003), thus proteasomal subunits were demonstrated not to be an exception from the above rule: there are at least three N-acetyltransferases which 20S proteasome subunits found to be modified by (NAT1, MAK3, and NAT3). The a1, a2, a3, a4, a7, and b3 subunits were acetylated with NAT1, the a5 and a6 subunits were acetylated with MAK3, and the b4 subunit was acetylated with NAT3. Moreover, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the a5 and a6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were N-a-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region (Kimura et al., 2000). Experiments performed on yeast mutants have shown significance of N-terminal propeptide of catalytic 20S proteasome b subunits for protection of N-terminal catalytic threonine against N-acetylation (Arendt and Hochstrasser, 1999). The yeast 20S proteasome purified from either N-a-acetyltransferase deletion mutants or normal strains exhibited similar hydrolytic activities suggesting that N-a-acetylation does not significantly affect proteasome function in yeast (Kimura et al., 2000). Huang and co-authors (2001) found two isoforms of a2 and a7 subunits from Trypanosoma brucei 20S proteasomes to be acetylated at serine residues. Recent data demonstrate evidence that 15 subunits of human 26S proteasomes are acetylated at their N-termini. Eight subunits, Rpt3, Rpt5, Rpn1, Rpn2, Gankyrin (a non-ATPase subunit), a5, a6, and b4, are acetylated at the first methionine residue, whereas seven subunits, Rpt4, Rpt6, Rpn6, Rpn13/ADRM1, a4, a7, and b3, underwent Met1 processing followed by N-acetylation of the next amino acid residue. The N-terminal modifications of 12 subunits (Rpt1, Rpt4, Rpt5, Rpn2, Rpn8, Rpn9, Gankyrin, Rpn13/ADRM1, a4, a6, b5, and b6) have been identified for the first time for mammalian proteasomes (Wang et al., 2007). 4.1.2.3. Other modifications of proteasomal subunits Mass-spectrometric studies of 26S proteasome subunit modifications revealed a glycine residue at the N-terminus of Rpt2 subunit to be modified by myristoylation, both in humans (Wang et al., 2007), mice (Gomes et al., 2006), yeast (Kimura et al., 2003) and rice (Shibahara et al., 2002; 2004). Although the function of

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N-myristoylation in Rpt2 is not clear, the conservation of this modification on Rpt2 from yeast to human suggests an important function for Rpt2 myristoylation. Since this modification promotes protein-protein and protein-membrane interactions, it is permissible to speculate about a role of Rpt2 in the interaction of the 26S proteasome with membranes or other proteins (Wang et al., 2007). Experiments performed on yeast proteasomes by Demasi and co-authors revealed another modification of sulphur-containing amino acids, namely glutationylation of cystein residues (Demasi et al., 2001, 2003). Chimotrypsinlike activity of 20S proteasomes was shown to be inhibited by S-glutationylation more significantly than trypsin-like, while caspase-like activity was not changed (Demasi et al., 2003). A number of cytoplasmic and nuclear proteins were shown to be modified by O-linked N-acetylglucosamine (Wells et al., 2002). Similar to phosphorylation, serine, threonine and tyrosine residues could be underwent to O-glycosylation. Furthermore, dynamism and reversibility of both these modifications have led to proposition about the role of glycosylation in reversible blocking of phosphorylation site, temporary terminating the last modification. While phosphorylation of many proteins is a ubiquitinylation signal following by further proteasome degradation, prevention of phosphorylation via N-acetylglucosamine binding can extend a protein’s half-life (Rechsteiner et al., 1993). Analysis of proteasomes purified from lenses of ageing humans revealed a number of subunits to be glycated, glyco-oxydated and carboxymethylated (Viteri et al., 2004). Such oxidative modifications were shown to inhibit proteolytic activities of proteasomes, and while trypsine- and chimotrypsinelike activities decreased partially, the caspase-like one fully vanished. Therefore, in aging eye lens accumulation of oxidized protein may occur, that could cause trouble to cell survival following oxidative stress (Viteri et al., 2004). Mass-spectrometric analysis of rat hepatocyte proteins interacting with N-acetylglucosamine-specific antibodies has revealed an a6 subunit of 20S proteasome among them (Wells et al., 2002). Immunoblotting with specific monoclonal antibodies allowed to recognize at least 8 of 19 subunits of PA700 regulatory complex and at least 8 of 14 components of proteasome 20S core complex purified from Drosophila cells to be O-glycosylated (Sumegi et al., 2003). Rpt2 ATPase subunit of 19S regulatory complex was found to be O-glycosylated both in vivo and in vitro, while appearance of this modification promoted a decrease of proteasome activity via inhibitory effect on the subunit’s ATPase activity (Zhang et al., 2003). Recent studies of Wang and co-authors (2007) have revealed that Rpn2 subunit of 19S regulator, when not N-acetylated, contains Met1 in oxidized form—methionine sulphoxide. However, Met1 of a1 subunit from archea Haloferax volcanii proteasome was shown to be oxidized when N-terminus is acetylated either. Moreover, both the same modification and methionine

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oxidation were found in b-type proteasome subunits of this organism (Humbard et al., 2006). Such modifications can lead to a shift of protein’s isoelectric point and to appearance of additional spots on 2D map. Detailed studies of yeast proteasomes allowed to detect methionine oxidation in such 20S core subunits as a1 (Met209), b1 (Met4, Met146), b4 (Met1), and b7 (Met14, Met181) (Iwafune, 2002). Recent proteomic studies of rat liver proteasomes have shown a4 subunit to consist of two distinct spots on a 2D map, one of which was proposed to correspond a protein containing oxidized Met71 (Schmidt et al., 2006). Moreover, the same authors have detected appearance of cysteine residues oxidation (cysteine sulphonic acid). The above modification is quite rare, and being not a product of electrophoretic sample preparation, it could be supposed to have a significant biological role in regulation of protein structure and function (Schmidt et al., 2006). The comprehensive overview on proteasome-associated proteins and their influence on stability, activities and other characterictics of the complexes is presented in the review by Glickman and Raveh (2005).

4.2. Regulation of proteasome abundance in the cell and cellular compartments 4.2.1. Regulation of proteasome subunit expression Unfortunately, current knowledge of regulation mechanisms of proteasome subunit expression, limited to several models described, seems to be insufficient and requires further studies. The Saccharomyces cerevisiae yeast were shown to have own unique system of coordinated control of proteasomal gene expression: the upstream activating cis-element called ‘‘proteasome-associated control element’’ being a target sequence for the transcription factor Rpn4 that activates genes encoding proteasomal subunits (Mannhaupt et al., 1999). The above transcription factor was found to be degraded by matured proteasomes (Xie and Varshavsky, 2001). Rpn4 was also demonstrated to participate in upregulation of all proteasomal subunits upon treatment with proteasome inhibitors (Fleming et al., 2002), so it is proposed to be a cellular tool responsible for the compensation of proteasome inhibition. Examples of regulation of proteasome expression were revealed also in mammals. An evidence was demonstrated that levels of proteasome expression depend on proliferative state of the cell. Thus, expression of proteasomes in actively proliferating hematopoietic malignancies was shown to be abnormally high (Kumatori et al., 1990), whereas in vitro terminal differentiation of immature leukemic cell lines led to significant decrease of proteasome expression independently of both cell type and differentiating agent (Shimbara et al., 1992). On the other hand, in normal cells up-regulation was demonstrated for mitogen-induced resting T-lymphocytes (Shimbara

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et al., 1992). Recent data showed proteasome expression to be up-regulated by action of proteasome inhibitors (MG132 and lactacystin) both in normal (primary culture of rat smooth muscle cells) and malignant (human HeLa, T2, HEK293 and green monkey COS7) cells (Meiners et al., 2003). The phenomenon observed is suggested to be general for all cells tested. The increase of proteasome subunit production was observed for both constitutive components of 20S core complex and proteins of 19S regulatory particles, whereas subunits of 11S proteasome activator and interferon-inducible subunits were not up-regulated at all. Authors showed that proteasomes are upregulated at both transcriptional and translational levels, so the data obtained suggest mammalian cells to have a compensatory mechanism lying in de novo proteasome formation in response to the action of proteasome inhibitors (Meiners et al., 2003). Experiment concerned with proteasomal subunit b5 overexpression in cultured human lens epithelial cells resulted in induction of expression of other 20S core catalytic subunits (b1 and b2), i.e., upregulation of proteasomes via transfection by a plasmid carrying b5 subunit sequence has been observed (Liu et al., 2007). The above model could be considered as a tool for prevention of age-related decline of proteasome activity in human lens epithelium, which can lead to cataract formation and is thought to contribute to the ubiquitinated and carboxymethylated proteins accumulation detected in elderly lenses (Viteri et al., 2004). However, the mechanism of proteasome expression regulation is not understood yet. The action of proteasome inhibitors did not affect expression of PA28 regulatory complex in a number of cell types (Meiners et al., 2003), whereas up-regulation of the above proteasome activator was observed during maturation of dendritic cells (Ossendorp et al., 2005). Moreover, in PA28b gene promoter, an NF-kB active site was found, so, therefore, expression of PA28b could be regulated by transcription factors of NF-kB family. Induction of PA28b subunit was shown to provide production of PA28 complex. In contrast to other cells, regulation PA28 expression in mature dendritic cells was demonstrated to be interferon-independent (Ossendorp et al., 2005). 4.2.2. Control of proteasome intracellular localization and assembly Proteasomes in eukaryotic cells are found both in nuclei and cytoplasm. Several proteasomal subunits were demonstrated to contain specific amino acid sequences called nuclear localization signals (NLSs) (Nederlof et al., 1995; Wang et al., 1997). In rat liver cells 16% of proteasomes revealed to be localized in nuclei, 14% detected to be associated with endoplasmic reticulum, whereas others were shown to be located in cytosol matrix (Rivett et al., 1992). However, studies of spermatozoa proteasomes detected 20S core and PA28g activator near the neck where centrosomes located,

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whereas no proteasomes were detected within nuclei (Wojcik et al., 2000). In contrast, in early stages of embryonal development and in malignant cells proteasomes were found to be accumulated in nuclei (Klein et al., 1990; Kumatori et al., 1990). In the cell nucleus, proteasomes are found not only in association with chromatin. During interphase, proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci (Rockel et al., 2005). Proteasomal activity has been detected in isolated nucleoplasmic cell fractions (Scharf et al., 2007), in isolated and purified nuclear proteasomes (Tsimokha et al., 2006) and nuclei of living cells (Scharf et al., 2007). Thus, microinjection of ectopic fluorogenic protein ovalbumin revealed that proteasomal protein degradation occurs in distinct nucleoplasmic foci (Rockel et al., 2005). Therefore, the proteasomes are proteolytically active in distinct nuclear domains. However, nuclear proteolysis remains mainly uninvestigated and little is known about the control of nuclear functions of proteasomes. In recent studies of Feist and co-authors (2007) performed on various cells, a suggestion was made that the distribution of proteasomes in the nucleus and cytoplasm is dependent on the cell type and cell cycle, where it may reflect changes in cell metabolism. Indeed, intracellular proteasome distribution is caused by requirements of certain cellular compartment. Thus, for example, respectively short cell cycle duration in yeast requires proteasomes to be abundant in those compartments where it is necessary to cleave a number of short-lived proteins known to control cell cycle progression and secretory and membrane protein biogenesis (Enenkel et al., 1999). When compared different immunochemical stainings of various cells, it turned out that the relative affinity with respect to nuclear and cytosolic proteasomes varies between different anti-proteasomal antibodies, probably due to different affinities regarding structural properties of proteasomal complexes or different interactions with regulatory proteins in examined compartments of the cell. The above observation shows a wide diversity of proteasome subsets within a single cell (Feist et al., 2007). The mechanisms controlling proteasome intracellular localization remain poorly studied at present time. However, the majority of the 20S core complex subunits was found to be phosphorylated (Gomes et al., 2006; Rush et al., 2005; Wang et al., 2007; Zong et al., 2006), while at least six of them underwent the above modification at tyrosine residues (Benedict et al., 1995; Iwafune et al., 2002; Rush et al., 2005; Wehren et al., 1996; Zong et al., 2006). Since tyrosine phosphorylation of proteasome subunits has been suggested to be involved in nucleo-cytoplasmic transfer of proteasomes (Tanaka et al., 1990; Wang et al., 1997), the specific kinases and phosphatases are considered to be candidates for the role of proteasome controller. Thus, experiments performed on fissing yeast discovered a

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complicated system of proteasomal nuclear localization control, containing heat-inducible regulatory protein Cut8, shown to be an upstream positive regulator of Cek1 kinase, which, in its turn, could subsequently control localization of proteasome via subunit phosphorylation. At the same time, Cut8, being very unstable, could be a substrate for nuclear proteasomes. Cut8 may be a feedback sensor for the amount of proteasome locating to the nucleus (Takeda and Yanagida, 2005; Tatebe and Yanagida, 2000). The authors (Takeda and Yanagida, 2005) suggest that regulation of nuclear proteasome abundance through ubiquitination and destruction of the sensor and anchor Cut8 is a conserved mechanism as a Cut8 homolog has been discovered in flies. Another localization controller, the internal one, was found in Schizosaccharomyces pombe: the Rpn5 subunit of 19S proteasome activator was shown to be involved in regulation of not only nuclear localization, but also proper assembly of proteasome complex ( Yen et al., 2003). The activity of purified nuclear proteasomes (isolated from control and apoptotic human K562 cells) is regulated by phosphorylation of proteasome subunits (Tsimokha et al., 2006, 2007a,b,c). The other enzyme, poly(ADPribose) polymerase (PARP), is also involved in the regulation of drug (adryamycin)-induced nuclear proteasome activation (Ciftci et al., 2001). Proteasome disassembly under severe stress is one of the mechanisms of proteasome’s abundance control (Glickman and Raveh, 2005). 4.2.3. Export from the cells Proteasomes were shown to be identified in various cellular compartments (see Section 4.2.2). Besides different intracellular populations, thoroughly studied by investigators all over the world, the extracellular one was detected in early 90th ( Wada et al., 1993). This proteasome population was found in the blood serum of both healthy subjects and patients with such hematologic malignancies as acute leukemia, chronic myelogenous leukemia, non-Hodgkin’s lymphoma, and multiple myeloma (Feist et al., 2007; Jakob et al., 2007; Lavabre-Bertrand et al., 2001; Wada et al., 1993). The estimated serum proteasome levels in control group were significantly lower than in patients. The data obtained suggest the elevated levels of serum proteasomes in patients to be derived from tumor cells ( Wada et al., 1993). The above results are in accordance with previously reported evidence of an abnormally high expression level of proteasomes in human leukemic cells first demonstrated by Kumatori and co-authors (1990). More recent studies recognized circulating proteasomes in blood serum of patients suffering various autoimmune disorders—systemic lupus erythematosus, dermatomyositis/polymyositis, primary Sjogren’s syndrome, undifferentiated connective tissue disease, etc. (Egerer et al., 2002; Feist et al., 1996, 1999, 2007; Zoeger et al., 2006), and authors consider extracellular proteasomes to be a suitable marker of numerous pathological processes listed below. Serum proteasome population was supposed to be a sensitive indicator for tissue injury and cellular turnover reflecting the chronic and destructive

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activity of rheumatic diseases (Egerer et al., 2002). Lavabre-Bertrand and co-authors (2001) proposed plasma proteasome level measurement to be an instrument for monitoring patients with neoplastic disease. They suppose that circulating proteasomes could be a kind of nonspecific warning signal which will allow detection of a still unknown neoplastic process: moreover, the abnormally high level may prompt a search for myeloid disorders or solid tumors. In contrast, a low level may suggest a lymphoid disorder. The plasma proteasome level also could be a kind of prognostic factor for disease burden detection ( Jakob et al., 2007). In patients with lymphoid disorders, an increase in the circulating proteasome level may lead to the disease transformation into an aggressive form of lymphoma (Lavabre-Bertrand et al., 2001). And, finally, plasma proteasome level determination may help to quantify patient response to chemotherapy. One also might hypothesize that plasma proteasome evaluation may help to monitor treatments based on the use of proteasome inhibitors (Lavabre-Bertrand et al., 2001). However, recent data of the same research group (Stoebner et al., 2005) suggest circulating proteasomes to represent themselves a marker more of nonspecific inflammation than of early cancer. Zoeger and co-authors (2006) have shown chromatographically that circulating proteasomes could be subdivided into at least six subpopulations, whereas subpopulations of intracellular ones purified from various blood cells encounter not more than five subtype peaks. Circulating proteasomes were shown to be intact both in healthy persons and patients with autoimmune disorders; they were enzymatically active, too (Zoeger et al., 2006). However, qualitative differences between cytoplasmic and extracellular proteasome populations and specificity of exported population were demonstrated by Kulichkova and colleagues (2004b). Thus, serum population of proteasomes retained enzymatic activities characteristic for these particles, whereas their both proteolytic and RNase activities differ from those obtained for intracellular proteasomes. Specific features of subunit content were also found in extracellular proteasomes. And finally, activities of these proteasome populations were shown to depend on physiological state of the cell. Thus, induction of apoptosis led to export of specific proteasome subpopulation with peptidase activities different from ones of extracellular proteasomes from control cells (Kulichkova et al., 2004b).

5. Reprogramming of Proteasomes at Immune Response, Differentiation and Apoptosis It was mentioned above that the proteasomes play important roles in most cellular processes. To perform their functions, proteasomes must be under a tight regulatory control and change their subunit composition and

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enzymatic activities to adapt them to the requirements of the each of the cellular processes during the cell life. Phosphorylation at serine, threonine, or tyrosine residues of regulatory proteins is the key event in signal transduction and cell cycle progression (Coux et al., 1996). Moreover, various protein kinases are involved in induction of apoptosis (Bandyopadhyay et al., 2004; Contri et al., 2005; Jin et al., 2005; Sabatini et al., 2004; Wang et al., 2005). Phosphorylation of substrates and enzymes also plays important roles in the ubiquitin-proteasome pathway (Glickman and Ciechanover, 2002; Wojcik and DeMartino, 2003). The 26S proteasome is posttranscriptionally modified by phosphorylation as well as by N-acetylation, glycosylation, 4-hydroxy-2-nonenal-alkylation in various species (Bose et al., 1999; Claverol et al., 2002; Farout et al., 2006; Fernandez Murray et al., 2002; Kimura et al., 2000; Schmid et al., 1993; Sumegi et al., 2003; see Chapter 4.1). These modifications of proteasomal subunits are intensively studied at present (see Section 4.1.2). Moreover, at present the changes of proteasomes proteolytic activity are widely investigated during various cell processes and under the action of different stimuli on a cell (Abramova et al., 2005; Ahn et al., 1991; Beyette et al., 1998; Ebisui et al., 1995; Hayashi and Goto, 1998; Kulichkova et al., 2004b; Low et al., 2001; Shibatani et al., 1996). Next, we consider in more detail changes in proteasomes during immune response, differentiation and apoptosis.

5.1. Changes of proteasome at immune response Under conditions of an intensified immune response, many eukaryotic cells adapt ubiquitin–proteasome system to the protein-breakdown process for optimized generation of antigenic peptide epitopes (see Section 3.4). Thus, as already mentioned above, during an immune response to pathogens, the pro-inflammatory cytokine INFg and tumor necrosis factor TNF-a are released and induce the replacement of constitutively expressed catalytic subunits of the proteasomes (b1, b2, b5) with subunits LMP2, LMP7, and MECL-1. Proteasome activity is further changed by the IFNg-mediated induction of the proteasome regulator PA28a/b (Groettrup et al., 2001b; see Section 3.4). IFNg greatly increases the levels of the mRNAs encoding LMP2 and LMP7, and the composition of the proteasome is changed in response to stimulation by IFNg, due to assembly of newly synthesized subunits (Aki et al., 1994). Interestingly, IFNg dramatically stimulates the trypsin-like and chymotrypsin-like proteasomal activities and inhibits the peptidyl-glutamyl-peptide hydrolyzing activity (Aki et al., 1994). After treatment of human embryonic lung L-132 cells with IFNg, the level of 26S proteasomes decreases and at once there is an increase in proteasome subcomplexes PA28. At that, free 19S regulatory complexes are not detected (Bose et al., 2001). It is significant that

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after IFNg treatment, the level of phosphorylation of proteasome subunits is decreased (Bose et al., 2001). Thus, the IFNg induces decrease of the phosphorylation of a3 (C9) and a7 (C8) proteasomal subunits (Bose et al., 2001). The decrease in phosphorylation of 19S component–ATPase subunit Rpt2 (S4) after IFNg treatment was also found (Rivett et al., 2001). Thus, modification of the proteasomes subunit composition and their functions under the action of IFNg suggests the reprogramming of the proteasomes, directed on specific function during immune response.

5.2. Proteasome reprogramming at differentiation Several studies have also shown the proteasome reprogramming during differentiation. For example, the molecular assembly and the proteolytic activity of certain 26S proteasome subunits undergo changes during starfish oocyte maturation induced by a maturation-inducing hormone, L-methyladenine (Sawada et al., 1999). Moreover, at least one component of 26S proteasomes changes during Xenopus oocyte maturation (Tokumoto et al., 1999a). Furthermore, the reprogramming of proteasomes at cellular differentiation is evidenced by changes in subcellular distribution and in the subunit composition of proteasomes during myogenic differentiation of satellite cells (Foucrier et al., 1999) and human lymphoblastic and monoblastic U937 cell line (Baz et al., 1997; Bureau et al., 1997; Henry et al., 1997). Other studies were focused on human proerythroleukaemic cell line K562 (Mittenberg et al., 2002a,b, 2007). The induction of erythroid differentiation of these cells by hemin is accompanied by changes in the phosphorylation state of several proteasome subunits at tyrosine, threonine, and serine residues (Mittenberg et al., 2007). Interestingly, treatment of K562 cells leads to redistribution of proteasomes and their migration mainly to the cytoplasm (Mittenberg et al., 2002b). The endoribonuclease activity of proteasomes also undergoes changes during differentiation of K562 cells (Mittenberg et al., 2002a,b, 2007).

5.3. Apoptosis-induced changes of proteasomes In thymocytes, changes in proteasomal proteolytic activity during glucocorticoid dexamethasone-induced apoptosis have been found (Beyette et al., 1998). So the dexamethasone treatment causes a decrease of the peptidylglutamyl peptide hydrolase, trypsin- and chymotrypsin-like activities of proteasomes. It is significant that the decreases of two proteasomes activities (peptidylglutamyl peptide hydrolase and trypsin-like) were canceled by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased

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further in the presence of this caspase inhibitor. The authors suggest that these changes of proteasomal proteolytic activity during the apoptosis in thymocytes may be responsible for the turnover of specific proteins, leading to apoptosis (Beyette et al., 1998). In tobacco hawkmoth, Manduca sexta after the appearance of the adult insect from its pupal cuticle, the intersegmental muscles undergo apoptotic regression in response to declining levels of the steroid hormone (Schwartz and Truman, 1983). At the same time, the proteasomal proteolytic activity increased about ninefold (Dawson et al., 1995; Jones et al., 1995). This increase of proteasomal proteolytic activity is correlated with the increase of the cellular amounts of the proteasomes and with the incorporation of new subunits into the 20S proteasomes (Dawson et al., 1995; Low et al., 1997). Furthermore, the proteasomes isolated from condemned muscles contained several new unidentified subunits that were not detected in the purified proteasomes from precommitment muscles (Dawson et al., 1995; Jones et al., 1995). Such changes in both proteasomal proteolytic activity and subunit composition during development of Manduca sexta show a reprogramming of the proteasomes that might result in an enhanced proteasomal degradation of certain ubiquitinated cellular anti-apoptotic proteins, leading to apoptosis of intersegmental muscle cells (Naujokat and Hoffmann, 2002). Interestingly, the accumulation of proteasomes in intersegmental muscle cells and thymocytes after induction of the apoptosis has been found (Beyette et al., 1998; Dawson et al., 1995; Jones et al., 1995). The increase of the proteasomes concentration together with regulatory reprogramming may facilitate the rapid proteolysis of proteins which function as anti-apoptotic regulators (Dawson et al., 1995; Low et al., 2001). Thus, the changes in proteasome subunit composition and proteolytic activity showing reprogramming of the proteasome have been observed during apoptosis of normally growing tissues. However, for a long time there have not been any reports on the changes of structure and function of the proteasomes in neoplastic tissues undergoing apoptosis. Recently, several works devoted to reprogramming of proteasomes during the apoptosis in leukemic cells have appeared. The proteasome proteolytic activity is changed under the action of the apoptotic inductors (doxorubicin [DR], diethylmaleate [DEM]) (Tsimokha et al., 2006, 2007a,b,c). At that, the apoptosis induction changes the specificity of the proteolytic activity of the proteasomes, isolated from both cytoplasm and nuclei of K562 cells. Thus, the trypsine- and chymotrypsine-like activity of proteasomes isolated from nuclei and cytoplasm of DR-induced cells is increased compared to the activity of these proteasomes from control cells K562 (Tsimokha et al., 2007a,c). Moreover, the endoribonuclease activity of proteasomes isolated from cytoplasm and nuclei of K562 cells undergoing apoptosis is also changed (Kulichkova et al., 2004b; Mittenberg et al., 2007; Toktarova et al., 2004;

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Tsimokha et al., 2006, 2007a,b,c). The characteristics of RNase activity of different proteasomal subpopulations differ. These changes of RNase activity of the proteasomes suggest the participation of proteasomal ribonuclease in the regulation of mRNA stability during the induction of apoptosis. For example, proteasomes can control the level of the apoptotic proteinsregulators both by ubiquitin-dependent proteasomal proteolysis and by the specific endonucleolysis of mRNAs encoding these proteins. And, thus, the cell can more quickly and effectively inactivate the anti-apoptotic genes and/or activate the pro-apoptotic genes during the realization of the death program. Treatment of K562 cells with the apoptotic inductor (DEM or DR) leads to yet unidentified modifications of proteasomal subunits (a1-a7 and b2, b3, b7) including catalytic subunits associated with proteolityc (b2) and RNase (a5/zeta, a1/iota/alpha type 6) activities (Tsimokha et al., 2006, 2007a,b,c). Moreover, the proteasomes isolated from control and apoptosisinduced K562 cells differ in the phosphorylation state of a number of subunits on threonine, serine and tyrosine residues (Tsimokha et al., 2006, 2007a,b,c). The specific changes of phosphorylation state of nuclear and cytoplasmic proteasomes and regulation of their phosphorylation state under the action of apoptotic inductors suggest the existence of specific regulatory cellular pathways, involving specific protein kinases and phosphatases. The observed changes in the phosphorylation state of specific subunits suggest that these modifications are necessary for the specific proteasomal functions in apoptosis in K562 cells. Thus, the subunit composition and enzymatic activities of proteasomes are changed in K562 cells undergoing apoptosis, and therefore the apoptosis of neoplastic cells also involves reprogramming of proteasomes.

6. Proteasomes in Regulation of Different Levels of Gene Expression 6.1. Action at multiple stages of transcription process A growing body of evidence reveals that proteasomes are involved in the control of different levels of gene expression: transcription process, messenger RNA stability and translation. The physical and functional association of subunits of the 20S core and 19S regulatory proteasome subparticles with approximately 6400 yeast genes has been studied (Sikder et al., 2006). The results revealed the crosslinking of the intact 26S proteasomes to most genes, while several hundred genes interacted with either the 20S or 19S subparticles. Correlation of many of these associations with gene expression levels and the presence of RNA polymerase II has been demonstrated. These data suggest

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independent functions for whole proteasome particles and for the proteasomal subparticles in transcription. Indeed, genetic and molecular biology evidence briefly summarized below, confirms that the intact 26S proteasome as well as their subparticles and subunits are involved in transcriptional regulation. The proteasomes participate in the regulation of multiple stages of gene transcription process through proteolytic and nonproteolytic activities; however, few targets of proteasomal regulation have been studied in detail. Proteasomes are implicated in transcription factor processing, proteolysis of co-activators, chromatin modifications, control of elongation, termination and other stages of transcription. Proteolytic roles of the proteasome in transcription involve the timely regulated stimulation of transcription factors via their processing or degradation of inhibitory proteins. The activity of a number of transcription factors and their regulation depends on this process, named ‘‘regulated ubiquitin/proteasome-dependent processing.’’ Proteins of the mammalian NF-kB family and the yeast proteins SPT23 and MGA2 are controlled by this pathway (see review of Rape and Jentsch, 2004). The proteolytic activity of the proteasome is also necessary for the stable recruitment of RNA pol II at promoters and re-initiation of transcription possibly through the proteolysis of the transcriptional activators by the proteasome. For example, repression of proteasome proteolytic activity blocks the ability of the yeast activator Gcn4 to recruit RNA pol II to promoters (Lipford et al., 2005). Accurate transcription termination depends on proteolytic activity of 26S proteasomes as indicated by the increased read-through of a transcription termination site in result of their activity inhibition (Gillette et al., 2004). Interestingly, the 20S proteasome catalytic b subunit LMP2 (low molecular mass polypeptide 2), associated with peptidylglutamyl peptidase activity, interacts directly with the steroid receptor coactivator (SRC)-interacting proteins. The recruitment of the 20S b subunit LMP2 by SRC coactivators is necessary for cyclic association of estrogen receptor (ER)-regulated transcription complexes on ER targets (Zhang et al., 2006). The proteasome proteolytic activity is required also for continued hormone response. This activity modulates glucocorticoid hormone receptordependent gene transcription by regulating turnover and recycling of receptor/transcriptional-DNA complexes (Kinyamu et al., 2007). The involvement of proteasomal proteolytic activity in transcriptioncoupled DNA repair is indicated by ubiquitin-mediated proteolysis of the elongating form of the RNA polymerase II in response to DNA damage (Kleiman et al., 2005; Krogan et al., 2004; Reid and Svejstrup, 2004; Somesh et al., 2005). The destruction of RNA pol II blocks transcription until DNA damage can be repaired.

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The 19S proteasome subunits, particularly the base ATPases, affect transcription of a number of genes through a nonproteolytic mechanism. The 19S complex is crucial for efficient elongation of RNA polymerase II in vitro and in vivo. Thus, yeast strains carrying mutant alleles of genes SUG1 (Rpt6) and SUG2 (Rpt4), encoding ATPases of 19S compplex, exhibit phenotypes indicative of elongation defects. Moreover, in vitro transcription was inhibited by immunodepletion of subunit Sug1, and the elongation was restored by addition of immunopurified 19S complex. The physical interaction of the elongation factor Cdc 68 with the 19S subparticle is suggested by the results of their coimmunoprecipitation. Inhibition of the proteasomal proteolytic activity did not affect elongation of transcription (Ferdous et al., 2001). Proteasome implication in dissociation of elongation complexes is indicated by the enrichment of proteasomes and RNA polymerase II binding at ribosomal protein genes in yeast proteasome mutant (Auld et al., 2006). The 19S subunits exhibit DNA-helicase and protein-chaperone activities. ATPase subunit SUG1 is a 30 -50 DNA-helicase whose activity depends on the intact ATP binding domain (Fraser et al., 1997). Therefore, 19S subcomplex can influence interactions of components of the transcription machinery with DNA via DNA-helicase activity, particularly, change protein-DNA interactions. The hypothesis has been offered that subunits of the 19S complex might affect transcription elongation and other stages of transcription process through their protein-chaperone activity, influencing protein folding/unfolding (Collins and Tansey, 2006). The 19S proteins can act at early steps in the transcription process: in the preinitiation interaction of co-activator (‘‘SAGA’’ complex containing 15 subunits, among them histone acetyltransferase) with a target promoter during activation of several genes. The authors (Lee et al., 2005) suggest that the 19S proteasome regulatory particle, possibly due to its chaperone activity, somehow alters SAGA conformation to stimulate its recruitment to transcriptional activator. Thus, the ATPase components of the 19S subparticle were shown to be nesessary to facilitate SAGA recruitment to promoters by transcriptional activators such as Gal4p (Lee et al., 2005). More recently (Ferdous et al., 2007), a new nonproteolytic activity of the proteasomal ATPases, i.e., the active destabilization of activatorpromoter complexes (between Gal4-VP16 and Gal4 binding sites) was discovered. This reaction depends on the presence of the activation domain and ATP. The obtained data suggest that proteasomal ATPase 19S subunits are required for active turnover of the activator-promoter complexes. Yeast regulatory proteasome 19S subparticle was found to be physically associated with many general transcription factors, including components of yeast FACT (Cdc68/Pob3), TFIID, TFIIH, and the RNA polymerase II holoenzyme; moreover, the whole 26S proteasome interacts with regions of the yeast inducible genes GAL1, GAL10, and HSP82, including the 30 ends, in a transcription-dependent fashion. These results demonstrate that

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proteasome subunits can interact both with components of the transcriptional machinery and gene regions (Sun et al., 2002). Chromatin structure is also affected by 19S subunits. For example, ATPases subunits of yeast 19S complex: Rpt4 and Rpt6 are necessary for methylation of histone H3 at lysine residues 4 and 79 that is a signal defining active sites of transcription (Ezhkova and Tansey, 2004). The suggestion has been put forward (Collins and Tansey, 2006) that bound 19S subunits affect local chromatin structure to facilitate recruitment of appropriate methyltransferases to their target sites on the histone molecules. Interestingly, binding of 19S subunits to sites of active transcription depends on ubiquitylation of histone H2B (Ezhkova and Tansey, 2004). Whether the subunits of 19S interact with DNA (due to their helicase activity) or histones (via chaperone activity) or both remains to be studied as well as the time of their appearance at gene regions. Chromatin immunoprecipitation assays have demonstrated contradictory results: subunits of the 19S complex were found at promoter sequences when the genes are already active (Gonzalez et al., 2002) as well as prior to activation (Ezhkova and Tansey, 2004). This recruited complex does not contain subunits of the 20S core particle. Furthermore, in reorganization of chromatin structure, proteolytic activity of proteasome is also involved. Thus, during glucocorticoid – induced transcription, inhibition of this activity increases tri-methyl histone H3K4 levels with a corresponding accumulation of this modification on glucocorticoid receptor-regulated promoters (Kinyamu et al., 2007). Probably, both proteolytic and nonproteolytic activities of the proteasome are involved in remodeling of chromatin structure. Moreover, proteasome association with chromatin, and transcription factor processing are interrelated. Thus, the transcription factor Gcn4 is destructed only when bound to the target promoter (Lipford et al., 2003). Finally, transcriptional regulation by the proteasome is highly complex, these particles are involved at multiple stages of the process, and, moreover, they act at the same stages through different mechanisms. The recent evidence also suggest that proteasomes and their subparticles might be independently involved in different steps of transcription of different genes. Although the main data were obtained with yeast cells, the evidence of the recent works with higher eukaryotic cells, cited above, and the homology between eukaryotic and yeast transcription factors allows assuming occurrence of similar mechanisms in higher eukaryotes.

6.2. Participation in the regulation of posttranscriptional stages of gene expression 6.2.1. Translation The data suggesting proteasome engagement in coordinated regulation of protein translation and degradation, although via two different mechanisms, have been obtained.

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Proteasomes have been shown to participate in selective alteration of different messenger RNA (mRNA) translation (Baugh and Pilipenko, 2004). The translation initiation factors eIF4G, a subunit of eIF4F, and eIF3a, a subunit of eIF3, are degraded by ubiquitin-proteasome pathway. The cleavage of eIF4G or eIF3a selectively affects the assembly of ribosomal preinitiation complexes on different cellular and viral mRNAs in an in vitro system probably due to competition of different RNA molecules for translation factors. Inhibition of proteasomal proteolytic activity prevents degradation of both factors, restores assembly of ribosomal complexes in vitro, and differentially affects translation of different mRNAs in vivo. The results of these studies allow suggesting the involvement of the changes in proteolytic activity of proteasomes in coordination of selective protein synthesis and degradation. Proteasome implication in the regulation of transcription of ribosomal protein (RP) genes allowed assuming a direct feedback mechanism of maintaining homeostasis between protein synthesis and protein degradation (Auld et al., 2006). In conditions under which cellular proteins accumulate, including proteasome mutants (Auld et al., 2006) and during inhibition of proteasomal proteolytic activity (Dembla-Rajpal et al., 2004; Jang and Wek, 2005), the decrease of the expression of RP genes in yeast cells was found. This observation suggests that proteasomes are required to activate transcription of the RP genes. Auld and Silver (2006) has supposed that the presence and activity of the proteasome at ribosomal protein genes could provide a cellular mechanism of responding to defects in protein degradation by slowing down transcription of the protein biosynthesis machinery. The changes in the activity of proteasomes would influence coordinative degradation of cellular proteins, of ribosomal proteins’ transcription and the affectivity of protein synthesis in the cells. 6.2.2. Messenger RNA stability At present there is no information about the role of the RNase activity of proteasomes in the cell (see Section 2.2). However, regulation of this activity in response to extracellular stimuli argues for its possible involvement in controlling the life time of mRNA molecules by these stimuli or, in other words, for a new mechanism controlling RNA stability in the cell. The phosphorylation dependence of the RNase activity of proteasomes suggests that these particles can represent a link between signalling pathways and mRNA stability; however, the cellular signalling pathways controlling ribonuclease activity of proteasomes remain to be elucidated. One of the key levels of gene expression control is the regulation of mRNA degradation rates. It becomes increasingly clear that signal transduction pathways affect mRNA stability. Specific cis elements in mRNA molecules necessary for mRNA turnover are recognized by signal responsive

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trans-active factors that influence signal-regulated RNA decay (Garneau et al., 2007; Shim and Karin, 2002; Tourriere et al., 2002). The RNA-binding activity of a number of trans-acting factors is modulated by phosphorylation (Tourriere et al., 2001). The mRNA decay rate can be regulated not only by the activity of protective proteins, but also may be directly controlled by changing activity of ribonucleases (Tourriere et al., 2001). Phosphorylation could also modulate activity of specific endonucleases. For instance, G3BP protein harbors the endoribonuclease activity that cleaves mouse c-myc 30 UTR in the phosphorylation-dependent manner. Besides, the phosphorylation status of G3BP in the cell is modulated by RasGAP cascade (Tourriere et al., 2003). The RNase activity associated with 26S proteasomes represents the new example of regulated endoribonuclease activity. Moreover, Laroia and co-authors (1999, 2002) have obtained data favoring the other mechanism of proteasome involvement in the control of the messenger RNA molecules lifespan. These authors have shown that 26S proteasomes are involved in the control of stability of specific mRNAs through proteolysis of mRNA-binding trans-acting proteins: AUFs. Thus, it is possible that proteasomes could participate in the both processes (proteolysis and nucleolysis) determining mRNA stability regulation. Furthermore, the evidence obtained so far allows proposing the proteasomes involvement in the coordinated control of degradation of proteins and mRNAs encoding these proteins. However, much additional work is needed, in particular, in vivo studies, to elucidate function of the proteasome-associated RNase activity in cellular processes. In all, the growing body of evidence favours the proteasome involvement in coordinated regulation of multiple stages of gene expression, placing the mechanisms of the control of proteasomal activity in the nodes of converge of gene expression regulatory pathways.

7. Concluding Remarks Recent evidence show that proteasomes are involved in the control of the main cellular processes as well as in the main stages of gene expression and that these complexes themselves are subjected to tightly organized regulation. However, the cellular pathways of this regulation (enzymes responsible for modifications of subunits, control pathways of their activity, mechanisms of the regulation of subunit expression, of cellular localization and others) remain mainly uninvestigated. Furthermore, despite great progress in proteasome studies, many other questions remain unanswered, and among them is the problem of specificity of proteasome involvement in transcription and other levels of gene

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expression—whether or not there is selectivity in the action of specifically modified proteasome subpopulations in the expression of different genes. Studies of specialized subpopulations of these complexes participating in responses to different external and internal signals and in various cellular processes are at the initial stage at the moment. Moreover, at present there is no information about the role of the RNase activity of proteasomes in the cell and the functional significance of the proteasome export from the cells.

ACKNOWLEDGMENTS This work was supported by Russian Foundation for Basic Research (project No. 08-0400834) and St. Petersburg’s Scientific Center of Russian Academy of Sciences.

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Permissive and Repulsive Cues and Signalling Pathways of Axonal Outgrowth and Regeneration Sheng T. Hou,* Susan X. Jiang,* and Robert A. Smith† Contents 1. Introduction 2. Neuritogenesis and Developmental Patterning 2.1. Role of neurotrophins 2.2. Extracellular matrix cues 2.3. Establishing polarity and axonal specification 3. Repulsive Guidance Cues in Axonal Pathfinding 3.1. Repulsive ligands 3.2. Receptors for repulsive guidance cues 3.3. Intracellular signalling pathways for repulsive guidance cues 4. Guidance Cues in Axonal Damage and Neuronal Death 4.1. Ischemic neuronal death and axonal damage 4.2. Semaphorin/neuropilin in neuronal death 4.3. Netrin-1/UNC/DCC in neuronal death (the dependence receptor theory) 4.4. RGM/Neogenin dependence receptors in neuronal death 4.5. CRMP in neuronal death and survival 4.6. CRMP modulation by calpain and CaMK during neuronal death 5. Guidance Cues and Synaptic Plasticity in Stroke Brains 6. Evidence for Guidance Cues as Therapeutic Targets 7. Concluding Remarks and Future Perspectives Acknowledgment References

* {

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Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, K1A 0R6, Canada Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, University of Glasgow, G12 8QQ, Scotland

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00603-5

# 2008 Elsevier Inc. and Her Majesty the Queen in right of Canada All rights reserved.

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Abstract Successful axonal outgrowth in the adult central nervous system (CNS) is central to the process of nerve regeneration and brain repair. To date, much of the knowledge on axonal guidance and outgrowth comes from studies on neuritogenesis and patterning during development where distal growth cones constantly sample the local environment and respond to specific physical and trophic influences. Opposing permissive (e.g., growth factors) and hostile signals (e.g., repulsive cues) are processed, leading to growth cone remodelling, and a concomitant restructuring of the cytoskeleton, thereby permitting pioneering extension and a potential for establishing synaptic connections. Repulsive cues, such as semaphorins, ephrins and myelin-secreted inhibitory glycoproteins, act through their respective receptors to affect the collapsing or turning of growth cones via several pathways, such as the Rho GTPases signalling which precipitates the cytoskeletal changes. One of the direct modulators of microtubules is the family of brain-specific proteins, collapsin response mediator protein (CRMP). Exciting evidence emerged recently that cleavage of CRMPs in response to injury-activated proteases, such as calpain, signals axonal retraction and neuronal death in adult post-mitotic neurons, while blocking this signal transduction prevents axonal retraction and death following excitotoxic insult and cerebral ischemia. Regeneration is minimal in injured postnatal CNS, albeit the occurrence of some limited remodelling in areas where synaptic plasticity is prevalent. Frequently in the absence of axonal regeneration, there is not only an inevitable loss of functional connections, but also a loss of neurons, such as through the actions of dependence receptors. Deciphering the cues and signalling pathways of axonal guidance and outgrowth may hold the key to fully understanding nerve regeneration and brain repair, thereby opening the way for developing potential therapeutics. Key Words: Axonal development, Growth cone, Growth factors, ECM, Guidance cues, Apoptosis, Regeneration, Myelin-secreted inhibitory molecules. ß 2008 Elsevier Inc. and Her Majesty the Queen in right of Canada

1. Introduction The failure of axons of the adult mammalian central nervous system (CNS) to regenerate after lesion or damage does not represent an intrinsic inability of CNS axons to grow, but rather the non-permissive nature of the CNS environment. For a brief period, the CNS is able to support sprouting of axons at the lesion site, but the growth cones soon adopt a swollen dystrophic morphology typical of growth inhibition (Dickson, 2002; Schnorrer and Dickson, 2004; Liu et al., 2006). Evidence derived from genetic and in vitro studies has demonstrated that CNS environment may

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exert four kinds of actions on growth cones, namely, chemorepulsion, chemoattraction, contact independent repulsion and contact dependent attraction. For example, injured CNS axons are arrested in the adult injured brain partly due to the presence of growth inhibitory chemorepulsive and contact independent repulsive ligands secreted from oligodendrocytes/ myelin, reactive astrocytes and fibroblasts in the scar tissue. Neurons must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction and neuronal death. It is therefore not surprising that strategies to promote regenerative axonal growth in the CNS after brain injury are thwarted by the plethora of inhibitory ligands and the ligand promiscuity of some of their receptors (Carmeliet and Tessier-Lavigne, 2005; De Wit and Verhaagen, 2003; Pasterkamp and Kolodkin, 2003; Pasterkamp and Verhaagen, 2001). In the context of cerebral ischemia-induced brain damage, the molecular and biochemical mechanisms involved in the retraction and collapse of the axonal network remains unclear. One of the early morphological changes accompanying excitotoxicity-induced cell death in cultured neurons is the retraction/collapse of the neuritic network (S. T. Hou and S. X. Jiang, unpublished observations), which strongly argues that axonal damage occurs before the emergence of the typical morphological hallmarks of neuronal death (Deckwerth and Johnson, 1993, 1994). Typically, axonal degeneration is manifested by irregular blebbing of axons with thinning and fragmentation, followed by retraction and collapse of the axonal network. While axonal damage may be an outcome of the death process occurring within the cell body, more importantly, it may, in and of itself, be a trigger for death of the whole neuron. In studies of white matter damage, axonal injury in response to ischemia is associated with increased axonal membrane permeability with excess Naþ and/ or Ca2þ influx into the axon (Stys and Jiang, 2002; Stys, 2004). This imbalanced Ca2þ influx activates deleterious cascades of locally localized intracellular proteases and subsequent breakdown of cytoskeletons and disturbance of axon transport leading to degeneration and neuronal death (Aarts and Tymianski, 2004; Chan and Mattson, 1999; Hara and Snyder, 2006). Given that the expression of chemorepulsive signals, such as semaphorin-3A, Ephrins (Ephs) (Beck et al., 2002; Fujita et al., 2001; Goldshmit et al., 2006) and their respective receptors are elevated in the brain following cerebral ischemia, it is easy to envisage that the affected neurons may undergo repulsive guidance cue-mediated axonal retraction/ collapse and neuronal death. For example, class 3 semaphorins are inhibitory ligands which are secreted by the scar tissue and neurons in adult brains following traumatic injury and cerebral ischemia (Beck et al., 2002; Fujita et al., 2001; Zhang et al., 2001). However, the precise pathological significance of semaphorin-3A expression in vivo and its relationship with axonal damage, regeneration and neuronal death remain unclear.

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Stroke is the result of sudden onset of loss of neurological function because the blood flow to the brain has been cut off (Hou and MacManus, 2002). Functional outcome of brain ischemia is the result of a complex interplay between permanent damage and long-term plasticity, which may be beneficial or detrimental. Accordingly, limiting tissue damage and promoting useful plasticity are the two pillars of modern stroke management (Baron, 2005). However, no clinical effective neuroprotective or regenerative therapeutic compounds are currently available (Buchan and Kennedy, 2007; Papadakis and Buchan, 2006; Zivin, 2007). Investigation of repulsive guidance cues and their pathological role in neuronal regeneration failure may shed light on ways to design therapeutics for preserving axons, neurite networks and encouraging axonal outgrowth and regeneration. In this review, efforts are made to highlight the current understandings of inhibitory molecules and their roles in axonal degeneration and regeneration in the context of stroke-induced brain damage and neuronal death. We aim to present an overview of the literature on in vitro studies of the effects of the guidance cues that have elucidated many of the mechanisms underlying neuronal devlopment, and further highlight their important in vivo relevance in order to provide readers with insights into the basis of axonal guidance molecules and their potential prospects for modulation in brain regeneration and repair, particularly following stroke.

2. Neuritogenesis and Developmental Patterning The principal mechanisms involved in both neurite extension and axonal pathfinding in the developing nervous system rely upon the reorganization of their cytoskeletal elements, induced by a number of microtubule-associated proteins (MAPs) (Bouquet et al., 2004; Dehmelt and Halpain, 2004; Dent and Gertler, 2003) and Rho-GTPases (Govek et al., 2005; Li et al., 2006; Luo, 2000; Riederer, 2007; Thies and Davenport, 2003), and which result from the stimulation of an array of the surface receptors that bind ephrins, netrins, semaphorins and other major ligands (Gallard et al., 2005; Huber et al., 2003). Growth cones, the motile enlargements at the distal tips of the extending processes, first described by Ramon y Cahal over a century ago (1890), are instrumental in detecting these spatially and temporarily distributed (and frequently repulsive) molecular guidance cues (for review, see Gordon Weeks, 2004; Letourneau et al., 1991; Mueller et al., 2006), thereby ensuring the successful establishment of neuronal connections both in the embryo, and in early post-natal development in vertebrates (Tessier-Lavigne et al., 1996). Much of our knowledge concerning growth cone mobility and neurite extension has come from the

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study of cultured neurons (Smith and Jiang, 1994) since, in addition to monitoring fixed material (Lein et al., 1992), their dynamic properties can be visualized in living cells by use of sophisticated microscopy and automated time-lapse video imaging (Anderson et al., 2006; Keenan et al., 2006; Parpura et al., 1993), and by fluorescent dye techniques (Davenport et al., 1993; Taylor et al., 2005). Studies of living growth cones allow observations of the responses associated with changes in environmental cues (Kuhn et al., 1995), with developmental stage (Blackmore and Letourneau, 2006), and following CNS axonal injury and regeneration (Taylor et al., 2005). The growth cone consists of a central (C-) cytoplasmic domain, rich in microtubules and organelles, which is surrounded by a peripheral (P-) cortical domain from which protrude numerous fine finger-like projections, known as filopodia (abundantly rich in actin filaments), and flat, sheet-like lamellipodia (Gordon-Weeks, 2004). Unlike in the neurite shaft, where cytoskeletal microtubules exist in straight bundles, on entering the C-domain they mainly become de-fasciculated, and are present singly, either retaining a straight form or becoming curved and bent (Yamada et al., 1971). Microtubules exist as either short or long, hollow filaments composed of a- and b-tubulin heterodimers that bind GTP. They are intrinsically polarized (see Sections 2.3 and 3.3), with distally oriented ‘‘plus’’ (or fast-growing) and more centrally oriented ‘‘minus’’ (slow-growing) ends (Heidemann et al., 1981). The microtubules undergo cycles of slow, continuous growth due to a dynamic instability, and due to hydrolysis of GTP bound to the b-tubulin heterodimers, which permits restructuring and growth cone shape changes (Gordon-Weeks, 2004). A role for tubulin cofactor B (TBCB), localized in the transition zone between growth cones and the neurite shaft, in regulating the dynamics and stabilization of the microtubules has been shown recently by gene silencing studies, where enhanced axonal growth was observed and, conversely, where TBCB overexpression was seen to lead to neuronal degeneration (LopezFanarraga et al., 2007). A number of microtubule binding proteins are also important in ensuring that bending and turning movements of the growing neurites can occur (Dent and Gertler, 2003). Microtubules from the C-domain are also frequently aligned with the long axis of the filopodia where they become associated with the core of actin filaments (Letourneau, 1983), playing a role in permitting growth cones to turn in response to environmental cues (Zhou et al., 2004), and in regulating the cytoskeleton during neuronal outgrowth (Morii et al., 2006). The form of both the lamellipodia and filopodia are dependent on the polymerization and organization of F-actin filaments which, once formed, are retrogradely transported to the centre of the growth cone to be rapidly broken down into subunits for further recycling distally, thereby allowing consequent forward extension of the growth cone (Gallo and Letourneau, 2004). The filopodia therefore represent transient structures

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which undergo cycles of contractile activity forming, extending and retracting, and in consequence acting as antenna-like sensors as they explore and sample the local environment over a wide radius (Davenport et al., 1993). Recent studies, employing small interfering RNA silencing approaches, have demonstrated that the balance between the antagonistic forces of dynein (a microtubule transporter) and myosin-II (which retrogradely transports actin) is fundamental in controlling the interaction between long microtubules and actin, and in consequence underpinning growth cone turning and axonal retraction in the pursuit of pathfinding (Myers et al., 2006). Complex interactions exist between the growth cone and an array of guidance cues and inhibitory molecules, such as the ephrins and the semaphorins (Tamagnone et al., 2004; Wu et al., 2005) (see Section 3.1). The signalling cascades which are stimulated by these are in turn modulated by a variety of environmental signals including growth factors (and especially the neurotrophins (for review, see Huang and Reichardt, 2001; Lykissas et al., 2007; Markus et al., 2002), components of the extracellular matrix (ECM) (such as laminin and fibronectin; Kuhn et al., 1995), cell adhesion molecules (CAMs) (Skaper, 2005), neurotransmitters (van Kesteren and Spencer, 2003), electrical stimulation (McCaig et al., 2002; Ming et al., 2001; Rajnicek et al., 2006), calcium signalling (Bolsover, 2005; Gomez and Zheng, 2006; Henley and Poo, 2004), and a number of other influences that establish chemotactic gradients to guide axonal pathfinding (Ming et al., 2002). Intrinsic differences develop between young and old neurons during embryonic life, however, which can greatly restrict the ability of axons to regenerate, even in permissive environments. This was recently demonstrated by Blackmore and Letourneau (2006) in a study of organotypic cocultures of chick brainstem and spinal cord, where explants from E15 animals regenerated about 90% fewer axons, which also extended much more slowly on permissive substrates, than was the case in preparations from younger embryos (E9). A major goal of future research will be to elucidate how the interplay of the intrinsic and extrinsic factors can be manipulated successfully, and how the efficacy of positive signals early in embryonic life can be reactivated following insult to overcome the negative control by inhibitory cues, if regeneration in adult neurons is to become routine.

2.1. Role of neurotrophins Neurotrophins [the nerve growth factor (NGF) family] are important regulators with a multifunctional role, not only in neuronal survival in the developing nervous system but also acting in stimulating axonal growth and directing the establishment of functional patterning (Huang and Reichardt, 2001; Lykissas et al., 2007). They act via tyrosine kinase receptors (TrkA, TrkB, TrkC), and by a common low affinity receptor (p75NTR) (Barbacid, 1995). In the adult, neurotrophins are frequently responsible for maintaining a

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differentiated phenotype in both the central and peripheral nervous systems (Smith and Jiang, 1994; Markus et al., 2002). Neurotrophins, and other trophic factors, such as GDNF (glial cell line-derived neurotrophic factor) family members, are also of interest with regard to their therapeutic potential in stimulating axonal sprouting in response to injury or disease (Airaksinen and Saarma, 2002; Love et al., 2005). Neurite extension in sensory neuronal populations is particularly responsive to NGF ( Jiang et al., 1995; LeviMontalcini, 1987; Lindsay, 1988). The neurotrophins are thought to act by mediating rapid changes in growth cone responses to collapsin-1, although chronic exposures with NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) caused a differential sensitivity to the inhibitory guidance molecule if co-applications are given to neuronal cultures (Tuttle and O’Leary, 1998). Trophic factor interactions with components of the ECM are also of significance in regulating growth cone activity. In a study of axonal growth from embryonic sensory neurons, neurites were seen to extend towards each other in the presence of NT-3, but not NGF, which caused growth cone collapse and neurite repulsion when plated on laminin surfaces, whilst intermingled processes were observed when maintained on fibronectin or polylysine substrates (Hari et al., 2004). In the CNS, differential regulation has been demonstrated with NT-3 and BDNF affecting extension of axons in cultured embryonic rat hippocampal pyramidal neurons, whilst neurotrophin-4 (NT-4) stimulated growth only of undifferentiated minor neurites (Labelle and Leclerc, 2000). Cerebellar granule neurons from 2-day-old rats have also been shown to respond differently, with BDNF and NT-4 significantly increasing neurite length, and the speed of growth cone migration, whilst NT-3 was not effective (Tanaka et al., 2000), although NT-3 enhanced outgrowth and branching in 50% of neurites from embryonic hippocampal neurons (Morfini et al., 1994). In organotypic slice cultures, taken from the visual cortex of P14 ferret brains, BDNF and NT-3 exerted opposing effects on dendritic outgrowth in pyramidal neurons in layers 4 and 6, with BDNF having a positive effect in layer 4, but inhibiting outgrowth mediated by NT-3, whilst in layer 6 NT-3 inhibited the growth stimulated by BDNF (McAllister et al., 1997). One possible explanation to account for this opposing effect could be competitive binding at the Trk B receptor, although, since the inhibition occurs at very low doses, the authors speculated that subsequent stages of signal transduction were involved (McAllister et al., 1997). This certainly appeared to be the case in a study of BDNF signalling in adult pig retinal ganglion regeneration where treatments with either the PI3K (phosphatidylinositol 3-kinase) inhibitor Wortmannin, or the MAPK inhibitor, U0126, significantly reduced neurite extension after 5 days’ culture (Bonnet et al., 2004). Interestingly, Pfenninger and co-workers (2003) found that whilst BDNF enhanced neurite growth and elongation in embryonic rat hippocampal neurons, the actual initiation of membrane expansion within the distal growth cone was stimulated by insulin-like growth

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factor 1 (IGF-1). Further studies, incorporating siRNA silencing and antibody blocking strategies, have highlighted that it is indeed IGF-1, rather than BDNF, that activates PI3K (Sosa et al., 2006), in a manner that is quasi independent of the neuron’s perikaryon (Laurino et al., 2005). Synergistic responses to combinations of trophins are probably commonplace in embryonic development; the loss of such cooperative mechanisms in the adult may account for the reduced success in regenerative potential in the adult following neuronal trauma. Glial cell line-derived neurotrophic factor also signals via PI3-K, as demonstrated by Edstrom and Edstrom (2003), who applied the inhibitor LY294002 to cultures of adult mouse DRG explants, and who noted significant decreases in axonal outgrowth. Transient phosphorylation of mitogenassociated protein kinase kinase was also seen following GDNF treatments of cultured ventral mesencephalic (VM) neurons, whilst BDNF induced a longer acting signalling response (Feng et al., 1999). Total neurite length increased twofold in E18 rat VM dopaminergic and calretinin-expressing neurons following culture in medium supplemented with 10 ng/ml GDNF for 7 days (Schaller et al., 2005). Enhancement of sprouting also continues into adulthood for dopaminergic neurons in the substantia nigra, whilst neurturin (another closely related member of the GDNF family) failed to sustain fiber outgrowth both in vitro and in vivo lesioning, and in grafting experiments (Akerud et al., 1999). The effects of GDNF in neural transplantation strategies have been encouraging indeed, with enhanced fiber outgrowth into host striatum demonstrated in rodent models for Parkinson’s disease (McLeod et al., 2006); and in a clinical report where GDNF infusion into the brain of a 62-year-old man for a period of 43 months stimulated fiber sprouting (Love et al., 2005) and clearly warrants further investigation. A number of other growth factors are known to regulate axonal dynamics in the embryo, and include transforming growth factor b (TGFb) (Unsicker et al., 1991), epidermal growth factor (EGF) (Kenigsberg et al., 1992), and fibroblast growth factors (FGF) (Walicke et al., 1986), including FGF-2 (which increased extension and branching of neurites in cortical neurons by over 50% by increasing actin polymeristation, and the formation of microtubule loops in growth cones; Dent et al., 2004). Clearly a plethora of trophic factors influence both neurite extension and growth cone activity, acting either singly or in combination in embryonic neurons, and in addition with a role in maintaining the differentiated state in mature cells in health and potentially playing a role in regeneration.

2.2. Extracellular matrix cues The role of the surrounding permissive physical environment in orchestrating growth cone dynamics, resulting from adhesive interactions with cellular receptors (including Ig-cell adhesion molecules, integrins and cadherins;

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Huber et al., 2003) is well documented, and is vital in ensuring growth cone precision in pathfinding (Letourneau, 1975; Luckenbill-Edds, 1997; Smith and Jiang, 1994). During development, the growing neurite must contact, sample, and either accept or reject ECM cues in order to reach and establish functional connections that will lead to the complex circuitries present in the adult. Nanoscale fabrication technology has now provided the means to produce high-resolution patterned surfaces which can demonstrate in vitro that substrate geometry, including three-dimensional patterning (Li and Folch, 2005), is instrumental in regulating growth cone ‘‘choices’’ and ‘‘decisions’’ ( Johansson et al., 2006; Kleinfeld et al., 1988; Vogt et al., 2004; Withers et al., 2006). For instance, whether growth cones from hippocampal neurons pause or accelerate at specific space intersections, and whether they extend straight or branch over the surface, depends on the width of the grooves (Withers et al., 2006). The importance of the chemical composition of the ECM, in influencing neuronal behavior, particularly axonal outgrowth and guidance, has been well documented. Many studies of cultured neurons have benefited from adapting the patterned substrates and developing ‘‘stripe assays’’, where one or more ECM component (such as glycoproteins) is bound to restricted longitudinal lanes alongside uncoated regions of the substratum. The alternating striped patterns permit the observation of any differential axonal behavioral responses at borders with or without the component of interest (see Freire et al., 2002; LuckenbillEdds, 1997; Myers et al., 2006; Nguyen-Ba-Charvet et al., 2001; Snow et al., 2002; Turney and Bridgman, 2005). Early work focused on identifying the actual ECM molecules that promoted axonal sprouting. The glycoproteins, laminin and fibronectin, which bind to integrin receptors (Ivins et al., 2000; Luckenbill-Edds, 1997; Powell and Kleinman, 1997), were demonstrated to be two of the key molecules in in vitro and in vivo studies of both peripheral and central neurons (for example, see Baron-Van Evercooren et al., 1982; Manthorpe et al., 1983; Orr and Smith, 1988; Rogers et al., 1983, 1987; Smith and Orr, 1987), with laminin having the most significant effect on directional growth (Luckenbill-Edds, 1997; Sanes, 1989). Other glycoproteins, such as wnt protein, have also been shown to be of importance in morphogen signalling and in regulating axonal outgrowth in the embryonic development of CNS patterning (Sanchez-Camacho et al., 2005). More recent work has focused on elucidating the mechanisms involved. Kuhn and his colleagues (1995) showed, by using polystyrene beads coated with the glycoproteins, that laminin guideposts induced a preferential, rapid and sustained response in neurites extending from sensory neurons. Filopodial dynamics were altered causing the growth cones to change their direction and advance, with increased velocity, towards laminin sources, compared to fibronectin signals. Binding to b1-integrin sites, and

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subsequent activation of calcium-dependent protein kinase C (PKC) intracellular signalling, was implicated as shown by the addition of a number of selective inihibitors to the cultured cells (Kuhn et al., 1995). Stimulation of axonal growth by IGF-1 and laminin, via integrin receptor activation, in cortical neurons has been shown to involve phosphorylation of the transmembrane signal regulatory protein, SIRPa (Wang and Pfennigner, 2006). Responses to laminin also result with changes in growth cone myosin II activity, as shown in studies using the myosin II inhibitor, blebbistatin, and from culturing neurons from wild type and myosin II knockout mouse embryos (E13.5) (Turney and Bridgman, 2005). Loss of myosin II would affect the recycling of actin in growth cone remodelling (Myers et al., 2006). Others have shown that the level of neuritogenesis in cortical and retinal neurons is varied by the pH at which the laminin matrices are assembled, and moreover that in consequence distinct signalling pathways are activated (Freire et al., 2002). Neurite outgrowth was two to three times greater on acidic (pH 4) laminin, and with the growth cones exhibiting lamellipodia and filopodia extending for large areas, rather than on neutral (pH 7) laminin where the size of the growth cones was reduced and filopodia often absent. Interestingly, staurosporine (a wide-spectrum inhibitor of protein kinases, but with particular effects on PKC) reduced neurite lengths by 40% on neutral substrates, whilst neuritogenesis was not significantly affected on the acidic matrix by this inhibitor. However, the protein kinase A inhibitor H-89 significantly reduced outgrowth on acidic laminin, but not on the neutral substrate (Freire et al., 2002). Such subtle alterations in the organization of individual ECM components, and/or the downregulation of integrin receptors, or at least a reduction in their activation state (Ivins et al., 2000), could be important in the responsive switches which occur as development proceeds (Blackmore and Letourneau, 2006), thereby regulating neuritogenesis, and could also explain differences that contribute to the impairment of regeneration in the adult central nervous system following trauma. The interactions of the proteoglygans, and particularly the chondroitin sulfate proteoglycans (CSPGs; present in the ECM and also expressed on the surface of oligodenrocytes), have a bearing on the efficacy of the glycoproteins in regulating axonal pathfinding, since CSPGs are known to typically cause an inhibition of growth cone behavior and neurite outgrowth (Cole and McCabe, 1991; Niedero¨st et al., 1999; Wilson and Snow, 2000). Niedero¨st and co-workers (1999) demonstrated the CSPGs significantly inhibited fiber extension by both cerebellar granule cells and DRG neurons in the presence of laminin, and that oligodenrocytes maintained in the presence of proteoglycan inhibitors are less effective in blocking neurite growth. In many instances the extent of the opposing effects appears to depend upon the ratio of CSPGs to laminin, when the concentration of

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CSPCs (<50 mg/ml) exceeds that of laminin (25 mg/ml), by up to twofold, neurite outgrowth in a range of forebrain, dorsal root ganglion and retinal ganglion neurons is inhibited, and by 1000 mg/ml growth on the substratum is totally absent. With a higher ratio of laminin (25 mg/ml) to CSPGs (25 mg/ml), however, the proteoglycans are incapable of blocking outgrowth stimulated by laminin (Snow et al., 2002). In hippocampal neurons, CSPG type proved significant in how growth cone behavior was modified with respect to crossing, stalling or turning behavior on encountering different lanes of a stripe assay (Wilson and Snow, 2000), whilst earlier workers had reported that chondroitin sulfate type C specifically modified neurite extension patterns on laminin substrata from cultured embryonic thalamic, but not hippocampal, neurons (Fernaud-Espinosa et al., 1994). Others have shown that heparin chondroitin sulfate actually favored axonal growth, whilst dermatan sulfate stimulated both dendritc and axonal growth in embryonic rat mesencephalic neurons (Lafont et al., 1992). Such interplay between glycoprotein and proteoglycan components of the ECM therefore represents a potentially fundamental mechanism that can fine-tune what is permissive, and what is repulsive, for fiber generation as the embryo’s nervous system develops, and thereby precisely establishes complex neuronal circuitry. In a recent elegant study of embryonic murine cerebellar granule neurons, Manzini and co-workers (2006) have highlighted that the signal which arrests growth, and prevents invading pontine mossy fibers from contacting immature cells in the external germinal layer during development, is dependent on heparin-binding factors. More mature internal granule layer cells were not affected by heparin treatments and permitted pontine mossy fiber axonal growth to establish synaptic connections yielding correct patterning (Manzini et al., 2006). Laminin modulation, on interacting with a number of other cues which regulate axonal growth, appears to involve a variety of signalling mechanisms—for instance, DRG axons avoid the non-permissive glycoprotein, Slit2, in the presence of laminin, following stimulation of cGMP signalling pathways (Nguyen-Ba-Charvet et al., 2001). Activation of cGMP signalling is also sufficient to cause growth cone collapse when dentate granule neurons are exposed to nitric oxide releasing reagents, suggesting a regulatory role for the diffusible molecule that is also known to be involved in synaptic plasticity in these cells (Yamada et al., 2006). Growth cone pauses and collapse stimulated by Eph B proteins, in retinal ganglion cell axon pathfinding in laminin environments, on the other hand, were shown to result from a redistribution of filopodial microtubules, and a reduction in the levels of the microtubule destabilising protein, SCG10 (Suh et al., 2004), rather than specifically triggering changes in actin via Rho GTPases—a common occurrence during growth cone collapse (Luo, 2000)—although cross-talk between the longer microtubules which enter the outer actin-rich P-domain remains a probable scenario (Myers et al., 2006).

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2.3. Establishing polarity and axonal specification The means by which neurons establish polarity during differentiation, involving a coordinated control of cytoskeletal organization and membrane trafficking in the different regions of the cell, thereby generating functional axons and dendrites emanating from their perikarya, must be the most significant and fundamental event in neural development. Neuronal polarity produces axons in which a loose cytoskeletal meshwork of F-actin is associated with microtubules [with their (þ) ends directed away from the perikaryon; Sargent, 1989]. The stability and polymerization of the microtubules, together with associated local actin instability, within axonal processes depends upon the phosphorylative state of certain low molecular weight MAPs: tau proteins and collapsing response mediator protein 2 (CRMP-2) that preferentially enrich axons, and which are regulated by the Rho GTPases (Arimura et al., 2004; Dehmelt and Halpain, 2004; Govek et al., 2005; Wiggin et al., 2005). Dendritic processes on the other hand, have a preponderance of MAP-2 and lack tau protein (Dotti et al., 1988). Following polarization, synaptic connections are made, so that electrical activity generation and functional maturation are achieved. The underlying mechanisms have been extensively studied in vitro, particularly in cultures of hippocampal neurons (e.g., Da Silva and Dotti, 2002; Dotti et al., 1988; Jiang et al., 2005), such that our current understanding of the regulation of neuronal polarity is very good. Despite an increasing number of in vivo studies which on the whole corroborate those in vitro, much still remains to be verified in the whole brain, where external interactive cues from other cells and the local environment would be expected to add to the complexity as seen at the individual neuronal level (Wiggin et al., 2005). The five stages in the establishment of polarity have been documented in detail by Dotti and his co-workers (1988), and the sequence they described remains, with minor modifications, one of the most useful to date. Initially in stage 1 the neuronal sphere is broken as local buds, with characteristic lamellipodia and filopodia, form. During the next 24 hours (Stage 2) many such buds sprout neurites over the surface and these extend approximately 20 mm in length. The microtubule cytoskeleton of the developing neurites becomes stabilized by an up-regulation of MAPIB protein, and further extension is promoted distally (Bouquet et al., 2004; Li et al., 2006; Yu et al., 2001). In Stage 3, one of the neurites becomes dominant, extending rapidly as the future axon, whilst the others are arrested until several days later, when they resume growing and acquire a dendritic phenotype (Stage 4). Finally in Stage 5, both axon and dendrites mature with a full complement of marker proteins present, and by now they express full functional competence. Interestingly, even when differentiation has occurred, neurons retain a capacity for polarity re-programming in that transection of the axonal process of a Stage 3 neuron triggered one of the other neurites to transform

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into a new axon (Bradke and Dotti, 2000a). In this instance the former axon was seen to grow only 15 mm after axotomy and Tau-1 protein was lost whilst it became immuno-positive for MAP-2. Concomitantly, the ‘‘new’’ axon changed morphologically to resemble an axon and at the same time became Tau-1 positive (Bradke and Dotti, 2000a). The intracellular signalling pathways recruited to execute this ordered sequence in establishing polarity, and particularly the role of the Rho GTPase family members (Govek et al., 2005), are covered in more detail in Section 3.3, and so will only be considered briefly here. Much interest however, has focussed recently on a number of gene products, originally identified in Caenorhabditis elegans as conferring cellular asymmetry, known as PAR (partitioning-defective) proteins, since they play a crucial role in axonal specification (for review, see Wiggin et al., 2005), and may have relevance to neurodegeneration and regeneration. Indeed, Par-1/MARK phosphorylates tau protein in regulating normal axonal cytoskeletal dynamics, although hyperphosphorylation may lead to an aggregation of tau tangles in some human neurodegenerative diseases (Fortini, 2004). At Stage 2, prior to the appearance of the dominant axonal process, PAR-3 and PAR-6 have a global distribution within the neuron. By Stage 3 polarity is established, and the PAR proteins, which can bind the Rho family members, CDc42 and Rac1, become selectively restricted to the axon (Shi et al., 2003). This occurs downstream of PI3-kinase activation (Sosa et al., 2006), and appears to involve adenomatous polyposis coli (APC) and kinesin (KIF) 3A in protein-mediated transport which is regulated by the phosphorylation (the inactive form) of GSK-3b, glycogen synthase kinase-3b ( Jiang et al., 2005; Shi et al., 2004). In addition to regulating APC, GSK-3b also modulates CRMP-2, and in consequence the assembly of tubulin microtubules along with the actin network of the axonal cytoskeleton (Arimura et al., 2004; Yoshimura et al., 2005). The exact upstream signalling pathways involved in regulating GSK-3b are not fully determined. Indeed, others are also probably of significance, with some evidence that Wnt signalling at least has a vital part to play in establishing polarity (Sanchez-Camacho et al., 2005).

3. Repulsive Guidance Cues in Axonal Pathfinding 3.1. Repulsive ligands As seen, many guidance cues have bifunctional roles in growth cone guidance in that, dependent on changes in the level of intracellular cAMP/ cGMP concentrations; they can either attract or repel growth cones.

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For ease of the rest of this review, we will be focusing on the inhibitory aspects of these cues. 3.1.1. Semaphorins Semaphorins are secreted, transmembrane, and glycosyl-phosphatidylinositol (GPI)-anchored proteins, defined by cysteine-rich semaphorin protein domains, which have important roles in a variety of tissues (Table 3.1) (Fujisawa, 2004; Yazdani and Terman, 2006). Initially found to play an important role in axonal guidance during development of the nervous system, semaphorins are now known to be involved in the formation of cardiovascular, endocrine, immune, reproductive and respiratory systems. So far there are eight known classes of semaphorins with more than 28 members described by the Semaphorin Nomenclature Committee (1999), and the majority of the family members are found in mammalian cells (Yazdani and Terman, 2006) (Table 3.1). Semaphorins are, however, only found in animals, not in plants. During nervous system development, semaphorins are best known as repellents to growing axons, but semaphorin-3A can also function as a chemoattractant depending on the intracellular level of cyclic nucleotides (Nishiyama et al., 2003; Song et al., 1997). Semaphorin-3A, a prototypical class 3, is both a secreted and neuronal-expressed chemorepulsive molecule which consists of an N-terminal signal peptide followed by the Sema domain, and an IgG domain of 70 amino acids (Luo et al., 1993, 1995; Puschel et al., 1995, 1996; Steup et al., 1999). A basic domain is present at the carboxyl end of the molecule. A common theme of the mechanism of semaphorin function is through alteration of the cytoskeleton, such as the actin filaments and microtubule network (Morita et al., 2006; Rohm et al., 2000). These effects occur primarily through binding of semaphorin to their receptors, neuropilin and plexin family of transmembrane proteins. Semaphorin-3A plays a key role in axonal guidance during development through induction of growth cone collapse (Pasterkamp and Kolodkin, 2003). The process occurs at the tip of the growth cone and is manifested by depolymerization and loss of F-actin. The downstream pathways by which semaphorins exert their actions are still unclear, but are known to include the neuropilin and plexin receptors as well as intracellular collapsin response mediator proteins (CRMPs) and G-proteins (Kawasaki et al., 2002; Sahay et al., 2005). The biological activities of the repulsive axons guidance molecule semaphorin-3A are known to be responsible for the elimination of neurons during development where axons are still too far away from reaching the target (Beck et al., 2002; Pasterkamp and Kolodkin, 2003). The cellular target of semaphorin-3A also appears to be selective since it inhibits the outgrowth of a specific set of neurons such as spinal motor neurons and neurons in the embryonic dorsal root ganglion and sympathetic ganglion (Nakamura et al., 1998, 2000; Sandvig et al., 2004).

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Table 3.1 Semaphorins and neuropilin/plexins Ligands/Receptors

Organisms

Names

Characteristics

Ligands Semaphorins

Invertebrates

Sema-1a

Transmembrane proteins

Vertebrates

Virus Receptors Neuropilins and Plexins

Vertebrates

Sema-1b Sema-2a Sema-2b Sema-3A-G Sema-4A-G Sema-5A-C Sema-6A-D Sema-7A SemaVA and VB Neuropilin 1 Neuropilin 2

Plexin A1-4

Secreted proteins Secreted proteins Trans-membrane proteins

GPI-anchored

Short intracellular domain without tyrosine kinase domain. Forms homo- or hetero-dimers to interact with other receptors Long intracellular domain with tyrosine kinase domain

Plexin B1-3 Plexin C1 Plexin D1

3.1.2. Netrins Netrins are guidance cues for commissural neurons and they act as chemoattractants that guide axons to cross midline by binding to receptors of the DCC (deleted in colorectal carcinoma) family (Kennedy et al., 1994, 2006; Herincs et al., 2005). Netrins were purified from homogenates of embryonic chick brain using an in vitro assay designed to identify soluble cues that promote the outgrowth of commissural axons, mimicking the activity of the floor plate. Most of the components of the netrin family are secreted proteins, and surprisingly it is only recently that the diffusible gradient of netrin proteins has been directly visualized in the developing

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spinal cord confirming the widely accepted mechanism of axon guidance by diffusible chemoattractants (Kennedy et al., 2006). As a recurring theme, the netrin proteins are bi-functional signals that are chemoattractive for some neurons and chemorepellent for others, and can act as long or short range signals (Dickson, 2002; Hedgecock et al., 1990; Kennedy et al., 1994; Tessier-Lavigne et al., 1988; Tessier-Lavigne and Goodman, 1996). Midline cells express netrins and defects in the expression of netrin ligands and their receptors cause abnormalities in the reaching and crossing of the midline. Once crossing the midline, the response of axons to netrin must be silenced to prevent re-crossing the midline. This inactivation of netrin’s attraction is through binding to DCC by the slit/Robo complex (Giger and Kolodkin, 2001; Kaprielian et al., 2000, 2001; Plump et al., 2002). It has been shown that netrin-mediated axonal repulsion is through the Unc5 family proteins by forming DCC-Unc5 heterodimers (Hedgecock et al., 1990; Hong et al., 1999; Keleman et al., 2005). The secondary protein structure of the netrin family is highly conserved in all species. The N-terminal signal peptide is followed by domains VI, V-1, V-2 and V-3 which share sequence homologies with the globular domain VI of laminin and the EGF repetitions found in region V of the laminin chains, respectively (Barallobre et al., 2005; Brankatschk and Dickson, 2006). The C-terminal domain sequences are rich in basic amino acids, which act as binding sites for heparin, heparan sulfate proteoglycans or membrane glucolipids, thereby allowing interaction with components of the extracellular matrix or cell surface. Therefore, the diffusion of secreted netrins is determined by both their level of expression and by the concentration of binding sites in the surrounding tissue. The netrin family members and their receptors are summarized in Table 3.2. 3.1.3. Ephrins Ephrins (Ephs) represent a major class of short-range axon guidance molecules. First identified as contact repulsive guidance cues during development of the retino projections, Ephs were later found to also have attractant functions to axons (Davy and Soriano, 2005; Huot, 2004; Tessier-Lavigne, 1995). There are nine known mammalian Eph ligands and 16 Eph receptor tyrosine kinases (Eph RTK, Table 3.3). The nine ephrin ligands consist of ephrin A1-A6, which are GPI-anchored membrane proteins, and ephrin B1-B3, which are typical transmembrane proteins with a single cytoplasmic domain (Carmeliet and Tessier-Lavigne, 2005; Davy and Soriano, 2005; Dickson, 2002; Goldshmit et al., 2006; Huot, 2004; Price et al., 2006; Tessier-Lavigne, 1995; Zhang and Hughes, 2006). EphAs and EphBs appear to have opposing roles in neurite outgrowth in specific regions of the brain. For example, EphA4 knockout mice hippocampal neurons show morphologically disorganized dendritic spines which are long and overlapping each other, whereas triple knockout of EphB1/2/3 leads to failure for hippocampal neurons to

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Table 3.2 Netrins and DCC/UNC receptors Ligands/Receptors

Organisms

Names

Characteristics

Netrin 1

Midline cell secreted bifunctional proteins which attract or repel axons

Ligands

Netrins

Receptors DCC/UNC

Vertebrates

Netrin 4b

Netrin G1-2 NTN2L Vertebrates

DCC1-2

Dependence receptors (induce apoptosis)

UNC5 (C. elegance) UNC5H1-4 Table 3.3 Ephrins and Eph receptors Ligands/Receptors

Organisms

Names

Characteristics

Ligands Ephrins

Vertebrates

Receptors Eph RTK

Ephrin A1-6 Ephrin B1-3

GPI-anchored Transmembrane

Vertebrates

Eph A1-10

GPI-anchored proteins binds to A class Ephrins Transmembrane proteins binds to B class Ephrins

Eph B-1-6

produce spines, and the neurons appear to be long and immature (Dickson, 2002; Davy and Soriano, 2005; Goldshmit et al., 2006). The functions of Ephs are complex and intriguing in that Ephs can transmit not only a forward signal through interacting with Eph receptors, but this ligand-receptor interaction also causes transduction of a reverse signal into the Eph bearing cells. The Eph receptor and ligand interaction specificity is far from clear and some promiscuities within the same subclass of Eph family have

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been documented (Blits-Huizinga et al., 2004; Mendes et al., 2006). To add to the complexity, Ephs are found not only widely expressed in the developing and adult nervous system to participate in tissue patterning, axonal guidance and synaptic plasticity, but they are also involved in modulating angiogenesis and vascular remodelling (Zhang and Hughes, 2006). Readers are directed to the above excellent review articles for specific details. 3.1.4. Slits Slit protein was identified as a ligand for the Roundabout (Robo) receptor family (Brose and Tessier-Lavigne, 2000). Again as a recurring theme for axon guidance cues, the interaction of Slits with Robo can act as repellents or attractants for branching and elongation of axons. Three distinct Slit genes have been identified encoding slits1-3 in mammals (Table 3.4). Slit1 and Slit2 proteins have been found to control the development of the lateral olfactory tract (Nguyen-Ba-Charvet et al., 2002). All Slits are large extracellular matrix-associated glycolproteins of about 200 kD which are characterized by the presence of unusual tandem of four leucine-rich repeat domains in their N-terminal sequence. Slit2 is proteolytically processed into 140 kD N-terminal and 55–60 kD C-terminal fragments (Nguyen Ba-Charvet et al., 2001). Slit2 cleavage fragments appear to have different characteristics, with the smaller C-terminal fragment being more diffusible and the larger N-terminal and uncleaved fragments being more tightly cell associated. Subsequent expression analysis has shown that the cleaved fragments have distinct axonal guidance properties (Plump et al., 2002; Nguyen-Ba-Charvet et al., 2002). Most importantly, Slits function to prevent midline crossing during nervous system development. At the midline, Slit is expressed by glia cells Table 3.4 Slits and Robos Ligands/Receptors

Organisms

Names

Characteristics

Ligands Slits

Vertebrates

Slits 1-3

Midline crossing repellent guidance cues

Vertebrates

Robo 1-3/Rig1 Robo4

Midline crossing (Magic Robo), vascular specific (binds to slit 2) Modulate slit presence

Receptors Roundabout (Robo)

Heparan Sulfate

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and is diffused away from the midline, thus setting up the gradient required for guidance (Hohenester et al., 2006). In addition to the well characterized role of Slits in the regulation of axon crossing at the midline, Slits are also found to specify the lateral and dorsoventral positioning of longitudinal axonal pathways, contribute to the formation of commissures by channelling axons into particular regions. As a recurring theme, in a similar fashion to Ephs and semaphorins, Slits also provide guidance for the vascular and immune system formation (Fouquet et al., 2007; Hohenester et al., 2006; Lopez-Bendito et al., 2007). 3.1.5. Repulsive guidance molecule The repulsive guidance molecule (RGM) cues represent another family of contact dependent repulsive factors comprising RGMa, RGMb, and RGMc (Table 3.5) (Matsunaga et al., 2004; Matsunaga and Chedotal, 2004; Monnier et al., 2002). RGMa and RGMb are abundantly expressed in the developing mouse nervous system, while RGMc expression is restricted to the muscles and blood cells. It has been discovered recently that RGMs play a key role in the guidance of optic nerve projection and crossing of the chiasma. The full-length RGMa is a GPI-anchored protein which is proteolytically processed into a 33 kD C-terminal fragment (C-RGMa) and an 11 kD N-terminal (N-RGMa) fragment (Matsunaga et al., 2004; Matsunaga and Chedotal, 2004; Monnier et al., 2002). Published studies demonstrated that C-RGMa inhibits axonal outgrowth through binding to its receptor, Neogenin. Noncleavable mutants of RGMa do not interact with Neogenin and do not have any function in axonal guidance unless it is proteolytically cleaved (Monnier et al., 2002; Brinks et al., 2004; Hata et al., 2006). Interestingly, most of the time, N-RGMa is linked with C-RGMa by a disulphide bond and it is still not

Table 3.5

RGM and Neogenin

Ligands/Receptors

Organisms

Names

Characteristics

Ligands RGM

Vertebrates

RGMa RGMb

Optic nerve projection and crossing of the chiasma Expressed in muscles and blood

RGMc Receptors Neogenin

Vertebrates

Neogenin

Dependence receptor (induce apoptosis)

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Table 3.6 Myelin-secreted inhibitory glycoproteins Ligands/ Receptors

Organisms

Names

Characteristics

Ligands Nogo

Vertebrates

Nogo-A Nogo-B Nogo-C Myelin-associated protein

GPI anchored proteins all contain Nogo-66 domain A sialic-dependent immunoglobulinlike protein GPI anchored protein

MAG

OMag

CSPG Receptors Nogo Vertebrates receptors

Oligodendrocytemyelin glycoprotein Myelin-associated chondroitin sulfate proteoglycans NRc and p75NTR

ECM

Nogo receptor is GPI anchored and has to function as a co-receptor for p75

yet known whether the N-RGMa is also an inhibitor to axonal outgrowth, and, if this is the case, what the receptor that N-RGMa interacts with is in mediating axon outgrowth inhibition. 3.1.6. Myelin, reactive glial and scar-derived axon growth inhibitors It has been demonstrated that scarring is a major impediment to successful repair of CNS connections after injury. Oligodendrocytes, myelin and reactive astrocytes within the scar tissue secrete inhibitory molecules which interact with growth cone receptors to arrest injured axons. Voluminous literature has been produced in the last few years describing factors such as Nogo, myelinassociated protein (MAG), oligodendrocyte myelin glycoprotein (OMag), and myelin-associated chondroitin sulfate proteoglycans (CSPGs) (Sandvig et al., 2004). Because of their apparent relevance to brain injury, extensive attempts have also been made to therapeutically modulate the expression and function of some of these inhibitors for brain repair, albeit with limited success (Domeniconi and Filbin, 2005; He and Koprivica, 2004; Johansson, 2007; Lenzlinger et al., 2005; Papadopoulos et al., 2002, 2006; Spencer et al., 2003). Nogo has three different spliced variants with the highest amino acid (aa) sequence homology towards the C-terminal ends. Nogo-A (1162 aa),

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Nogo-B (373 aa) and Nogo-C (199 aa) all share two hydrophobic domains close to the C-terminus (transmembrane domain) and a common 66 aa extracellular inhibitory domain (Nogo-66) in between the two hydrophobic motifs (He and Koprivica, 2004; Sandvig et al., 2004). MAG is secreted by myelin sheaths of oligodendrocytes and Schwann cells, and it is a sialic-dependent immunoglobulin-like protein with an extracellular domain, a transmembrane domain and a short intracellular domain. MAG is also known to be a bifunctional protein demonstrating a neurite outgrowth promotion function in developing young neurons, but causing neurite outgrowth inhibition in older neurons. The function of MAG appears to be regulated by the changes in the intracellular levels of cAMP (Mimura et al., 2006; Quarles, 2007). OMag is also a GPI-anchored inhibitor for neurite outgrowth and it signals neurite growth inhibition through Nogo receptors and p75 via Rho GTPases (Mikol and Stefansson, 1988; Wang et al., 2002a,b). Little is known yet about the functions of Omag, for although it is highly expressed on oligodendrocyte membranes and on neurons, the functions of neuronal expressed OMag are not clear. Although these molecules appear to be very diverse in structure, they share one common feature which is that they are all heavily glycosylated through either N- or O-linked glycosylation. It is not yet known whether modulation of glycosylation of these inhibitors would have any effects on their pathological roles in axonal regeneration. Detailed reviews of the structures and functions of these molecules have recently appeared, and readers are directed to the excellent treatises of others which are available (He and Koprivica, 2004; Sandvig et al., 2004; Quarles, 2007).

3.2. Receptors for repulsive guidance cues 3.2.1. Semaphorin receptors: Neuropilins Cellular receptors for semaphorins are neuropilins (He and Tessier-Lavigne, 1997; Kolodkin et al., 1997). Neuropilin contains two family members: neuropilin-1 and neuropilin-2. Both of them comprise an extracellular domain of two CUB motifs, next to two domains with homology to coagulation factors V and VIII, an MAM domain, a single transmembrane domain, and a short intracellular domain of 39 amino acids lacking any known signalling motifs. Neuropilins are non-tyrosine kinase transmembrane proteins. Their short intracellular segments lack cytoplasmic signal transduction domains. Therefore, neuropilins participate in signal transduction as co-receptors with plexins for axonal guidance and with vascular endothelial growth factor (VEGF) receptors for vascular guidance during development. Neuropilin-1 is a cell surface glycoprotein expressed on axons (Fujisawa, 2002; Kawasaki et al., 2002), and functions as a receptor for axon guidance

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factors such as semaphorin-3A during the process of axonal pathfinding. Peptides antagonizing the MAM domain have been found to inhibit neuropilin-1mediated inhibition of axonal outgrowth (Nakamura et al., 1998; Williams et al., 2005). The nature of the downstream effectors of neuropilin signal transduction remains elusive. One of the first cytosolic protein families identified to link neuropilin receptor complex to the cytoskeleton are the collapsin response mediator proteins (CRMPs), members of a small family of brain specific proteins (Liu and Strittmatter, 2001). There appears to be some selectivity in Neuropilin-1 and Neuropilin-2 interaction with their respective ligands. Neuropilin-1 binds to all classes of semaphorin 3, whereas neuropilin-2 binds selectively to the secreted semaphorins with the exception of semaphroin-3A. The specificity and selectivity of neuropilins are determined not only by their associated co-receptor proteins—plexins, but also by the way the neuropilin dimers are formed. For example, signalling via neuropilin-1 is mediated principally by plexin-A4, whereas signalling via neuropilin-2 is mediated principally by plexin-A3. Neuropilin-1 homodimers function as selective ligand-binding receptors for semaphorin-3A, while neuropilin-2 homodimers act as receptors for semaphorin-3F which is a promoter for axonal outgrowth (Pasterkamp and Kolodkin, 2003). In addition to functioning as guidance cue to axons, Neuropilins also guide developing blood vessels through interaction with VEGFs (Carmeliet and Tessier-Lavigne, 2005; Kim et al., 2006a; Schnorrer and Dickson, 2004). Furthermore, neuropilin interaction with VEGF165, but not Flt1 or KDR, enhances the survival of synviocytes from apoptosis by rapidly triggering phospho-AKT and phospho-ERK activities (Kim et al., 2006a). 3.2.2. Netrin receptors: DCC and UNC5 receptors DCC (Deleted in Colorectal Cancer) is a receptor for netrin-1 that mediates axonal growth cone turning dependent on the level of intracellular secondary messengers such as cAMP levels (Ming et al., 1997; Nishiyama et al., 2003). Elegant work from Dr. Poo’s lab has shown that Xenopus spinal neurons exhibited chemoattractive turning toward the source of netrin-1, but showed chemorepulsive responses in the presence of a competitive analog of cAMP or an inhibitor of protein kinase A, all of which are dependent on the Netrin receptor DCC (Ming et al., 1997). Recent studies also showed that DCC interacts with focal adhesion kinase in Netrin-1 signalling which revealed that a novel role of focal adhesion kinase in axonal guidance and also demonstrated the complexity in netrin downstream signalling (Ren et al., 2004) More importantly, the DCC and UNC5H receptors are also found to act as ligand dependent receptors in that etrin-1 is a survival factor through its receptors DCC and UNC5H (Furne et al., 2006; Herincs et al., 2005; Llambi et al., 2001, 2005; Mehlen and Llambi, 2005; Thiebault et al., 2003). Activation of DCC in the absence of netrin-1 induces apoptosis, while the presence

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of netrin-1 blocks DCC-mediated apoptosis (Arakawa, 2005; Tanikawa et al., 2003). It has therefore been proposed that DCC is a ‘‘conditional’’ tumor suppressor that is dependent on netrin-1. Similarly, UNC5H is also a regulator of p53-dependent apoptosis in the absence of netrin-1 (Arakawa, 2005). Further, netrin-1 itself is also a direct target of the p53 gene. Collectively, these findings indicate a close link between axon-guidance molecules and cellular death/survival machinery so that targeting this pathway may have an additional therapeutic effect in cancer treatment (Mehlen, 2005; Mehlen and Bredesen, 2004; Porter and Dhakshinamoorthy, 2004). 3.2.3. Ephrin receptor tyrosine kinases (Eph RTK) The vertebrate Eph RTK subfamily is by far the largest known subfamily of receptor tyrosine kinases and contains 16 receptor members EphA1–A10 and EphB1–B6 (Goldshmit et al., 2006). Although the molecular structures of the Eph RTK are well documented in recent reviews (Blits-Huizinga et al., 2004; Goldshmit et al., 2006; Huot, 2004; Pasquale, 2005), it is important to point out the key signalling motif which contains the two tyrosine residues near the juxtamembrane region that serves as the major autophosphorylation site involving receptor signalling. The tyrosine kinase domain functions as the binding site for activation of the GTPases which signal through the Rho kinase pathway to modulate cytoskeleton and axon guidance. Eph RTK activation is through conformational changes during Eph binding, autophosphorylation of the two tyrosine residues, and clustering of Eph receptorligand complexes. It has been shown that transcription regulation also plays a role in Eph RTK signalling. Mechanisms have been identified in that a sequence in the 30 -UTR of EhpA2 allows the mRNA to be translated only after it had crosses the midline, providing evidence to show another level of regulation of Eph (Brittis et al., 2002). 3.2.4. Slits receptors: Robos Advances in understanding the functions of Slits proteins come from the identification of Slits cellular receptors. Genetic studies have identified two types of cellular receptors for Slits: Robos (Roundabout)/Rig1 (Brose et al., 1999), and the heparan sulfate (HS) proteoglycan syndecan (Hohenester et al., 2006; Hussain et al., 2006). Several distinct Robos have been identified recently encoding Robo1, Robo2, Robo3 (also known as Rig1) and Robo4 (vascular specific) (Table 3.4). For the structures of these receptor proteins, refer to the excellent detailed recent review by Dickson and Gilestro (2006). The function of Robo receptor for Slits in midline crossing during development is relatively understood in that as the axon approaches the midline attracted by netrin, Robo is ‘‘silenced’’ by Slits, which keeps the Robo level low through an intracellular mechanism of preventing the Robo expression at the cellular membrane (Marillat et al., 2004; Sabatier et al., 2004).

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When the axon crosses the midline, this inhibition ceases and the reappearance of Robo on the cell surface enable repulsion to prevent re-entry of the growing axon. Remarkably, genetic disorder of Robo expression causes human diseases called ‘‘horizontal gaze palsy with progressive scoliosis’’ (HGPPS) ( Jen et al., 2004). Mutagenesis of Robo3 gene expression leads to abnormalities in reticular formation pathway to cross the midline which may explain the observed symptom, i.e., the lack of coordinated horizontal eye movement. The precise function of Slit interaction with HS is not absolutely clear, but their interactions may be important for shaping the presumed Slit gradient or presenting Slit at its target cell surface during midline crossing (Whitford et al., 2002). Much remains to be learned about this. 3.2.5. RGM receptors: Neogenin The receptor for RGMa is Neogenin. It is not yet clear how Neogenin signals growth cone collapse, and information on RGMa/Neogenin guidance of axon in vivo is also scant (Matsunaga et al., 2006; Wilson and Key, 2006a,b). During early embryonic development, RGM/Neogenin interaction repels axons. RGMa possesses activities similar to Ephrins which cause growth cone collapse and inhibits the outgrowth of temporal retinal ganglion cell axons (Matsunaga et al., 2006; Monnier et al., 2002). Recent studies showed that RGMa-induced growth cone collapse is also mediated by activation of the small GTPase RhoA and its downstream effector Rho kinase and PKC (Conrad et al., 2007). Neogenin is also a dependence receptor in that in the absence of its ligand, RGMa, Neogenein undergoes Caspase-3mediated cleavage to signal neuronal apoptosis (Matsunaga et al., 2004, 2006) (Table 3.2). From a mechanistic point of view, Neogenin induces death through formation of an RGMa-dependent complex that includes specific caspases such as caspase 3. In the absence of RGMa, activation of caspase 3 cleaves Neogenin thereby releases a pro-apoptotic peptide. Albeit the fact that it is still unclear how and in what order Neogenin cleavage and caspase activation occurs, it is believed that RGMa is a pro-survival factor and can be targeted for neuroprotection in stroke or traumatic brain injury (Doya et al., 2006; Matsunaga et al., 2004, 2006; Matsunaga and Chedotal, 2004; Rajagopalan et al., 2004; Schwab et al., 2005a,b). Caspase cleaves Neogenin at amino acid 1323 which leads to the production of two fragments, a 180 transmembrane fragment (N-terminal part) and an intracellular 23 kD fragment (C-terminal fragment) (Matsunaga and Chedotal, 2004). To add further to the complexity of RGM signaling, a hypothesis/ model is emerging that interaction of RGMa with Neogenin at the level of growth cone leads to growth cone collapse, while on the cell soma, their interaction promotes cell survival (Fitzgerald et al., 2006; Matsunaga et al., 2004; Matsunaga and Chedotal, 2004). Such an understanding of RGM

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function may have therapeutic implications in that it can be envisaged—for example, intravitreal injection delivering of RGMa to the retinal cell soma may promote retinal cell survival against optic nerve transection since the regenerating axons are not exposed to the negative effects of RGMa on the growth cone. 3.2.6. Receptors for myelin-secreted inhibitory glycoproteins The Nogo receptor is a 473 aa protein with a GPI anchor. Because it has no intracellular domain, it is understandable that Nogo receptor acts as a linker with another protein to transduce repulsive signals. It is now clear that Nogo receptor interacts with p75NTR which has transmembrane polypeptide sites. P75NTR is a member of the tumor necrosis factor superfamily which contains several important signal transduction domains such as the type II death domain which contains a GTPase-activating domain to activate the RhoA pathway (He and Koprivica, 2004; Wang et al., 2002a). Both MAG and OMag also bind to Nogo receptors with high affinity and require p75NTR as a co-receptor for signaling of MAG-mediated inhibition of axon growth and intracellular calcium elevation. Both MAG and Nogo bind to the same extracellular domain of the Nogo receptor (Wang et al., 2002b). Collectively, the complete trajectory of a given class of axons is likely to result from the effects of multiple positive and negative guidance cues that act at long and short ranges (Kaprielian et al., 2000, 2001; Plump et al., 2002). The growth cone must be able to integrate these signals and to modulate its responsiveness en route. Although the major mechanisms underlying this plasticity may be determined by the specific and selective intracellular signalling pathways (see the following section), it has come to light recently that even a single cyclic nucleotide, such as cAMP or cGMP, can modulate distinct responses of growth cones. For example, netrin-1 is sensitive to the levels of cAMP or protein kinase A (PKA) activity, while others including Sema 3A are modulated by cGMP and protein kinase G (PKG). It is generally believed that lowering of cAMP or cGMP levels, or inhibiting PKA or PKG activities, converts an attractive response to repulsive one, whereas elevating cAMP or cGMP, or activating PKA or PKG switches responses to attraction (Kao et al., 2002; Ming et al., 1997, 2002; Nishiyama et al., 2003; Song et al., 1997). It is therefore crucial to understand completely the relationship and interaction of these cues with their canonical receptors before the development of effective regenerative therapeutics can be advanced.

3.3. Intracellular signalling pathways for repulsive guidance cues The molecular signal transduction pathways for growth cone collapse and axonal guidance are hot topics of current research. The identification of the vast amount of guidance cues and the diverse range of molecules in response

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to the guidance cues towards their intended targets have also raised the question as just how all these proteins work together to cause growth cones to grow, turn and collapse in a very specific and precise manner. Although not completely understood, a large body of literature is out there suggesting that guidance of axonal growth is the result of activation of several distinct parallel signal transduction pathways and the cross-talk between these pathways. Three such intracellular pathways are highlighted in Figure 3.1, include the pathways of the ‘‘Rho GTPases’’, the ‘‘PI3K-GSK’’ and the ‘‘Fyn-Cdk5’’. 3.3.1. The ‘‘Rho GTPases’’ pathway The Rho family of GTPasaes includes Cdc42, Rac, and Rho, which are important regulators of the actin cytoskeleton affecting the shape and movement of the cells. Several interesting studies have shown that small GTPases of the Rho family are the major regulators of signalling pathways that link the extracellular cues (such as growth factors or repulsive factors mentioned above) to the assembly of focal adhesions and associated structures (Best et al., 1996; Kozma et al., 1996). Through a cascade of phosphorylation activation of downstream effectors, such as ROCK, PAK and coffilin, the Rho GTPases pathway ultimately affects the integrity of actins and tubulins. Rho GTPases play a pivotal role in many aspects of neuronal development, influencing neuritogenesis, axonal pathfinding and regulating dendritic spine formation (for review, see Govek et al., 2005). More recently, Rho family of GTPases has been shown be involved in excitotoxic neuronal death through directly modulating functions of the SAPKs (Semenova et al., 2007). For example, Cdc42 and Rac are activators of the c-Jun N-terminal kinase ( JNK) and p38 SAPKs and Rho A selectively mediates calcium-dependent activation of p38a to induce excitotoxic neuronal death (Semenova et al., 2007). Over-expression of dominant negative Cdc42 and Rac provides neuroprotection against neuronal death caused by the withdrawal of trophic support which strongly supports the idea that cdc42/Rac and their associated proteins contribute to the survival of neurons (Bazenet et al., 1998). These studies reinforce the idea that aberrant functions of the guidance signalling molecules may tip the balance to maintain growth and survival thereby causing neuronal death. 3.3.2. The ‘‘PI3K-GSK’’ pathway The phosphoinositide 3-kinase (PI3K)/AKT1 pathway is acknowledged as a key component of cell survival. More recently, PI3K is now viewed as an important player in many aspects of cell motility and adhesion (hence it contributes to metastatic/invasive phenotypes of various cancer cells). Inhibitors to PI3K affects both axon formation and elongation (Yoshimura et al., 2006). The PI3K/AKT activation leads to activation of glycogen synthase

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kinase-3a and b (GSK-3a and b) through specific phosphorylation of Ser9 of GSK-3b by AKT, a kinase involved in many signalling pathways. PTEN, the lipid phophatase that breaks down that product of PI3K, prevents PI3K mediated activation of AKT therefore having an opposite effect on axonal outgrowth. PI3K/GSK in turn modulates the function of CRMP2 through

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specific phosporylation to affect the assembly of the cytoskeleton (Kim et al., 2006b; Yoshimura et al., 2005). Knockdown of both GSK-3a and b markedly reduced axon growth in dissociated cultures and slice preparations, led to the suggestion that GSK-3 is a downstream convergent point for many axon growth regulatory pathways (Kim et al., 2006b). Another group of PI3K downstream effectors of polarity setting molecules are the Par-3/Par-6/ aPKC polarity complex. However, major gaps exist in the picture as to whether Pars interact with AKT/GSKs/CRMPs (Fig. 3.1). Nevertheless, GSK has now been actively exploited as a drug target for brain therapeutics (Bhat et al., 2004). 3.3.3. The ‘‘Fyn-Cdk5’’ pathway Another pathway is emerging which involves Fyn (Morita et al., 2006) and Cdk5. Fyn is a member of the Src family of non-receptor tyrosine kinases and plays important roles in neuronal network building and behavior pattern determination. Cdk5 is a member of the serin/threonine kinase family which, due to the neuron-specific expression of the p35 regulatory subunit, has important roles in laminar formation of the cerebral cortex through regulation of neuronal migration, axon elongation and maintenance as well as stability and steering of their growth cones (Morfini et al., 2004; Nguyen and Bibb, 2003). In the cultured cortical neurons from fyn (-/-) and Cdk5, mutant (Tyr15 to Ala) mice, dendrites bear few spines and their response to Sema3A is also attenuated (Morita et al., 2006; Sasaki et al., 2002). Recent intense investigation of Cdk5 has also revealed a much larger role of this molecule in neuronal death and survival (Cheung and Ip, 2004; O’Hare et al., 2005). So how exactly do Fyn and Cdk5 regulate cytoskeleton reorganization to affect growth cone collapse? As illustrated in Figure 3.1, Sema3A activation of Plexin-As transduces signals to Cdk5 through activation of Fyn which is always associated with Plexins. Increased phosphoryaltion of Tyr15 of Cdk5 in the growth cone by Fyn Src kinase activates Cdk5 which leads to increased phosphorylation of the cytoskeleton system, such as Tau, thereby affecting the collapse of growth cone (Sasaki et al., 2002; Uchida et al., 2005). Alternatively, Cdk5 may modulate CRMPs and microtubules through GSKs to affect axonal guidance (Uchida et al., 2005). It is unclear at this time as to relatively how much weight each pathway contributes to the end result of growth cone guidance. One thing that is clear though is how these proximal pathways converge on several key points to impact on the integrity of the cytoskeleton system, and that one of the key converging molecules is the family of collapsin response mediator proteins (CRMPs). CRMPs are members of a small family of brain specific proteins consisting of five highly related members (CRMP-1 to -5) (Bretin et al., 2005; Wang and Strittmatter, 1996). They are homologues of Unc33 whose

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mutation in C. elegans causes an ubiquitous impairment in the formation of neural circuits and results in severely uncoordinated locomotion (Hedgecock et al., 1985, 1990; Li et al., 1992). CRMPs are also heavily involved in the regulation of axonal path-finding during development. During semaphorin 3A-mediated growth cone collapse, CRMPs act as cytosolic messengers through neuropilin receptors (Liu and Strittmatter, 2001). The most studied member of the family is CRMP-2 which is expressed in growth cones and distal parts of the growing axons (Arimura et al., 2000; Inagaki et al., 2001; Suzuki et al., 2003). It has be shown that CRMP-2 modulates axonal length by modifying F-actin filaments, microtubules and cytoplasmic flow (Bradke and Dotti, 1999, 2000b; Charrier et al., 2003; Franken et al., 2003). Although how CRMP-2 precisely modulates cytoskeleton to induce axonal collapse remains not well understood, it is known that CRMP-2 acts as a common intracellular target by integrating both positive and negative effects on axon extension possibly through distinct upstream activators. For example, CRMP-2 is phosphorylated in vitro and in vivo by the Rho family GTPase and a Rho effecter, Rhokinase (ROCK). ROCK-dependent and independent pathways exist for growth cone collapse through CRMP-2 phosphorylation (Arimura et al., 2000). Cdk5, as a result of association with Fyn, also targets CRMP2 through increasing phosphorylation of CRMP-2, thereby inactivating CRMP-2 to mediate growth cone changes (Uchida et al., 2005). Importantly, CRMPs have also recently been implicated in the death of neurons in a number of neurodegenerative diseases as described in the following section, which further support the notion that misbehave of the guidance signaling molecules may lead to neuronal death.

4. Guidance Cues in Axonal Damage and Neuronal Death 4.1. Ischemic neuronal death and axonal damage Glutamate receptor-mediated excitotoxicity is a major mechanism of neuronal death in various pathological conditions including cerebral ischemia. Cerebral ischemia-induced interruption of the supply of energy (glucose) to neurons leads to a reduction in ATP levels causing depolarization of the presynaptic membrane. Depolarization of the pre-synaptic membrane increases the release of glutamate with disturbed Ca2þ homeostasis, causing neuronal death. In many cases, glutamate toxicity can be attributed to excessive stimulation of the NMDA (N-methyl-D-aspartic acid) subtype glutamate receptors. NMDA receptors are important for both normal transmission and pathological damage, and it appears that the locations of the activated receptors determine the consequence of NMDA signalling (Aarts and

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Tymianski, 2004; Hou and MacManus, 2002; Kirino, 2000; Kobayashi and Mori, 1998; Love, 2003). Fascinating evidence emerged recently that growth and survival signals may in fact be derived from synaptic NMDA receptor complexes, whereas cell death signals are derived from extrasynaptic NMDA receptors (Hardingham, 2006; Hardingham et al., 2002; Hardingham and Bading, 2002, 2003; Riccio and Ginty, 2002). Distinct regulation of neuronal survival or death gene expression through synaptic versus extrasynaptic NMDA receptors, respectively, represents a fundamental and yet unexplored mechanism for neuronal response to excitotoxic insult including cerebral ischemia. Since NMDA receptors interact with many intracellular molecules, extrasynapse-to-nucleus signalling mechanisms are far from being clearly understood. A single second messenger, calcium, controls gene expression triggered by neuronal activity, and the spatial properties of calcium signalling determine the type of transcriptional response (Hardingham and Bading, 2003). In contrast to influx through synaptic NMDA receptors, calcium influx through extrasynaptic NMDA receptors is coupled to the cell death pathway. Identification of novel mediators responsible for death signal transduction in response to extrasynaptic NMDA receptor signalling will provide new insight into the mechanism of excitotoxicity-mediated neuronal death and provide the basis for designing novel drugs to achieve neuroprotection in diseases like cerebral ischemia. In this context, it is of significant interest to know if NMDA signalling requires the interaction with guidance pathways. Glutamate receptormediated Ca2þ influx modulation of cerebellar granule neuronal migration and EphB2 regulation of postnatal NMDA-dependent synaptic function are both especially highly suggestive of the existence of interactions of guidance pathway with mediators of excitotoxicity (Henderson et al., 2001; Yacubova and Komuro, 2003).

4.2. Semaphorin/neuropilin in neuronal death The role of semaphorins in the adult nervous system is far less clear. In addition to questions such as why are there so many related semaphorins, what are the underlying mechanisms of their complex regulation in expression patterns, and what of the poorly understood molecular mechanisms of semaphorin signalling, much remains to be learned about the importance of semaphorin in brain pathology and disease (De Winter et al., 2002a,b; De Wit and Verhaagen, 2003; Giger et al., 1998; Goshima et al., 2000; Pasterkamp et al., 1998). Because of these inhibitory roles of semaphorin-3A during development, it is not surprising to see researchers implicating an underlying role for semaphorins in a number of neurodegenerative diseases including Alzheimer’s, motor neuron degeneration and injuries caused by cerebral ischemia [as reviewed by De Winter and co-workers (2002b)].

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Semaphorin-3A expression is elevated in both neurons and components of the scar tissues such as glial cells in the injured adult brain (Beck et al., 2002; Fujita et al., 2001; Zhang et al., 2001). Our studies also demonstrated an increased expression of receptors for semaphorins, neuropilin1 and 2, during postnatal development and in the ischemic side of the brain (Fig. 3.2). The precise pathological significance of the induction of semaphorin-3A and neuropilins in the adult ischemic brain is unknown. In particular, little is known about the functions of the neuronal-expressed semaphorin-3A. However, it is highly possible that semaphorin-3A, both neuronal expressed and glial secreted from the scar tissue, form an inhibitory gradient to repel regenerating axons through interaction with its neuronal receptor neuropilin-1 resulting in the loss of connection and neuronal death (Fujisawa, 2002; He and Tessier-Lavigne, 1997; Kolodkin et al., 1997; Nakamura et al., 2000; B

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Figure 3.2 Characterization of neuropilin expression. Brain mRNAs were extracted using the Trizol reagent and equal amount of the mRNA was reverse-transcribed into cDNA for PCR amplification. An internal control, b-actin, was used to indicate equal cDNA loading. As shown in panels A and B, the level of neuropilin 1 and 2 (NRP1/2) mRNA increased in the adult mice brains, and the level of NRP1/2 also increased in adult mouse brain after 1 h middle cerebral artery occlusion (MCAO) and followed by 2 and 4 h reperfusion (panels C and D). * in panel D, represents statistical significance with p <.01 (Student’s t-test).

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Pasterkamp et al., 1998). Interestingly, a recent in vitro study has demonstrated that neuronal-expressed semaphorin-3A causes the death of activated microglial thereby providing neuroprotection in the injured brain (Majed et al., 2006). Direct evidence showing semaphorin-3A’s involvement in neuronal degeneration comes from in vitro studies in that dopamine treatment of cultured sympathetic neurons up-regulates the expression of semaphorin-3A which eventually causes neuronal death (Shirvan et al., 1999, 2000, 2002), while blocking semaphorin-3A provides protection against neuronal death (Shirvan et al., 2002). These studies suggest that semaphorin-3A may transmit a death signal through neuropilins. Detailed investigation is urgently needed to unveil the role of semaphorin-3A in vivo following cerebral ischemia.

4.3. Netrin-1/UNC/DCC in neuronal death (the dependence receptor theory) It has been found that the netrin-1 receptor UNC5B is a molecular target of p53 (Arakawa, 2005; Tanikawa et al., 2003). Through bioinformatics searches and sequence comparison analysis, it has been shown that UNC5 contains the classical death domain sequence similar to that found in Fas (APO-1) and TNF receptors (Arakawa, 2005). Studies also found that Fas, the death receptor, can indeed mediate the pruning of neuronal branches during development (Zuliani et al., 2006), arguing that the death domain may be involved in microfilament re-organization and subsequently may lead to apoptosis. UNC5B is one of four related receptors for netrin-1 and all of which are type-1 transmembrane proteins. The proposed mechanism of p53 activating UNC5B in response to death stimuli is that membrane localized UNC5B is cleaved, possibly by activated caspases since the intracellular fragment of UNC5B contains a classical caspase-cleavage sequence DXXD, thereby releasing the peptides containing the death domain. This fragment then in turn interacts with DAPK and/or NRAGE which subsequently activates more capsases to initiate apoptosis. Therefore, UNC5B-induced apoptosis represents a pathway which is dependent on its interaction with netrin-1, but independent of mitochondrial and death receptor pathways (Arakawa, 2005c). These receptors have been dubbed dependence receptors (Mehlen, 2005; Mehlen and Bredesen, 2004; Mehlen and Goldschneider, 2005) because, in the absence of ligand availability, they induce programmed cell death, whereas in the presence of their trophic ligands, programmed cell death is inhibited as illustrated in Figure 3.3. So far, more than 10 such dependence receptors have been identified (Fig. 3.3). For example, p75NTR, the common neurotrophin low affinity receptor; the netrin-1 receptors DCC, UNC5H1, UNC5H2, and UNC5H3; the androgen receptor (AR); RET, the receptor for GDNF (glial cell line-derived

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Figure 3.3 The dependence receptor theory. In the presence of ligand, the dependence receptor mediates signal transductions to distal axonal guidance response. However, in the absence of appropriate guidance cues, apoptotic signals activate caspases or calpain to cleave the guidance receptor. The truncated membrane-anchored receptor and/or the cleavage product in turn activate apoptotic death pathways to induce neuronal death (A). The known possible ligands of dependence receptors are listed in B. (Modified and updated based on Mehlen and Bredesen, 2004; Mehlen, 2005.)

neurotrophic factor); integrins such as avb3 and a5b1, and the receptor for sonic hedgehog, patched (Ptc) (Mehlen and Bredesen, 2004).

4.4. RGM/Neogenin dependence receptors in neuronal death Works by Monnier and colleagues (Monnier et al., 2002; Schwab et al., 2001, 2005a,b) have shown that RGMa is highly expressed in the human adult nervous system and at the site of CNS injury. For example, following focal cerebral ischemia and traumatic brain injury, RGMa was found to

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increase in expression in the lesion site and in the penumbral area, both in neurons and in leucocytes infiltrating the lesion site (Schwab et al., 2005a,b). One week after insult, as the lesion matures, RGMa expression was observed in the glial scar on reactive astrocytes. The fact that RGMa is present in the lesion site has led to the suggestion that RGMa might exert inhibitory effects to the regenerating axons. However, questions remain as to what exactly causes the expression of RGMs, in particular, whether inflammation induces RGM expression in the injured brain is of significant interest. At least one report suggested that directly injected complete Freund’s adjuvant, although inducing spinal cord inflammation, did not elicit the up-regulation of RGMa (Doya et al., 2006; Hata et al., 2006). More experimental data are definitely required.

4.5. CRMP in neuronal death and survival Although the role of CRMPs in mediating growth cone collapse is well established, confounding evidence exists in the literature as to whether CRMPs modulate the death or survival of postmitotic neurons. The indication that CRMPs may be involved in neuronal death comes from the studies of dopamine-induced death of cerebellar granule neurons (CGNs) (Shirvan et al., 1999). Using the non-subjective differential display technique, it was found that CRMP-2 expression was up-regulated during the early stages of dopamine-induced neuronal apoptosis (Shirvan et al., 1999). Antibodies to Semaphorin 3A blocked CRMP-2 activities and neuronal death, whereas Semaphorin 3A-derived peptides induced apoptosis of cultured neurons (Gagliardini and Fankhauser, 1999; Shirvan et al., 2000). There is an increasing amount of evidence demonstrating that CRMPs participate in neuronal death both during development and in injured adult brains (Charrier et al., 2003; Franken et al., 2003; Hou et al., 2006; Zhang et al., 2007). In particular, our studies showed that a 2 h middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion caused a dramatic increase in the expression of CRMPs in injured neurons located in the infarct area on the ischemic side of the brain, but not in the contralateral side of the brain (see Fig. 3.4) (S. Jiang, J. Kappler, B. Zurakowski, A. Desbois, A. Alysworth and S. T. Hou, unpublished data; Hou et al., 2006). In response to glutamate toxicity in cultured CGNs, the expression of CRMP-3 increased dramatically in dying neurons (Hou et al., 2006). The full-length CRMP-3 (p63) was cleaved by calpain to produce a N-terminally truncated form of p54. MK801 and calpain inhibitors (calpastatin and ALLN) prevented CRMP-3 cleavage and neuronal death evoked by glutamate. Small interfering RNA to CRMP-3 (siRNA) significantly protected axons and neurons against glutamate toxicity. Over-expression of the full-length CRMP-3 (p63) in human HEK293 cells did not cause cell death, but over-expression of the N-terminally truncated p54 induced

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Figure 3.4 CRMP cleavage in response to MCAO reperfusion. Mice were subjected to 2 h MCAO and followed by 2^24 h reperfusion. Brains were collected at the time point as indicated in panel A.Western blotting was performed on these proteins using specific antibodies to CRMP 1^5. A clear cleavage band is visible in all MCAO brain samples. CRMP5 antibody to the C-terminus of the protein did not detect any cleavage suggesting that CRMP5 was cleaved towards the C-terminus of the protein. GAPDH was used as an internal control to show equal protein loading. The CRMP cleavage band was quantified using densitometry and normalized against that of GAPDH. As shown in panel B, the relative expression of the cleavage bands are plotted demonstrating that CRMP4 cleavage sharply increased after 6 h reperfusion.

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significant amounts of cell death. These studies demonstrated a potential role of CRMP-3 in modulating axonal damage and neuronal death (Hou et al., 2006). As shown in Figure 3.4, all CRMPs are targeted for cleavage during cerebral ischemia, and this cleavage can be inhibited by calpain inhibitors (S. Jiang and S. T. Hou, unpublished data), and ubiquitin proteasome system (UPS) inhibitor MG132, but not by protein translation inhibitor cychloheximide (Fig. 3.5), which strongly argues for the fact that posttranslational modification is very important for CRMP-mediated signal transduction in neuronal death. What is the evidence supporting the role of CRMPs role in neuronal survival? In normal adult brains, the expression of CRMPs is differentially regulated. Some members of CRMPs are significantly reduced in the level of expression (Bretin et al., 2005; Wang and Strittmatter, 1996) and are only found in areas undergoing neurogenesis and/or plasticity such as the hippocampus, the olfactory system and the cerebellum (Charrier et al., 2003; Kee et al., 2001). Over-expression of CRMP-2 has been found to accelerate nerve regeneration (Pasterkamp and Verhaagen, 2001; Suzuki et al., 2003), suggesting that CRMP-2 is associated with the survival and plays a maintenance role in postmitotic neurons. Following ischemic injury in the brain, induction of CRMP-4 expression was found in the ischemic brains and the level of CRMP-4 expression was associated with neurons having an

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intact morphology. These observations have led to the suggestion that CRMP-4 is a survival factor and may be involved in neuronal regeneration (Kee et al., 2001). Much remains to be learned as to the exact role of CRMPs in adult brain neurons during stress and injury.

4.6. CRMP modulation by calpain and CaMK during neuronal death The role of CRMPs in axonal guidance has been shown to be regulated by upstream kinases through increasing/decreasing phosphorylations. How CRMPs are modulated during neuronal death caused by excitotoxicity is very different. It appears that calcium activates calpain and CaM kinases are two very important candidates as summarized below. 4.6.1. Calpain targets CRMPs Excitotoxicity-mediated neuronal death involves the activation of Ca2þdependent proteases such as calpain. Calpains are a highly conserved family of calcium-dependent proteases. The ubiquitous -calpain and m-calpain are heterodimeric regulatory enzymes consisting of an 80 kD catalytic subunit and a 30 kD regulatory subunit. Whereas the two forms differ in their calcium requirements for in vitro activation, their substrate specificities are almost identical (Chan and Mattson, 1999; Lee et al., 1997). In almost all cases, the activation of calpain has been measured indirectly as increased proteolysis of endogenous -calpain substrates such as spectrin or MAP-2. It is important to note that calpain-mediated spectrin breakdown is specifically coupled to Ca2þ entry through the NMDA receptors. Nonspecific Ca2þ influx via ionomycin or KCl-mediated depolarization failed to activate the enzyme. The importance of calpains is underscored by the fact that mice deficient in the 30 kD small regulatory subunit suffer from embryonic lethality (Carragher, 2007; Hara and Snyder, 2006; Kuchay and Chishti, 2007). In the CNS, calpains are widely expressed and their activities are modulated by an endogenously expressed inhibitory protein calpastatin. Calpain has been reported to cleave a large number of substrates including cytoskeletal proteins and important regulatory proteins such as cyclindependent protein kinase 5 (CDK -5) activator p39 producing a C-terminal truncated fragment at a molecular weight of 29 kD (Chan and Mattson, 1999; Hara and Snyder, 2006; Hou et al., 2006; Kulkarni et al., 2002). Expression of calpain inhibitor calpastatin is neuroprotective to many neurological diseases such as Parkinson’s and cerebral ischemia. Therefore identification of targets of calpain in response to NMDA receptor activation will shed light on the molecular mechanism of glutamate-induced neuronal death. Our recent studies showed that the activation of calpain through extrasynaptic NMDA receptors (by adding glutamate) induced proteolytic cleavage of CRMP-3 (Hou et al., 2006). Furthermore, the calpain inhibitor

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ALLN and calpastatin blocked the breakdown of CRMP-3 in response to glutamate toxicity. The CRMP-3 amino acid sequence indeed contained a putative calpain cleavage site between amino acid 73 and 85. Taken together, these data are highly suggestive that calpain activated in a calcium-dependent manner through NMDA receptors targets CRMP-3 during glutamatemediated neuronal death. Calpain also associates with focal adhesion proteins in platelets and regulates the attachment of modulatory proteins to the cytoskeleton and relaxes the retraction of fibrin clots. Because focal adhesion and stress-fiber formation are processes dependent on Rho GTPases, calpain has been postulated to regulate these processes (Kulkarni et al., 1999, 2002). Expression of Rho or Rac constructs overcomes the effect of calpain inhibition of adhesion and stress fiber formation; strongly suggesting that calpain regulates Rho and Rac. Moreover, calpain also cleaves RhoA to modulate actin filaments (Kulkarni et al., 1999, 2002). It is therefore highly possible that calpain modulation of guidance molecules causes neuronal death during excitotoxicity. 4.6.2. CaMK II targets CRMPs A potential alternative activator of CRMP-3 in response to calcium influx through NMDA receptors is the Ca2þ/calmodulin-dependent protein kinase II (CaMK II). Indeed, recent mass spectrometry studies have identified more than 30 proteins at the postsynaptic density (PSD) to be substrates of CaMK II (Fink et al., 2003; Fink and Meyer, 2002; Silva et al., 1992; Yoshimura et al., 2004) and, interestingly, CRMP-2 is one of them (Yoshimura et al., 2004). CRMPs are present in relatively high concentrations in the PSD fraction. However, whether CaMK II targets CRMPs to regulate plasticity and death of neurons in adult brains remains unknown. CaMK II functions as a link between Ca2þ stimuli and neuronal death caused by NMDA receptor activities. When calcium increases in the postsynaptic component, CaMK II is autophosphorylated and activated. Activated CaMK II is translocated to the PSD to target NR2B, a major component of the extrasynaptic NMDA receptors. The importance of CaMK II in neural functions is underscored by the fact that mice lacking CaMK II show numerous deficiencies in learning and neuronal plasticity (Silva et al., 1992). Loss of CaMK II activity also results in increased damage to neurons in response to both focal and global ischemia in mice (Hajimohammadreza et al., 1995; Takano et al., 2003; Waxham et al., 1996). However, numerous reports have shown that inhibitors to calmodulin and CaMK II are potently neuroprotective (Hajimohammadreza et al., 1995; Takano et al., 2003), which argue for a role of CaMK II in modulating neuronal death. Given that CaMK II has many intracellular targets, it is necessary to determine mechanistically whether CaMK II may target CRMP-3 to mediate neuronal death or survival.

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5. Guidance Cues and Synaptic Plasticity in Stroke Brains Glutamate over-activation of NMDA receptors increases intracellular Ca2þ concentrations which in turn activates Ca2þ-dependent proteases resulting in degradation of key structural and regulatory proteins and which ultimately leads to neuronal death. Even before the final demise of the injured neuron, distally located neurites undergo rapid physiological and structural alterations consisting of focal swelling and spine loss (Calabresi et al., 2003; Hasbani et al., 2001). It has also been shown that during cerebral ischemia reperfusion, the spine synapses can re-emerge in neurons indicating synaptic plasticity. Indeed, energy deprivation and anoxia evoke longterm potentiation (LTP), which also constitutes to synaptic plasticity (Calabresi et al., 2003; Smith and Jiang, 1994). Together, it has been postulated that ischemia-induced synaptic plasticity may be a crucial factor in determining the ensuing delayed neuronal death after ischemic injury to neurons. Although NMDA receptors (NMDAR) are known to cause ischemia-induced synaptic plasticity, the identities of mediators of NMDAR in ischemia-induced synaptic plasticity remain unknown. Nevertheless, it has been suggested that growth cone guidance molecules are intimately involved in synaptic plasticity as a result of local protein synthesis or in response to specific calcium channel activities. For example, local protein synthesis occurring in the mature axons has recently been confirmed and shown to be necessary for axonal guidance regulation and synaptic plasticity in neuronal physiology and pathology (Calabresi et al., 2003; Martin, 2004; Martin and Zukin, 2006; Otis et al., 2006; Thompson et al., 2004). Local translation of RhoA mRNA occurs in response to Sema3A-mediated growth cone collapse (Wu et al., 2005). With the design of novel growth cone selective expression vectors, many more growth cone guidance molecules are found to be expressed locally and in response to specific stimuli (Martin, 2004). EphA2 mRNA was found to be translated only after it has crossed the midline (Brittis et al., 2002). Moreover, BDNF potentiates neurotransmitter release from the developing synapse which requires local protein synthesis (Tyler et al., 2006; Zhang and Poo, 2002). More interestingly, protein degradation systems such as caspases (Williams et al., 2006), calpain and UPS are all found to play important roles in growth cone guidance and synapses during pruning of dendrites and neuronal death in nerve growth factor withdrawal (Zhai et al., 2003). Work from our own laboratory also showed that synaptosomal cleavage of growth cone guidance molecules such as CRMP also occurs during cerebral ischemia (Hou et al., 2006) (see the following section).

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There is also emerging evidence to suggest that transient receptor potential channels (TRPCs) may be an important target of NMDAR-mediated synaptic remodelling. TRPCs are members of a large family of TRP channels with conserved six-transmembrane domains forming nonselective cation-permeable channels (Ramsey et al., 2006). TRPCs mediate the transmembrane flux of cations down their electrochemical gradients, thereby raising intracellular Ca2þ and Naþ concentrations and depolarizing the cell. A striking number of biological functions have already been assigned to the various TRPC proteins, interestingly, with several members having similar functions in chemotropic axon guidance (TRPC1 and TRPC3) and axonal growth (TRPC5). TRPCs are widely expressed in the brain and are activated by the G protein–coupled receptors and receptor tyrosine kinases (RTKs). In the central nervous system, many trophic factors involved in modulating synaptic plasticity are RTKs, thereby implicating TRPCs in synaptic plasticity. Recent studies from Dr. Wang’s lab in Shanghai have shown that TRPCs are required in neuronal growth cone steering by the brain-derived neurotrophic factor (BDNF), confirming the function of TRPCs in axonal guidance and response to chemotaxis (Li et al., 2005). BNDF receptor TrkB contributes to dendritic spine development, synaptic strength and LTP induction (Lu, 2003; Tyler et al., 2002, 2006; Tyler and Pozzo-Miller, 2001) which further implicates TRPCs in modulating synaptic functions. Significantly, recent studies from Dr. Wang’s laboratory showed that TRPC3 and 6 are important in mediating neuronal survival during serum withdrawal. Although it is not clear if this protection involves synaptic plasticity, the fact TRPC may signal protection through CREB strongly argues for a role in synaptic and survival response.

6. Evidence for Guidance Cues as Therapeutic Targets The functional outcome of ischemic brain injury is the result of a complex interplay between permanent damage and long-term plasticity which can be beneficial or detrimental. Accordingly, limiting tissue damage and promoting useful plasticity are the two pillars of modern stroke management. It is therefore not surprising that efforts have been made to target the repulsive guidance cues and indeed in vivo evidence strongly support the hypothesis that blocking inhibitory guidance cues and their pathways have protective utilities in limiting brain damage. For example, our own work shows that specific polypeptides targeted against semaphorin-3A and neuropilin-1 (Williams et al., 2005) indeed protect neurons from oxygenglucose-deprivation induced neuronal death ( Jiang et al., 2007). A smallmolecule inhibitor of semaphorin-3A (SM-216289, isolated as a natural

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product from fungal fermentation) has also been shown to promote CNS repair in adult rats subjected to spinal cord transaction (Kaneko et al., 2006; Kikuchi et al., 2003). Treatment with SM-216289 increased regeneration of injured axons, Schwann cell-mediated myelination, enhanced angiogenesis and decreased apoptotic cell death. Delivery of Nogo antibody (IN-1) through direct injection into rat brains is also effective in improving neuroanatomical and functional recovery following middle cerebral artery occlusion (Papadopoulos et al., 2002, 2006). Inhibition of intracellular signalling pathways is also neuroprotective. Rho kinase inhibition has been shown to enhance axonal plasticity and confer neuroprotection (Lehmann et al., 1999; McKerracher and Higuchi, 2006; Ramer et al., 2004).

7. Concluding Remarks and Future Perspectives Significant progress has been made in understanding the role of growth promoting and inhibitory guidance molecules in both axonal initiation and specification during development, but also axonal degeneration and neuronal death in disease states and following trauma. A goal of future research which could have far-reaching consequences should focus on how the plethora of intrinsic and extrinsic factors, that act in promoting axonal growth, guidance and in establishing successful contacts in embryonic life, might be reactivated in the adult and thereby enhance regeneration. Degeneration of neurites occurring during neuronal death is controlled by events confined to the neurites and which occurs autonomously from the neuronal soma (Deckwerth and Johnson, 1994). Encouragingly, inhibitors blocking the inhibitory cues have been shown in proof-of-principle to be effective in facilitating axonal regeneration and providing neuroprotection. However, questions remain as to how these inhibitory molecules are activated in the injured brain. Recently, semaphorin 6A has been shown to be induced by interferon-g and defines an activation status of Langerhans cells (Gautier et al., 2006). Do cytokines regulate the guidance molecules in the injured CNS? Strong evidence exist that activated microglial causes neuronal toxicity by synthesizing and releasing neurotoxic compounds in response to neuronal injury thereby potentiating neuronal loss and synaptic dissolution, a typical response mediated by cytokines (Schwartz, 2003; Schwartz and Moalem, 2001). Emerging evidence also strongly supports the notion that immune response in the injured CNS may be beneficial to the repair and regrowth of the injured neurons (Banati et al., 1996; Kreutzberg, 1996; Schwartz, 2003) and it appears that the timing of the immune response is critical in determining the outcome. Future efforts will reveal the role of cytokines in the regulation of the production of guidance molecules.

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ACKNOWLEDGMENT We would like to thank Melissa Sheldrick and Angele Desbois for technical assistance. This work was supported by grants-in-aid from the Heart and Stroke Foundation of Ontario to S.T.H. (NA5393 and T5760). S.T.H. and R.A.S. thank the British Council for funding a number of Researcher Exchange Awards which enabled them to make reciprocal visits to each others laboratories whilst planning and writing this review.

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Wilson, M. T., and Snow, D. M. (2000). Chondroitin sulfate proteoglycan expression pattern in hippocampal development: Potential regulation of axon tract formation. J. Comp. Neurol. 424, 532–546. Wilson, N. H., and Key, B. (2006a). Neogenin interacts with RGMa and netrin-1 to guide axons within the embryonic vertebrate forebrain. Dev. Biol. 296, 485–498. Wilson, N. H., and Key, B. (2006b). Neogenin: One receptor, many functions. Int. J Biochem. Cell Biol. 39, 874–878. Withers, G. S., James, C. D., Kingman, C. E., Craighead, H. G., and Banker, G. A. (2006). Effects of substrate geometry on growth cone behavior and axon branching. J. Neurobiol. 66, 1183–1194. Wu, K. Y., Hengst, U., Cox, L. J., Macosko, E. Z., Jeromin, A., Urquhart, E. R., and Jaffrey, S. R. (2005). Local translation of RhoA regulates growth cone collapse. Nature 436, 1020–1024. Yacubova, E., and Komuro, H. (2003). Cellular and molecular mechanisms of cerebellar granule cell migration. Cell Biochem. Biophys. 37, 213–234. Yamada, K. M., Spooner, B. S., and Wessells, N. K. (1971). Ultrastructure and function of growth cones and axons of cultured nerve cells. J. Cell Biol. 49, 614–635. Yamada, R. X., Matsuki, N., and Ikegaya, Y. (2006). Nitric oxide/cyclic guanosine monophosphate- mediated growth cone collapse of dentate granule cells. NeuroReport 17, 661–665. Yazdani, U., and Terman, J. R. (2006). The semaphorins. Genome Biol. 7, 211. Yoshimura, T., Arimura, N., Kawano, Y., Kawabata, S., Wang, S., and Kaibuchi, K. (2006). Ras regulates neuronal polarity via the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway. Biochem. Biophys. Res. Commun. 340, 62–68. Yoshimura, T., Kawano, Y., Arimura, N., Kawabata, S., Kikuchi, A., and Kaibuchi, K. (2005). GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120, 137–149. Yoshimura, Y., Yamauchi, Y., Shinkawa, T., Taoka, M., Donai, H., Takahashi, N., Isobe, T., and Yamauchi, T. (2004). Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatographytandem mass spectrometry. J. Neurochem. 88, 759–768. Yu, W. Q., Ling, C. Y., and Baas, P. W. (2001). Microtubule reconfiguration during axogenesis. J. Neurocytol. 30, 861–875. Zhai, Q., Wang, J., Kim, A., Liu, Q., Watts, R., Hoopfer, E., Mitchison, T., Luo, L., and He, Z. (2003). Involvement of the ubiquitin-proteasome system in the early stages of wallerian degeneration. Neuron 39, 217–225. Zhang, J., and Hughes, S. (2006). Role of the ephrin and Eph receptor tyrosine kinase families in angiogenesis and development of the cardiovascular system. J. Pathol. 208, 453–461. Zhang, X., and Poo, M. M. (2002). Localized synaptic potentiation by BDNF requires local protein synthesis in the developing axon. Neuron 36, 675–688. Zhang, Z., Ottens, A. K., Sadasivan, S., Kobeissy, F. H., Fang, T., Hayes, R. L., and Wang, K. K. (2007). Calpain-mediated collapsin response mediator protein-1, -2, and -4 proteolysis after neurotoxic and traumatic brain injury. J. Neurotrauma 24, 460–472. Zhang, Z. G., Tsang, W., Zhang, L., Powers, C., and Chopp, M. (2001). Up-regulation of neuropilin-1 in neovasculature after focal cerebral ischemia in the adult rat. J. Cereb. Blood Flow Metab. 21, 541–549. Zhou, F. Q., and Cohan, C. S. (2004). How actin filaments and microtubules steer growth cones to their targets. J. Neurobiol. 58, 84–91. Zivin, J. A. (2007). Clinical trials of neuroprotective therapies. Stroke 38, 791–793. Zuliani, C., Kleber, S., Klussmann, S., Wenger, T., Kenzelmann, M., Schreglmann, N., Martinez, A., del Rio, J. A., Soriano, E., Vodrazka, P., Kuner, R., Groene, H. J., et al. (2006). Control of neuronal branching by the death receptor CD95 (Fas/Apo-1). Cell Death. Differ. 13, 31–40.

C H A P T E R

F O U R

New Insights into Mechanism and Regulation of Actin Capping Protein John A. Cooper* and David Sept† Contents 1. Introduction 2. Background 2.1. Physical and chemical properties 2.2. Biochemical activities 2.3. Cellular studies 2.4. Sequence conservation and isoforms 3. Mechanism of Binding Actin 3.1. Structural studies 3.2. Mobility of the C-terminal regions of subunits 4. Capping Protein Inhibitors and Uncapping 4.1. Contrasting results with CARMIL and V-1 4.2. Interaction of polyphosphoinositides with capping protein 4.3. CARMIL, CKIP-1 and CD2AP—A motif for inhibition of capping protein 4.4. Regulators that antagonize capping protein by indirect effects 4.5. Other interactors 5. Role of Capping Protein in Complex Cellular Processes 5.1. Actin-based motility at the plasma membrane 5.2. Z-line of the sarcomere in striated muscle 5.3. Drosophila development 5.4. Dynactin 6. Concluding Remarks and Future Directions Acknowledgments References

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Abstract The heterodimeric actin capping protein, referred to here as ‘‘CP,’’ is an essential element of the actin cytoskeleton, binding to the barbed ends of actin

* {

Department of Cell Biology, Washington University, St. Louis, MO, 63110 Department of Biomedical Engineering and Center for Computational Biology, Washington University, St. Louis, MO, 63130

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00604-7

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filaments and regulating their polymerization. In vitro, CP has a critical role in the dendritic nucleation process of actin assembly mediated by Arp2/3 complex, and in vivo, CP is important for actin assembly and actin-based process of morphogenesis and differentiation. Recent studies have provided new insight into the mechanism of CP binding the barbed end, which raises new possibilities for the dynamics of CP and actin in cells. In addition, a number of molecules that bind and regulate CP have been discovered, suggesting new ideas for how CP may integrate into diverse processes of cell physiology. Key words: Actin, Polymerization, Cell motility, Cell migration, Capping protein, Sarcomere. ß 2008 Elsevier Inc.

1. Introduction The actin capping protein (CP) was discovered, defined and named based on its ability to bind to the barbed ends of actin filaments, i.e., to ‘‘cap’’ them. The presence of CP at the barbed end inhibits the addition and loss of actin subunits at that end. In cells, CP is important for the dynamics of actin filament assembly, and this is important for the control of cell shape and movement. CP was called b-actinin when first characterized and purified from muscle by Maruyama and colleagues in the 1960s and 1970s in a remarkably prescient series of studies (Maruyama, 1966, 2002; Maruyama et al., 1977; Maruyama and Obinata, 1965). Nonmuscle CP was purified to homogeneity from Acanthamoeba in 1980 and shown to cap barbed ends (Isenberg et al., 1980). CP has continued to be an active subject of research, in part because it is found in essentially every eukaryotic organism and every metazoan cell type. Recent studies have produced new insights into the biochemistry of the interaction of CP with the actin filament, the mechanism of how this interaction can influence the architecture of actin filaments nucleated by Arp2/3 complex, the role of CP’s actin-binding activity in cells, and the identities and roles of molecules that bind and regulate CP. This review focuses on these recent discoveries. Other reviews of CP include the following: (Cooper et al., 1999; Schafer and Cooper, 1995; Wear et al., 2000; Wear and Cooper, 2004b).

2. Background 2.1. Physical and chemical properties CP is an a/b heterodimer with each subunit having a mass of 30 kDa. Individual subunits are unstable, but the heterodimer is very stable. The heterodimer remains folded in 0.6 M KI or 1% nonionic detergent (Wear and Cooper, 2004a), and it melts at 58  C in a single irreversible transition (Sizonenko et al., 1996). Individual subunits expressed in bacteria are largely insoluble, but they can be renaturated as heterodimers from

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urea (Remmert et al., 2000). Simultaneous expression of both subunits in bacteria produces large quantities of soluble active protein; the development of this expression system was a major technological advance in the field (Soeno et al., 1998). CP remains soluble, folded and active for capping actin under a variety of physiological conditions, including the presence or absence of divalent cation, and in a variety of salt concentrations, osmolality and pH. The CP molecule has the shape of a mushroom (Yamashita et al., 2003). The two subunits have very similar secondary structures, which is remarkable given their essentially complete lack of sequence similarity. The secondary structural elements of the subunits are arranged such that the molecule has a pseudo-two-fold axis of rotational symmetry down the center of the mushroom (Fig. 4.1A) (Yamashita et al., 2003). On the top surface of the mushroom, both subunits have C-terminal amphipathic a helixes, which appear to bind actin (Wear et al., 2003).

2.2. Biochemical activities CP was named for its ability to inhibit growth of the actin filament at the barbed end, i.e., to ‘‘cap’’ that end (Isenberg et al., 1980). CP binds to barbed ends with sub-nanomolar affinity (Wear et al., 2003). The presence of CP at the barbed end prevents the loss of the terminal actin subunit at the end of the filament, thus preventing depolymerization of the filament from that end. The critical concentration for actin polymerization is lower at the barbed end than at the pointed end, and the rate constants for actin elongation are higher at the barbed end than at the pointed end. These facts mean that capping of barbed ends by CP leads to an increase in the critical concentration, i.e., the actin monomer concentration at steady state. Cell cytoplasm has a high concentration of unpolymerized actin, for which capping of barbed ends is probably necessary. One molecule of CP appears to be sufficient to bind and attach a filament barbed end to an object, based on direct observation of single actin filaments by light microscopy (Bearer, 1991), including recent TIRF microscopy (Pavlov et al., 2007). TIRF microscopy confirms that the presence of CP at the barbed end abrogates the addition and loss of actin subunits (Kim et al., 2007). CP was one of the proteins found to be required for the reconstitution of motility based on actin assembly from pure proteins, in a landmark study (Loisel et al., 1999). Other molecules that cap barbed ends, not only CP, can serve this function (Revenu et al., 2007). One idea about the essential role of CP in the reconstitution system is that CP caps barbed ends that are older and thus located away from the surface of the object to be moved. By preventing actin subunits from adding in these undesired locations, the

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A

B

Figure 4.1 Illustrations of a model for the interaction of V-1 with CP and how their interaction inhibits actin capping activity. (A) The structure of the proposed molecular interaction between capping protein and V-1/myotrophin.The a subunit of capping protein is yellow, and its C-terminal actin-binding region is teal. The b subunit of capping protein is red, and its C-terminal actin-binding region is green. V-1/myotrophin is in pink. (B) The binding of V-1/myotrophin prevents capping protein from binding to actin filament barbed ends, i.e.,‘‘capping.’’ Growth of free barbed ends is an essential of the dendritic nucleation model for actin assembly, which involves Arp2/3 complex as the nucleating and branching agent. The color scheme is similar to the one in panel A, with Arp2/3 complex as the green oval at an end-to-side branch point for two actin filaments, whose subunits are teal.

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addition of actin subunits in the optimal locations is promoted, which can be considered as ‘‘funneling’’ of the subunits to these locations (Carlier and Pantaloni, 1997). Recent studies with synthetic systems show that CP can cause the shell of Arp2/3-nucleated actin filaments that assemble around a bead to break symmetry, which is necessary to produce polarity and movement (Orkun Akin and Dyche Mullins, personal communication). In reconstituted systems, high concentrations of CP lead to decreased actin assembly, making the plot of actin assembly or motility versus CP concentration a bell-shaped curve (Loisel et al., 1999). This biphasic nature of the effect of CP makes it difficult to interpret results in complex systems, such as mixtures of multiple actin regulators or even the cell cytoplasm. Mathematical modeling can help make predictions in such cases. For example, the concentration dependence of actin assembly on CP in the presence of Arp2/3 complex provided information about the end versus side nature of Arp2/3-mediated branching, based on predictions from mathematical modeling of the alternatives (Carlsson et al., 2004).

2.3. Cellular studies The abilities of CP to cap barbed ends and to tether barbed ends to objects appear to be a physiologically relevant in cells. The concentration of CP in cells is in the micromolar range, comparable to the number of actin filament barbed ends, and the binding affinity is in the sub-nanomolar range (Cooper et al., 1984; Wear et al., 2003). Analysis of a set of CP mutants in yeast showed a correlation of capping activity with the ability to rescue the null mutant phenotype (Kim et al., 2004). In cultured myotubes, injection of an anti-CP mAb that inhibited the actin-binding ability of CP caused a disruption in the early steps in myofibrillogenesis, as did expression of a mutant form of the CP b subunit that caps actin poorly (Schafer et al., 1995). In the mouse heart, expression of a capping-deficient CP b subunit during development caused disruption of myofibril architecture (Hart and Cooper, 1999). Other potential functions for CP are discussed below.

2.4. Sequence conservation and isoforms One of the most interesting and surprising features of the CP crystal structure was the two-fold rotational similarity between the tertiary structures of the two subunits (Yamashita et al., 2003). In vertebrates, the sequence similarity between the a and b subunits is very low, and given the lack of symmetry at the end of the actin filament, there was little reason to expect such structural symmetry in the CP heterodimer. When comparing the individual subunits in different organisms, sequence similarity is much higher. BLAST searches readily reveal apparent homologs of both subunits in vertebrates, invertebrates, plants, fungi, insects and protozoa (Fig. 4.2A, B). The sequences

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of the b subunits appear to be more strongly conserved than those of a subunits (Fig. 4.2C). The regions of conservation and variability are localized in a complementary manner on the two subunits. Within the b subunit, the actinbinding C-terminal region, the b tentacle, shows the highest sequence

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Figure 4.2 Phylogenetic analysis of CP subunits. Amino acid sequences were identified in a wide range of eukaryotes by BLAST.The sequences were aligned with CLUSTALW. (A) Phylogenetic trees for the a and b subunits are remarkably similar. Vertebrates have up to three isoforms of each subunit, while invertebrates and lower organisms have single isoforms of each subunit. Vertebrate isoforms 1 and 2 represent nearly all the CP outside of germ cells, they cluster into distinct groups. (B) Phylogenetic analysis of CP subunits compared with cofilin and profilin, other actin-binding proteins.The a and b subunits of CP are not more similar to each other than they are to the cofilin and profilin families, despite their similar secondary structures and interactions with actin. (C) For each organism, the similarity of its b subunit to the b subunits of other organisms is plotted versus the similarity of its a subunit to the a subunits of other organisms. For vertebrates, only a single isoform of each subunit was included per species. The results show that the b subunit sequences are more similar to each other than are those of the a subunits.

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variability. In contrast, the body of the a subunit is more weakly conserved than is the C-terminal region, giving rise to an inherent asymmetry in the molecule where the half of the molecule containing the b subunit and a tentacle is more conserved than the half with the a subunit and the b tentacle. This asymmetry may have implications for how CP interacts with the barbed end of the actin filament, and it appears to be consistent with structural studies discussed next. Organisms other than vertebrates have single genes encoding each of the CP subunits. Vertebrates, in contrast, have two somatically expressed isoforms of each subunit and one additional male germ-cell specific isoform (Hart et al., 1997b; Hurst et al., 1998; Schafer et al., 1994; von Bulow et al., 1997). For the a subunit, the somatic isoforms, termed a1 and a2, are encoded by different genes (Hart et al., 1997a), while the b subunit isoforms are produced from a single gene by alternative splicing (Schafer et al., 1994). The sequences of the a1 and a2 isoforms are conserved across vertebrates, as are those of the b1 and b2 isoforms, suggesting that they have distinct functions in vertebrates. Little evidence exists regarding specific functions of the a isoforms, but they are expressed at varying ratios in different cells and tissues (Hart et al., 1997b). The b1 isoform is located specifically at the Z-disc of the sarcomere of striated muscle; b2 is also present in the same cells, but it localizes elsewhere (Schafer et al., 1994). The b1 and b2 isoforms were not able to substitute for each other in muscle cells, supporting the hypothesis of distinct functions (Hart and Cooper, 1999). The biochemical nature of the functional difference has not been discovered. One would suspect that the b1 isoform interacts specifically with one or more components of the Z-disc. CP isoforms appear to bind equally well to actin and nebulin, as purified proteins in vitro (Pappas et al., 2008; Schafer et al., 1994), and other components remain to be tested.

3. Mechanism of Binding Actin 3.1. Structural studies An x-ray crystal structure of CP shows that the molecule has the shape of a mushroom and that the two subunits are arranged with a pseudo-two-fold axis of rotational symmetry (Yamashita et al., 2003). The N-termini of the subunits are located at the base of the stalk of the mushroom, and the subunits are extensively intertwined, with a large b sheet at the core of the mushroom cap structure. On the top surface of the mushroom, each subunit has an extended a helix oriented perpendicular to the strands of the

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b sheet on which it lies. Each subunit ends with a C-terminal amphipathic a helix on the top surface of the mushroom. Truncation and point mutations of the C-terminal regions reveal that both are important for high-affinity capping (Wear et al., 2003), with the C-terminal region of the a subunit being more important than that of the b. CP binds to the barbed end of the actin filament with high affinity, generally less than 1 nM. The second-order association rate constant is high, approaching the range of the diffusion limit, and the first-order dissociation rate constant is accordingly low. CP containing only one (either one) of the C-terminal regions is able to cap, and the C-terminal region of the b subunit alone is sufficient to cap, with decreased affinity. The binding affinity and rate constants have been inferred largely from actin polymerization experiments, and physical binding studies would provide a valuable confirmation of those results. Recent cryoEM work from the Mae´da lab has resulted in a low˚ ) structure of CP on the barbed end of an actin filament resolution (23 A (Narita et al., 2006; Narita and Maeda, 2007). CryoEM analysis of actin filament binding proteins that decorate the sides of the filament can benefit from helical averaging. Here, only one molecule of CP was present on the barbed end of each filament, so this analysis depended on the collection and averaging of single-particle images, a challenging task. A novel method for combining the images that were collected produced a new model for the structure of the CP-capped filament. This structure was able to unambiguously identify the a and b subunits, and their positions with respect to the actin protomers at the barbed end suggest that the body of the b subunit, along with the a subunit C-terminal region, make the primary contacts with the last two protomers of the filament. This finding is supported by the sequence conservation data discussed above. Mutational analysis confirmed the importance of residues in the C-terminal region of the a subunit and on the top surface. Computational modeling analysis showed that the b subunit C-terminus, a tentacle-like amphipathic helix, can bind to a hydrophobic cleft on the actin subunit, in a manner comparable to that of a WH2 domain (Dominguez, 2004; Hertzog et al., 2004). Based on these results, the authors proposed a model, shown in Figure 4.3, in which CP binds to the barbed end in two steps, first by the a subunit C-terminus and surrounding residues and second by the flexible b subunit C-terminus (Narita et al., 2006). This model raises the possibility that CP bound to the barbed end of the actin filament might dissociate from the a subunit site and thus be attached only by the b subunit tentacle. If this were to occur, the mobility of the b tentacle might allow the body of CP to move, or ‘‘wobble,’’ in place. If another molecule would bind to CP in this wobble state, then the presence of that molecule might inhibit rebinding and thus favor complete detachment, or ‘‘uncapping.’’

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Figure 4.3 A model for the binding of CP to the actin filament barbed-end proposed by Narita and colleagues (Narita et al., 2006) kindly provided by the authors and reproduced with their permission. First, basic residues on the CP a C-terminal region (blue) are attracted to acidic residues on the barbed end of the actin filament (red).These acidic residues include ones from the terminal and the penultimate protomers of the filament, labeled B and B-1. Next, the mobile b tentacle searches for its binding position on the filament. Finally, the hydrophobic surface (yellow) of the amphipathic b tentacle binds to the hydrophobic cleft (yellow) on the terminal protomer, B.

Uncapping will be an important subject for further study, because of its potential relevance in cells. In vitro, the dissociation rate for CP to leave the barbed end is quite long relative to the time scale on which actin filaments assemble and disassemble in cells and to the time scale on which CP appears to dissociate from the actin cytoskeleton in cells (Iwasa and Mullins, 2007; Miyoshi et al., 2006). Inducing uncapping might be a mechanism for cells to induce assembly or disassembly of the filament network, depending on other conditions in the local environment.

3.2. Mobility of the C-terminal regions of subunits As described above, the C-terminal region of the CP a subunit is an amphipathic helix that lies on the top surface of the protein in the crystal structure. Its hydrophobic side is oriented toward the body of the protein

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(i.e., the top of the mushroom) (Yamashita et al., 2003), and in molecular dynamics simulations, this region remains in that position on the mushroom surface (Bhattacharya et al., 2006). Residue Trp271 of the amphipathic helix occupies a hydrophobic pocket on the surface of the body (Yamashita et al., 2003). A short peptide corresponding to part of this same region of the CP a subunit was found to bind to the protein S100 (Ivanenkov et al., 1995, 1996), and an NMR structure of the S100 peptide complex showed that the Trp residue corresponding to position 271 occupies a hydrophobic pocket in S100 (Inman et al., 2002). This peptide from CP a can bind S100, and full-length unfolded CP a can bind S100, but native CP was found not to bind S100 (Schafer et al., 1996; Wear and Cooper, 2004a). Treatment of CP with high concentrations of nonionic detergent enabled S100 to bind weakly (Wear and Cooper, 2004a). Thus, the C-terminal region of the a subunit, which is necessary for binding actin, appears to be immobile in the native solution structure, as implied by the crystal structure and molecular dynamics results. Mutating the Trp271-analogous residue of yeast CP to Ala caused a large loss of capping activity, which may be due to alteration of the structure of the amphipathic helix (Kim et al., 2004). In the crystal structure, the C-terminal region of the CP b subunit is also an amphipathic helix, but the helix extends out from the body of the protein, surrounded by solvent ( Yamashita et al., 2003). In molecular dynamics simulations, this region is highly mobile, as expected (Bhattacharya et al., 2006). The mobilities of the C-terminal regions of the subunits are incorporated into the current model for CP binding to the barbed end of the actin filament proposed by Mae´da and colleagues (Narita et al., 2006; Narita and Maeda, 2007). In terms of the wobble hypothesis, the mobilities of the C-terminal actin-binding regions helps to predict that CP will not wobble when it is bound to a barbed end only by the C-terminal region of the a subunit, i.e., when the b subunit’s C-terminal tentacle is not bound to actin. In contrast, CP will wobble if the a subunit C-terminal region dissociates, leaving only the b tentacle attached (Bhattacharya et al., 2006).

4. Capping Protein Inhibitors and Uncapping 4.1. Contrasting results with CARMIL and V-1 CARMIL and V-1/myotrophin are two different proteins that can bind to CP and inhibit its ability to bind to the barbed end of the actin filament, i.e., to cap. When CP is already present on the barbed end, CARMIL appears to be able to remove it. This conclusion is based on physical and

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functional assays. First, CP is found in the supernatant after sedimentation of actin filaments following the addition of CARMIL (Uruno et al., 2006). Second, the concentration of free barbed ends increases rapidly on addition of CARMIL (Uruno et al., 2006; Yang et al., 2005). This increase occurs on the time scale of 10 s (Uruno et al., 2006; Yang et al., 2005), while the spontaneous dissociation rate of CP from the barbed end appears to be on the time scale of 10 min (Schafer et al., 1996). This difference suggests that CARMIL can bind to the CP/barbed-end complex in some manner, and we hypothesize that this interaction occurs in the wobble state. In support of this hypothesis, titration of CP with increasing concentrations of CARMIL in an actin-capping assay does not lead to complete inhibition of CP (Yang et al., 2005). Less than complete inhibition can be explained by the CARMIL/CP complex having a very low level of capping activity, suggesting that CARMIL and CP can co-exist in a ternary complex with the barbed end. In the future, identification of the CARMIL-binding site on CP should provide an important test of the wobble hypothesis, and imaging of single CP molecules on actin filaments should further our understanding of uncapping. V-1/myotrophin provides an important and interesting contrast with CARMIL. V-1 also binds to CP and inhibits its ability to cap the barbed end (see Fig. 4.1) (Bhattacharya et al., 2006; Taoka et al., 2003). However, V-1 has little or no uncapping activity in functional assays. That is, addition of a high concentration of V-1 to CP-capped actin filaments, at a level sufficient to inhibit all the CP in the reaction, produces little increase in the number of free barbed ends (Bhattacharya et al., 2006). Another difference between CARMIL and V-1 is that high concentrations of V-1 completely inhibit the capping activity of CP (Bhattacharya et al., 2006). Thus, both lines of evidence fail to indicate that V-1 can bind to CP that is bound to the barbed end. In the hypothetical wobble state, CP is attached to the barbed end only by the b subunit’s C-terminal region, i.e., the tentacle. V-1 requires the C-terminal region of the b subunit but not that of the a subunit for optimal binding to CP (Bhattacharya et al., 2006), suggesting that V-1 may interact with the b tentacle, among other parts of CP. Thus, V-1 would not be predicted to bind to the hypothetical wobble state, and this predicts that V-1 should not be able to uncap, which is the case. The binding site on CP for V-1 appears to include an area at the base of the b tentacle, as indicated in Figure 4.1A. This conclusion is based on several results. First, truncation of the CP a subunit C-terminal region was found to weaken the interaction of CP with V-1 by a small amount (Bhattacharya et al., 2006). The simplest interpretation of this observation alone would be a direct interaction between V-1 and the a subunit C-terminus. However, the dynamics of the a C-terminus are coupled to the rest of the protein, in contrast to the situation for the highly mobile ‘‘tentacle’’ of the b subunit. Molecular dynamics simulations of a CP

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molecule from which the C-terminal region of the a subunit was truncated revealed decreased dynamics of the a subunit at locations distant from the C-terminus, near the base of the b tentacle (Bhattacharya et al., 2006). The surface residues at the affected location were also part of a V-1 binding site identified on CP by computational docking analysis. In addition, these residues are among the relatively few that differ between the a1 and a2 isoforms, and V-1 binds slightly differently to the a isoforms. To account for all these observations, we suggest that V-1 binds to CP as depicted in Figure 4.1A. Structural data about the CP/V-1 complex should provide valuable information to test this model.

4.2. Interaction of polyphosphoinositides with capping protein Polyphosphoinositides, including PIP2, can bind to CP and inhibit its capping activity (Heiss and Cooper, 1991). PIP2 can also cause rapid uncapping, demonstrated recently by observations of the polymerization of single actin filaments by TIRF microscopy (Kim et al., 2007). In those studies, addition of PIP2 to a flow chamber with actin filaments that were capped by CP and thus not able to polymerize resulted in the rapid and complete conversion of ends from the nongrowing to the growing state. Computational docking analysis suggested that PIP2 binds to three conserved basic residues on the surface of CP near the a subunit C-terminus, and mutations of those residues weakened the affinity of PIP2 for CP, measured with functional and physical assays (Kim et al., 2007). Some of these residues were predicted to sit at the interface between CP and actin based on the cryoEM CP/actin filament structure (Narita et al., 2006), and mutations of these residues affected the ability of CP to cap actin (Kim et al., 2007; Narita et al., 2006). Thus, the PIP2 and actin binding sites on CP may overlap. In addition, these observations are consistent with the wobble model in that they suggest that the region of the a C-terminus is available for PIP2 binding when CP is on the barbed end, i.e., in the wobble state. Little recent work addresses the physiological significance of PIP2/CP interaction, but in older work, studies of the actin assembly that accompanies platelet activation suggested that an early step was uncapping of CP-capped actin filaments by polyphosphoinositides (Barkalow et al., 1996). In many other cells systems, CP appears to terminate actin assembly by capping free barbed ends that are created by other mechanisms. The actin assembly that results from treatment of Dictyostelium cells with chemoattractant appears to be such a case (Eddy et al., 1997). Since polymerization of free barbed ends at membranes appears to drive the movement of those membranes, one attractive hypothesis is that PIP2 generated in the membrane helps to inhibit capping by CP near the membrane.

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4.3. CARMIL, CKIP-1 and CD2AP—A motif for inhibition of capping protein 4.3.1. Motif for inhibition of capping protein The existence of a motif for binding and inhibiting CP has been suggested by comparative analysis of the sequences and biochemical properties of the proteins CARMIL, CKIP-1 and CD2AP (Bruck et al., 2006; Canton et al., 2005, 2006; Uruno et al., 2006). Each of the three proteins was found to bind directly to CP and inhibit the actin capping activity of CP. Structurefunction analysis of each protein revealed an essential region with a common set of essential amino-acid residues. The potential CP-binding motif appears to be LXHXTXXRPK(6X)P (Bruck et al., 2006). 4.3.2. CARMIL Acan125 was the original name for CARMIL when the protein was discovered in amoeba as a binding partner for the SH3 domain of certain class I myosins (Xu et al., 1995). Later, this protein was found to bind CP and Arp2/3 complex as well, leading to the acronym CARMIL ( Jung et al., 2001). The protein is relatively large, with a long leucine-rich repeat (LRR) region of unknown function (Xu et al., 1997). The LRR region may participate in autoinhibition of the CP-binding activity of CARMIL (Uruno et al., 2006). CARMIL binds tightly to CP, with a Kd in the nanomolar range (Yang et al., 2005). CARMIL purified from Acanthamoeba contains CP in near-stoichiometric amounts (Remmert et al., 2004), but the large majority of Acanthamoeba CP in cell extracts is free and able to cap actin (Cooper et al., 1984). CARMIL is important for actin-based motility, based on knockout and knockdown studies in Dictyostelium and cultured vertebrate cells ( Jung et al., 2001; Yang et al., 2005). The multiple biochemical functions associated with CARMIL raise many possibilities for its mechanism of action in cells. Loss of the CP-binding site, by internal deletion of 100 aa residues, produced a mutant form of CARMIL unable to rescue the knockdown phenotype in cultured vertebrate cells (Yang et al., 2005). 4.3.3. CKIP-1 CKIP-1 was discovered as an interaction partner for casein kinase 2, helping to recruit CK2 to the plasma membrane (Olsten et al., 2004). CKIP-1 was also found to interact biochemically with CP in cultured cells (Canton et al., 2005). The binding of CKIP-1 and CK2 to CP inhibits capping activity (Canton et al., 2005), and CKIP-1 expression in cultured cells causes changes in cell morphology and the actin cytoskeleton that depend on its interaction with CP (Canton et al., 2006). CK2 can phosphorylate CP by CK2 (Canton et al., 2005), which may be a novel regulatory mechanism.

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4.3.4. CD2AP CD2-associated protein (CD2AP) and its relative Cin85 (Cbl-interacting protein) have been found to bind to CP and inhibit its capping activity (Bruck et al., 2006; Hutchings et al., 2003). CD2AP and Cin85 appear to be adaptor proteins that provide signaling pathway connections from membrane receptors to the actin cytoskeleton (Dustin et al., 1998; Lynch et al., 2003; Shih et al., 1999). CD2AP and Cin85 also interact with cortactin (Lynch et al., 2003; Nam et al., 2007), which promotes actin assembly via Arp2/3 complex, so these adaptors have multiple potential connections to actin assembly.

4.4. Regulators that antagonize capping protein by indirect effects Formin proteins act as competitors of CP at the actin filament barbed end. Formins are a large family of proteins, with a diversity of structures and functions that have yet to be understood fully (Goode and Eck, 2007; Staiger and Blanchoin, 2006). A formin dimer can bind to an actin filament barbed end, which can inhibit the binding of CP (Fig. 4.4) (Zigmond et al., 2003). However, the presence of formin at the barbed end still allows an actin subunit to add to that barbed end. Most remarkably, one formin dimer can remain bound to the barbed end while more and more actin subunits add over time; the formin essentially ‘‘surfs’’ with the growing barbed end of the actin filament. Thus, formins are very effective anti-cappers, promoting the growth of barbed ends. A number of cellular studies of formins support the relevance of this mechanism in vivo (Goode and Eck, 2007; Staiger and Blanchoin, 2006). VASP also functions as an antagonist of CP, via interactions with the actin filament. VASP is a member of the Ena/VASP family of proteins, which have been implicated in actin-based motility and morphogenesis. VASP antagonizes the capping activity of CP in vitro (Barzik et al., 2005). The anti-capping effect of VASP is not specific for CP in that other barbed-end cappers, such as gelsolin, are also antagonized by VASP. Ena/VASP proteins are found at the tips of filopodia, where they may prevent capping, which would allow barbed ends of actin filaments to grow and filopodia to elongate (Fig. 4.4) (Applewhite et al., 2007; Mejillano et al., 2004).

4.5. Other interactors Twinfilin was characterized as a protein that binds and sequesters actin monomers, thus inhibiting actin polymerization (Goode et al., 1998; Lappalainen et al., 1998; Palmgren et al., 2002). Twinfilin binds directly to CP, and that interaction does not affect the interaction of either protein

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B Membrane Membrane protrusion

CP active CP inhibited

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+ F-actin (+; barbed / −, pointed) Arp2/3 complex-mediated

Arp2/3 complex CP

Filament nucleation

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Figure 4.4 Illustration of potential modes of actin assembly in cells with respect to CP, based on one created and provided by Dr. MartinWear (Wear and Cooper, 2004b). (A) When CP is active and actin nucleation is Arp2/3-mediated, lamellipodial assembly predominates. Newly created free barbed ends are near the membrane.They elongate to push the membrane forward and /or the actin filament network backward. After some time, CP caps those barbed ends, which would seem to be efficacious because the ends are no longer near the membrane and their further growth would not produce useful work. (B) In this setting, when CP is inactivated in one location, by any of several potential inhibitors, then the filaments in that small region may continue to grow, producing a thin protrusion that contains a bundle of actin filaments. (C) An alternative mechanism that may produce actin filament bundles, perhaps not associated with a plasma membrane, is the nucleation of actin polymerization by a formin. Formins allow actin subunits to add and do not allow CP to add.Thus, the filaments continue to grow.

with actin (Falck et al., 2004). In yeast, twinfilin’s ability to bind CP and its ability to bind actin are both necessary for its function in actin dynamics (Falck et al., 2004). Twinfilin alone can also cap barbed ends, with a preference for ADP-actin (Helfer et al., 2006; Paavilainen et al., 2007),

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raising important questions about the relative contributions of these various biochemical activities to cell physiology. Extracts of neutrophils were found to contain a low-molecular-weight inhibitor of CP (Huang et al., 2005), which has not yet been identified. This inhibitor was able to inhibit and reverse capping of barbed ends by purified CP in functional assays. The biochemical properties of the inhibitor indicated that it was not PIP2, V-1, CARMIL or VASP. In an earlier study with neutrophil extracts, Cdc42-induced actin polymerization was found to be insensitive to CP, relative to polymerization induced by actin seeds (Huang et al., 1999). In retrospect, this Cdc42-induced anti-capper may have been a formin.

5. Role of Capping Protein in Complex Cellular Processes 5.1. Actin-based motility at the plasma membrane In metazoan cells in culture, the ability to form lamellipodial type protrusions was found to depend on CP in siRNA knockdown studies (Iwasa and Mullins, 2007; Mejillano et al., 2004). In mouse melanoma cells, inhibition of lamellipodial assembly was accompanied by an increase in filopodia formation (Mejillano et al., 2004), consistent with older results in Dictyostelium with antisense (Hug et al., 1995). In contrast, filopodia were not increased on CP knockdown in Drosophila cultured S2 cells (Iwasa and Mullins, 2007). In the mouse melanoma cells, the increased filopodia formation depended on VASP (Mejillano et al., 2004), so the Drosophila S2 cells may have lacked sufficient activity of VASP or some other filopodial component (Iwasa and Mullins, 2007). The need for CP in lamellipodial assembly supports the relevance of a key element of the dendritic nucleation model proposed to account for the assembly of branched networks of actin filaments associated with membranes and Arp2/3 complex (Nicholson-Dykstra et al., 2005). In that model, the reason why capping of barbed ends by CP is important has been proposed to be to ‘‘funnel’’ actin assembly to the new filament ends at the membrane, as described above, or to keep the actin filaments short and highly branched, to strengthen the network (Fig. 4.4). Testing these ideas will likely require mathematical modeling and measurements of physical parameters on a microscopic time scale with high time resolution. Speckle and single-molecule fluorescence imaging of lamellipodial regions of cultured cells reveals that CP binds to the actin filament network very near the membrane and that it dissociates from the network after a short time and distance (Iwasa and Mullins, 2007; Miyoshi et al., 2006), also consistent with the proposed role for CP in the dendritic nucleation model.

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CP dissociation may result from severing-induced depolymerization of filaments or it may be the direct effect of an uncapper. Distinguishing these possibilities will require observing the behavior of CP in cells carrying a specific defect in severing or uncapping, which will require careful biochemical characterization of mutant proteins. A challenge for these studies will be identifying which of several potential uncappers or severing agents are the relevant actors in a given cell system. For CP, the ability to alter protein activity protein locally and rapidly should provide powerful information for testing predictions of models. Acute inactivation of CP has been achieved in fibroblasts by laser inactivation of GFP-CP. The result was a local increase in the concentration of free barbed ends, the polymerization of actin and the formation of actinbased protrusive structures (Vitriol et al., 2007). These results support the notion that the CP caps barbed ends and that barbed-end capping prevents actin polymerization. Note that intuitive reasoning from the dendritic nucleation model seems capable of explaining the increase in actin-based protrusions in this experiment but also the decrease in lamellipodial protrusions in the set of knockdown experiments discussed above. One can rationalize the opposing predictions for the effect of the loss of CP activity in these two experiments on the basis of the laser inactivation effect being local and acute, while the knockdown inhibition effect is global and chronic. The rigor and certainty of the conclusions would be greatly enhanced by the application of mathematical modeling so that predictions are based on more than intuition and rationalization. Other membrane movements, in addition to lamellipodial protrusions of the plasma membrane, appear to be based on actin assembly. Endocytosis is another good example, based on recent studies in yeast and vertebrate systems (Engqvist-Goldstein and Drubin, 2003; Kaksonen et al., 2006). The endocytic process is composed of multiple steps of actin assembly and actin-based movement. Membrane receptors, endocytic adaptors, and actin-binding proteins, including Arp2/3 complex, are involved, with distinct roles at various steps in the process. Yeast CP null mutants showed a decrease in the initial movement of the cortical actin patch, the site of endocytosis, away from the plasma membrane, and the actin filaments of the patch still assembled (Kim et al., 2006), all of which appears to be consistent with the dendritic nucleation model. In contrast, other steps of endocytic traffic showed little to no effect from the loss of CP, so the model may not apply in these cases.

5.2. Z-line of the sarcomere in striated muscle CP purified from skeletal muscle was called ‘‘CapZ’’ because of its presence at the Z-disc of the sarcomere (Casella et al., 1987). The barbed ends of the actin-based thin filaments are also located at the Z-disc, and one molecule of

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CapZ appears to cap each barbed end (Schafer et al., 1993). The reason for capping the barbed end may be to help anchor the thin filament to the Z-disc, or it may be to prevent the growth of the thin filament into the adjacent sarcomere. CapZ and its actin-binding activity appear to be important for assembly of the sarcomere, as noted above. Recent studies have uncovered a biochemical interaction between nebulin, a giant protein of the sarcomere, and CapZ (Pappas et al., 2008; Witt et al., 2006). Nebulin knockdown in developing muscle cells leads to decreased accumulation of CapZ at the Z-disc and poor alignment of thin filament barbed ends (Pappas et al., 2008), consistent with a role for nebulin as a ‘‘ruler’’ specifying thin filament length by interacting with CapZ as the capper of the barbed end. The location of the CapZ binding site in nebulin suggests a model for the Z-disc in which nebulin connects one thin filament with an adjacent one, thus serving as a structural cross bridge to impart strength to the disc (Fig. 4.5).

5.3. Drosophila development In Drosophila, CP is essential for viability of the organism, and loss-offunction mutants die as embryos (Hopmann et al., 1996). In the bristles of the adult fly, actin bundles underlie and define the surface structure of the bristle, and the assembly of these actin bundles depends on CP and other actin regulators, including profilin and Arp2/3 complex (Frank et al., 2006; Hopmann and Miller, 2003). The effects of CP and the other proteins on the actin filament bundles appears to be an indirect one, mediated by their effects on a separate dynamic pool of actin filaments termed snarls (Frank et al., 2006). Studies of eye and wing development have also revealed an important role for CP, most likely through effects on actin assembly and morphogenesis (Iwasa and Mullins, 2007; Janody and Treisman, 2006). Nebulin

Z-disc Cap z a-actinin

∗M160M164

M177M181

SH3

Thin filament

Figure 4.5 An illustration of a new model for the structure of the Z-disc, provided by Dr. Carol Gregorio. Based on a structure-function analysis of the interaction of nebulin with CapZ (Pappas et al., 2008), the model proposes that the nebulin molecule crosses from one actin-based thin filament to another one, within the Z-disc.

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5.4. Dynactin CP is a biochemical component of dynactin, a multi-subunit complex necessary for the function of dynein (Schroer, 2004). Dynactin contains an actin-like filament composed largely of Arp1 (actin-related protein 1) and 1 mol per mol of CP. Single-particle EM image averaging of purified dynactin reveals lobes at the barbed end of the actin-like filament, which are likely to correspond to the subunits of CP (Hodgkinson et al., 2005; Imai et al., 2006). The presence of CP may be important to control the number of Arp1 subunits in the filament, which is remarkably constant among dynactin molecules. Whether the presence of CP affects the function of dynactin in cells has not been thoroughly tested. In yeast, CP null mutations produce no measurable effect on dynein function (Moore et al., 2008). Null mutations of some other dynactin subunits, including Arp1, produce a complete loss of dynein function. Biochemical approaches have not revealed CP to be a component of dynactin. Therefore, CP is either not an important component of dynactin in yeast or not a component at all. Yeast dynactin may lack other subunits of dynactin as well (Moore et al., 2008). To our knowledge, no tests of the functional role of CP have been done in other systems, which would be useful.

6. Concluding Remarks and Future Directions The emerging multiplicity of molecules that interact with CP and the diversity of their biochemical actions raise many new questions about how CP functions and is regulated in cells. The potential complexity is amplified by the cases where the interactors are proteins with multiple domains and may serve as adaptors with other molecules. Dissecting the individual roles of these interactions in biochemical and physiological terms will be an important challenge. New insight into the dynamic nature of the interaction of CP with the actin filament barbed end raises interesting possible mechanisms for the action of CP in the rapid assembly and disassembly of actin in cells. The potential existence of a wobble state needs to be established with direct physical methods, which will allow one to test whether the wobble state is part of the mechanism of uncapping. CP is present in essentially all cells and tissues of vertebrates, and actin filaments are proving to have multiple distinct roles in various cell settings. The roles that CP may play in these settings, especially the possibility of different roles for the conserved vertebrate isoforms of CP, will be an important avenue for the future.

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ACKNOWLEDGMENTS The authors are grateful to Drs. Carol Gregorio, Martin Wear, Yuichiro Mae´da, and Akihiro Narita for providing illustrations. Research in this area in the authors’ laboratories is supported by NIH GM 38542 to J.A.C. and GM 67426 to D.S.

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Eddy, R. J., Han, J., and Condeelis, J. S. (1997). Capping protein terminates but does not initiate chemoattractant-induced actin assembly in Dictyostelium. J. Cell Biol. 139, 1243–1253. Engqvist-Goldstein, A. E., and Drubin, D. G. (2003). Actin assembly and endocytosis: From yeast to mammals. Annu. Rev. Cell Dev. Biol. 19, 287–332. Falck, S., Paavilainen, V. O., Wear, M. A., Grossmann, J. G., Cooper, J. A., and Lappalainen, P. (2004). Biological role and structural mechanism of twinfilin-capping protein interaction. EMBO J. 15, 3010–3019. Frank, D. J., Hopmann, R., Lenartowska, M., and Miller, K. G. (2006). Capping protein and the Arp2/3 complex regulate nonbundle actin filament assembly to indirectly control actin bundle positioning during Drosophila melanogaster bristle development. Mol. Biol. Cell 17, 3930–3939. Goode, B. L., Drubin, D. G., and Lappalainen, P. (1998). Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein. J. Cell Biol. 3, 723–733. Goode, B. L., and Eck, M. J. (2007). Mechanism and function of formins in the control of actin assembly. Annu. Rev. Biochem. 76, 593–627. Hart, M. C., and Cooper, J. A. (1999). Vertebrate isoforms of actin capping protein beta have distinct functions in vivo. J. Cell Biol. 6, 1287–1298. Hart, M. C., Korshunova, Y. O., and Cooper, J. A. (1997a). Mapping of the mouse actin capping protein alpha subunit genes and pseudogenes. Genomics. 3, 264–270. Hart, M. C., Korshunova, Y. O., and Cooper, J. A. (1997b). Vertebrates have conserved capping protein alpha isoforms with specific expression patterns. Cell Motil. Cytoskeleton 2, 120–132. Heiss, S. G., and Cooper, J. A. (1991). Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids. Biochemistry 30, 8753–8758. Helfer, E., Nevalainen, E. M., Naumanen, P., Romero, S., Didry, D., Pantaloni, D., Lappalainen, P., and Carlier, M. F. (2006). Mammalian twinfilin sequesters ADP-Gactin and caps filament barbed ends: Implications in motility. EMBO J. 25, 1184–1195. Hertzog, M., van Heijenoort, C., Didry, D., Gaudier, M., Coutant, J., Gigant, B., Didelot, G., Preat, T., Knossow, M., Guittet, E., and Carlier, M. F. (2004). The beta-thymosin/ WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly. Cell 117, 611–623. Hodgkinson, J. L., Peters, C., Kuznetsov, S. A., and Steffen, W. (2005). Three-dimensional reconstruction of the dynactin complex by single-particle image analysis. Proc. Natl. Acad. Sci. USA 10, 3667–3672. Hopmann, R., Cooper, J. A., and Miller, K. G. (1996). Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila. J. Cell Biol. 133, 1293–1305. Hopmann, R., and Miller, K. G. (2003). A balance of capping protein and profilin functions is required to regulate actin polymerization in Drosophila bristle. Mol. Biol. Cell 14, 118–128. Huang, M., Pring, M., Yang, C., Taoka, M., and Zigmond, S. H. (2005). Presence of a novel inhibitor of capping protein in neutrophil extract. Cell Motil. Cytoskeleton 62, 232–243. Huang, M., Yang, C., Schafer, D. A., Cooper, J. A., Higgs, H. N., and Zigmond, S. H. (1999). Cdc42-induced actin filaments are protected from capping protein. Curr. Biol. 9, 979–982. Hug, C., Jay, P. Y., Reddy, I., McNally, J. G., Bridgman, P. C., Elson, E. L., and Cooper, J. A. (1995). Capping protein levels influence actin assembly and cell motility in Dictyostelium. Cell 81, 591–600. Hurst, S., Howes, E. A., Coadwell, J., and Jones, R. (1998). Expression of a testis-specific putative actin-capping protein associated with the developing acrosome during rat spermiogenesis. Mol. Reprod. Dev. 1, 81–91.

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Hutchings, N. J., Clarkson, N., Chalkley, R., Barclay, A. N., and Brown, M. H. (2003). Linking the T cell surface protein CD2 to the actin-capping protein CAPZ via CMS and CIN85. J. Biol. Chem. 25, 22396–22403. Imai, H., Narita, A., Schroer, T. A., and Maeda, Y. (2006). Two-dimensional averaged images of the dynactin complex revealed by single particle analysis. J. Mol. Biol. 359, 833–839. Inman, K. G., Yang, R., Rustandi, R. R., Miller, K. E., Baldisseri, D. M., and Weber, D. J. (2002). Solution NMR structure of S100B bound to the high-affinity target peptide TRTK-12. J. Mol. Biol. 5, 1003–1014. Isenberg, G., Aebi, U., and Pollard, T. D. (1980). A novel actin binding protein from Acanthamoeba which regulates actin filament polymerization and interactions. Nature 288, 455–459. Ivanenkov, V. V., Dimlich, R. V. W., and Jamieson, G. A. (1996). Interaction of S100a(0) protein with the actin capping protein, CapZ—Characterization of a putative S100a(0) binding site in CapZ-alpha-subunit. Biochem. Biophys. Res. Commun. 1, 46–50. Ivanenkov, V. V., Jamieson, G. A., Jr., Gruenstein, E., and Dimlich, R. V. (1995). Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ. J. Biol. Chem. 24, 14651–14658. Iwasa, J. H., and Mullins, R. D. (2007). Spatial and temporal relationships between actinfilament nucleation, capping, and disassembly. Curr. Biol. 17, 395–406. Janody, F., and Treisman, J. E. (2006). Actin capping protein alpha maintains vestigialexpressing cells within the Drosophila wing disc epithelium. Development 133, 3349–3357. Jung, G., Remmert, K., Wu, X., Volosky, J. M., and Hammer, J. A., 3rd. (2001). The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains. J. Cell Biol. 7, 1479–1497. Kaksonen, M., Toret, C. P., and Drubin, D. G. (2006). Harnessing actin dynamics for clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 7, 404–414. Kim, K., Galletta, B. J., Schmidt, K. O., Chang, F. S., Blumer, K. J., and Cooper, J. A. (2006). Actin-based motility during endocytosis in budding yeast. Mol. Biol. Cell 17, 1354–1363. Kim, K., McCully, M. E., Bhattacharya, N., Butler, B., Sept, D., and Cooper, J. A. (2007). Structure/function analysis of the interaction of phosphatidylinositol 4,5-bisphosphate with actin-capping protein: Implications for how capping protein binds the actin filament. J. Biol. Chem. 282, 5871–5879. Kim, K., Yamashita, A., Wear, M. A., Maeda, Y., and Cooper, J. A. (2004). Capping protein binding to actin in yeast: Biochemical mechanism and physiological relevance. J. Cell Biol. 4, 567–580. Lappalainen, P., Kessels, M. M., Cope, M. J., and Drubin, D. G. (1998). The ADF homology (ADF-H) domain: A highly exploited actin-binding module. Mol. Biol. Cell 9, 1951–1959. Loisel, T. P., Boujemaa, R., Pantaloni, D., and Carlier, M. F. (1999). Reconstitution of actin-based motility of Listeria and Shigella using pure proteins. Nature 6753, 613–616. Lynch, D. K., Winata, S. C., Lyons, R. J., Hughes, W. E., Lehrbach, G. M., Wasinger, V., Corthals, G., Cordwell, S., and Daly, R. J. (2003). A cortactin-CD2-associated protein (CD2AP) complex provides a novel link between epidermal growth factor receptor endocytosis and the actin cytoskeleton. J. Biol. Chem. 24, 21805–21813. Maruyama, K. (1966). Effect of beta-actinin on the particle length of F-actin. Biochim. Biophys. Acta 126, 389–398. Maruyama, K. (2002). beta-Actinin, Cap Z, connectin and titin: What’s in a name? Trends Biochem. Sci. 27, 264–266. Maruyama, K., Kimura, S., Ishi, T., Kuroda, M., and Ohashi, K. (1977). Beta-actinin, a regulatory protein of muscle. Purification, characterization and function. J. Biochem. (Tokyo) 81, 215–232.

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Maruyama, K., and Obinata, T. (1965). Presence of beta-actinin in the soluble fraction of the muscle cells of the chick embryo. J. Biochem. (Tokyo) 57, 575–577. Mejillano, M. R., Kojima, S., Applewhite, D. A., Gertler, F. B., Svitkina, T. M., and Borisy, G. G. (2004). Lamellipodial versus filopodial mode of the actin nanomachinery; pivotal role of the filament barbed end. Cell 3, 363–373. Miyoshi, T., Tsuji, T., Higashida, C., Hertzog, M., Fujita, A., Narumiya, S., Scita, G., and Watanabe, N. (2006). Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: Evidence of frequent filament severing. J. Cell Biol. 175, 947–955. Moore, J. K., Li, J., and Cooper, J. A. (2008). Dynactin function in mitotic spindle positioning. Traffic 9, 510–527. Nam, J. M., Onodera, Y., Mazaki, Y., Miyoshi, H., Hashimoto, S., and Sabe, H. (2007). CIN85, a Cbl-interacting protein, is a component of AMAP1-mediated breast cancer invasion machinery. EMBO J. 26, 647–656. Narita, A., and Maeda, Y. (2007). Molecular determination by electron microscopy of the actin filament end structure. J. Mol. Biol. 365, 480–501. Narita, A., Takeda, S., Yamashita, A., and Maeda, Y. (2006). Structural basis of actin filament capping at the barbed-end: A cryo-electron microscopy study. EMBO J. 25, 5626–5633. Nicholson-Dykstra, S., Higgs, H. N., and Harris, E. S. (2005). Actin dynamics: Growth from dendritic branches. Curr. Biol. 15, R346–357. Olsten, M. E., Canton, D. A., Zhang, C., Walton, P. A., and Litchfield, D. W. (2004). The Pleckstrin homology domain of CK2 interacting protein-1 is required for interactions and recruitment of protein kinase CK2 to the plasma membrane. J. Biol. Chem. 40, 42114–42127. Paavilainen, V. O., Hellman, M., Helfer, E., Bovellan, M., Annila, A., Carlier, M. F., Permi, P., and Lappalainen, P. (2007). Structural basis and evolutionary origin of actin filament capping by twinfilin. Proc. Natl. Acad. Sci. USA 104, 3113–3118. Palmgren, S., Vartiainen, M., and Lappalainen, P. (2002). Twinfilin, a molecular mailman for actin monomers. J. Cell Sci. Pt 5, 881–886. Pappas, C. T., Bhattacharya, N., Cooper, J. A., and Gregorio, C. C. (2008). Nebulin interacts with CapZ and regulates thin filament architecture with in the Z-disc. Mol. Biol. Cell (in press). Pavlov, D., Muhlrad, A., Cooper, J., Wear, M., and Reisler, E. (2007). Actin filament severing by cofilin. J. Mol. Biol. 365, 1350–1358. Remmert, K., Olszewski, T. E., Bowers, M. B., Dimitrova, M., Ginsburg, A., and Hammer, J. A., 3rd. (2004). CARMIL is a bona fide capping protein interactant. J. Biol. Chem. 4, 3068–3077. Remmert, K., Vullhorst, D., and Hinssen, H. (2000). In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein. Prot. Exp. Purif. 1, 11–19. Revenu, C., Courtois, M., Michelot, A., Sykes, C., Louvard, D., and Robine, S. (2007). Villin severing activity enhances actin-based motility in vivo. Mol. Biol. Cell 18, 827–838. Schafer, D. A., and Cooper, J. A. (1995). Control of actin assembly at filament ends. Annu. Rev. Cell Dev. Biol. 11, 497–518. Schafer, D. A., Hug, C., and Cooper, J. A. (1995). Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments. J. Cell Biol. 1, 61–70. Schafer, D. A., Korshunova, Y. O., Schroer, T. A., and Cooper, J. A. (1994). Differential localization and sequence analysis of capping protein b-subunit isoforms of vertebrates. J. Cell Biol. 2, 453–465. Schafer, D. A., Waddle, J. A., and Cooper, J. A. (1993). Localization of CapZ during myofibrillogenesis in cultured chicken muscle. Cell Motil. Cytoskeleton 4, 317–335. Schafer, D. A., Jennings, P. B., and Cooper, J. A. (1996). Dynamics of capping protein and actin assembly in vitro: Uncapping barbed ends by polyphosphoinositides. J. Cell Biol. 135, 169–179.

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Schroer, T. A. (2004). Dynactin. Annu. Rev. Cell Dev. Biol. 20, 759–779. Shih, N. Y., Li, J., Karpitskii, V., Nguyen, A., Dustin, M. L., Kanagawa, O., Miner, J. H., and Shaw, A. S. (1999). Congenital nephrotic syndrome in mice lacking CD2-associated protein. Science 5438, 312–315. Sizonenko, G. I., Karpova, T. S., Gattermeir, D. J., and Cooper, J. A. (1996). Mutational analysis of capping protein function in Saccharomyces cerevisiae. Mol. Biol. Cell 1, 1–15. Soeno, Y., Abe, H., Kimura, S., Maruyama, K., and Obinata, T. (1998). Generation of functional beta-actinin (CapZ) in an E. coli expression system. J. Muscle Res. Cell Motil. 6, 639–646. Staiger, C. J., and Blanchoin, L. (2006). Actin dynamics: Old friends with new stories. Curr. Opin. Plant Biol. 9, 554–562. Taoka, M., Ichimura, T., Wakamiya-Tsuruta, A., Kubota, Y., Araki, T., Obinata, T., and Isobe, T. (2003). V-1, a protein expressed transiently during murine cerebellar development, regulates actin polymerization via interaction with capping protein. J. Biol. Chem. 278, 5864–5870. Uruno, T., Remmert, K., and Hammer, J. A. (2006). CARMIL is a potent capping protein antagonist: Identification of a conserved CARMIL domain that inhibits the activity of capping protein and uncaps capped actin filaments. J. Biol. Chem. 281, 10635–10650. Vitriol, E. A., Uetrecht, A. C., Shen, F., Jacobson, K., and Bear, J. E. (2007). Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins. Proc. Natl. Acad. Sci. USA 104, 6702–6707. von Bulow, M., Rackwitz, H. R., Zimbelmann, R., and Franke, W. W. (1997). CP b-3, a novel isoform of an actin-binding protein, is a component of the cytoskeletal calyx of the mammalian sperm head. Exp. Cell Res. 1, 216–224. Wear, M. A., and Cooper, J. A. (2004a). Capping protein binding to S100B: Implications for the tentacle model for capping the actin filament barbed end. J. Biol. Chem. 279, 14382–14390. Wear, M. A., and Cooper, J. A. (2004b). Capping protein: New insights into mechanism and regulation. Trends Biochem. Sci. 8, 418–428. Wear, M. A., Schafer, D. A., and Cooper, J. A. (2000). Actin dynamics: Assembly and disassembly of actin networks. Curr. Biol. 24, R891–R895. Wear, M. A., Yamashita, A., Kim, K., Maeda, Y., and Cooper, J. A. (2003). How capping protein binds the barbed end of the actin filament. Curr. Biol. 13, 1531–1537. Witt, C. C., Burkart, C., Labeit, D., McNabb, M., Wu, Y., Granzier, H., and Labeit, S. (2006). Nebulin regulates thin filament length, contractility, and Z-disk structure in vivo. EMBO J. 25, 3843–3855. Xu, P., Mitchelhill, K. I., Kobe, B., Kemp, B. E., and Zot, H. G. (1997). The myosin-Ibinding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins. Proc. Natl. Acad. Sci. USA 8, 3685–3690. Xu, P., Zot, A. S., and Zot, H. G. (1995). Identification of Acan125 as a myosin-I-binding protein present with myosin-I on cellular organelles of Acanthamoeba. J. Biol. Chem. 43, 25316–25319. Yamashita, A., Mae´da, K., and Mae´da, Y. (2003). Crystal structure of CapZ: Structural basis for actin filament barbed end capping. EMBO J. 7, 1529–1538. Yang, C., Pring, M., Wear, M. A., Huang, M., Cooper, J. A., Svitkina, T. M., and Zigmond, S. H. (2005). Mammalian CARMIL inhibits actin filament capping by capping protein. Dev. Cell 2, 209–221. Zigmond, S. H., Evangelista, M., Boone, C., Yang, C., Dar, A. C., Sicheri, F., Forkey, J., and Pring, M. (2003). Formin leaky cap allows elongation in the presence of tight capping proteins. Curr. Biol. 20, 1820–1823.

C H A P T E R

F I V E

Effects of Environmental Estrogens and Antiandrogens on Endocrine Function, Gene Regulation, and Health in Fish Mary Ann Rempel* and Daniel Schlenk* Contents 1. Introduction 2. Mechanisms of Estrogenic and Antiandrogenic Effects 2.1. Steroid receptor 2.2. Steroid synthesis 2.3. Steroid distribution 2.4. Metabolic clearance 2.5. Hypothalamus-pituitary-gonad axis 2.6. Indirect mechanisms of estrogenic and antiandrogenic effects 3. Consequences of Impaired Reproductive Endocrine Function 3.1. Impaired gene regulation 3.2. Effects of unscheduled protein synthesis 3.3. DNA damage 3.4. Intersex/sex reversal 3.5. Reproductive failure 4. Summary and Concluding Remarks References

208 209 209 219 223 226 230 233 236 236 237 238 239 241 243 243

Abstract A number of studies have indicated widespread reproductive endocrine disruption in wild fish populations. A number of laboratory studies have been conducted to determine the sources and to elucidate potential mechanisms of the disruption. This review explores the varied mechanisms of estrogenic and antiandrogenic effects in fish including effects at the steroid receptor level, effects on steroid synthesis, distribution, and excretion, actions up the hypothalamuspituitary-gonad axis, as well as indirect mechanisms including thyroid and growth hormone disruption. Consequences of reproductive endocrine disruption

*

Department of Environmental Sciences, University of California, Riverside, CA 92521

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00605-9

#

2008 Elsevier Inc. All rights reserved.

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will be touched on including non-reproductive responses such as impaired gene regulation, effects of unscheduled protein synthesis and DNA damage, and reproductive responses such as intersex, sex reversal and reproductive failure. Key words: Endocrine function, Endocrine disruption, Environmental, Estrogen, Antiandrogen, Fish. ß 2008 Elsevier Inc.

1. Introduction In the 1990s, local fishermen in the United Kingdom noted the presence of hermaphroditic fish in lagoons receiving wastewater-treatmentplant effluent. The presence of hermaphroditic fish was confirmed in a follow-up survey, leading to speculation that there could be a link between the state of the fish and exposure to the effluent from sewage-treatment works (STWs) (Purdom et al., 1994). In order to test this link, Purdom et al. (1994) conducted a study in which caged male trout were placed in the sewage effluent where the hermaphroditic fish were observed, as well as at other STWs and several control locations. Exposure to sewage-treatment effluent, but not water at control locations, led to the induction of the synthesis of a female-specific protein, the egg-yolk-precursor protein vitellogenin, in male fish. Production of this protein was previously shown to be under the control of estrogens (Clemens, 1978). This was the first published study to demonstrate that exposure to exogenous estrogens in the environment could lead to endocrine disruption in fish. Since that time a number of studies were conducted in the United Kingdom demonstrating that endocrine disruption was widespread in wild fish populations. Wild roach (Rutilus rutilus) were sampled from upstream (where possible) and downstream of eight STWs as well as five reference locations. The occurrence of intersex, in which developing eggs are present in the testes, and vitellogenin induction was highest at locations downstream of the STWs, lower at the upstream sites, and lowest at the reference locations ( Jobling et al., 1998). Similar results were demonstrated in another freshwater species, the gudgeon (Gobio gobio), demonstrating that the phenomenon was not species specific (van Aerle et al., 2001). Endocrine disruption extended even to saltwater species. Flounder (Platichthys flesus) in United Kingdom estuaries were found to have increased levels of vitellogenin in fish from contaminated estuaries over fish from reference estuaries (Allen et al., 1999). In the most severely contaminated estuary vitellogenin levels in males were found to be as high or higher than levels in reproducing females, more than six-fold higher than found in control males. In the same area 17% of the male fish sampled had intersex gonads, whereas none were present in the reference fish.

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Endocrine disruption in wild fish populations is not exclusive to the United Kingdom. Estrogenic effects, including high levels of vitellogenin, intersex, depressed levels of testosterone and low gonadosomatic index, were demonstrated in carp (Cyprinus carpio) collected near a sewage outfall in the Ebro River in Spain (Lavado et al., 2004). Coho salmon (Oncorhynchus kisutch) in Lake Erie, United States, had depressed testosterone, 11-ketotestosterone (the major androgen in fish), cortisol, and gonadotropin concentrations in plasma, demonstrated a high rate (91%) of precocious sexual maturation, yet lacked development of secondary sexual characteristics in males (Leatherland, 1993). Studies in Hong Kong have indicated that abiotic factors such as hypoxia can also lead to endocrine disruption in fish (Wu et al., 2003). There has even been some speculation that fish residing in the open ocean may be experiencing endocrine disruption, perhaps through exposure to toxicants from the food chain (Scott et al., 2006). In response to the numerous studies reporting reproductive endocrine disruption in fish, researchers have put forth an effort to determine which compounds are reproductive endocrine disruptors and how they act. This review will provide an overview of the various mechanisms of action of estrogenic and antiandrogenic compounds, as well as provide specific examples of compounds that act in each manner. It will also summarize the varying consequences of reproductive endocrine disruption.

2. Mechanisms of Estrogenic and Antiandrogenic Effects The most studied paradigm of estrogenic action is one in which compounds that have a similar structure as the endogenous estrogen 17b-estradiol (E2) bind to its receptor(s) and elicit downstream effects. Estrogen mimicking however is only one of many mechanisms of action. Inhibition of androgens can lead to estrogenic effects. Compounds can affect steroid synthesis, distribution, and excretion. Actions higher up the hypothalamus-pituitarygonad (HPG) axis can elicit downstream, estrogenic effects. Compounds can also affect the reproductive endocrine axis indirectly through other axes. Each one of these mechanisms is explored in further detail below.

2.1. Steroid receptor 2.1.1. Estrogen receptors in fish Nuclear estrogen receptors (ER) have six functional domains, as illustrated in Figure 5.1. The A and B domains have ligand-independent transactivation function AF-1. The C domain is the DNA binding-domain and is also involved in dimerization. The E domain is the ligand-binding domain and is

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NH2

A

B

C

D

E

F

COOH

Figure 5.1 Generic structural domains of nuclear estrogen receptors.

involved in dimerization as well (Menuet et al., 2005). The D domain acts as a hinge between the C and E domains (Urushitani et al., 2003). The function of the F domain remains unclear at this time. However, data suggest that it might have a role in modulation of ER activity (Menuet et al., 2005). Once a ligand, such as E2, binds to the ER, the receptor dimerizes with another receptor, translocates to the nucleus, and binds to estrogen-response elements (ERE) in the promoter region of estrogen-responsive genes to regulate transcription. Teleosts, in similarity to mammals, have two main subtypes of ER, ERa, and ERb. Rainbow trout (Oncorhynchus mykiss) have been shown to possess two subtypes of ERa derived from separate genes, ERa1 and ERa2 (Nagler et al., 2007), as well as two isoforms derived from alternative splicing, ERa-long and ERa-short. ERa-long has a structure similar to that found in mammals. ERa-short appears to be truncated by 45 residues at its N-terminus, in the A domain (Pakdel et al., 2000). The A domain in the absence of a ligand interacts with the C-terminal region, leading to an inhibition of the AF1 activity of the B domain (Metivier et al., 2000). The truncation of the A domain removes this interaction, increasing the activity of the receptor in the absence of a ligand. The short form has a ligandindependent transcriptional activity representing 30% of the E2-induced activity (Pakdel et al., 2000). ERa-short is expressed only in the liver, whereas ERa-long is expressed in a wide variety of tissues, demonstrating a tissue-specific distribution of the two isoforms (Menuet et al., 2001). It is conjectured that the short form is involved in the sustained production of vitellogenin necessary during reproduction (Menuet et al., 2001). There are also two subtypes of ERb, ERba (also known as ERg) and ERbb. Although the two subtypes of ERb are transcribed from separate genes, they share a higher degree of amino acid homology with each other than they do with ERa. They are believed to have arisen from the duplication of an ancestral ERb gene early in the teleost lineage (Hawkins and Thomas, 2004). Studies performed in largemouth bass (Micropterus salmoides), and sea bream (Sparus auratus) have indicated differential expression of the two isoforms, with ERbb being more prominently expressed in the liver than ERba, even though both are equally present in the gonads, pituitary and the male kidney (Pinto et al., 2006; Sabo-Attwood et al., 2004). Distribution of the two subtypes in the brain of the Atlantic croaker (Micropogonias undulates) and zebrafish (Danio rerio) is also tissue-specific (Hawkins et al., 2005; Menuet et al., 2002). Variability in tissue distribution between subtypes and isoforms of ER might be linked to the pleiotropic effects of estrogens in the body.

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While most of the amino acids in the binding pocket of ERs that are responsible for ER-estrogen interactions are conserved, there are some key differences between species and subtypes. For example, in human ERa there is a leucine in the binding pocket that interacts with the A ring of E2. This leucine is changed to a methionine in fish, and this change is found in fourteen fish ERas to date (Hawkins and Thomas, 2004). In the channel catfish there are three amino acid substitutions between ERa and ERbb in positions known to be important for ligand binding (Pinto et al., 2006). There are two amino acid changes between ERa and ERba (Y526 to H495 and C530 to M499) that are conserved in nine fish ERs designated as ERba. But Y526 is changed to a serine, not a histidine, and C530 is changed to an arginine, not a methionine, in all teleost ERbb identified to date. C530 is conserved, however, between tetrapod ERa and ERb (Hawkins and Thomas, 2004). The variability in the amino acid sequence in the ligand-binding domain between species is believed to lead to variations observed in ligand binding affinity. There also appears to be differences in inherent activity between the short and long forms of ERa. Recently a membrane-bound, G-protein
coupled estrogen receptor has been identified in the gonads of croaker. Similar to GPR30 in mammals, it is thought to lead to the rapid reactions to steroid hormones observed in the body (Thomas et al., 2006). Also, estrogen receptor-related receptors (ERRs) have been characterized in the killifish (Fundulus heteroclitus) (Tarrant et al., 2006). While ERRs do not bind E2, their expression appears to be regulated by E2 to some extent. In particular, expression of ERRa in the female heart was downregulated upon exposure to E2. Taking all of the variability in binding and response to estrogens into account, it may be important to take into consideration the type of estrogen receptor and the species in which it resides when determining whether a compound is ‘‘estrogenic’’ by its ability to bind to the ER. 2.1.2. Common biomarkers of estrogen receptor activation Researchers use several methods to demonstrate activation of the estrogen receptor. The most direct measurements are receptor-binding assays using either hepatic cytosolic/nuclear extractions of estrogen binding sites or receptor clones (Denny et al., 2005; Latonnelle et al., 2002; Nimrod and Benson, 1997; Segner et al., 2003; Tollefsen et al., 2002; Urushitani et al., 2003). MCF-7, an estrogen-sensitive breast cancer cell line, is used for both ER binding and cell proliferation assays (Souttou et al., 1993; Vanderburg et al., 1992). There are also assays using cell lines with reporter genes linked to estrogen receptors. The yeast estrogen screen (YES) assay utilizes a yeast cell into which the gene for human ER and ERE tied to the reporter gene lac-Z have been incorporated (Routledge and Sumpter, 1996). The estrogen receptor-mediated, chemical activated luciferase reporter gene expression (ER-CALUX) assay was developed in the T47D breast cancer

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cell line (Legler et al., 1999). A luciferase reporter gene was also transfected into the MCF-7 cell line (Kramer et al., 1997). More popular but also more indirect assays measure the activation of genes or quantify the products of genes known to be under the control of the ER. The most common female-specific genes targeted in fish are the vitellogenin and choriogenin (egg envelope) genes in the liver (Hansen et al., 1998; Lee et al., 2002). The difficulty with this type of assay is the inability to determine whether the compound of interest is directly binding to the ER (an ER mimic) or eliciting effects through more indirect routes. 2.1.3. Estrogen mimics Estrogen mimics are compounds that bind to the ER and elicit similar, downstream effects as the endogenous estrogen itself. There are a wide variety of compounds that have been shown to bind to the ER, including the more obvious pharmaceuticals and estradiol metabolites, to the less obvious phytochemicals, surfactants, pesticides, plasticizers and even sunscreen agents. Structures of some representative estrogenic compounds are presented in Figure 5.2. Many estrogenic compounds contain a phenol ring, though some, like o,p0 -DDT may have a halogen in place of the hydroxyl group on the aromatic ring and others, like dibutyl phthalate, may have an ester linkage. Even if a parent compound does not have the proper structure to bind to the ER, degradation and/or metabolism of the parent compound might lead to the formation of a ligand, as exemplified by the hydroxylated polychlorinated biphenyls (PCBs) OH-PCB 30 and OH-PCB 61 (Carlson and Williams, 2001). Although a compound may have a structure that enables it to bind to the ER, it does not necessarily mean that the compound will activate the ER. It may actually act as an inhibitor of ER in some tissues, such as tamoxifen and its metabolite, 4-hydroxytamoxifen (Sasson, 1991). Relative binding affinities for various compounds to fish estrogen receptors are presented in Table 5.1. The table includes endogenous estrogens and metabolites (E2, estrone, estriol, estradiol glucoronide, estradiol sulfate), pharmaceutical estrogens (diethylstilbesterol, ethinylestradiol, mestranol), estrogen antagonists (tamoxifen, 4-hydroxytamoxifen, ICI 182, 780), phytochemicals (genistein, zearalenone, coumesterol, daidzein, equol, formononetin), surfactants (nonylphenol, tert-octylphenol, bisphenol A), pesticides and degradation products of pesticides (methoxychlor, demethylated methoxychlor, o,p0 -DDT, o,p0 -DDE, chlordecone), placticizers (dibutyl phthalate, di[2-ethylhexyl]halite, dicyclohexyl phthalate), a sunscreen agent (benzophenone), industrial waste (octachlorostyrene), and an antifouling agent (tributyltin chloride). Many of these compounds are present in the environment, and given the proper dose and sufficient uptake could bind to the ER and elicit downstream, feminizing effects. Though the ER shows some binding capacity for all of the compounds listed, there is a wide variation in the affinity of the ER for each compound. The structure of the

OH OH

OH CH3

CH3

CH3

CH3

N

H

H

H

O

HO

H

H H

CH

OH

O

H3C

O

OH

HO

HO

17b -Estradiol Endogenous estrogen

Ethinylestradiol Pharmaceutical estrogen

Genistein Phytochemical

4-hydroxytamoxifen Pharmaceutical antiestrogen

Cl Cl

CH3

HO

CH3

Cl Cl

CH3

H3C O

O

OH

O

H3C H3C

OH

O O

OH

HO

CH3

Cl

o,p⬘-DDT p-tert-Octylphenol Surfactant degradation product Organochlorine pesticide

Dibutyl phthalate Plasticizer

Benzophenone-2 Sunscreen agent

Figure 5.2 Chemical structure of representative compounds that have been demonstrated to bind to the estrogen receptor.

Table 5.I estradiol

Relative binding affinities for estrogen receptor in various fish species. All binding affinities are expressed as a percentage relative to 17b-

Chemical

17b-Estradiol Estrone Estriol Estradiol glucoronide Estradiol sulfate Diethylestilbesterol Ethinylestradiol Mestranol Tamoxifen 4-Hydroxytamoxifen ICI 182, 780 Genistein Zearlenone Coumesterol Daidzein Equol Formononetin Nonylphenol tert-Octophenol Bisphenol A

Rainbow trouta (Denny et al., 2005)

100 5.0 4.0

Rainbow troutb (Laton nelle et al., 2002)

Rainbow trout a (Tollefse n et al., 2002)

Fathead minnowa (Denny et al., 2005)

Siberian sturgeonb (Laton nelle et al., 2002)

Atlantic salmon a (Tollefse n et al., 2002)

100

100

100

100

100 3.9

0.1 <0.07

0.039

0.07 0.05

0.012

Carpa (Seg ner et al., 2003)

Channel catfisha (Nim rod and Benson, 1997)

100

100

100

5.2

179 88.9 0.34

6.1

1.7

70 1.2

390 84

583 166

28.4 490

4.2

0.02

1.4

8.5 2.3

3.6 2.5

204 55

94

90 516 5.4 0.31

25

1.75 0.077 0.13 2.69 0.03 0.009

Mummichogc (Urushitani et al., 2003)

0.48 0.70

3.5 0.006 0.06 0.5 0.001 0.01

0.13 0.013

0.012 0.020

0.42 0.65

0.049 0.010 0.12

Methoxychlor Demethylated Methoxychlor o, p0 -DDT o, p0 -DDE Chlordecone Dibutyl phthalate Di(2-ethylhexyl)halate Dicyclohexyl phthalate Benzophenone Octachlorostyrene Tributyltin chloride a b c

0.028

ER derived from hepatic cytosol extraction. ER derived from hepatic nuclear extraction. ERa clone.

0.010

0.0038

0.015 0.10 0.0011

0.012 0.005 0.027 0.010 0.017 0.014 0.0075 0.019 0.17

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compound, in particular its similarity to E2, plays a large role in determining the affinity. Despite this variation the interspecies and intraspecies (interlab) ER affinity for each compound is surprisingly consistent, in most cases varying by an order of magnitude or less. Differences may be due in part to the source of the ER used by the authors. The study by Urushitani et al. (2003) used an ERa clone, whereas other studies used either hepatic cytosol or nuclear extraction to derive the ER used in the assay. The extraction method may be providing several ER subtypes and/or isoforms, not just one form of ERa, and as shown earlier, subtypes and isoforms may have differences in ligand-binding pocket structure and thus different affinity for estrogenic compounds. This may be why the ERa clone seems to show higher affinity for the surfactants nonylphenol and tert-octophenol than the extracted ER used in the other studies (Denny et al., 2005; Nimrod and Benson, 1997; Tollefsen, 2002; Urushitani et al., 2003). 2.1.4. Androgen receptors in fish Much less is known about androgen receptors (AR) than ER in fish. The functional regions of the nuclear AR are similar to other nuclear receptors, with regions A and B involved in transactivation, region C acting as the DNA binding domain, and region E as the ligand binding domain (Olsson et al., 2005). Most vertebrates are believed to have one active form of nuclear AR with a high specificity for the androgen 5a-dihydrotestosterone (DHT), whereas there appear to be two subtypes of the AR in some teleosts, ARa and ARb, differentially expressed in tissues, with high affinities for either testosterone (T) or DHT (Olsson et al., 2005). Two forms have been isolated in the Japanese eel (Anguilla japonica) testes, Nile tilapia (Oreochromis niloticus) testes, brain and ovary of Atlantic croaker and rainbow trout, which appears to have only one active form (Ikeuchi et al., 2001; Pasmanik and Callard, 1988; Sperry and Thomas, 1999; Takeo and Yamashita, 1999). One form of nuclear AR has been isolated from goldfish (Carassius auratus), red sea bream (Pagrus major), Japanese medaka (Oryzias latipes) and three-spined stickleback (Gasterosteus aculeatus). There are two splice variants, ARb1 and ARb2 in the stickleback (Olsson et al., 2005; Pasmanik and Callard, 1988; Touhata et al., 1999). In addition, a membrane AR was isolated and characterized recently in the ovary of the Atlantic croaker (Braun and Thomas, 2004). Analyses of the structure of teleost ARs in comparison to the human AR seem to indicate that the amino acids important for ligand binding are well conserved. An amino acid thought to be important for the architecture of the AR, R779 in humans, however, is not conserved. Mutational assays have indicated that the replacement of this arginine results in complete inactivation of the AR; however, in teleosts, where both R779S and R779T are found, the AR is still functional (Olsson et al., 2005). Therefore, even

Estrogenic and Antiandrogenic Actions in Fish

217

though the ligand-binding domains are similar between teleosts and mammals, they are not identical. Most of the ARs that have been assessed for binding affinity have a higher affinity for DHT or T than the major endogenous androgen in fish, 11-ketotestosterone (11-KT) (Olsson et al., 2005; Wells and Van Der Kraak, 2000). However, when the three-spined stickleback ARb2 was assessed in reporter-gene transfection experiments in two different cell lines, even though 11-KT had a lower binding affinity it was more effective than DHT in activating the luciferase gene, indicating that 11-KT preferentially activates ARb2. Human AR, however, was not preferentially activated by 11-KT (Olsson et al., 2005). 2.1.5. Common biomarkers of androgen receptor interaction/suppression Similar assays are used in measurement of AR activation as are used in ER activation analysis, including binding affinity assays and reporter-gene assays (Bauer et al., 1998; Olsson et al., 2005; Wells and Van Der Kraak, 2000). The reporter-gene assays have the added benefit of providing information not just on binding to the receptor but also activation of the receptor, important for determining whether the compound is acting as an inhibitor or an activator. One gene in some teleosts that has been shown to be under control of 11-KT is the gene for spiggin, a protein produced by the kidney of male fish that acts as glue in nest building once released from the body. An assay has recently been developed for the effects of antiandrogens on the production of this male-specific protein in three-spined sticklebacks (Katsiadaki et al., 2006). However, from this type of assay, it is unclear whether the compound is acting directly through the androgen receptor or through another, indirect route. The same is true for measuring other, higher order effects such as effects on gonadal development. 2.1.6. Antiandrogens Most antiandrogens are believed to exert their effects by occupying the AR without activating it (there are other mechanisms that will be described later). The result is an inactivation of the receptor because the endogenous androgens are unable to bind to it, and this leads to a demasculinization effect in the fish, or feminization by default. Environmental antiandrogens that have been identified include p,p0 -DDE, a DDT degradation product; dicarboximide fungicides such as procymidone and the M1 and M2 metabolites of vinclozolin; linuron, a herbicide; and interestingly methoxyclor, demethylated methoxychlor, and o,p0 -DDT, which have also been shown to bind to the ER (see earlier) (Katsiadaki et al., 2006; Kiparissis et al., 2003; Wells and Van Der Kraak, 2000). Structures of some antiandrogens are presented in Figure 5.3, including the model antiandrogen, flutamide, used to treat prostate cancer. There seems to be some variation in the species and

OH CH3

O CH3

Cl

H

O

CH3

Cl

Cl

F

O

H3C H

H Cl

O

O

11-ketotestosterone Endogenous androgen

Procymidone Dicarboximide fungicide

F

O

N

CH3

Cl

Cl

p,p⬘-DDE Organochlorine pesticide (DDT) metabolite

F

N H

+

N

CH3

Flutamide Pharmaceutical antiandrogen

Figure 5.3 Chemical structures of endogenous fish androgen,11-ketotestosterone, and representative antiandrogens.

O

–

Estrogenic and Antiandrogenic Actions in Fish

219

tissues in which AR binding of the earlier antiandrogens occur. Vinclozolin and its metabolites and procymidone have been shown to bind to mammalian AR, but failed to bind to ARs extracted from the cytosolic fractions of rainbow trout brains and goldfish brains, ovaries and testes (Wells and Van Der Kraak, 2000). o,p0 -DDT, p,p0 -DDE, methoxychlor and demethylated methoxychlor did bind to goldfish testes AR in addition to mammalian AR. As there are differences in sequence homology between AR in different teleost species (Olsson et al., 2005), data from receptor binding assays from more species is needed to determine whether chemicals identified as antiandrogens in mammals indeed pose a threat to fish exposed in the wild.

2.2. Steroid synthesis 2.2.1. Reproductive steroidogenesis Sex steroids are primarily, but not exclusively, produced in the gonads, specifically the thecal and granulosa cells of the ovary and the leydig cells of the testis. The chief estrogenic product produced is E2, and the chief androgenic products are T and 11-KT. In mammals T is converted to a more potent form for activity, DHT. While fish are capable of producing DHT as well, its role in reproduction is currently unknown (Thibaut and Porte, 2004). Another steroid important in the final maturation process of both male and female gametes is 17a,20b-dihydroxypregn-4-en-3-one, also known as maturation inducing hormone (MIH). As shown in Figure 5.4, steroidogenesis is initiated via a signal from a pituitary hormone, either follicle-stimulating hormone (also known as gonadotropin I) or luteinizing hormone (also known as gonadotropin II). Once the gonadotropins bind to their receptor, a signal is sent via an adenosine 30 ,50 -cyclicmonophospate (cAMP) and protein kinase A (PKA) pathway, which triggers the release of free cholesterol from a cholesterol ester. Free cholesterol is then transported to the mitochondria via the steroidogenic acute regulatory protein (StAR), where the enzyme P450 side-chain-cleavage (P450scc) produces pregnenolone from cholesterol. Pregnenolone then diffuses back into the cytosol where a host of P450 enzymes work to produce the final active reproductive steroids. While it is unclear whether the entire reproductive steroidogenic pathway is present in extragonadal tissues, some key enzymes are present in multiple regions of the body. P450 aromatase (CYP19), the enzyme responsible for the conversion of T to E2, is present in two forms in fish. The CYP19A1 is present mainly in the gonads, and the CYP19A2 is present mainly in the brain, although both are found in other tissues as well (Piferrer and Blazquez, 2005). The presence of 11b-hydroxysteroid dehydrogenase (11b-HSD) is relatively widespread in fish tissues, albeit levels are highest in gonadal steroidogenic cells and head kidney interrenal cells (Kusakabe et al., 2003). The enzyme is responsible for conversion of 11b-hydroxytestosterone to

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Mary Ann Rempel and Daniel Schlenk

FSH, LH Gonadotropin receptor cAMP

ATP

Cholesterol Ester

Cholesterol Ester Hydrolase

PKA

H3C CH3

StAR CH3

H

H

O CH3

Free cholesterol H 3C CH3

CH3

3b-HSD

CH3

H

HO

P450scc on inner mitochondrial CH3 membrane

P450C17

HO

3b-HSD

CH3

CH3

O

CH3 H HO

17b-HSD

H

H

5a -reductase

P45011b OH CH3

HO

O H

H

5a-dihydrotestosterone

O

H

17b-estradiol

HO

g -HCH

OH CH3

O

H

11b-hydroxytestosterone

H

CH3

11b-HSD

H

CH3 H

H

H

Testosterone

CH3

CH3

H

H

O

H

OH

EE

P450 aromatase

H

OH

CH3

OH

H

CH3

H

Dicofol, DBP, DBT

EtOH, EE, NP, BaP, atrazine? CH3

Androstenediol

HO

O

Androstenedione

17b-HSD 3b-HSD

17a,20bdihydroxypregn-4-en-3-one

H

O

OH CH3 H

CH3

Η

O

H

CH3

Dehydroepiandrosterone

CH3 OH

Η

CH3 Η

CH3

H

20b-HSD

17ahydroxyprogesterone

P450C17

3b-HSD

OH

H

O

H

HO CH3

O

H

H

17ahydroxypregnenolone

P450C17

NP, dicofol, p,p⬘-DDE H3C CH3

H

H

Dicofol

Progesterone

P450C17 CH3

H

H

Η

O

OH O CH3

CH3

O

H

H

Pregnenolone

HO

CH3 CH3

H

H

O

11-ketotestosterone

Figure 5.4 Reproductive steroidogenic pathway in teleosts. Enzymes are labeled in red. Potential sites of action of estrogenic and anti-androgenic compounds (labeled in blue) are represented by lightning rods. White lightning rods indicate stimulation. Black lightning rods indicate inhibition. FSH, follicle-stimulating hormone; LH, luteinizing hormone; NP, nonylphenol; EtOH, ethanol; EE, ethinylestradiol; BaP, benzo(a) pyrene; DBP, di-n-butyl phthalate; DBT, dibutyltin; g-HCH, g-hexachlorocyclohexane.

11-KT, as well as the conversion of cortisol to cortisone. In some fish species it appears that the testes secrete 11b-hydroxytestosterone, which is later converted to 11-KT outside of the gonad (Young et al., 2005). There has also been some speculation that cortisol can be metabolized to 11-KT, providing a second function for 11b-HSD in the head kidney (Ozaki et al., 2006).

Estrogenic and Antiandrogenic Actions in Fish

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Successful reproduction requires proper timing and level of expression of the range of enzymes responsible for steroid biosynthesis, perhaps in more than one tissue. Improper inhibition or stimulation of enzymes can lead to an imbalance of steroid hormones, resulting in reproductive impairment. Impacts on steroidogenesis primarily by estrogenic or antiandrogenic compounds will be discussed here. 2.2.2. Common biomarkers of steroidogenic impairment Measurement of impairment of steroidogenic enzymes can include quantifying the level of expression, or mRNA levels, for a specific enzyme in the tissue of interest (Hecker et al., 2005; Kazeto et al., 2004). One can also measure the activity of an enzyme of interest by measuring the amount of its substrate converted per unit time per amount of protein (Hecker et al., 2005; Kim et al., 2003; Thibaut and Porte, 2004). Reporter-gene assays have also been employed (Fan et al., 2007). Steroid levels in plasma or tissues and ratios of estrogens to androgens are also commonly measured to confirm in vivo correlation to the enzyme assays (Hecker et al., 2005; Kim et al., 2003). 2.2.3. Steroidogenic impairment linked to estrogenic/ antiandrogenic effects A sampling of enzymes demonstrated to be impacted by estrogenic or antiandrogenic compounds is shown in Figure 5.4 and described in more detail below. It is believed that E2 production is regulated at least in part by regulating CYP19 expression at the transcriptional level (Young et al., 2005). CYP19A2 expression has been linked to sexual differentiation, and suppression may be necessary for proper testicular differentiation (Piferrer and Blazquez, 2005). CYP19A1 mRNA is detected in ovaries during ovarian maturation, when both E2 and vitellogenin levels are high, and cannot be detected at other times. Stimulation of CYP19 by exogenous compounds could lead to varying results in fish depending on the isoform affected and the stage of development, from sex reversal and impaired sexual differentiation to production of vitellogenin in males. The pharmaceutical estrogen ethinylestradiol and the surfactant nonylphenol have been demonstrated to stimulate CYP19A2 expression in zebrafish (Kazeto et al., 2004). In contrast, ethinylestradiol was shown to inhibit CYP19A1, while nonylphenol had no effect (Kazeto et al., 2004). The endogenous estrogen E2 upregulates CYP19A2 (Young et al., 2005). Half and full sites for the ERE have been found in the regulatory region of the CYP19A1 and CYP19A2 genes, respectively, in several fish species. In addition, there has been some speculation that posttranscriptional regulation of the CYP19 genes may also be performed by estrogens (Piferrer and Blazquez, 2005). As ethinylestradiol and nonylphenol are known to bind

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to the ER (see earlier), it is possible that their modulation of CYP19 may be through activation of the ER. Interestingly, benzo(a)pyrene, a polycyclic aromatic hydrocarbon not generally thought of as a xenoestrogen, also simulated CYP19A2 expression in zebrafish (Kazeto et al., 2004). An aryl hydrocarbon receptor/aryl hydrocarbon nuclear translocator (AhR/ARNT) response element has been found in the regulatory region of the CYP19A1 gene in zebrafish, but not the CYP19A2 gene. Therefore, benzo(a)pyrene does not appear to be affecting CYP19A2 through binding of the AhR (Piferrer and Blazquez, 2005). However, at high concentrations, it is possible that benzo(a)pyrene and its metabolites may act through the ER (Kazeto et al., 2004). Ethanol has also been shown to increase expression of CYP19 as well as shift the ratio of estrogens
androgens toward estrogens in tilapia (Oreochromis mossambicus) (Kim et al., 2003). This has interesting implications in toxicity testing, as ethanol is commonly used as a solvent for estrogens in aquatic assays and therefore might affect estrogenic endpoints. Atrazine, a ubiquitous herbicide, has been implicated in the demasculinization of frogs, primarily through a decrease in T and an increase in E2 (Hayes et al., 2006; Hecker et al., 2005). Similar effects on steroid levels have been demonstrated in goldfish (Spano` et al., 2004). It has been postulated that stimulation of CYP19 by atrazine is the cause of the shift in steroid ratios (Hayes et al., 2006). However, support of this hypothesis has been spotty in fish and in other species (Fan et al., 2007; Hecker et al., 2005; Kazeto et al., 2004). Recently it was demonstrated that atrazine will bind to the orphan nuclear receptor, steroidogenic factor 1 (SF-1). Atrazine only seems to affect CYP19 expression in human cancer cell lines that express SF-1 and utilize an SF-1
dependent promoter, ArPII (Fan et al., 2007). Therefore it appears that atrazine may affect CYP19 indirectly, through SF-1. Response elements for SF-1 are present in the regulatory region of the genes for both isoforms of CYP19 in several fish species (Piferrer and Blazquez, 2005). However, it is unclear whether SF-1 is consistently coexpressed with CYP19. Therefore, the variability in response upon exposure to atrazine may be influenced by SF-1 levels in cells. 20b-Hydroxysteroid dehydrogenase (20b-HSD) is the enzyme responsible for producing MIH from 17a-hydroxyprogesterone. An increase in the activity of 20b-HSD is triggered by a luteinizing hormone surge during the final maturation stage of reproduction in both male and female fish. In females an increase in MIH is usually accompanied by a decrease in E2 synthesis. The shift toward MIH production causes a resumption of meiosis in the oocytes followed by ovulation. In males a similar decrease in T and 11-KT accompanies an increase in MIH. Upon this signal the gonads transition from spermatogenesis to spermiation. The reproductive repercussions of an unscheduled shift from the production of E2, 11-KT and testosterone to MIH are unclear and merit further research.

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The estrogenic compounds nonylphenol and dicofol (an organochlorine miticide) and the antiandrogenic compound p,p0 -DDE have been shown to stimulate the enzyme 20b-HSD and increase the production of MIH. In addition the androgenic compounds di-n-butyl phthalate (a placticizer) and fenarimol (a fungicide) also had a similar effect (Thibaut and Porte, 2004). Dicofol also had an inhibitory effect on the activity of 17b-HSD and the 5a-reductase (Thibaut and Porte, 2004). 17b-HSD converts dehydroepiandrosterone to androstenediol and also androstenedione to T. Inhibition of 17b-HSD could lead to decreased T, and perhaps demasculinization. 5aReductase is responsible for the production of DHT from T. DHT is generally more potent at the AR than T, therefore inhibition of the formation of DHT could lead to demasculinization. Although fish can produce DHT, it has not been shown that it is a major androgen in teleosts. Therefore, the implications of the inhibition of 5a-reductase in fish are unclear. Two known androgens, di-n-butyl phthalate and dibutyltin, also were shown to inhibit production of DHT by 5a-reductase, further clouding the issue (Thibaut and Porte, 2004). The organochlorine pesticide g-hexachlorocyclohexane (lindane) was shown to increase concentrations of T but decrease concentrations of 11-KT in goldfish testes (Kime and Singh, 1996). The pattern was seen regardless of whether endogenous or exogenous precursor was used, and led the authors to believe that the decreased level of 11-KT was likely due to a decrease in conversion from 11-hydroxytestosterone, indicating inhibition of 11b-HSD. 11-KT plays an important role in spermatogenesis and also perhaps sexual differentiation (Bhandari et al., 2006). Thus, decreases in 11-KT could lead to demasculinization of fish.

2.3. Steroid distribution 2.3.1. Sex steroid binding protein Sex steroid hormones, both estrogens and androgens, are transported in the plasma of many species bound to a carrier protein, or sex steroid binding protein (SBP). SBPs are thought to have several roles in fish, including protection of steroids from rapid metabolic clearance, modulation of steroid availability and uptake by target tissues, and control of rates of release and uptake across gills (Scott et al., 2005). In mammals unbound SBP binds to cell-surface receptors. Once bound SBP accepts a steroid ligand, which then activates a cellular response through cAMP pathways (Hryb et al., 1990; Nakhla et al., 1999). It is unknown, however, whether SBP serves the same function in fish. SBP is a glycoprotein that forms a homodimer, both units of which likely contain a binding site (Miguel-Queralt et al., 2004). In zebrafish, SBP could first be detected in 5- to 6-day-old larvae. It was detected in the liver and gut in larvae, and also in the testes in small amounts in the adults.

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Even though the liver is the main organ responsible for the synthesis of SBP, it was not readily detected in hepatocytes, most likely due to rapid secretion (Miguel-Queralt et al., 2004). There appears to be little difference in the levels of SBP in males and unreproductive females. However, in some species the binding capacity increases in vitellogenic females, purportedly to help maintain the high levels of estradiol necessary for vitellogenesis (Hobby et al., 2000). 2.3.2. Measurement of xenoestrogen binding to sex steroid binding protein To determine the potential for exongenous chemicals to interact with SBP, binding affinity assays are performed, similar to measurement of nuclear receptor interaction. The assays are usually performed on charcoal-treated plasma to remove endogenous steroids that may interfere with the assay. Both the disassociation constant, Kd, and the maximum binding capacity, Bmax, can be calculated. The disassociation constant gives a measurement of binding affinity, and the Bmax gives a measurement of effects on the concentration of SBG. It does not appear to date that exogenous chemicals have a marked impact on Bmax. Therefore that endpoint will not be discussed further in this paper. Relative binding affinities are calculated from the concentration of substrate required to displace 50% of bound estradiol, or the IC50 (Kloas et al., 2000; Tollefsen, 2002; Tollefsen et al., 2004). They can also be calculated by directly comparing inhibition constants (Ki) between estradiol and the compound of interest (Gale et al., 2004). 2.3.2.1. Steroid distribution and estrogenic effects The role of SBP in the endocrine system can have several impacts on the effects of endocrine disrupting compounds in fish. A study performed on Tinca tinca found that the rate of uptake of steroids from water was not only influenced by their concentration in the water versus the body, and their hydrophobicity, but also their binding affinities for SBP (Scott et al., 2005). SBP may also alter the diffusion gradient for endocrine disrupting compounds, increasing their bioconcentration. Once in the system, the amount of free versus bound compound will be a factor of both the relative binding affinity and the relative concentration compared to endogenous steroids. If the concentration or relative binding affinity is high for the compound, it could displace endogenous steroids, making them more available to exert their actions. If the compound is carried to a location or tissue in which the level of steroids is high (e.g., gonads), the steroids could displace the compound, leaving it free to exert its actions on the local tissue; a sort of shuttling effect (Tollefsen, 2002). Relative binding affinities for endogenous steroids and estrogenic compounds to SBP of four fish species are listed in Table 5.2. There is variation, sometimes marked, between the binding affinities of a given compound in

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Estrogenic and Antiandrogenic Actions in Fish

Table 5.2 Relative binding affinities (%) for endogenous steroids and estrogen mimics to steroid-binding proteins in plasma of various fish species. All binding affinities relative to 17b-estradiol

Compound

Common carp (Kloas et al., 2000)

Steroids 17b-Estradiol 100 Testosterone 104 11-Ketotestosterone 51 Progesterone 198 Pharmaceuticals Ethinylestradiol Diethylstilbestrol Phytochemicals Genistein Zearalenone Industrial Chemicals 4-Nonylphenol 0.6 4-Octylphenol 0.32 Bisphenol A 0.29 n-Butyl Benzyl Phthalate Pesticides o,p0 -DDT Endosulfan

Rainbow trout (Tollefsen, 2002)

Arctic charr Channel catfish (Gale (Tollefsen et al., 2004) et al., 2004)

100 86.6 13.3 0.22

100

100

0.77 0.014

307

0.25 0.035

0.018 0.017

0.0051 0.0018 0.00015

0.0012 0.018 0.03 0.1

0.0074 0.0091

0.00056 0.02

different fish (e.g., ethinylestradiol). As the Tollefsen studies with rainbow trout and Arctic charr have low variation in binding affinities, it is more likely that the differences are due to interlaboratory variability rather than species differences. Also noticeable is the discrepancy between the binding affinity of a compound to the ER versus SBP. The binding affinity of diethylstilbestrol to both the ER and SBP was measured in rainbow trout by the Tollefsen group. The binding affinity to the ER (relative to E2) was 390%, versus 0.014% for SBP (Tollefsen, 2002; Tollefsen et al., 2002). It appears that the binding properties between nuclear receptors and SBP are quite different, and binding affinities cannot be inferred between the two. In general, it appears that the exogenous compounds tested have much lower affinity for SBP than the endogenous steroids. Therefore, it may be unlikely, unless concentrations are very high, that the xenoestrogens exert their effects by displacing steroids from SBP and making them more

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bioavailable. It is more likely that if effects are occurring through SBP, it is by aiding in bioconcentration of estrogenic compounds from water, and by shuttling those compounds to areas of high steroid concentration, such as gonadal tissue (Kloas et al., 2000).

2.4. Metabolic clearance 2.4.1. Steroid metabolism Metabolism of steroids involves phase I and phase II metabolism. Phase I metabolism is catalyzed by P450 enzymes and most often results in the hydroxylation of the steroid, rendering it more polar. Phase II metabolism is catalyzed by transferases and results in the conjugation of the steroid with groups such as glucuronic acid and sulfates. Phase I and II reactions may occur separately or in conjunction and result in the efficient elimination of the steroid. Testosterone phase I reaction products in fish include 6a/b-, 16a/b-, 2b-, 15a/b-, and 12b-hydroxytestosterone (OHT) and androstenedione (Arukwe et al., 1997; Baldwin et al., 2005; Hasselberg et al., 2004; Kime, 1980; Kime and Saksena, 1980; Smeets et al., 2002). The major phase I reaction products are 6b-OHT, produced primarily by CYP3A and CYP2K, 16b-OHT, produced primarily by CYP3A, CYP2K and CYP1A, and androstenedione (Arukwe et al., 1997; Baldwin et al., 2005; Smeets et al., 2002). Phase II products are conjugations with glucuronic acid, either of the phase I metabolites or of unmetabolized T, the conjugation performed by the enzyme UDP-glucuronosyltransferase (Kime, 1980; Kime and Saksena, 1980; Thibaut and Porte, 2004). Free T may also be eliminated. 11-KT is eliminated in an unconjugated or conjugated (with glucuronic acid) form (Kime, 1980). Estradiol phase I reaction products in fish include 2-, 4-, 16b-, 7b-, and 6a/b-hydroxyestradiol (OHE2), and estrone (Butala et al., 2004; Hansson and Rafter, 1983; Willett et al., 2006). The major phase I reaction products are 2-OHE2, produced primarily by CYP3A and CYP1A, and estrone (Butala et al., 2004; Lin et al., 2002). 4-OHE2, a genotoxic metabolite of E2, is primarily produced by CYP1B (Willett et al., 2006). Phase II reaction products include conjugations of E2 with glucuronic acid and sulfates, utilizing UDP-glucuronosyltransferase or sulfotransferase (Thibaut and Porte, 2004). Catechol-O-methyltransferase, which adds a methyl group to 2-OHE2 and 4-OHE2, is present but its activity is low, at least in the ovary of the catfish (Heteropneustes fossilis) (Mishra and Joy, 2006). The clearance of steroids is dependent upon the level of expression and activity of phase I and II enzymes, and modulation of these enzymes by some xenoestrogens is thought to lead to some of the estrogenic effects observed in fish.

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2.4.2. Common biomarkers of steroid metabolism impairment Measurement of steroid metabolism impairment uses many of the same methods as measuring effects on steroidogenesis. Researchers have measured both mRNA and protein levels for enzymes in tissues (Arukwe et al., 1997; Baldwin et al., 2005; Mortensen et al., 2006; Navas and Segner, 2001; Willett et al., 2006). Researchers also measure the activity of enzymes in the presence of varying concentrations of inhibitor or activator by measuring the level of product produced when a given substrate is added. Common assays are T biotransformation and 7-ethoxy-O-deethylase (EROD) activity. These assays can either be run with tissue extracts (liver and gonad) or with enzymes expressed in bacteria (Arukwe et al., 1997; Hasselberg et al., 2004; Lin et al., 2002; Smeets et al., 2002; Thibaut and Porte, 2004). Exposure to the inhibitor or activator of enzyme activity can either be in vitro or in vivo. Analysis of inhibitor/enzyme complexes has also been carried out to determine the type of inhibition occurring (Lin et al., 2002). Levels of steroid hormones and products of metabolism in vivo have also been measured as confirmation of effect on enzymes under varying conditions (Arukwe et al., 1997; Kime, 1980; Kime and Saksena, 1980). 2.4.3. Impairment of steroid metabolism linked to estrogenic compounds Modulation of CYP1A expression and activity by estrogenic compounds has been demonstrated by several groups. Mortensen et al. (2006) examined effects on CYP1A1 expression in Atlantic salmon hepatocytes upon exposure to ethinylestradiol and hydroxylated PCBs. Ethinylestradiol decreased expression of CYP1A1 with doses of 0.01 to 0.1 mM, but at a concentration of 1 mM expression of CYP1A1 rose again, although not reaching control levels. A similar pattern was seen for the 4-hydroxy form of the PCB congener 187 (4-OH-PCB 187), where concentrations of 0.06 to 0.6 nM suppressed CYP1A1 but 60 nM significantly elevated CYP1A1 expression levels. The hydroxlated metabolites of congeners 107, 138 and 146 supressed CYP1A1 expression over all treatment levels. Navas and Senger (2001) noted a decrease in basal levels of CYP1A mRNA and CYP1A activity (measured by EROD) upon exposure to 0.01 to 1 mM E2 in rainbow trout hepatocytes. If the ER antagonist tamoxifen was added, it mitigated the effect of E2, indicating an ER-mediated effect of E2 on CYP1A. If the CYP1A was induced by the AhR agonist b-napthoflavone, the effect of E2 was also mitigated, which led the authors to believe that the effect of E2 was not through an ER-AhR interaction. It is possible that ethinylestradiol and the hydroxylated PCBs are acting through a similar mechanism as E2, at least over low doses. There is some evidence that ethinylestradiol also acts as a suicide inhibitor of CYP3A (see later) (Lin et al., 2002), if the same is occurring for CYP1A the result may be an increase in expression over high doses to compensate for the loss of active enzyme.

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Alkylphenols, surfactant degradates purported to be estrogenic, increased expression of CYP1A in male Atlantic cod, but had no effect on enzyme activity in vivo (Hasselberg et al., 2004). In females there was no change in expression of CYP1A, but there was a significant albeit transient increase in enzyme activity, indicating a potential difference in response between sexes. In vitro the alkylphenols inhibited enzyme activity, and the strength of inhibition increased with the length of the alkyl chain (Hasselberg et al., 2004). As CYP1A metabolizes both E2 and T, modulation of CYP1A by estrogenic chemicals may be thought to affect circulating levels of active hormones. Induction of CYP1A however had little effect on T metabolism in flounder (Platychthis flesus) or dab (Limanda limanda) hepatocyctes, which may indicate that CYP1A is a minor enzyme in T metabolism in fish and effects on CYP1A may not effect circulating T levels significantly (Smeets et al., 2002). CYPIA may have effects on E2 levels, however. Treatment of rainbow trout liver cells with CYP1A inducers decreased vitellogenin induction upon administration of E2, although whether the response was due to increased E2 clearance was in question (Anderson et al., 1996). Treatment of channel catfish (Ictalurus punctatus) microsomes with the CYP1A inducer benzo(a)pyrene did however increase the concentrations of 2- and 4-OHE2 (Butala et al., 2004). This may indicate that suppression of CYP1A expression and activity by the estrogenic chemicals described earlier decreases metabolism of E2, thereby increasing the amount of the active hormone in circulation, adding to any estrogenic effect. In addition to suppression, induction of CYP1A may also have estrogenic implications. Methoxychlor, an organochlorine pesticide, is believed to exert its estrogenic effects primarily through its demethylated metabolites. The metabolites are believed to be produced by CYP1 and CYP3 enzymes (Stuchal et al., 2006). Therefore induction of CYP1A may increase the estrogenicity of methoxychlor. In addition to CYP1A induction, benzo(a)pyrene exposure increased levels of CYP1B mRNA in the liver, gonad and blood of channel catfish (Willett et al., 2006). The co-induction of CYP1A and CYP1B altered the ratio of 2- and 4-OHE2 toward more 4-OHE2, the genotoxic metabolite, which could have implications on fish health (Butala et al., 2004). CYP3A and CYP2K are believed to be the most active P450 enzymes involved in steroid metabolism (Arukwe et al., 1997). Nonylphenol treatment of winter flounder (Pleuronectes americanus) produced an increase in 6b-OHT (nonsignificant) and 16b-OHT (significant) levels, both products of CYP3A and CYP2K (Baldwin et al., 2005). There was also a corresponding significant increase in hepatic CYP3A protein levels. CYP2K protein was not measured. Nonylphenol has been shown to bind to the pregnane-X-receptor (PXR) to induce CYP3A in rodents (Masuyama et al., 2000). CYP2 enzymes are activated by the constitutive

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androstane receptor (CAR) in mammals, but CAR has not been identified in fish. However, zebrafish PXR has similarities to CAR, and it was suggested that fish PXR is a precursor to both CAR and PXR in mammals (Moore et al., 2002). If this is the case, CYP2K may be activated by fish PXR, as is CYP3A. A dose-dependent increase in CYP3A protein levels in male Atlantic cod was also observed following exposure to alkylphenols (Hasselberg et al., 2004). However, Arukwe et al. (1997) found a different response in Atlantic salmon exposed to nonylphenol. At a dose of 1 mg/kg there was an increase in 6b-hydroxylation, as in the Baldwin study, and also a decrease in plasma E2 levels, indicating an increase in steroid clearance. However, there was no effect on CYP2K and CYP3A-like protein levels. At a dose of 125 mg/kg, there was a decrease in 6b-hydroxylation activity, no effect on plasma E2 levels, and a decrease in CYP2K and CYP3A-like protein levels, indicating inhibition on some level, not stimulation. The high dose in the Arukwe experiment was similar to the dose used in the Baldwin experiment (125 mg/kg body weight versus 100 mg/kg body weight) but the Arukwe experiment lasted 14 days, whereas the Baldwin experiment lasted 2 days. Therefore, the difference in response may be due to the duration of the exposure. It is possible that nonylphenol regulates CYP3A expression not only through PXR but also other receptor coactivators/ repressors, and the interaction between regulatory pathways is dependent on concentration and/or duration of exposure (Meucci and Arukwe, 2006). Ethinylestradiol, in contrast to nonylphenol, strongly decreased expression of CYP3A at 0.01 mM (Mortensen et al., 2006). The effect, while still significant, was weaker with increasing dose. The loss of effect at higher doses may be due to compensatory increases in transcription of the enzyme, because ethinylestradiol has also been shown to be a mechanism-based irreversible (suicide) inactivator of CYP3A, albeit in humans (Lin et al., 2002). The metabolite of ethinylestradiol covalently modifies the apoprotein, and also modifies and/or destroys the heme moiety. This demonstrates two very different effects by two xenoestrogens on phase I steroid metabolism enzymes. There appears to be a general inhibition of phase II enzymes by endocrine disrupting chemicals. Estrogenic chemicals including hydroxylated PCBs, ethinylestradiol, nonylphenol, and dicofol all decreased glucuronidation of T and/or E2 (Mortensen et al., 2006; Thibaut and Porte, 2004). In addition androgenic chemicals such as triphenyltin, tributyltin, and fenarimol had similar effects. Nonylphenol, triphenyltin, and tributyltin also decreased sulfation of estradiol (Thibaut and Porte, 2004). Temperature appears to increase the production of glucuronidated T and 11-KT, as well as the production of unconjugated 11-KT, in goldfish (Kime, 1980). This may have interesting implications on the role of temperature in sexual differentiation of fish.

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2.5. Hypothalamus-pituitary-gonad axis 2.5.1. Description of the hypothalamus-pituitary-gonad axis While reproduction is largely under the control of reproductive steroids, the production and release of those steroids is under the regulation of a suite of neurotransmitters and hormones that make up the hypothalamuspituitary-gonad (HPG) axis (Trudeau, 1997; Weltzein et al., 2004). In response to external and internal stimuli, neurotransmitters are released into the preoptic area of the hypothalamus. Dopamine, serotonin, GABA, and neuropeptide Y all appear to be involved in this signal transduction, dopamine as an inhibitor, and serotonin, GABA, and neuropeptide Y as stimulants (Corio et al., 1991; Khan and Thomas, 1993; Trudeau, 1997; Yu et al., 1991). Some of the neurotransmitters also act directly on the pituitary. Upon stimulation the decapeptide gonadotropin-releasing hormone (GnRH) is released from the hypothalamus. The neurons that produce GnRH directly innervate the proximal pars distalis region of the pituitary, releasing GnRH to bind to G-protein
coupled receptors on the surface of gonadotropic cells. The gonadotropin hormones subsequently released are either LH or FSH, each of which is produced in a distinct cell type. The gonadotropins consist of two subunits, an a subunit which they share, and a distinct b subunit. The released gonadotropins are carried through the bloodstream to the gonads. The hormones bind to G-protein
coupled receptors on the surface of gonadal cells. Both LH and FSH can bind to GTH-R1 (FSH) receptors located on Sertoli cells in the testes and thecal and granulosa cells in the ovaries. Only LH can bind to the GTH-R2 (LH) receptors that appear on Leydig cells of the testes and granulosa cells of the ovary during the final stages of gamete maturation ( Janz and Weber, 2000; Weltzein et al., 2004). FSH appears to play an active role in early vitellogenesis and spermatogenesis, most likely through stimulation of steroidogenesis and sertoli cell proliferation in the gonadal cells. LH is also involved in early steroidogenesis. Just prior to spawning there is a shift in the production of steroids, leading to a decrease in estrogens and androgens and an increase in MIH. This shift corresponds to an increase in LH and LH receptor levels, although the actual role of LH in the shift is unclear ( Janz and Weber, 2000). Steroid hormones can affect all levels of the HPG axis through feedback loops. The effects of neuropeptide Y on GnRH and the gonadotropins is potentiated by T and E2 (Trudeau, 1997). The effects of GABA are enhanced by T and inhibited by E2 in reproductively active fish. However the opposite is true in regressed fish. T, through aromatization to E2, increases GnRH responsiveness in the pituitary. 17a,20b-Dihydroxypregn-4-en-3-one, which not only acts as MIH but also as a potent pheromone in some fish species, stimulates GnRH neurons to cause a release of LH, and decreases

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pituitary dopamine turnover. Pituitary dopamine turnover is enhanced, however, by T and E2 in regressed fish (Trudeau, 1997). It appears that the feedback loops of steroids are likely mediated to some extent through nuclear receptors. ARa, ARb, ERba and ERbb have all been found in GnRH-producing preoptic area neurons in fish (Harbott et al., 2007; Hawkins et al., 2005). ARa, ARb, ERa, ERbb, and, to a lesser extent, ERba appear to be present in the pituitary as well (Choi and Habibi, 2003; Harbott et al., 2007). E2 increases ER mRNA in the preoptic area, and several putative hormone response elements have been found for GnRH. Therefore, it appears that steroids may cause feedback to some extent through effects on transcription (Harbott et al., 2007; Salbert et al., 1993). Steroids can also regulate pituitary hormone secretion though the stabilization of the mRNA of peptide hormones. Stabilization of mRNA increases the half-life of mRNA in cells, sometimes on the order of days, which in turn increases translation rates, and therefore protein levels. Progesterone stabilizes the mRNA of the b subunit of LH, and this stabilization is augmented by estrogen treatment (Ing, 2005). Androgens have been shown to stabilize the mRNA of the b subunit of FSH. Estradiol also stabilizes the mRNA of its own receptor. Thus, there could be multiple modes of regulation occurring in the same cell. 2.5.2. Estrogenic and antiandrogenic effects on the hypothalamus-pituitary-gonad axis Given the multiple feedback loops in steroid endocrinology, only a few of which were described here, the HPG axis appears more like a web, with multiple inducers and inhibitors acting on multiple levels to finely tune the reproductive process in fish. It is clear that any disturbance in one part of the axis is likely to cause effects on other parts, both upstream and downstream. Endocrine disruptors have been shown to cause effects on all levels of the HPG axis, and for some a clear path down the axis ultimately to reproductive effects has been demonstrated. The actions of a few endocrine disruptors are described below. The antidepressant mianaserin is a selective serotonin reuptake inhibitor (SSRI). It is an antagonist for serotonin receptors, and by blocking reuptake increases the effects of serotonin. Effects of mianaserin on zebrafish were evaluated using an oligo microarray. In the gonads of both sexes there was upregulation of genes associated with egg and embryo development, including genes encoding vitellogenin and zona pellucida glycoproteins, which are necessary for egg envelope formation. In the brain of males there was also upregulation of the same category of genes, at both 2 and 14 days. In the females at 2 days there were more genes down regulated than upregulated in this category, but at 14 days the opposite was true. The overall magnitude of response was greater in the males than in the females (Van der Ven et al., 2006a). This may indicate sex differences in the

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response of zebrafish to mianaserin, which could perhaps be brought about by differences in the feedback loops of each sex. LH was found to be downregulated in female brains in this study, which is in contrast with another study in which serotonin was shown to increase LH secretion, albeit in another species (Khan and Thomas, 1992). The effects of PCBs on the HPG axis have been well studied in Atlantic croaker, from the central nervous system neurotransmitters down through the hypothalamus and pituitary, with effects demonstrated in the gonad as well. In male Atlantic croaker, PCB 77, a coplanar congener present in the Aroclor 1254 mixture, was shown to decrease tryptophan hydroxylase activity, the rate-limiting enzyme in serotonin synthesis. Two non-coplanar congeners (PCB 47 and PCB 153) also present in Aroclor 1254 had no effect (Khan and Thomas, 2006). Aroclor 1254 decreased GnRH content in the preoptic area of the hypothalamus and also decreased GnRH receptors in the pituitary. The decrease was concomitant with a decrease in LH secretion (Khan et al., 2001). The male fish were exposed during their gonadal maturation stage, starting at the early-recrudescing stage and ending 30 days later when the controls were spermiating. The PCB-treated fish had hormone levels similar to those of early-recrudescing fish, indicating a possible inhibition of sexual maturation, which indeed appeared to be the case as the gonadosomatic index was significantly lower in the PCB-treated fish. It appears that the PCB mixture Aroclor 1254, through the actions of its coplanar congeners, inhibits serotonin synthesis, decreasing GnRH signaling, which in turn decreases LH secretion, which seems to ultimately have an effect on gonadal maturation in male Atlantic croaker. Nonylphenol has been mentioned to have effects in almost every category described thus far, from receptor interactions to steroid metabolism. Modulation of hormones up the HPG axis has been demonstrated as well. In juvenile Atlantic salmon both nonylphenol and estradiol increased mRNA levels of the b subunit of LH in females, but not in males, most likely because the basal levels were already high (Yadetie and Male, 2002). There were corresponding increases in zona radiata (an egg envelope protein) and vitellogenin mRNA. There was no measurable effect on FSHb mRNA levels, which could be because the background levels were highly variable. In a study with female rainbow trout going through gonadal maturation increases in plasma LH were demonstrated upon exposure to nonylphenol for 6 to 12 weeks; however, the difference between control and treatment disappeared at 18 weeks (Harris et al., 2001). Plasma FSH levels were suppressed over all time periods. Both LH and FSH pituitary protein concentrations and mRNA levels were depressed at 18 weeks. Plasma vitellogenin concentrations were elevated over all time periods as expected, but E2 levels were depressed, and gonadal development ceased. E2 appears to cause positive feedback on LHb and negative feedback on FSHb in salmonids undergoing gonadal maturation, supporting the idea that

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nonylphenol is acting through classic estrogen pathways on the HPG axis, at least in the short-term (Dickey and Swanson, 1998). However, the E2 exposure lasted a maximum of 7 days, whereas the nonylphenol exposure lasted for 18 weeks. It is possible that the effect of E2 on LH wanes over the duration of the maturational period, in a similar manner to the effects of nonylphenol.

2.6. Indirect mechanisms of estrogenic and antiandrogenic effects Reproduction in fish is also regulated by factors outside of the reproductive endocrine axis. Tangential endocrine, paracrine and autocrine systems can be involved in the signals leading to puberty in fish as well as potentiating processes involved in the cyclical reproductive cycle. Two examples are the growth hormone axis and the thyroid hormone axis. The effects of these axes on the reproductive axis are summarized in Figure 5.5, and presented in more detail below. Both axes can be influenced by exogenous chemicals, which may lead indirectly to reproductive dysfunction. 2.6.1. Growth hormone axis Growth hormone (GH) is produced primarily in the rostral pars distalis region of the pituitary. Its release is stimulated by GnRH, and inhibited by somatostatin and through negative feedback loops by itself and insulin-like growth factor (IGF) ( Janz and Weber, 2000). GH is also produced in the gonad, but its function there is likely autocrine or paracrine, and not GH +

GnRH

−

+ +

IGF-I +

LH

− +

+

T3

+

+

FSH

+

T

Arom

E2

Figure 5.5 Influence of growth hormone (GH), insulin-like growth factor I (IGF-1) and thyroid hormone (T3) on the reproductive endocrine axis in fish. GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone;T, testosterone; E2,17b-estradiol; Arom, P450 aromatase; þ, positive regulation;
, negative regulation.

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released into the general circulation (Filby and Tyler, 2007). GH release leads to an increase in transcription and release of two forms of IGF: IGF-1 and IGF-2. IGFs have a similar structure and function to insulin. IGF-1 has highest expression in the liver, but is also expressed in adipose tissue, brain, heart, kidney, muscle, spleen and the gonads ( Janz and Weber, 2000). GH and IGFs primarily affect somatic growth and osmoregulatory adaptation in fish. However, effects on the reproductive axis have also been demonstrated. GH potentiates LH-stimulated steroidogenesis and stimulates ovarian aromatase activity in seatrout. GH is high when the gonadosomatic index is increasing in goldfish (Trudeau, 1997). IGF-1 has been linked to increases in GnRH expression in the hypothalamus, and increases in LH and FSH content in the pituitary, as well as LH release (Baker et al., 2000; Hiney et al., 1996; Huang et al., 1998). Studies in red seabream and Coho salmon demonstrated that IGF-1 decreased the production of T in thecal cells, but increases the production of E2 in granulosa cells (Kagawa et al., 2003; Maestro et al., 1997). Aromatase activity and expression in granulosa cells were also increased upon exposure to IGF-1, although the effect was only present prior to final maturation of the oocytes. The decrease in T and increase in E2 seemed to balance each other, so that the increase in E2 production could only be demonstrated upon addition of exogenous T. This may indicate that endogenous IGF-1 does not act on steroidogenesis in the gonad in an endocrine manner, as the result on steroidogenesis would be null, but rather a paracrine or an autocrine manner, perhaps eliciting effects on each type of cell at a different time during reproductive development. In sexually maturing female sturgeon (Acipenser ruthenus), the levels of IGF-1 in the gonad increased to the same as in the liver, and IGF receptor levels were 20-fold higher in the gonad than in the liver (Wuertz et al., 2007). The increase appeared to be temporary, and the actions believed to be paracrine. The lowest expression of IGF-1 in the ovary was in arrested, pre-vitellogenic follicles and the highest in late vitellogenic follicles. In female trout, a peak in plasma IGF-1 a few months before the first spawn was observed, indicating IGF-1 signals the beginning of puberty in salmonids (Taylor et al., 2008). In fathead minnows (Pimephales promelas) there was apparent sexual dichotomy in the expression of GH, IGF-1, and GH receptors in gonads (Filby and Tyler, 2007). Expression of mRNA was in general higher in ovaries than in testes, and the levels of GH and IGF-1 mRNA increased progressively in ovaries but not in testes during gonadal development. However, despite the higher expression in ovaries than testes, IGFs still play a role in testes by acting directly on premeiotic germinal cells to induce proliferation (Le Gac et al., 2001). Prochloraz and nonylphenol ethoxylates have been shown to target IGF’s role in spermatogenesis of male trout. Both compounds increased the cellular binding capacity for IGF-1 in germ cells, and also noncompetitvely inhibited binding of IGF-1 to those sites (Le Gac et al., 2001). They also decreased IGF-stimulated DNA synthesis in germ cells. Similar results

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were demonstrated using Triton X-100 (a surfactant), but not E2, which the authors postulated might indicate that the inhibition of spermatogenesis is not caused by estrogen mimicking, but rather through the lipophilic or detergent properties of the compounds. Estrogenic compounds may act on other levels of the axis as well. E2, o,p0 -DDT and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) all increased GH expression in rainbow trout pituitary (Elango et al., 2006). The mechanism of action was believed to be ER-mediated for E2 and o,p0 -DDT, and through AhR/ER receptor cross-talk for TCDD. Bisphenol A has been shown to modulate somatostatin levels in the diencephalic regions of the brain of Coris julis and may affect GH levels by affecting somatostatin’s ability to inhibit GH release (Alo et al., 2005). 2.6.2. Thyroid hormone axis Release of thyroid-stimulating hormone (TSH) from the pars distalis region of the pituitary appears to be under hypothalamic control although the actual controlling factors in fish have not been well elucidated. Negative feedback from thyroid hormones also affects TSH synthesis and release. TSH has a similar structure as the gonadotropins, sharing the same a subunit but having a distinct b subunit. The function of TSH is to increase iodide uptake by thyroid follicles, and increase the synthesis and release of thyroxines. Thyroid follicles are not in glands as in mammals, but rather located diffusely in the ventral pharyngeal region of fish. There are two forms of thyroid hormone: L-thyroxine, T4, which is mostly inactive, and triiodothyronine, T3, which is the active form. Conversion of T4 to T3 mainly occurs in peripheral tissues by the action of deiodinases ( Janz and Weber, 2000). The primary functions of T3 are to increase lipid mobilization, protein synthesis, and metabolism. Thyroid hormone also increases GH mRNA expression, GH release from the pituitary and increases hepatic IGF-1 production. Thyroid hormone receptors are highly expressed in the brain and pituitary, and there is sexually dimorphic expression in the gonad and intestine, with higher expression in females (Filby and Tyler, 2007). The effects of thyroid hormones on reproduction are highly dependent on the species of fish, the sex of the fish, the reproductive cycle (e.g., daily spawner versus annual), and the stage of the given reproductive cycle. In medaka T3 promoted gonadotropin-stimulated E2 production in the ovary during vitellogenesis (Soyano et al., 1993). T3 and T4 levels peak 12 and 36 hours before spawning, followed by a peak in E2 4 hours later. T3 was also shown to increase gonadotropin sensitivity in ovaries of vendace (Coregonus albula) during final maturation (Tambets et al., 1997). The effects of compounds on the thyroid axis are as varied as the effects of the thyroid axis itself, and depend on exposure regimen, species, maturity stage, sex, etc. Different groups have seen different responses in fish with the

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same compound. A large variety of compounds have been shown to affect the thyroid axis, including PCBs, organophosphate pesticides, ammonia, perchlorate and pharmaceuticals as reviewed by Brown et al. (2004). There are fewer studies showing links between contaminant exposure, thyroid effects, and reproductive dysfunction. Propylthiouracil, while not necessarily an environmental contaminant, is a model antithyroid agent known to decrease production and conversion of T4 and T3. In a zebrafish partial life cycle test, the compound decreased the levels of circulating thyroid hormones, increased the total number of eggs, and decreased the size of mature eggs in exposed adults (Van der Ven et al., 2006b). Exposure of medaka starting at the egg stage to b-hexachlorocyclohexane (a byproduct of lindane production) was shown to influence both thyroid function and reproduction, though whether there was a cause-effect relationship is unclear (Wester and Canton, 1986). After 3 months’ exposure, medaka thyroid follicles decreased in size and content, indicative of activation. The number of TSH cells in the pituitary increased, and the cytoplasmic content was reduced, also indicative of activity. There was increased vitellogenin production in fish, and males treated with 0.18 to 1.0 mg/L presented ova-testes.

3. Consequences of Impaired Reproductive Endocrine Function Up to this point we have been focusing on the mechanisms through which compounds can cause estrogenic or antiandrogenic actions to occur. At this point, we focus on the consequences.

3.1. Impaired gene regulation The ER is involved in regulation of a large number of genes such as the P450 enzymes described earlier. The ER also regulates genes not directly involved in reproduction. Activation of the ER by estrogen mimics can lead to either upregulation or downregulation of genes which may in turn alter protein processing, homeostasis, and even enhance the toxicity of certain compounds. In addition antiandrogens appear to impair gene regulation in a sex specific manner different than estrogen mimics. Although there are many genes in mammals that have been determined to be under control of the ER, less is known in teleosts. Osteonectin is a glycoprotein in bone and fish scales that binds calcium and collagen, and its expression is markedly downregulated by E2, leading to calcium mobilization (Lehane et al., 1999). The fish orthologue to transforming growth factor beta
binding protein three is another protein whose gene has been determined to be under estrogenic control. The protein is upregulated by E2, which

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can modulate the effects that the growth factor has on early developmental processes (Andersson and Eggen, 2006). Flavin-containing monooxygenases (FMO) are a group of enzymes that are involved in the biotransformation of some exogenous compounds. FMOs sulfoxidate carbamate pesticides, such as aldicarb, rendering the pesticides more toxic. E2 has been shown to increase expression of FMO1-like and FMO3-like proteins in the gills of female medaka, enhancing the toxicity of aldicarb to the fish (El-Alfy and Schlenk, 2002). It is interesting to note the differences between gene expression profiles of E2, the estrogen mimic nonylphenol and the antiandrogen p,p0 -DDE in largemouth bass. E2 upregulated vitellogenin and choriogenin mRNA expression, as expected, and also mRNAs of aldose reductase and aspartic protease, genes involved in sugar metabolism and post-translational protein modification, respectively (Larkin et al., 2002). E2 downregulated transferrin, a protein involved in iron transport. Nonylphenol had a similar profile to E2, although it also upregulated expression of signal peptidase, an enzyme that cleaves signal sequences off secreted proteins, as well. Nonylphenol may be acting through an alternate pathway in addition to the ER, which has been demonstrated earlier. P,p0 -DDE upregulated expression of vitellogenin and choriogenin in males, but downregulated the genes in females. Several other genes were downregulated in females as well, including the androgen receptor. The dichotomy in expression between the sexes upon exposure to the same contaminant indicates that the responses to exposure will vary depending on the sex, and most likely the reproductive status, of the fish.

3.2. Effects of unscheduled protein synthesis Vitellogenin has served as an excellent biomarker for exposure to and uptake of estrogenic chemicals in oviparous animals such as fish. The production of the protein generally is not seen as a toxicological endpoint per se, but there has been some indication that production of large quantities of unusable vitellogenin can lead to pathological effects. In several fish species the unscheduled production of vitellogenin in the liver has been associated with moderate increases in hepatosomatic index as well as histopathology including thickening of nuclear and cell membranes, proliferation of rough endoplasmic reticulum, accumulation of clear eosinophilic material (most likely vitellogenin itself ), damaged cellular disarray, and reduction of glycogen deposits (Mills et al., 2001; Wester and Canton, 1986; Zha et al., 2007). Effects have been demonstrated in the kidney as well, with increased renal-somatic index and histopathology including lesions characterized by severe hemorrhaging in kidney tubules, the Bowman’s space and renal interstitium, and hypertrophy, degeneration and necrosis of tubular epithelia (Zha et al., 2007). Eosinophilic material accumulated in the head kidney, similar to in the liver. In general it appears that males are more affected pathologically by the accumulation of vitellogenin than females,

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most likely because the females can potentially incorporate some of the excess vitellogenin into oocytes if they are actively reproducing. There may be some effects on growth associated with production of vitellogenin (Wester and Canton, 1986; Zha et al., 2007), perhaps due to the shunting of energy and materials to the production of an unnecessary protein. This is supported by the depletion of glycogen stores in the liver.

3.3. DNA damage A metabolite of E2, 4-OHE2, is a genotoxicant. The mechanism of toxicity is discussed in detail in Cavalieri et al. (2000). Briefly, both 2-OHE2 and 4-OHE2 can be metabolized further to semiquinones by peroxidases or P450 enzymes. Further transformation to a quinone form can occur either by the same enzymatic process, or through redox cycling in the presence of oxygen. The quinone form can react directly with DNA, forming depurinating adducts. Redox cycling can also produce hydrogen peroxide, leading to formation of hydroxyl radicals that then react with DNA to produce oxidized DNA bases. In the presence of lipids, hydroxyl radicals can aid in the formation of lipid hydroperoxide-derived aldehyde-DNA adducts (Fig. 5.6). Generally, the 4-OHE2 form of catehol estrogen is more toxic than the 2-OHE2 form because the 2-OHE2 form is more readily conjugated and excreted before semiquinones and quinones can form. Also the quinone produced by 2-OHE2 produces stable adducts instead of Reductase CYP1A, peroxidases CYP1A, peroxidases 4-hydroxyestradiol Estradiol-3,4-semiquinone -OrEstradiol-3,4-quinone Non-enzymatic DNA CYP1B 17b -estradiol

O2

CYP3A, CYP1A

. Depurinating adducts O2 SOD

H2O2

2-hydroxyestradiol

Fe2+or Cu+ Lipid hydroperoxides DNA Aldehyde DNA adducts

Lipids

•OH DNA

Oxidized DNA bases

Figure 5.6 Activating metabolism pathway for 17b-estradiol metabolite 4-hydroxyestradiol. SOD, superoxide dismutase. Adapted from Cavalierei et al., 2000.

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depurinating adducts, which are less prone to cause mutation. The ratio of 4-OHE2 to 2-OHE2 formed and the level of detoxifying enzymes present in a given tissue can determine the tissue-specific genotoxicity of estrogens. While DNA damage caused by exogenous estrogens has been well studied in mammals, relatively little data is available on fish. Exposure to exogenous E2 has been shown to increase DNA damage in fish erythrocytes, albeit transiently and nonsignificantly (Teles et al., 2005). The estrogenic pesticide dieldrin was linked to oxidative DNA damage in gilthead seabream liver (Rodriguez-Ariza et al., 1999). The area deserves further research as a potential impact of estrogen exposure, especially in light of environmental contaminants like benzo(a)pyrene which shift E2 metabolism toward production of 4-OHE2 (see earlier).

3.4. Intersex/sex reversal Fish exhibit gender plasticity. Some fish, like the California sheephead (Semicossyphus pulcher), start life as one sex, in this case female, and switch to the other sex in adulthood under appropriate environmental stimuli. For other fish, the sex is determined at a very early age, soon after hatching, termed the sexual differentiation period. Medaka begin life with undifferentiated gonads which then differentiate into either male or female gonads ¨ rn et al., 2006). Zebrafish on within the first couple of months post-hatch (O the other hand all start out with ovary-like gonads which form at about 3 to 4 weeks post hatch, and then in males the ovaries morph into testes between ¨ rn et al., 2003). While many fish have genetic sex 4 to 5 weeks post-hatch (O determination, it can be overridden or partially reversed by exposure to steroids, endocrine disrupting compounds or even extremes in temperature, especially when the exposure occurs at a critical ‘‘window’’ in sexual differentiation. The critical time varies depending on the species of fish. Sticklebacks exposed to E2 and ethinylestradiol within 2 weeks after hatching presented both sex reversed and intersex males (Hahlbeck et al., 2004). However, exposure of the egg to E2 before hatching had no effect. Incidence of intersex was highest in medaka when exposure to octylphenol occurred at 3 days post hatch versus seven or 21 days (Gray et al., 1999). However, in a study exposing 5- to 8-day-old medaka to E2 for 28 days, all fish became phenotypic females, indicating that the fish were still sensitive at an older age (Nimrod and Benson, 1998). Genetically female Japanese flounder (Paralichthys olivaceus) exposed to elevated temperature between 30 and 100 days after hatching had suppressed expression of P450 aromatase and became phenotypic males (Kitano et al., 1999). Zebrafish exposed to the fungicide prochloraz (both a steroidogenesis inhibitor and an androgen receptor antagonist) from 1 to 60 days post-hatch had a higher ratio of males and the presence of intersex individuals (Kinnberg et al., 2007).

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However, exposure of zebrafish to 2 to 10 ng/L ethinylestradiol from 20 to 60 days post-hatch also produced sex reversal, this time to females, ¨ rn et al., 2003). indicating that the critical period may occur after 20 days (O Interestingly, the effect was reduced at 25 ng/L. No intersex individuals were observed at any concentration. Response to endocrine disruptor or steroid exposure during sexual differentiation and beyond also varies with the species. Both medaka and zebrafish were exposed to ethinylestradiol for 1 to 60 days post-hatch. In zebrafish, the sex ratio became 100% female at a concentration of ¨ rn et al., 2006). 10 ng/L, and complete mortality occurred at 100 ng/L (O No intersex occurred, similar to the experiment earlier. In medaka there was no change in sex ratio at 10 ng/L, but a small percentage of fish were intersex. At 100 ng/L the sex ratio became predominantly female, and a higher percentage of fish were intersex. Unlike sticklebacks, exposure of medaka to E2 in the egg, as little as 1 day beginning right after fertilization, can cause phenotypic sex reversal to females (Kobayashi and Iwamatsu, 2005). Medaka seem particularly sexually labile, as exposure even into adulthood can lead to intersex. Exposure to the herbicide oryzalin for 21 days led to intersex in 7-month-old, sexually mature medaka (Hall et al., 2007). Spontaneous intersex in untreated medaka has also been reported, and the incidence of intersex increased with age (Grim et al., 2007). While altered steroids levels, both androgens and estrogens, clearly have an effect on sexual differentiation, the trigger or mechanism of sexual differentiation still has not been elucidated. It was mentioned earlier that aromatase expression was suppressed during sexual differentiation in phenotypic Japanese flounder males. A study was performed on medaka exposing them for 14 days post-hatch to the estrogenic compound o,p0 -DDT with and without the aromatase inhibitors fadrozole (a pharmaceutical) and tributyltin (an antifouling agent). Even though aromatase activity was significantly suppressed in the presence of the inhibitors, 100% phenotypic sex reversal to females still occurred (Kuhl and Brouwer, 2006). A study with roach exposed to environmentally relevant concentrations of ethinylestradiol found a concentrationdependent increase in both ERa and ERb, with highest induction of ERa, alongside sex reversal (Katsu et al., 2007). If sexual differentiation is at least in part ER-mediated, then perhaps the presence of sufficient quantities of a compound that can interact with the ER are enough to elicit feminization and aromatase inhibition can be overcome. There seems to be some question whether intersex fish are still reproductively viable. Balch et al. (2004) indicated that intersex individuals were still able to reproduce successfully. However, Nash et al. (2004) believed that the intersex individuals were not sexually viable. The Nash intersex fish had other, extensive abnormalities including malformation of the sperm ducts, which could have had a greater impact on gonadal function than the presence of oocytes, however.

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While it is clear that if all males in a population were sex-reversed it would likely result in reproductive failure, the consequences of sex reversal in mixed populations is cloudier. Aquaculture studies with sex-reversed tilapia (Oreochromis aureus) demonstrated that phenotypic female/genetic male (pseudofemale) fish were still capable of reproducing, but the production of fry was lower due to reduced spawning frequency and a higher percentage of nonspawning pseudofemales versus control females (Desprez et al., 1995). The percentage of male offspring was also much higher (almost 100%) for pseudofemales (Desprez et al., 1995; Me´lard, 1995). Tilapia have ZZ (male) and ZW (female) genetic sex determination. Pairing of sex-reversed males with untreated males are likely to generate all ZZ, or male genotypes. Sex reversal in XX/XY sex-determined fish will likely provide different results. Pseudofemales paired with untreated males will likely result in XX, XY, and YY genotypes. Crossing YY males with females from various genetic backgrounds led to all male populations, as expected, but few of the offspring were sterile (Bongers et al., 1999). Therefore, even though pseudofemales are still capable of reproducing (albeit at a lower rate for tilapia), there may be an impact on the sex ratios of later generations.

3.5. Reproductive failure Estrogenic and antiandrogenic compounds have been shown in several species to have deleterious effects on reproduction. Treatment of rainbow trout with 280 ng/L nonylphenol for 60 days led to a decrease in semen quantity, a decrease in the percentage of eggs surviving to the eyed stage, and a decrease in the percentage of larvae that survived to the end of the yolk-sac stage (Lahnsteiner et al., 2005). Guppies (Poecilia reticulata) treated with at least 100 mg/L octylphenol and 1 mg/L E2 for 30 to 60 days had decreased testes growth but an increase in sperm count (Toft and Baatrup, 2001). There was a decrease in male secondary sexual characteristics upon treatment with both compounds. E2 decreased the birth rate, but the results were variable with octylphenol. Ninety days after the cessation of exposure there were still significant effects on sperm count and secondary sexual characteristics, indicating slow recovery. The antiandrogen flutamide decreased fecundity in fathead minnows treated for 21 days with 651 mg/L ( Jensen et al., 2004). The decrease in fecundity was caused by both a decrease in the number of spawns per female, and the number of eggs per spawn. There was a concomitant increase of atresia and a delay in egg development. Spermatocyte degeneration and necrosis was also present in males. At least two long-term reproductive studies have been conducted in the laboratory to try to determine the potential for population-level effects of exposure to environmentally relevant ethinylestradiol concentrations. Balch et al. (2004) exposed medaka from 2 to 5 days post-hatch until sexual maturity (about 4 to 6 months). Males had feminized secondary sexual

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characteristics, disorganized testicular tissue, and some intersex. There were no alterations to ovarian development in females, yet there was a trend toward a decrease in fecundity. It appeared that fish that copulated were able to produce fertilized eggs, but the number of copulations was decreased both in males paired with untreated females and females paired with untreated males; therefore, the impact on reproduction was more behavioral than physiological. The impact on behavior could be due to the feminized secondary sexual characteristics in males, and perhaps a decrease in pheromones produced by females. While cessation of exposure for six months did not alter gonadal deformities, effects on secondary sex characteristics decreased, indicating that a population may be able to recover somewhat and reproduce. Nash et al. (2004) performed a multigenerational study with zebrafish. Exposure of the F0 generation to 50 ng/L ethinylestradiol for up to 15 days led first to a decrease in egg production and viability, and eventually complete reproductive failure. Seven months’ exposure of the F1 generation to 5 ng/L led to the production of no viable eggs. There were no phenotypic males, no expressible sperm, and, therefore, no fertilization. Ethinylestradiol at 0.5 ng/L caused a decrease in the proportion of viable eggs, but the surviving larval integrity was not impaired. Therefore, it appears that long-term exposure to environmentally relevant concentrations of ethinylestradiol can impair or completely block reproduction. However, if there are surviving larvae there are no transgenerational effects. Partial recovery was also demonstrated upon removal of ethinylestradiol in this study. True population-level effects are extremely difficult to reproduce in the laboratory because it is difficult to unequivocally state that estrogenic and/or antiandrogenic compounds at environmentally-relevant concentrations will cause the demise of a fish population. However, a study was recently conducted in the field in which a lake was treated with environmentally realistic concentrations of ethinylestradiol over a period of 3 years (Kidd et al., 2007). The fathead minnow population in the lake was monitored. Increases in vitellogenin in both sexes were evident, along with testicular abnormalities in males (delayed spermatogenesis, fibrosis, malformed tubules, intersex, lowered gonadosomatic index) and females (delayed ovarian development, follicular atresia). Catch-per-unit-effort decreased consistently over time compared to two reference lakes. In the fall after the second season of ethinylestradiol additions, the fathead minnow population collapsed because of a loss of young-of-the-year animals. The reproductive failure continued through the third year of chemical additions, and an additional two years post-treatment. Another species in the lake, the pearl dace (Margariscus margarita), did not experience the same population crash perhaps because this species is longer lived. Therefore, it appears that populations of short-lived fish species may be at risk from chronic exposure to estrogenic compounds in the environment.

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4. Summary and Concluding Remarks There are many compounds found in the environment with estrogenic or antiandrogenic properties. There are several different types of ER and AR receptors within a species, as well as differences between species, and the binding properties and downstream responses vary. Therefore, the interaction of a given compound with a given receptor may not indicate how it will react with another. There are many other mechanisms for estrogenicity in addition to receptor interactions, including effects on steroid synthesis, distribution and excretion, effects on other parts of the HPG axis, and also indirect mechanisms. Some compounds can act through a myriad of different mechanisms to bring about an estrogenic response. And the response itself can vary, from non-reproductive effects on gene regulation, DNA damage, and liver and renal histopathology to reproductive effects such as gonadal histopathology, sex reversal and eventually reproductive and even population failure. While there are many in vitro tools available to assess the mechanisms of estrogenicity, relying on them to determine the magnitude of response in a whole animal may be narrow and misleading. In addition even whole animal responses will vary depending on species, sex, age, and reproductive status. Therefore, it is imperative to utilize endpoints throughout the biological hierarchy to better understand mechanisms of action, with the ultimate benefit being more accurate risk assessments for compounds of this nature.

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Roles of P21-Activated Kinases and Associated Proteins in Epithelial Wound Healing Mirjam Zegers* Contents 1. Introduction 2. Biology of Wound Healing in Different Model Systems 2.1. Developmental models 2.2. Scrape wound healing 2.3. Wound healing and cancer 2.4. Epithelial plasticity during wound healing 3. Rho GTPases and Epithelial Morphogenesis During Wound Healing 3.1. Steps in wound healing 3.2. Rho GTPases 4. P21-Activated Kinases 4.1. Background of P21-activated kinases 4.2. Structure 4.3. Mechanism of activation 4.4. Inactivation 5. Pak Activation During Wound Healing and Epithelial Sheet Migration 5.1. Background 5.2. Activation of Pak by wounding-associated signals 5.3. Kinase-independent functions and Pak-interacting proteins 5.4. The PIX-GIT complex 6. Regulation of Wound Healing Downstream of Pak 6.1. Cell motility and sheet migration 6.2. Regulation of cell proliferation by Pak, PIX and GIT 7. Concluding Remarks Acknowledgments References

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Department of Surgery, University of Chicago, Chicago, IL 60637

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00606-0

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Abstract The primary function of epithelia is to provide a barrier between the extracellular environment and the interior of the body. Efficient epithelial repair mechanisms are therefore crucial for homeostasis. The epithelial wound-healing process involves highly regulated morphogenetic changes of epithelial cells that are driven by dynamic changes of the cytoskeleton. P21-activated kinases are serine/threonine kinases that have emerged as important regulators of the cytoskeleton. These kinases, which are activated downsteam of the Rho GTPases Rac and cd42, were initially mostly implicated in the regulation of cell migration. More recently, however, these kinases were shown to have many additional functions that are relevant to the regulation of epithelial wound healing. Here, we provide an overview of the morphogenetic changes of epithelial cells during wound healing and the many functions of p21-activated kinases in these processes. Key words: Epithelial morphogenesis, Wound healing, Rho GTPases, p21-activated kinase, Cell adhesion, Cell migration. ß 2008 Elsevier Inc.

1. Introduction Epithelial cells are organized in sheets of adherent cells that are polarized, meaning that they have distinct apical and basolateral surfaces. An important function of polarized epithelial cells is to form barriers between distinct physiological environments (Nelson, 2003; O’Brien et al., 2002; Zegers et al., 2003b). Examples of such barriers are the skin and the luminal surfaces of internal organs such as the respiratory, gastrointestinal and uritogenitary tracts, as well as the mammary, prostate and other exocrine glands. The functions of epithelia rely entirely on the ability of epithelial cells to form a polarized monolayer. It is therefore essential that epithelia have efficient mechanisms to repair injuries induced by trauma, surgery, inflammation and toxic or ischemic insults. In general, epithelial repair can be divided into a start and a stop phase ( Jacinto et al., 2001). In the start phase, cells adjacent to injured areas partially dedifferentiate and migrate into the site of injury. These cells migrate by extending protrusions and lamellipodia into the wound, while pulling along cells located further back from the wound edge. In large wounds, cell proliferation is stimulated to replace lost or damaged cells. In the stop phase, newly formed cell-cell contacts block cell migration and proliferation. In this review I will discuss intracellular signaling pathways that control the diverse morphogenetic changes that epithelial cells undergo during the different stages of epithelial wound healing. In vivo wound healing is a complex and highly regulated process that involves a wide range of

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extracellular signals and epithelial cells, stromal and inflammatory cells. Interestingly, the signaling pathways that control epithelial behavior during injury repair appear to be highly conserved and bear strong resemblance with related behaviors that are observed during embryonic development. One of the conserved features for epithelial morphological changes is the crucial role of the cytoskeleton. Rho GTPases in particular, are ubiquitous intracellular signaling intermediates critical for cytoskeletal regulation and wound healing in a wide variety of models. In this review I will first discuss general aspects of epithelial morphogenesis during wound healing. Next, I will focus on the role of one particular effector family of Rho GTPases, the p21-activated kinases, and their role in epithelial cell behavior during wound healing.

2. Biology of Wound Healing in Different Model Systems Epithelial wound healing in vivo has been most widely studied in the context of the skin (Martin, 1997; Singer and Clark, 1999). Cutaneous wounds in adult tissue are temporarily repaired by the formation of a fibrinrich blood clot to which platelets bind. The clot acts as a reservoir for growth factors and cytokines secreted by platelets and damaged keratinocytes, such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), the transforming growth factor a (TGF-a), and members of the transforming growth factor b (TGF-b) family. These factors attract neutrophils and fibroblasts, which also secrete growth factors and proteases such as matrix metalloproteases (MMPs). Upon secretion, MMPs degrade specific components of the extracellular matrix, thereby allowing matrix remodeling, while at the same time releasing additional growth factors that had been linked to the extracellular matrix. This complex mix of these growth factors generated in the stroma of wounded tissue will also induce angiogenesis, by stimulating endothelial cells to proliferate and form new capillaries into the wound stroma. A prime objective of wound healing is to restore epithelial function by inducing the epithelial cells to undergo sheet movements in which migration, proliferation and cell adhesion processes are highly coordinated. The released growth factors and proteases in the wound stroma profoundly affect the epithelial cells at the wound edge and induce phenotypical changes that resemble an epithelial-mesenchymal transition (EMT, see below). As a result, the interaction of epithelial cells with the underlying basement membrane and neighboring cells is reduced and cells migrate as a sheet over the provisional wound matrix. This provisional matrix forms under the clot and contains fibronectin, vitronectin and other matrix molecules.

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These morphological changes and rapid stimulation of epithelial cell migration is generally followed by sharp increase in cell proliferation to replace lost cells (Martin, 1997; Singer and Clark, 1999). The initial events at the ‘‘start phase’’ of wounding, which includes the inflammatory, angiogenic, migratory and mitogenic responses, need to be inhibited at the ‘‘stop phase’’ of wound closure ( Jacinto et al., 2001). Presently, the downregulation of these different responses remains poorly understood. In epithelial cells, ‘‘contact inhibition,’’ a mechanism that inhibits cell motility and proliferation upon reaching high density and/or establishment of cell-cell contacts (Abercrombie, 1979; Fagotto and Gumbiner, 1996; Middleton, 1972; Stoker and Rubin, 1967) is likely to be involved, but the molecular mechanisms underlying contact inhibition are still largely unknown. The basic wounding response in adult mammalian epithelia other then the skin appears to be generally similar to cutaneous wound healing. Some differences, however, exist dependent on the nature of the insult, the specific tissue involved, or the developmental stage of the organism. As has become clear from in vivo model systems, wound healing is a complex process that involves many different cell types and cellular behaviors. In addition, it generates a complex mixture of secreted growth factors, proteases and matrix molecules, which in turn will act on a wide array of membrane receptors and adhesion molecules. For this reason, the signaling pathways that underlie epithelial cell behavior at the cellular level have been difficult to decipher in mammalian in vivo models. For this reason, investigators have used a variety of alternative in vivo and in vitro model systems to study the mechanisms that control epithelial repair.

2.1. Developmental models The forward movement and fusion of epithelial sheets that occur during wound healing is not unique to wound repair, but is in fact a common phenomenon in many other morphogenetic processes, in particular during development. Examples are eyelid closure, in which fetal mouse eyelids move toward the center of the eye and tightly fuse which each other, only to open again 2 weeks after birth (Harris and McLeod, 1982). During late Drosophila embryogenesis, retraction of the germ band results in an epithelial hole, which is closed by lateral sheets of epithelia which move towards each other and fuse at the dorsal midline in a process called dorsal closure (Harden, 2002; Martin and Parkhurst, 2004). Embryonic tissue movements similar to Drosophila dorsal closure occur in the worm C. elegans, where the epidermis spreads from the dorsal surface of the C. elegans embryo, until the epithelial sheets encloses the embryo and seal at the ventral midline (Ding et al., 2004). As the molecular mechanism that drive these tissue movements appear to be largely conserved ( Jacinto et al., 2001; Martin and

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Parkhurst, 2004), aspects of epithelial sheet migration during embryonic development can serve as a model for epithelial wound healing. Not all aspects of wound healing are recapitulated during development, as these models for instance lack inflammatory responses. Nevertheless, the study of epithelial sheet movements during development has provided many insights into the regulation of sheet migration.

2.2. Scrape wound healing One of the simplest models systems for epithelial repair in vitro are scrape wound healing assays, in which closure of a monolayer is analyzed following the removal of a few rows of cells from a confluent monolayer of cells grown in culture dishes. Although this approach is obviously reductionistic, the advantages of these scrape wound models are the ease of experimental manipulation and the fact that such model systems only comprise epithelial cells. Thus, scrape wound healing assays allows investigators to study the intrinsic epithelial response to wounding in the absence of the complex mix of factors contributed by the stroma, and allows an analysis of the role of the individual components of this mix. In fact, most of our current knowledge on both the intracellular signaling pathways and the molecular machinery required for epithelial sheet migration during wound healing has been elucidated using scrape wound healing assays. Even though not all regulatory factors identified in scrape wound healing assays appear to be crucial in vivo (DiPersio, 2007), the mechanisms that drive wound healing in scrape wound healing assays have been found to be recapitulated to a remarkable extent in the different in vivo and developmental models (Van Aelst and Symons, 2002). This suggests that the epithelial wound healing response is driven by robust and conserved signaling pathways.

2.3. Wound healing and cancer Based on the similarities in both histology and signaling process that promote tumor progression, tumors have been proposed to behave as ‘‘wounds that never heal’’ (Dvorak, 1986). This notion has been further supported by recent genomic analyses comparing carcinoma cells and cells engaging in or mimicking a wounding response (Chang et al., 2004; Iyer et al., 1999; Pedersen et al., 2003). Data from those studies not only demonstrate significant overlap between transcriptional profiles of both cell types, but also indicate that increased similarities correlate with a tendency of tumor cells to metastasize (Chang et al., 2004). Thus, the extensive analysis of cellular behaviors of cancer cells, in particular those concerning regulation of motility and epithelial dedifferentiation, is highly relevant for understanding epithelial wound healing. Clearly, the opposite is

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equally true, in that understanding the epithelial wound healing response may yield novel insights in cancer progression.

2.4. Epithelial plasticity during wound healing As discussed above, epithelial wound healing is accompanied by dramatic cell shape changes of wound edge cells. In intact epithelia, epithelial cells have apical-basolateral polarization and their lateral membranes tightly interact through specialized structures such as tight junctions and E-cadherin-based adherens junctions. At the basal side, cells interact with basement membrane (a specialized form of the extracellular matrix) through adhesion receptors like integrins. Several of these epithelial characteristics will largely disappear at sites of injury. Cells will lose apical-basolateral polarization and tight cell-cell and cell-matrix interactions will be downregulated, weakened or altered. These morphological changes recapitulate aspects of epithelial-mesenchymal transition (EMT). EMT is a process mainly found during embryonic development in which epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype, allowing cells to migrate and invade the stroma. EMT is characterized by a down-regulation of epithelial-specific proteins, such as E-cadherin and the acquisition of mesenchymal-specific proteins like vimentin (Grunert et al., 2003; Hay and Zuk, 1995). The opposite process, mesenchymal-epithelial transition (MET), in which mesenchymal cells revert to cells with an epithelial phenotype, also exist and is instrumental for kidney development (Hay and Zuk, 1995). The term EMT has recently also been used to describe many types of epithelial plasticity. As a consequence, the initial stages of wound healing and sealing of epithelial sheets in the final stages of wound repair is sometimes suggested to represent EMT and MET, respectively. EMT in the strict sense however, is characterized by the ability of individual cells to leave the epithelial monolayer entirely, which does not occur during wound healing. Moreover, EMT is mainly regulated by transcriptional programs through transcriptional regulators such as Snail family proteins (Thiery and Sleeman, 2006) and Twist (Yang et al., 2004), and it is currently unclear to what extent these transcription factors play a role in normal epithelial wound healing. At least in in vitro scrape wound healing assays, wound healing can occur in the absence of protein synthesis (Altan and Fenteany, 2004), which would argue against a crucial role of transcriptional regulation, at least during some stages of epithelial wound healing. On the other hand, stromal growth factors such as TGF-b, which are released during wound healing and play important roles during the process, are well known to induce EMT. Furthermore, wounded epithelial cells are more susceptible to TGFb-induced EMT (Masszi et al., 2004), and EMT has been implicated in

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pathological wound healing processes such as kidney fibrosis in chronically injured kidney epithelia (Boutet et al., 2006; Zeisberg et al., 2003). Therefore, it is possible that wound healing shares the same signaling events with the initial stages of EMT, but that does normally not progress to a complete EMT.

3. Rho GTPases and Epithelial Morphogenesis During Wound Healing 3.1. Steps in wound healing Epithelial wound healing critically depends on the ability of cells to migrate. Cell migration can be regarded as a cyclical process in which the following distinct steps are distinguished (Ridley et al., 2003): I. Polarization. In response to migration-inducing factors, cells polarize and form protrusions towards the direction of migration. These protrusions can be lamellipodia, which are large and sheet-like and driven by formation of actin meshworks, or filopodia, which are spike-like and driven by actin bundles. II. Traction at the leading edge. The polarized protrusions at the leading edge are stabilized by adhesion to the ECM though transmembrane adhesion receptors which link to the actin cytoskeleton. The formation of such focal contacts allows the cell to generate traction force at the leading edge that the cell uses to move forward. III. Retraction at the trailing edge. Focal contacts at the trailing edge will disassemble and the tail of the cell will retract. These steps in cell migration are found in wide variety of cells and although they have been mainly characterized in single cells, epithelial sheet migration during wound healing appears to proceed in a similar fashion, with many cells acting in concert (Farooqui and Fenteany, 2005).

3.2. Rho GTPases Local rearrangements of the cytoskeleton drive the specific cell morphological changes that accompany cell migration. The small GTPases of the Rho family are crucial regulators of the actin cytoskeleton and it is therefore not surprising that these molecules are involved in the distinct steps of cell migration. With regard to epithelial wound healing, the role of Rho GTPase is not limited to regulation of migration, as these GTPases have also been implicated in many other aspects of this process, such as the regulation of cell-cell adhesion, apical-basolateral cell polarization and cell

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cycle control. Many excellent reviews on the role of Rho GTPases in these processes are available (Hall, 1998; Jaffe and Hall, 2005; Jaffer and Chernoff, 2004; Kaibuchi et al., 1999; Marshall, 1999; Schmidt and Hall, 2002; Schmitz et al., 2000; Settleman, 2000; Van Aelst and Symons, 2002). Rho GTPases act as switches between extracellular signals and intracellular effector molecules. Rho GTPases can be activated by activated growth factor receptors or in response to cell-cell or cell-matrix adhesion. With regards to the latter types of activation, they participate in bidirectional signaling with both cadherins (Kaibuchi et al., 1999) and cell-matrix receptors like integrins (Keely et al., 1998), meaning that they are not only activated through these adhesion receptors, but they also regulate their adhesive functions. Rho GTPases are activated via guanine nucleotide exchange factors (GEFs), which replace the GDP bound to the GTPase with GTP. Upon activation, Rho-GTPases activate different effector molecules, thereby stimulating signaling cascades that regulate a variety of cellular processes. Currently, over 20 Rho GTPase members have been identified in mammalian cells ( Jaffe and Hall, 2005). Most research however has focused on the prototypical family Rho GTPase family members Rac1, RhoA and cdc42. Rac, Rho and cdc42 all have been implicated in regulation of wound healing and sheet migration. Although their respective roles in these processes are to some extent cell type and tissue-specific, the roles of Rac1 appear the most widely conserved. In Drosophila, loss of function or inhibition of Rac leads to defects in dorsal closure, likely by an inhibition of lamellipodia and filopodia and inhibition of actin-myosin contractility (Hakeda-Suzuki et al., 2002; Harden et al., 1995, 1999; Woolner et al., 2005). In vitro scrape wound healing in the epithelial Madin-Darby canine kidney (MDCK) monolayers is blocked when dominant-negative Rac1 is microinjected in the first three rows of cells at the wound edge, whereas dominant-negative RhoA or cdc42 essentially have no effect (Fenteany et al., 2000). Studies in bronchial epithelial cells demonstrated similar requirement for Rac1, but in these cells wound healing also depends on RhoA (Desai et al., 2004). Together, these studies indicate a crucial role for Rac in wound healing. This was recently confirmed in vivo, as inhibition or deletion of Rac1 in mouse skin was shown to inhibit incisional epidermal wound healing (Tscharntke et al., 2007). The mechanism responsible for these wound healing defects likely involves the intrinsic inhibition of keratinocyte migration and proliferation (Castilho et al., 2007; Tscharntke et al., 2007), but may also involve the depletion of follicular stem cells (Benitah et al., 2005). Other in vivo studies however demonstrated that Rac1 deletion only inhibits hair follicle development by follicular stem cell depletion, but does not affect epidermal development and maintenance (Castilho et al., 2007; Chrostek et al., 2006).

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4. P21-Activated Kinases 4.1. Background of P21-activated kinases Rho GTPases, such as Rac, mediate their biological functions by activating effector molecules, which are mostly, but not always, kinases that initiate cellular behaviors by phosphorylating downstream substrates which may initiate signaling cascades. In order to understand the role of Rac in the regulation of wound healing and sheet migration, it is therefore crucial to identify and characterize the specific Rac effector molecules. P21-activated kinases (Paks) were the first identified binding partners of GTP-bound Rac and cdc42 (Manser et al., 1994) and are among the best characterized of the many Rho GTPase effector molecules currently known. Indeed, Paks were named after this characteristic, as the p21 in their name stands for Rac and cdc42, which, as all Rho GTPases, have a molecular weight around 21 kDa. Initial reports showed that Paks specifically interact with GTP-bound forms of Rac1 and cdc42, but not with the GDP-bound versions of these protein (Bagrodia et al., 1995; Knaus et al., 1995; Manser et al., 1994; Martin et al., 1995). More recently, several additional small GTPases of the Rac and cdc42 subfamilies (Bustelo et al., 2007) were found to activate Pak, including Rac2 (Knaus et al., 1998), Rac3 (Mira et al., 2000), Chp (Aronheim et al., 1998; Weisz Hubsman et al., 2007), TC10 (Neudauer et al., 1998) and Wrch-1 (Tao et al., 2001). Paks are not activated by Rho A-G or by Ras superfamily members (Bokoch, 2003). To date, six Pak family members have been identified. The human Pak1 (rat aPak), human Pak2 (rat gPak) and human Pak3 (rat bPak) are now classified as conventional, group I or group A Paks. In addition, there are the nonconventional, group II or group B Paks, which are named Pak4, Pak5 (sometimes described as Pak7) and Pak6 (Bokoch, 2003; Dan et al., 2001; Hofmann et al., 2004; Jaffer and Chernoff, 2002; Zhao and Manser, 2005). The structure and regulation of group II Paks differs significantly from the group I Paks and a detailed understanding of their roles is only beginning to emerge. For these reasons, this review will focus on Pak1-3.

4.2. Structure Pak1-3 share several conserved characteristic features. As shown in Figure 6.1, their general structure comprises a regulatory N-terminal domain and a catalytic C-terminal domain. The N-terminal domain contains a p21-binding domain (PBD), which interacts with the active, GTP-bound forms of Rac and cdc42. Partially overlapping with the PBD is an autoinhibitory domain (AID). A large part of the combined PBD and

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Grb2 Nck

Pak Rac/cdc42

PIX

PBD AID Regulatory domain

Catalytic domain

Figure 6.1 Structural features of Pak kinases. Image represents the domain structure of Pak1. Pak1-3 contain an N-terminal regulatory domain and a C-terminal serinethreonine kinase domain. Several domains within the regulatory domain are indicated: The dashed areas represent the canonical proline-rich, SH3-binding domains. The adaptor proteins Nck and Grb2 bind to the first and second proline-rich domain, respectively. In black, the non-canonical, proline-rich PIX-binding domain is indicated. In dark-grey, the p21-binding domain (PBD) is indicated, which binds GTP-bound forms of Rac and cdc42. Partially overlapping with the PBD is the autoinhibitory domain (AID) in light-grey, which inhibits Pak activity in trans by binding to the catalytic domain of another Pak molecule.

AID comprises the so-called ‘‘inhibitory switch domain’’ (Lei et al., 2000), which is crucial for the activation of Pak. The N-terminus furthermore contains several proline-rich domains with canonical PxxP SH3 binding domains (five in Pak1, two in Pak2 and four in Pak3) and a conserved nontypical proline-rich PxP SH3 domain which binds the GEFs of the PIX/Cool family (Bokoch, 2003; Jaffer and Chernoff, 2002). The serine/ threonine kinase domain of Pak is at the C-terminus of the protein and is at least 93% identical in Pak1, 2 and 3 ( Jaffer and Chernoff, 2002).

4.3. Mechanism of activation 4.3.1. Activation by Rho GTPases The mechanism of Pak activation has been analyzed at the molecular level in Pak1, for which a crystal structure of both the inactive and active kinase domain has been resolved (Lei et al., 2000; Lei et al., 2005). Based on the crystal structure, it was concluded that Pak1 exists as a dimer in a transautoinhibitory conformation in which the inhibitory switch domain of one Pak1 molecule inhibits the catalytic domain of the other. It is believed that Pak1 exist in this form both in solution and in unstimulated cells (Buchwald et al., 2001; Lei et al., 2000; Parrini et al., 2002). Binding of GTP-bound Rac or cdc42 induces a series of conformational changes, which results in a disruption of the dimer and ends with the kinase domain in a stable catalytically active conformation. Central to Pak1 activation is the phosphorylation of the Thr423 residue in the activation loop of Pak1. Thr423 is exposed upon Rac/cdc42 binding and its phosphorylation allows for kinase activation and stabilization of the active conformation. It furthermore allows for autophosphorylation of several other sites, which also contribute to kinase activation (Chong et al., 2001; Frost et al., 1998; Hoffman and Cerione, 2000; Lei et al., 2000, 2005; Tu and Wigler, 1999; Zenke et al., 1999; Zhao et al., 1998).

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Though Thr423 phosphorylation in solution can occur through autophosphorylation (Parrini et al., 2002), in cells it may be mediated by 3-phosphoinositide
dependent kinase 1 (PDK1), perhaps through a mechanism that depends on the membrane lipid sphingosine (Bokoch et al., 1998; King et al., 2000a,b). 4.3.2. Rho GTPase
independent activation Even though Paks are considered bona fide downstream targets of active Rac/cdc42, several Rac/cdc42-independent activation mechanisms have been reported as well. Initial studies that characterized Pak activity in vitro, had shown that proteolytic cleavage of Pak, which removes its N-terminus, yields a highly active Pak in solution (Benner et al., 1995; Roig and Traugh, 2001). Interestingly, proteolytic cleavage was demonstrated to be a physiological mechanism of Pak activation during apoptosis, when Pak2 is cleaved by caspase-3 (Rudel and Bokoch, 1997; Walter et al., 1998). Pak can be recruited to the plasma membrane by several different mechanisms, which activates the kinase by a process that is not fully understood. Membrane recruitment of Pak1, either through binding to the adaptor protein Nck (Lu et al., 1997), or experimentally induced by introduction of a C-terminal isoprenylation sequence (Daniels et al., 1998) activates Pak1 kinase activity, possibly through a sphingosine- and PDK1dependent phosphorylation of Pak1. Phosphorylation and activation of Paks by the kinase Akt, either downstream of Ras (Sun et al., 2000; Tang et al., 2000) or downstream of the heterotrimeric G-protein b/g subunits (Menard and Mattingly, 2004) has been reported as well. These Pak phosphorylations by PKD1 or Akt occur in the presence of dominant-negative mutants of Rac1 and cdc42, suggesting that they are independent of these GTPases. However, since the activating phosphorylations occur at sites at the catalytic domain that are masked by the inhibitory switch domains in inactive Pak1 dimers, the question remains how these residues are accessible to PKD1 or Akt. As will be discussed in detail later, Paks can also be recruited to the membrane by an interaction with the Pak-interacting exchange factor (PIX). Pak binds directly to PIX, which in turn tightly interacts with the G protein-coupled receptor kinase-interacting target (GIT1). This Pak-PIX-GIT complex accumulates at focal adhesions in migrating cells where the (indirect) interaction of GIT1 with Pak can activate Pak1. As this activation also occurs in the presence of dominantnegative Rac1 or cdc42 or in a Pak1 mutant that cannot bind active cdc42, this activation appears to be independent of Rho GTPases (Loo et al., 2004). In mitotic cells, GIT1 can target Pak1 to the centrosome, which results in Pak1 activation at this site (Zhao et al., 2005). Interestingly, when Pak1 is targeted to the centrosome by the addition of a centrosomal targeting domain, Pak1 is activated at the centrosome as well, suggesting that targeting to the centrosome is sufficient to drive Pak activation.

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The molecular mechanisms underlying GTPase-independent Pak activation is still unclear. At this point, it cannot be excluded that initial disruption of the Pak1 dimers in the studies mentioned above is mediated by a Rac/cdc42 subfamily GTPase other than Rac1 or cdc42 (Lu and Mayer, 1999) and that Pak phosphorylation by PKD1/Akt or other yet unidentified kinases cooperate in Pak1 activation. A role for sphingosine in making the activation loop accessible to phosphorylations by other kinases is possible as well (Zenke et al., 1999). Alternatively, ‘‘dimer breathing’’ has been proposed, in which the kinase domain is temporarily released from the inhibitory switch domain, thus allowing activation in a Rho-GTPase independent manner (Loo et al., 2004).

4.4. Inactivation As in all signaling pathways, it is important for cellular homeostasis that activating signals are counteracted by inactivating signals. Consistent with this notion are findings that the activity of Pak is tightly regulated: Pak activation in response to stimuli peaks within 15 seconds and returns to base levels after 3 minutes (Huang et al., 1998). The initial Pak-activating signals, i.e., GTP-bound Rho-GTPases are rapidly inactivated through the action of GTPase activating proteins (GAPs). The inactivation of Rho GTPases is however unlikely to deactivate Pak, since autophosphorylated Pak has a decreased affinity for the GTP-bound forms, and GTP-bound Rho GTPases are thought to be released from Pak upon its activation (Manser et al., 1994). Indeed, it was shown that separate signals activate and deactivate Pak (Huang et al., 1998). Several proteins have been implicated in the negative regulation of Pak. Two serine/threonine phosphatases of the PP2C family, POPX1 and POPX2, directly interact with PIX and form a heterotrimeric complex with PIX and Paks. POPX1 and POPX2 dephosphorylate and downregulate Pak activity, most likely by dephosphorylating the Thr423 residue (Koh et al., 2002). The phosphorylation and activation of Pak may also target it for degradation by the proteosome. Interestingly, this process is mediated by the small GTPases Chp or cdc42 (Weisz Hubsman et al., 2007). Since these GTPases also activate Pak1, these findings suggesting a dual role for Chp and cdc42 as both activators and as negative feedback regulators of Pak1. A third mechanism of negative regulation of Pak can be accomplished by blocking its activation, which can be mediated by a number of proteins. Caveolin (Kang et al., 2006), nischarin (Alahari et al., 2004), CRIPak (Talukder et al., 2006) and hPIP (Xia et al., 2001) all bind to Pak and prevent the activation of Pak by GTPbound Rac/cdc42. Also, even though one study implicated G protein b/g subunits upstream of Pak activation (Menard and Mattingly, 2004), G protein b/g subunits have also been implicated in Pak inhibition (Wang et al., 1999). Currently, it is unknown how the activity and expression of

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any of the Pak inhibitors is regulated. Clearly, this knowledge is required to fully understand the roles of Pak in regulation of wound healing and other processes.

5. Pak Activation During Wound Healing and Epithelial Sheet Migration 5.1. Background Since Rac is crucial for wound repair, it seems likely that Pak kinases are important regulators of this process. Indeed, several lines of evidence have implicated Pak1 in the regulation of developmental epithelial sheet movements or wound healing. During Drosophila dorsal closure, the Pak family member Dpak accumulates in the leading edge cells (Harden et al., 1996) where it is required for the integrity of the actin cytoskeleton and for epithelial sealing (Conder et al., 2004). Even though loss-of-function Dpak mutants survive, they are sterile (Hing et al., 1999) and have various defects in the follicular epithelium that covers the egg chamber, including a loss of apical-basolateral polarity (Conder et al., 2007). In C. elegans embryos, the Pak homologue CePak is highly expressed at hypodermal cell boundaries and regulates embryonic body elongation by controlling an actin-dependent process called hypodermal fusion (Chen et al., 1996). Pak1 may also have a role in epithelial morphogenesis during mammalian embryonic development since high levels of a phosphorylated form of Pak1 have been found in developing epithelial organs such as the lung, kidney, intestine and skin (Zhong et al., 2003). Furthermore, many in vitro studies in mammalian cells have demonstrated a role for Pak in cell migration during scrape wound healing or in cell migration of single fibroblasts or epithelial cells. Together, these studies support the hypothesis that Paks play important roles in the regulation of the cytoskeleton. (Bokoch, 2003). One of the clues that Pak1 is involved the regulation of the actin cytoskeleton and cell motility came from observations that a constitutively active form of Pak1, the phosphomimetic Pak1-T423E, induces lamellipodia and increases migration in 3T3 fibroblasts (Sells et al., 1997). Later studies showed that silencing Pak1 expression with small interference RNA (siRNA) inhibits fibroblast migration (Rhee and Grinnell, 2006). Consistent with a role in regulating actin polymerization at the leading edge, Pak1 distributes from the cytosol to the cortical actin in lamellipodia in v-Src
transformed fibroblasts or in normal cells during wound healing or after PDGF stimulation (Dharmawardhane et al., 1997). In breast cancer cells, Pak1 relocalizes to the leading edge of motile cells and promotes invasiveness in response to heregulin treatment (Adam et al., 2000). Finally, numerous other studies have demonstrated that Paks also localize to focal

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contacts, which stabilize the protrusions at the leading edge (Brown et al., 2002; Frost et al., 1998; Manser et al., 1997; Obermeier et al., 1998; Sells et al., 1997, 2000; Stofega et al., 2004; Zegers et al., 2003a). As will be discussed in a later section, this localization likely reflects their regulatory role in the formation and turnover of focal adhesions.

5.2. Activation of Pak by wounding-associated signals Growth factors and cytokines that are released during epithelial wound healing are able to activate Pak. Numerous studies have reported stimulation of Pak by PDGF in many different cell types, likely by a PDGF-induced activation of Rac (Dharmawardhane et al., 1997; Sells et al., 2000; Yoshii et al., 1999). At least one study reported that Pak1 activation by PDGF relies on bPIX (Lee et al., 2001). PDGF may also activate Pak1 through transactivation of the EGF receptor by the active PDGF receptor (He et al., 2001). Direct stimulation of EGF receptor family receptors with heregulin (Adam et al., 1998) or EGF (Galisteo et al., 1996) also activates Pak1. In addition, other growth factors, including hepatocyte growth factor (Royal et al., 2000) and VEGF (Stoletov et al., 2001) have been reported to activate Pak in epithelial and endothelial cells respectively. Even though several different growth factors can activate Pak, the downstream signaling response appears to some extent dependent on the specific growth factor. For instance, stimulation of HeLa and NIH-3T3 cells with either PDGF or EGF leads to activation of Pak1 and Pak2, but only PDGF stimulation links Pak kinases to extracellular-regulated kinase (ERK) activation (Beeser et al., 2005). The strong correlation of Pak signaling downstream of PDGF signaling is of particular importance for wound healing from a clinical standpoint. PDGFBB, a recombinant form of PDGF comprising PDGF b-chain homodimers, is currently the only FDA-approved growth factor in clinical use to accelerate wound healing (Harrison-Balestra et al., 2002; Papanas and Maltezos, 2007). Understanding the roles of Pak downstream of PDGF-signaling is therefore highly relevant for the development of future options for the treatment of wounds. Interestingly, activation of Paks upon in vitro scrape wounding does not rely on the addition of exogenous growth factors, and it currently unclear which wounding-induced signals are responsible for Pak activation. It is possible that factors released from damaged cells are involved. As an alternative, shear stress, which activates integrin signaling, or alterations or absence of cell-cell and cell-matrix contacts may be involved as well. Integrinmediated signaling is well known to activate Rho-GTPases and b1-integrin mediated attachment to extracellular matrix is required for GTP-bound Rac to interact with Pak1 and to activate the kinase (Chaudhary et al., 2000; del Pozo et al., 2000; Howe, 2001; Price et al., 1998). Activation of Pak1 is

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specific for some matrix-integrin interactions. For instance, Pak1 activity is induced by shear stress in endothelial cells plated on fibronectin, but not in cells plated on Matrigel basement membrane or on collagen I (Orr et al., 2007). Furthermore, cell attachment to laminin-332 through a3b1 integrin activates Pak1, whereas a2b1 integrin-mediated attachment to collagen I by did not influence Pak1 activation (Zhou and Kramer, 2005). With respect to these two latter studies, it is relevant to note that fibronectin is an important component of the wound provisional matrix (Martin, 1997; Singer and Clark, 1999), whereas laminin-332 is an important regulator of wound healing which synthesis is induced by both shear stress (Avvisato et al., 2007) and upon epithelial injury in vitro and in vivo (Mak et al., 2006; Schneider et al., 2007). Taken together, it seems likely that integrin signaling is an important factor in wounding-induced Pak1 activation.

5.3. Kinase-independent functions and Pak-interacting proteins Presently, over 40 different kinase substrates of activated Paks have been identified (Bokoch, 2003; Kumar et al., 2006). Pak substrates comprise a diverse group of proteins, many of which have been implicated in the regulation of the cytoskeleton. Pak effectors also include several transcriptional regulators and signaling proteins involved in regulation of cell proliferation and cell death. As will discussed below, many of the Paks’ functions in wound healing-related processes depend on its functional catalytic domain and involve phosphorylation of specific substrates. In addition, Pak has functions that do not rely on its catalytic activity. Numerous studies have demonstrated that at least some of the Paks’ morphological effects, such as stimulation of cell motility or the formation of actin-based structures like lamellipodia, invadapodia and podosomes are kinase-independent (Furmaniak-Kazmierczak et al., 2007; Manser et al., 1997; Sells et al., 1997, 1999; Webb et al., 2005; Zegers et al., 2003a). In addition, kinaseindependent transcriptional regulation by Pak has been reported (Hullinger et al., 2001). The kinase-independent effects of Pak depend on SH3-domain
containing proteins that bind to one of the several PxxP-containing motifs (in which x is any amino acid) within the Pak N-terminus (Frost et al., 1998). Two of such proteins are the SH3/SH2-domain
containing proteins adaptor proteins Nck and Grb2, which link activated receptor tyrosine kinases to intracellular signaling molecules through their SH2 and SH3 domains respectively. Several growth factor receptors that play important roles in in vivo wound healing interact with Pak1 through these adaptor proteins. Nck interacts with the first PxxP motif of Pak1 and links it to tyrosine-phosphorylated PDGF or EGF receptors or to activated integrins,

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thereby recruiting the kinase to the plasma membrane (Bokoch et al., 1996; Galisteo et al., 1996; Howe, 2001; Lu et al., 1997). In the keratinocyte cell line HaCaT, Grb2 recruits Pak1 to the plasma membrane by coupling it to the activated EGF receptor. When the interaction of Pak1 with Grb2 is inhibited with an inhibitory SH3-containing peptide, EGF-mediated lamellipodia extension is blocked, indicating a crucial role for Pak1 in this process (Puto et al., 2003). Using a similar approach, it was shown that the Pak-Nck interaction is important for endothelial cell migration and angiogenesis (Kiosses et al., 2002). Though membrane recruitment by adaptor proteins has generally been implicated in Pak activation (Galisteo et al., 1996; Lu et al., 1997), it is possible that the kinase-independent effects of Pak are mediated by Pak acting as a scaffold. As Nck and Grb2 bind exclusively to the first and second PxxP motif of Pak1 (Bokoch et al., 1996; Galisteo et al., 1996; Puto et al., 2003), respectively, and PIX binds to a third central proline-rich domain in Pak (Manser et al., 1998), it is possible that Pak integrates different signaling pathways during wound healing. Consistent with this notion are data from several studies that demonstrated the formation and membrane recruitment of Nck-PakPIX
containing protein complexes in response to either PDGF-stimulation (Yoshii et al., 1999), or as a result of cell-matrix adhesion and integrin signaling (Brown et al., 2005; Zhao et al., 2000a). Furthermore, Nck and Grb2 likely indirectly associate with PIX via GIT, as both the Nck and Grb2 SH2 domains directly bind to GIT when GIT is tyrosine-phosphorylated (Brown et al., 2005).

5.4. The PIX-GIT complex Of all the Pak-interacting proteins, PIX and its binding partner GIT have been studied most extensively and appear to be crucial for many of the Paks’ functions. aPIX and bPIX (Pak-interacting exchange factor) and the identical p85cool-1 and p85cool-2 were first identified as Pak-binding proteins and interact with a specific proline-rich domain in Paks through a SH3 domain (Bagrodia et al., 1998; Manser et al., 1998). Based on the presence of tandem DH/PH (Dbl homology/Pleckstin homology) domains, a conserved characteristic of Rho-GTPase GEF proteins, the PIX proteins were predicted to exhibit GEF activity. However, while GEF activity of aPIX towards cdc42 and Rac could be readily demonstrated (Feng et al., 2002), it is still uncertain to what extent bPIX exhibits GEF activity towards these GTPases. In vitro GEF assays have indicated that bPIX contains an autoinhibitory domain, and GEF activity towards cdc42 could only be demonstrated upon deletion of this domain (Feng et al., 2002). Data from some studies suggest that phosphorylation of bPIX by Pak2 or protein kinase A can relieve bPIX from its autoinhibitory state and allows it to act

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as a GEF in vitro (Chahdi et al., 2005; Shin et al., 2002, 2004), but evidence for such a mechanism at the molecular level is still lacking. Resolving this question has been further complicated by findings that aPIX and bPIX can both homo- and heterodimerize and that specificity of GEF activity of aPIX depends on its dimerization state (Feng et al., 2002, 2004). Currently, it is unclear whether its ability to activate Rac and/or cdc42 is the main role of PIX proteins. As many Pak functions depend on its ability of Pak to interact with PIX, it is possible that one of PIX functions is to recruit Pak to specific intracellular sites. Furthermore, PIX appears to facilitate the formation of large oligomeric complexes that function in the regulation of focal adhesions (see V.A-2). Indeed, PIX tightly binds to the highly homologuos family of G protein-coupled receptor interacting target (GIT) proteins, which comprises GIT1 (or Cat1/p95-APP1), GIT2 (or Cat2) and p95PKL (or p95APP2) (Bagrodia et al., 1999; Di Cesare et al., 2000; Paris et al., 2003; Premont et al., 1998, 2000; Turner et al., 1999). GITs are multidomain proteins. At their N-terminus, they contain an ARF-GAP domain, which exhibits activity towards several different small GTPases of the ARF family. They furthermore contain a Spa2-homology motif, which is required for its interaction with PIX proteins, a coiled-coil motif that mediates homo- and heterodimerization of GIT1 and GIT2 and a C-terminal paxillin-binding site, which binds the focal adhesion protein paxillin. In many migrating cells, Pak is recruited to focal contacts through a complex that forms through the sequential interactions with PIX, GIT and paxillin (Bagrodia et al., 1998, 1999; Manser et al., 1998; Turner et al., 1999; Zhao et al., 2000b). This complex plays important roles in cytoskeletal dynamics and cell motility. The dynamic assembly and disassembly of the complex, which we will call here Pak-PIX-GIT complexes, is highly regulated. The functions of Pak-PIX-GIT complexes are still not completely understood, but they appear to be involved in many aspects of epithelial wound healing, as will be discussed in the following sections.

6. Regulation of Wound Healing Downstream of Pak Although many studies have implicated Pak in the regulation of epithelial wound healing and sheet migration, knowledge about the molecular mechanisms by which Pak controls these processes is only beginning to emerge. As discussed earlier, wound healing occurs via distinct steps, and depends on many different interconnected signaling pathways. Here, an attempt is made to review the specific molecular mechanisms by which Pak regulates these distinct steps.

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6.1. Cell motility and sheet migration 6.1.1. Cell polarization One of the first processes during wound healing is the polarization of the actin cytoskeleton. In response to an extracellular migration signal, cells polarize and extend protrusions such as lamellipodia or filopodia in the direction of migration. Migratory cell polarization involves numerous interconnected signaling pathways and both positive and negative feedback loops that involve integrins, growth factor receptors, Rho GTPases and phosphoinositidemediated signaling. Pak kinases regulate cell polarity in many different organisms. In yeast, the Pak homologs Ste20 and Cla4p are required for polarized actin assembly during bud formation and cytokinesis (Eby et al., 1998; Holly and Blumer, 1999), while Pak induces actin polarization during directed cell migration of Dictyostelium and Entemoebe amoebas. In mammalian organisms, Paks regulates polarized actin rearrangements during many different cellular processes, including cell migration and polarized actin assembly that occur at the immunological synapse and during neurogenesis (Bokoch, 2003). Together, these findings suggest a rather direct role of Pak at the level of the cytoskeleton. 6.1.1.1. Lamellipodia extension Protrusion of lamellipodia involves the formation of a newly assembled actin meshwork. This is mediated by the Arp2/3 complex, which binds to the side or tip of an existing actin filament and nucleates and branches new filaments at the leading edge (Pollard and Borisy, 2003). Pak1 phosphorylates the p41-Arc subunit of the Arp2/3 complex, and phosphorylation of p41-Arc regulates its association with the Arp2/3 complex at actin nucleation sites at the leading edge of the cells (Vadlamudi et al., 2004). As a non-phosphorylatable mutant of p41-Arc slows cell migration in breast cancer epithelial cells, these data indicate a functional role for Pak1 in Arp2/3-regulated actin branching and lamellipodia extension (Vadlamudi et al., 2004). However, Pak1 may also inhibit the Arp2/3 complex by phosphorylating caldesmon, which increases the ability of caldesmon to compete with the Arp2/3 complex for actin binding (Morita et al., 2007). It remains to be determined whether these differences reflect cell type-dependent differences or that they may reflect different levels of regulation. Clearly, the activity of the Arp2/3 complex in actin branching needs to be spatially restricted to the leading edge of migrating cells, but it is currently unclear if the these apparent opposite roles of Pak are involved in the spatial restriction of the active Arp2/3 complex. In addition to its proposed role in actin nucleation by acting on the Arp2/3 complex, Pak may stabilize actin filaments. The actin depolymerizing factor/cofilin destabilizes actin filaments by severing actin filaments and by actin depolymerization. LIM kinase is activated by Pak1, and, upon activation, phosphorylates and inactivates cofilin, thus promoting actin

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filament stability (Edwards et al., 1999). The same study showed that a kinaseinactive LIM kinase abolishes many of Pak1-induced cytoskeletal changes and membrane ruffling. This suggests that Pak1 stabilizes lamellipodia by a mechanism that involves LIM kinase-mediated inactivation of cofilin. Caldesmon and tropomyosin are two actin-filament stabilizing proteins (Gunning et al., 2005; Hai and Gu, 2006) that have also been implicated in Pak-mediated stabilization of actin. In breast cancer epithelial cells, the kinase-dead Pak1K299R stabilizes F-actin filaments by causing an increased association of tropomyosin and caldesmon with actin stress fibers (Adam et al., 2000). Furthermore, Pak induces caldesmon phosphorylation in Rous sarcomatransformed fibroblasts (Morita et al., 2007) and in response to wounding in CHO cells (Eppinga et al., 2006). In the latter cells, wound healing is impaired in cells that express either the Pak-phosphomimetic or a nonphosphorylatable form of caldesmon (Eppinga et al., 2006). 6.1.1.2. Regulation of microtubules The polarization of the cortical actin at the leading edge during cell migration is accompanied by reorganization of the microtubule cytoskeleton. Though most attention has been focused on the dynamics of the actin cytoskeleton, recent work provided evidence that directional migration depends on microtubules as well, and that both components of the cytoskeleton are in fact tightly integrated during cell migration (Siegrist and Doe, 2007; Watanabe et al., 2005). Microtubules nucleate from their minus ends, which are generally located at the microtubule organizing center (MTOC). At their plus ends, they undergo phases of growth and shrinkage, known as dynamic instability. Plus ends can be captured at specific targets, often associated with the actin cytoskeleton, which prevents shrinkage and stabilizes the microtubules. In migrating cells, microtubule plus ends are selectively stabilized at the leading edge, where they can interact with the cortical actin. In addition, the MTOC usually reorients towards to direction of migration. Though reorganization of microtubules is likely not required for the protrusion of leading edge and migration per se, it is thought be essential for the positioning of the leading edge and persistent directional movement by stabilizing cell polarization of the migrating cell. Similar to the regulation of actin dynamics, Rho GTPases play important roles in the reorganization of microtubules during cell migration. Though a detailed understanding of the cross-talk between microtubules and Rho GTPases is only beginning to emerge and is reviewed in detail elsewhere (Fukata et al., 2003; Raftopoulou and Hall, 2004; Siegrist and Doe, 2007; Small et al., 2002; Small and Kaverina, 2003; Watanabe et al., 2005), several lines of evidence support a role for Pak family kinases in the regulation of microtubules. One of the mechanisms by which Pak regulates microtubules is by phosphorylating stathmin. Stathmin, also called oncoprotein 18 (Op18), binds a/b-tubulin dimers, thereby preventing tubulin polymerization and

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causing catastrophe, the rapid shrinkage of microtubule plus ends (Cassimeris, 2002). Microtubule destabilization by stathmin is inhibited by its phosphorylation on Ser16, which prevents binding of stathmin to tubulin. Several studies demonstrated that Pak1 is required for this phosphorylation (Daub et al., 2001; Wittmann et al., 2003, 2004). A later study showed that stathmin is a direct substrate of Pak1 in vitro, and that its phosphorylation on Ser16 by Pak1 results in a decreased ability of stathmin to inhibit tubulin polymerization in an in vitro assay (Wittmann et al., 2004). In vivo however, additional factors appear to be involved in stathmin phosphorylation (Wittmann et al., 2004), which would be consistent with reports that Pak1 is required but not sufficient for Rac1-mediated stimulation of microtubule growth at the leading edge of migrating cells (Wittmann et al., 2003). Both Pak1 (Zenke et al., 2004) and Pak4 (Callow et al., 2005) phosphorylate GEF-H1. GEF-H1 is a GEF for Rho whose activity is suppressed by binding to microtubules. As GEF-H1 can bind both actin and tubulin, it may locally integrate regulation of the actin and microtubule cytoskeleton by a spatial control of Rho activation (Krendel et al., 2002). Phosphorylation of GEF-H1 by Pak4 causes its release from microtubules in NIH 3T3 cells, which co-incided with a dissolution of stress fibers (Callow et al., 2005). In contrast, phosphorylation of GEF-H1 by Pak1 on an analogous Ser residue did not affect the association of GEF-H1 with microtubules in a study using HeLa cells. Rather, this study showed that Pak1-mediated GEF-H1 phosphorylation results in binding of the scaffold protein 14-3-3 to GEF-H1, thereby recruiting 14-3-3 to microtubules, which could potentially affect GEF-H1 function (Zenke et al., 2004). In addition to the potential roles of Pak in microtubule stabilization, Pak has also been implicated in the regulation of centrosomes and the centrosomal MTOC during mitosis. Pak1 is targeted to the MTOC of mitotic cells, which leads to its activation, as revealed by immunofluorescent staining of an antibody specific for rat Pak1 phosphorylated at Thr422 (Zhao et al., 2005). Furthermore, inducible overexpression of an active analogous human Pak1 phosphomimetic (Pak1-T423E) in epithelial breast cancer cells induces mitotic spindle abnormalities such as multiple spindles (Vadlamudi et al., 2000). The aberrant spindles may be due to phosphorylation of tubulin cofactor B by Pak1, as overexpression of tubulin co-factor B, but not expression of forms that cannot be phosphorylated by Pak1, gives rise to a similar phenotype (Vadlamudi et al., 2005). Alternatively, the phenotype may be mediated through Aurora A. Aurora A is a kinase that has been implicated in centrosome maturation and centrosomal microtubule assembly (Brittle and Ohkura, 2005), and was recently shown to be activated by Pak1 at the centrosome (Zhao et al., 2005). Finally, Pak1, but not Pak2 or Pak3 (Thiel et al., 2002), can be phosphorylated on T212 via p35/cdk5 kinase in neuronal cells (Nikolic et al., 1998), or by cyclinB1/cdc2 in mitotic fibroblasts and other cells (Banerjee et al., 2002; Thiel et al., 2002). This phosphorylation,

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which does not affects Pak1 activity (Thiel et al., 2002), targets Pak1 to the MTOC, where it has been implicated in microtubule destabilization during mitosis (Banerjee et al., 2002). Pak’s effect on microtubule stability and its association with the MTOC would suggest important roles of the kinase on microtubule organization during cell migration. However, to date stathmin is the only Pak substrate directly implicated in the regulation of directional motility (Wittmann et al., 2003). It is currently unclear to what extent other Pak substrates are involved in the control of microtubule polarization and MTOC reorientation during migration and wound healing. Recent studies in fibroblasts and astrocytes have indicated that polarized microtubule stabilization is initiated by localized activation of Rac and Cdc42 at the leading edge but does not involve Pak. Instead, upon activation, Cdc42 mediates the reorientation of the MTOC through a pathway that involves the Cdc42 effector PAR6, which forms a complex with PAR3 and the atypical PKC-zeta (Cau and Hall, 2005; Etienne-Manneville and Hall, 2001). Formation of polarized actin-based protrusions in response to active Cdc42 on the other hand, was reported to be regulated independently of PAR3/PAR6/PKC-zeta, through a pathway that did depend on a Pak1-dependent recruitment of bPIX at the leading edge (Cau and Hall, 2005). The Pak-bPIX complex may then facilitate downstream activation of Rac and/or cdc42, which in turn may control spatial actin reorganization through downstream effectors, which may include Pak. It is possible that these data are cell type-dependent, considering the diverse functions of Pak in regulating microtubule stabilization in other cells. Also, as will discussed below, Pak may be involved in the polarization of microtubules through its interaction with bPIX. Furthermore, Pak can directly regulate atypical PKC-zeta in prostate carcinoma cells where PKC-zeta constitutively associates with Pak1 and is phosphorylated in a Pak1-dependent manner (Even-Faitelson and Ravid, 2006). Thus, these findings may suggest that PKC-zeta and Pak can integrate Cdc42 signaling to microtubules and filaments respectively. Finally, it is relevant to note that all studies discussed above were done in either non-epithelial cells or in epithelial cells that lacked apico-basolateral polarity. In such cells microtubules radiate out from a perinuclear MTOC, which is often, but not always, oriented towards the leading edge (Salaycik et al., 2005). In contrast, in polarized epithelial cells (i.e., cells with apicalbasolateral polarization), microtubules do not radiate from a centrosomal MTOC. Rather, they are organized in parallel arrays, in which the minus ends are associated with the apical membrane, and the plus ends extend towards the basal surface (Fukata et al., 2003; Luders and Stearns, 2007). This organization must undergo significant changes upon wounding since epithelial cells at wound edges exhibit a radial organization similar to that observed in non-polarized cells. How the transitions in microtubule organization during wound healing are regulated is unclear, and it would be

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important to know if the downstream effectors of Pak are involved in this process. In that respect, it is interesting to note that MARK2/Par-1 induces a change from a parallel organization to an organization in which microtubules nucleate from a single MTOC in the MDCK cell line (Cohen et al., 2004). In neuronal cells, MARK2/Par-1 destabilizes microtubules by phosphorylating tau, causing its dissociation from microtubules (Nishimura et al., 2004). Pak5 binds MARK2/Par-1 and when both molecules are overexpressed in CHO cells, Pak5 counteracts the function of MARK2/Par-1, thereby stabilizing microtubules. Though it is tempting to speculate that Pak may be involved in regulating parallel or radial microtubule organizations, the kidney-derived MDCK are unlikely to express Pak5 (Dan et al., 2002), and it remains to be established if other Pak forms are involved in this process. 6.1.1.3. The Pak-PIX-GIT complex in cell polarization PIX-GIT
containing complexes may regulate cell polarization by recruiting other cell polarity protein complexes. Three major protein complexes that localize at apical cell junctions and control epithelial polarization were initially identified in Drosophila and C. elegans. The general function and key components of these complexes are highly conserved in different vertebrate and invertebrate organisms. The Par3/Par6/aPKC complex is recruited to cadherin-based junctions and appears to initiate formation of the apical membrane. Maintenance of ‘‘apical identity’’ of the apical membrane is mediated by the Crb/Stardust complex, which antagonizes the function of the Lgl/Dlg/Scrib complex. This Lgl/Dlg/Scrib complex is proposed to generate and maintain basolateral identity by counteracting Par3/Par6/ aPKC function (Nelson, 2003). Although these three complexes have been mostly implicated in apical-basolateral polarization, it has become increasingly clear that they also function in other types of cell polarization. For instance, the Par3/Par6/aPKC and the Lgl/Dlg/Scrib complexes engage in bidirectional signaling with Rho GTPases and have been implicated in regulation of cell polarization during migration (Humbert et al., 2006). Mass spectrometry analysis of proteins that co-immunoprecipitate with Scrib in mammary epithelial cells identified bPIX (and associated GIT1) as a main binding partner of Scrib (Audebert et al., 2004). Recent studies indicate that Scrib plays a crucial role in directional motility and epithelial wound healing by a mechanism that depends on bPIX. Previously it was found that loss of the Drosophila forms of Scrib and Dlg results in defects of dorsal closure (Bilder et al., 2000), while mice that carry Scrib mutations exhibit defects in embryonic fusion events such as eyelid- and neural tube closure (Murdoch et al., 2003; Zarbalis et al., 2004). These observed effects on sheet migration suggest a potential role of these proteins in wound healing. Indeed, expression of mutant mammalian Scrib was recently shown to inhibit epidermal wound healing in an in vivo mouse model (Dow et al., 2007). It appears that deregulation of migratory polarity

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underlies these defects. In scrape wound healing assays, bPIX and Scrib are recruited to the leading edge in mammary epithelial cells (Dow et al., 2007) and astrocytes (Osmani et al., 2006). Knockdown of Scrib expression significantly interferes with migratory polarization of these cells; it blocks recruitment of bPIX, cdc42 (Osmani et al., 2006) and Rac (Dow et al., 2007) to lamellipodia and results in a loss of polarized actin and microtubule organization and directional motility. In astrocytes, it also inhibits wounding-induced activation of cdc42 (Osmani et al., 2006). Interestingly, whereas knockdown of Scrib abolishes sheet migration in mammary epithelial cells, it does not affect general rates of cell motility when the cells are subconfluent (Dow et al., 2007), suggesting functional cross-talk with cell-cell adhesions. Subsequent experiments suggested that the phenotype of Scrib knockdown cells depends on the interaction of Scrib with bPIX. This conclusion was based on findings that knockdown of bPIX or expression of bPIX mutants that lack the Scrib binding motif or the DH domain (required for GEF function) phenocopied the Scrib knockdown phenotype (Osmani et al., 2006). Taken together, these data are consistent with a model in which Scrib and bPIX-GIT1 complexes recruit and regulate the activation of Rac and cdc42 at the leading edge of migrating cells. The active Rac and cdc42 in turn, may then induce cytoskeletal rearrangements and lamellipodia formation by activating effector proteins, including, quite likely, Pak kinases. It must be noted that some of Scrib’s effects on cell migration may be context or cell-type dependent and/or appear to mediated by alternative mechanisms. For instance, while knockdown of Scrib results in a loss of directional migration in MDCK cells, it increases overall motility in these cells (Qin et al., 2005). In these cells, however, increased motility appeared to be caused by a destabilization of adherens junctions, which occurs independently of bPIX function. Thus, it is possible that Scrib-containing complexes with different compositions and/or distinct intracellular localizations have different and perhaps even opposite functions in directional migration. Such distinct functions have already been demonstrated for GIT1-containing complexes, which, depending on intracellular localization and molecular composition either promote or inhibit lamellipodia formation. Thus, while Pak1-PIX-GIT1
containing complexes, in association with paxillin, stimulate motility and protrusion of lamellipodia, likely by promoting activation of Rac at the leading edge of the cell, GIT1 inhibits Rac activation at the trailing edge when it is associated with a4 integrin through paxillin (Nishiya et al., 2005). As a consequence, GIT1 promotes cell polarization by mediating opposite effects at the leading and trailing edge of the cell. On a related note, even though the different GIT family members appear to interact equally well with paxillin and PIX, they may have distinct roles, as it was recently shown that GIT2, but not GIT1 represses motility in nontransformed mammary epithelial cells (Frank et al., 2006).

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6.1.2. Stabilization of cell protrusions and the dynamic regulation of focal contacts To promote cell migration, protrusions at the leading edge must be stabilized and anchored to the underlying extracellular matrix. The main proteins that mediate this process are integrins; heterodimeric matrix receptors that bind to different components of the extracellular matrix. At the inside of the cell, integrins link to the cytoskeleton. The connection of the extracellular matrix to the actin cytoskeleton allows the cells to exert traction forces, which are required to pull the cell forward. In addition, integrins are important signaling molecules that transmit intracellular signals upon binding to the extracellular matrix (‘‘outside in signaling’’), while their function is also being regulated by intracellular signals (‘‘inside out signaling’’). Key regulators of bidirectional integrin signaling are Rho GTPases (Schmitz et al., 2000; Wozniak et al., 2004; Yu et al., 2005). The formation and regulation of integrin-based adhesion sites is not completely understood. Upon adhesion, integrins are activated and cluster in focal complexes, in which many different multidomain proteins, including paxillin, interact and ultimately link to the actin cytoskeleton. Different types of integrin clusters exist: Focal complexes are relatively small, are found at the cell periphery, form by a mechanism that depends on Rac activity and exhibit high turnover rates (Ballestrem et al., 2001; Hall, 1998; Zaidel-Bar et al., 2003). Focal complexes are also thought to be the precursors of focal adhesions (Hall, 1998), which are larger, more stationary complexes that generally localize more distally and form in Rho-dependent manner. As differences between the two different integrin-based contact sites are not always obvious, I will use the term focal contact to refer to either complex. Focal contacts need to turn over to allow the cell to move forward and highly migratory cells tend to have many smaller focal contacts that turn over rapidly. Numerous studies have shown that Pak1-PIX-GIT
containing complexes are targeted to focal contacts and have implicated the complex in the dynamic regulation of these adhesion sites. The precise targeting mechanisms and functions of these proteins at focal contacts is still not entirely clear as apparently conflicting evidence have been reported. As mentioned earlier, Paks are activated in response to integrin-mediated adhesion (Chaudhary et al., 2000; del Pozo et al., 2000; Howe, 2001; Orr et al., 2007; Price et al., 1998; Zhou and Kramer, 2005). Activated Pak1 mutants localize to focal contacts (Kiosses et al., 1999; Sells et al., 1997; Stofega et al., 2004) and endogenous Pak1 is recruited to focal contacts upon its activation by PDGF or VEGF (Dharmawardhane et al., 1997; Sells et al., 2000; Stoletov et al., 2001) or following expression of active Rac1 or cdc42 mutants (Manser et al., 1997), although this latter study did not find that active Pak mutants localized to focal contacts (Manser et al., 1997). Conversely, many studies demonstrated that inactive Pak accumulates in focal adhesions. Thus, inhibition of Pak function by expression of the

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Pak-autoinhibitory domain or by expression of kinase-dead Pak results in recruitment of Pak in focal adhesions (Kiosses et al., 1999; Royal et al., 2000; Zegers et al., 2003a; Zhao et al., 2000a). How Pak is initially targeted to focal contacts is also matter of some debate. Pak can be recruited to focal contacts by both Nck (Kiosses et al., 1999; Zhao et al., 2000a) and PIX (Manser et al., 1998; Zegers et al., 2003a). PIX-dependent recruitment is likely mediated by sequential interactions of paxillin, GIT family proteins and PIX, in which GIT serves as a linker between paxillin and PIX (Brown et al., 2002, 2005; Manabe Ri et al., 2002; Turner et al., 1999; Zhao et al., 2000b). The precise function of the complex at focal contacts is still under investigation, but appears to be multifaceted. Overexpression of active Pak mutants leads to disassembly of focal contacts in some systems (Manser et al., 1997; Sells et al., 1997), but was not observed in endothelial or epithelial cells (Kiosses et al., 1999; Zegers et al., 2003a). In fact, Pak activity and recruitment to focal contacts is required for formation of these structures in VEGF-stimulated endothelial cells (Stoletov et al., 2001). Conversely, an increase of the number of large focal adhesions upon inhibition of Pak function has been widely reported in many cell types (Kiosses et al., 1999; Royal et al., 2000; Zegers et al., 2003a; Zhao et al., 2000a) (Fig. 6.2). In summary, although most studies are consistent with the hypothesis that active Pak promotes focal contact turnover, there is no straightforward correlation between the recruitment of Pak and its binding partners PIX and GIT to focal contacts, the formation of these structures, and the effect on cell motility. This may not be surprising, as motility depends on a tightly regulated balance of focal contact formation and breakdown. The functional effects of a disruption of this balance will likely depend on the spatial and molecular context of the complex. In that respect, it is relevant to note that the Pak-PIX-GIT complex is subject to different intermolecular interactions and posttranslational modifications. For instance, phosphorylation of Pak1 on Ser21 by Akt decreases the interaction of Pak1 with Nck, which leads to the release of Pak1 from focal contacts and an increase in cell motility (Zhou et al., 2003). Autophosphorylation of Pak1 also decreases its affinity for PIX and Nck binding (Manser et al., 1997; Zhao et al., 2000a), and induces disassembly of focal contacts and retraction of peripheral membrane, which suggests a potential inhibitory effect on migration. Finally, Pak1 phosphorylates paxillin on Ser273 (Nayal et al., 2006) and Ser709 (Webb et al., 2006), which increases the affinity of paxillin for GIT and promotes cell protrusion (Webb et al., 2006). Pak-mediated phosphorylation of paxillin also induces formation of small highly dynamic focal contacts that promote cell motility by a mechanism that depends on Pak-PIX and PIX-GIT interactions (Nayal et al., 2006). Taken together, the data appear to be consistent with the hypothesis that Pak-PIX-GIT complexes may promote formation of focal complexes by a

Pak / Vinculin

Pak

Wild type Pak1

Vinculin

Pak / Vinculin

Pak

Vinculin

Kinase-dead Pak1

Figure 6.2 Dominant-negative Pak1 accumulates in focal contacts in scrape-wounded epithelial cells. Image represents MDCK cells, which express wild type or a dominant-negative (Pak1-K299R, kinase-dead) under control of a tetracycline-regulatable promoter (Zegers et al., 2003a). Monolayers of MDCK cells expressing these HA-tagged forms of Pak1were scrape wounded.The next day, cells were fixed and stained using antibodies against the HA-tag and the focal contact marker vinculin. Note that wild type Pak1 localized to focal contacts to a limited extent, and localization is restricted to very peripheral focal contacts. In contrast, kinase-dead Pak1 accumulates at focal contacts and is found both peripheral and more distally within lamellipodia.

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mechanism that relies on the local, PIX-mediated activation of Rac at the leading edge. This mechanism may not require Pak activation, or perhaps relies on a partial activation, but could be mainly mediated by recruiting PIX to the leading edge. Full activation, mediated by the resulting local Rac activation, may subsequently lead to full Pak activation, which results in a release of Pak from focal contacts through its diminished affinity for PIX and Nck. Furthermore, activation of Pak will induce the degradation of focal contacts. How this latter process is regulated is still an open question. It is possible that degradation occurs through proteolytic cleavage by calpain, which is recruited to focal contacts by interacting with aPIX (Rosenberger et al., 2005). Also, as will discussed below, Pak can regulate myosin activity, which may be involved in adhesion disassembly (Crowley and Horwitz, 1995). Finally, microtubules have recently also emerged as regulators of focal adhesions (Palazzo and Gundersen, 2002). Hence, Pakmediated focal adhesion disassembly may also be regulated indirectly by through Pak’s diverse effects on microtubules. 6.1.3. Generation of traction forces The traction forces required to move cells forward are generated by the interaction of the non-muscle myosin II with actin filaments. Crucial to actin-myosin contractility is the phosphorylation of myosin II, which regulates both its association with actin and its motor activity. The phosphorylation of the regulatory myosin light chain (MLC) is controlled by myosin light chain kinase (MLCK). This kinase needs to be non-phosphorylated to be active, and phosphorylation of MLCK negatively inhibits the activity of the kinase. The role of Pak in the regulation of actin-myosin contractility has been somewhat controversial. One study provided evidence that Pak phosphorylates MLCK, thereby promoting dephosphorylation of MLC, thus potentially decreasing actin-myosin contractility (Sanders et al., 1999). Others however, showed that active Pak1 mutants lead to phosphorylation of MLC (Kiosses et al., 1999; Sells et al., 1999), and that MLC is a direct substrate of Pak1 (Bokoch, 2003). In addition to MLC phosphorylation, the myosin heavy chain (MHC) can be phosphorylated as well. The function of MHC phosphorylation in actinmyosin contractility is somewhat unclear, but at least for non-muscle myosin II-B, it may promote myosin filament assembly (Even-Faitelson and Ravid, 2006; van Leeuwen et al., 1999). Bradykinin-induced Rac activation results in MHC phosphorylation in PC12 cells, which is inhibited by dominantnegative Pak1. However, as active Pak1 mutants does not increase MHC phosphorylation, MHC may not be a direct substrate of Pak1 (van Leeuwen et al., 1999). In that respect, it was recently shown that Pak can mediate MHC phosphorylation through atypical PKC-zeta in the metastatic prostate carcinoma cell line TSU-pr1. In these cells, EGF stimulation drives the formation of protein complex containing Pak1, the atypical PKC-zeta, and the MHC of

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myosin II-B. Pak1 induces phosphorylation of PKC-zeta, and, upon stimulation with EGF, PKC-zeta phosphorylates myosin II-B directly, leading to slower filament assembly of myosin II-B (Even-Faitelson and Ravid, 2006). Since PKC-zeta has a clear role in directional cell migration (Cau and Hall, 2005; Etienne-Manneville and Hall, 2001), it would be of considerable interest to know how this interaction is regulated.

6.2. Regulation of cell proliferation by Pak, PIX and GIT 6.2.1. Positive regulation of mitogenic signaling Although the repair of minor wounds and other epithelial injuries relies on epithelial sheet migration and can occur independently of cell proliferation, healing of larger wounds is accompanied and critically depends on proliferation to replace lost cells (Mammen and Matthews, 2003; Martin, 1997; Singer and Clark, 1999). There is increasing evidence that Paks, in addition to their well-established roles in migration, play significant roles in the regulation of cell proliferation. As discussed previously, Paks are activated downstream of several mitogenic growth factors and interact with the EGF and PDGF receptors through adaptor proteins. Furthermore, Paks play important roles in growth factor-induced effects on cell migration. The canonical Raf!MEK!ERK pathway is well known for regulating cell proliferation in response to adhesion or growth factors, and appears to be regulated by Pak on several different levels. Pak is required for ERK activation and transformation by Ras (Tang et al., 1997), and both Raf-1 and MEK1 (King et al., 1998; Li et al., 2001; Slack-Davis et al., 2003; Sun et al., 2000) are believed to be direct substrates of Pak. ERK is activated upon scrape wounding in many cell types, and at least in some epithelial cells, the wounding-induced activation depends on an upstream activation of Src (Matsubayashi et al., 2004). Several groups showed that activated ERK localizes to focal contacts in fibroblasts and poorly differentiated epithelial cells (Fincham et al., 2000; Slack-Davis et al., 2003; Yin et al., 2005). Recently, it has become evident that Pak-PIX-GIT
containing complexes play a crucial role in recruiting and activating ERK at these sites. For instance, cell matrix-adhesion sequentially activates FAK, Src and Pak1. Active Pak1, in turn, phosphorylates MEK1 at S298, which primes MEK for its activation and allows for subsequent MEK and ERK activation (summary: adhesion!FAK!Src!Pak1!p-S298-MEK1!p-MEK1(S218/ S222, active)!p-ERK (T202,Y204, active) (Eblen et al., 2004; Slack-Davis et al., 2003). Moreover, GIT1, when phosphorylated by Src, is required for recruitment of ERK to focal adhesions and can bind both MEK1 and ERK2. Then, acting as a scaffold for MEK and ERK, GIT1 mediates sustained ERK activation at focal adhesions (Yin et al., 2004, 2005). Finally, Pak1 interacts with the MEK-ERK scaffold MP1, and this interaction is required for Pak1-mediated ERK activation (Pullikuth et al., 2005). The signaling pathway at focal contacts mentioned above and summarized

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here for simplicity as Src! GIT1/Pak1!MEK!ERK, is under control of several negative feedback steps. Specifically, ERK phosphorylates MEK on T292, which blocks the ability of Pak to activate MEK, and thus subsequent ERK activation. ERK also phosphorylates Pak1 on T212, which attenuates ERK signaling as well (Sundberg-Smith et al., 2005). On the other hand, ERK-dependent phosphorylation of bPIX and activation of Pak2 was also reported (Shin et al., 2002). Interestingly, available studies suggest that this type of Pak1-mediated ERK activation is particularly important in signaling downstream of cell-matrix adhesion. Although the same pathway has been reported to be activated in response to growth factors, the response seems specific for some, but not all growth factors that are known to activate ERK and may therefore be of lesser importance. Also, even though several groups have shown that Pak1 phosphorylates Raf-1 on S338, the most recent studies show that Raf is, at least in some systems, not required for Pak-mediated ERK activation (Beeser et al., 2005 and discussion therein). Mitogenic signaling by growth factors is under tight control. One of the negative regulators of EGF signaling is ubiquitin ligase Cbl, which binds and mono-ubiquitinates the activated EGF receptor (EGFR), thereby targeting it for endocytosis and degradation in the lysosome (Dikic, 2003). Recently, a series of studies showed that bPIX inhibits Cbl-mediated EGFR downregulation, thereby prolonging EGF signaling. EGF stimulation of cells induces a Src and Fak-dependent phosphorylation of bPIX on Tyr442, which stimulates its GEF activity toward Cdc42 (Feng et al., 2006), likely by releasing autoinhibitory constraints (Feng et al., 2002; Peterson and Chernoff, 2006). Upon phosphorylation, bPIX forms a complex with both activated Cdc42 and Cbl, thereby sequestering Cbl away from the EGFR, leading to an inhibition of EGFR endocytosis and degradation. As a result, EGFR-coupled signaling, such as the activation of ERK is sustained (Schmidt et al., 2006). Furthermore, these studies have provided evidence for an essential role of bPIX for cellular transformation and deregulated cell growth induced by either v-Src or Cdc42 (Feng et al., 2006; Wu et al., 2003). Though the effects on epithelial wound healing was not specifically addressed in these studies, others showed that either silencing of Cdc42 or overexpression of a Cbl mutant that cannot bind bPIX inhibits cell proliferation and wound closure in scrape wound healing assays in EGFR-overexpressing breast cancer cells (Hirsch et al., 2006). Based on these observations, a role for bPIX in EGF-stimulated wound healing seems likely. 6.2.2. Regulation of the cell cycle Many of the extracellular signals that regulate cell division are interpreted during the G1 phase of the cell cycle, which precedes DNA replication (S phase). Cyclin-dependent kinases are the major kinases that drive the cell cycle and are activated by cyclins. D-type cyclins, such as cyclin D1, control cyclin-dependent kinase 4 and are major regulators of cell cycle progression

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through the G1 phase. The expression of cyclin D1 is controlled by both mitogens and signaling pathways downstream of integrins. Studies with constitutive and dominant-negative Rho GTPases have shown that Rho GTPases are essential for progression through G1 (Olson et al., 1995). Rho GTPases appear to regulate the cell cycle through several different mechanism, which are not yet completely understood (Coleman et al., 2004). Several studies have indicated that Paks can induce cyclin D1. Pak is required for Ras-induced cyclin D1 expression (Nheu et al., 2004) and in normal and transformed epithelial cell lines, overexpression of wild-type and constitutively active Pak1 stimulates cyclin D1 promoter activity as measured by in vitro reporter assays. Furthermore, overexpression of active Pak induces an increase of cyclin D1 protein levels and accumulation of cyclin D1 in the nucleus (Balasenthil et al., 2004), whereas inhibition of Pak by siRNA-mediated knockdown, expression of the Pak1 autoinhibitory domain, or by a pharmacological inhibitor decreased cyclin D1 expression. Interestingly, cyclin D1 expression is also inhibited by a peptide that inhibits Pak-PIX interaction (Nheu et al., 2004), which may suggest a role for PIX in this process. On the other hand, another study demonstrated an inhibitory effect of Pak1 on cyclin D1 expression and cell cycle progression. The mechanism underlying this inhibition, which can be induced by overexpression of either wild-type or kinase-dead Pak1, is still unclear but is mediated by the domain that comprises the Pak-autoinhibitory domain (Thullberg et al., 2007). Surprisingly, the mechanism does not involve an inhibition of Pak kinase activity, since the same study showed that an inactive autoinhibitory domain elicits the same effect and that active Pak cannot rescue the defect. This suggests that inhibition of proliferation by the Pak autoinhibitory domain is mediated by a yet unidentified function of this domain in the control of cell proliferation. Pak phosphorylation on Thr212 is regulated in a cell cycle-dependent manner and markedly increases in mitotic cells (Banerjee et al., 2002; Li et al., 2002; Thiel et al., 2002). While it is unclear how this phosphorylation affects Pak activity, it is suggested to promote its association with centrosomes, as a Thr212-phosphomimic Pak peptide is targeted to centrosomes (Banerjee et al., 2002). Active endogenous Pak also localizes to centrosomes during metaphase where it phosphorylates the centrosomal Aurora A kinase (Li et al., 2002; Zhao et al., 2005). Finally, overexpression of an active Pak1 mutant interferes with normal spindle formation (Vadlamudi et al., 2000). Taken together, these data point to a regulatory role of Pak in spindle formation in mitotic cells. 6.2.3. Contact inhibition and Pak signaling at cell-cell contacts The roles of Pak in wound healing I have discussed in this review suggest that Pak functions primarily in the ‘‘start phase’’ of wound healing, i.e., the promotion of cell migration and proliferation upon initial wounding.

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However, in order to properly close a wound, this initial phase is temporary, and is followed by a ‘‘stop phase’’ ( Jacinto et al., 2001), in which cell migration and proliferation is inhibited and the epithelial cells regain their apico-basolateral polarization. Contact inhibition, the ability of cells to cease cell migration and proliferation upon the establishment of cell-cell contacts, is thought to be in important factor in the final stage of wound healing ( Jacinto et al., 2001). However, even though it has been known for over 50 years that cells can undergo contact inhibition, the mechanism underlying this process are still poorly understood (Abercrombie, 1979; Middleton, 1972; Stoker and Rubin, 1967). Most likely, signaling pathways that induce contact inhibition in epithelial cells are initiated by the E-cadherin-based adherens junctions, which form when adjacent cells come into contact with each other (Fagotto and Gumbiner, 1996). Rho GTPases are well known regulators of cell-cell junctional integrity and involved in many signaling pathways that are activated in response to cell-cell adhesion ( Jaffer and Chernoff, 2004). Thus, Rho GTPases may be important signaling intermediates in the regulation of contact inhibition. Several studies have indicated that Pak-PIX-GIT complexes are involved in adherens junction-related signaling. In epithelial and endothelial cell, Pak, PIX and GIT can be recruited to cell-cell contacts (Audebert et al., 2004; Orr et al., 2007; Stockton et al., 2007; Zegers et al., 2003a) but the precise functions of the complex at cell-cell contacts is still unclear and may be celltype dependent. In endothelial cells, shear stress induces recruitment of endogenous active Pak to cell junctions, where it promotes vascular permeability (Orr et al., 2007; Stockton et al., 2007). Others however proposed that activation of Pak and junctional recruitment of Pak, PIX and GIT in response to oxidized phospholipids enhances the barrier function of endothelial cells (Birukova et al., 2007a,b). Pak may negatively regulate contact inhibition by inhibiting the tumor suppressor protein Merlin. Merlin has high homology to members of the ERM (Ezrin-radixin-moesin) family of actin linker proteins, which link the cytoskeleton to the plasma membrane. When active, Merlin is in a closed conformation and acts as a growth suppressor by inducing contact inhibition through mechanisms that are not well understood (Okada et al., 2007). Pak phosphorylates Merlin on Ser518, which inactivates the protein and enables cell proliferation (Kissil et al., 2002; Xiao et al., 2002, 2005). Interestingly, Pak and Merlin engage in bidirectional signaling, as Merlin also inhibit Pak function and inhibits the recruitment and activation of Rac and Pak at the plasma membrane, which may be part of the mechanism by which Merlin regulates contact inhibition (Kissil et al., 2003; Okada et al., 2005; Shaw et al., 2001). Indeed, in endothelial cells, Pak1 activity reduces when cells reach confluency, and expression of an active membrane-targeted form of Pak1 is sufficient to release cells from contact inhibition of growth (Okada et al., 2005).

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On the other hand, Pak may also promote contact inhibition, since localization of Pak-PIX-GIT complexes at cell-cell contacts is required for the establishment of contact inhibition upon wound closure in epithelial cells (Zegers et al., 2003a). Using a model system of scrape-wounded MDCK cells, it was shown that the complex localizes to focal contacts in cells at wound edges, but is dramatically retargeted to areas of cell-cell contacts upon establishment of cell-cell contacts and wound closure. Inhibition of endogenous Pak1 blocks the ability of cells to undergo contact inhibition of proliferation by causing an accumulation of Pak1 and bPIX at focal contacts, which results in an inhibition of their recruitment to lateral membranes. Interestingly, although these cells are unable to undergo contact inhibition of proliferation, they still form adherens junctions and are able to polarize (Zegers et al., 2003a). This suggests that Pak-PIX-GIT complex may act as a sensor of extracellular environment, and acts as a signaling intermediate downstream of integrin in wounded epithelial cells, but downstream of E-cadherin upon wound closure. Such a dual role would be consistent with the apparent opposite roles of Rac, which is both necessary for cell migration (see Section 3.2) and for the establishment of adherens junctions (Van Aelst and Symons, 2002).

7. Concluding Remarks While it is obvious that Paks play important roles in the control of cellular behaviors that accompany and drive epithelial wound healing and sheet migration, many questions remain. An important outstanding issue is the spatiotemporal regulation of Pak function. This type of regulation likely entails both its spatiotemporal activation as well as the formation of distinct Pak-containing protein complexes. Clearly, many processes during wound healing need to be locally controlled, and a process of cell motility and cell polarization often depends on opposite behaviors at the leading versus the trailing edge of cells. As is obvious from the reviewed literature, a common theme of wounding-associated roles of Pak that are reviewed here are the often opposing effects that Pak appears to have in different cell types or under slightly different experimental conditions. One possible explanation of such findings is that most studies rely on approaches that modify Pak functions uniformly within the cell, and, it that light it may not be surprising that, depending on the context, opposite results can be obtained. A related question is how Pak is inactivated. While our knowledge of signals upstream of Pak is fairly extensive, our understanding of Pak inhibitors is scarce. Although several Pak inhibitory proteins, such as for instance the Pak phosphatase POPX, have been identified, insight into the regulation of these inhibitors is almost entirely lacking. Studies that specifically address

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Pak’s activation and deactivation in space and time, will undoubtedly lead to significant new insights in Pak biology and our understanding of epithelial wound healing.

ACKNOWLEDGMENTS I thank Martin ter Beest for critical comments on the manuscript. The work in my laboratory is funded by the NIH (GM076363) and the Concern Foundation.

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Tu, H., and Wigler, M. (1999). Genetic evidence for Pak1 autoinhibition and its release by Cdc42. Mol. Cell Biol. 19, 602–611. Turner, C. E., Brown, M. C., Perrotta, J. A., Riedy, M. C., Nikolopoulos, S. N., McDonald, A. R., Bagrodia, S., Thomas, S., and Leventhal, P. S. (1999). Paxillin LD4 motif binds Pak and PIX through a novel 95-kDa ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling. J. Cell Biol. 145, 851–863. Vadlamudi, R. K., Adam, L., Wang, R. A., Mandal, M., Nguyen, D., Sahin, A., Chernoff, J., Hung, M. C., and Kumar, R. (2000). Regulatable expression of p21-activated kinase-1 promotes anchorage-independent growth and abnormal organization of mitotic spindles in human epithelial breast cancer cells. J. Biol. Chem. 275, 36238–36244. Vadlamudi, R. K., Barnes, C. J., Rayala, S., Li, F., Balasenthil, S., Marcus, S., Goodson, H. V., Sahin, A. A., and Kumar, R. (2005). p21-activated kinase 1 regulates microtubule dynamics by phosphorylating tubulin cofactor B. Mol. Cell Biol. 25, 3726–3736. Vadlamudi, R. K., Li, F., Barnes, C. J., Bagheri-Yarmand, R., and Kumar, R. (2004). p41Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate. EMBO Rep. 5, 154–160. Van Aelst, L., and Symons, M. (2002). Role of Rho family GTPases in epithelial morphogenesis. Genes. Dev. 16, 1032–1054. van Leeuwen, F. N., van Delft, S., Kain, H. E., van der Kammen, R. A., and Collard, J. G. (1999). Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading. Nat. Cell Biol. 1, 242–248. Walter, B. N., Huang, Z., Jakobi, R., Tuazon, P. T., Alnemri, E. S., Litwack, G., and Traugh, J. A. (1998). Cleavage and activation of p21-activated protein kinase gamma-Pak by CPP32 (caspase 3). Effects of autophosphorylation on activity. J. Biol. Chem. 273, 28733–28739. Wang, J., Frost, J. A., Cobb, M. H., and Ross, E. M. (1999). Reciprocal signaling between heterotrimeric G proteins and the p21-stimulated protein kinase. J. Biol. Chem. 274, 31641–31647. Watanabe, T., Noritake, J., and Kaibuchi, K. (2005). Regulation of microtubules in cell migration. Trends Cell Biol. 15, 76–83. Webb, B. A., Eves, R., Crawley, S. W., Zhou, S., Cote, G. P., and Mak, A. S. (2005). PAK1 induces podosome formation in A7r5 vascular smooth muscle cells in a Pak-interacting exchange factor-dependent manner. Am. J. Physiol. Cell Physiol. 289, C898–C907. Webb, D. J., Kovalenko, M., Whitmore, L., and Horwitz, A. F. (2006). Phosphorylation of serine 709 in GIT1 regulates protrusive activity in cells. Biochem. Biophys. Res. Commun. 346, 1284–1288. Weisz Hubsman, M., Volinsky, N., Manser, E., Yablonski, D., and Aronheim, A. (2007). Autophosphorylation-dependent degradation of Pak1, triggered by the Rho-family GTPase, Chp. Biochem. J. 404, 487–497. Wittmann, T., Bokoch, G. M., and Waterman-Storer, C. M. (2003). Regulation of leading edge microtubule and actin dynamics downstream of Rac1. J. Cell Biol. 161, 845–851. Wittmann, T., Bokoch, G. M., and Waterman-Storer, C. M. (2004). Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1. J. Biol. Chem. 279, 6196–6203. Woolner, S., Jacinto, A., and Martin, P. (2005). The small GTPase Rac plays multiple roles in epithelial sheet fusion–dynamic studies of Drosophila dorsal closure. Dev. Biol. 282, 163–173. Wozniak, M. A., Modzelewska, K., Kwong, L., and Keely, P. J. (2004). Focal adhesion regulation of cell behavior. Biochim. Biophys. Acta 1692, 103–119. Wu, W. J., Tu, S., and Cerione, R. A. (2003). Activated Cdc42 sequesters c-Cbl and prevents EGF receptor degradation. Cell 114, 715–725.

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Xia, C., Ma, W., Stafford, L. J., Marcus, S., Xiong, W. C., and Liu, M. (2001). Regulation of the p21-activated kinase (Pak) by a human Gbeta-like WD-repeat protein, hPIP1. Proc Natl. Acad. Sci. USA 98, 6174–6179. Xiao, G. H., Beeser, A., Chernoff, J., and Testa, J. R. (2002). p21-activated kinase links Rac/Cdc42 signaling to merlin. J. Biol. Chem. 277, 883–886. Xiao, G. H., Gallagher, R., Shetler, J., Skele, K., Altomare, D. A., Pestell, R. G., Jhanwar, S., and Testa, J. R. (2005). The NF2 tumor suppressor gene product, merlin, inhibits cell proliferation and cell cycle progression by repressing cyclin D1 expression. Mol. Cell Biol. 25, 2384–2394. Yang, J., Mani, S. A., Donaher, J. L., Ramaswamy, S., Itzykson, R. A., Come, C., Savagner, P., Gitelman, I., Richardson, A., and Weinberg, R. A. (2004). Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117, 927–939. Yin, G., Haendeler, J., Yan, C., and Berk, B. C. (2004). GIT1 functions as a scaffold for MEK1-extracellular signal-regulated kinase 1 and 2 activation by angiotensin II and epidermal growth factor. Mol. Cell Biol. 24, 875–885. Yin, G., Zheng, Q., Yan, C., and Berk, B. C. (2005). GIT1 is a scaffold for ERK1/ 2 activation in focal adhesions. J. Biol. Chem. 280, 27705–27712. Yoshii, S., Tanaka, M., Otsuki, Y., Wang, D. Y., Guo, R. J., Zhu, Y., Takeda, R., Hanai, H., Kaneko, E., and Sugimura, H. (1999). alphaPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase. Oncogene 18, 5680–5690. Yu, W., Datta, A., Leroy, P., O’Brien L, E., Mak, G., Jou, T. S., Matlin, K. S., Mostov, K. E., and Zegers, M. M. (2005). b1-Integrin orients epithelial polarity via rac1 and laminin. Mol. Biol. Cell 16, 433–445. Zaidel-Bar, R., Ballestrem, C., Kam, Z., and Geiger, B. (2003). Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. J. Cell. Sci. 116, 4605–4613. Zarbalis, K., May, S. R., Shen, Y., Ekker, M., Rubenstein, J. L., and Peterson, A. S. (2004). A focused and efficient genetic screening strategy in the mouse: identification of mutations that disrupt cortical development. PLoS Biol. 2, E219. Zegers, M. M., Forget, M. A., Chernoff, J., Mostov, K. E., ter Beest, M. B., and Hansen, S. H. (2003a). Pak1 and PIX regulate contact inhibition during epithelial wound healing. EMBO J. 22, 4155–4165. Zegers, M. M., O’Brien, L. E., Yu, W., Datta, A., and Mostov, K. E. (2003b). Epithelial polarity and tubulogenesis in vitro. Trends Cell Biol. 13, 169–176. Zeisberg, M., Hanai, J., Sugimoto, H., Mammoto, T., Charytan, D., Strutz, F., and Kalluri, R. (2003). BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat. Med. 9, 964–968. Zenke, F. T., King, C. C., Bohl, B. P., and Bokoch, G. M. (1999). Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity. J. Biol. Chem. 274, 32565–32573. Zenke, F. T., Krendel, M., DerMardirossian, C., King, C. C., Bohl, B. P., and Bokoch, G. M. (2004). p21-activated kinase 1 phosphorylates and regulates 14-3-3 binding to GEF-H1, a microtubule-localized Rho exchange factor. J. Biol. Chem. 279, 18392–183400. Zhao, Z. S., Lim, J. P., Ng, Y. W., Lim, L., and Manser, E. (2005). The GIT-associated kinase Pak targets to the centrosome and regulates Aurora-A. Mol. Cell 20, 237–249. Zhao, Z. S., and Manser, E. (2005). Pak and other Rho-associated kinases—Effectors with surprisingly diverse mechanisms of regulation. Biochem. J. 386, 201–214. Zhao, Z. S., Manser, E., Chen, X. Q., Chong, C., Leung, T., and Lim, L. (1998). A conserved negative regulatory region in alphaPAK: inhibition of Pak kinases reveals their morphological roles downstream of Cdc42 and Rac1. Mol. Cell Biol. 18, 2153–2163.

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C H A P T E R

S E V E N

Biology and Biophysics of the Nuclear Pore Complex and Its Components Roderick Y. H. Lim,* Katharine S. Ullman,† and Birthe Fahrenkrog* Contents 1. Introduction 2. Nuclear Pore Complex Structure 2.1. Overall nuclear pore complex architecture 2.2. The nuclear pore complex at atomic level 2.3. Nuclear pore complex density and distribution 3. Nucleoporin Function(s) 3.1. FG-nucleoporins and nucleocytoplasmic transport 3.2. Nucleoporins and kinetochores 3.3. Nucleoporins and transcription 3.4. Nucleoporins, the immune system and Parkinson’s disease 4. Selective Cargo Translocation Across the Nuclear Pore Complex 4.1. The NPC as a selective gate 4.2. Current models of selective gating 4.3. In vitro studies of FG-domain function 4.4. In silico studies of the FG-domains and barrier function 4.5. Kinetic aspects of nucleocytoplasmic transport 4.6. Toward an understanding of FG-domain behavior in the NPC 5. Nuclear Pore Complex Assembly and Disassembly 5.1. Building a nuclear pore: Who’s on first? 5.2. Nuclear pore building blocks: The transmembrane proteins 5.3. Collaboration between nucleoporins in NPC assembly 5.4. Peripheral pore structures 5.5. Regulation of NPC assembly 5.6. Clues from a second site for NPC assembly 5.7. Nuclear pore assembly is never-ending 5.8. Deconstructing the NPC 6. Concluding Remarks

* {

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M.E. Mu¨ller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00632-1

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2008 Elsevier Inc. All rights reserved.

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Acknowledgments References

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Abstract Nucleocytoplasmic exchange of proteins and ribonucleoprotein particles occurs via nuclear pore complexes (NPCs) that reside in the double membrane of the nuclear envelope (NE). Significant progress has been made during the past few years in obtaining better structural resolution of the three-dimensional architecture of NPC with the help of cryo-electron tomography and atomic structures of domains from nuclear pore proteins (nucleoporins). Biophysical and imaging approaches have helped elucidate how nucleoporins act as a selective barrier in nucleocytoplasmic transport. Nucleoporins act not only in trafficking of macromolecules but also in proper microtubule attachment to kinetochores, in the regulation of gene expression and signaling events associated with, for example, innate and adaptive immunity, development and neurodegenerative disorders. Recent research has also been focused on the dynamic processes of NPC assembly and disassembly that occur with each cell cycle. Here we review emerging results aimed at understanding the molecular arrangement of the NPC and how it is achieved, defining the roles of individual nucleoporins both at the NPC and at other sites within the cell, and finally deciphering how the NPC serves as both a barrier and a conduit of active transport. Key words: Nuclear pore complex, Nuclear envelope, Nucleoporins, Nucleocytoplasmic transport, Transmembrane proteins. ß 2008 Elsevier Inc.

1. Introduction In interphase eukaryotic cells, transcription takes place in the cell nucleus while proteins are synthesized in the cytoplasm. Exchange of material between these two cellular compartments occurs via nuclear pore complexes (NPCs) located in the double membrane of the nuclear envelope (NE). NPCs support passive diffusion of small molecules and ions and facilitate receptor-mediated translocation of proteins and ribonucleoprotein complexes. Overall, the vertebrate NPC is a
120 MDa protein complex made up
30 different proteins called nucleoporins (or Nups) that are repetitively arranged as distinct subcomplexes to form the NPC (Cronshaw et al., 2002; Lim and Fahrenkrog, 2006; Rout et al., 2000; Schwartz, 2005; Tran and Wente, 2006). In the plane of the NE, the eightfold symmetric central framework of the NPC embraces a central pore that is
50 nm long and is narrowest (
40 nm) at the NE midplane (Beck et al., 2004, 2007; Stoffler et al., 2003). Attached to the central framework are cytoplasmic filaments and a nuclear basket (Fig. 7.1). We begin, here, by reviewing recent advances towards the elucidation of NPC

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architecture at the ultrastructural and atomic level by electron tomography and X-ray crystallography. These inroads into NPC structure lay important groundwork for understanding the function of the nuclear pore and we will overview progress that has been made in our understanding how the NPC acts as a selective barrier for macromolecular cargo. We will also discuss recent insights into the function of individual nucleoporins in nuclear organization that go beyond their well-characterized role in nucleocytoplasmic transport. Last but not least, we will review recent progress in addressing how the NPC disassembles and assembles at the beginning and end of mitosis, respectively.

2. Nuclear Pore Complex Structure 2.1. Overall nuclear pore complex architecture The NPC is a highly complex structure and electron microscopy (EM) and, more recently, cryo-electron tomography (CET) have proven to be the methods of choice to study intact NPCs at high resolution. The NPC consists of an approximately cylindrical central framework, eight cytoplasmic filaments and a nuclear basket composed of eight filaments that join into

Cytoplasmic filaments

Nuclear envelope

Nuclear basket

Central framework

100 nm

Figure 7.1 Electron micrograph with partially overlaid schematic representation of a cross-sectioned nuclear pore complex. The major structural components include the central framework, the cytoplasmic filaments and a nuclear basket.

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a distal ring (Fig. 7.1). Early EM studies provided 3D reconstructions of the central framework using negatively stained and frozen-hydrated NPCs from Xenopus laevis oocyte NEs (Akey and Radermacher, 1993; Hinshaw et al., 1992) or frozen-hydrated yeast cells (Yang et al., 1998). The central framework of the NPC (also called the spoke complex) resides between the inner and outer nuclear membranes, anchored where these parallel membrane bilayers curve to meet each other. Early structural studies showed that cytoplasmic and nuclear ring moieties are integral to the central framework. Recent CET studies in Xenopus oocyte isolated nuclei (Stoffler et al., 2003) and in intact, transport-competent nuclei isolated from Dictyostelium discoideum (Beck et al., 2004, 2007) have improved the resolution of the central framework to 89 nm and revealed the first reconstructions of peripheral, flexible components of the NPC, i.e., the cytoplasmic filaments and the nuclear basket. In Dictyostelium, the cytoplasmic filaments have a length of
35 nm and the nuclear basket is about 60 nm long. Together with the
50 nm central framework, the NPC therefore has a total length of
150 nm with the outer diameter of the structure being 125 nm (Beck et al., 2004, 2007). The overall linear dimensions of the NPC varies between species, whereas the overall 3D architecture appears to be evolutionarily conserved (Fahrenkrog et al., 1998; Kiseleva et al., 2004; Yang et al., 1998). Enclosed by the central framework is the hourglass-shaped central pore of the NPC with a diameter of 6070 nm at its cytoplasmic and nuclear periphery and
45 nm in the midplane of the NPC/NE (Beck et al., 2004, 2007; Pante´ and Kann, 2002; Stoffler et al., 2003). This central pore mediates all exchange between the cytoplasm and the nucleus and enables transport of macromolecules with diameters of up to 39 nm (Pante´ and Kann, 2002). Increasing concentrations of signal-carrying cargoes selectively interferes with the passage of other molecules that utilize a facilitated pathway, but not with the diffusion of inert molecules and vice versa, suggesting that passive and facilitated transport across the NPC proceed via routes that are sterically nonoverlapping (Naim et al., 2007). Whether these two routes exist in the central pore, i.e., facilitated transport along the walls of the central pore and passive diffusion through a hollow diffusion tube located at the pore center (Peters, 2005), or whether passive diffusion might additionally utilize peripheral channels (Akey and Radermacher, 1993; Beck et al., 2004; Hinshaw et al., 1992; Stoffler et al., 2003) remains to be seen. Peripheral channels of the NPC have a diameter of
8 nm and have been implicated in the diffusion of small molecules and ions (Feldherr and Akin, 1997; Hinshaw et al., 1992) and/or in trafficking of integral membrane proteins to the inner nuclear membrane (Soullam and Worman, 1995). However, the more recent observation that the cytoplasmic openings of the peripheral channels are not topologically continuous with the nuclear openings (Stoffler et al., 2003) challenges the view that they act as transport channels. Other potential roles have been proposed, such as in

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maintenance the NE electrical conductance (Danker et al., 1999; Enss et al., 2003; Mazzanti et al., 2001; Shahin et al., 2001) or as buffer zones that accommodate deformations of the central framework upon translocation of large cargoes (Fahrenkrog and Aebi, 2003).

2.2. The nuclear pore complex at atomic level Based on secondary structure prediction, nucleoporins can be grouped into three classes (Devos et al., 2006). The transmembrane group, which contains transmembrane a-helices and a cadherin-fold, comprises the outermost features of the NPC central framework and at least some members of this group are thought to help anchor the NPC in the NE. The second group of nucleoporins contain b-propeller and a-solenoid folds and these nucleoporins localize towards the inside of the NPC, whereas the third class harbors the conserved sequence motif of phenylalanine-glycine (FG)repeats (see Sections 3 and 4) in combination with a coiled-coil fold and may contribute to the formation of the NPC’s inner central framework and the peripheral structures (Devos et al., 2006; Schwartz, 2005; Tran and Wente, 2006). Other less frequent structural motifs found in nucleoporins are zinc-finger domains as in Nup153 and RanBP2/Nup358 (Higa et al., 2007) or RNA-recognition motifs as in Nup35 (Handa et al., 2006). b-Propellers are predicted in a third of the nucleoporins, and in fact sevenbladed b-propellers have been resolved from the N-terminal domains (NTD) of the human nucleoporins Nup133 and Nup214 and its yeast homologue Nup159p by X-ray crystallography (Berke et al., 2004; Napetschnig et al., 2007; Weirich et al., 2004). Proteins with b-propeller folds participate in diverse cellular functions and serve as platforms for multiple dynamic protein– protein interactions. Along this line, yeast Nup133p and Nup159p both play roles in mRNA export from the nucleus, and deletion or mutations in their NTDs impair their functions in mRNA export, probably by preventing the association of multiple mRNA export factors with the NPC (Berke et al., 2004; Weirich et al., 2004). The NTD of human Nup133 furthermore contains an amphipathic a-helical motif capable of sensing membrane curvature (Drin et al., 2007). This motif corresponds to an exposed loop, which connects two blades of the b-propeller and folds into an a-helix upon interacting with small liposomes. Whether the membrane curvature sensor in Nup133 serves to recognize the topology of the nuclear pore membrane to anchor the NPC during interphase or to recognize vesicles or tubules containing NE fragments critical for NE reassembly after mitosis, or both, remains to be seen (Drin et al., 2007). The NTD of human Nup214, in comparison to its yeast homologue Nup159p, consists of two distinct structural elements: the b-propeller and a 30-residue C-terminal extended peptide segment (Napetschnig et al., 2007).

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This extension binds to the bottom of the b-propeller with low affinity and has been suggested to play an ‘‘autoinhibitory’’ role in NPC assembly. The first crystal structure obtained for a nucleoporin was the NPC targeting domain of human Nup98 (Hodel et al., 2002). This domain, similar to the nuclear pore targeting domain of its yeast homologue Nup116p, consists of a six-stranded b-sheet sandwiched against a two-stranded b-sheet and flanked by two a-helical regions (Hodel et al., 2002; Robinson et al., 2005). This domain exhibits multiple conformations and is stabilized only when bound to a ligand, i.e., Nup96 and Nup145p-C in the case of Nup98 and Nup116p, respectively (Robinson et al., 2005). Conformational diversity might allow Nup98 and Nup116p to bind to multiple targets within the NPC or to associate and dissociate fast from the NPC to increase the mobility of the nucleoporins, as described for Nup98, which shuttles in an transcriptiondependent manner (Griffis et al., 2002, 2004). The attempt to crystallize the first subcomplex of the NPC, the Nup62 complex, yielded the structure of the a-helical coiled-coil domain of one of its components, rat Nup58/45 (Melcak et al., 2007). Nup58/45 forms tetramers in the crystal structure consisting of two antiparallel dimers. Each dimer consists of two a-helices that are connected by a short loop. The intradimer interactions are of hydrophobic nature, whereas two dimers associate through hydrophilic residues. The tetramer can adopt various conformations leading to a lateral displacement between tetramers suggesting an intermolecular sliding mechanism (Melcak et al., 2007). The Nup62 complex has recently been mapped to the cytoplasmic periphery of the NPC’s central pore (Schwarz-Herion et al., 2007), so that sliding of Nup58/45, and most likely of Nup62 and Nup54 as well, could contribute to modulating the diameter of the central pore in response to transport activity (Melcak et al., 2007).

2.3. Nuclear pore complex density and distribution The number of NPCs per cell varies greatly with cell size and activity. Yeast cells have
200 NPCs, proliferating human cells
30005000 and a mature Xenopus oocyte
5  107 (Gorlich and Kutay, 1999). A comprehensive ultrastructural study using freeze-fracture EM of yeast cells in combination with 3D reconstruction has shown that the distribution of yeast NPCs in the NE is not equidistant, but rather clustered into regions of higher density (Winey et al., 1997). The number of NPCs was found to increase steadily, beginning in G1- and peaking in S-phase of the cell cycle, suggesting that NPC assembly occurs continuously throughout the cell cycle (see Section 5) (Winey et al., 1997). Similarly, the density of NPCs increases through-out the cell cycle in HeLa S3 cells (Maeshima et al., 2006). Interestingly, these HeLa S3 cells exhibit large subdomains in the NE devoid of NPCs. These ‘‘pore-free islands’’ are present in telophase and

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G1 nuclei and are enriched in the inner nuclear membrane proteins emerin and lamin A/C, but not lamin B (Maeshima et al., 2006). Knock-down of lamin A/C by RNAi resulted in the disappearance of the pore-free islands, whereas upregulation of lamin A/C facilitated the formation of pore-free islands. Although the physiological relevance of pore-free islands remains to be elucidated, in HeLa cells the presence of such regions correlates with lower proliferative activity. Consistent with this, embryonic cells lack lamin A/C and have a high density of NPC along with high proliferative activity (Maeshima et al., 2006; Maul et al., 1980). Further indication of a relationship between lamin expression and pore density was obtained using Xenopus oocytes, whose giant nuclei lack lamin A/C and exhibit a high density of NPCs. Overexpression of human lamin A in these oocyte nuclei leads to the appearance of stretches in the NE that are devoid of NPCs (B. Fahrenkrog and B. Maco, unpublished results; Fig. 7.2). Recent studies using mouse embryonic stem (ES) cells addressed the adaptation of NPC structure and density during cardiac differentiation (Perez-Terzic et al., 2003, 2007). Accordingly, NPC density increases somewhat when ES cells differentiate into proliferative cardiomyocytes.

Figure 7.2 Electron micrographs of cross sections along a nuclear envelope of isolated Xenopus oocyte nuclei. (A) The nuclear envelope of a stage 6 nucleus is characterized by a high density of nuclear pore complexes (black arrows). (B) Overexpression of human lamin A in these Xenopus oocyte nuclei causes a decrease in nuclear pore complex (black arrows) density and a thickened nuclear lamina (gray arrowheads). c, cytoplasm; n, nucleus. Scale bar,100 nm.

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With a few significant exceptions, genes encoding components of the nucleocytoplasmic transport machinery, i.e., nuclear transport receptors, nucleoporins and Ran-related factors, were found to be broadly down regulated in cardiomyocytes derived from ES cells compared to the undifferentiated cells, supporting the notion that changes in transport occur concomitantly (and maybe help to drive) differentiation. Further differences have been observed between stem cellderived cardiomyocytes and adult heart-isolated cardiomyocytes, which have a density of
15 NPCs/mm2 and
28 NPCs/mm2, respectively. While the overall diameter and height of the NPC are similar in both cell types, there is greater central density in the NPCs of stem cellderived cardiomyocytes, indicative of greater transport activity (Perez-Terzic et al., 2003). Drosophila Nup154, the homologue of rat Nup155, is essential for gametogenesis (Gigliotti et al., 1998) and regulated expression of a testisspecific isoform of RanBP2/Nup358, BS-63, and Nup50/Npap60 may also influence gamete/testis maturation (Hogarth et al., 2005). Another case in which the nuclear transport machinery appears to be involved in cellular fate is found in malignant cells resistant to chemotherapy (Lewin et al., 2007). Multidrug resistance commonly limits efficiency in treating malignant cells with chemotherapy and is classically described as a plasma membrane phenomenon. However, multidrug-resistant cells specifically exclude chemotherapeutic drugs from the nucleus and have now been shown to exhibit an increased number of NPCs compared to drug-sensitive cells (Lewin et al., 2007). The increase in NPC number somehow correlates with the exclusion of chemotherapeutic drugs from the nucleus, suggesting that nuclear export is selectively enhanced in the resistant cells. The mechanism by which NPCs export chemotherapeutic drugs remains elusive, but inhibition of nucleocytoplasmic transport with injection of wheat germ agglutinin can reverse multidrug resistance in these cells (Lewin et al., 2007). All together, these data indicate that regulation of NPC number, composition and nucleocytoplasmic transport may drive and influence more cellular processes than previously assumed.

3. Nucleoporin Function(s) 3.1. FG-nucleoporins and nucleocytoplasmic transport FG-repeat domains are found in about one third of the nucleoporins and mediate the interaction between soluble transport receptors loaded with signal-bearing cargo and the NPC. These FG-repeat domains also likely contribute to the selective barrier that limits diffusion through the NPC (see section 4). Atomic structures of FG-repeat peptides in complex with, for

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example importin b, NTF2 or the mRNA export factor TAP/NXF1, have consistently shown that the interaction between FG-repeats and the different transport receptors involves primarily the phenylalanine ring of the FG-repeat core and hydrophobic residues on the surface of the receptor. Hydrophilic linker between individual FG-motifs, which constitute the majority of amino acid mass in the overall FG-domain, appear to influence the strength of the binding and allow simultaneous binding of several FGcores to the receptor (Liu and Stewart, 2005). Based on biophysical measurements, the FG-repeat domains of yeast nucleoporins were found to be natively unfolded, i.e., having no or only little secondary structure. Similarly, FG-repeat domains of human, fly, worm and other yeast species are most likely disordered based on their amino acid composition (Denning and Rexach, 2007). This notion is further supported by immuno-EM studies on two vertebrate FG-repeat nucleoporins, Nup153 and Nup214, which suggested that FG-repeat domains are flexible and mobile within the NPC (Paulillo et al., 2005, 2006). Atomic force microscopy (AFM) studies on recombinantly expressed FG-repeat domain of human Nup153 further revealed that this
700 residue domain in fact is an unfolded molecule with a length of
180 nm, resembling an unfolded polypeptide chain (Lim et al., 2006b). Nup153 and Nup214 are both known to play roles in distinct nucleocytoplasmic transport pathways and to interact with a number of nuclear transport receptors via their FG-repeats (Ball and Ullman, 2005; Bernad et al., 2006; Hutten and Kehlenbach, 2006; Sabri et al., 2007; van Deursen et al., 1996). The location of the FG-repeat domains of Nup153 and Nup214 in the NPC shifts in a transport-dependent manner, further supporting their role in nucleocytoplasmic transport (Paulillo et al., 2005). Systematic deletion of FG-repeat regions in yeast nucleoporins revealed, however, that yeast NPCs are able to compensate the loss of
50% of their FG-repeats with only little effect on distinct nuclear transport pathways, indicating that FG-repeats are highly redundant within the NPC, that individual FG-nucleoporins appear critical for specific nuclear transport pathways but not for bulk nucleocytoplasmic transport and/or that other interaction sites for transport receptors exist within the NPC. Besides playing important roles in nucleocytoplasmic transport, FG-repeat domains may have other functions as well. The crystal structure of the RRM domain of mouse Nup35 revealed that all three FG-sequences of this nucleoporin are in ordered secondary structure elements and consistent with these FG-sequences do not interact with transport receptors, such as importin b, but rather with, for example, the integral membrane protein Ndc1. Thus, the FG-sequences of Nup35 may contribute to the formation of the NPC’s central framework (Handa et al., 2006).

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3.2. Nucleoporins and kinetochores A well-studied and conserved subcomplex of the NPC is the vertebrate Nup107-160 complex and its yeast homologue the Nup84p complex. The Nup107-160 complex is composed of nine nucleoporins and resides on both sides of the central framework of the NPC (Belgareh et al., 2001; Krull et al., 2004; Loiodice et al., 2004; Orjalo et al., 2006). The Nup107-160 complex seems to represent the core element of the central framework, since depletion of any member of this NPC subcomplex in nuclear reconstitution assays or by RNAi led to the assembly of NPC-free nuclei or nuclei with severe deficiencies in NPC formation (Boehmer et al., 2003; Harel et al., 2003b; Loiodice et al., 2004; Walther et al., 2003a) (see Section 5). A fraction of the Nup107-160 complex is targeted to kinetochores from prophase to late anaphase (Belgareh et al., 2001; Loiodice et al., 2004), to spindle poles and proximal spindle fibers in prometaphase mammalian cells and throughout reconstituted spindles in Xenopus egg extracts (Orjalo et al., 2006). Anchoring of the human Nup107-160 complex to kinetochores is mediated by the Ndc80 complex, which is part of the outer kinetochore and involved in formation and maintenance of stable kinetochoremicrotubule (MT) interaction, and CENP-F, which is also involved in MT attachment (Zuccolo et al., 2007). Kinetochores depleted of the Nup107-160 complex fail to establish proper MT attachment, which leads to a checkpoint-dependent mitotic delay (Zuccolo et al., 2007). Another nucleoporin recruited to kinetochores and the spindle in mitosis is RanBP2/Nup358 in complex with RanGAP1 ( Joseph et al., 2004, 2002; Matunis et al., 1998). RNAi approaches revealed that the RanBP2/ RanGAP1 complex is involved in chromosome congression and segregation, stable kinetochore-MT association, and kinetochore assembly (Askjaer et al., 2002; Joseph et al., 2004; Salina et al., 2003). The nuclear export receptor CRM1 provides the anchoring site for RanBP2 and RanGAP1 at the kinetochores (Arnaoutov et al., 2005), and the Nup107-160 complex in turn is required for the recruitment of CRM1, RanBP2 and RanGAP1 to the kinetochores (Zuccolo et al., 2007). Therefore, the Nup107-160 complex helps to recruit distinct kinetochore subcomplexes required for stable kinetochore-MT interaction.

3.3. Nucleoporins and transcription In the past few years it became evident that the NPC plays a role in chromatin organization in the nucleus. In this context, the nuclear periphery and the NPCs have been considered a zone of gene repression caused by the presence of heterochromatin and silencing factors (Brown and Silver, 2007). Consistently, in S. cerevisiae, two nucleoporins, Nup60p and Nup145p-C, are required for repression of the silent mating type loci

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HML and HMR and for the proper silencing of telomeres (Brown and Silver, 2007; Feuerbach et al., 2002; Galy et al., 2000). However, yeast NPCs can also positively regulate gene expression by preventing the spread of heterochromatin regions and by recruiting actively transcribed genes to the nuclear periphery (Cabal et al., 2006; Casolari et al., 2004, 2005; Ishii et al., 2002; Luthra et al., 2007; Menon et al., 2005; Schmid et al., 2006), indicating a function for nucleoporins in transcription activation as well as gene silencing. Most important, the same set of nucleoporins, namely Nup2p and the Nup84p complex, can have repressive and activating functions (Dilworth et al., 2005; Ishii et al., 2002; Menon et al., 2005; Schmid et al., 2006; Therizols et al., 2006), and it will be interesting to see how their dual functions in gene expression are regulated at the molecular level. In higher eukaryotes, the first clues to function of nucleoporins in transcription came from studies with the chimeric NUP98-HOXA9 protein, a chromosomal translocation product that occurs in myelodysplastic syndromes and acute myeloid leukemia. These studies consistently showed that NUP98-HOXA9 acts as an aberrant transcription factor, with the N-terminal FG-repeat domain of Nup98 enhancing the transcriptional activity of the DNA binding domain derived from the transcription factor HoxA9 (Ghannam et al., 2004; Kasper et al., 1999). The NUP98 gene has been found to fuse with 19 different fusion partners causing different forms of acute and myeloid leukemia. Recently, it became evident that NUP98 fusions can also act as trans-repressors of transcription (Bai et al., 2006). The intranuclear localization of these fusion proteins (Kasper et al., 1999) suggests that transcription regulation by the NUP98 fusion may not occur at the NPC or the NE. Additionally, it is not yet clear whether transcriptional regulation is the sole role of Nup98 sequences in oncogenic fusions or how this ability to modulate transcription relates to the role of endogenous Nup98. It is notable, however, that in yeast, human Nup98 was found capable of stimulating the transcription of a reporter gene at the nuclear periphery (Menon et al., 2005). Nup153 and Nup98 both dynamically interact with the NPC and their mobility within the cell appears transcription-dependent (Griffis et al., 2002, 2004). The transcription factor PU.1, which is expressed in several hematopoietic cell lineages and plays a pivotal role in the differentiation of myeloid cells and lymphocytes, is proposed to be imported into the nucleus via direct interaction with Nup153 (Zhong et al., 2005). Binding of PU.1 to Nup153 is stimulated by RanGTP but is independent of any nuclear transport receptor of the karyopherin family. In the presence of a source of energy (and presumably elevated RanGTP levels), PU.1 associates with the nuclear side of the NPC (Zhong et al., 2005), suggesting that PU.1dependent active genes might be targeted to the NPC, at least in part. More direct evidence that Nup153 in fact targets genes directly to the NPC came from a recent study on dosage compensation in Drosophila, a phenomenon

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that is distinguished by the hypertranscription of the male X chromosome (Mendjan et al., 2006). Proteins of the dosage compensation complex (DCC) were found to associate with NPC components, in particular Nup153 and Mtor, the Drosophila homologue of the mammalian nucleoporin Tpr. Nup153 and Mtor/Tpr are part of the NPC’s nuclear basket, and knock-down of Nup153 or Mtor resulted in a shift of DCC localization away from the nuclear rim, coinciding with a loss of dosage compensation in male cells (Mendjan et al., 2006). The putative transcription factor ELYS was identified as binding partner of the Nup107-160 complex and found to localize to NPCs during interphase and to kinetochores in mitosis (Rasala et al., 2006). ELYS, and its homologue in C. elegans named Mel-28, is required for NPC assembly (see Section 5; Fernandez and Piano, 2006; Franz et al., 2007; Galy et al., 2006; Rasala et al., 2006), and as a DNA binding protein it potentially targets active genes to the NPC. Taken together, recent results lend support to the notion that NPCs act in gene gating (Blobel, 1985) and that locating genes directly to the NPC is indeed a ubiquitous, evolutionary conserved mechanism for regulating gene expression.

3.4. Nucleoporins, the immune system and Parkinson’s disease The nucleoporin Nup96 is autocatalytically cleaved from a Nup98/Nup96 precursor protein, which results in the two nucleoporins Nup96 and Nup98 (Enninga et al., 2003). Nup96, like Nup98, localizes to both sides of the NPC, and is a component of Nup107-160 complex (Enninga et al., 2002). Both Nup96 and Nup98 are induced by interferons (Enninga et al., 2002). Heterozygous Nup96þ/ mice show downregulation of interferon-regulated genes and defects in the mRNA export of major players of immune response, MHCI and MHCII gene products, coinciding with alterations in MHCrelated T cell function. Additionally, B cell function is impaired in Nup96þ/ mice, resulting in Nup96þ/ cells and mice highly susceptible to viral infection. Therefore, Nup96 appears to function in antiviral response and in innate and adaptive immunity (Faria et al., 2006). A homologue of Nup96 has recently been identified in Arabidopsis thaliana, but in contrast to vertebrates the AtNup98 and AtNup96 genes locate to different chromosomal regions (Mans et al., 2004; Zhang and Li, 2005). AtNup96 is required for basal defense and constitutive resistance response to pathogens (Li et al., 2001; Zhang et al., 2003), indicating a conserved function of Nup96 in immune response. Similarly conserved is the function of Nup96 in mRNA export: plants depleted for Nup96 accumulate polyadenylated RNA within their nuclei (Parry et al., 2006). Moreover, plants depleted for Nup96 and Nup160, another component of the Nup107-160 complex, exhibit pleiotropic growth defects implicating

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these nucleoporins in hormone signaling (Parry et al., 2006). In different contexts, other roles for nucleoporins in signaling and development have been documented: a genome-wide RNA interference screen in Drosophila identified Nup153 and Nup98 as positive regulators of the Hedgehog signaling pathway (Nybakken et al., 2005). Additionally, Drosophila Nup154 was found to play a critical role in oogenesis due to its interaction with the germline specific protein Cup, which is implicated in multiple aspects of female gametogenesis (Grimaldi et al., 2007). A pleiotropic role in cell function has also been suggested for the nucleoporin RanBP2/Nup358. RanBP2 is a large modular protein and several molecular partners with distinct functions interacting with specific domains of RanBP2 have been identified. Several roles of RanBP2 have emerged that implicate RanBP2 in nucleocytoplasmic transport (Bernad et al., 2004; Yokoyama et al., 1995), protein biogenesis (Ferreira et al., 1996, 1997), the formation of the mitotic spindle and NE assembly (Askjaer et al., 2002), and the integration of NE breakdown with kinetochore formation and maturation during early mitotic progression (Salina et al., 2003). Some protein partners interact with RanBP2 in a tissue-specific manner, such as a subset of G proteincoupled receptors, the red/green opsin, in photosensory neurons (Ferreira et al., 1996, 1997) or the kinesins KIF5B and KIF5C selectively in the central nervous system (CNS) (Cai et al., 2001). CNS-selective effects of RanBP2 may underlie the pathogenesis of certain neuropathies, in particular Parkinson’s disease (PD). The Parkin protein, which has E3 ubiquitin ligase activity, has been implicated in autosomal recessive juvenile Parkinsonism, and RanBP2 has been identified as target for Parkin leading to the ubiquitination of RanBP2 and its subsequent proteosomal degradation (Um et al., 2006). Abnormal processing of RanBP2 by Parkin might therefore play a role in PD pathogenesis. RanBP2 itself possesses SUMO-E3 ligase activity (Pichler et al., 2002), and it will therefore be interesting to see if RanBP2-mediated sumoylation or the loss of it contributes to PD progression, in particular since NPC-regulated sumoylation appears to also play a role in other cellular processes, such as DNA repair and cytokinesis (Makhnevych et al., 2007; Palancade et al., 2007). Haploinsufficient RanBP2þ/ mice show a selective reduction of hexokinase type I (HKI) in the CNS, whereas skeletal muscle, spleen and liver levels of HKI remained largely unaffected. HKI is a key player in glucose metabolism and ATP production, and RanBP2 appears to prevent the inhibition of HKI and its degradation by binding the HKI antagonist COX 11 (Aslanukov et al., 2006). Haploinsufficiency in RanBP2 consequently promotes the destabilization and degradation of HKI, decreases ATP production and, hence, reduces the responsiveness of neurons. These observations may help to explain how targeting of RanBP2 for degradation by Parkin (Um et al., 2006) contributes to the pathophsyiological mechanisms underlying Parkinsonism and other neurodegenerative disorders.

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Together, mouse models as for Nup96 and RanBP2 present a powerful tool to link cellular pathways, as well as pathophysiological states, to the NPC—and, in doing so, expose roles for nucleoporins that had not been anticipated.

4. Selective Cargo Translocation Across the Nuclear Pore Complex Whereas small molecules (e.g. H2O and ions) can diffuse freely through the NPC, large cargoes (>40 kDa) require the assistance of soluble transport receptor molecules, known collectively as karyopherins (Kaps; also called importins, exportins and transportins), to be effectively chaperoned through the NPC (Stewart, 2007). Appropriate macromolecules (i.e., cargo) are identified through a short sequence of residues known as nuclear localization/export signals (i.e., NLS/NES), which exhibit binding interactions with the Kaps. Import of NLS-cargo into the nucleus usually entails the use of importin a, which acts as an adaptor to importin b through an importin bbinding (IBB) domain (Gorlich et al., 1996). However, there are some proteins that can bind directly with importin b. The directionality of nucleocytoplasmic transport is driven by an asymmetric distribution of the two nucleotide states of Ran (GTP/GDP) (reviewed in Gorlich and Kutay, 1999; Macara, 2001; Weis, 2002). RanGTP is found predominantly in the nucleus and functions to release NLS-cargo from its import receptor by binding to the import receptor itself (Gorlich et al., 1996). The importin-RanGTP complex is then recycled back into the cytoplasm. Similarly, trimeric complexes formed by an export receptor, its cargo and RanGTP, are ferried to the cytoplasm. Once in the cytoplasm, RanGAP1 (together with RanBP1 and RanBP2/Nup358) catalyzes GTP hydrolysis, which drives the disassembly of the complexes (reviewed in Gorlich and Kutay, 1999; Macara, 2001; Weis, 2002). In this manner, the receptors are recycled while a large pool of RanGDP in the cytoplasm is constantly replenished (Stewart, 2007; Weis, 2002). A current controversy remains as to how passage through the NPC is obstructed for non-NLS/NES harboring molecules that do not bind to the karyopherins or have a noncanonical means of transport (Paine et al., 1975). Hence, the selection criterion for transport through the NPC is not simply based on size exclusion per se and alludes to the presence of a selective gating mechanism within the NPC that simultaneously prevents the passive passage of molecules while promoting the translocation of receptor-mediated cargo. In this section, we will review the various concepts and supporting evidence that have led to the current understanding of selective gating, as

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well as highlight outstanding aspects of the NPC which need to be addressed in order to provide for a more refined description of NPC function.

4.1. The NPC as a selective gate Initial EM-based structural studies linked the biophysical origin of the selective gate to the presence of a ‘‘central plug’’ or ‘‘transporter’’ module located within the NPC (Akey, 1990; Feldherr and Akin, 1997). With its nanometerresolution imaging capability and its ability to be used in physiologically relevant environments, AFM was subsequently used to resolve the basis of the central plug, but these studies resulted in controversy ( Jaggi et al., 2003a,b; Mooren et al., 2004; Stoffler et al., 1999; Wang and Clapham, 1999). Today, by using state-of-the-art CET, it has been shown that the central plug most likely represents cargo caught in transit (Beck et al., 2004, 2007; Stoffler et al., 2003). Several lines of evidence now indicate that the key constituents of the NPC selective gate consist of FG-repeat nucleoporins and reside at both the cytoplasmic and nuclear peripheries surrounding the central pore (Rout et al., 2000). Cargo selection relies on binding interactions that occur between karyopherins and the FG-motifs (Bayliss et al., 2000, 2002; Bednenko et al., 2003; Liu and Stewart, 2005). Instead of possessing any well-defined structure, the FG-repeat domains exhibit large Stokes radii and are natively unfolded (Denning et al., 2003). Accordingly, AFM-based stretching experiments (i.e., single molecule force spectroscopy [SMFS]) show that the FG-repeat domains exhibit a highly flexible entropic elasticity (Lim et al., 2006a, 2007). Interestingly, studies reveal a high level of functional redundancy between the various FG-repeat domains in the NPC: 1) the asymmetric FG-domains have been shown to be dispensable for nucleocytoplasmic transport (Strawn et al., 2004; Zeitler and Weis, 2004); 2) the direction of transport through the NPC can be inverted by reversing the gradient of RanGTP (Nachury and Weis, 1999); 3) active transport is able to proceed in NPCs lacking cytoplasmic filaments (i.e., FGrich RanBP2/Nup358) (Walther et al., 2002); and perhaps most tellingly, 4) the selective gating mechanism has been found to remain functional even after 50% of the FG-repeats have been depleted (Strawn et al., 2004).

4.2. Current models of selective gating The manner in which the FG-repeat domains contribute to the selective gating of the NPC is widely speculated and has been the subject of several reviews (Fahrenkrog and Aebi, 2003; Lim et al., 2006b; Stewart, 2007; Suntharalingam and Wente, 2003; Weis, 2003). As illustrated in Figure 7.3, it is generally agreed that the FG-repeat domains form the physical

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Figure 7.3 Main models of selective gating in the NPC. (A) The Brownian/virtual gating model (Rout et al., 2000, 2003) predicts that the entropic fluctuations of the unfolded FG-domains form an effective barrier to passive cargo. Although the central pore appears unobstructed, the highly stochastic motion of the elongated FG-domains (shaded area) generates a high-density FG-domain entropic barrier or ‘‘cloud’’ that surrounds and extends beyond the immediate peripheries of the NPC (dark)(Lim et al., 2006a). (B) The selective phase model predicts that hydrophobic interactions between the FG-repeats drive the FG-domains to form an randomly interconnected gel-like meshwork within the central pore that acts as a sieve to passive, hydrophilic cargo (Ribbeck and Gorlich, 2002). Receptor-cargo complexes can dissolve through and negotiate the meshwork by breaking the‘‘links’’ between the FG-domains via receptorFG interactions. The gray area denotes the ‘‘range’’ of the meshwork while three FGdomains are drawn in red to emphasize that the FG-domains have to be elongated in order to cross-link with each other. (C) By combining aspects of Brownian gating and the selective phase, the two-gate model suggests that the more central GLFG-domains form a cohesive meshwork in the central pore while the peripheral FxFG-domains give rise to an entropic barrier (Patel et al., 2007).The shaded areas represent the locations of the two gates.

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constituents of the underlying barrier (Ben-Efraim and Gerace, 2001; Macara, 2001; Ribbeck and Gorlich, 2002; Rout et al., 2000). The Brownian affinity gating model (Rout et al., 2000) or virtual gating (Rout et al., 2003) proposes that the entropic behavior of peripheral FG-repeat domains acts as a substantial barrier to inert cargo. Translocation is anticipated for receptor-mediated cargoes due to interactions between the FG-repeats and the transport receptors (Bayliss et al., 2000, 2002) which increases the residence time and probability of entry into the NPC. In a similar manner, the ‘‘oily-spaghetti’’ model (Macara, 2001) postulates that noninteracting FG-repeat domains are pushed aside by cargo complexes but otherwise obstruct the passage of passive cargo. The selective phase model (Ribbeck and Gorlich, 2002) predicts that FG-repeat domains attract each other via hydrophobic inter-FG-repeat interactions to form a hydrophobic gel or meshwork. This interpretation is based on experiments which show that the addition of hydrophobic solvents disrupts the meshwork and triggers a nonselective opening of the central pore (Ribbeck and Gorlich, 2002; Shulga and Goldfarb, 2003). Hence, it is predicted that passive, more hydrophilic material is obstructed while hydrophobic cargo complexes are able to ‘‘dissolve’’ through the sieve-like meshwork. Most recently, Patel et al. (2007) have proposed a two-gate model that combines elements of both Brownian gating and the selective phase. Based on the observation that the centrally located yeast FG-repeat domains (i.e., GLFG) exhibited cohesion as opposed to the peripheral yeast FG-repeat domains (i.e., FxFG) that did not, the authors deduced that the more centralized FG-repeat domains formed a cohesive meshwork while the peripheral FG-repeat domains functioned as an entropic barrier.

4.3. In vitro studies of FG-domain function Despite progress in characterization of the NPC and its individual components, an accurate picture of how selective gating is achieved by the FGrepeat domains remains unclear due to a general lack of information about FG-domain behavior in the context of the NPC. The source of this ambiguity stems in part from the difficulty in trying to visualize the FG-repeat domains in vivo, which is evident given the lack of resolution, even when using state-ofthe-art structural techniques such as CET, to detect the FG-repeat domains (Beck et al., 2004, 2007; Stoffler et al., 2003). Direct imaging of the NPC with AFM is also limited in resolution and chemical sensitivity due to the complexity of the NPC and its cellular environment ( Jaggi et al., 2003a,b; Mooren et al., 2004; Stoffler et al., 1999; Wang and Clapham, 1999). Presently, only immunogold-EM has been able to provide positional information of the FG-repeat domains and has been used to show that the FG-repeat domains of Nup153 and Nup214 (Paulillo et al., 2005) appear diffuse and mobile within the nuclear and cytoplasmic peripheries of the NPC, respectively.

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The FG-repeat domains themselves have so far been directly visualized only in isolation as individual biopolymers by AFM (Lim et al., 2006b). To reproduce the contextual dimensions of the NPC (i.e., the FGrepeat domains are anchored to the NPC surface and not free-floating in solution), Lim et al. (2006a) developed an experimental platform that allowed for the collective, biophysical behavior of surface tethered FGrepeat domains to be probed at the nanoscopic level. In support of the Brownian affinity model (Rout et al., 2000, 2003), they found that FGdomain clusters of Nup153 (termed cNup153) are entropically dominated and resemble a polymer brush (Halperin et al., 1992; Milner, 1991; Zhao and Brittain, 2000). Being surface-anchored, the molecular chains exhibit a predisposed net directionality normal to the surface because of lateral packing constraints, which causes them to stretch away from the surface, i.e., forming a brush. This provided an explanation as to how FG-repeat domains could give rise to an effective repulsive entropic barrier in and around the NPC. The observation that the extended brush-like conformation of the FG-repeat domains collapses in hexanediol provides an explanation as to why NPCs appear to reversibly open and close when the same reagent is added/removed (Patel et al., 2007; Ribbeck and Gorlich, 2002; Shulga and Goldfarb, 2003). This was substantiated with SMFS-AFM analysis, which showed that individual Nup153 FG-domain molecules could be reversibly stretched and relaxed without any change to its intrinsic entropic elasticity, resembling a worm-like chain (Bustamante et al., 1994; Marko and Siggia, 1995). These measurements indicate a lack of intra-FG interactions within each individual FG-repeat domain and provide a nanomechanical verification of the natively unfolded conformation of this domain. In comparison, SMFS analysis detected an interaction between importin b and cNup153 when importin bmodified AFM tips were used (Lim et al., 2007) and, further, provided evidence for multiple points of contact of importin b with the cNup153 region. This is in agreement with the fact that importin b consists of five hydrophobic FG-binding sites (Bayliss et al., 2000, 2002; Bednenko et al., 2003; Liu, and Stewart, 2005) (with an additional five binding sites predicted by molecular dynamics (MD) simulations (Isgro and Schulten, 2005)) that can be simultaneously occupied (Isgro and Schulten, 2005). This led to the suggestion that 1) cooperativity between FG-repeat domains arises from FG-receptor interactions instead of FG-FG interactions, and 2) binding promiscuity allows for a ‘‘capture’’ mechanism that involves the coiling or wrapping of the FG-domain(s) around receptor molecules (Lim et al., 2007). At the macroscopic level Frey et al. (2006) showed that the yeast FGnucleoporin, Nsp1p, can be cast in the form of a macroscopic hydrogel to lend support to the ‘‘selective phase’’ model (Ribbeck and Gorlich, 2002). Remarkably, the authors showed that a saturated hydrogel made of Nsp1p FG-repeats can reproduce the permeability properties of NPC (Frey and

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Gorlich, 2007). In order to investigate the cohesiveness of different FGnucleoporins, Patel et al. devised a low affinity assay, which could detect the binding of CFP-nucleoporins to GST-nucleoporins immobilized on Sepharose beads (Patel et al., 2007). Interestingly, they found that only GLFG-domains showed weak cohesive interactions whereas FxFGdomains (such as in Nsp1p) did not. By overexpressing the FG-repeat domains in yeast cells, the authors observed a similar pattern of interactions in vivo, albeit not visualized in the context of the NPC. A systematic depletion of FG-repeat domains in yeast showed, however, that the NPCs displayed similar qualitative ‘‘leakiness’’ in all the cases studied, which indicated that the FG-repeat domains in both peripherally and centrally anchored nucleoporins play an important role in maintaining the selective gating mechanism. These findings led to the conclusion that FxFG domains on both faces of the NPC act as an entropic repulsive barrier while the GLFG-domains form a cohesive meshwork in the NPC’s central pore (Patel et al., 2007).

4.4. In silico studies of the FG-domains and barrier function Computational studies have also been useful in providing additional insight into the possible aspects of FG-repeat domain behavior. By modelling a cross-linked network, Bickel and Bruinsma (2002) showed that a receptor molecule would have a lower, and not higher, mobility than a passive molecule due to its attachments to the FG-repeat domains. In agreement with the cohesive properties observed between GLFG-domains (Patel et al., 2007), Kustanovich and Rabin (2004) predicted that FG-repeat domains would exhibit low equilibrium affinities for each other. In support of brushlike behavior for FG-domains (Lim et al., 2006a), Nielsen et al. (2006) showed that the conformational entropy of non-interacting FG-repeat domains was enough to provide for a robust barrier around the NPC by modelling the FG-repeat domains as surface grafted, polymeric random coils. By solving a rigorous mathematical model of transport through the NPC, Zilman et al. (2007) showed that selectivity, efficiency, directionality, and robustness of nucleocytoplasmic transport could be explained by combining the interaction strengths of binding to the flexible FG-repeat domains with the physics of diffusion inside a channel. Besides finding that NPC selectivity arises from a balance between the probability of forming receptor-mediated cargo complexes and their speed, they propose that the competition between specific receptor-mediated versus nonspecific interaction for FG-binding also contributes to the selectivity of the NPC mechanism. In contrast to predictions of the selective phase model, the authors find the inherent flexibility of the FG-repeat domains to play a key

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role in maintaining the high throughput and relative robustness (i.e., insensitive to FG-repeat deletions) of the NPC. Using molecular dynamics simulations, Isgro and Schulten predicted that additional hydrophobic binding spots could exist on the transport receptors importin b (Isgro and Schulten, 2005), NTF2 (Isgro and Schulten, 2007a) and the Cse1p:Kap60p:RanGTP complex (Isgro and Schulten, 2007b) that could enhance receptor interactions with the FG-repeats. Besides requiring several binding spots, the authors predict that close physical proximity between binding spots on each receptor molecule is likely to be an important criterion for transport selection. Furthermore, by using identical FG-repeat peptides derived from both FxFG domains (i.e., Nsp1p) and GLFG-domains (i.e., Nup116p) in all three studies, these simulations show that both classes of FG-domains interact with overlapping binding spots on importin b, NTF2 and Cse1p, respectively. Indeed, such overlap has been experimentally observed previously for importin b (Bayliss et al., 2002).

4.5. Kinetic aspects of nucleocytoplasmic transport Selective gating appears to be a rapid process given the relatively short residence times of receptor-cargo complexes at the NPC as shown by single molecule fluorescence microscopy (Kubitscheck et al., 2005; Yang et al., 2004). By directly monitoring the transport of a model protein substrate (i.e., NLS-2xGFP) through individual NPCs in permeabilized HeLa cells, Yang et al showed that movement through the NPC is bidirectional, resembling a random walk whereby the import substrate spends the majority of its
10 ms interaction time within the central pore (Yang et al., 2004). Kubitscheck et al. (2005) obtained kinetic data regarding the dwell times of the nuclear transport receptors NTF2 (5.8 ms) and transportin (7.2 ms) at their respective NPC binding sites. They observed that the dwell times decreased from 5.8 ms to 5.2 ms for NTF2 and 7.2 ms to 5.6 ms for transportin when each respective transport receptor was bound to specific transport substrates, indicating that translocation is accelerated for receptorcargo complexes. By comparing their data with known bulk transport rates, they suggested that nucleocytoplasmic transport proceeds via multiple parallel pathways within each NPC. More recently, Yang and Musser (2006) showed that the transport efficiency and import time of cargo was modulated by importin b concentration and suggested that in vivo mechanisms that altered the expression levels of the receptor could dramatically affect transport rates. In addition, the recent findings of Paradise et al. (2007) and Timney et al. (2006) revealed that karyopherins compete nonspecifically with other cytosolic structures and proteins. By colliding with a large number of nonspecific partners in the crowded cytosolic environment, karyopherins could be ‘‘shielded’’ from the FG-repeat domains. Indeed, Timney et al. (2006) reported that nonspecific competition

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resulted in each karyopherin having to take
10 s to search for and successfully import the appropriate cargo—100 times longer than previous estimates, which ignored such effects. Based on the 510 ms residence/dwell time at the NPC (Kubitscheck et al., 2005; Yang et al., 2004), this led the authors to suggest that the limiting factor in cargo transport arises from the receptor having to seek out specific partners (i.e., cargo) in the milieu of nonspecific interactions instead of the actual process of translocation through the NPC. To obtain a direct thermodynamic perspective of nuclear transport, Kopito and Elbaum (2007) conducted quantitative transport measurements in reconstituted nuclei and showed that nuclear accumulation follows Michaelis-Menten first-order kinetics as a function of the cytoplasmic cargo concentration. Importantly, this suggests that 1) the fate of a protein population led by receptor-mediated transport is dictated by the NLS, and 2) individual molecules are free to shuttle back and forth through the NPC.

4.6. Toward an understanding of FG-domain behavior in the NPC While the biophysical behavior of the FG-repeat domains in vivo remains unsubstantiated, the effect of nonphysiological reagents (e.g., hexanediol) to abolish the NPC barrier as observed in transport assays (Patel et al., 2007; Ribbeck and Gorlich, 2002; Shulga and Goldfarb, 2003) provides an important clue to their physiologically relevant conformations. By observing that the FG-repeat domains ‘‘collapse’’ in hexanediol (Lim et al., 2006a), it can be inferred that the FG-repeat domains are predominantly extended to a degree in the NPC in the midst of ongoing receptor-FG interactions. How then does the movement of karyopherins (and cargo) through the NPC occur? To achieve a rational mechanistic picture of how the NPC selectively gates nuclear transport, it will be essential to understand how the different FGdomain conformations (i.e., gel vs. brush) simultaneously prevent the passage of passive molecules while promoting the translocation of receptor-cargo complexes through the NPC (i.e., definition of selective gating) at the observed transport rates (
5 ms). Thus, newer structural/biophysical techniques will be required to elucidate even finer dynamic, molecular details of FG-repeat domain behavior within the NPC and how they respond to the biochemical interactions that govern nucleocytoplasmic transport.

5. Nuclear Pore Complex Assembly and Disassembly Consideration of the massive, ornate structure of the NPC and its central role in creating distinct nuclear and cytoplasmic environments leads to the question of how this macromolecular machine is assembled with each

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cell division. In a proliferating human cell, thousands of NPCs are formed de novo during each cell cycle. NPCs are assembled both concomitantly with membrane recruitment to the newly forming nuclei, as well as after the chromatin is fully enclosed by the two lipid bilayers that comprise the NE. Whether these are truly distinct modes of assembly remains to be determined. If so, in post-mitotic cells or in organisms with a ‘‘closed mitosis’’ only the latter path is relevant. The process of NPC assembly is extremely rapid; in the Xenopus egg extract system, NPCs are estimated to form at the rate of
140 per minute (D’Angelo et al., 2006). Given the observation that during an ‘‘open mitosis’’ NPC components disperse into subunits and individual components, as well as evidence that formally rules out NPC splitting to create new NPCs (D’Angelo et al., 2006), a pathway of selfassembly clearly exists. Many key players and steps in this process are now known, although significant gaps remain to be elucidated.

5.1. Building a nuclear pore: Who’s on first? One strategy to set the stage for understanding NPC formation has been to delineate the order of nucleoporin recruitment during post-mitotic nuclear assembly (Bodoor et al., 1999; Haraguchi et al., 2000). Certain nucleoporins are thought to be present, albeit initially on a restricted region of the chromatin surface, from the very beginning since they reside at the kinetochore during mitosis (see Section 3.2). These include the Nup107-Nup160 complex (Belgareh et al., 2001) and a newly identified associated protein, ELYS/MEL28, as well as RanBP2/Nup358 ( Joseph et al., 2002). Broader recruitment of Nup107 and Nup133 has been reported to also occur very early—during anaphase, similar to Nup153 and before Nup62 (Belgareh et al., 2001). Membrane recruitment naturally brings with it integral membrane proteins of the NPC, although notably the recruitment of transmembrane proteins (or at least their stable association with the nuclear rim) does not occur simultaneously. Recruitment of POM121 and likely Ndc1, which appears to localize to the same vesicle population in Xenopus egg extracts (Mansfeld et al., 2006), is an early event, whereas gp210 does not accumulate until later in the nuclear assembly process (Bodoor et al., 1999). mAb414 reactivity is detected relatively late during nuclear reconstitution in egg extracts (Antonin et al., 2005); although this antibody recognizes at least four nucleoporins, the bulk of its reactivity usually reflects Nup62 levels. Nup214 was noted to arrive after Nup62 when these nucleoporins were tracked individually in NRK cells (Bodoor et al., 1999). Nup155 arrives relatively late in the assembly process as well, although in its absence nucleoporins that get recruited earlier do not accumulate at the rim, suggesting that Nup155 plays a critical role in stabilizing interactions that lead to NPC formation (Franz et al., 2005). Tpr is also a late-arriving

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nucleoporin (Bodoor et al., 1999; Haraguchi et al., 2000; Hase and Cordes, 2003), but in this case its presence is dispensable for the core structure of the NPC (Frosst et al., 2002; Hase and Cordes, 2003; Shibata et al., 2002). Another approach to understanding the early steps of nuclear pore formation has been to observe this process at high resolution, using electron microscopy. Analysis of nuclear assembly using transmission EM led to the notion that an intermediate structure, termed a ‘‘pre-pore’’, forms on the surface of chromosomes independent of membranes (Sheehan et al., 1988). Further visual analysis in both Xenopus and Drosophila systems, using scanning EM, has provided a more detailed conceptual framework of the structural stages of pore assembly (Goldberg et al., 1997; Kiseleva et al., 2001). The molecular composition of NPC assembly intermediates is not yet known, although there is speculation that the Nup107-160 complex is a good candidate for forming a pre-pore-type structure (Walther et al., 2003a). Revisiting these structures in combination with immuno-detection techniques will result in a more integrated picture of nucleoporin recruitment and the step-wise assembly of the NPC (Drummond et al., 2006).

5.2. Nuclear pore building blocks: The transmembrane proteins Integral membrane proteins of the nuclear pore are predicted to play unique and essential roles in NPC formation, as such proteins seem likely to be involved in facilitating creation of the pore itself (or, described from a different perspective, in joining the inner and outer nuclear membranes) and in anchoring the soluble NPC building blocks to this site of the NE. In the Xenopus egg extract system, depletion of POM121-containing vesicles results in an early block to NE assembly: vesicles appear to bind the chromatin, but do not fuse (Antonin et al., 2005). This phenotype precludes direct analysis of the role for POM121 in nuclear pore formation. Nonetheless, an interesting layer of regulatory cross-talk between POM121 and the Nup107-160 subcomplex was observed (see below). Knockdown approaches in mammalian cells have not led to a unified view on the role of POM121; the degree of impairment in NE/NPC assembly may depend on the extent of depletion (Antonin et al., 2005; Imreh et al., 2003; Stavru et al., 2006b). In any case, the observation that POM121 appears to be restricted to vertebrates indicated a priori that another transmembrane protein would likely play a pivotal role. Indeed, the integral membrane protein Ndc1 (Chial et al., 1998) has emerged in several studies as an important player in NPC assembly (Lau et al., 2004; Madrid et al., 2006; Mansfeld et al., 2006; Stavru et al., 2006a). Even depletion of Ndc1, however, does not lead to an absolute defect in NPC assembly; this was best illustrated by a C. elegans strain bearing a deletion that disrupts the ORF of NDC1. This mutant strain has high embryonic and larval mortality and dramatically

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reduced mAb414 reactivity at the nuclear rim, but rare survivors can be propagated (Stavru et al., 2006a). RNAi-directed depletion of the third metazoan transmembrane protein, gp210, in mammalian cells did not have a significant effect on NPCs in some studies (Eriksson et al., 2004; Mansfeld et al., 2006; Stavru et al., 2006b), but did alter NPC and NE phenotype in another case (Cohen et al., 2003). In one study, simultaneous knockdown of gp210 and Ndc1 was found to have a synergistic effect (Mansfeld et al., 2006). Likewise, in yeast, Ndc1p was found to be partially redundant with POM152p (Madrid et al., 2006). Results in Xenopus egg extracts have been complicated by initial mis-identification of the C-terminus of this orthologue (Antonin et al., 2005; Drummond and Wilson, 2002). In C. elegans, gp210 is required for viability, but the NPC phenotype associated with its depletion is primarily a problem in NPC positioning rather than formation (Cohen et al., 2003). Tissue-specific expression patterns for gp210 (Olsson et al., 2004) indicate that it may be more likely to modulate rather than to dictate NPC structure. One overriding theme of these studies is that redundancy is built into the NPC assembly pathway, ensuring that NPC formation is a robust biological process (Kitano, 2004; Stavru et al., 2006a).

5.3. Collaboration between nucleoporins in NPC assembly Understanding how the transmembrane components of the nuclear pore are linked to the soluble pore building blocks is key to understanding NPC assembly. One such connection exists between Ndc1 and Nup35 (sometimes referred to as Nup53 as it is the homologue of yeast Nup53p), which associate in a manner independent from the Nup35-Nup93 interaction (Mansfeld et al., 2006). Nup35 itself has been implicated as an important player in NPC assembly (Hawryluk-Gara et al., 2005). Although this nucleoporin is not a transmembrane protein, it appears to be in close apposition to the nuclear membrane and associates with lamin B. Indeed, overexpression of the yeast homologue, Nup53p, causes accumulation of extramembrane structures within the nucleus, in which Nup53p is found (Marelli et al., 2001). Ndc1p is also targeted to these extra-membrane structures and the C-terminal region of Nup53p that is functionally implicated in membrane recruitment is also important for the interaction with Ndc1p. A complex relationship appears to exist between the Nup107-160 complex and POM121 during NE formation. The observations are 1) when the Nup107-160 complex is depleted, POM121 is no longer recruited to the NE during assembly (Harel et al., 2003b) and 2) the arrest in NE assembly seen when membrane vesicles are depleted of POM121 is dependent on the Nup107-160 complex (Antonin et al., 2005). Although there is no evidence for a direct interaction between the Nup107-160 complex and POM121

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itself, these interesting interdependent phenotypes have been proposed to suggest that chromatin-associated Nup107-160 complex exerts negative feedback on NE formation, which is relieved by the presence of POM121. Whatever the exact nature of this regulatory relationship, the Nup107160 complex is clearly playing an important collaborative role in creating the NPC. When this NPC subunit is depleted, the membranes that enclose chromatin in an in vitro nuclear assembly system completely lack NPCs (Antonin et al., 2005; Harel et al., 2003b; Walther et al., 2003a) and knockdown of Nup107 in mammalian cells leads to severe NPC defects as well (Boehmer et al., 2003; Walther et al., 2003a). This NPC subunit is thought to eventually create an important core aspect of the nuclear pore. Interestingly, this complex, which shares two members with COPII and possesses additional general features similar to coatomer complexes, is hypothesized to form a coat-like structure at the pore membrane (Devos et al., 2004; see also Antonin and Mattaj, 2005).

5.4. Peripheral pore structures Tpr is proposed to be the critical building block of the nuclear pore basket in vertebrates, with its recruitment to the NPC dependent on Nup153 (Frosst et al., 2002; Hase and Cordes, 2003; Krull et al., 2004). The role of Nup153, however, may be complicated by the presence of more than one population of this nucleoporin at the NPC. One possibility is that a stably associated population of Nup153 at the nuclear ring moiety is dedicated to tethering Tpr to form the core basket, while another population dynamically associates with the NPC (Ball and Ullman, 2005; Fahrenkrog and Aebi, 2003). The basket structure itself is conserved in yeast (Fahrenkrog et al., 1998; Kiseleva et al., 2004), although its composition is not welldefined in this organism. Tpr homologues in S. cerevisae, Mlp1p/Mlp2p, have been described as being on intranuclear filaments connected to the nuclear pore (Strambio-de-Castillia et al., 1999), leaving open the question of how the basket structure itself is formed in this case. These proteins may have the potential for dual (multiple) localization, as Tpr has been reported to be on intranuclear filaments/channels in certain metazoan cells (Cordes et al., 1997; Fontoura et al., 2001; Zimowska et al., 1997) and Mlp2p binds to the spindle pole body in yeast (Niepel et al., 2005). Based on knockdown analysis in cultured human cells, the partner proteins Nup214 and Nup88 are proposed to play a key role in recruiting RanBP2/Nup358 to create the cytoplasmic filaments of the NPC (Bernad et al., 2004). This does not seem to be the case in Drosophila cells, where knocking down Nup214 leaves functions ascribed to RanBP2/Nup358 intact (Forler et al., 2004). Likewise in nuclei assembled in Xenopus egg extract, depletion of Nup214 does not interfere with RanBP2/Nup358 targeting to the NPC (Walther et al., 2002). Corresponding ultrastructural

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analysis suggested that Nup214 and RanBP2/Nup358 localize to distinct structures on the cytoplasmic face of the NPC (Walther et al., 2002). The apparent discrepancy in the interdependence of Nup214 and RanBP2/ Nup358 targeting may be due to the role of Nup88, which is proposed to anchor each of these nucleoporins. Nup88 levels are closely linked to those of Nup214, but in certain cases, Nup88 may be present in enough excess to recruit RanBP2/Nup358 to the NPC when Nup214 (and certain amounts of Nup88) are depleted. A central role for Nup88 in organizing the features of the cytoplasmic face of the NPC is underscored by its role in anchoring Nup98 to this site as well; in contrast, interactions with Nup96, a member of the Nup107-160 complex, target Nup98 to the nuclear face of the nuclear pore (Griffis et al., 2003). Only a fraction of known interactions between nucleoporins have been highlighted here, but almost all of such contacts ultimately contribute to NPC assembly. Gaining an even more complete map of this interaction network is an important step in understanding the process of NPC assembly and its overall structure.

5.5. Regulation of NPC assembly NPC assembly is regulated at several levels. One particularly striking observation is that the calcium chelator BAPTA completely prevents NPC assembly in the Xenopus nuclear reconstitution assays (Macaulay and Forbes, 1996). The early observation that EGTA does not phenocopy BAPTA (Macaulay and Forbes, 1996) suggests that it is not absolute calcium levels, but rather a burst of calcium, which may not be quickly enough quenched by EGTA, that is required during NPC assembly. Whether this reflects a calcium flux requirement for inner and outer membrane fusion or some earlier step has not been formally addressed. Among many possibilities, calcium could be involved in a SNARE-related event (Baur et al., 2007) or in modulating the calciumbinding protein Cdc31p/centrin, which was identified as a component of yeast nuclear pores (Rout et al., 2000) but not found, as yet, at the vertebrate NPC. A role for calcium, though intriguing, is ill-defined and there is evidence against a requirement for lumenal calcium stores during the process of NE/ NPC assembly (Marshall et al., 1997). Indeed, the dearth of information on the molecular events that underlie BAPTA inhibition leave open the possibility that this small molecule in fact exerts its effects on nuclear pore assembly via a mechanism distinct from calcium chelation. Whatever the mechanism, an important clue may lie in the observation that depletion of the Nup107-160 complex gives rise to a similar morphological phenotype as BAPTA inhibition. In addition to post-translational modification, which will be discussed below, association with transport receptors has proven to provide another important layer of regulation for nucleoporins. Considered in this context, the transport receptors serve as chaperones and, in doing so, guide the spatial

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and temporal order of nuclear pore protein recruitment. This chaperone role of transport receptors has also been proposed to be important to formation of an FG-domain based hydrogel within the confines of the NPC (Frey and Gorlich, 2007). Just as in the case of nucleocytoplasmic trafficking, the small GTPase Ran works as a switch to regulate nucleoporin association with importin b, the transport receptor best studied in the context of nuclear pore assembly. In fact, this role of Ran is intimately linked to the role of chromosomal DNA as the surface on which the NE and NPCs are assembled. This is because the guanine nucleotide exchange factor (GEF) for Ran, RCC1, is targeted to chromatin and its activity is stimulated by histones (Nemergut et al., 2001). Thus, RanGEF activity is high in the vicinity of chromosomes, in turn creating a gradient of RanGTP even in the absence of a nuclear membrane (Kalab et al., 2002, 2006; see also Gorlich et al., 2003). RanGTP modulates the binding activity of importin b, promoting the release of associated proteins, such as Nup107, Nup153, and RanBP2/Nup358 (Walther et al., 2003b), as well as (presumably) other factors required for fusion of the NE and assembly of nuclear pores. Yet, additional regulation of importin b appears to be at play: RanQ69L, which reverses the inhibitory effects of excess importin b on nuclear membrane fusion, does not reverse its ability to inhibit NPC insertion in a preassembled NE (Harel et al., 2003a). A role for importin b, and for Ran, in NPC assembly is also found in yeast (Ryan et al., 2003, 2007), suggesting similarity in fundamental regulatory mechanisms despite certain differences in NPC assembly due to the open vs. closed configuration of mitosis. A chaperone-like role is not restricted to importin b: in yeast, Kap121p helps to target Nup53p and is involved in NPC remodeling at mitosis (Lusk et al., 2002; Makhnevych et al., 2003); transportin may aid in escorting Nup153 to the NPC (Nakielny et al., 1999). Other components of the reforming nucleus impinge on nuclear pores as well. This was recently illustrated by the observation that patches of newlyformed NEs are initially pore-free and correspond to regions that are enriched in underlying lamin A/C and have lower levels of lamin B (Maeshima et al., 2006) (see Section 2.3). Whether this is a case of inhibiting NPC assembly in particular regions or of preventing NPC anchorage in certain domains of the lamina network remains to be addressed. And, in either case, the molecular mechanism and mediating proteins have yet to be explored. Nonetheless, this report underscores the many levels of nuclear assembly that are simultaneously orchestrated and cross-regulated.

5.6. Clues from a second site for NPC assembly Although chromatin is typically the favored scaffold on which to build NPCs, an alternate site for NPC assembly exists in the annulate lamellae (AL). These cytoplasmic membrane cisternae house tightly arrayed NPCs.

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AL formation is more pronounced in rapidly proliferating cells and has been proposed to be a storage site for NPC components. These NPC-like complexes might seem poised to contribute to NPC formation at the NE itself during membrane expansion, but at least under certain circumstances, this does not appear to be the case. Specifically, in Drosophila embryos the excess nucleoporins, presumably the stores for new NPC formation, were found to be largely soluble rather than AL associated (Onischenko et al., 2004). There was no decrease in AL-associated pore complexes concomitant with increases in NPC numbers at the NE. Although NPC formation at the AL may be a separable event, rather than a prelude to the appearance of NPCs at the NE, understanding what initiates this chromatin-independent assembly process and how it differs at this site is a way of gaining insight into the pathways that converge to create NPC structure. Experimental manipulations that lead to increased AL formation include increasing levels of RanGTP (Harel et al., 2003a; Walther et al., 2003b). Similarly, Ran-coated beads are sufficient to direct formation of double membrane bilayer replete with nuclear pores (Zhang et al., 2002), consistent with the notion that local levels of RanGTP direct NE/NPC formation to the chromatin surface and are capable of driving this process at other sites as well. Another interesting observation is that inhibition of microtubule formation and/or kinesin function prevents NPC assembly at the NE, but does not perturb formation of a nuclear envelope or NPC-containing AL (Ewald et al., 2001). This suggests that delivery of certain components, perhaps a vesicle population, to the chromatin surface is facilitated by microtubules whereas this same component is either not needed at the AL or is incorporated independently of microtubules.

5.7. Nuclear pore assembly is never-ending Beyond the fact that new NPCs are assembled throughout much of the cell cycle, the dynamic nature of NPC components reveals that this structure is not assembled to a static end-point but rather is continuously remodeled. Dynamic association with the NPC was first observed for Nup153 (Daigle et al., 2001) and for Nup98 (Griffis et al., 2002). An extensive survey of this property with respect to nucleoporins later revealed that several nucleoporins move on and off the NPC structure (Rabut et al., 2004; Tran and Wente, 2006). Interestingly, this movement has been shown in certain cases to be dependent on ongoing transcription (Griffis et al., 2002, 2004). NPC structure has additional layers of dynamics as well. For instance, largescale conformational rearrangements have been observed by scanning EM (Goldberg et al., 2000; Kiseleva et al., 1996, 1998), by AFM (Shahin et al., 2001, 2005), and by CTE (Beck et al., 2004). In addition, domains within individual pore proteins have been found to be (or have the potential to be) flexibly arranged within the NPC (Fahrenkrog et al., 2002; Paulillo et al.,

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2005, 2006; Schwarz-Herion et al., 2007), indeed, the dynamic arrangement of these domains is likely central to trafficking and the selectivity of the nuclear pore (see Section 4). Functional alterations of the NPC, such as the greater upper limit of cargo diameter in proliferating vs. quiescent cells, may also reflect specific reconfiguration of NPC structure (Feldherr and Akin, 1990, 1991).

5.8. Deconstructing the NPC Several pore proteins are targets of phosphorylation at mitosis. These include both integral membrane proteins of the NPC, such as Ndc1 (Mansfeld et al., 2006; Stavru et al., 2006a) and gp210 (Favreau et al., 1996), as well as nonmembrane anchored, such as Nup35, Nup153, Nup98, Nup62, Nup214, and Nup107-160 complex members (Belgareh et al., 2001; Glavy et al., 2007; Lusk et al., 2007; Macaulay et al., 1995; Walther et al., 2003b). A phosphorylation-dephosphorylation switch has long been thought to aide in driving NPC disassembly and re-assembly. Indeed, when isolated nuclei from Drosophila embryos are incubated with cdc2-cyclin, several pore proteins are released (Onischenko et al., 2005). It is difficult to formally prove that this is due to nucleoporins being direct targets of cdc2-cyclin, but clearly they are responsive to a mitotic signaling cascade driven by phosphorylation events. Hallberg and colleagues also recently demonstrated that a phosphomimetic mutation in gp210, at a serine (1880) known to be phosphorylated at mitosis, interfered with its incorporation into the NPC (Onischenko et al., 2007). Consistent with these observations, phosphatase activity is implicated in the process of post-mitotic nuclear pore assembly. In Drosophila embryos, okadaic acid inhibits NPC assembly, suggesting that PP1 and/or PP2A are involved (Onischenko et al., 2005). In certain organisms, nuclear pore remodeling at mitosis is limited to discrete, but still significant, rearrangements. In Aspergillus nidulans these changes lead to an increase in the diffusion cut-off of the NPC, whereas in S. cerevisae mitotic changes at the nuclear pore appear more subtle and selectively alter particular transport paths (De Souza et al., 2004; Marelli et al., 1998). In organisms that undergo open mitosis, changes in the diffusion cut-off of the NPC herald the prophase-to-prometaphase transition (Lenart and Ellenberg, 2006; Lenart et al., 2003) and correspond to an early wave of nucleoporin exit from the NPC (Lenart et al., 2003), perhaps stimulated by phosphorylation. Extensive dispersal of the NPC components ensues and, concomitantly, the membranes that enclose the nucleus are also remodeled, allowing complete intermixing between cytoplasmic and the nuclear space. Although Ran is not implicated in the initial steps of NPC remodeling, it has recently been shown to have a regulatory role in mitotic nuclear membrane remodeling (Muhlhausser and Kutay, 2007).

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Mitotic membrane remodeling brings up the question of the fate of integral membrane proteins of the nuclear pore at mitosis. Direct observation of a GFP fusion with the inner nuclear membrane protein LBR in COS-7 cells indicated that this protein is ultimately intermixed with endoplasmic reticulum (ER) at mitosis (Ellenberg et al., 1997), as was also the case for two other residents of the inner nuclear membrane (LAP1 and LAP2) as well as for the nucleoporin gp210 (Yang et al., 1997). This intermixing could be explained by a passive process in which interactions that normally serve to keep the nuclear membranes distinct from the ER are lost at mitosis, allowing lateral diffusion between these membrane domains. In another study, however, LAP1 and LAP2 were found to have distinct localizations at mitosis (Maison et al., 1997). Whether differing results are due to the timing of detection within mitosis or to the specific isoforms detected or to some technical issue is not clear. There is independent evidence from HeLa cells for distinct vesicle populations (Chaudhary and Courvalin, 1993); however, an alternate interpretation is that such vesicles are derived artificially from microdomains within a contiguous ER-like network (Collas and Courvalin, 2000). Studies using nuclei reconstituted in Xenopus egg extract have implicated the coatomer complex, COPI, in nuclear disassembly (Cotter et al., 2007; Liu et al., 2003; Prunuske et al., 2006). Given the role of COPI in forming vesicles at the Golgi (Bethune et al., 2006), this brings up the possibility of a more active mechanism for dismantling the nuclear membrane and, at least in some cases, delivering it to the ER. It is possible that COPI-mediated vesicle formation occurs only local to the nuclear pores and represents one of two distinct routes of membrane disassembly (the second being passive lateral diffusion). Indeed, Allen and colleagues saw evidence of both vesicles and ER-like tubules when they examined the mitotic breakdown of reconstituted nuclei by field emission scanning EM (Cotter et al., 2007). Vesicles enriched in certain proteins such as POM121 and Ndc1, but not necessarily the highly mobile pore membrane protein gp210, may have escaped observation in earlier studies, but could account for the presence of distinct vesicle populations present in Xenopus egg extract. Alternatively, COPI components may play a noncanonical role, perhaps participating in the establishment or maintainance of microdomains within the ER network. Interestingly, impairing the function of many nucleoporins results in NE abnormalities, and likewise alterations in particular membrane proteins that reside in the NE/ER results in phenotypic differences at the NPC (Franz et al., 2005; Lewis et al., 2007; Liu et al., 2007; Miao et al., 2006; Stavru et al., 2006a). A recent example of the latter is the protein Apq12p; when this gene is deleted in yeast, NPCs appear to be embedded in only the inner nuclear membrane (Scarcelli et al., 2007). This advance in identification of important players in proper NPC assembly in fact exposes the general gap in our understanding of how fusion between the inner and outer nuclear

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membranes is coordinated as the core framework of the NPC coalesces. Future study of membrane dynamics at the nuclear envelope and how this is integrated with NPC assembly and disassembly will yield new insight into this important central question.

6. Concluding Remarks Although it is not yet possible to build a complete molecular picture of the nuclear pore complex, the structures of individual nucleoporins and NPC subcomplexes are indispensable to our growing understanding of NPC assembly and nucleocytoplasmic transport. Being able to merge this information with CET on intact nuclei will allow for detection of distinct functional states of the NPC and may reveal changes in the configuration of individual NPC subcomplexes during nucleocytoplasmic transport. Single molecule approaches in combination with immunogold-labelling will provide additional insight into the molecular composition of individual NPCs and variation among tissues or even from NPC to NPC. Newer structural and biophysical techniques in combination with molecular simulations will be required to elucidate even finer molecular details of the NPC, such as the identity of the anchoring sites and the exact numbers of FG-repeat domains per NPC and FG-repeat domain behavior in a cellular context. Recent data also points to intriguing roles for nucleoporins that are distinct from, but perhaps coordinated with, their roles at the NPC. As nucleoporins are studied in further detail, it is likely that the scope of their roles will continue to hold surprises. Finally, as more detailed knowledge of how the NPC is put together emerges, this will go hand-in-hand with a deeper understanding of the molecular architecture of the NPC and in turn will aide in building a comprehensive model of trafficking through the NPC.

ACKNOWLEDGMENTS This work was supported by a grant from the Swiss National Science Foundation (to B.F.), the M.E. Mu¨ller Foundation and the Kanton Basel Stadt, and from the N.I.H. (GM61275) and the Leukemia and Lymphoma Society (to K.U.).

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Endocytosis and the Actin Cytoskeleton in Dictyostelium discoideum Francisco Rivero* Contents 1. Introduction 2. Tools to Study Endocytosis 3. Role of D. discoideum Actin and ABPs in Endocytosis 3.1. Actin 3.2. The F-actin nucleation machinery 3.3. Monomeric actin binding proteins 3.4. Severing and capping proteins 3.5. Actin crosslinking proteins 3.6. Lateral ABPs 3.7. Membrane-associated ABPs 3.8. Actin-based molecular motors 4. Molecular Events During the Uptake Phase: A Simplified Model 5. Concluding Remarks Acknowledgments References

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Abstract Endocytosis, an essential process of all eukaryotic cells, requires the actin cytoskeleton for proper functioning. The soil amoeba Dictyostelium discoideum is well known for its contribution to the actin cytoskeleton field. The genetic tractability and the availability of appropriate tools have made of Dictyostelium an attractive model for studies of endocytosis and vesicle trafficking as well. These tools include a large palette of fluorescent protein fusions and the combination of improved fractionation methods with high throughput techniques along with the recently propagated use of the amoeba a host for microbial pathogens. In this review I discuss in a comprehensive manner the evidence accumulated in the literature towards a participation of components of the

*

The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, United Kingdom

International Review of Cell and Molecular Biology, Volume 267 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)00633-3

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2008 Elsevier Inc. All rights reserved.

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microfilament system of D. discoideum in endocytic trafficking and conclude with a model that describes the sequence of events and the components involved during the well-investigated uptake phase of the endocytic process in the soil amoeba. Key Words: Dictyostelium, Actin, Actin-binding protein, Phagocytosis, Pinocytosis, Cytoskeleton. ß 2008 Elsevier Inc.

1. Introduction Endocytosis, the uptake of fluid or solid phase material, is an essential process of all eukaryotic cells. In single cell organisms, such as Dictyostelium discoideum, endocytosis serves primarily a nutritive function in vegetative cells, whereas in the multicellular stage a small population of sentinel cells serves a defensive purpose (Chen et al., 2007). Dictyostelium is a notorious professional phagocyte: cells are able to engulf and internalize particles of various sorts and sizes, ranging from bacteria to yeast and apoptotic cells of the same species, as well as synthetic beads. Commonly used laboratory strains are also able to grow in synthetic liquid media. In Dictyostelium the bulk of fluid is taken up by macropinocytosis, which takes place at crownlike protrusions apparent at the dorsal and lateral cell surface of adherent cells (Hacker et al., 1997). Additional pathways of fluid uptake also exist in Dictyostelium, like clathrin-dependent or independent micropinocytosis, but they are less well understood and will not be considered further (Neuhaus et al., 2002; O’Halloran and Anderson, 1992). The endocytic pathway of Dictyostelium has been operationally divided into three major steps: uptake at the plasma membrane, transit through endosomal compartments and finally release of indigestible components by exocytosis (Maniak, 2002, 2003). The first step consists in the formation of a cell surface protrusion that engulfs a particle or an aliquot of surrounding medium, involves a considerable amount of signaling and is driven by actin remodeling. This step is accomplished very rapidly: within approximately 1 min after internalization the actin coat that surrounds the nascent endosome begins to dissociate and the endosome is captured by peripheral microtubules. During its transit through the endosomal pathway the endosome matures, a process that requires numerous fusion and fission events. Concomitantly with the dissociation of the actin coat, fusion with vesicles that carry vacuolar HþATPases results in acidification of the endosomal lumen, lasting for approximately 30 min. Several sets of lysosomal enzymes are then delivered, allowing digestion of the endosomal contents. The vacuolar HþATPase and the lysosomal enzymes are retrieved and recycled. The pH of the endosome returns to a neutral value, which enables homotypic fusion. Large neutral endosomes form that acquire a coat of filamentous actin. The release of the late endosomal content by exocytosis occurs

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then within seconds. Judging from population studies, the whole process takes about one hour, but recent studies based on tracking of individual cells are beginning to challenge this view, indicating that at least early steps of endosome maturation occur more rapidly than inferred from population studies (Clarke and Maddera, 2006). In what follows I will present in a comprehensive manner the evidence that has accumulated in the literature towards a participation of components of the microfilament system of D. discoideum in endocytic trafficking (Tables 8.1 and 8.2). Details on structure, biochemical activities and other functions of these proteins not given here can be found in (Rivero and Eichinger, 2005). Due to space constrains, aspects like signaling to the cytoskeleton (in particular by small GTPases) and other signaling pathways as well as the role of calcium and phospholipids, which are important regulators of numerous ABPs, are not treated here in detail. This aspects have been covered elsewhere (Cardelli, 2001; Maniak, 2002; Vlahou and Rivero, 2006). More recently Dictyostelium has begun to be used as a model system for the study of pathogenic bacteria that manipulate the endocytic pathway to create a favorable environment for replication and dissemination (Farbrother et al., 2006; Steinert and Heuner, 2005). The relevant information has been incorporated into Tables 8.1 and 8.2, but this aspect is not treated in depth here. To close this review I will make an attempt to integrate most of the information into a model that describes the sequence of events and the components involved during the uptake phase, which is the best investigated phase of the endocytic process in Dictyostelium.

2. Tools to Study Endocytosis One fundamental advantage of Dictyostelium discoideum as a model organisms for studies of endocytosis and vesicle trafficking, apart from the genetic tractability and the availability of a fully sequenced and assembled genome, is the availability of tools that allow the investigation of almost every step of the endocytic pathway. Laboratory strains of Dictyostelium relay on an efficient uptake of both particles and fluid as the predominant way to bring into the cell nutrients needed for growth and multiplication, therefore measuring the growth rate in a bacterial suspension or in axenic medium or the expansion of a plaque on a bacterial lawn are first approximations to whether a strain is capable of normal endocytosis or not. The major drawback of these determinations is that the rate of cell multiplication also depends on the efficiency of cell division, which may be controlled independently of endocytic performance. For a precise analysis, it is therefore instrumental to quantify the uptake of cargo directly. Phagocytosis is commonly quantitated using fluorescent particles (latex beads, yeasts or bacteria), whereas pinocytosis, exocytosis and changes in endosomal pH are most frequently quantitated

Table 8.1 Cytoskeleton components reportedly or putatively involved in endocytosis in Dictyostelium Protein Class

Gene

Monomeric actin-binding Profilin proA, (I and II) proB

CAP

cap

Capping and/or severing Capping protein acpA, (Cap32/34, acpB aginactin)

Cofilin 1

cofA

Severin

sevA

GRP125

gnrA

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Phagocytic cup (IF, beads). Isolated phagosomes.

Normal (double KO; latex beads)/ increased (double KO; latex beads, bacteria)

Decreased (double KO)

Decreased (double KO)

Decreased (double KO)

Cortex (IF, GFP).

Normal (KO; bacteria, yeast)

Decreased (KO)

ND

ND

Cortex.

ND

ND

ND

ND

Crowns, phagocytic cup (IF, GFP, beads). Isolated phagosomes. Phagosomes (IF, bacteria). Isolated phagosomes. Vesicles (IF).

Normal (OE; latex beads)

ND

ND

ND

ND

ND

ND

ND

KO described but not assayed.

ND

ND

ND

ND

KO described but not assayed.

Notes

Single KOs unimpaired. KO cells are more susceptible to infection with Legionella and Legionella-like bacteria. proA gene downregulated at 3 and 6 hours upon infection with Legionella.

Localization inferred from studies in A. castellani. No KO of any of the subunits described; OE and UE strains described but not assayed. KO is lethal.

vilA

Golgi, ER vesicles (GFP, IF).

Normal (KO; yeast)

Normal

ND

ND

KO showed reduced uptake and intracellular growth of pathogenic Legionella.

arpB, arpC, arcA, arcB, arcC, arcD, arcE

Cortex, crowns, macropinosomes, phagocytic cup (yeast), postlysosomal endosomes (IF, fluorescent proteins). Isolated phagosomes (Arp3)

ND

ND

ND

ND

Scar

scrA

Cytosolic, weakly cortical (IF, GFP).

Decreased (KO; beads, bacteria)

Decreased (KO)

Decreased (KO)

Decreased (KO)

No KO of any of the subunits described. An Arp2 hypomorphic strain described but not assayed. GFP fusions of Arp3 and p41-Arc, mRFP fusion of p41-Arc, and antiArp3 and anti-p21-Arc antibodies used to monitor the complex. Additive defects in triple mutants of Scar and profilins I and II.

CARMIL VASP

carmil vasP

Crowns (IF). Tips of filopods (GFP).

Decreased (KO) ND

ND ND

ND ND

Formin C

forC

Crowns (GFP).

ND Normal (KO; beads) ND

ND

ND

ND

Formin H

forH

Tips of filopods (GFP).

ND

ND

ND

ND

Cortex, phagocytic cup (IF; bacteria, beads). Isolated phagosomes.

Increased (KO; yeast)

Normal (KO)

ND

ND

Villidin

Nucleation Arp2/3 complex

Cross-linking ABP34 abpB

Reduced particle (beads) adhesion in KO. KO strain described but not assayed. KO strain described but not assayed. Effector for Rac1a. Reduced pinocytosis in ABP34/a-actinin double KO. Normal pinocytosis in ABP34/filamin double KO. Increased phagocytosis (yeast), normal pinocytosis and exocytosis in ABP34/ fimbrin double KO.

(continued)

Table 8.1

(continued)

Protein Class

Gene

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Fimbrin

fimA

Cortex, phagocytic cup (IF, GFP; yeast), macropinosomes (IF, GFP).

Normal (KO)

Normal (KO)

Normal (KO)

ND

Filamin (gelation factor, ABP120)

abpC

Cortex. Phagocytic cup (IF, beads).

Decreased (KO, beads, bacteria)/ normal (KO) (depending on parental strain)

Normal (KO)

ND

ND

a-actinin

abpA

Late phagosomes (IF). Isolated phagosomes (weakly or absent).

Normal (KO)

Normal (KO)

ND

ND

Cortexillin 1 and 2

ctxA, ctxB

Cortex, crowns (IF, GFP). Isolated phagosomes.

ND

ND

ND

ND

Notes

Increased phagocytosis (yeast), normal pinocytosis and exocytosis in ABP34/ fimbrin double KO. Reduced phagocytosis (bacteria) and pinocytosis in filamin/ a-actinin double KO. Normal pinocytosis in ABP34/filamin double KO. Reduced uptake and intracellular growth of L. pneumophila in filamin/a-actinin double KO. Reduced phagocytosis (bacteria) and pinocytosis in filamin/ a-actinin double KO. Reduced pinocytosis in ABP34/a-actinin double KO. Reduced uptake and intracellular growth of L. pneumophila in filamin/a-actinin double KO. KO (singles and double) reported but not assayed.

Dynacortin

dct

eEF1A (ABP50) efaAI eEF1B

efa1B

Lateral actin-binding Coronin corA

Aip

wdpA

Coactosin

coaA

Abp1 LimC

abpE limC

LimD1 (LimD) limD1

Cortex, crowns (IF, GFP).

ND

ND

ND

ND

Cortex (IF). Isolated phagosomes. Cortex (IF). Isolated phagosomes.

ND

ND

ND

ND

Both genes upregulated after 3 hours upon infection with L. pneumophila. Strain expressing almost no dynacortin (gene silencing) described but not assayed. No KO described.

ND

ND

ND

ND

No KO described.

Phagocytic cup (IF, GFP; yeast, bacteria), crowns, macropinosomes (IF, GFP) Isolated phagosomes. Post-lysosomal vesicles. Phagocytic cup (IF, GFP), macropinosomes (IF, GFP). Isolated phagosomes.

Decreased (KO; yeast, bacteria)

Decreased (KO)

ND

ND

Reduced uptake of L. pneumophila in KO, but more permissive for intracellular growth of L. pneumophila and Mycobacterium marinum.

Decreased (KO; yeast) Increased (OE, yeast) ND

Decreased (KO) Normal (OE)

ND

ND

ND

ND

ND

Cortex (IF). Cortex, macropinosomes, phagocytic cup (GFP; yeast).

Normal (KO) Normal (yeast; KO)

Normal (KO) Normal (KO)

ND ND

ND ND

Cortex, macropinosomes, phagocytic cup (GFP; yeast). Isolated phagosomes.

Normal (KO; yeast)

Normal (KO)

ND

ND

No KO, no localization studies. Double KO limC/limD also unimpaired. Reduced uptake and growth of L. pneumophila in double KO. Double KO limC/limD also unimpaired. Reduced uptake and growth of L. pneumophila in double KO.

(continued)

Table 8.1 Protein Class

(continued)

Gene

LimE (DdLim) limE

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Cortex, macropinosomes (IF, GFP), Phagocytic cup (GFP; yeast). Vesicles, phagocytic cups (YFP, yeast).

ND

ND

ND

ND

KO and OE strains described but not assayed.

Decreased (KO; yeast) Increased (OE yeast) Increased (KO; yeast) Decreased (OE; yeast)

Normal (KO, OE)

ND

ND

Double KO limF/CH-lim behaves like limF null.

Normal (KO, OE)

ND

ND

Double KO limF/CH-lim behaves like limF null.

KO and OE strains described but not assayed. MhkA gene downregulated at 6 hours upon infection with L. pneumophila. Effector for Rac1a, not RacC, RacE.

LimF

limF

CH-Lim

ChLim

Cortex, vesicles, phagocytic cups, phagosomes (GFP, yeast).

MHCK A

mhkA

Macropinosomes (GFP), phagocytic cup (IF; yeast).

ND

ND

ND

ND

DGAP1

rgaA

Cortex (IF, GFP).

ND

ND

ND

RacGEF1

gxcA

Cortex (GFP).

Normal (KO; yeast) Decreased (OE; yeast) ND

ND

ND

ND

Notes

KO strain and diverse overexpressors described but not assayed. Exchange factor for RacB, weakly for Rac1b, not for RacC, RacE, RacG.

Trix

gxcB

Cortex (GFP; yeast).

Normal (KO)

ND

Decreased (KO)

ND

GxcDD

gxcDD

ND

Normal (KO, yeast)

Normal (KO)

ND

ND

Cortex (GFP), tips of filopods.

Decreased (KO; yeast, bacteria, beads)

Normal (KO)

ND

ND

Membrane-associated Talin A talA (filopodin)

Comitin

comA

Golgi, vesicles (IF). Isolated phagosomes.

Decreased (yeast, bacteria); normal (latex beads) (KO)

Normal (KO)

Normal (KO)

ND

Ponticulin A

ponA

Normal (KO; bacteria, beads)

Normal (KO)

ND

ND

Ponticulin B Annexin C1

ponB nxnA

ND Normal (KO)

ND Normal (KO)

ND ND

Annexin C2

nxnB

PM, Golgi vesicles, phagocytic cup (yeast) (IF). PM, vesicles (IF). PM, nucleus, vesicles (IF, GFP). Isolated phagosomes. PM, Golgi, vesicles (GFP).

ND

ND

ND

ND Normal (KO) Reduced on low Ca ND

N-terminal fragment (CH domains) accumulates at (presumably) late endosomes. No exchange factor for Rac1a, RacC, RacE. Interacts with Rac1a, RacA, RacC, RacE, RacH, RacI; not with RacB, RacD. Reduced particle adhesion in KO. TalA gene upregulated at 24 hours upon infection with L. pneumophila. KO more permisive to infections with L. pneumophila and Legionella-like bacteria; delayed degradation of S. enterica.

No KO described.

No KO described.

(continued)

Table 8.1

(continued)

Protein Class

Gene

Localization

Phagocytosis

Pinocytosis

Exocytosis

Secretion of lysosomal hydrolases

Hisactophilins

hatA, hatB

ND

ND

ND

ND

KO and OE strains described but not assayed.

Interaptin

abpD

PM (IF, GFP), phagocytic cup (IF; yeast). Isolated phagosomes. Nuclear envelope, ER, Golgi (IF, GFP).

ND

ND

ND

ND

Motors Conventional myosin (Myosin II)

KO and OE strains described but not assayed.

mhcA

Cortex, vesicles (IF). Phagocytic cup (GFP; yeast). Isolated phagosomes.

Normal (KO, beads)

Normal (KO)

ND

ND

Crowns (MyoB, MyoC, MyoK, IF; MyoE, YFP). Phagocytic cup (MyoB, IF, bacteria and GFP, yeast; MyoE, YFP, yeast; MyoK, IF, yeast). Purified early pinosomes (MyoB).

See Table 9.2

See Table 9.2

See Table 9.2

See Table 9.2

Strong inhibition of phagocytosis and pinocytosis upon inactivation with blebbistatin. MhcA gene upregulated at1 and 3 hours upon infection with L. pneumophila. See Table 9.2

Class I myosins myoA, myoB, myoC, myoD, myoE, myoF, myoK

Notes

Myosin-I

myoI

Cortex, tips of filopods, phagocytic cup (yeast; GFP).

Decreased (beads, yeast, bacteria; KO)

Normal (KO)

Normal (KO)

ND

Myosin-J Myosin-M

myoJ myoM

ND Cortex, crowns, macropinosomes (GFP).

ND Normal (KO)

Normal (KO) ND

ND ND

ND ND

Reduced particle adhesion in KO KO permissive to infection by acapsular strains of the pathogen fungus Cryptococcus neoformans.

The table is a compilation of ABPs reportedly involved in endocytic trafficking based on functional studies or likely to be involved in this process by virtue of their subcellular localization or by analogy with homologs in other species. Note that some Dictyostelium proteins have been described but not analyzed in terms of localization or role in endocytosis, like protovillin, WASP, WIPa, enlazin and most formins, and have therefore been excluded from the table. See main text for references on localization and functional studies. For details on domain architecture and biochemical properties, see Rivero and Eichinger (2005). Additional information can also be retrieved from dictybase (www.dictybase.org) using the gene name. Data on infection studies are compiled from Steinert and Heuner (2005) and Farbrother et al. (2006). Where the protein has been localized at phagosomes and macropinosomes, this is indicated. In many cases this has not specifically been investigated, therefore missing information in the table should not be taken as to imply that the protein is not localized at relevant structures. It should also be considered that, although not indicated in the table, many proteins are (sometimes predominantly) cytosolic but show some enrichment at relevant places. Only localization data determined for the full-length protein has been considered. Data on phagocytosis and pinocytosis is based exclusively on quantitative analyses of particle or fluid phase uptake, not on defects in growth on bacterial lawn, growth in suspension with bacteria or growth in nutrient medium. Where reported, the particle (yeasts, bacteria, latex beads) used for localization and phagocytosis studies is indicated. IF, immunofluorescence; GFP, green fluorescent protein fusion; RFP, red fluorescent protein fusion; YFP, yellow fluorescent protein fusion; PM, plasma membrane; ER, endoplasmic reticulum; KO, knockout strain; OE, overexpressor strain; ND, not determined.

Table 8.2 Mutant strains of class I myosins and endocytosis in Dictyostelium Strai n

Phagocytosis

Pinocytosis

Exocytosis

Othe r phenotypes

References

ND

Normal

ND

(Neuhaus and Soldati, 2000; Novak et al., 1995; Peterson et al., 1995)

myoB

# 30% (bacteria) # 37% (beads)

Normal

Normal/#

myoBþ

ND

# 66%

Normal clearing (5h) of bacterial suspension. Oversecretion of lysosomal enzymes. Normal membrane recycling from early endosomes. Slow growth on bacterial lawn. Normal clearing (5h) of bacterial suspension. Normal intravesicular pH. Normal intracellular retention time. Impaired membrane recycling from early endosomes. Oversecretion of lysosomal enzymes. Deficient pressureinduced rocketing of phagosomes.

ND

myoA



(Clarke et al., 2006; Jung and Hammer III, 1990; Jung et al., 1996; Neuhaus and Soldati, 2000; Novak et al., 1995; Temesvari et al., 1996)

(Novak and Titus, 1997)

myoBþSH3

ND

Normal

ND

myoBþS332A ND

Normal

ND

myoC

# 36% (beads)

Normal

Normal

myoCþ myoD myoEþ

ND Normal " 40% (yeast) ND # 30% (yeast) (adherent cells)

# 60% Normal # 30%

ND ND ND

Normal Normal

ND ND

myoF myoK

Fails to complement the pinocytosis defect of myoA/ myoB. Localization not altered. Fails to complement the pinocytosis defect of myoA/ myoB. Localization not altered. Normal clearing (5h) of bacterial suspension. Normal intravesicular pH. Normal intracellular retention time. Normal secretion of lysosomal enzymes

(Novak and Titus, 1997; Novak and Titus, 1998)

(Novak and Titus, 1997; Novak and Titus, 1998)

( Jung et al., 1996; Novak et al., 1995; Peterson et al., 1995; Temesvari et al., 1996)

(Dai et al., 1999) ( Jung et al., 1996) (Du¨rrwang et al., 2005)

Normal steady state levels of particle uptake

(Titus et al., 1995) (Schwarz et al., 2000)

(continued)

(continued )

Table 8.2 Strain

myoK

þ

myoA/ myoB

myoB/ myoC

Phagocytosis

Pinocytosis

Exocytosis

Other phenotypes

References

# 30% (yeast) (adherent cells) ND

ND

ND

Normal steady state levels of particle uptake

(Schwarz et al., 2000)

# 60%

ND

(Clarke et al., 2006; Neuhaus and Soldati, 2000; Novak et al., 1995; Solomon et al., 2000; Temesvari et al., 1996)

ND

# 60%

Normal

Normal clearing (5h) of bacterial suspension. Slow growth in suspension, low saturation density. Delayed and decreased membrane internalization. Impaired membrane recycling from early endosomes. Oversecretion of lysosomal enzymes. Deficient pressureinduced rocketing of phagosomes. Increased intracellular growth of L. pneumophila. Normal clearing (5h) of bacterial suspension.

(Novak et al., 1995; Temesvari et al., 1996)

myoB/ myoDAS myoA/ myoF myoB/ myoCAS/ myoD

# 37% (beads) ND

# 39%

##

ND

ND

# 37% (beads)

# 60%

###

Slow growth in suspension, low saturation density. Delayed and decreased membrane internalization. Normal intravesicular pH. Normal intracellular retention time. Oversecretion of lysosomal enzymes. Slow growth in suspension. Only motility analyzed. Slow growth in suspension. Increased intracellular retention time.

( Jung et al., 1996) (Falk et al., 2003) ( Jung et al., 1996)

Data on phagocytosis and pinocytosis is based exclusively on quantitative analyses of particle or fluid phase uptake. Where reported, the particle (yeasts, bacteria, latex beads) used for phagocytosis studies is indicated. Unless otherwise indicated, initial rates of phagocytosis in suspension are given. In two strains the levels of one myosin have been reduced by antisense techniques (indicated with AS); in all other cases homologous recombination has been employed. A myoE knockout has not been described; ND, not determined.

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Francisco Rivero

using fluorescently labeled dextran. A detailed methodological discussion can be found in Rivero and Maniak (2006). Biochemical, immunological and microscopy tools allow monitoring the localization and dynamics of molecules and organelles along the endocytic pathway. In vivo studies using fusions of fluorescent proteins have played and will continue to play a major part in elucidating the role of the cytoskeleton in endocytic trafficking. Such studies were pioneered in Dictyostelium, with coronin being one of the first proteins whose dynamics during particle uptake was monitored in vivo (Maniak et al., 1995), soon followed by actin (Westphal et al., 1997) and many other proteins. In fact, the use of fluorescent protein fusions can be considered as de rigueur in the functional characterization of any novel protein. Early attempts to identify Dictyostelium proteins associated with endosomes were performed more than ten years ago and yielded a reduced amount of information due to technical limitations (Adessi et al., 1995; Rezabek et al., 1997). More recently the combination of improved fractionation methods with high throughput techniques is allowing dissection of the phagosome maturation pathway at increased resolution in several organisms (Griffiths and Mayorga, 2007). In these studies actin, actin-binding proteins (ABPs) and numerous signaling components directly associated with the actin cytoskeleton are consistently found. An important milestone has been the publication of proteomes over the course of maturation of Dictyostelium latex beads phagosomes (Gotthardt et al., 2006). Such studies are revealing a hitherto unexpected complexity of the phagosome maturation process. While diverse approaches have been employed to address the role of a particular protein in the endocytic pathway, the limitations of each method should always be taken into account. In many cases localization studies suggest a role in endocytosis, yet disruption of the corresponding gene results in no overt quantitative alteration of this process, frequently due to functional compensation by one or more other genes. Conversely, disruption of a particular gene may result in impaired endocytosis in the absence of biochemical or immunohistochemical association of the corresponding protein with endosomal compartments. In general it can be said that only a combination of several approaches will provide a complete picture on the role of a protein in endocytosis.

3. Role of D. discoideum Actin and ABPs in Endocytosis 3.1. Actin Because filamentous actin can be easily stained with fluorescent derivatives of phalloidin, the accumulation of actin and its colocalization with numerous ABPs during formation of phagosomes and macropinosomes is reported

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359

almost routinarily. Indeed, actin is a major protein of isolated endocytic vesicles and phagosomes (Adessi et al., 1995; Nolta et al., 1994; Rezabek et al., 1997; Rodriguez-Paris et al., 1993; Yuan and Chia, 1999). Formation of an actin coat is essential for endocytosis, as shown in studies using the actin polymerization inhibitor drug cytochalasin A. Cytochalasin A has a dosisdependent and reversible effect on phagocytosis of yeast particles (Maniak et al., 1995) and on fluid-phase uptake (Hacker et al., 1997). Nevertheless, the requirements for actin in pinocytosis and phagocytosis seem to differ, because the 50% inhibitory concentration of cytochalasin A for pinocytosis is one order of magnitude lower than for phagocytosis. Also the effects of latrunculin A, a drug that sequesters monomeric actin, distinguish phagocytosis from pinocytosis: latrunculin A exerts a stimulatory effect on phagocytosis whereas pinocytosis becomes inhibited (Konzok et al., 1999). Because introduction of fluorescent probes in Dictyostelium cells, either by microinjection or electroporation, is notoriously cumbersome, monitoring the in vivo behavior of actin had to await the advent of GFP technology. As already mentioned (Section 2), coronin-GFP was the first probe introduced (Maniak et al., 1995), followed by GFP-actin (Westphal et al., 1997) and fluorescent protein fusions that allow specific visualization of actin filaments, like the actin-binding domain of filamin (Lee and Knecht, 2002; Pang et al., 1998), the N-terminus of LimE (LimE△coil) and the C-terminal 63 kDa fragment of talin A (TalC63). LimE△coil in particular is a sensitive probe for rapid assembly of new filaments because it labels newly polymerized filaments more strongly that older filaments (Diez et al., 2005). TalC63 has been used as a trap for actin filaments because it forms complexes with F-actin that dissociate slowly enough to be carried with the flow of actin, allowing visualization of transient actin flows (Weber et al., 2002). Actin accumulates at the cell cortex during formation of the phagocytic cup around solid particles. Once the particle is engulfed, the actin coat disassembles within 1 min. (Konzok et al., 1999; Maniak et al., 1995; Peracino et al., 1998). A similar behavior has been reported during macropinosome formation, where actin accumulates at crowns, remains transiently associated with the nascent macropinocytic vesicle and finally dissociates within 1 min after internalization (Lee and Knecht, 2002). In many cases disassembly of actin is visibly more rapid from the side of the phagosome facing the cytosol. Studies with the TalC63 probe showed that during formation of macropinosomes or phagosomes an actin flow is induced at the border of the cup and filaments disassemble at the base of the cup (Weber et al., 2002). Recent in vivo studies using bacteria instead of yeasts and multiple fluorescent probes have allowed a more accurate description of actin’s behavior during particle uptake and the role of the Arp2/3 complex in assembly and of coronin in disassembly of actin filaments (Sections 3.2.1 and 3.6.2) (Clarke and Maddera, 2006; Lu and Clarke, 2005). Such studies have

360

Francisco Rivero

revealed the formation, shortly after detachment of actin from the phagosome, of an actin comet tail between the phagosome and the plasma membrane that propels the phagosome away from the site of uptake. Actin-powered movement lasts for few seconds to up to 30 sec before actin again dissipates and the phagosome is captured by microtubules. A comet-like burst of actin accumulation after engulfment of yeast particles has went unnoticed probably due to the large size of the phagosome, but has also been reported recently, overlapping with accumulation of vacuolar proton pumps (Pikzack et al., 2005). Actin-mediated rocketing of phagosomes can also be induced mechanically by compressing the cells so as to bring the phagosome into contact with the plasma membrane, but the mechanism that triggers actin polymerization and the functional implications of this phenomenon are not clear (Clarke et al., 2006). Actin is present at vesicles along the entire postlysosomal pathway (Rauchenberger et al., 1997). Treatment of cells with latrunculin A or expression of a post-lysome targeted cofilin disrupts the actin coat of postlysosomal vesicles and results in vesicle clustering, indicating that one function of the actin coat is keeping the late endosomes in a disperse state throughout the cytoplasm (Drengk et al., 2003). Because cytochalasin A has an inhibitory effect on exocytosis, the actin coat may facilitate the association of the exocytotic vesicle with the cell cortex (Rauchenberger et al., 1997). The in vivo actin dynamics during exocytosis has been seldom reported, in great part because, contrary to uptake, release of endocytosed material cannot be readily synchronized and is therefore difficult to capture. Using GFP-ABD as a probe, Lee and Knecht (2002) have shown that postlysosomal vesicles with a weak actin coat persist within the cell for long periods of time, an indication that actin coating does not by itself trigger the exocytotic process. Rather, vesicle docking is followed by release of contents and, apparently, a concomitant increase of actin polymerization while the vesicle collapses. Accumulation of actin (as monitored by the actin-binding protein LimC) during exocytosis of yeast particles has also been reported (Khurana et al., 2002a).

3.2. The F-actin nucleation machinery Local generation of actin filaments in response to signals is tightly regulated. Two major protein complexes are employed by the cell to initiate new actin filaments, the Arp2/3 complex and formins (Pollard, 2007). The Arp2/3 complex produces branched filaments and remains attached to the pointed end; it is a stable equimolar assembly of seven subunits consisting of two actin related proteins (Arp2 and Arp3) and five more proteins (ARPCs). The complex is intrinsically inactive and requires so-called nucleation promoting factors, like WASP/Scar family proteins and CARMIL and in yeast (but not in Dictyostelium) myoI and Abp1. Formins, on the contrary,

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361

form ring-shaped flexible dimers that associate to barbed ends, where they antagonize capping proteins and allow processive elongation of unbranched actin filaments. 3.2.1. The Arp2/3 complex The Arp2/3 complex plays a primary role in the nucleation of actin filaments in many cellular processes. It is required for phagosome formation in macrophages (May et al., 2000) and for clathrin-mediated endocytosis in yeast and mammalian cells (Kaksonen et al., 2006). In Dictyostelium the localization of the complex has been investigated using Arp3 and p21-Arc specific antibodies and fluorescent protein fusions of Arp3 and ARPC1 (p41). The complex is localized diffusely in the cytoplasm, occasionally with a punctate pattern, and accumulates at the cell cortex, crowns and, in a discontinuous pattern, at large vesicles (Insall et al., 2001; Jung et al., 2001). In vivo studies revealed that the complex accumulates at sites of particle attachment and later on at the border of the extending phagocytic cup until the membrane seals around the particle. A similar behavior was observed during formation of macropinosomes. The complex is stripped off the endosome shortly after closure, coincident with acidification of its lumen, and re-associates with postlysosomal endosomes, suggesting that the Arp2/3 complex participates in reorganization of the actin system both during uptake and exocytosis (Insall et al., 2001). In isolated phagosomes Arp3 can be detected predominantly during phagosome formation (Gotthardt et al., 2006). The Arp2/3 complex accumulates also at comet tails that propel phagosomes around ingested bacteria after the phase of actin assembly and disassembly that follows uptake (Clarke and Maddera, 2006). In an in vivo study in which actin-mediated rocketing of yeast-containing phagosomes was induced by compression, mRFP-p41 and GFP-Arp3 were found at places where the phagosome contacts the plasma membrane, almost completely overlapping with an actin probe. Interestingly, when the phagosome moved away a ring or track of GFP-Arp3 was left behind and dissipated (Clarke et al., 2006). There are no molecular genetics studies on the Arp2/3 complex of Dictyostelium to date. Because each component of the complex is encoded by a single gene, it is very likely that targeting of any of them results in a lethal phenotype. 3.2.2. WASP/Scar family In Dictyostelium this family consists of Scar, WASP (Wiskott-Aldrich syndrome protein) and two uncharacterized WASP-related proteins. These proteins share a central proline-rich region and a C-terminal region composed of one WH2 (WASP-homology 2) domain that binds actin monomers followed by one acidic region that interacts with the Arp2/3 complex. The proline-rich region binds profilin as well as SH3 domains from a variety of proteins (Takenawa and Suetsugu, 2007). Both Scar and WASP are positive regulators of actin polymerization in Dictyostelium (Myers et al., 2005;

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Seastone et al., 2001). WASP plays important roles in motility and chemotaxis, however its subcellular localization and participation in endocytic processes have not been addressed. By contrast, a requirement for WASP is well established in other organisms. For example, it is recruited to nascent FcgR-mediated phagosomes in macrophages, as well as to Golgi vesicles and to clathrin coated vesicles, where it induces actin polymerization (Takenawa and Suetsugu, 2007). It is conceivable that WASP, similar to other cytoskeleton components like class I myosins (Section 3.8.2), plays dual roles in cell motility and endocytosis both in aggregation competent and vegetative cells, respectively. Although it remains to be verified experimentally, WASP might be targeted to sites of particle or fluid phase uptake through binding to phosphoinositides. WASP binds in vitro to phosphatidylinositol (4,5) bisphosphate (PIP2) and phosphatidylinositol (3,4,5) trisphosphate (Myers et al., 2005), the latter accumulating transiently at phagocytic cups and macropinocytic crowns (Dormann et al., 2004). Interaction with phosphoinositides and Rho GTPases present at the plasma membrane would then result in a conformational change that renders WASP active. WASP is able to interact with several Rho GTPases, and RacC, a Rho GTPase involved in phagocytosis, appears to be the major regulator (Han et al., 2006), providing a link of WASP to endocytic processes that needs further analysis. One more regulator of WASP is the recently described WIPa (WASP-interacting protein a), a member of the verprolin family of proline-rich ABPs. Verprolins act as scaffolds that interact with many SH3 domain-containing proteins as well as with profilin and, through its C-terminal region, with WASP. In Dictyostelium WIPa is important for actin remodeling during chemotaxis, but a role in vegetative cells has not been explored (Myers et al., 2006). Unlike WASP, the role of Scar in endocytosis has been extensively investigated in a knockout cell line (Seastone et al., 2001). These cells have reduced rates of phagocytosis (80%), pinocytosis (40%) and exocytosis, delayed transit from the lysosomal to the post-lysosomal compartment and defective secretion of lysosomal enzymes. The levels of F-actin are reduced by 50% and macropinocytic crowns are absent. Scar is a predominantly cytosolic protein only weakly enriched at the cell cortex at actin-rich protrusions. It does not associate stably with endolysosomes, however the association might be transient and might be needed to trigger actin polymerization, because endolysosomes of Scar null cells lack an F-actin coat. Consistent with this role, inhibition of actin polymerization with cytochalasin A results in similar phenotypes as those described above for the Scar null mutant. Disruption of Scar in a profilin I and II null background results in additive effects on fluid phase uptake and release as well as on secretion of lysosomal enzymes, consistent with profilin participating in the F-actin nucleation process through binding to the proline-rich region of Scar. Like its mammalian homolog WAVE (WASP family verprolin homology protein), Scar exist as a multimolecular complex with PIR121, Nap1,

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Abi2 and HSPC300 (Blagg et al., 2003; Vlahou and Rivero, 2006). Strains deficient in PIR121 or Nap1 have been described, but only their roles in motility and filopod formation, respectively, have been investigated (Blagg et al., 2003; Steffen et al., 2006). Although lacking a Rho GTPase binding domain WAVE is subject to regulation by Rac, but the exact mechanism and the role of other components of the complex in targeting and activation of Scar/WAVE remain controversial. 3.2.3. CARMIL CARMIL (p116; capping protein, Arp2/3 and myosin I linker) was identified in a search for proteins interacting with the SH3 domain of class I myosins MyoB and MyoC ( Jung et al., 2001). It turned out to be homologous to Acanthamoeba p125, and similar proteins have been subsequently found in metazoa. Members of the CARMIL family have a C-terminal half that harbors a region functionally related to the WH2 and acidic domains of WASP family proteins. The C-terminus binds all subunits of the Arp2/3 complex and displays Arp2/3 dependent actin nucleation activity, although weaker than WASP. This region is followed by a proline-rich region that does not contain runs of five or six consecutive proline residues, and therefore is not expected to bind profilin; by contrast, it contains PXXP motifs that bind SH3 domains. This proline-rich region is immediately followed by a CAH3 domain (CARMIL homology domain 3), a region responsible for binding of both subunits of the capping protein (CP). In fact, CARMIL proteins are potent antagonists of CP (Section 3.4.2) (Remmert et al., 2004; Yang et al., 2005). CARMIL localizes in dynamic actin-rich extensions, like macropinocytic crowns, where components of the complex like MyoC and Arp3 also localize. Consistent with a role in pinocytosis, CARMIL deficient cells show a 45% reduction of the rate of fluid phase uptake. Mutant cells display defective formation of macropinocytic crowns and a smaller intracellular endocytic compartment ( Jung et al., 2001). It has been proposed that the predominant role of CARMIL would be as a regulator of the functional levels of CP. The interaction with SH3-bearing class I myosins would be used to translocate and concentrate the complex at the vicinity of the plasma membrane, facilitating the extension of actin filaments and the formation of protrusions. Studies on the Acanthamoeba ortholog suggest that CARMIL forms homodimers that might exist in an autoinhibited state (Remmert et al., 2004). In this respect, CARMIL would behave like the other nucleators described in this section, and even activation by Rho GTPases has been postulated (Uruno et al., 2006). 3.2.4. Formins, VASP and IQGAP Evidence is accumulating, particularly in mammalian cells, towards a participation of formins in endocytosis. Some formins localize on endosomes and are apparently required for their motility, although there are still many

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open questions regarding targeting and regulation of these proteins (Faix et al., 1996). Only four of the 10 formins of Dictyostelium (ForA to ForJ) have been characterized to some extent (Rivero et al., 2005). ForH localizes predominantly at the tips of filopods, along with VASP (vasodilator-stimulated phosphoprotein), with which it interacts, and both are required for formation of filopods. In addition to this role, VASP is required for particle and substrate adhesion, although cells lacking VASP were able to take up latex beads at a normal rate (Han et al., 2002). Mutants lacking ForA, ForB or both have been described as showing no detectable growth phenotype. Finally, cells lacking ForC, a formin that plays roles at late developmental stages, grew normally in nutrient medium and in bacterial lawns, indicating that this formin might be dispensable for endocytosis (Kitayama and Uyeda, 2003). However, in vivo experiments with a GFP fusion showed that ForC accumulates at macropinocytic crowns and that the N-terminal region is sufficient for targeting the protein to places of active actin reorganization, like macropinosomes, phagocytic cups and cell-to-cell contacts (Kitayama and Uyeda, 2003). Several formins might conceivably play redundant roles, which would explain the absence of detectable phenotypes in some mutants. Clearly, much work is still needed to delineate the roles of formins in endocytosis and other processes in Dictyostelium and other organisms, and how localization and activity are regulated, in particular by Rho GTPases, for which formins function as effectors. More recently IQGAP1 has been identified as interaction partner of the formin Dia1, and this interaction appears to be required for phagocytic cup formation in macrophages (Brandt et al., 2007). IQGAPs are scaffolding proteins that interact with cytoskeletal and signal transduction proteins and are primarily involved in cell adhesion, binding to microtubules and cytokinesis (Vlahou and Rivero, 2006). A possible implication in endocytic processes has been investigated only in one of the four IQGAPs of Dictyostelium, DGAP1, which accumulates at the cell cortex and is an effector of Rac1a. Cells lacking DGAP1 display unaffected uptake of yeasts, and overexpression of the protein results in a moderately (25%) reduced rate of particle uptake (Faix et al., 1998). This alteration might be consequence of the reduced levels of F-actin and the absence of F-actin at cortical protrusions found in the overexpressor cells, which in turn might result from misregulated formin activity.

3.3. Monomeric actin binding proteins ABPs that bind G-actin are essential both to maintain and regenerate the pool of unpolymerized actin and to make the actin monomers available for filament elongation by constituting part of the nucleation machinery discussed above (Section 3.2). Profilin and the cyclase-associated protein are

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the best characterized ABPs of this class, which also includes the unexplored actobindins and twinfilin. 3.3.1. Profilin The abundant profilins catalyze the exchange of ADP bound to G-actin for ATP. In complex with ATP-actin profilins then efficiently promote elongation of F-actin barbed ends, particularly in association with proteins that contain poly-L-proline-rich motifs, like WASP, Scar, VASP and formins. In addition, profilins bind with high affinity phosphoinositides (in particular PIP2), causing an inhibition of their hydrolysis by phospholipase C. The Dictyostelium genome encodes three profilins of which two have been studied in detail (Haugwitz et al., 1991). Profilin I and II display a uniform cytoplasmic localization and concentrate and colocalize with actin at nascent phagosomes (Lee et al., 2000; Yuan and Chia, 1999). This localization at early phagosomes seems independent of the ability to bind poly-L-proline motifs, as shown with a profilin II mutant (W3N) with abolished poly-L-proline binding capacity (Lee et al., 2000). Profilin I and II have been identified as constituents of isolated early phagosomes, from where they detached together with actin and cofilin during phagosome processing (Yuan and Chia, 1999), although more sophisticated profiles of phagosome proteins show a more persistent localization of profilin along the endocytic pathway (Gotthardt et al., 2006). Although profilins I and II differ in their biochemical properties (Haugwitz et al., 1991), they appear to be functionally equivalent. Single mutants lacking either of them show an unaltered phenotype, which to some extent can be explained by compensatory increased expression of the remaining isoform. Double mutants, by contrast, displayed a severe phenotype. They have increased levels of F-actin with notable defects in uptake and transit of fluid phase: low rate of pinocytosis, less numerous macropinosomes of small size, delayed progress during the acidification phase, defective secretion of lysosomal enzymes and delayed exocytosis (Haugwitz et al., 1994; Seastone et al., 2001; Temesvari et al., 2000). Phagocytosis, by contrast, has been reported as normal (Haugwitz et al., 1994) or increased (Temesvari et al., 2000). The phenotypes elicited by loss of profilin may result not only from an altered actin polymerization, but also from an altered phosphoinositide turnover. An increased availability of PIP2 in the double knockout mutant has been invoked to explain the normal or increased rate of phagocytosis versus the profoundly reduced rate of fluid uptake. PIP2 is a substrate of phospholipase C, which is specifically required for phagocytosis (Duhon and Cardelli, 2002). Moreover, it is interesting that most of the vesicle transport defects of the profilin deficient mutant are partially rescued upon disruption of the gene encoding DdLIMP, a glycoprotein of the

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endolysosomal pathway (Karakesisoglou et al., 1999; Temesvari et al., 2000). DdLIMP belongs to the CD36/LIMP-II (lysosomal integral membrane protein) family, which includes plasma membrane receptors and lysosomal membrane proteins of unclear function in the endolysosomal pathway (Kuronita et al., 2002). DdLIMP does not bind profilin or actin, but it binds PIP2, and therefore DdLIMP and profilin might compete for PIP2 binding on endolysosomal membranes. 3.3.2. Cyclase-associated protein The cyclase-associated protein (Srv2/CAP) is a multifunctional protein composed of two domains separated by a central proline-rich stretch and a conserved WH2 domain of unclear role. The C-terminal domain is responsible for binding to monomeric actin, whereas the N-terminal domain inhibits this activity in a PIP2-dependent manner (Gottwald et al., 1996). The function of CAP appears to be more complex than simple monomer sequestering. CAP forms high molecular weight complexes with actin that function as monomer processing intermediates that accelerate cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhance nucleotide exchange by profilin (Balcer et al., 2003; Paavilainen et al., 2004). CAP accumulates at the cell cortex (Gottwald et al., 1996). Using GFP fusions of CAP and several domain combinations it was shown that targeting to the cell cortex is mediated by the N-terminal domain and that the proline-rich central region is dispensable for targeting (Noegel et al., 1999). In homologs from other organisms the proline-rich region binds to SH3 domains. The yeast protein (Srv2), for example, is targeted to cortical actin patches through binding of the SH3-domain containing protein Abp1p (Hubberstey and Mottillo, 2002). The proline-rich region of Dictyostelium CAP is less prominent than in yeast or mammalian homologs: there is only one motif that could potentially bind SH3 domains, but this has not been analyzed. For the same reason, a direct interaction with profilin reported in Srv2 is unlikely in Dictyostelium CAP. Compatible with a role at the cell cortex, a mutant strain that expresses less than 5% of the endogenous protein showed a pinocytosis defect but phagocytosis was not affected (Noegel et al., 1999). Additionally, CAP plays roles at the interface between the actin cytoskeleton and the endolysosomal system. CAP displays partial colocalization with components of the endocytic pathway, like V-ATPase, N-ramp1 and vacuolin. It binds to subunits of the V-ATPase and the CAP mutant has reduced levels and altered distribution of this proton pump. The function of the V-ATPase is also altered, resulting in a slightly higher basal endolysosomal pH, but unaffected time course of acidification of endosomes and relocalization of the V-ATPase during phagocytosis (Sultana et al., 2005).

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3.4. Severing and capping proteins Severing and capping of actin filaments are essential activities during remodeling of actin-based structures. Capping of barbed ends prevents addition of actin monomers and growth of filaments, whereas severing facilitates disassembly of filaments and subsequent release of actin monomers. 3.4.1. Cofilin and Aip Two of the five functional Dictyostelium cofilins have been investigated. Cofilin-1 is expressed in vegetative cells and during early development, and is apparently essential (Aizawa et al., 2001, 1995). Cofilin-2 predominates in the aggregation stage and is absent in vegetative cells; disruption of the corresponding gene does not cause any apparent phenotype (Aizawa et al., 2001). I will focus therefore on cofilin-1. It binds to and depolymerizes actin filaments in a pH-dependent and phosphoinositide-regulated manner and has a relatively weak monomer sequestering activity. Phosphorylation of a serine residue close to the N-terminus appears to be a major inhibitor of cofilin function in mammals and A. castellani, but such a regulatory mechanism is apparently absent in Dictyostelium cofilin-1 (Aizawa et al., 1997; Yuan and Chia, 1999). Cofilin-1 distributes uniformly in the cytoplasm and at sites of active remodeling of actin networks, such as crown-like protrusions, phagocytic cups and leading edges of migrating cells, where the protein rapid and transiently accumulates (Aizawa et al., 1995, 1997; Konzok et al., 1999; Yuan and Chia, 1999). Cofilin-1 was also identified as a constituent of early phagosomes, from which it detached together with actin and profilins during phagosome processing (Yuan and Chia, 1999). Cells that overexpress cofilin-1 have increased levels of F-actin, organized in actin bundles, but otherwise grow normally in nutrient medium and perform normal phagocytosis (Aizawa et al., 1996). Nevertheless, the fact that cofilin-1 appears to be essential is consistent with an absolute requirement for actin remodeling during food uptake. Cofilin activity is also regulated by interaction with other components of the actin cytoskeleton, like actin interacting protein (Aip) and CAP. Aip1 is a nine WD40-repeat protein that enhances the severing activity of cofilin (Aizawa et al., 1999) with which it associates to form a barbed end cap that prevents reannealing of severed filaments (Balcer et al., 2003). Aip1 localizes to regions of active microfilament remodeling like phagocytic cups and macropinosomes, where it colocalizes with actin (Aizawa et al., 1999; Konzok et al., 1999). The enrichment of Aip at these structures is transient; at phagosomes in particular, Aip detaches within 1 min after internalization. Consistent with a role in the rapid remodeling of the cortical actin meshwork, Aip1 null cells were strongly impaired in phagocytosis and fluid-phase uptake, and engulfment of yeast particles was prolonged, as monitored using GFPactin. The localization of cofilin during phagocytosis was not altered in Aip

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null cells. Overexpression of the protein did not affect fluid phase uptake, but increased the rate of yeast particle uptake (Konzok et al., 1999). 3.4.2. Capping protein Capping protein (CP; aginactin, Cap32/34) caps but does not sever or nucleate actin filaments and thereby prevents the addition or loss of actin subunits at the barbed filament end. It is composed of two subunits, each encoded by a distinct gene (Schleicher et al., 1984). The subcellular localization of CP in Dictyostelium has not been reported, but since the A. castellani homolog localizes in the cytoplasm and accumulates in dynamic actin-rich regions like the cell cortex and filopods (Cooper et al., 1984), a similar localization in Dictyostelium can be inferred. Strains deficient in one or both subunits of the CP have not been described, but the function of the heterodimer has been investigated using strains where simultaneous expression of both subunits is upregulated or downregulated. These studies have shown that CP is the major regulator of the number of free barbed ends and contributes to terminating the actin polymerization response upon chemoattractant stimulation (Hug et al., 1995). One would expect endocytosis to be severely affected in those strains, and although uptake of fluid or particles has not been investigated, underexpressors grew slowly both in nutrient medium and in bacterial suspension, whereas overexpressors displayed no defects. The capping activity of CP is inhibited by PIP2 (Haus et al., 1991) and by proteins harboring a CAH3 domain (Section 3.2.3). 3.4.3. Gelsolin family Members of this family are characterized by the presence of several copies (three to seven) of the conserved gelsolin repeat. Of the eight members of this family, four have been characterized to some extent: severin, protovillin (Cap100), villidin and GRP125. The most extensively studied is severin, an abundant protein that displays nucleating, severing and capping activity in addition to G-actin binding and is regulated by Ca2þ, phosphoinositides and phosphorylation (Eichinger et al., 1998, 1991; Eichinger and Schleicher, 1992). Severin appears diffusely distributed in the cytosol, with some enrichment at pseudopods and phagosomes (Andre et al., 1989; Brock and Pardee, 1988), and it has also been reported at isolated phagosomes (Gotthardt et al., 2006). Cells deficient in severin grow normally both in nutrient medium and on bacterial lawns, indicating that endocytosis is not severily impaired in this mutant (Andre et al., 1989). Protovillin displays a strong capping activity and binds G-actin. Its main function might be in regulating the G- and F-actin pools, but there are no data on the subcellular localization of this protein and functional studies are also lacking (Hofmann et al., 1992). Villidin and GRP125 share many features (Gloss et al., 2003; Stocker et al., 1999). Both lack the first gelsolin repeat and may not display nucleating, severing and capping activities, and

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both localize at vesicular compartments. Villidin however has a coroninrelated module in the N-terminus that seems to be the major actin-binding region of this protein. Villidin and GRP125 have been proposed as links between membranes and the cytoskeleton, but appear dispensable for endocytosis (Gloss et al., 2003; Stocker et al., 1999).

3.5. Actin crosslinking proteins F-actin crosslinking proteins stabilize three-dimensional networks or densely packed bundles of actin filaments, depending on the spatial arrangement of their actin-binding sites and the length and flexibility of spacer elements that separate the actin-binding sites. This class of proteins contains some of the most extensively characterized ABPs, which will be discussed first. 3.5.1. Filamin, a-actinin, ABP34 and fimbrin Filamin (ddFLN, formerly gelation factor or ABP-120), a-actinin, the 34 kDa actin-bundling protein (ABP34) and fimbrin are abundant regulators of the organization of cortical microfilaments. Whereas ABP34 is apparently unique to Dictyostelium and related species, fimbrin, filamin and a-actinin are widely distributed among the eukaryotes. ABP34 and fimbrin are the only calcium inhibited ABPs that function primarily to induce the formation of F-actin bundles (Fechheimer and Taylor, 1984; Prassler et al., 1997). Filamin crosslinks actin filaments to form branched networks, whereas a-actinin, also a calcium inhibited ABP, gives rise to lateral arrays (Condeelis et al., 1984). ABP34, fimbrin and filamin have been found enriched in the cell cortex, including protrusions like pseudopods and filopods (Carboni and Condeelis, 1985; Johns et al., 1988; Ogihara et al., 1988; Prassler et al., 1997). By contrast, a-actinin displays a diffuse and patchy distribution throughout the cytoplasm and is also enriched in pseudopods and at the leading edge (Brier et al., 1983). ABP34 colocalizes with actin during formation of phagocytic cups and dissociates as the phagosome matures (Furukawa et al., 1992; Furukawa and Fechheimer, 1994). The protein has also been identified in isolated phagosomes (Rezabek et al., 1997). Fimbrin localizes at phagocytic cups and macropinosomes, where it also colocalizes with actin (Pikzack et al., 2005). The enrichment of fimbrin at these structures is transient, and detachment takes place within 1 min after internalization, as revealed in in vivo studies with GFP-fimbrin and deletion mutants (Pikzack et al., 2005). Filamin colocalized with actin at phagocytic cups (Cox et al., 1996) whereas a-actinin did not consistently accumulate at the phagocytic cup (Maniak et al., 1995), rather it was found to associate with phagosomes at later stages (after ABP34 has been recruited) and to remain associated with the maturing phagosome after ABP34 has dissociated (Furukawa and Fechheimer, 1994). In agreement with a role at late stages of phagocytosis, other studies

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show only very low amounts (Schreiner et al., 2003) or complete absence (Maniak et al., 1995) of the protein in phagosomal fractions. From the localization and the presumed role of actin crosslinkers in stabilizing the actin coat a prominent role during the uptake phase would be anticipated. Early experiments in which a specific monoclonal antibody that blocks the interaction of ABP34 with actin in vitro were loaded into cells specifically inhibited, but did not abolish phagocytosis, supporting a specific implication of ABP34 in phagocytosis (Furukawa et al., 1992). However, single knockout mutants of a-actinin, ABP34 and fimbrin did not present obvious defects in the rates of phagocytosis or fluid phase uptake, ABP34 null cells even displayed increased phagocytosis in quantitative tests (Pikzack et al., 2005; Rivero et al., 1996a, 1996b, 1999; Wallraff et al., 1986). Mutants deficient in filamin have been generated in different genetic backgrounds with conflicting outcomes. Whereas those generated in the AX2 strain (either by chemical mutagenesis or by homologous recombination) lacked any obvious phenotype (Brink et al., 1990; Rivero et al., 1996b, 1999; Witke et al., 1992), those generated in AX3 displayed abnormal formation of phagocytic cups and 50% reduced uptake of bacteria or latex beads, but normal rates of uptake of a fluid phase marker (Cox et al., 1996). The mild phenotype of the fimbrin null mutant is in contrast to the situation in yeast, where fimbrin is essential for endocytosis (Kubler and Riezman, 1993) and illustrates very nicely the caveats of extracting universal conclusions from studies on a particular organism. Precisely yeast is equipped with a rather limited repertoire of ABPs, and gene disruptions lead more frequently to overt phenotypes. The absent or mild phenotypes of single mutants has prompted several studies on strains deficient in combinations of two proteins aimed at verifying the hypothesis that some extent of functional redundancy exists among actin crosslinkers. Some combinations of double knockouts, like a-actinin/ filamin or a-actinin/ABP34, were found more deleterious than others, like ABP34/filamin or ABP34/fimbrin (Pikzack et al., 2005; Rivero et al., 1996b, 1999). These studies have revealed a complex network of unique and shared roles among actin crosslinkers. Nevertheless, the roles of additional yet uncharacterized crosslinkers uncovered after sequencing of the Dictyostelium genome need to be investigated in order to have a better picture of the contribution of this class of ABPs to endocytosis. 3.5.2. Other actin crosslinkers Cortexillins and dynacortin are best known for their roles in maintaining cortical viscoelasticity both in interphase and during cytokinesis (Girard et al., 2004; Simson et al., 1998). In interphase cells both cortexillin isoforms and dynacortin are enriched at the cell cortex and macropinocytic crowns (Faix et al., 1996; Robinson et al., 2002), and cortexillins have been identified in isolated phagosomes both in Dictyostelium (Gotthardt et al., 2006),

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and in E. histolytica (Marion et al., 2005). However, the behavior of these proteins during endocytosis has not been addressed, and endocytosis studies in knockout or overexpression mutants have not been reported. Some transcriptional elongation factors constitute important physical and functional links between the translational machinery and the actin cytoskeleton. The best studied is elongation factor 1A (ABP50, eEF1A) an abundant protein that binds G-actin and bundles F-actin, but also eEF1B displays actin-binding properties. Both proteins distribute diffusely throughout the cytosol and are enriched at actin-rich regions of the cell cortex (Dharmawardhane et al., 1991; Furukawa et al., 2001). Noncanonical roles of eEF1A have been established for example in studies with yeast eEF1A, where mutants with reduced actin bundling activity cause alterations in actin cytoskeleton organization (Gross and Kinzy, 2005). It remains speculative, albeit not unlikely, that elongation factors are implicated in actin remodeling during endocytic traffic, but this aspect has not been addressed specifically and mutants are not available. In fact these proteins have been repeatedly identified in isolated phagosomes in several organisms, including Dictyostelium (Gotthardt et al., 2006) and E. histolytica (Okada et al., 2005).

3.6. Lateral ABPs In general the ABPs included in this section function as scaffolds for the recruitment of multiprotein complexes that frequently include signaling molecules, like small GTPases. These proteins therefore constitute important elements at the interface between signal transduction, remodeling of the actin cytoskeleton and vesicle trafficking. 3.6.1. ADF/cofilin family The ADF (actin depolymerization factor) is found in cofilin and in several proteins that bind to filamentous actin without displaying severing activity. Two of them, coactosin and Abp1, have been reported. For coactosin only in vitro data is available: the protein binds but does not bundle actin and appears to counteract the capping activity of CP and severin (Rohrig et al., 1995). Coactosin was identified as a protein associated to pre-spore vesicles, along with profilin in a proteomics study (Srinivasan et al., 2001) and as a component of isolated phagosomes (Gotthardt et al., 2006) but the subcellular localization of this protein has not been addressed in detail and there are no functional studies, therefore the cellular function of coactosin remains unclear. Abp1 is a conserved protein and has homologs in metazoa (drebrin F) and yeast (Abp1p). In Abp1p the ADF domain is followed by an acidic region, a proline-rich domain and a SH3 domain. In yeast and mammals Abp1 constitutes an important functional link between the endocytic machinery and actin filament dynamics: the SH3 domain interacts with

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numerous proteins all involved in endocytosis, including dynamin, synaptojanin, CAP and the class I myosin Myo5 (Qualmann and Kessels, 2002). While binding to Dictyostelium CAP is unlikely (Section 3.3.2), class I myosins appear as relevant binding partners of Abp1, and indeed an interaction with MyoK has been reported (Soldati, 2003). It was also shown that Abp1p binds and activates the Arp2/3 complex presumably via its acidic region (Higgs and Pollard, 2001), but in the Dictyostelium homolog the acidic region cannot be recognized and it remains to be seen whether it has a similar function as the yeast Abp1p. In Dictyostelium Abp1 localizes at the cell cortex of vegetative cells where it co-localizes with actin to a large extent, but in depth studies addressing the localization of Abp1 during endocytosis are missing. Work on strains lacking or overexpressing Abp1 has revealed a role during early aggregation, where the protein regulates the number of pseudopods, but the protein seems to be dispensable for pinocytosis and phagocytosis (Wang and O’Halloran, 2006). 3.6.2. Coronins Coronins are proteins of the WD repeat family that apart from binding F-actin are also able to bind to and inhibit the nucleation activity of the Arp2/3 complex, although this last aspect has been addressed biochemically only in yeast and mammalian coronin (Uetrecht and Bear, 2006). Coronin strongly accumulates at crown-like surface extensions (hence the name of the protein) as well as at phagocytic cups and other regions of active actin remodeling (de Hostos et al., 1991). GFP-coronin accumulates at the phagocytic cup and at crowns, and is gradually released within 1 min after closure of the phagosome or macropinosome (Hacker et al., 1997; Lu and Clarke, 2005; Maniak et al., 1995). Coronin is also present at actin-coated vesicles of the post-lysosomal compartment prior to accumulation of vacuolin (Rauchenberger et al., 1997). More accurate observations using GFPcoronin and mRFP-LimE△coil show binding of coronin to actin filaments a few seconds after they have formed. At comet tails that form after ingestion of bacteria or when actin-driven rocketing of phagosomes is induced by compression of the cell to bring the phagosome into contact with the plasma membrane, coronin is recruited to the end of the actin tails, where actin filaments disassemble. All this is consistent with an inhibitory role of coronin on the Arp2/3 complex (Clarke and Maddera, 2006; Clarke et al., 2006). Mutants lacking coronin display markedly decreased rates of phagocytosis and macropinocytosis (de Hostos et al., 1993; Hacker et al., 1997; Maniak et al., 1995). Interestingly, coronin deficient cells, contrary to many cytoskeleton mutants, are more permissive for intracellular growth of Legionella pneumophila and Mycobacterium marinum (Fajardo et al., 2004; Solomon et al., 2000, 2003). These studies might point towards roles of coronin at later steps of the endocytic pathway. For example, in macrophages coronin 1A associates with the p40phox subunit of the NADPH oxidase complex

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(Grogan et al., 1997). If Dictyostelium coronin functions in a similar way and is required for the assembly of the NADPH oxidase complex, its absence would favor survival of the ingested pathogens. Besides a typical coronin, two more members of the coronin family are encoded by the Dictyostelium genome, villidin (Section 3.4.3) and a coronin 7/POD-1 homolog. Because roles in intracellular trafficking have been proposed for members of the coronin 7 family (Uetrecht and Bear, 2006), it will be of great interest to study the Dictyostelium homolog. 3.6.3. LIM proteins Many LIM proteins associate directly or indirectly with the actin cytoskeleton (Khurana et al., 2002b), and several of the LIM proteins of Dictyostelium participate in vesicle trafficking processes. LimC, LimD and LimE (DdLim) have a similar pattern of subcellular localization: they localize preferentially at the cell cortex, where they colocalize with actin, and are transiently recruited to macropinosomes and phagocytic cups (Khurana et al., 2002a; Prassler et al., 1998; Schneider et al., 2003). Recruitment of LimC to the cell cortex during exocytosis has also been reported, and LimD has been identified in isolated phagosomes (Khurana et al., 2002a). Fluid phase and particle uptake of single and double mutants of LimC and LimD are unimpaired under standard conditions. Rather, these proteins appear more important for maintenance of cortical strength, establishment of cell polarity and chemotaxis (Khurana et al., 2002a). LimE appears to exist in a complex with activated Rho GTPases, but the implications of this association are unknown (Prassler et al., 1998). Although knockout and overexpressor strains of LimE have been described, these studies address mainly the role of the protein in cytokinesis. LimD is more closely related to LimE than to LimC, therefore it is likely that they play redundant roles in endocytosis. It would be interesting to study the phenotype of a LimD/LimE double mutant. A clear role in phagocytosis has been assigned to two more LIM proteins, LimF and CH-Lim, that interact with each other and with Rab21 (Khurana et al., 2005). Both proteins appear enriched at intracellular vesicles, but the identity of these vesicles has not been investigated. In addition, CH-Lim accumulates at the cell cortex as well as at phagocytic cups, and remains attached as the yeast particle traffics within the cell. The CH domain of this protein is probably responsible for association with actin at the cell cortex and during particle uptake, but the LIM domain region, which alone is purely cytosolic, seems to be required for prolonged association of the whole protein to the phagosome. LimF appears also enriched at phagocytic cups. Interestingly, Rab21 also displays a cytosolic and vesicular localization, but it was not determined whether all three components of the complex localize at the same vesicles. Extensive quantitative analyses performed on knockout and overexpressor strains of both LIM proteins in connection with Rab21 have revealed that all three components act

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cooperatively. Knockout of LimF and CH-Lim have opposite effects. Rab21 has a positive effect on phagocytosis, but both LIM proteins are required for Rab21 to function. Because LimF and CH-Lim null cells are not defective in adhesion, it is probable that the complex is not needed for particle attachment and uptake, but rather operates at the interface of the actin cytoskeleton and vesicle fusion during phagosome formation, in agreement with the localization of the components at vesicles. The interaction of these proteins with Rab21 is independent of the activation state of the GTPase, indicating that these LIM proteins are not effectors but might regulate availability or targeting of Rab21. It would be interesting to investigate the nature of these vesicles and their behavior in mutants of the complex. 3.6.4. ABPs containing a Rho GTPase exchange factor domain Three ABPs harboring one or more calponin homology (CH) domains and a Rho GTPase exchange factor (RhoGEF) domain have been reported very recently: RacGEF1, Trix and GxcDD. RacGEF1 associates partially with the cell cortex through a CH domain and plays roles in chemotaxis; a participation in endocytosis has not been addressed (Park et al., 2004). Trix contains an N-terminal region with three CH domains that binds and bundles actin filaments. Its localization has been studied with the help of GFP fusions of the full length protein or the N-terminal region. Both fusions accumulate at the cell cortex. Dynamics studies of yeast uptake with the N-terminal fusion have revealed that the protein does not enrich at the phagocytic cup, but progressively and very intensely accumulates after 45 min of incubation with yeast, presumably at late endosomes. It has not been determined, however, whether this pattern of distribution reflects the behavior of the full length protein. Further in support of a role in the regulation of late steps of the endocytic pathway, presumably exocytosis, cells deficient in Trix displayed normal rates of yeast uptake, but 30% reduction in exocytosis (Strehle et al., 2006). In the multidomain protein GxcDD the single CH domain functions as a membrane association domain, whereas an ArfGAP (GTPase activating protein for Arf )-PH (plekstrin homology) tandem colocalizes with actin at the cell cortex and accumulates at phagocytic cups. The targeting of the ArfGAP-PH tandem may be mediated by binding to phosphoinositides. As for Trix, however, the behavior of the full length protein has not been reported. A GxcDD deficient strain displayed normal pinocytosis and phagocytosis rates (Mondal et al., 2007).

3.7. Membrane-associated ABPs The establishment of links between membranes and the actin cytoskeleton is essential to endocytosis, and many of the ABPs discussed above are targeted to membranes and become regulated by phospholipids. In this

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section I will discuss several families of proteins for which the association to membranes is the most salient feature. Many unconventional myosins are able to establish links between actin filaments and membrane lipids, however myosins are discussed separately below. 3.7.1. Talin Talins exist as homodimers and are components of focal adhesions in metazoan cells where they act as links between integrins and the actin cytoskeleton (Critchley, 2005). Two talins have been characterized in Dictyostelium, talin A (TalA, originally called filopodin) and talin B (TalB). They are 50% similar to each other, but apparently functionally distinct, because neither talin compensates the defects of the other. This indicates that each isoform plays unique roles, TalA at the vegetative and TalB at the multicellular stage (Tsujioka et al., 1999). TalA accumulates at the tips of filopods, in the cortex and in dot-like structures on the ventral surface but is apparently excluded from podosome-like structures known as eupodia (Fukui and Inoue, 1997; Hibi et al., 2004; Kreitmeier et al., 1995). Although talin is enriched in phagocytic cups during Fc and complement receptor mediated particle uptake in macrophages (Lim et al., 2007), an enrichment of TalA during phagocytosis has not been reported in Dictyostelium (Weber et al., 2002). Cells lacking TalA showed defects in adhesion to the substrate, Ca2þdependent cell-to-cell adhesion and phagocytosis (Gebbie et al., 2004; Niewohner et al., 1997). The phagocytosis defect was traced down to the initial phases of uptake that require adhesion, because uptake of yeast particles improved with shaking at low frequency and only bacterial species devoid of carbohydrate moieties of the cell surface lipopolysaccharides are efficiently taken up by the TalA mutant. In addition, macropinocytosis, which does not depend on adhesion, was unaffected, and actin distribution to macropinosomes was not altered in TalA null mutants (Gebbie et al., 2004; Weber et al., 2002). There is genetic and biochemical evidence that TalA and MyoI function in the same pathway. Both null mutants display the same phenotype and both co-immunoprecipitate (Tuxworth et al., 2005). However, TalA is not needed for targeting of MyoI and vice versa, therefore a model has been put forward in which the interaction induces a conformational change in each protein; in the absence of one partner the other can no longer promote formation or stabilization of adhesion complexes. The membrane anchor of these complexes might be provided by Sib proteins, members of a family of type I transmembrane proteins with features found in integrin b. The cytosolic domain of these proteins interacts with TalA and a mutant defective in one of the Sib proteins (SibA) behaves in many respects like the TalA mutant (Cornillon et al., 2006). This mechanism of action resembles in many respects that of mammalian talin, that is specifically required for uptake through complement receptor 3

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(which is identical to integrin aMb2) through direct binding of the cytosolic domain of the b2 subunit and activation of the receptor (Lim et al., 2007). 3.7.2. Comitin Comitin is an unusually basic homodimeric protein with some relationship to mannose-specific plant lectins. By virtue of its mannose binding activity comitin might bind to vesicle membranes exhibiting mannosylated glycans ( Jung et al., 1996a). Comitin is present on Golgi membranes as well as on vesicles of unclear affiliation, probably of the ER and endosomes, although not lysosomes (Weiner et al., 1993). An enrichment in isolated early phagosomes has been reported, although immunolocalization studies have failed to reveal a specific enrichment around phagosomes (Gotthardt et al., 2006; Schreiner et al., 2003). Comitin null cells are impaired in the early steps of phagocytosis of yeast particles or bacteria, but not of latex beads, and accumulation of actin at phagosomes and subsequent dissociation was found unaffected. Other processes like pinocytosis, exocytosis and maturation of glycoproteins, were also found unaltered (Schreiner et al., 2003). The interpretation has been put forward that comitin associates with (and is needed for proper targeting) of a particular class of receptors. In support of this, comitin is enriched in the Triton-insoluble floating fraction (TIFF), where it co-caps, among others, with the cell adhesion molecule gp80 (contact site A) (Harris et al., 2003). In addition, comitin null cells are more pemissive to infections with Legionella pneumophila and Legionellalike bacteria; the mutation also delays degradation of Salmonella enterica (Skriwan et al., 2002). All this indicates a reduced efficiency of phagosome processing. This is in agreement with the proposal that comitin, by virtue of its biochemical activities and its localization, acts as a linker between intracellular membrane vesicles and the actin cytoskeleton ( Jung et al., 1996a). Nevertheless, the genome sequence has uncovered an uncharacterized comitin-related gene, therefore it is expected that the available data do not reveal the full spectrum of comitin’s roles in the cell. 3.7.3. Ponticulin Ponticulins constitute a large family of small atypical integral membrane proteins with a C-terminal glycosylated lipid anchor. Two ponticulins have been reported, ponticulin A and B, and only ponticulin A has been characterized extensively. Ponticulin A is abundant in axenically growing cells and during early development, where it accounts for most of the actinbinding activity of the plasma membrane (Hitt et al., 1994). Ponticulin A was found throughout the plasma membrane and in vesicles of the Golgi apparatus and, like comitin, it is enriched in the TIFF fraction (Harris et al., 2003). During phagocytosis the protein is present, although not enriched, in phagocytic cups and also associates with intracellular vesicles around the engulfed particle (Wuestehube et al., 1989). However, although membranes

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of ponticulin A null cells displayed only 10% of the high affinity actinbinding of wild type cells, the cells displayed unaffected pinocytosis and phagocytosis (Hitt et al., 1994), suggesting that the activities of ponticulin A are dispensable for endocytosis. Interestingly, ponticulin B, which exhibits 50% sequence similarity to ponticulin A and also localizes at the plasma membrane and in vesicles, is only expressed in cells grown on bacterial lawns and may play a clearer role in phagocytosis (Hitt et al., 2003). 3.7.4. Other membrane-associated ABPs Dictyostelium expresses two members of the annexin family of calcium and phospholipid binding proteins, annexin C1 (originally described as annexin VII or synexin) and annexin C2 (Marko et al., 2006). Annexins have been proposed to participate in the regulation of membrane organization, membrane trafficking and Ca2þ homeostasis. A number of annexins have been described as F-actin-binding proteins, and it has been suggested that they could participate in regulating membrane-cytoskeleton dynamics (Gerke et al., 2005). However, a direct binding of actin to any of the two annexins of Dictyostelium has not been demonstrated yet, therefore these proteins will not be discussed further. Hisactophilins by contrast are well documented ABPs targeted to the plasma membrane by virtue of myristoylation and positive charges of their numerous histidine residues (Hanakam et al., 1996). It has been proposed that hisactophilin acts as a pH sensor at the plasma membrane and reversibly connects the cortical actin network to the membrane in response to local changes of the proton concentration (Hanakam et al., 1996; Stoeckelhuber et al., 1996). During attachment and uptake of particles hisactophilin remains associated to the plasma membrane, where it re-shuffles after internalization (Maniak et al., 1995). Not surprisingly, histactophilin has been identified in isolated phagosomes (Gotthardt et al., 2006). Further evidence linking hisactophilins to endocytosis processes is missing: although a hisactophilin1/2 double mutant and an overexpressor strain have been generated, those processes have not been specifically addressed in these strains, and the Dictyostelium genome harbors a third hisactophilin gene that awaits characterization. Interaptin is an ABP of the a-actinin superfamily specifically targeted to the nuclear envelope, endoplasmic reticulum and Golgi apparatus (Rivero et al., 1998). It constitutes the Dictyostelium member of the metazoan nesprin family of proteins functioning as bridges that connect the nuclear matrix with the actin cytoskeleton (Ma¨a¨tta¨ et al., 2004). Despite its ER and Golgi localization, there is no evidence for interaptin playing roles in vesicle trafficking processes. Dictyostelium also has an uncharacterized member of the Sla2p/ Hip1 family of talin-related proteins. Proteins of this family bind to actin and clathrin at endocytic sites in yeast and mammals, functioning as adaptors that link the actin cytoskeleton to vesicle transport (Hyun and Ross, 2004).

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3.8. Actin-based molecular motors Myosins are actin-based motor proteins that convert the energy of ATP hydrolysis into movement. Dictyostelium expresses one conventional and twelve unconventional myosin heavy chains, along with up to eight myosin light chains (Kollmar, 2006). The roles of these myosins in diverse cellular processes and the pathways involved in their regulation have been the subject of several excellent reviews (Bosgraaf and van Haastert, 2006; de la Roche and Cote, 2001; Ma et al., 2001; Soldati, 2003). Here I will focus on the evidence linking Dictyostelium myosins (referring to the heavy chains only) to endocytosis and membrane traffic. 3.8.1. Myosin II and the myosin heavy chain kinase A The single conventional myosin of Dictyostelium has the characteristic long coiled-coil tail and assembles into bipolar filaments in a phosphorylationdependent manner. Myosin II accumulates at the cleavage furrow of dividing cells and at the rear end of migrating cells, and plays well established roles in cytokinesis, cell motility, chemotaxis and multicellular development. The evidence in support of a role of Dictyostelium myosin II in endocytosis and vesicle trafficking is scarce. The need for such studies was not appreciated because first, myosin II was not found to localize at phagosomes in immunofluorescence studies (Yumura et al., 1984) and second, it was soon realized that cells in which the myosin II heavy chain gene was disrupted were able to phagocytose bacteria but displayed more severe and attractive phenotypes (De Lozanne and Spudich, 1987; Manstein et al., 1989). By contrast, the participation of myosin II in phagocytosis is well established in other organisms. Myosin II is present in isolated phagosomes in E. histolytica, where it is recruited around the nascent phagosome during erythrophagocytosis. A strain that expresses a dominant negative myosin II displays a reduced erythrophagocytosis activity (Marion et al., 2005). It must be noted however that E. histolytica has a very limited repertoire of myosins (only a class I myosin, apart from myosin II), leaving little room for functional compensation. Myosin II has been also identified in phagosomes isolated from mouse macrophages (Garin et al., 2001). In these cells myosin II plays roles during CR3 and Fcg receptor-mediated phagocytosis, being required for phagosome closure (Araki et al., 2003; Olazabal et al., 2002). Is there any evidence linking Dictyostelium myosin II to any step of endocytosis? Electron microscopy studies on isolated cortices revealed an association of myosin II with unidentified cytoplasmic vesicles (Ogihara et al., 1988). Myosin II accumulates below ConA patches and moves with the patches into the cap (Carboni and Condeelis, 1985), a process that takes place less efficiently in cells lacking myosin II (Pasternak et al., 1989). This led to propose that this myosin participates in maintaining the resting-state cortical stiffness. Myosin II has been found in isolated early phagosomes,

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from which it is lost upon maturation (Rezabek et al., 1997), and in isolated early pinosomes, probably associated to actin filaments via the head domain (Neuhaus and Soldati, 2000). In one study with a GFP fusion myosin II was found at the base of phagocytic cups (but not at the protruding rim or at the entire phagosome), where it co-localized with PAKa (Mu¨llerTaubenberger et al., 2002). A shorter myosin II (HMM-140) unable to assemble into thick filaments does not localize at phagocytic cups (Fukui et al., 1990). Interestingly, accumulation of enzymatically inactive myosin II (either by treatment of wild type cells with the specific inhibitor blebbistatin, or by introducing the E476K mutant in myosin II deficient cells) results in strong inhibition of pinocytosis and phagocytosis, but this is probably due to formation of cytoplasmic aggregates, without altering the distribution of actin (Shu et al., 2005). In summary, the evidence gathered so far assigns a secondary part to myosin II in the endocytosis play, and the reason can be suspected at least in one of the proteins involved in its regulation, the myosin heavy chain kinase (MHCK) A. MHCK A is one of four closely related atypical kinases that phosphorylate the tail of Dictyostelium myosin heavy chain on specific threonine residues, driving disassembly of myosin filaments. These kinases have in common a catalytic domain followed by a WD repeat domain that binds directly to myosin II. Only MHCK A, which is unique to Dictyostelium, has an N-terminal coiled-coil extension responsible for homooligomerization and for binding and bundling of actin filaments (Bosgraaf and van Haastert, 2006). This coiled-coil region is necessary and sufficient for translocation of the protein to F-actin-rich structures (Steimle et al., 2001) and binding to F-actin leads to a 40-fold increase in MHCK activity. This might prevent myosin II filament accumulation at sites of actin-based protrusive activity, like the leading edge of migrating cells, but also the phagocytic and macropinocytic cup, where MHCK A accumulates transiently (Steimle et al., 2001). Knockout and overexpressor strains of MHCK A have been reported, but only the effects on myosin assembly have been studied (Kolman et al., 1996). It would be interesting to investigate how myosin II behaves during phagocytosis in the knockout mutant, where an increased accumulation of myosin II at the phagocytic cup is to be expected. 3.8.2. Class I myosins A large number of unconventional myosins belong to class I, probably reflecting their important roles in processes involving membrane dynamics. In fact, roles for these myosins in several steps of the endocytic pathway are well established in several fungal and mammalian species (Soldati, 2003). Three of them (MyoB, C and D) carry tails with the characteristic three regions TH1, TH2 and TH3, whereas three more (MyoA, E and F) have only the TH1 region. The TH1 region is a basic phospholipid-binding domain involved in attachment to membranes (Senda et al., 2001), the

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TH2 region constitutes an ATP-independent F-actin binding site and the TH3 region harbors an SH3 domain. One more class I myosin, MyoK has virtually no tail, but ends in a prenylation motif that targets the protein to the plasma membrane. This myosin has a TH2-like insertion in the motor domain that represents an additional F-actin-binding site, and contains poly-L-proline motifs that might bind profilin and SH3-domain containing proteins. Several class I myosins of Dictyostelium have been extensively studied, and single and multiple mutants have been characterized, in some cases extensively (Table 8.2 and references therein). From these studies a role in maintaining cortical tension, rather than in facilitating vesicle movement, has emerged (Dai et al., 1999; Schwarz et al., 2000). This role as ‘‘cortical managers’’ (Soldati, 2003) has been invoked to explain the motility and endocytosis defects observed in several myosin 1 mutants. Class I myosins are predominantly cytosolic, but a significant fraction (less than 15%) associates with the plasma membrane, as reported for MyoA, B, C and D in a detailed fractionation study (Senda et al., 2001). Several class I myosins accumulate at macropinocytic crowns (Du¨rrwang et al., 2005; Jung et al., 2001; Novak et al., 1995; Schwarz et al., 2000) and some have been also described at phagocytic cups (Du¨rrwang et al., 2005; Fukui et al., 1989) and in purified early pinosomes (Neuhaus and Soldati, 2000). Class I myosins have also been found to accumulate at phagocytic cups in Acanthamoeba, Entamoeba and macrophages (Allen and Aderem, 1995; Baines et al., 1992; Voigt et al., 1999). Extensive studies on single, double and triple mutants of class I myosins in several combinations have revealed a complex network of redundant, shared and non-redundant roles in endocytosis (Table 8.2 and Titus, 2000). For example, elimination of one class I myosin does not result in fluid phase uptake or exocytosis defects, whereas elimination of two or three leads to substantial (and in cases additive) pinocytosis and exocytosis defects, and consequently reduced growth rates in suspension. Phagocytosis is in general less affected: 30–40% reduction of the initial rate of particle uptake (but otherwise normal steady state levels) has been reported for MyoB and MyoC null mutants (but not for MyoA or MyoD null), and this defect does not become more intense in double or triple mutants. MyoA and MyoB null mutants display increased secretion of a-mannosidase and acid phosphatase, suggesting that these myosins play some role in recycling or retention of lysosomal enzymes. Some short tailed mammalian class I myosins associate to lysosomes and are important for delivery to internalized molecules (Raposo et al., 1999), but there is no evidence of association of Dictyostelium myosins with lysosomes (Senda et al., 2001). MyoB, but not MyoA, seems to be important for efficient recycling of membrane components from early endosomes to the plasma membrane (Neuhaus and Soldati, 2000). Perhaps all these defects explain why the MyoA/B double mutant is more permissive for intracellular growth of L. pneumophila (Solomon et al., 2000).

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Detailed analyses of endosomal trafficking performed in some myosin I mutants allowed also to conclude that these myosins are probably not involved in intracellular movement of vesicles, but rather at events that take place at the cell cortex. Here class I myosins are needed for achieving a balanced cortical tension; changes in one or the other direction result in a similar phenotype of deficient pinocytosis or phagocytosis, as demonstrated with Dictyostelium strains that lack or overexpress MyoB, C or K, or with an E. histolytica strain that overexpresses MyoIB, the unique unconventional myosin of this organism (Voigt et al., 1999). One aspect that has not been addressed in depth in the studies summarized above is the contribution of each class I myosin to endocytosis and other processes in terms of intracellular concentration and functional significance. MyoB and MyoC are more abundant than MyoD, which would explain in part why MyoD null mutants phagocytose normally compared to MyoB and MyoC null mutants ( Jung et al., 1996b). Another aspect to be considered is the ratio of phosphorylated (active) versus unphosphorylated (inactive) myosin. Class I myosins require phosphorylation of the so-called TEDS site of the motor domain for actin-activated ATPase activity (de la Roche and Cote, 2001). The functional relevance of phosphorylation has been addressed with a S332A mutant of the TEDS site of MyoB. This mutant failed to complement the pinocytosis and growth defects of a MyoA/B double mutant. Phosphorylation of class I myosins is performed by kinases of the PAK family, which are in turn regulated, among others, by GTPases of the Rho family. This is just one line of evidence that links unconventional myosins with the actin nucleation and elongation machinery (Soldati, 2003). Studies on class I myosins from several organisms highlight the functional relevance of the SH3 domain. SH3 domains interact specifically with proline-rich motifs and both SH3 domains and proline-rich motifs are found in numerous proteins involved in actin dynamics and endocytic trafficking. In Dictyostelium a truncated MyoB lacking the SH3 domain is unable to revert the growth and endocytosis defects of a MyoA/B double mutant, and when overexpressed it did not provoke the reduced rate of pinocytosis of the wild type myosin (Novak and Titus, 1997; Novak and Titus, 1998). As already mentioned (Section 3.2.3), the SH3 domains of MyoB and MyoC interact with the C-terminus of the scaffolding protein CARMIL, which in turn is an activator of the Arp2/3 complex ( Jung et al., 2001). On the other hand, the TH2-like insertion of MyoK harbors proline-rich motifs able to recruit profilin-actin and to interact with the SH3 domain of Abp1 (Soldati, 2003). Some class I myosins would be therefore needed for translocating multiprotein complexes involved in actin dynamics towards the barbed end of existing filaments, concentrating the complex in the vicinity of the plasma membrane. In support of such a role, MyoB is enriched at the plasma membrane close to phagosomes before the onset of actin-driven rocketing

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induced by compression of the cell. In MyoB null and MyoA/B double knockout cells rocketing is inefficient (Clarke et al., 2006). 3.8.3. MyoI and other unconventional myosins MyoI was initially described as belonging to class VII myosins, but recent sequence analyses seem to indicate that this myosin might constitute a common ancestor of several classes of myosins whose tail consists of a tandem repeat of TH4-FERM domains separated by an SH3 domain (Kollmar, 2006). The same architecture is found in the as yet uncharacterized MyoG. A GFP fusion of MyoI localizes diffusely in the cytosol and accumulates at the tip of filopods and some areas of the cell cortex. During particle uptake MyoI is enriched at the phagocytic cup, but only during the initial engulfment stages (Tuxworth et al., 2001). As already mentioned (Section 3.7.1), MyoI deficient cells exhibit a phenotype similar to that of TalA deficient cells: defective Ca2þ-dependent cell-to-cell adhesion, adhesion to the substrate and phagocytosis, but normal rates of fluid phase uptake and exocytosis (Titus, 1999; Tuxworth et al., 2001). The phagocytosis defect is severe, has been tracked down to the initial step of uptake and is caused by reduced adhesion of particles. In contrast to the TalA defect, the phagocytosis defect persisted when the assay was performed on cells attached to a surface or when a more adherent bacterial strain was used (Titus, 1999). When successful, extension of the phagocytic cup proceeded normally (Tuxworth et al., 2001). MyoI might promote the formation of links between the cytoskeleton and receptors at the plasma membrane needed for adhesion to the substrate and for engulfment of particles (Tuxworth et al., 2001). MyoM defines a novel class of myosins involved in Rho signaling. Its tail harbors a short coiled-coil stretch followed by a proline-rich region and a RhoGEF-PH domain combination. Such architecture has not been identified thus far in myosins of other organisms, while in higher eukaryotes myosins with a RhoGAP domain (class IX) exist (Geissler et al., 2000; Oishi et al., 2000). The motor domain of MyoM contains a phosphorylatable serine residue at the TEDS rule site, suggesting a mechanism of activation in common with class I myosins. In fixed cells a GFP fusion of MyoM appeared enriched at the cell cortex, crowns and large vesicles, probably macropinosomes. MyoM deficient cells grow normally (indicating that pinocytosis is not severely impaired) and perform phagocytosis normally. Two myosins, MyoJ and MyoH, are closely related to class V myosins (Kollmar, 2006). These myosins possess a tail with a coiled-coil dimerization domain and a large globular domain. Class V myosins are processive mechanoenzymes, which fits with their well established activity as vesicle motors in yeast and mammals, involved in transport of a broad repertoire of cargo organelles (Wu et al., 2000). MyoJ deficient cells did not present altered pinocytosis and apparently also phagocytosis, indicating that its

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function might be compensated by the uncharacterized MyoH (Peterson et al., 1996), but clearly more work is needed to establish the role of these interesting myosins.

4. Molecular Events During the Uptake Phase: A Simplified Model I will now make an attempt to incorporate the information summarized above into a simplified model that describes the sequence of molecular events that take place during formation and closure of the phagosome (Fig. 8.1). A similar sequence of events is likely to occur during formation of macropinosomes, with the difference that macropinocytosis does not need the local trigger of particle attachment. 1. Attachment of the particle (Fig. 8.1A). Phagocytosis frequently begins with the contact of filopods to the particle. Components of a complex formed by TalA and MyoI are required for this step. TalA is linked to the plasma membrane by interaction with integrin-related Sib proteins. MyoI probably also interacts with receptors at the plasma membrane. Proteins like VASP and formins are needed for the formation of filopods, but at least VASP is dispensable for particle uptake. Although some more membrane proteins essential for substrate adhesion and particle uptake have been described, it remains unclear how these proteins elicit the signaling changes required for recruitment and activation of the actin polymerization machinery. 2. Activation of the actin polymerization machinery (Fig. 8.1B). Although many details are still unclear, a hierarchy of signaling events involving heterotrimeric G proteins, GTPases of the Ras family and activation of phosphatidylinositol 3-kinases leads to de novo F-actin formation at the site of particle contact. Phosphoinositides play important roles for the recruitment of proteins bearing PH and other domains, like RhoGEFs. Recruitment and activation of small GTPases of the Rho family are instrumental for the activation of the actin polymerization machinery. Several multimolecular assemblies may be involved in positioning and activating the Arp2/3 complex in the vicinity of the plasma membrane. MyoB, MyoC and presumably also MyoD recruit CARMIL, whose main role is to inhibit CP and therefore enable elongation of actin filaments. CARMIL also activates, albeit weakly, the Arp2/3 complex. A more potent activator of the complex is WASP, in turn dependent on Rho GTPases and phosphoinositides for its activation. The role of WASP has not been addressed in Dictyostelium but is very likely, and the participation of the other major Arp2/3 complex activator, Scar, is

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A

?

Sib

Myol

Talin A

VASP P

P

Formin F-actin Rac B WIPa

CAP

Rac

P

WASP P P

Scar

MyoB,C,D

PAK Arp2/3 complex

IQGAP

CARMIL

Formin P

MyoA,E,F MyoM Abp1

MyoK P

CP

F-actin

ABP34 Fimbrin Filamin

MHCKA

LimC LimD LimE

LimF

CH-Lim Ponticulin Hisactophilin

Rab21

Other crosslinkers

Myosin II C

Severin Arp2/3 complex

F-actin

Cofilin Coronin

CP

Aip

ADP

ATP

C

P CAP

V-ATPase

Figure 8.1 Model of the role of actin cytoskeleton components during the uptake phase. See Section 4 of the main text for a detailed description. Although Rac occupies a central place in this model, this is not intended as to underplay the role of other signaling pathways. To avoid unnecessary complexity or because of incompleteness of the available information, some interactions are oversimplified. For example, it is not specified which Racs, formins or PAKs are involved and which specific myosins are targeted. Note that many more interactions are possible, particularly between SH3 domainbearing proteins and proteins with proline-rich regions, but only those interactions are depicted here that have been demonstrated in Dictyostelium or appear very likely by analogy to what we know from other organisms. While the role of WASP, WIPa, formins and IQGAP during uptake has not been specifically addressed in Dictyostelium, these components have been included based on data from other organisms. (A) Components

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unclear. In any case, there must be some overlapping in nucleation activity because none of the single knockouts of Scar, WASP or CARMIL is lethal nor results in abolished F-actin polymerization and actindependent functions. Additional unconventional myosins may also be required to position multimolecular complexes like MyoK (that binds profilin and Abp1) and MyoM (an activator of Rho GTPases). Class I myosins and MyoM would become activated by phosphorylation through kinases of the PAK family, which are in turn effectors of Rac. The exact role of one or more formins at this stage, eventually in complex with proteins of the IQGAP family, has not been defined yet. Profilin plays an important role in this phase, supplying actin monomers freshly charged with ATP to the nucleators. Profilin acts synergistically with cofilin and CAP to increase actin filament turnover at the phagocytic cup. 3. Progression and closure of the phagocytic cup. De novo assembly of actin filaments pulls the borders of the rim of the phagocytic cup following the contour of the engulfed particle. The forces exerted by de novo actin polymerization are probably sufficient for closure of the phagocytic cup. Myosin II is not required for phagosome closure in Dictyostelium, probably an effect of MHCKA, which is recruited to the actin meshwork and ensures that myosin II remains in a phosphorylated, inactive state. New actin filaments are crosslinked by fimbrin, ABP34, filamin and other crosslinkers, thus contributing to the stabilization of the meshwork. Numerous other ABPs with largely unknown roles also interact with F-actin at the phagocytic cup. 4. Detachment of the actin coat (Fig. 8.1C). While the polymerization machinery progresses from the base to the rim of the phagocytic cup, coronin begins to inactivate the Arp2/3 complex from the base of the phagosome. Removal or inactivation of CARMIL would enable repositioning of CP at barbed ends, preventing actin filament elongation. In this region of the phagocytic cup the activity of cofilin and severin dominates, leading to severing and depolymerization of filaments. CAP and Aip regulate the activity of cofilin. Detachment of actin and ABPs from the phagosome is completed usually within one minute after phagosome closure. In many cases residual actin polymerization at the point of phagosome closure is visible as a comet tail that pushes the phagosome away from the involved in the formation of filopods and attachment to the particle. (B) Components involved in activation of the actin polymerization machinery, subsequent elongation of actin filaments and stabilization of F-actin networks. (C) Components involved in disassembly of the actin coat. Black arrows indicate interactions (a discontinuous line means that the interaction is indirect or has not been proved in Dictyostelium). Green arrows indicate interaction and/or activation. Red arrows indicate interaction and/or inhibition. Gray arrows indicate interaction and/or activation. Open arrows indicate interaction and/or inhibition. P, profilin; C, cofilin.

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plasma membrane. As occurs during formation of clathrin-coated vesicles, actin polymerization takes place at the plasma membrane and not at the membrane that surrounds the vesicle. Away from the plasma membrane, the phagosome is captured by microtubules. Concomitant with the detachment of the actin coat, delivery of proton pumps and acidification of the phagosome starts.

5. Concluding Remarks One observation that emerges after examining the literature on actin cytoskeleton components is that a large number of those play dual roles, participating in endocytosis during the vegetative phase and in cell migration during the aggregation phase. In fact, the actin remodeling machinery used during the uptake phase is probably the same as needed for motility during chemotaxis (Gerisch et al., 1999), therefore many components whose role in chemotaxis is well established may be anticipated to play roles during uptake as well and vice versa. Although phagocytosis and macropinocytosis proceed in similar ways, either process has different requirements for particular sets of proteins as becomes apparent from inspection of Table 8.1, with macropinocytosis depending more strongly on G-actin binding proteins (Maniak, 2002). If a comparison could be made, it is as if phagocytosis and micropinocytosis (and cell motility too) were variations on the same musical theme played with the same set of instruments but with different arrangements. Dictyostelium is traditionally well known for its contribution to the actin cytoskeleton field. It is therefore not surprising that a large body of literature covering diverse aspects of the actin cytoskeleton, endocytosis among them, has accumulated over the years. In that way the roles of many components have been defined, although clearly, the roles of many others remain to be addressed. With the advent of the genome era we can now easily see whether homologs of components already studied in other organisms are also present in Dictyostelium and play similar roles. Nevertheless, a key task that needs to be accomplished in the future is to define networks of interactions, elucidate the timing with which events occur along the endocytic pathway and determine how these events are coordinated by regulatory elements. Further refinements of the imaging and high throughput technologies will help to overcome the current imbalance in our knowledge of the endocytosis process, in which a very well studied uptake phase contrasts with the poorly understood transit and exocytosis phases. These approaches along with the recently propagated use of Dictyostelium as a genetically tractable model for infectious disease promise to yield substantial contributions to a better general understanding of the endocytosis process.

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ACKNOWLEDGMENTS This review was elaborated in part while at the Institute for Biochemistry, Medical Faculty, University of Cologne. Support by the Deutsche Forschungsgemeinschaft and the Ko¨ln Fortune Programme of the Medical Faculty, University of Cologne is acknowledged.

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Index

A Acan125. See CARMIL proteins Acanthamoeba, 184, 195, 363, 380 Acipenser ruthenus, 234 Actin based molecular motors, 378–383 crosslinking proteins, 369–371 cytoskeleton components, model of, 383–386 in endocytosis, 358–360 related proteins, 360 Actin-bundling protein (ABP), 369 lateral ADF/cofilin family, 371–372 coronins, 372–373 LIM proteins, 373–374 RhoGEF, 374 membrane-associated, 374–375 comitin, 376 ponticulin, 376–377 talin, 375–376 Actin capping protein (CP) biochemical activies inhibiting molecular interaction, 186 motility reconstitution, role in, 185, 187 cellular studies, 187 CKIP-1, 196 isoforms, 189 physical and chemical properties, 184–185 sequence conservation BLAST, 187, 189 phylogenetic analysis, 188 in vertebrates, 187 a-Actinin, 369–371 b-Actinin, 184 Actin interacting protein (Aip), 367 Active transcription factor NF-kB, 72 Adenine nucleotide carrier (ANC) and phosphate carrier (PC) ratio, 28 Adenomatous polyposis coli (APC), 137 Adryamycin-induced nuclear proteasome activation, 95 AhR/ER receptor cross-talk for TCDD, 235 Aip protein, 367–368 Aldicarb, sulfoxidate carbamate pesticides, 237 Alkylphenols, surfactant, 228 Androgen receptors (AR) biomarkers of, 217 in fish, 216–217

Angiostatin, 41 Anguilla japonica, 216 Annexin protein, 377 Annulate lamellae (AL), 325 Antiandrogenic effects, mechanisms of metabolic clearance of biomarkers of, 227 steroid metabolism and, 226 steroid distribution sex steroid binding protein, 223–224 xenoestrogen binding measurement of, 224–226 steroid receptor androgen receptors, 216–217 antiandrogens, 217–219 biomarkers of estrogen receptor activation, 211–212 estrogen mimics, 212–216 estrogen receptors in, 209–211 steroid synthesis biomarkers of, 221 estrogenic/antiandrogenic effects, 221–223 reproductive steroidogenesis, 219–221 Antiandrogens, 217–219 Anti-apoptotic regulatory proteins, 72 Antigen-presenting cells (APCs) of immune system, 75–76 apoA-I and inhibition of ATP hydrolysis, 41 Apoptosis, 141, 143, 146–148, 156–158 regulatory proteins, 70 Arabidopsis thaliana, 310 Arp2/3 complex, in endocytosis, 361 Aryl hydrocarbon receptor/aryl hydrocarbon nuclear translocator (AhR/ARNT), 222 Aspergillus nidulans, 327 Atomic force microscopy (AFM), 20, 30, 307, 313, 315–316, 326 ATP synthase supramolecular organization, model for, 29 ATP synthesis by F1F0-ATPase, 5 Axonal development extracellular matrix in adhesive interactions, 132 glycoproteins and laminin, 133–135 nanoscale fabrication technology, 133 proteoglygans, 134–135 neuronal polarity in, 136–137 neurotrophins in differential regulation, 131–132

399

400

Index

Axonal development (cont.) glial cell line-derived neurotrophic factor, 132 receptors support, 130 with trophic factors, 131 Axon regeneration, 130, 132, 134, 145, 165 B Bacterial e subunit, ratchet mechanism, 10 BAPTA inhibition, 324 B-Cell chronic lymphocytic leukaemia (B-CLL), 72 BDNF. See Brain-derived neurotrophic factor 20b-Hydroxysteroid dehydrogenase (20b-HSD), 222 Binding actin mechanism C-terminal regions, mobility, 192 structural studies complete detachment, 191, 193 cryoEM analysis, 190–191 crystal structure, 189 truncation and point mutations, 190 Binding-change mechanism for steady-state catalysis, 6 Bovine ATP synthase from peripheral stalk, crystal structure of, 13–14 Bovine F1 bound with inhibitor dicyclohexylcarbodiimide (DCCD), 9 Brain-derived neurotrophic factor (BDNF), 131 Burkitt’s lymphoma, 73 C 2þ

Ca /calmodulin-dependent protein kinase II (CaMK II), 162 Caenorhabditis elegans, 137 Calpain protein, 158, 161–162, 279 Calponin homology (CH), 374 CaMK II. See Ca2þ/calmodulin-dependent protein kinase II CAP. See Cyclase-associated protein Capping protein (CP), 363, 368 actin polymerization inhibition, 198 antagonist regulators formin protein, 196 VASP protein, 196–197 complex cellular processes Cap Z, 200 dendritic nucleation model, 198–199 drosophila development, 201 dynactin, 201 lamellipodial regions, cultured cells, 199 Z-disc, sarcomere, 200 inhibition motif CARMIL proteins, 195 CD2AP and CKIP-1, 196 polyphosphoinositides interaction

computational docking analysis, 194 PIP2 capping activity, 194–195 protein inhibitors CARMIL and V-1 myotrophin, 193 molecular dynamics simulations, 194 Carassius auratus, 216 CARMIL protein, in endocytosis, 363 Caspase inhibitor benzyloxycarbonyl-Val-AlaAsp(OMe)-fluoromethylketone, 98 Caveolae/lipid rafts and eAS localisation, 37 Cell adhesion, 255, 259, 275, 283 Cell cycle regulation, 282 Cell cycle targets for ubiquitin-dependent proteasomal degradation, 67 Cell migration endothelial cells, VEGF-stimulated, 277 focal complex formation, 277, 279 focal contacts, regulation of, 276 traction force generation, 279 Cell motility based on actin assembly, 185 CARMIL, 195 vs. CP concentration, 187 Cell proliferation EGF signaling, negative regulators, 281 Merlin on Ser518 role in, 283 Pak-PIX-GIT–containing complexes and Raf-MEK-ERK pathway, 280 Cerebral ischemia, 156–157, 160 calpain inhibitor calpastatin in, 161 induced brain damage, 127 neuron death, 153–154 reperfusion, 163 CET. See Cryo-electron tomography CF6 expression in HUVECs, 40 Chlamydomonas reinhardtii, 24, 27 Chondroitin sulphate proteoglycans (CSPGs), 134–135, 144 Chromatin structure reorganization, 103 CKIP-1 protein, 196 Cofilin protein, 367–368 Collapsin response mediator protein (CRMP) in neuron death, 158–159 in neuron survival, 160–161 Comitin protein, 376 Computational docking analysis, 194 Coronin-GFP, 359 Coronins protein, 372–373 Cortexillins protein, 370–371 CRMP. See Collapsin response mediator protein Cryo-electron tomography, 301 CryoEM analysis, 190 Cyclase-associated protein, 366 Cyclin dependent kinases (CDKs), 65 CYP1A1 expression in Atlantic salmon hepatocytes, 227 Cyprinus carpio, 209 Cytochalasin A drug, 359–360

401

Index D Danio rerio, 210 DCC. See Dosage compensation complex DCC receptor, 146–147 Deleted in colorectal cancer receptor. See DCC receptor DHT. See 5a-Dihydrotestosterone Dicofol, estrogenic chemicals, 229 Dictyostelium discoideum, actin and endocytosis, 344 ABPs lateral, 371–374 membrane-associated, 374–377 actin, 358–360 F-actin nucleation machinery, 360–364 molecular motors, 378–383 monomeric actin binding proteins, 364–366 proteins actin crosslinking, 369–371 severing and capping, 367–369 Diethylmaleate (DEM), apoptotic inductors, 99 5a-Dihydrotestosterone (DHT), 216 Dosage compensation complex (DCC), 310 Doxorubicin/diethylmaleate-induced apoptosis of cell line, 88 Doxorubicin (DR), apoptotic inductors, 99 Dynacortin, 370–371 Dynactin, 201 Dynein, 130, 201 E ECM. See Extracellular matrix E. coli F1, central stalk protein complex from, 9 E. histolytica, 371, 378, 381 eIF4G translation initiation factors and ubiquitin-proteasome pathway, 104 ELYS/MEL28 protein, 320 Endocrine disruption, in fish, 208–209 Endocrine function consequences of, 236–237 DNA damage and, 238–239 intersex/sex reversal, 239–241 reproductive failure and, 241–242 unscheduled protein synthesis effects of, 237–238 Endocytosis analysis of cytoskeleton components, 346–353 myosins, mutant strains of, 354–357 Dictyostelium discoideum actin, role of ABPs lateral, 371–374 membrane-associated, 374–377 actin, 358–360 F-actin nucleation machinery, 360–364 molecular motors, 378–383 monomeric actin binding proteins, 364–366 proteins

actin crosslinking, 369–371 severing and capping, 367–369 Endocytosis, analysis of, 345 Endothelial cell proliferation and differentiation, 40 Enterostatin and fat intake regulation, 40 and inhibition of ATP synthesis, 41 Ephrin receptor tyrosine kinases (Eph RTK), 147 Ephrins (Ephs) molecules, 140 receptors, 141 Eph RTK. See Ephrin receptor tyrosine kinases Epidermal growth factor (EGF), 255 Epithelial cells barrier formation, 254 repair process outline, 254–255 Epithelial-mesenchymal transition (EMT), 258 Epithelialmorphogenesis, 259–260 ER-CALUX. See Estrogen receptor-mediated, chemical activated luciferase reporter gene expression ERE. See Estrogen-response elements ERM (Ezrin-radixin-moesin), 283 17b-Estradiol metabolite 4-hydroxyestradiol activating metabolism pathway, 238 Estradiol phase I reaction products in fish, 226 Estrogenic effects, mechanisms of steroid receptor androgen receptors, 216–217 antiandrogens, 217–219 biomarkers of estrogen receptor activation, 211–212 estrogen mimics, 212–216 estrogen receptors in, 209–211 Estrogen mimics, 212–216 Estrogen receptor, 209 activation, biomarkers of, 211–212 mediated, chemical activated luciferase reporter gene expression, 211 regulated transcription complexes on ER targets, 101 related receptors, 211 Estrogen receptor-mediated, chemical activated luciferase reporter gene expression (ER-CALUX), 211 Estrogen-response elements, 210 Ethinylestradiol, estrogenic chemicals, 229 Eukaryotes cell cycle and cyclindependent kinases (CDKs) activation, 65 Eukaryotes protein modifications and Co-translational N-a-acetylation by N-acetyltransferases, 90 Excitotoxicity-induced cell death, 127 External ATP synthase (eAS), 36 Extracellular ATP synthesis on HUVECs, 41–42 Extracellular matrix (ECM) role in axonal growth

402

Index

Extracellular matrix (ECM) (cont.) adhesive interactions, 132 glycoproteins in, 133, 135 laminin in, 133–135 nanoscale fabrication technology, 133 proteoglygans in, 134–135 F F-actin filaments, 129 nucleation machinery, 360–364 Fadrozole, aromatase inhibitors, 240 F1 and apolipoprotein A-1 on tumour cells complex, 41 Fas receptor-ligand apoptotic signaling pathway, 73 Fenarimol, androgenic chemicals, 229 F1F0-ATP synthases enzyme, 2 extra-mitochondrial expression of, 36 FG-FG interactions, 316 FG-nucleoporins, 306–307 Fibroblast growth factors (FGF), 132 Filamin protein, 369–371 Filopodia, 129 Fimbrin protein, 369–371 Flavin-containing monooxygenases (FMO), 237 Formin, 196 in endocytosis, 360–361, 363–364 Fundulus heteroclitus, 211 Fyn-Cdk5 pathway, in guidance cue, 152–153 G Gasterosteus aculeatus, 216 Gcn4 yeast activator and proteasome proteolytic activity, 101 GDNF. See Glial cell line-derived neurotrophic factor GEF. See Guanine nucleotide exchange factor Gelsolin protein, 368–369 Glial cell line-derived neurotrophic factor (GDNF), 131–132 GnRH expression in hypothalamus, 234 Gobio gobio, 208 Gonadotropin-releasing hormone (GnRH), 230 G-Proteins, 138 Growth cones composition of, 129 in guidance cue detection, 128 interaction with guidance cues, 130 Growth factors, 130, 132 GTH-R1 (FSH) receptors, 230 Guanine nucleotide exchange factors (GEFs), 260, 325 Guidance cues, repulsive axonal damage and neuronal death calpain in, 161–162 CaMK effects and CRMP modulation, 161

cerebral ischemia mediated, 153–154 collapsin response mediator protein, 158–159 CRMP in neuronal death and survival, 158 dependence receptor theory, 156 netrin mediated, 156–157 repulsive guidance molecule mediated, 157–158 RGM/Neogenin dependence receptors, 157 semaphorins and neuropilin mediated, 154–156 axonal pathfinding ephrins, 140–142 Fyn-Cdk5 pathway, 152–153 intracellular signalling pathways, 149 myelin, reactive glial and scar-derived inhibitors, 144–145 netrins, 139–141 PI3K-GSK pathway, 150–152 receptors for, 146–149 Rho GTPases pathway, 150 semaphorins, 138–139 slit protein, 142–143 future research potential, 165 and synaptic plasticity, 163 as therapeutic agents, 164 H HDL apolipoprotein A-I, 40 HeLa S3 cells, 304 Hemin and proteasome subunits dephosphorylation, 89 Heparan sulphate (HS), 147 Heparin-binding factors, 135 Hermaphrodism, in fishes, 208 Hexokinase type I (HK1), 311 Hisactophilins protein, 377 HKI. See Hexokinase type I Horizontal gaze palsy with progressive scoliosis (HGPPS), 148 HPG. See Hypothalamus-pituitary gonad Human umbilical vein endothelial cells (HUVECs), 39–40 Hydroperoxide-derived aldehyde-DNA adducts, 238 Hypodermal fusion, 265 Hypothalamus-pituitary-gonad axis, 209 description of, 230–231 estrogenic and antiandrogenic effects on, 231–233 indirect mechanisms of, 233–236 I IBB. See Importin b-binding IF1 inhibited ATP hydrolysis activity of eAS and HDL endocytosis, 42

403

Index

Importin b-binding, 312 Inhibitor protein (IF1), 5 Insulin-like growth factor I (IGF-1), 131–132, 233–234 Integrin receptors, 133 Interaptin protein, 377 Intracellular signaling pathways, in guidance cue Fyn-Cdk5 pathway, 152–153 P13K-GSK pathway, 150–152 Rho GTPase pathway, 150 IQGAP, in endocytosis, 363–364 Ischemia-induced synaptic plasticity, 163 Ischemic neuronal death, 157. See also Neuronal death K Karyopherins, 312 11-Ketotestosterone (11-KT), 217 Kringle 1–5, plasminogen fragments, 40–41 L Laminin, 131, 133–135, 140 Latrunculin A drug, 359–360 Leber hereditary optic neuropathy (LHON), 20 Legionella pneumophila, 372, 376, 380 Leigh syndrome, 20, 34 Limanda limanda, 228 LimEDcoil, 359 LIM proteins, 373–374 LMP7-dependent degradation of POMP, 75 LMP2/LMP7/MECL-1-dependent epitopes in inflammatory sites, 75 M mAb414 protein, 320 Madin-Darby canine kidney (MDCK), 260 MAG. See Myelin associated glycoprotein Malignancies, hematologic, 95 MAM. See Mitochondria-associated membrane Mammalian cells mtATPase, subunit composition, genetic specification and stoichiometry, 4 Manduca sexta, 99 MAPIB protein, 136 MAPs. See Microtubule-assosiated proteins Maternally inherited Leigh syndrome (MILS), 20 Mdm2 RING-finger ubiquitin ligase, 73 Merlin, 283 Mesenchymal-epithelial transition (MET), 258 MG132 proteasome inhibitor, 72 Micropogonias undulates, 210 Micropterus salmoides, 210 Microtubule-assosiated proteins (MAPs), 128, 136 Microtubule organizing center (MTOC), 271–274

Microtubules, 129, 271 Mitochondria-associated membrane (MAM), 39, 145–146 Mitochondrial ATP synthase (mtATPase) F0 sector, function and structure of, 18–22 F1 sector, 7 central stalk, 9–10 inhibitor protein (IF1), 10–12 function and structure of, 3 mitochondrial membranes, arrangement in, 29–31 mtATPase oligomerisation, role of, 31–34 peripheral/Stator stalk, 12–13 bacterial and bovine OSCP, 13–18 rotary catalysis of, 3–7 Monomeric actin binding proteins, 364–365 cyclase-associated protein, 366 profilin, 365–366 mtATPase oligomerisation, role of, 29–34 MTOC. See Microtubule organizing center Multi-subunit mtATPase complex, 37 Mutant strains of class I myosins and endocytosis in, 354–357 Mycobacterium marinum, 372 c-Myc oncoprotein accumulation, 73 Myelin associated glycoprotein (MAG), 144–145 Myelin-secreted inhibitory molecule, 144, 149 Myosin class I, 379–382 II, 378–379 MyoM, 382 Myosin heavy chain kinase (MHCK) A, 378–379 Myosin heavy chain (MHC), 279 Myosin-II, 130, 378, 385 Myosin light chain kinase (MLCK), 279 Myosin light chain (MLC), 279 N þ

2þ

Na and Ca influx, 127 Nanoscale fabrication technology, 133 Neogenin receptors, 148–149 Netrin proteins in axon pathfinding, 139–140 and DCC/UNC receptors, 141 in neuron death, 156–157 Netrin receptors, 146–147 Neuritogenesis, 128, 134, 150 Neurogenic ataxia and retinitis pigmentosa (NARP), 20 Neuronal death, 127–128, 150 CRMP in, 158–161 CRMP modulation by calpain and CaMK, 161–162 Netrin-1/UNC/DCC in, 156–157

404 Neuronal death (cont.) RGM/Neogenin dependence receptors in, 157–158 semaphorin/neuropilin in, 154–156 Neuronal regeneration, 128, 161 Neuron polarity, in axonal growth, 136–137 Neuropilin-1 glycoprotein, 145 Neuropilin receptor in axon pathfinding, 145–146 in neuron death, 154–156 Neurotrophin-4 (NT-4), 131 Neurotrophins, axonal growth differential regulation, 131–132 glial cell line-derived neurotrophic factor, 132 receptors support, 130 with trophic factors, 131 NK cell-mediated cytotoxicity of tumour cells, 41 N-methyl-D-aspartic acid (NMDA), 153–154 Nogo receptor, 149 Nonylphenol, estrogenic chemicals, 229 NPCs. See Nuclear pore complexes Nsp1p nucleoporin, 316 NTD. See N-terminal domains N-terminal domains, 12–15, 22, 261, 303, 366 Nuclear envelope (NE), 300 Nuclear estrogen receptors, domains of, 209–211 Nuclear pore complexes (NPC), 300 agreement and disagreement, 319–320 interpretation of, 327–329 nuclear pore, 320–321 nuclear pore building blocks, 321–322 nucleoporins, 322–323 peripheral pore structures, 323–324 regulation of, 324–325 cargo translocation in, 312–313 FG-domain behavior, 319 FG-domain function, 315–317 FG-domains and barrier function, 317–318 nucleocytoplasmic transport, 318–319 selective gating, 313–315 structure of architecture of, 301–303 atomic level, 303–304 density and distribution, 304–306 Nucleation promoting factors, 360 Nucleoporins (Nups) function nucleocytoplasmic transport, 306–307 nucleoporins and kinetochores, 308 nucleoporins and transcription, 308–310 Parkinson’s disease, 310–312 Nup107–160, 323 Nup133, 303 Nup214, 303–304 Nup107–160 complex, 308 NUP98 gene, 309 Nup35-Nup93, 322 Nup153, role of, 323

Index O 4-OHE2, genotoxicant, 238 Oncoprotein 18 (Op18). See Stathmin Oncorhynchus kisutch, 209 Oncorhynchus mykiss, 210 Oreochromis aureus, 241 Oreochromis niloticus, 216 Oryzias latipes, 216 Osteosarcoma cell line plasma membrane (PM), 37 P PA28a/b proteasome regulator, 97 PA700 activator protein, 62 P21-activated kinases activation, mechanism of Rho GTPase–independent activation, 263–264 by Rho GTPases, 262–263 background functions, 261 inactivation, 264–265 structure of, 261–262 PA28 expression in mature dendritic cells, 93 Pagrus major, 216 Pak activation wound healing and epithelial sheet migration background functions, 265–266 kinase-independent functions, 267–268 PIX-GIT complex, 268–269 wounding-associated signals, 266–267 Paralichthys olivaceus, 239 Paramecium multimicronucleatum, 23 Parkin protein, 311 Parkinson’s disease, 310–312 Partitioning-defective proteins (PAR), 137 Phosphatidylinositol (4,5) bisphosphate, 362 Phosphoinositide 3-kinase (PI3K), 150 Phosphorylation, in class I myosins, 381 Platelet-derived growth factor (PDGF), 255 Platichthys flesus, 208, 228 Plexin-A4 and A3, 146 Poecilia reticulata, 241 Poly(ADPribose) polymerase (PARP), 95 Polymerization, 194 actin inhibiting, 198–199 in binding actin mechanism, 190 in biochemical activities, 185 PIP2 uncapping in, 194 Ponticulin protein, 376–377 Pore-free islands’’, 305 Pregnane-X-receptor (PXR), 228 Prochloraz and spermatogenesis, 234 Profilin protein, 365–366 b-Propellers, 303–304 Proteasome enzyme antiapoptotic functions of, 71–74 and apoptosis, 68–70

405

Index

apoptosis-induced changes of, 98–100 catalytic activities of peptidase activity and, 63 RNase activity and, 64 in cell regulation of and proteasome subunit expression, 92–93 composition modulation of heterogeneity in cell, 76–78 N-terminus of Rpt2 subunit by myristoylation, 90–91 post-translational, 78–86 gene expression and posttranslational stages, 103–105 transcription process, 100–103 immune response and, 74–76 N-acetylation and N-terminal propeptide processing of, 89–90 proapoptotic function of, 70–71 reprogramming at differentiation, 98 19S regulatory complex, 62–63 structure of, 61 subunits phosphorylation of, 86–89 Proteasome maturation protein (POMP), 75 Proteasome proteolytic activity, 103 Proteasome-ubiquitin pathway, 60 Protein cross-bridge at F1-F1 interface of dimeric structure, 11 Protein kinase C (PKC), 40, 134, 148, 273, 279–280 Proton-driven ATP synthesis by F1F0-ATPase, 6 Protovillin, role in G-actin, 368 p53 tumor suppressor and apoptosis induction, 73 R Rac/cdc42, 262–264 RanBP2 protein, 303, 308, 311–312, 324 RanGTP, 309, 312, 318, 325–326 Repulsive guidance molecule (RGM) in axon pathfinding, 143–144 and Neogenin, 143 in neuron death, 157–158 RGM. See Repulsive guidance molecule Rho GTPase exchange factor (RhoGEF), 374 Rho GTPase pathway, in guidance cue, 150 Rho-GTPases protein, 128, 135, 259 RNA silencing, 130 Robo receptor, 142, 147–148 Roundabout receptor. See Robo receptor Rpn5 subunit of 19S proteasome activator and proteasome complex, 95 Rutilus rutilus, 208 S Salmonella cerevisiae, 308, 323, 327 Salmonella enterica, 376 Sarcomere

in striated muscle, 200 Z-disc, located at, 189 Semaphorin protein in axon pathfinding, 138–139 in neuron death, 154–156 and neuropilin/plexins, 139 semaphorin-3A, 138, 155–156 Semicossyphus pulcher, 239 Serotonin reuptake inhibitor (SSRI), 233–234 Sewage-treatment works, 208 Sex steroid binding protein (SBP), 223–226 SH3/SH2-domain, 267 Single-molecule experiments and rotational catalysis in F1F0-ATP synthase, 6 Single molecule force spectroscopy, 313 siRNA silencing, 132 Sjogren’s syndrome, 95 Skp1/Culin/F-box protein (SCF) protein-ubiquitin ligase, 66 Slit protein, 142–143 SMFS. See Single molecule force spectroscopy SMFS-AFM analysis, 316 Snail family proteins, 258 Sparus auratus, 210 Stathmin, 271–272 Stator stalk, subunit i/j forms, 22 Steroid receptor coactivator (SRC)-interacting proteins, 101 Stroke, 128 STWs. See Sewage-treatment works Supramolecular ATP synthase dimers and oligomers, 23–25 e, g and k, dimer specific subunits, 25–28 Supramolecular structures and respiratory complexes, 35–36 Synaptic plasticity, 163–164 T Talin protein, 375–376 Tau protein, 136–137, 152, 274 TBCB. See Tubulin cofactor B T Cell receptor-induced apoptosis, 71 Testosterone phase I reaction products in fish, 226 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), 235 Thr423 residue, 262–264 Thyroid-stimulating hormone (TSH), 235 TIFF. See Triton-insoluble floating fraction TIRF microscopy, 185, 194 TM2 swivelling, protonation/deprotonation cycle, 20 TNF-a-induced apoptosis, 71 Tpr nucleoporin, 320–321 Transforming growth factor a (TGF-a), 255 Transforming growth factor b (TGF-b), 132, 255 Transient receptor potential channels (TRPC), 164

406

Index

Tributyltin, androgenic chemicals, 229 Trigger caspase-mediated apoptosis, 40–41 Triphenyltin, androgenic chemicals, 229 Triton-insoluble floating fraction, 376 Triton X-100, surfactant, 235 TRPC. See Transient receptor potential channels Trp271 yeast residue, 192 a-and b-Tubulin, 129 Tubulin cofactor B (TBCB), 129 Twinfilin protein, 198, 365 Two serine/threonine phosphatases, 264 U Ubiquitin-dependent proteasomal proteolysis, 99 Ubiquitin-proteasome system, 66 UDP-Glucuronosyltransferase, 226 UNC5 receptor, 146–147 V Vascular endothelial cells and stress-induced ATP release, 37 Vascular endothelial growth factor (VEGF), 145, 255 VASP protein, in endocytosis, 363–364 Vitellogenin, biomarker, 237 V-1, myotrophin protein, 193 W WASP/Scar family, in endocytosis, 361–363 Wobble hypothesis, 192–193 Wound healing actin-myosin, regulation of MHC phosphorylation, function of, 279 role of Pak in, 279 cell-cell contacts contact inhibition by Pak, 284 scrape-wounded MDCK cells, 284 stop phase, apico-basolateral polarization, 283 tumor suppressor protein (Merlin) inhibition, 283 cell migration cell protrusion stabilization, 276 endothelial cells, VEGF-stimulated, 277

focal complex formation, 277, 279 focal contacts, regulation of, 276 traction force generation, 279 cell polarization cell protrusion stabilization, 276 lamellipodia protrusion, 270–271 microtubule cytoskeleton reorganisation, 271 microtubule stabilization, 272–273 Pak-PIX-GIT complex, 274–275 polarized vs. nonpolarized cells, 273–274 Scrib complex proteins, 274–275 traction forces, generation of, 279–280 developmental models, 256–257 epithelial plasticity apical-basolateral polarization, 258 future research directions Pak inhibitory proteins, 284–285 spatiotemporal regulation of Pak function, 284 mitogenic signaling, regulation of, 280–281 regulation of cell motility and sheet migration, 270–275 cell proliferation by Pak, PIX and GIT, 280–284 mitogenic signaling, 280–281 Rho GTPases and epithelial morphogenesis, 259–260 scrape wound healing, 257 steps involved in, 259 wound healing and cancer, 257–258 X Xenopus laevis, 68, 302 Y Yeast estrogen screen (YES), 211 Yeast F1 structure, 7 Yeast mtATPase, 4 regulation, 12 Yeast regulatory proteasome 19S subparticle, 101–102