International Review of Cell and Molecular Biology, Volume 280

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INTERNATIONAL REVIEW OF

CELL AND MOLECULAR BIOLOGY

INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY Series Editors

GEOFFREY H. BOURNE JAMES F. DANIELLI KWANG W. JEON MARTIN FRIEDLANDER JONATHAN JARVIK

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ISAIAH ARKIN PETER L. BEECH ROBERT A. BLOODGOOD DEAN BOK KEITH BURRIDGE HIROO FUKUDA RAY H. GAVIN MAY GRIFFITH WILLIAM R. JEFFERY

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KWANG W. JEON Department of Biochemistry University of Tennessee Knoxville, Tennessee

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Front Cover Photography: Cover figure by Anders Lydik Garm Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 32 Jamestown Road, London NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands First edition 2010 Copyright # 2010, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@elsevier. com. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material. Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress For information on all Academic Press publications visit our website at elsevierdirect.com

ISBN: 978-0-12-381260-5

PRINTED AND BOUND IN USA 10 11 12 10 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors

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1. Natriuretic Peptides in the Regulation of the Hypothalamic–Pituitary–Adrenal Axis

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Andrea Porzionato, Veronica Macchi, Marcin Rucinski, Ludwik K. Malendowicz, and Raffaele De Caro 1. Introduction 2. Biology of Natriuretic Peptides and Their Receptors 3. Expression of Natriuretic Peptides and Their Receptors in the HPA Axis 4. Effects of Natriuretic Peptides on the HPA Axis 5. Natriuretic Peptides and Pathophysiology of HPA Axis 6. Concluding Remarks Acknowledgment References

2. Evidence for Multiple Photosystems in Jellyfish

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¨m Anders Garm and Peter Ekstro 1. Multiple Photosystems 2. Photosensitivity in Cnidarians 3. Photosensory Organs in Hydromedusae 4. Photosensory Organs in Scyphomedusae 5. Photosensory Organs in Cubomedusae 6. Multiple Opsins in Cnidarians—Multiple Photosystems? 7. Conclusion Acknowledgments References

3. Membrane Trafficking in Protozoa: SNARE Proteins, H+-ATPase, Actin, and Other Key Players in Ciliates

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Helmut Plattner 1. Introduction 2. Factors Involved in the Regulation of Vesicle Trafficking 3. Features of SNAREs

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Contents

4. Exocytosis and Endocytosis 5. Possible SNARE Arrangement in Microdomains and Membrane Fusion 6. Phagocytosis 7. Calcium-Binding Proteins and Calcium Sensors 8. Additional Aspects of Vesicle Trafficking 9. Emerging Aspects of Vesicle Trafficking in Ciliates 10. Concluding Remarks Acknowledgments References

4. New Insights into the Types and Function of Proteases in Plastids

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Yusuke Kato and Wataru Sakamoto 1. Introduction 2. Overview 3. Major Proteases 4. Processing Peptidases 5. Intramembrane Proteases 6. Other Proteases 7. Concluding Remarks References

5. Impact of ATP-Binding Cassette Transporters on Human Immunodeficiency Virus Therapy

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Johanna Weiss and Walter Emil Haefeli 1. Introduction 2. Drug Therapy of HIV-1: Drug Classes and Site of Action 3. ABC-Transporters Influencing Drug Therapy of HIV-1 Infections 4. Cell Models Investigating the Impact of ABC-Transporters for HIV-1 Therapy 5. Anti-HIV-1 Drugs as Substrates, Inhibitors, and Inducers of ABC-Transporters: In Vitro and In Vivo Findings 6. Clinically Relevant Drug Interactions with Anti-HIV-1 Drugs Attributed to ABC-Transporters 7. ABC-Transporters, ‘‘Cellular’’ Resistance, and Therapeutic Success 8. ABC-Transporter Polymorphisms and HIV-1 9. Concluding Remarks References

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6. New Insights into the Circadian Clock in Chlamydomonas

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Takuya Matsuo and Masahiro Ishiura 1. Introduction 2. Behavioral and Physiological Circadian Rhythms in Chlamydomonas 3. Circadian Oscillator in Chlamydomonas 4. Input Pathways to the Circadian Oscillator in Chlamydomonas 5. Output Pathways from the Circadian Oscillator in Chlamydomonas 6. Concluding Remarks Acknowledgments References Index

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CONTRIBUTORS

Raffaele De Caro Department of Human Anatomy and Physiology, University of Padova, Padova, Italy ¨m Peter Ekstro Department of Cell and Organism Biology, Lund University, Lund, Sweden Anders Garm Department of Comparative Zoology, University of Copenhagen, Copenhagen, Denmark Walter Emil Haefeli Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Heidelberg, Germany Masahiro Ishiura Center for Gene Research and Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Yusuke Kato Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, Japan Veronica Macchi Department of Human Anatomy and Physiology, University of Padova, Padova, Italy Ludwik K. Malendowicz Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland Takuya Matsuo Center for Gene Research and Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan Helmut Plattner Department of Biology, University of Konstanz, Konstanz, Germany Andrea Porzionato Department of Human Anatomy and Physiology, University of Padova, Padova, Italy

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Marcin Rucinski Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland Wataru Sakamoto Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, Japan Johanna Weiss Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Heidelberg, Germany

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Natriuretic Peptides in the Regulation of the Hypothalamic– Pituitary–Adrenal Axis Andrea Porzionato,* Veronica Macchi,* Marcin Rucinski,† Ludwik K. Malendowicz,† and Raffaele De Caro* Contents 1. Introduction 2. Biology of Natriuretic Peptides and Their Receptors 2.1. Natriuretic peptides 2.2. Natriuretic peptide receptors and their signaling mechanisms 3. Expression of Natriuretic Peptides and Their Receptors in the HPA Axis 3.1. Hypothalamus 3.2. Pituitary gland 3.3. Adrenal cortex 3.4. Adrenal medulla 4. Effects of Natriuretic Peptides on the HPA Axis 4.1. Hypothalamus 4.2. Pituitary gland 4.3. Adrenal cortex 4.4. Adrenal medulla 5. Natriuretic Peptides and Pathophysiology of HPA Axis 5.1. Adrenocortical adenomas and carcinomas 5.2. Pheochromocytomas 6. Concluding Remarks Acknowledgment References

2 2 2 4 4 4 8 10 11 12 12 15 18 20 21 21 22 22 24 24

Abstract Atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides act by binding to three main subtypes of receptors, named NPR-A, -B, and -C. NPR-A and NPR-B are coupled with guanylate cyclase. Not only NPR-C is involved in removing natriuretic * Department of Human Anatomy and Physiology, University of Padova, Padova, Italy Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland

{

International Review of Cell and Molecular Biology, Volume 280 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)80001-2

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2010 Elsevier Inc. All rights reserved.

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peptides from the circulation but it also acts through inhibition of adenylyl cyclase. NPR-A binds ANP and BNP; NPR-B preferentially binds CNP; and NPR-C binds all natriuretic peptides with similar affinities. All natriuretic peptides and their receptors are widely present in the hypothalamus, pituitary, adrenal cortex, and medulla. In the hypothalamus, they reduce norepinephrine release, inhibit oxytocin, vasopressin, corticotropin-releasing factor, and luteinizing hormone-releasing hormone release. In the hypophysis, natriuretic peptides inhibit basal and induced ACTH release. Conversely, the effects of natriuretic peptides on secretion of growth, luteinizing, and follicle-stimulating hormones are not clear. Natriuretic peptides are known to inhibit basal and stimulated aldosterone secretion, through an increase of intracellular cGMP, and to inhibit the growth of zona glomerulosa. Inhibition or stimulation of glucocorticoid secretion by adrenocortical cells has been reported on the basis of the species involved, and an indirect effect mediated by adrenalmedullary cells has been hypothesized. In the adrenal medulla, natriuretic peptides inhibit catecholamine release and increase catecholamine uptake. It appears that natriuretic peptides may play a role in the pathophysiology of adrenocortical neoplasias and pheochromocytomas. Key Words: Natriuretic peptides, Hypothalamic–pituitary–adrenal axis, ACTH secretion, Catecholamine secretion, Pheochromocytomas. ß 2010 Elsevier Inc.

1. Introduction Numerous neuropeptides control the hypothalamic–pituitary–adrenal (HPA) axis, acting on both its central and peripheral branch. Natriuretic peptides are known to be included in this group of regulatory peptides, but only a few review articles have been published regarding the role of natriuretic peptides in the HPA axis, and mainly with reference to specific structures or specific pathological conditions (Gutkowska et al., 1997; Wiedemann et al., 2000). A comprehensive and updated review on the role of natriuretic peptides in all the levels of the HPA axis is still lacking. Thus, after a synthetic account on the biology of the natriuretic peptides system, we will herein review data indicating how natriuretic peptides and their receptors are expressed in all the anatomical components of the HPA axis, and are involved in the functional regulation of HPA axis under both physiological and pathological conditions.

2. Biology of Natriuretic Peptides and Their Receptors 2.1. Natriuretic peptides Natriuretic peptides represent a family of three hormones called atrial natriuretic peptides (ANP) (Kangawa and Matsuo, 1984), brain natriuretic peptides (BNP) (Sudoh et al., 1988), and C-type natriuretic peptides (CNP) (Sudoh

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et al., 1990). ANP is a 28-amino acid peptide which has first been isolated from human atrial extract (Kangawa and Matsuo, 1984). BNP and CNP have been identified in the porcine brain (Sudoh et al., 1988, 1990). Figure 1.1 shows the sequences of natriuretic peptides. All peptides contain the conserved sequence FGXXXDRIGXXSGL. The flanking cysteines form a 17-amino acid disulfide-linked ring that is required for biological activity. In some tissues, CNP-53 is cleaved to CNP-22.

ANP 1

26

124

151

103

134

105

126

pro-ANP

ANP-28 URO BNP 1

27 pro-BNP

g-BNP (pro-BNP, in blood) BNP-32 CNP 1

24 pro-CNP

CNP-53

CNP-22

Figure 1.1 Natriuretic peptide expression (prepro-ANP, -BNP, and -CNP). Each oval represents 1-amino acid residue: yellow—the signal sequence; blue—part removed during processing of propeptide to mature peptide; and red—mature peptide. Alternative processing of pro-ANP generates a 32-residue peptide called urodilatin (URO, renal natriuretic peptide). Two variants of BNP are known: mature BNP-32 and in the blood g-BNP (pro-BNP). CNP also is known in two variants: CNP-53 and CNP-22.

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2.2. Natriuretic peptide receptors and their signaling mechanisms The biological activity of the natriuretic peptides occurs via the activation of three different receptors, which have been cloned and pharmacologically characterized: NPR-A, NPR-B, and NPR-C. The first two receptors are coupled with guanylate cyclase. They consist of an extracellular ligand-binding domain, a short transmembrane region, a juxtamembranous protein kinasehomology domain, an alpha-helical or hinge region, and a C-terminal guanylyl cyclase catalytic domain, receptor dimerization being essential for the activation of the catalytic domain (reviewed in Anand-Srivastava and Trachte, 1993; Kuhn, 2003; Maack, 1992; Potter et al., 2006, 2009). Alternative splicing of NPR-A has recently been found to produce an isoform which does not bind ANP and may inhibit ligand-inducible cGMP generation by forming heterodimers with the wild-type receptor (Hartmann et al., 2008). NPR-A is activated by ANP and BNP, ANP being more effective than BNP in stimulating cGMP production. NPR-B binds with higher affinity CNP (Fig. 1.2). All natriuretic peptide receptors are also known to be internalized and to some extent recycled as a result of ligand binding (reviewed in Pandey, 2009). NPR-C binds all three natriuretic peptides with relatively similar affinities (Maack, 1992). It is a disulfide-linked homodimer with a single transmembrane domain which lacks the intracellular guanylate cyclase domain but is able to internalize natriuretic peptides after binding. Thus, it has first been considered to be involved in removing natriuretic peptides from the circulation (Fig. 1.2). Nevertheless, following studies suggested that NPR-C contains a 37-amino acid intracellular domain which is able to inhibit the adenylyl cyclase and activate phospholipase C, through activation of Gi proteins. Moreover, NPR-C may also inhibit the mitogenactivated protein kinase pathway (signaling pathways of NPR-C reviewed in Anand-Srivastava, 2005).

3. Expression of Natriuretic Peptides and Their Receptors in the HPA Axis 3.1. Hypothalamus ANP has first been identified in the rat hypothalamus by radioimmunoassay (Glembotski et al., 1985; Tanaka et al., 1984) and its release in vitro from rat hypothalamus has also been demonstrated (Shibasaki et al., 1986a; Tanaka and Inagami, 1986). Although it must be considered that some authors reported cross-reactions with neurophysins in immunohistochemistry of the rat hypothalamus, suggesting absence of ANP immunostaining in the hypothalamus (Nilaver et al., 1989), ANP has been identified by immunohistochemistry in

ANP-28

BNP-32

COOH

H 2N

CNP-22

H2N

COOH

H2N

COOH

NPR-B NPR-C

NPR-A

KHD KHD cGMP GTP

cGMP

GTP GC

GC GTP GTP

cGMP

cGMP

Figure 1.2 Interaction of ANP, BNP, and CNP natriuretic peptides with receptors NPA-R, NPB-R, and NPC-R. NPR-A and NPR-B are membrane-bound guanylyl cyclases, NPR-C—not coupled to guanylyl cyclase—is involved in clearance and metabolism of natriuretic peptides. KHD, kinase homology domain.

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neurons of several mammal hypothalamic and nonhypothalamic brain structures, such as the septum, anteroventral region of the third ventricle (AV3V), subfornical organum, paraventricular nucleus (PVN), preoptic, supraoptic (SON), infundibular and ventromedial nuclei, lateral hypothalamus, organum vasculosum lamina terminalis, median eminence, lamina terminalis, periaqueductal gray matter, parabrachial nucleus, solitary tract nucleus, tegmental lateral dorsal nucleus, and periventricular regions (e.g., Chriguer et al., 2001; Gutkowska et al., 1997; Jirikowski et al., 1986; Kawata et al., 1985; Raidoo et al., 1998; Standaert et al., 1986a; Tanaka et al., 1984). Most ANP-immunoreactive neurons in the PVN belong to the parvocellular division ( Jirikowski et al., 1986; Kawata et al., 1985), but colocalization of ANP and oxytocin (OT) immunostaining has also been reported in some magnocellular neurons of the magnocellular division of the PVN and SON (Chriguer et al., 2001; Gutkowska et al., 1997; Jirikowski et al., 1986; Kawata et al., 1985). The densest terminal fields of ANP-containing fibers have been reported in the PVN of the hypothalamus, the bed nucleus of the stria terminalis, the interpeduncular nucleus, and the median eminence (Standaert et al., 1986a), where ANP may modulate the release of anterior pituitary hormones (Franci et al., 1990, 1992; Gutkowska et al., 1997). It has also been reported that ANPcontaining neurons in the PVN are the major source of ANP-containing nerve terminals in the median eminence (Palkovits et al., 1987). ANP-immunoreactive fibers have also been observed in close proximity with oxytocinergic fibers in the median eminence (Chriguer et al., 2001). In the hypophyseal portal blood, ANP has been found in 3–4 times higher concentrations than in the peripheral blood and the predominant species of IR-ANP in extracts of portal blood from adult rats is ANP(5–28), whereas in peripheral blood is ANP(1–28) (Lim et al., 1994). ANP mRNA has also been identified in the rat hypothalamus (Chen et al., 1992; Dagnino et al., 1991; Gardner et al., 1987; Komatsu et al., 1992). The distribution of mRNA encoding prepro-ANP has also been investigated in rat brain by in situ hybridization and the highest relative concentrations have been detected in the anteromedial preoptic nucleus of the medial preoptic area (Gundlach and Knobe, 1992; Ryan et al., 1997). Analysis through RT-PCR in the rat and monkey hypothalamus did not identify BNP mRNA (Abdelalim et al., 2006; Langub et al., 1995). However, radioimmunoassay studies have detected BNP in porcine (Ueda et al., 1988), canine (Itoh et al., 1989), rat (Sone et al., 1991), human (Takahashi et al., 1992), and ovine (Pemberton et al., 2002) hypothalamus. BNP-immunoreactive fibers are also present in the PVN of the hypothalamus and many BNP-positive neurons have been retrogradely labeled in the tuberomammillary nucleus of the hypothalamus and in the pedunculopontine and laterodorsal tegmental nuclei (Moga and Saper, 1994; Saper et al., 1989). An immunohistochemical study on monkey hypothalamus revealed BNP-like immunoreactivity in the form of clusters of granules in the PVN, SON, and periventricular area (Abdelalim et al., 2006). These BNP-positive dots were located in neurons,

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oligodendrocytes, astrocytes, and microglial cells. It has been suggested that BNP granules in the hypothalamus are originated from outside the hypothalamus and reach the hypothalamus through the subfornical organ (Abdelalim et al., 2006) as high-density binding sites for BNP have been observed by autoradiography in rat subfornical organ, SON, and paraventricular hypothalamic nucleus (Brown and Czarnecki, 1990) and NPR-A mRNA has been found in the subfornical organ (Langub et al., 1995). CNP has also been identified in the human hypothalamus in both high and low molecular weight forms by using radioimmunoassay (Totsune et al., 1994a). In the ovine hypothalamus, the concentration of CNP is much higher than that of ANP, similar amounts of CNP-53- and CNP-22-like immunoreactive-CNP being present (Yandle et al., 1993). In the rat hypothalamus, the highest CNP tissue concentrations have been found in the arcuate nucleus and PVN (Herman et al., 1993; Minamino et al., 1993). Hybridization signals of lower intensity were reported in the medial, median, and periventricular preoptic area; the SON; dorsomedial, ventral premammillary, and lateral mammillary nuclei; and in the posterior hypothalamic area (Herman et al., 1993). Through in situ hybridization, prepro-CNP mRNA has also been detected in the rat hypothalamus, particularly in the anteromedial preoptic nucleus of the medial preoptic area (Ryan et al., 1997). CNP synthesis has also been identified in immortalized luteinizing hormone-releasing hormone (LHRH) neurons using RT-PCR, immunocytochemistry, and electron microscopic immunohistochemistry and in these cells CNP also elevated LHRH production in an autocrine manner (Middendorff et al., 1997). The concentration of CNP in the cerebrospinal fluid has been reported to be one order of magnitude greater than that of ANP (Kaneko et al., 1993). Gibson et al. (1986) have found the highest levels of ANP binding in the rat subfornical organ, area postrema and olfactory apparatus; moderate ANP binding has been found throughout the brainstem and low levels in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. ANP-binding sites have been identified in hypothalamic and nonhypothalamic structures in both rat and guinea pig (Mantyh et al., 1987). ANP-binding sites have been identified in cerebral circumventricular organs, including the subfornical organ and organum vasculosum of the lamina terminalis (Mendelsohn et al., 1987). ANP-binding sites have also been reported in the SON and in the magnocellular and parvocellular subdivisions of the PVN in rat (Castre´n and Saavedra, 1989). In particular, high numbers of ANP-binding sites have been reported in the circumventricular organs (the organon vasculosum laminae terminalis, subfornical organum, and area postrema) and selected hypothalamic (SON, median preoptic, and paraventricular) nuclei (Kurihara et al., 1987). ANP-binding sites have also been reported in the median eminence, pineal gland, subfornical organ, choroid plexus, but not in the magnocellular hypothalamic nuclei (Gerstberger et al., 1992).

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NPR-B mRNA has been observed to be expressed throughout the hypothalamus, in the magnocellular and parvocellular paraventricular, the arcuate, and the SON, the median preoptic, anteroventral periventricular, tuberomammillary, ventromedial, and suprachiasmatic nuclei (Langub et al., 1995). The three receptors have been identified in astrocyte glial and neuronal cultures from the hypothalamus and brain stem of 1-day-old rats, with astrocytes containing predominantly the ANP-A subtype and neurons predominantly the ANP-B subtype (Sumners and Tang, 1992). NPR-A and -B mRNA have also been identified in the GT1-7 cell line, an immortalized LHRH neuronal cell line. All the natriuretic peptides elevated cGMP production in this cell line with the following rank order of potency: CNP > ANP > BNP (Olcese et al., 1994). NPR-C expression has also been found in mammalian hypothalamus (Peng et al., 1996; Sumners and Tang, 1992). In the human, ovine, and rat hypothalamus, higher expression of CNP and NPR-B have been found than of ANP, BNP, and NPR-A (Herman et al., 1993, 1996a; Komatsu et al., 1991; Langub et al., 1995; Minamino et al., 1993; Pemberton et al., 2002). Natriuretic peptide expression in the rat hypothalamus has also been studied with reference to postnatal maturation. It has been found through radioimmunoassay that ANP concentrations show a first increase in the postnatal days 0–5 and a second one in the postnatal days 10–20, for a 16fold final increase ( Jankowski et al., 2004). Increments of ANP mRNA have also been found by in situ hybridization in the septohypothalamic, lateral, periventricular, and arcuate nuclei from postnatal day 4 until postnatal days 21–28 (Ryan and Gundlach, 1998). In rat SON and suprachiasmatic nuclei, ANP peptide and mRNA have been identified starting from the 18th day of the fetal life (Lipari et al., 2005, 2007). CNP concentrations, instead, increased steadily until postnatal day 60, when they were 3.7-fold higher than at birth ( Jankowski et al., 2004). As regards concentrations of the transcripts of the natriuretic peptides receptors in adult versus newborn rats, higher NPR-A concentrations, lower NPR-C concentrations, and no differences in NPR-B concentrations were found ( Jankowski et al., 2004).

3.2. Pituitary gland ANP has been identified in the rat anterior pituitary by radioimmunoassay (Gutkowska and Cantin, 1988) and ANP and BNP mRNA have been identified in human pituitary by PCR (Gerbes et al., 1994). The presence of all the three natriuretic peptides has been reported through radioimmunoassay in the ovine pituitary, CNP (15.84 pmol/g wet weight) showing higher concentrations than ANP and BNP (0.25 and 0.26 pmol/g wet weight) (Pemberton et al., 2002). In the ovine hypophysis, the CNP-53like IR-CNP was mainly present (Yandle et al., 1993). CNP has been identified by radioimmunoassay in the anterior lobe and neurointermediate lobe of the pituitary (Komatsu et al., 1991). ANP-like immunoreactivity has

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been detected in the rat posterior hypophysis (Gutkowska et al., 1987). In particular, a low molecular weight peptide with a RP-HPLC pattern similar to that of the synthetic rat 28-amino acid C-terminal (Ser 99-Tyr 126) ANP was found, together with an unidentified higher molecular weight peptide (Gutkowska et al., 1987). An immunohistochemical study on rat pituitary gland has found ANP-, BNP-, and CNP-immunoreactive cells in the anterior lobe but not in the intermediate lobe of fetal and maternal glands on day 21 of gestation, fetal samples showing fewer and weakly stained cells (Chatelain et al., 2003). ANP has been localized by immunohistochemistry (Gutkowska and Cantin, 1988; McKenzie et al., 1985) and in situ hybridization (Morel et al., 1989a) in rat gonadotroph cells. Its expression has also been reported through RT-PCR in LbT2 cells and primary mouse pituitary tissue (Thompson et al., 2009). An in vivo ultrastructural autoradiographic approach through intravenous injection of 125 I-ANP has also demonstrated internalization of extracellular ANP by gonadotroph cells (Morel et al., 1989a). BNP has not been found to be expressed in gonadotroph aT3-1 and LbT2 cells and rat and mouse pituitaries (Thompson et al., 2009). Conversely, CNP has been localized in rat and mouse LH-positive cells of the anterior pituitary and in aT3-1 and LbT2 cells (McArdle et al., 1994; Thompson et al., 2009). Putative processing enzymes of CNP (Furin and peptidyl a-amidating monoxygenase enzymes) have also been found to be expressed in aT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that CNP expression is responsive to GNRH action in a protein kinase C and calcium-dependent manner (Thompson et al., 2009). The CNP promoter has been reported to work effectively also in somatomammotroph or somatotroph GH3 cells but not in corticotroph AtT20 cells (Ohta et al., 1993). ANP-binding sites have also been reported in the anterior pituitary in rabbit (Gerstberger et al., 1992) and rat (Agui et al., 1989) and in the posterior pituitary in guinea pig (Mantyh et al., 1986) and rabbit (Gerstberger et al., 1992). NPR-A and -B have been isolated from a human pituitary cDNA library (Chang et al., 1989; Wilcox et al., 1991). In situ hybridization study in the anterior pituitary of rhesus monkey has revealed NPR-A and NPR-B mRNA (Wilcox et al., 1991). NPR-B mRNA has been identified in some cells of the anterior pituitary and in pituicytes in the neural lobe (Herman et al., 1996a). Northern blot analysis identified all three natriuretic peptide receptors in the mouse pituitary (Guild and Cramb, 1999). Analysis in alpha T3-1 and AtT-20 cell lines did not confirm the presence of NPR-A mRNA, suggesting cGMP accumulation occurring via NPR-B (Gilkes et al., 1994; McArdle et al., 1994). Ohta et al. (1993) have identified NPR-B in rat pituitary somatotroph and somatolactotroph progenitor cells. In situ hybridization in rat anterior pituitary gland has revealed NPR-A, -B, and -C mRNA in lactotroph, corticotroph, and gonadotroph cells, but not in somatotroph or tyreotroph ones

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(Grandcle´ment et al., 1995; Thompson et al., 2009). NPR-C mRNA has been identified by in situ hybridization not only in the rat anterior lobe but also in the intermediate one (Herman et al., 1996b). Pituicytes cultured from adult rat neurohypophyses have been found to possess high-affinity binding sites for ANP, but ANP has been found not to modulate the basal or electrically stimulated release of OT or vasopressin (VP) from the isolated neurohypophysis in vitro (Luckman and Bicknell, 1991). NPR-B mRNA has also been found in the pars intermedia and posterior of the pituitary gland in the monkey (Wilcox et al., 1991) and rat (Konrad et al., 1992). NPR-B mRNA was also observed in the neural lobe of the pituitary gland, suggesting expression by pituicytes (Langub et al., 1995).

3.3. Adrenal cortex Although Morel et al. (1988) did not report the presence of ANP mRNA in the rat adrenal cortex and Lee et al. (1994) did not report BNP mRNA and protein in the adrenal cortex by in situ hybridization and immunohistochemistry, ANP and BNP mRNA have been identified in human adrenal gland (without distinction between cortex and medulla) by PCR (Gerbes et al., 1994). Moreover, Lai et al. (2000) detected ANP mRNA and protein by in situ hybridization and immunohistochemistry in the rat zona glomerulosa and outer region of the zona fasciculata, but not in the remaining part of the zona fasciculata and in the zona reticularis. In bovine, CNP mRNA has also been demonstrated by RT-PCR in the zona glomerulosa tissue and cultured cells and CNP immunoreactivity has been localized in the outermost region of the adrenal cortex but not in the inner portion of the zona fasciculata and zona reticularis (Kawai et al., 1996). ANP-binding sites have been identified in the rat, guinea pig, rabbit, bovine, and tree shrew adrenal zona glomerulosa (e.g., Chai et al., 1986; De Le´an et al., 1984; Fuchs et al., 1986; Gerstberger et al., 1992; Lynch et al., 1986; Mantyh et al., 1986; Mendelsohn et al., 1987; Morel et al., 1989b). In particular, internalization of ANP in rat adrenal glomerulosa cells was also demonstrated (Morel et al., 1989b). ANP-binding sites have also been observed in the rat zona fasciculata (Chai et al., 1986) and in the tree shrew and bovine zona fasciculata and reticularis (Fuchs et al., 1986; Nunez et al., 1990). Lynch et al. (1986) also reported the presence of ANP-binding sites in the rat zona fasciculata and reticularis, although at lower levels. Developmental changes have also been reported in the expression of ANP receptors as in the 16-day-old rat ANP-binding sites are present throughout the cortical area but at 20 days gestation and 1 day postpartum ANP receptors are more numerous in the peripheral region (Scott and Jennes, 1989). Conversely, rat adrenocortical autotransplants regenerated from capsular-tissue fragments implanted in the musculus gracilis have been found not to significantly bind 125I-ANP (Belloni et al.,

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1993). BNP-binding sites have also been identified in bovine adrenocortical membrane fractions (Higuchi et al., 1989). In the rat zona glomerulosa cells, mRNA of the three natriuretic peptide receptors have been identified (Grandcle´ment et al., 1997; Nagase et al., 1997; Vaillancourt et al., 1997). The amount of NPR-A mRNA has been found to be the highest (Grandcle´ment et al., 1997) and Western analysis using polyclonal anti-NPR-A and anti-NPR-B antibodies revealed the presence of NPR-A but not of NPR-B proteins (Vaillancourt et al., 1997). Wilcox et al. (1991) reported the presence of NPR-A but not NPR-B in the monkey zona glomerulosa by in situ hybridization and observed clusters of NPR-C-positive cells suggestive of endothelial, not necessarily secretory, cells. In the rat zona fasciculata cells, NPR-A but not NPR-B and -C receptor’s mRNA has been identified (Mulay et al., 1995; Vaillancourt et al., 1997). In the monkey zona fasciculata and reticularis, mRNA of the three receptors was not identified in secretory cells (Wilcox et al., 1991). NPR-A has also been identified in the H295R human adrenocortical cell line (Bodart et al., 1996). Plasma ANP concentrations are known to decrease after water deprivation or hemorrhage and to increase after blood volume expansion. Conversely, data concerning plasma ANP concentrations in response to salt-overloading are contradictory. Water deprivation increases total number of ANP receptors in the adrenal gland of adult and maternal rats, but not of fetal ones (Deloof et al., 1999; Lynch et al., 1986). In particular, the density of NPR-C but not of NPR-B has been found to be increased (Deloof et al., 1999). Most studies, with few exceptions (Deloof et al., 2000) reported downregulation of the ANP receptors in the adrenal glands after salt-overloading (Lynch et al., 1986; Sessions et al., 1992).

3.4. Adrenal medulla ANP, BNP, and CNP have been identified in rat, bovine, porcine, and human adrenal medulla cells (e.g., Babinski et al., 1992; Dagnino et al., 1991; De Le´an et al., 1985; Komatsu et al., 1991; Lai et al., 2000; Lee et al., 1993, 1994; McKenzie et al., 1985; Minamino et al., 1993; Morel et al., 1988; Nawata et al., 1991; Nguyen et al., 1990; Wolfensberger et al., 1995; reviewed in Kobayashi et al., 1998). In situ hybridization identified ANP mRNA in noradrenergic cells while immunohistochemistry identified ANP protein in both noradrenergic and adrenergic cells, suggesting ANP synthesis in noradrenergic cells and internalization in adrenergic ones (Morel et al., 1988). It has also been reported that the majority ANP-immunoreactive chromaffin cells are the adrenergic ones (Wolfensberger et al., 1995). Electrical stimulation of the splanchnic nerves has been found to cause the release of ANP-like immunoreactive material in isolated perfused calf adrenal glands (Duntas et al., 1993; Edwards et al., 1990) and enhance the uptake of ANP by chromaffin cells (Edwards et al., 1990).

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It has been hypothesized that ANP produced in the adrenal medulla may act on the adrenal cortex (Lee et al., 1993, 1994; Nawata et al., 1991) and may be involved in the regulation of blood flow and even in the zonation of the adrenal cortex (Lee et al., 1994). 125I-ANP-binding sites have been identified by in vivo autoradiography in rat adrenal medulla and by in vitro autoradiography in bovine, guinea pig, tree shrew, rabbit, and rat adrenal medulla (Bormann et al., 1989; Fuchs et al., 1986; Gerstberger et al., 1992; Konrad et al., 1992; Maurer and Reubi, 1986; Morel et al., 1988; Niina et al., 1996). Specific binding sites for ANP have been identified in the phaeochromocytoma cell line PC12 (Boumezrag et al., 1988). 125I-ANP-binding sites, instead, have not been identified in mouse, hamster, monkey, human, and in other studies in bovine, guinea pig, and rat (Chai et al., 1986; Lynch et al., 1986; Mantyh et al., 1986; Maurer and Reubi, 1986; Stewart et al., 1988). In rat, 125I-BNP and125I-[Tyr0]-CNP-binding sites have also been identified (Konrad et al., 1992). The number of ANP-binding sites has also been found to increase regularly in fetal (day 17 of gestation and term) and neonatal (weeks 1 and 4) rats (Deloof et al., 1994). NPR-A and NPR-B mRNA, but not NPR-C mRNA, have been identified by in situ hybridization in adrenal chromaffin cells of monkey (Wilcox et al., 1991). This finding is in keeping with displacing of 125I-ANP and125I-BNP bindings by ANP and BNP but not by selective analogues for NPR-C in rat and bovine (Konrad et al., 1992; Niina et al., 1996). In rat adrenal medulla, the mRNA of the three subtypes has been found by in situ hybridization, the amount of NPR-A mRNA being the highest (Grandcle´ment et al., 1997). The above receptors were selectively present in adrenaline-containing chromaffin cells and not in the noradrenalinecontaining ones (Grandcle´ment et al., 1997). NPR-A mRNA expression has also been reported to be significantly increased in the adrenal medulla of adult pro-ANP gene-disrupted mice (O’Tierney et al., 2007).

4. Effects of Natriuretic Peptides on the HPA Axis 4.1. Hypothalamus ANP has been found to modulate the membrane excitability of neurons of the lateral septal nucleus, lateral paraolfactory area, bed nucleus of the anterior commissure, and medial preoptic area (Wong et al., 1986). ANP has been found to produce significant increases in blood pressure and heart rate when injected into the preoptic suprachiasmatic nucleus, suggesting it may play an important role in central cardiovascular regulatory mechanisms (reviewed in Oparil et al., 1996). Moreover, intracerebroventricular injection of ANP has

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been found to inhibit dehydration- and angiotensin II-induced water intake in conscious, unrestrained rats (Antunes-Rodrigues et al., 1985). ANP, BNP, and CNP have been found to reduce both spontaneous and acetylcholine, Kþ and angiotensin II-evoked norepinephrine release in slices of rat hypothalamus (Giridhar et al., 1992; Vatta et al., 1996). ANP has been found to increase neuronal norepinephrine uptake in hypothalamus (Fernandez et al., 1993) and in organum vasculosum lamina terminalis and organum subfornical (Vatta et al., 1995) of rat. BNP and CNP have also been found to increase neuronal norepinephrine uptake in slices of rat hypothalamus and, particularly, independently of the hypothalamic nucleus involved (preoptic, periventricular, paraventricular, SON, and arcuate nuclei; median eminence) (Rodriguez Fermepin et al., 2000; Vatta et al., 1996). ANP has been found to diminish monoamine oxidase activity, but not catechol-O-methyl transferase activity and the formation of deaminates metabolites, in rat hypothalamus slices (Vatta et al., 1998). Moreover, centrally applied ANP has been reported to increase the hypothalamic content of NE, diminish its utilization and turnover, inhibit basal and KCl-evoked tyrosine hydroxylase activity, and increase cyclic GMP levels (Vatta et al., 1999). Experimental studies on rats have shown that ANP microinjections into the third ventricle do not change basal levels of OT but attenuate the increase in OT secretion induced by hyperosmolarity (Chriguer et al., 2001; Gutkowska et al., 1997; Lewandowska et al., 1992; McCann et al., 1996; Poole et al., 1987). ANP has also been found to markedly inhibit OT release in vitro from the isolated neurointermediate lobe both under basal condition as well as during stimulation (Lewandowska et al., 1992; Poole et al., 1987). ANP has been proven to be a potent inhibitor of VP neurons of the PVN in anesthetized rats (Okuya and Yamashita, 1987; Standaert et al., 1987). Intravenous infusion of ANP has been found to reduce dehydration and hemorrhageinduced VP release in the rat (Samson, 1985). ANP has been reported to inhibit the basal and stimulated release of VP in hypothalamo-neurohypophyseal slice preparations and in superfused rat posterior pituitary gland ( Januszewicz et al., 1986; Obana et al., 1985). ANP has also been found to inhibit VP release in vitro from the neurointermediate lobes both under basal condition as well as during stimulation (Lewandowska et al., 1992; Poole et al., 1987). Intracerebroventricular injections of ANP, BNP, or CNP have been found to show inhibitory effects on the VP secretion (e.g., Iitake et al., 1986; Lewandowska et al., 1992; Makino et al., 1992; Poole et al., 1987; Samson et al., 1991; Shirakami et al., 1993). The three natriuretic peptides have also been reported to inhibit the basal secretion of VP from rat SON neurons in dissociated cell preparations, CNP being the most potent inhibitory factor (Yamamoto et al., 1997). Reduction of VP plasma levels due to central ANP stimulus has been observed in both euhydrated and dehydrated sheeps (Lee et al., 1987) and rats (Manzanares et al., 1990). In rats, inhibition of VP secretion was not accompanied by modifications in the concentrations of 3,4-dihydroxyphenylacetic

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acid and dopamine, indicating that ANP-induced suppression of VP secretion is not mediated by tuberohypophysial or tuberoinfundibular dopaminergic neurons (Manzanares et al., 1990). Conversely, in dehydrated but not in euhydrated rabbits, infusion of ANP has also been found to inhibit secretion of VP (Gerstberger et al., 1992). ANP and BNP have also been found to decrease the firing rate and hyperpolarize the membrane potential in phasically firing (putative VP) but not in nonphasically firing (putative OT) neurons of SON; inhibition of cGMP synthesis was also reported in neurons of SON (Akamatsu et al., 1993). ANP and BNP have been found to inhibit AV3V neurons, suggesting direct actions of the peptides on drinking, and in the SON, these peptides inhibited selectively putative VP neurons but not putative OT neurons, suggesting direct actions of the peptides on VP secretion (Yamamoto et al., 1995). The central inhibition of OT and VP release from the magnocellular neurosecretory cells by ANP has been suggested to be mediated by presynaptic inhibition of glutamate release from osmoreceptor afferents derived from the organum vasculosum lamina terminalis (Richard and Bourque, 1996). Experiments through injection of highly specific antiserum against ANP into the third cerebral ventricle of rats also showed that the inhibitory role in suppressing ACTH release during stress is in part mediated by inhibition of VP release (Franci et al., 1992). Conversely, it has also been reported an increase of the plasma VP response to acute moderate hemorrhage after intracerebroventricular injection of CNP (Charles et al., 1995). It has also been demonstrated that CNP has a potent and selective inhibitory effect on magnocellular cells of SON and PVN, which is mediated by NPR-C (Rose et al., 2005). Moreover, since NPR-C binds all natriuretic peptides with equal affinity (Levin et al., 1998), it has been suggested that this receptor could mediate the hypothalamic effects by the other natriuretic peptides (Rose et al., 2005). It has been reported that intracerebroventricular injection of ANP in rats does not modify tuberoinfundibular dopaminergic neuronal activity and serum prolactin levels, but it attenuates the stimulatory effects of angiotensin II on tuberoinfundibular dopaminergic neuronal activity, negatively modulating also the inhibitory effect on serum prolactin level (Yen and Pan, 1997). ANP and BNP have been reported to cause a dose-dependent increase in dopamine accumulation in cultured rat hypothalamic cells through an increase in intracellular cGMP concentration (Kadowaki et al., 1992). Franci et al. (1992) also reported a role by ANP in augmenting the prolactin release in stress through a hypothalamic action. On the other hand, CNP has been found to stimulate prolactin secretion in rats by a hypothalamic site of action (Huang et al., 1992a; Samson et al., 1995). In rat, ANP has been found to inhibit acetylcholine- and KCl-induced release of corticotrophin-releasing factor in vitro (Grossman et al., 1993; Ibanez-Santos et al., 1990; Takao et al., 1988) and to increase its immunoreactivity in the hypothalamus in vivo (Biro´ et al., 1996). In humans,

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intranasal administration of ANP has been shown to inhibit secretion of ACTH stimulated by hypoglycemia but not by CRH/VP, suggesting inhibition of central nervous mechanisms of HPA activation, probably at the level of the hypothalamus (Perras et al., 2004). High doses of BNP and CNP have been found to increase and decrease, respectively, corticotropinreleasing factor immunoreactivity in the hypothalamus (Gardi et al., 1997). Charles et al. (1992) reported suppression of the adrenocortical secretion in the sheep after intracerebroventricular injection of CNP, while ANP had no significant effect. The same research group in a following experiment reported increase of the plasma cortisol response to acute moderate hemorrhage after intracerebroventricular injection of CNP, although the plasma ACTH response was not significantly different, probably for feedback inhibition (Charles et al., 1995). Intracerebroventricular injections of BNP and CNP have been found to inhibit the stress-induced corticosterone response, without changes of the basal secretion, thus suggesting a hypothalamic actions of these hormones ( Ja´szbere´nyi et al., 1998, 2000). Experiments through injection of highly specific antiserum against ANP into the third cerebral ventricle of rats to immunoneutralize hypothalamic ANP showed that ANP inhibits basal but not stress-induced GH release. The same study did not find a modulatory role by ANP in thyroid-stimulating hormone release (Franci et al., 1992). ANP and CNP have been reported to inhibit LHRH release (Huang et al., 1992b; Samson et al., 1992, 1993). Microinjection of antisera against ANP into the third cerebral ventricle of rats produced elevation of plasma LH levels (Franci et al., 1990). Conversely, some authors reported a slight increase in LH serum levels after applying ANP into rat PO/AH by means of push–pull cannula, probably through reduction of preoptic GABA release rates (Rodriguez Lopez et al., 1993). Recent studies involving ricin A chain conjugated ANP suggest that ANP binding to clearance receptors in the hypothalamus displaces CNP from the shared clearance receptor, making more CNP available to inhibit LHRH release through binding to the ANPR-B receptor (Samson et al., 1992, 1993). The perfusion of hypothalamo-neurohypophysial complex with ANP has also been found to increase the beta-endorphin concentration, whereas such an effect was not reported in isolated neurointermediate lobes of rat pituitary (Ikeda et al., 1989).

4.2. Pituitary gland Although a first study in the rat did not report inhibition of basal and CRF-, VP-, and angiotensin II-induced ACTH release by ANP both in vivo and in vitro (Hashimoto et al., 1987), following studies in the adult rat, reported inhibition by ANP of ACTH release both in vivo (Antoni et al., 1992; Fink et al., 1991; Kova´cs and Antoni, 1990) and in vitro (King and Baertschi, 1989;

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Kova´cs and Antoni, 1990; Shibasaki et al., 1986b). In cultured ovine and rat anterior pituitary cells, CRF- and VP-stimulated, but not basal, ACTH secretion has also been found to be inhibited by rat ANP (Dayanithi and Antoni, 1990; Engler et al., 1990). This effect was also confirmed for all three natriuretic peptides in vitro in mouse hemipituitary preparations over a concentration range of 10 12  10 10 M (Guild and Cramb, 1999) and in vivo in humans (Kellner et al., 1992). Inhibition of ACTH release was accompanied by stimulation of cGMP accumulation (Guild and Cramb, 1999). Conversely, it must also be considered that in the work by Ur et al. (1991) significant differences were not found in mean peak cortisol and ACTH levels between ANP and placebo infusion. In young healthy men exposed to ANP infusion and stimulation of ACTH secretion by CRH and/ or VP, Bierwolf et al. (1998) reported inhibition of ACTH/cortisol secretory responses within the first hour after stimulation with secretagogues and augmentation of ACTH/cortisol response during the third hour after stimulation. The early suppression was ascribed to direct inhibitory actions of ANP on both adrenal release of cortisol and pituitary release of ACTH; the late effect was ascribed to secondary hypovolemic actions. Natriuretic peptides have been found to stimulate cGMP accumulation in AtT-20 cell line, CNP being the most effective hormone (Fowkes and McArdle, 2000), but not to affect basal or CRF-stimulated ACTH secretion (Gilkes et al., 1992, 1994). In AtT-20 cells, ANP has also been found to reduce POMC mRNA content, together with a modest reduction in the release and cell content of betaendorphin-like immunoreactivity (Tan et al., 1994). ANP, BNP, and CNP have also been reported to inhibit CRF-stimulated ACTH secretion and proopiomelanocortin mRNA expression in in vitro fetal rat pituitary gland in late gestation (Chatelain et al., 2003). The three natriuretic peptides are equipotent in inhibiting the CRF-stimulated ACTH release (Chatelain et al., 2003; Guild and Cramb, 1999). Intracerebroventricular administration of BNP has also been found to suppress endothelin-induced ACTH secretion in rat (Makino et al., 1990). Other studies, instead, have not reported inhibition on ACTH secretion by ANP in cultured pituitary cells of rat, sheep, and horse (e.g., Bowman et al., 1997; Mulligan et al., 1997). Horvath et al. (1986) also reported a small but significant stimulation of ACTH release by ANP in superfused rat pituitary cells. Mulligan et al. (1997) also reported absence of inhibition on ACTH secretion by CNP in horse cultured pituitary cells. Such differences may be explained with reference to different in vitro models or concentrations of ANP. The three natriuretic peptides have been reported to cause increases in cGMP content in GH3 cells (McArdle et al., 1993). Experimental studies on rat pituitary have reported ANP suppression of basal, growth hormone releasing factor-stimulated and stress-induced GH secretion (Shibasaki et al., 1986b). Conversely, other studies on superfused anterior pituitary cells did not revealed any effect by ANP on GH release (Horvath et al., 1986;

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Shimekake et al., 1994) and central administration of ANP in rats (Murakami et al., 1988) stimulated GH release. In other studies, stimulation of GH release by natriuretic peptides has been reported from rat cultured anterior pituitary cells, such as GH3 cell line, ANP and CNP being the most effective hormones (Fowkes and McArdle, 2000; Hartt et al., 1995). Shimekake et al. (1994) reported stimulation of GH release by CNP, but not ANP, from GH3 cells. In conclusion, effects of natriuretic peptides on GH release seem to be equivocal. ANP has been found to produce cGMP accumulation in rat anterior pituitary cells in culture, basal, and ANP-induced cGMP levels being higher in cell populations enriched in gonadotrophs compared to gonadotrophimpoverished preparations, but alteration of LH release was not reported (Simard et al., 1986). ANP, BNP, and CNP have also been found to stimulate cGMP accumulation in primary cultures of rat pituitary cells and aT3-1 and LbT2 gonadotroph-derived cells, ANP and CNP being the most effective hormones in stimulating LbT2 and aT3-1 cells, respectively (Fowkes and McArdle, 2000; McArdle et al., 1993; Thompson et al., 2009). Moreover, aT3-1 cells produced significantly more cGMP in response to CNP than other cell lines, that is, GH3, TtT-GF, and AfT-20 cells (Fowkes and McArdle, 2000). CNP has been found to inhibit GnRH-stimulated calcium mobilization in aT3-1 gonadotroph-derived cells (Fowkes et al., 1999). Moreover, CNP has been reported to stimulate the human glycoprotein hormone a-subunit promoter in LbT2 cells, although not in aT3-1 ones (Thompson et al., 2009). However, CNP had no measurable effects on basal and GnRH-stimulated LH release and on cell proliferation (McArdle et al., 1993; Thompson et al., 2009). Conversely, stimulation of LH and FSH release had been reported by ANP in anterior pituitary cells of rats (Horvath et al., 1986), although recently not confirmed (Thompson et al., 2009). Intracerebroventricular injection of ANP has also been reported to induce an increase in plasma LH levels without significantly affecting prolactin release (Steele, 1990). On the other hand, Standaert et al. (1986b) had reported in vivo inhibition of the release of LH by ANP. In conclusion, effects of natriuretic peptides on LH and FSH release will have to be better clarified in the future. ANP has been found not to affect thyrotropin and PRL release from dispersed rat anterior pituitary cells, but central administration of high doses in rats has been reported to cause significant inhibition of PRL release (Horvath et al., 1986; Samson and Bianchi, 1988). ANP, BNP, and CNP have also been found to stimulate cGMP accumulation in TtT-GF cell line, a pituitary folliculo-stellate-like cell line derived from an isologously transplantable pituitary thyrotropic tumor line, CNP being the most effective hormone (Fowkes and McArdle, 2000). Synthetic rat ANP has also been found to attenuate, in a dose-dependent manner, basal and CRF-induced secretion of

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proopiomelanocortin-derived peptides from cultured intermediate lobe cells of rat pituitary (Shibasaki et al., 1986b).

4.3. Adrenal cortex In the cells of the mammal adrenal zona glomerulosa, ANP has been found to inhibit basal and angiotensin II-, Kþ -, PACAP-, calcium ionophores and ACTH-stimulated aldosterone secretion via a cGMP-mediated mechanism (e.g., Atarashi et al., 1984; Bodart et al., 1997; Chartier et al., 1984; Cozza et al., 1993; Deloff et al., 1992; Elliott et al., 1993; Isales et al., 1989; Kudo and Baird, 1984–1985; Lotshaw et al., 1991; Mazzocchi et al., 1987; Naruse et al., 1987; Nawata et al., 1991; Nussdorfer et al., 1988–1989; Spiessberger et al., 2009; Vesely et al., 1995; reviewed in Ganguly, 1992; Nussdorfer, 1996). Inhibition of aldosterone production in adrenal zona glomerulosa cells has also been reported by a specific ligand for NPR-C (Isales et al., 1992). Inhibition of angiotensin II-induced aldosterone production by a NPR-A agonist has also been demonstrated in H295R human adrenocortical cell line (Bodart et al., 1996). On the other hand, the pro-ANP 1–30 and 31–67 have been found not to affect angiotensin II-stimulated aldosterone secretion in calf adrenal cells (Denker et al., 1990). ANP-induced inhibition of aldosterone secretion has also been found to be mediated by inhibition of T-type calcium channels (McCarthy et al., 1990). ANP has also been found to diminish cAMP levels in glomerulosa cells through stimulation of a phosphodiesterase by cGMP (MacFarland et al., 1991; Nikolaev et al., 2005; Spiessberger et al., 2009). Moreover, ANP has been found to have no effect on ACTH-stimulated aldosterone levels in mice with a homozygous inactivation of the cGMPdependent protein kinase II, suggesting involvement of this enzyme in ANPmediated inhibition of aldosterone expression (Spiessberger et al., 2009). Inhibition of the phosphorylation of the myristoylated alanine-rich C-kinase substrate (MARCKS) and the synthesis and phosphorylation of the steroidogenic acute regulatory protein (StAR) has also been found to play a pivotal role in inhibition of aldosterone production (Calle et al., 2001; Cherradi et al., 1998). ANP has also been found to inhibit the phosphorylation of histone H3 in bovine adrenal glomerulosa cells (Elliott, 1990). In cultured human and bovine adrenal cells, BNP has also been found to increase intracellular cGMP and inhibit ACTH- and angiotensin II-stimulated aldosterone secretion (Hashiguchi et al., 1989; Higuchi et al., 1989; Nawata et al., 1991). In calf adrenal zona glomerulosa cells in culture, BNP has also been found to inhibit AII-, Kþ,- and ACTH-stimulated increase in aldosterone, while CNP showed only weak effects (Cozza et al., 1993). In bovine adrenal zona glomerulosa cells in culture, CNP has also been found to increase the basal secretion of cGMP and inhibit ACTH-stimulated increase in aldosterone (Kawai et al., 1996). In primary human adrenocortical cells investigated through intracellular cGMP assay and cDNA microarray, BNP has been

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reported to induce cGMP synthesis and oppose 49% of ANGII-regulated genes, with particular reference to genes involved in cell growth and differentiation, steroid synthesis, and cholesterol synthesis and transfer (Liang et al., 2007). Treatment with BNP alone, instead, produced downregulation only of a small number of genes. Moreover, BNP inhibited ANGII-induced stimulation of the binding of LDL and HDL and of the release of aldosterone, cortisol, and estradiol (Liang et al., 2007). ANP has also been reported to inhibit the growth of rat zona glomerulosa (Mazzocchi et al., 1987; Rebuffat et al., 1988; Trejter et al., 2002); this action has been reported with both ANP and ANP antagonist, suggesting a nonreceptor-mediated mechanism of action (Trejter et al., 2002). ANP has been found to decrease basal and ACTH-stimulated glucocorticoid production from cultured human and cow zona fasciculata cells (Carr and Mason, 1988; Hashiguchi et al., 1989; Naruse et al., 1987; Nawata et al., 1991). This effect was also observed in the Y1 mouse adrenocortical tumor cell line (Heisler et al., 1989). Other studies did not show effects of ANP on glucocorticoid secretion in rat (Cantin and Genest, 1985; Ganguly, 1992). It has also been found that isolated fasciculata cells of rat adrenal cortex, when incubated with ANP, stimulated the levels of cyclic GMP and corticosterone production in a concentration-dependent manner ( Jaiswal et al., 1986). ANP treatment for 6 days has been reported to increase plasma concentrations of cortisol by about 20% in normal guinea pigs and by about 3.5-fold in dexamethasone/captopril administered animals, indicating a direct action on the adrenal gland. Although ANP has been found not to affect cortisol secretion from dispersed guinea pig zona fasciculata-reticularis cells, a raise in cortisol production has been reported in guinea pig adrenocortical slices containing adrenomedullary tissue, suggesting an indirect effect, mediated by medullary chromaffin cells, under the secretagogue action of ANP (Raha et al., 2006). In fact, the bulk of evidence indicates that catecholamines are able to stimulate steroidogenesis through binding beta-adrenoreceptors on adrenocortical cells (Lightly et al., 1990; Mazzocchi et al., 1998; Nussdorfer, 1996) and various peptides, such as neuromedin U (Malendowicz et al., 1994, 2009), VIP and PACAP (Nussdorfer and Malendowicz, 1998a), neuropeptide-Y (Spinazzi et al., 2005), tachykinins (Nussdorfer and Malendowicz, 1998b), endothelins (Malendowicz et al., 1998; Nussdorfer et al., 1999), and adrenomedullin (Nussdorfer, 2001), have been found to stimulate cortisol secretion by indirect action on the medullary chromaffin cells. Lastly, in the evaluation of ANP effects on adrenal gland, relevant species-specific differences must be considered. BNP has also been found to inhibit basal and ACTH-stimulated cortisol production in cultured human, bovine, and guinea pig adrenal cells (Hashiguchi et al., 1989; Higuchi et al., 1989). Inhibition of basal and ACTH-stimulated dehydroepiandrosterone production by ANP and BNP, although less potent, has also been demonstrated in human and bovine adrenal cell cultures (Higuchi

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et al., 1989; Nawata et al., 1991). Many in vitro studies have been performed on transformed cell lines and different findings in studies performed on cell cultures may derive from the fact that transformed cell lines may not appropriately reflect the features of primary human adrenal cells (Liang et al., 2007).

4.4. Adrenal medulla In the literature, the three natriuretic peptides have been reported to increase cGMP content in the rat and bovine adrenal chromaffin cells (Ferna´ndez et al., 1997; Fe´thie`re et al., 1993; Tsutsui et al., 1994; Yanagihara et al., 1991). ANP has been reported to inhibit catecholamine release by adrenal medulla cells (e.g., Babinski et al., 1995; Ferna´ndez et al., 1997; Papouchado et al., 1995; Vatta et al., 1994; reviewed in Kobayashi et al., 1998). In particular, ANP has been demonstrated to inhibit acetylcholine-induced membrane currents in bovine chromaffin cells (Bormann et al., 1989) and to enhance activity of potassium conductance (Ganz et al., 1994). ANP mediates also indirect sympathoinhibitory effects through antagonism of the renin-angiotensin (Atlas and Maack, 1987) and endothelin (Emori et al., 1993; Neuser et al., 1993) systems, which modulate catecholamine release from the adrenal medulla (Armando et al., 2004; Lange et al., 2000). ANP has also been found to reduce monoamine oxidase activity, but not catechol-O-methyl transferase activity and the formation of deaminates metabolites, in rat adrenal medulla slices (Vatta et al., 1998). In cultured bovine adrenal medullary cells ANP increases phosphorylation and activity of tyrosine hydroxylase (Yanagihara et al., 1991), whereas, in rat adrenal medulla, inhibition of both spontaneous and KCl-evoked TH activity has been reported (Ferna´ndez et al., 1997). In rat adrenal medullary cells, ANP has also been found to increase noradrenaline uptake (Vatta et al., 1992) and endogenous content and to diminish noradrenaline utilization (Ferna´ndez et al., 1997). Pro-ANP gene-disrupted mice have also found to show an increase in circulating catecholamine levels (Melo et al., 1999) and upregulation of tyrosine hydroxylase expression in sympathetic ganglia and adrenal medulla (O’Tierney et al., 2007). Conversely, in some studies ANP has been found to potentiate catecholamine secretion due to low concentrations (3 mM) of nicotine in bovine adrenal chromaffin cells (O’Sullivan and Burgoyne, 1990) and to enhance catecholamine release from bovine adrenomedullary cultured cells of guinea pigs (Raha et al., 2006). BNP has also been show to stimulate tyrosine hydroxylase activity, in cultured adrenomedullary cells (Yanagihara et al., 1991), and to decrease spontaneous and KCl-induced norepinephrine release and enhance noradrenaline uptake in rat adrenal medulla slices (Vatta et al., 1996, 1997). It has been suggested that BNP may contribute to increase adrenal tyrosine hydroxylase expression in ANP/ mice due to elevated levels of NPR-A (O’Tierney et al., 2007).

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CNP has also been found to inhibit catecholamine secretion stimulated by nicotine (10 mM), acetylcholine (50 mM), or KCl (30 mM) in bovine chromaffin cells, through cGMP-dependent and -independent mechanisms (Babinski et al., 1995; Rodriguez-Pascual et al., 1996). Inhibition of spontaneous and KCl-induced catecholamine release has also been demonstrated in rat adrenal medulla slices, together with enhancement of noradrenaline uptake (Vatta et al., 1997). CNP has also been reported to stimulate catecholamine synthesis, through increasing of intracellular cGMP content and activation of tyrosine hydroxylase, in cultured bovine adrenal medullary cells (Tsutsui et al., 1994).

5. Natriuretic Peptides and Pathophysiology of HPA Axis 5.1. Adrenocortical adenomas and carcinomas Plasma levels of ANP and BNP have been found to be higher in patients with primary aldosteronism due to aldosterone-producing adrenal adenoma or bilateral adrenal hyperplasia, reduced levels being found after adenoma resection ( Jakubik et al., 2006; Kato et al., 2005; Lapinski et al., 1991; Naruse et al., 1994; Tunny and Gordon, 1986; Yamaji et al., 1986). BNP was more closely correlated with blood volume, being a more sensitive marker of cardiac load or volume status in patients with primary aldosteronism (Kato et al., 2005). In human adrenocortical tumors, CNP has been found by radioimmunoassay in concentration of 0.69  0.19 pmol/g wet tissue, with respect to 0.49  0.22 pmol/g wet tissue in normal adrenal glands (cortex and medulla mixed) (Totsune et al., 1994b). BNP has been found by radioimmunoassay in concentrations of 0.203  0.061 pmol/g wet tissue in normal adrenal glands (cortex and medulla mixed), 0.230  0.062 pmol/g wet tissue in aldosteronomas, and 0.180  0.054 pmol/g wet tissue in adrenocortical adenomas with Cushing’s syndrome (Totsune et al., 1996). Multiple molecular forms of BNP have been reported in aldosteronomas (Totsune et al., 1996). Significant differences in the allelic frequencies of restriction fragment length polymorphisms in the ANP gene have been found between angiotensin II-unresponsive and -responsive aldosteroneproducing tumors (Tunny et al., 1994). Enhanced expressions of ANP and BNP from adrenal medulla surrounding aldosteronomas have also been reported (Lee et al., 1993, 1994). The inhibitory effect of natriuretic peptides on aldosterone production from aldosteronomas has been found to be less potent or even absent (Hirata et al., 1985; Mantero et al., 1987; Naruse et al., 1987; Nawata et al., 1991; Rocco et al., 1989; Shionoiri et al., 1988, 1989). Moreover, ANP has been found not to inhibit basal and ACTH-stimulated cortisol secretion in tissue

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slices of Cushing’s adenoma (Shionoiri et al., 1989). Shionoiri et al. (1988, 1989) did not report NPR-A presence in aldosteronoma by binding assay and immunohistochemistry. mRNA of the three NPRs has been found in the aldosteronomas (Chen et al., 1995; Sarzani et al., 1999). NPR-B and -C mRNA, but not NPR-A mRNA, have been reported to be downregulated in aldosteronomas by Chen et al. (1995), while Sarzani et al. (1999) did not report significant differences. Moreover, ANP-binding sites have also been reported to be reduced in aldosteronomas (Ohashi et al., 1991; Sarzani et al., 1999).

5.2. Pheochromocytomas In patients with pheochromocytoma, higher plasma ANP concentration has been found with respect to controls and patients with essential hypertension and ANP concentrations has been reported to decline after removal of the tumor, suggesting that catecholamines produced by the chromaffin tumor induce ANP secretion through stimulation of adrenergic receptors (Stepniakowski et al., 1992). In human pheochromocytomas, BNP and CNP have been found in concentrations of 0.205  0.037 pmol/g wet tissue (Totsune et al., 1996) and 0.54  0.40 pmol/g wet tissue, respectively (Totsune et al., 1994b). Multiple molecular forms of BNP have been reported in pheochromocytomas (Totsune et al., 1996). Nakamaru et al. (1989) reported increases in plasma levels of catecholamines after intravenous infusion of ANP in patients with pheochromocytoma but they did not observe modifications of the basal release of catecholamines from isolated superfused pheochromocytoma tissue. Release of catecholamines from tissue slices of pheochromocytoma has been found to be inhibited by hANP in a dose-dependent manner, binding assays using 125I-ANP have revealed a single class of high-affinity binding sites for ANP and immunohistochemistry has also revealed the presence of ANP receptors (Shionoiri et al., 1987, 1989).

6. Concluding Remarks The preceding sections of the paper have shown that a huge mass of data strongly suggests that natriuretic peptides play an important role in the regulation of the function of the HPA axis, although some important topics have not yet received adequate answers. The above data and doubts may be synthesized as follows. Natriuretic peptides and their receptors are widely expressed in the hypothalamus, although some doubts still remain if BNP is locally expressed or internalized through receptor binding. In the hypothalamus, natriuretic peptides play different roles: reduction of norepinephrine

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release; inhibition of OT, VP, corticotropin-releasing factor, growth hormone, and LHRH release. All the natriuretic peptides and their receptors are present in the hypophysis; in particular, the three subtypes of receptors seem to be present in lactotroph, corticotroph, gonadotroph cells, equivocal data being present regarding somatotroph ones. Nevertheless a huge mass of studies investigating the effects of natriuretic peptides on pituitary cell populations, some doubts are still present. Majority of literature (although not all the literature) reported inhibition of both basal and induced ACTH release by natriuretic peptides. Instead really contrasting data are present regarding effects of natriuretic peptides on GH, LH, and FSH release, being reported inhibition, stimulation, or absence of effects in different studies. Such differences may be explained with reference to different in vitro or in vivo models but surely request further analyses in the future. Natriuretic peptides have mainly been identified in the zona glomerulosa and adrenal medulla. They are known to inhibit aldosterone secretion and growth of zona glomerulosa. More problematic are data regarding effects of natriuretic peptides in the zona fasciculata. Inhibition or stimulation of glucocorticoid secretion by adrenocortical cells has been reported and these contradictory data may be explained with reference to the different species considered. Lastly, in the adrenal medulla, natriuretic peptides inhibit catecholamine release and increase catecholamine uptake. Despite the extensive experimental investigations of the natriuretic peptide biology under both normal and pathological conditions many interesting problems remain to be addressed in the next years. It will have to be better investigated how the central nervous system control the natriuretic peptide system in the central and peripheral branches of the HPA axis. Moreover, natriuretic peptides modulate different hormonal systems and further experiments are needed to better ascertain the functional interrelationships between these systems. Data reviewed in Sections 5.1 and 5.2 indicate that the natriuretic peptide system is involved in the pathophysiology of adrenal cortical and medullary neoplasias but further studies will be necessary. The study of these and many other basic topics, along with the development of new potent and selective agonists and antagonists of the different receptors, not only will open new frontiers in the knowledge of the physiology of the HPA axis, but also will shed light on new therapeutical perspectives. Moreover, in recent years new technologies have been developed which could be used in order to specifically study the expression and action of natriuretic peptides in the different components of the HPA axis. Laser-capture microdissection has recently been applied to obtain homogeneous cell populations from nervous and endocrine structures, such as the hypothalamus (Segal et al., 2005) and pituitary gland (Lloyd et al., 2005). Microarray and proteomic analyses have also been performed on mRNA and proteins extracted from these cell populations. Laser-capture

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microdissection in conjunction with microarray analysis may allow genome-wide screening of transcripts from homogeneous cell populations of hypothalamus and pituitary in order to better analyze the expression of natriuretic peptides and receptors and to specifically study the effects of these peptides on different cell types. Microarray and proteomics studies could also provide complete and accurate profiles of expression in response to various environmental stimuli.

ACKNOWLEDGMENT We thank Alberta Coi for secretarial support and invaluable help in the provision of bibliographic items.

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Pandey, K.N., 2009. Ligand-mediated endocytosis and intracellular sequestration of guanylyl cyclase/natriuretic peptide receptors: role of GDAY motif. Mol. Cell. Biochem. 2009 Nov 26 [Epub ahead of print]. Papouchado, M.L., Vatta, M.S., Bianciotti, L.G., Ferna´ndez, B.E., 1995. Effects of atrial natriuretic factor on norepinephrine release evoked by angiotensins II and III in the rat adrenal medulla. Arch. Physiol. Biochem. 103, 55–58. Pemberton, C.J., Yandle, T.G., Espiner, E.A., 2002. Immunoreactive forms of natriuretic peptides in ovine brain: response to heart failure. Peptides 23, 2235–2244. Peng, N., Oparil, S., Meng, Q.C., Wyss, J.M., 1996. Atrial natriuretic peptide regulation of noradrenaline release in the anterior hypothalamic area of spontaneously hypertensive rats. J. Clin. Invest. 98, 2060–2065. Perras, B., Schultes, B., Behn, B., Dodt, C., Born, J., Fehm, H.L., 2004. Intranasal atrial natriuretic peptide acts as central nervous inhibitor of the hypothalamo-pituitary-adrenal stress system in humans. J. Clin. Endocrinol. Metab. 89, 4642–4648. Poole, C.J., Carter, D.A., Vallejo, M., Lightman, S.L., 1987. Atrial natriuretic factor inhibits the stimulated in-vivo and in-vitro release of vasopressin and oxytocin in the rat. J. Endocrinol. 112, 97–102. Potter, L.R., Abbey-Hosch, S., Dickey, D.M., 2006. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr. Rev. 27, 47–72. Potter, L.R., Yoder, A.R., Flora, D.R., Antos, L.K., Dickey, D.M., 2009. Natriuretic peptides: their structures, receptors, physiologic functions and therapeutic applications. Handb. Exp. Pharmacol. 191, 341–366. Raha, D., Tortorella, C., Neri, G., Prasad, A., Raza, B., Raskar, R., et al., 2006. Atrial natriuretic peptide enhances cortisol secretion from guinea-pig adrenal gland: evidence for an indirect paracrine mechanism probably involving the local release of medullary catecholamines. Int. J. Mol. Med. 17, 633–636. Raidoo, D.M., Narotam, P.K., van Dellen, J., Bhoola, K.D., 1998. Cellular orientation of atrial natriuretic peptide in the human brain. J. Chem. Neuroanat. 14, 207–213. Rebuffat, P., Mazzocchi, G., Gottardo, G., Meneghelli, V., Nussdorfer, G.G., 1988. Further investigations on the atrial natriuretic factor (ANF)-induced inhibition of the growth and steroidogenic capacity of rat adrenal zona glomerulosa in vivo. J. Steroid Biochem. 29, 605–609. Richard, D., Bourque, C.W., 1996. Atrial natriuretic peptide modulates synaptic transmission from osmoreceptor afferents to the supraoptic nucleus. J. Neurosci. 16, 7526–7532. Rocco, S., Opocher, G., D’Agostino, D., Leone, L., Mantero, F., 1989. Lack of aldosterone inhibition by atrial natriuretic factor in primary aldosteronism: in vitro studies. J. Endocrinol. Invest. 12, 13–17. Rodriguez Fermepin, M., Vatta, M.S., Bianciotti, L.G., Wolovich, T.J., Fernandez, B.E., 2000. B-type and C-type natriuretic peptides modify norepinephrine uptake in discrete encephalic nuclei of the rat. Cell. Mol. Neurobiol. 20, 763–771. Rodriguez Lopez, P., Ehlerding, A., Leonhardt, S., Jarry, H., Wuttke, W., 1993. Effects of angiotensin II and atrial natriuretic peptide on LH release are exerted in the preoptic area: possible involvement of gamma-aminobutyric acid (GABA). Exp. Clin. Endocrinol. 101, 350–355. Rodriguez-Pascual, F., Miras-Portugal, M.T., Torres, M., 1996. Effect of cyclic GMPincreasing agents nitric oxide and C-type natriuretic peptide on bovine chromaffin cell function: inhibitory role mediated by cyclic GMP-dependent protein kinase. Mol. Pharmacol. 49, 1058–1070. Rose, R.A., Anand-Srivastava, M.B., Giles, W.R., Bains, J.S., 2005. C-type natriuretic peptide inhibits L-type Ca2þ current in rat magnocellular neurosecretory cells by activating the NPR-C receptor. J. Neurophysiol. 94, 612–621.

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C H A P T E R

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Evidence for Multiple Photosystems in Jellyfish ¨ m† Anders Garm* and Peter Ekstro Contents 42 42 44 45 46 46 49 51 54 56 56 60 64 67 70 72 73 73

1. Multiple Photosystems 1.1. General-purpose versus special-purpose eyes 1.2. Multiple photopigments 1.3. Visual channels 2. Photosensitivity in Cnidarians 2.1. Behavioral evidence of photoreception in cnidarians 2.2. Photosensitive structures in cnidarians 3. Photosensory Organs in Hydromedusae 4. Photosensory Organs in Scyphomedusae 5. Photosensory Organs in Cubomedusae 5.1. The rhopalium 5.2. Eye types and visual properties 5.3. Presence of different photoreceptor types? 5.4. Neuronal organization 6. Multiple Opsins in Cnidarians—Multiple Photosystems? 7. Conclusion Acknowledgments References

Abstract Cnidarians are often used as model animals in studies of eye and photopigment evolution. Most cnidarians display photosensitivity at some point in their lifecycle ranging from extraocular photoreception to image formation in cameratype eyes. The available information strongly suggests that some cnidarians even possess multiple photosystems. The evidence is strongest within Cubomedusae where all known species posses 24 eyes of four morphological types. Physiological experiments show that each cubomedusan eye type likely constitutes a separate photosystem controlling separate visually guided behaviors. Further, the visual system of cubomedusae also includes extraocular * Department of Comparative Zoology, University of Copenhagen, Copenhagen, Denmark Department of Cell and Organism Biology, Lund University, Lund, Sweden

{

International Review of Cell and Molecular Biology, Volume 280 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)80002-4

#

2010 Elsevier Inc. All rights reserved.

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photoreception. The evidence is supported by immunocytochemical and molecular data indicating multiple photopigments in cubomedusae as well as in other cnidarians. Taken together, available data suggest that multiple photosystems had evolved already in early eumetazoans and that their original level of organization was discrete sets of special-purpose eyes and/or photosensory cells. Keywords: Cnidarians, Eyes, Ocelli, Photopigments, Opsins, Vision, Cubomedusae. ß 2010 Elsevier Inc.

1. Multiple Photosystems In recent years, cnidarians (Fig. 2.1) have attracted increasing interest as model animals for evolutionary studies, in particular regarding gene evolution, cell differentiation, and patterning processes during embryonic development (Chiori et al., 2009; Jacobs et al., 2007; Kozmic et al., 2008b; Martindale, 2005). Among the more surprising findings is the demonstration of large numbers of opsin and opsin-like genes in cnidarians—also in species that have no eyes (Suga et al., 2008). What does this mean? Do cnidarians possess multiple photosystems, possibly encompassing multiple light-sensitive properties that are currently unknown from other animals? And, in that case, what distinguishes one photosystem from another?

1.1. General-purpose versus special-purpose eyes The complex camera eyes of vertebrates, as well as the compound eyes of arthropods, serve many purposes like wavelength (‘‘color’’) discrimination, luminance perception, edge detection, and movement detection (Land and Nilsson, 2002)—all functions that we normally link with ‘‘eyes’’ and ‘‘vision.’’ However, in many animals there are photosensory structures with more limited capabilities. One example from vertebrates is the pineal organ, which in many fishes functions basically as a dosimeter that distinguishes day from night, and signals ‘‘night’’ with synthesis of the neurohormone melatonin (Ekstro¨m and Meissl, 2010; Falcon, 1999). In dipterans, ocelli mediate stabilizing reflexes during flight (Van Kleef et al., 2008; Warrant et al., 2006). Many invertebrates possess simple pigment cup eyes that function as simple detectors of light coming from directions limited by their screening pigment (Arendt and Wittbrodt, 2001). For example, the simple eye-spot ocellus of larval ascidians mediates phototactic swimming behavior (Kusakabe and Tsuda, 2007). Pineal organs, ocelli, and pigment cup eyes may be characterized as special-purpose (or limited-purpose) eyes, whereas vertebrate camera eyes then are general-purpose eyes; all (or most) visual functions are served by one eye type. In animals with special-purpose

A

Octocorallia

Anthozoa Acropora millepora Hexacorallia Nematostella vectensis

Stylocoronella riedli

Stauromedusae

Chiropsella bronzie

Chirodropida

Alatina moseri Carybdea marsupialis Carybdeida Carybdea rastoni Tamoya bursaria Tripedalia cystophora

Staurozoa

Cubozoa

Coronata

Discomedusae: Semaeostomeae

Cassiopea xamachana Mastigias

Discomedusae: Rhizostomeae

Scyphozoa

Medusozoa

Aurelia aurita Cyanea capillata

Trachylina

Tiaropsis multicirrata

Hydroidolina: Leptothecata

Capitata: Cladonema radiatum Hydra sp. Polyorchis penicillatus Sarsia tubulosa

Filifera: Bougainvillia principis Hydractinia echinata Leuckartiara octona Podocoryne carnea

B

Hydrozoa Hydroidolina: Anthoathecata

C

Figure 2.1 Cnidarians. (A) The phylogenetic relationships of the cnidarian species discussed in this chapter, according to the hypothesis of Collins et al. (2006). Data on photosensitivity are lacking for Octocorallia, Coronata, and Trachylina (grey branches). Nomenclature according to WoRMS (World Register of Marine Species: http://www.marinespecies.org/index.php). (B) A medusa of a box jellyfish, Carybdea sivickisi, which has an advanced visual system (arrowheads). (C) An example of an anthozoan polyp (unknown species), which in general has the simplest photosystem within cnidarians.

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eyes sensu stricto each eye type supports one visual task, for example responding to the shadow of a presumptive predator (Land and Nilsson, 2006). Thus, a special-purpose eye with one function would constitute one photosystem. In contrast, a vertebrate camera eye or an arthropod compound eye contains multiple photosystems.

1.2. Multiple photopigments Animal photoreceptors are characterized by their photopigment and their phototransduction mechanism, as well as by their morphology (Arendt and Wittbrodt, 2001). There are two basic morphological types, characterized by their surface enlargements that provide a large surface area for the photopigments. Ciliary photoreceptors use internal or external extensions of the ciliary membrane, while rhabdomeric photoreceptors have their cell surface enlarged by microvilli. Interestingly, morphology is tightly linked with opsin type and phototransduction system. Thus, there are ciliary opsins (c-opsins) and rhabdomeric opsins (r-opsins), with their specific G proteincoupled transduction mechanisms (Arendt and Wittbrodt, 2001; Fernald, 2006). These are the basis of the photopigments that are used in what we generally mean with ‘‘vision,’’ that is, by the photoreceptor cells of generalpurpose eyes, but they are also used in simpler special-purpose eyes. There are six opsin subfamilies: four of which contain opsins that transduce light using different G protein-coupled mechanisms, one contains photoisomerases, and one (neuropsins) has functions that remain to be determined (Shichida and Matsuyama, 2009). Recently, numerous opsins have been sequenced in cnidarians (Kozmic et al., 2008a; Plachetzki et al., 2007; Suga et al., 2008), one of which belongs to a new subfamily that utilizes a Gs-based mechanism (Koyanagi et al., 2008; Fig. 2.2). In addition to the opsin-based photopigments, cryptochromes may be used in the circadian clock gene machinery (Partch and Sancar, 2005), and recently cryptochromes that are probably involved in lunar-controlled mass spawning were described in the anthozoan coral Acropora millepora (Levy et al., 2007). Furthermore, it has recently been suggested that cytochrome c oxidase may be the photopigment mediating a shadow response in sponge larvae (Bjo¨rn and Rasmusson, 2009). Multiple photopigments form the basis of color vision. However, multiple photopigments can be used for simpler responses like wavelengthspecific behaviors (Land and Nilsson, 2002). Sets of multiple photopigment/phototransduction systems also may serve the purpose of extending the sensitivity range and give different temporal characteristics (vertebrate rods and cones; Kawamura and Tachibanaki, 2008). Photopigments may be used for specific ‘‘nonvisual’’ perception, like melanopsin for entrainment of circadian rhythms and pupillary reflex control in mammals (Hankins

45

Vertebrate visual opsins and pinopsin

VA and VAL opsins

Parapinopsin

Parietopsin

Encephalopsin

TMT opsin

Arthropod c-opsins

Platynereis c-opsin

Gt (transducin) coupled

Cnidarian c-opsins

Lophotrochozoan r-opsins

Ecdyzozoan r-opsins

Melanopsin

Retinochrome

RGR

Go opsins

Photoisomerases Gq coupled

Peropsin

Go

Neuropsin

Gs Gs opsins (cnidops)

Outgroup GPCRs

Multiple Photosystems in Jellyfish

Figure 2.2 Cnidarian opsins (red branches) and a hypothetical view of their phylogenetic relationship to other opsins. The phylogenetic tree is based on those presented by Koyanagi et al. (2008), Kozmic et al. (2008a; Supporting information), Plachetzki et al. (2007), and Suga et al. (2008; Supplemental data). There are major uncertainties regarding the phylogenetic position of neuropsins and photoisomerases, and their relationship to Go opsins. Koyanagi et al. place Go opsins as a separate branch basal to the Gs opsins. Kozmic et al. (2008a) and Suga et al. (2008) place Gq opsins basal to the other main groups, the Go opsin/neuropsin/photoisomerase group, and the Gt opsins. However, in all scenarios the cnidarian Gs opsins (cnidops) is basal to all other opsins (except possibly Go opsins), and the cnidarian Gt opsins is basal to all other Gt opsins. See on-line version for color figure.

et al., 2008). Thus, one photosystem may comprise multiple photopigments (color vision), or only one photopigment.

1.3. Visual channels Visual channels describe pathways of signal transfer in the visual system. A channel may be likened to a matched filter (Wehner, 1987) that extracts and conveys a certain aspect of the visual information. In the vertebrate retina, a ‘‘rod pathway’’ transmits signals from rod photoreceptors via bipolar cells to ganglion cells whereas ‘‘cone pathways’’ denote the pathways, from cone photoreceptors via bipolar cells to ganglion cells, which form the circuitry that is the basis of color opponency and center-surround mechanisms

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(Dacey, 1996; Sharpe and Stockman, 1999). Visual channels may converge and diverge, thereby providing a basis for complex information processing. For the pathways from rods to ganglion cells, the combination of a high convergence rate with a more modest divergence rate gives high absolute sensitivity combined with signal averaging that reduces the synaptic noise (Sterling et al., 1988; Taylor and Smith, 2004). It is clear that a general-purpose eye encompasses a multitude of visual channels and that further processing by higher visual centers in the brain depends on parallel transfer of visual information by visual channels and on extensive crosstalk—that is, interaction—between different visual channels. It is the total outcome of this interaction that determines the processing capacity of a given visual system. Thus, each visual channel serves to convey a particular aspect of the visual world. In this sense, a visual channel may be seen as a separate photosystem, and it is in this sense the question of multiple photosystems in cnidarians will be discussed.

2. Photosensitivity in Cnidarians Cnidarians constitute the most basal group of recent metazoans with a nervous system. Present-day cnidarians all have a well-developed nervous system, which in several cases includes a proper central nervous system (Garm et al., 2007c; Mackie, 2004). Cnidarians also have differentiated sensory cells that in some cases organize to form sensory organs. Singlecelled mechano- and chemoreceptors are found scattered across the body of both polyps and medusae but may form fairly complex structures to control cnidocyte discharge (Kass-Simon and Scappaticci, 2002; Scappaticci and Kass-Simon, 2008). Photoreception plays a major role in the life of many cnidarians, especially in the medusa stage. This importance is reflected in the complexity and diversity of their photosensitive structures spanning from muscle cells to camera-type eyes, the latter being the best defined cnidarian organ. In addition, a multitude of opsin and opsin-like genes have been found, some of them expressed in structures with not yet demonstrated inherent photosensitivity. Thus, it would appear that cnidarians use multiple photosystems. Notably, it is the medusa ( jellyfish; cf. Fig. 2.1A) stage that possesses differentiated eyes alongside other photosensory structures, and this review will focus on photosystems in cnidarian medusae.

2.1. Behavioral evidence of photoreception in cnidarians Many cnidarians display photosensitivity and the supporting behavioral evidence dates back at least to the nineteenth century when Romanes, Conant, and Berger studied the physiology, behavior, and morphology of

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a number of different medusae (Berger, 1898, 1900; Conant, 1898; Romanes, 1876, 1877). Romanes’ studies of locomotion in the hydromedusa Sarsia tubulosa showed that this species exhibits positive phototaxis and, by simple ablation experiments, that photosensitivity was located to their ocelli (Romanes, 1876). Also, he showed that a light stimulus increased their rate of swim pulses (Romanes, 1877). Berger (1900) and Conant (1898) did similar experiments with cubomedusae and showed that they also responded to light but that both darkness and strong light seemed to inhibit their swimming. Since these early reports on photoreception several experiments have documented a long list of different cnidarian behaviors influenced by light. It is not within the scope of the present review to list all these reports, but in this section we instead provide an overview of lightguided behaviors to indicate the diversity and point to where we can expect multiple photosystems among cnidarians. There are several cases of extraocular photoreception within cnidarians. Some of the first reports stems from the eyeless Hydra, where it was shown that the ambient light influences rhythmic contractions in the body wall (Passano and McCullough, 1962). Later it was shown that this photic entrainment depends on the spectral composition and intensity of the light (Taddei-Ferretti et al., 2004). Extraocular photosensitivity can be present in any of the cnidarian life stages and is well documented in the planula larva of Hydractinia echinata, which displays positive phototaxis (Katsukura et al., 2004). Another important behavioral response controlled by extraocular photosensitivity is found in some anthozoans where the reproductive cycle is timed by the moonlight, resulting in mass spawning events (Levy et al., 2007). In general, extraocular photosensitivity constitutes the simplest photosystems in cnidarians and seems to support only a single behavior in each case. The more advanced light-guided behaviors are all found in the medusa stage. Many scyphomedusae display simple phototaxis but some species are also able to perform horizontal migrations controlled by the light environment. It has been found that the common moon jelly, Aurelia aurita, migrates according to the solar position and similar results have been obtained for members of the genus Mastigias (Hamner and Hauri, 1981; Hamner et al., 1994). This shows that these medusae are able to at least locate a point source. Sudden changes in the ambient light intensity also initiate strong behavioral responses in many cnidarians. Since Romanes’ discovery that light can influence the rate of swim contractions in Sarsia this behavior has been studied in more details in other hydromedusae (Arkett, 1985; Arkett and Spencer, 1986a,b; Yoshida and Ohtsu, 1973). These studies led to the definition of a ‘‘shadow response’’ or ‘‘shadow reflex’’ where a sudden decrease in light intensity causes the medusa to make a few rapid swim contractions. It has been suggested that this behavior serves either to control vertical migrations (Arkett, 1985) or to avoid predators

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(Anderson and Mackie, 1977). A somewhat different shadow response has also been described from cubomedusae, where the response is stronger and longer lasting and is thought to enable the medusae to optimize their time spent feeding in light shafts (Garm and Bielecki, 2008). Cubomedusae are very agile swimmers and are typically found in habitats where other medusae are not able to thrive (Coates, 2003) and it is so far only in cubomedusae that behaviors requiring true vision with spatial resolution have been documented. Medusae of the Caribbean species Tripedalia cystophora and the Australian species Chiropsella bronzie perform an obstacle avoidance response that is triggered when the obstacle takes up a certain angle of the visual field (Garm et al., 2007b; Fig. 2.3). Further, T. cystophora has recently been shown to use the canopies of nearby

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Figure 2.3 Obstacle avoidance in the box jellyfish Chiropsella bronzie. The medusae were tested in a flow chamber of 10  35 cm with two obstacles in the downstream end and their swim trajectories were map with obstacles of different visual appearance. With black obstacles giving a high contrast to the opaque white background the medusae stayed away from them and had very few contacts (A). If the obstacles were transparent the medusae failed to respond to them and kept bumping into them (B).

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mangrove trees when navigating toward their habitat between the mangrove roots (Garm et al., in preparation). This they do by looking into the terrestrial environment through Snell’s window (an optical phenomenon caused by the difference in refractive index between air and water resulting in a compression of the entire terrestrial hemisphere into a cone of 97.2 when seen from under the water). As already evident from the behavioral data, advanced photic behaviors that possibly involve multiple photosystems have been observed only in medusae. They occur in hydrozoan, scyphozoan, and cubozoan medusae, but cubomedusae have the most elaborate behavioral repertoire involving spatial vision and therefore make the best candidates for possessing multiple photosystems. The following sections will show that this is supported by the available morphological and physiological data.

2.2. Photosensitive structures in cnidarians The structures underlying the photosensitivity within Cnidaria vary greatly from structurally nonspecialized cells to proper eyes that are structurally similar to cephalopod and vertebrate eyes (Martin, 2002). In the following survey of the morphological diversity we will distinguish between three main categories: extraocular receptors, ocelli, and proper eyes. Extraocular receptors are defined as any cell giving a physiological response to light but which is not associated with any kind of screening pigment. Specialized photoreceptors organized together with screening pigment but without optics are considered ocelli. Ocelli may be multicellular or single-celled and multicellular ocelli may or may not have spatial resolution. Proper eyes include some kind of image-forming optics and spatial resolution. 2.2.1. Extraocular photoreception As we saw earlier, photic behaviors are not restricted to species with ocelli or eyes, and extraocular photoreception is well documented within cnidarians. Still, little is known about the structures underlying these behaviors, which can be observed in the planula larvae as well as in the polyp and medusa stages. The responsive cells are notoriously difficult to identify, since they often lack the structural specialization usually seen in photoreceptors. This is clearly demonstrated in Hydra where photosensitivity is very well documented and thoroughly examined but the receptors are still unknown (Musio et al., 2001; Taddei-Ferretti and Musio, 2000; TaddeiFerretti et al., 2004). Extraocular photoreception in cnidarians may be found outside the nervous system. In some sea anemones, it has been shown that the muscle cells are light-sensitive and in general contract upon a light stimulus (Marks, 1976). In other cases, it seems to be epithelial cells that are light-sensitive. For example, the mass spawning events of many anthozoans is controlled by

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the moon phases, which are probably detected using cryptochrome-based photosensitivity. Immunocytochemical staining along with in situ hybridization have located the cryptochrome to more or less the entire layer of epithelial cells in the ectoderm (Levy et al., 2007). In some cases, it has been shown that parts of the nervous system itself are photosensitive. These photosensitive neurons can be situated at different places in the nervous system. In the hydromedusa Polyorchis penicillatus, there is electrophysiological evidence that they form part of the central nervous system, the outer nerve ring (Arkett and Spencer, 1986a,b). In the case of several sea anemones, photosensitivity has been suggested to at least partly occur in the general nerve net (Marks, 1976; Sawyer et al., 1994). Photosensitivity has also been proven in the eyeless planula larvae of the hydrozoan H. echinata (Plickert and Schneider, 2004). The responsive cells have not been identified but immunocytochemical staining against the neuropeptide RFamide, which is often associated with photosensory structures in cnidarians, suggests neurons at the frontal end of the larva (Plickert and Schneider, 2004). 2.2.2. Ocelli Ocelli are found in all cnidarian subgroups except Anthozoa. They are almost exclusively found in the motile medusa stage but very simple ocelli, which are structurally similar to the general ocellus type of hydromedusae, have been described from the polyps of the staurozoan Stylocoronella riedli (Blumer et al., 1995). The detailed structure of cnidarian ocelli varies greatly and the only structurally unifying character is that their receptor cells are of the ciliary type. The most common ocellus type in scyphomedusae and hydromedusae is cup-shaped and has relatively few nonpigmented photoreceptors, normally 10–50, arranged in an everse retina (Singla, 1974; Singla and Weber, 1982a; Yamamoto and Yoshida, 1980; Yamasu and Yoshida, 1976). The receptor cells lie in between nonsensory pigment cells that form the screening pigment of the cup. In cubomedusae, the ocelli differ somewhat from this general picture. Firstly, they contain several hundred very small photoreceptors and secondly, their photoreceptors also contain the screening pigment (Garm et al., 2008; see later for more details). It has not yet been proven but the cup shape of some of the cnidarian ocelli might provide them with crude spatial resolution. A peculiar kind of ocellus is found in the planula larvae of the cubozoan T. cystophora (Nordstro¨m et al., 2003). Here 20–25 single-celled ocelli are found in the posterior end of the larva and within the subcellular pigment cup microvilli are found along with an unmodified motor cilium. Even though the evidence is purely morphological, it is believed that the microvilli contain a photopigment and that the degree of illumination directly controls the activity of the cilium, whereby the behavior of the larva is

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controlled. Together the several ocelli could work as a compound eye that lacks neural connections between the photosensory units. Each ocellus would constitute an independent part of the motor system. 2.2.3. Eyes Proper eyes are found in all cubomedusae and possibly also in a few species of hydromedusae. In many ways these eyes resemble the ocelli described above but they are larger and have an additional lens-like structure (Nilsson et al., 2005; Piatigorsky et al., 1989; Weber, 1981). In cubomedusae specialized cells filled with crystalline form the lens and in the center of the lens they lack clear organelles, similar to what is seen in the vertebrate lens. To our knowledge, the only documented lens-like structure in hydromedusae is found in Cladonema radiatum. Here the lens is formed by apical outgrowths of the nonsensory pigment cells (Weber, 1981). Image-forming capacities have been shown for the cubozoan lenses (Nilsson et al., 2005) but have so far not been examined in C. radiatum.

3. Photosensory Organs in Hydromedusae Hydrozoans display the largest diversity of life styles, ecological niches, and lifecycles among cnidarians, and this is also reflected in the visual equipment of their medusae. It spans the range from photosensitive muscle cells to eyes that somewhat resemble the camera-type eyes of vertebrates and cephalopods, and which may provide the carrier with at least some spatial resolution. It is important to notice that the diversity is between species and that usually each species carries only one single type of ocellus or eye. However, there is evidence that some hydromedusae have extraocular photoreceptors in addition to ocelli (Arkett and Spencer, 1986b). A large number of hydromedusan species have ocelli of some kind (Hyman, 1940; Minchin et al., 1900). In almost all cases, the ocelli are situated on or at the basis of each tentacle, but in at least one species, Tiaropsis multicirrata, they are found directly on the subumbrella in close association with statocysts (Singla, 1974). The number of ocelli can vary from four to several hundred. Ocelli from a little more than a handful of species have been examined in ultrastructural details and these studies have pinpointed a number of shared features (Singla, 1974; Singla and Weber, 1982a,b; Yamamoto and Yoshida, 1980; Yamasu and Yoshida, 1973). Within a specimen the size of the ocelli may vary but otherwise they are of the same structure. They are small (typically <100 mm in diameter) and their putative photoreceptors are of the ciliary type and unpigmented (Fig. 2.4). The receptors are of ectodermal

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Figure 2.4 Ultrastructure of an ocellus from Sarsia tubulosa. The ocelli of S. tubulosa represent the typical hydromedusan ocellus. It is cup-shaped with the cup filled by the outer segments (OS) of the photoreceptors (A). The OS are modified cilia with membranes forming numerous microvilli (B, arrowheads indicate cilia). The ocellus is made of two cell types: photoreceptors (PRs) and pigment cells (PCs) (B, C). The PRs are slim whereas the PCs are about 10 mm in cross section (B, arrowheads indicate the ciliary rootlets of the PRs).

origin and they are 30–80 mm long including the outer segment and 5–10 mm in diameter at the cell body. The shape of the ocelli differs somewhat between species, and range from a flattened structure like in Leuckartiara octona to a deep pit as seen in Bougainvillia principis (Singla, 1974). Although it has not been experimentally proven, the shape is likely to have a major impact on the ability to obtain spatial information. There also seems to be a difference in their sensitivity,

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since the outer segment in L. octona is a more or less unmodified cilium without microvilli extensions. In all the other examined species the outer segment dissolves in microvilli, which expand the membrane surface and thereby probably increase the amount of photopigment and hence sensitivity. Quite surprisingly, the number of outer segments also varies: In most species, there is one modified cilium protruding from each photoreceptor but in B. principis there is a variation between one and three cilia per photoreceptor (Singla, 1974). The number of receptor cells per ocellus has only been estimated for two species, C. radiatum and T. multicirrata, where each ocellus contains about 20 and 400–500 photoreceptors, respectively (Singla, 1974; Weber, 1981). The large number of receptor cells in Tiaropsis has the effect that their outer segments are relatively small with only few microvilli. The nonsensory pigment cells that form the screening pigment also vary in their structural details. Their cell bodies are of similar size ( 10  20 mm) and so are their pigment granules, but in some species these cells also have an apical outgrowth. The outgrowths extend between the outer segments of the photoreceptors and constitute a large part of the internal space of the pigment cup. In C. radiatum they form a lens-like structure (Weber, 1981), giving the appearance of a proper eye, but the optical properties of the lens-like structure have never been examined. In most cases the pigment cells are also of ectodermal origin and lie in between the receptor cells making the retina everse, but in T. multicirrata they are of endodermal origin and are separated from the rest of the ocellus by a thin layer of mesoglea. Here the photoreceptor outer segments are invaginated into the pigment cup making the retina inverse (Singla, 1974). Since Romanes’ (1876) pioneering work more than 130 years ago many neurophysiological studies have been done on hydrozoans. Unfortunately, few of these have concerned the ocelli and their photoreceptors. Electroretinograms (ERGs) are only available from the ocelli of S. tubulosa and P. penicillatus and they have shown that the receptors are relatively slow and have a single sensitivity peak in the blue-green part of the light spectrum (Weber, 1982a,b). Further, the ERGs support the morphological data showing that the retina of hydromedusan ocelli and eyes consists of a homogeneous population of photoreceptors. This would indicate the presence of a single photosystem only. However, electrophysiological experiments have shown that P. penicillatus also has additional extraocular photoreceptors, even though their identity is not agreed upon (Anderson and Mackie, 1977; Arkett and Spencer, 1986b; Satterlie, 1985b). This might indicate that at least in this particular species two photosystems are present, but so far the two groups of photoreceptors have only been shown to serve the same function mediating the shadow response (Arkett and Spencer, 1986a,b). Taken together, the available data suggest that hydromedusae in general possess a single photosystem only.

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4. Photosensory Organs in Scyphomedusae Several scyphomedusae have been shown to respond to light. If they have ocelli these are always situated on their sensory structures, the rhopalia, a feature shared with cubomedusae (see below). Alongside the ocelli the rhopalia are equipped with chemo- and mechanoreceptors, and a large distally placed crystal that may serve as a statolith. Little data exist on the detailed structure of scyphomedusan ocelli and almost all of it stem from two species only, the common moon jelly A. aurita and the very specialized Cassiopea xamachana (Bouillon and Nielsen, 1974; Schewiakoff, 1889; Yamasu and Yoshida, 1973). In the case of Aurelia each rhopalium carries two ocelli, a larger one on the aboral side and a smaller one on the oral side (Fig. 2.5C–E). Interestingly, these two eyes differ substantially in the detailed anatomy. The larger ocellus is of ectodermal origin and consists of a single cell type only, pigmented receptor cells (Yamasu and Yoshida, 1973). It is flattened and has a diameter of about 150 mm (Schewiakoff, 1889). The putative photoreceptors of this flat ocellus each have a single cilium with little structural specializations. If this is indeed a light-sensitive organ (for which there is no experimental data) it will be a mere light meter and have no spatial resolution. The smaller ocellus of Aurelia (40–50 mm in diameter) is of both endodermal and ectodermal origin, similar to the ocelli of the hydromedusa T. multicirrata. The pigment cells are endodermal and are separated from the ectodermal receptor cells by a thin layer of mesoglea (Yamasu and Yoshida, 1973). The pigment cells forming the cup of the ocellus are of two types: one with electron dense pigment granules and one with more electron lucent pigment granules. The two cell types are of similar size ( 20  8 mm). A rather unusual feature is that the receptor cells fill the internal cup almost entirely with their cell bodies. As a consequence, their small outer segments, with very little microvilli extensions, lie against the mesoglea at the very bottom of the cup (Yamasu and Yoshida, 1973). Due to this arrangement, the individual outer segments will probably be shaded by the pigment screen in specific ways that might provide this eye with spatial resolution. The rhopalia of C. xamachana also carry two ocelli each but they are similar in structure. Like the large ocellus of Aurelia they are both ectodermal, situated on the oral side of the rhopalium, and consist entirely of pigmented photoreceptors (Bouillon and Nielsen, 1974). At the medusae stage, Cassiopea is a specialized bottom dweller that displays very little behavior (Passano, 2004). The ocelli probably only support light intensity measurements, ensuring that the medusa with its photosynthetic zooxanthellae is situated in an area with enough light. There are less neurophysiological data from scyphomedusae than from hydromedusae, and none of it concerns the ocelli and their photoreceptors

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Figure 2.5 The rhopalia of Tripedalia cystophora and Aurelia aurita. All cubomedusae have four similar rhopalia situated along the rim of the bell in invaginations, the rhopalial niches (A, arrowheads). The rhopalia hang by a flexible stalk and have six eyes of four morphological types: the upper lens eye (ULE), the lower lens eye (LLE), a pair of slit ocelli (SOc), and a pair of pit ocelli (POc). In the distal part a heavy crystal is found (B). Scyphomedusae also have rhopalia but they vary in appearance and not all carry eyes or ocelli. The eight rhopalia of the scyphomedusa A. aurita are situated in indentations of the bell margin (C). Here two ocelli are found: a small ocellus (SO) on the oral side and a larger ocellus (LO) on the aboral side (D, E). Like for cubomedusae the scyphomedusan rhopalium has a large crystal distally (D, E).

directly. Following in the footsteps of Romanes (1876, 1877), most of the studies have concerned the pacemakers that control the rate of swim pulses (Passano, 1982; Satterlie, 2002). The identity of the pacemaker cells is still unknown but they are located in the rhopalia and in A. aurita their frequency is influenced by the light conditions. Darkness decreases the pacemaker frequency and an increase in light intensity increases the frequency (Horridge, 1959). This response is governed by the input to the small ocelli, which was shown by ablation experiments (Passano, 1982). In the eyeless Cyanea no response to light could be seen (Horridge, 1959). When it comes

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to spectral sensitivity or other physiological properties used to identify different receptor classes nothing is known from scyphomedusae. The sparse morphological and physiological data from photosensitive structures in scyphozoans support what has been suggested by their behavioral repertoire, namely that their photosensory structures are relatively simple and contain only a single or maybe two photosystems each. In A. aurita the two ocelli types can be considered two separate photosystems that probably support separate behaviors. The larger ocellus is a mere light meter that might control phototaxis. The smaller ocellus possibly has some spatial resolution, which could allow detection of the position of a point source (like the sun) and thereby form the sensory basis for navigation by the solar position (Hamner et al., 1994). Interestingly, the small ocellus also has light meter function that is used for pacemaker control (Passano, 1982).

5. Photosensory Organs in Cubomedusae When considering the visually guided behaviors, structural complexity, or physiology of the photoreceptors, cubomedusae without doubt possess the most advanced visual system among cnidarians. They are agile swimmers and cannot be considered plankton organisms as other medusae. When observing them in their natural habitat their swim patterns resemble a shoal of fish with directional swimming and fast 180 turns. They perform visually guided obstacle avoidance and also use their eyes for navigational purposes. Further, all known species of cubomedusae possess a similar set of 24 eyes of four distinct morphological types, some resembling vertebrate eyes. More importantly for this review, ultrastructural data, along with physiological and immunocytochemical data suggest that multiple classes of photoreceptors might by present in some cubomedusae. The following sections will describe this photosensory system in details.

5.1. The rhopalium Like in scyphomedusae most of the senses of cubomedusae are gathered on rhopalia, which seem to serve only sensory functions. Following the general quadral symmetry of the cubomedusae there are always four rhopalia evenly spaced along the bell margin (Fig. 2.5). There are large and functionally significant differences between the rhopalial structure of cubomedusae and scyphomedusae, though. The rhopalia of cubomedusae are more complex and this structural complexity has a large impact on the visual perception. For this reason, we will start the description of the cubozoan visual system by giving a detailed account of their rhopalia.

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Cubomedusae are more or less square in cross section, with the tentacles gathered on structures called pedalia at the corners of the bell. The rhopalia are situated in invaginations of the exumbrella, the rhopalial niches, on the sides of the bell between the pedalia (Fig. 2.5A). Here they hang from the roof of the niches in thin and flexible stalks (Fig. 2.5B). The stalk is attached to the backside of the rhopalium (defined as the opposite side to the ocelli and eyes) such that the side with the eyes will be facing toward the center of the bell when the animal is vertically oriented. The center of the stalk is a branch of the gastrovascular cavity that extends into the rhopalium where it expands. The epidermis of the stalk has some regional specializations. The proximal two thirds have myoepithelial cells forming longitudinal muscle fibers suggesting that the rhopalia can be moved actively. Further, on the upper side of the stalk concentrations of nerve fibers are found in both the epidermis and gastrodermis (Garm et al., 2006). The epidermal nerve is a part of the ring nerve, which runs into the stalk and connects with the rhopalial neuropil where the visual information is probably integrated (Parkefelt et al., 2005; see also later section). The gastrodermal nerve projects down from the bell and stops just before it enters the rhopalium, and its ultrastructural features suggest that it has a mechanosensory function (Garm et al., 2006). The stalk inserts at the apical end of the rhopalium. The distal part of the rhopalium contains a large crystal (Fig. 2.5B), which has been referred to as a statocyst (e.g., Laska and Hu¨ndgen, 1982; Satterlie, 2002). Still, there has been no documentation of a gravity sensing function and our microscopical studies have not indicated any sensory cells in association with the crystal (A. Garm and P. Ekstro¨m, unpublished results). Berger (1898) suggested that the crystal might help to stabilize the rhopalium. We have recently shown that his idea was correct and that the weight of the crystal and the flexible stalk together ensure that the rhopalium is always oriented in the same way relative to gravity, independent of the orientation of the medusae (Garm et al., in preparation). As a consequence the visual fields of the eyes and ocelli are also kept constant along the vertical axis. Such a system of semiconstant visual fields is to our knowledge unique to cubomedusae and is probably a clear example of matched filtering (Wehner, 1987). The filter allows the jellyfish to ‘‘know’’ where in the visual environment information is gathered without having to actually recognize the area. In all known species of cubomedusae each of the four rhopalia contains a similar set of six eyes and ocelli (Figs. 2.6B and 2.7). These eyes and ocelli are of four morphological types and their gross morphology is fairly constant among cubozoan species. The rhopalia are bilaterally symmetric (Parkefelt and Ekstro¨m, 2009; Parkefelt et al., 2005; Skogh et al., 2006) and along the midline are two complex camera-type eyes: the upper and lower lens eyes. In addition, two pairs of smaller ocelli, the pit and slit ocelli, are situated on either side of the lens eyes. The morphology, optics, and physiology of these eyes and ocelli will be dealt with in the next section.

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Figure 2.6 Morphology of cubozoan eyes and ocelli. The two lens eyes (the upper lens eye—ULE and lower lens eye—LLE) are complex eyes resembling vertebrate eyes (A). The largest cubozoan eye is the LLE of C. alata, which reaches 800 mm in diameter (B). In transverse section through this eye, it is seen that the vitreous space (VS) is relatively broad, which could give higher spatial resolution (B). The slit ocelli are surprisingly complex and made of four cell types: pigmented photoreceptors (PPR), nonpigmented photoreceptors (not shown), nonsensory pigment cells (NSPC), and vitreous cells (VC) (C, T. cystophora; E, C. alata). The vitreous cells shield the outer segments (OS) of the receptors and may have a filtering effect. The pit ocelli are simple ocelli and in C. alata they almost lack a pigment screen (D) leaving them with no spatial resolution.

The entire rhopalium is covered by a monociliated epithelium, which also forms the cornea of the lens eyes. Our structural and immunocytochemical data suggest that the epithelium also contains sensory cells, possibly mechanoreceptors, which are situated on the backside of the rhopalium under the stalk (Skogh et al., 2006). The function of these putative mechanoreceptors is entirely unknown and has to our knowledge never been experimentally tested. The backside of the rhopalium normally faces the opening of the rhopalial niche where sensory cells would be exposed to waterborne vibrations entering the niche from the outside. Under the epithelium on the backside of the rhopalium several layers of what seems like undifferentiated cells are found (Garm et al., 2006). This large number

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Figure 2.7 Visual control of the pacemaker activity. When stimulating the different eyes and ocelli in the box jellyfish Tripedalia cystophora different effects on the pacemaker frequency are obtained. Stimulating the lower lens eye results in a strong inhibition at light-on and a strong but short-lived activation at light-off (A). A light stimulus on the upper lens eye has close to the opposite effect, and here light-on stimulates the pacemaker and light-off makes it return to basis activity (B). Illumination of the pit ocelli elicits responses that are similar but not identical to those of the upper lens eye. At light-off the pacemaker is inhibited and falls below basis activity (C). Red lines indicate pacemaker frequencies when individual eyes are stimulated and black lines indicate the basis frequency (dark reference). The inserts indicate the eye type being stimulated.

of putative precursor cells could indicate a high turnover rate in the rhopalial nervous system as seen in the CNS of Hydra (Koizumi et al., 1992). The bulk of the rest of the rhopalium is filled by the rhopalial neuropil, which will be accounted for in details later in this review.

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5.2. Eye types and visual properties One of the characteristics of all known cubomedusae is the presence of multiple eye types on each rhopalium. They differ not only in size, shape, and orientation but also in complexity, number of receptor types, physiology, and probably also in the type of photopigment they use. The differences are mostly seen between the different eye types but when examined in details differences within each type are also seen between species. 5.2.1. Cubozoan lens eyes The most advanced of the four types are the two unpaired lens eyes. They are found along the midline of the rhopalium and are the largest of the eye types. In Carybdea alata, which has the largest eyes, the lower lens reaches a diameter of 800 mm. The diameters of the lens eyes of our model species, T. cystophora from the Caribbean, are 150 and 250 mm for the upper and lower lens eyes, respectively (Fig. 2.6A). The gross morphology of these eyes is similar in all the examined species and they also have similar cellular components. They have a thin cornea made of monociliated epithelial cells, a cellular lens that may be oval or spherical, a thin vitreous space, and a hemisphere-shaped everse retina with pigmented photoreceptors of the ciliary type (Fig. 2.6; Laska and Hu¨ndgen, 1982; O’Connor et al., 2009; Yamasu and Yoshida, 1976). Further, in the two species examined in this respect, T. cystophora and C. bronzie, the frontal part of the pigment screen in the lower lens eye is specialized as an adjustable iris (Nilsson et al., 2005; O’Connor et al., 2009). This structural complexity exceeds that of all other cnidarian photosensitive structures and has a striking resemblance to what is seen in the camera-type eyes of vertebrates and cephalopods. The obvious question is what image quality is produced by these eyes, and in that respect there seems to be some difference between the different species of cubomedusae. One of the surprising findings is that the lenses of T. cystophora are of very high quality. When examined with interference microscopy they were found to have graded refractive indices creating close to an aberration free image especially for the upper lens eye lens (Nilsson et al., 2005). Surprisingly, the focal length exceeds the distance to the retina by far, resulting in unfocused images with poor spatial resolution. Depending on the eye, the area of the retina, and whether the pupil is opened or closed (for the lower lens eye) the half-width of the receptive fields of individual receptors varies between 10 and 25 (Nilsson et al., 2005). Interestingly, the lenses together with the orientation of the receptor outer segments creates asymmetric receptive fields not unlike receptive fields of motion detectors in brain centers of other animals (Suder et al., 2002). In C. bronzie, which is the only other species where it has been examined, the spatial resolution is even worse. Here the lower lens eye has a maximum spatial resolution of 20 and 50 depending on whether the pupil

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is closed or open, respectively (O’Connor et al., 2009). The lens in the upper lens eye of C. bronzie seems to have little if any optical function and spatial resolution can only be produced in this eye through selective shading by the screening pigment (O’Connor et al., 2009). As mentioned above, the cubozoan rhopalia have the special feature that they are always oriented in the same way relative to vertical. This means that the upper lens eye always looks straight upwards and the lower lens eyes looks downward at an angle of about 40 . With a total visual field matching Snell’s window the upper lens eye can monitor the terrestrial world, and in T. cystophora this ability is used for navigational purposes (Garm et al., in preparation). It is likely that this is also the case for C. bronzie (O’Connor et al., 2009). C. alata has the largest lower lens eye of all examined cubomedusae, about 800 mm in diameter in a fully grown specimen, but our preliminary data indicate that the lens is not equally large. This would imply a longer distance between the lens and the photoreceptor outer segments and possibly a better-focused image with higher resolution. Thorough optical and morphological examinations of the eyes in this species are needed to test this hypothesis. The ecology of C. alata is largely unknown and therefore we cannot speculate about the possible need of higher visual acuity in this species. The physiology of the lens eyes has been examined in a few species using ERG recordings providing information on spectral and temporal properties of these eyes. Spectral sensitivity data have been obtained from T. cystophora, Carybdea marsupialis, and C. bronzie (Coates et al., 2006; Ekstro¨m et al., 2008; Garm et al., 2007a). In all three cases, the spectral sensitivity curve matches the presences of a single opsin implying that the visual systems in the lens eyes are monochromatic and hence color blind. For T. cystophora and C. bronzie the data also indicate that the photoreceptors of the upper and lower lens eyes contain the same opsin. The peak sensitivity for both eyes in both species was close to 500 nm (Garm et al., 2007a). The spectral sensitivity of the lower lens eye of C. marsupialis is shifted toward the blue part of the spectrum and peaks at about 485 nm (Ekstro¨m et al., 2008). The temporal resolution of the lens eye receptors has been investigated both indirectly (half-width and time to peak of the response) and directly (flicker fusion frequency—fff ) from the ERG recordings. The receptors were found to be slow with minimum half-widths of approximately 30 and 55 ms for T. cystophora and C. bronzie, respectively (Garm et al., 2007a), which is comparable to slow and night-active insects (Howard et al., 1984). These results have recently been supported by measurements of the fff of the lens eyes of T. cystophora (O’Connor et al., 2010b). Here it is found that the upper lens eye had a fff of 10 Hz and the lower lens eye 8 Hz. The eyes also differed in the frequency to which they had the highest sensitivity. These are the first physiological results showing that the photoreceptors of the two lens eyes have different response patterns.

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A prominent feature of the cubomedusan nervous system is the presence of pattern generators in the shape of swim pacemakers that control the swim contractions in a one-to-one manner (Satterlie and Spencer, 1979). We have shown that in T. cystophora this pacemaker system is in turn controlled by the visual input (Garm and Bielecki, 2008). An increase in the ambient light intensity results in a decrease in the pacemaker frequency and vice versa. Interestingly, the pacemaker system is influenced by multiple photosystems in a complex way (Garm and Mori, 2009). Stimulation of the lower lens eye alone causes effects on the pacemaker frequency that are similar, but not identical, to whole rhopalia stimulations (Fig. 2.7). Stimulation of the upper lens eye, the pit ocelli, or the rhopalial neuropil causes effects that are almost opposite to the effects obtained by stimulating the lower lens eye (Garm and Mori, 2009). It is notable that although the responses obtained by stimulation of the upper lens eye, the pit ocelli and the neuropil are in general similar, there were consistent differences in the details of the responses of the different eyes (Fig. 2.7). Besides stressing the complexity of the behavioral control involving the pacemaker system in T. cystophora, our neurophysiological data also provide the first direct evidence that visual input from several eyes and ocelli is processed and integrated within the rhopalial nervous system. Such integration has earlier been suggested from neuroanatomical data (Parkefelt et al., 2005). Further, the pacemaker control is the first evidence of a behavioral control system in cubozoans that involves multiple photosystems. Interestingly, both eyes with spatial resolution as well as the ocelli without spatial resolution and extraocular photoreceptors are involved. Pacemaker control in the hydromedusa P. penicillatus also involves both ocelli and extraocular photoreceptors (Arkett and Spencer, 1986a,b). However, neither of these two systems is understood well enough to explain why several photosystems are involved. Navigation in T. cystophora is the only cubozoan behavior so far known to be supported by the upper lens eye, and it has not been examined whether the pacemaker control is involved in this behavior (Garm et al., in preparation). So far, no behavior has been connected with the pit ocelli (see below for more details). 5.2.2. Cubozoan ocelli Compared to the lens eyes much less is known about the two ocelli types in cubomedusae, and they have only been examined in structural detail in T. cystophora, C. bronzie, and Tamoya bursaria (Garm et al., 2008; O’Connor et al., 2009; Yamasu and Yoshida, 1976). As mentioned earlier the pit ocelli are rather simple ocelli composed of pigmented photoreceptors only. Although smaller, the photoreceptors are very similar to those from the lens eyes. The cell bodies are only 1–2 mm in diameter and the ciliary outer segment less than 10 mm long (Garm et al., 2008). They form a hemisphereshaped pigment pit with an outer diameter of 30–50 mm. The opening of the

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pit is typically 10–20 mm and is covered by a layer of epithelial cells. One interesting aspect of these small ocelli is their large number of receptor cells. In T. cystophora, the pit ocelli contain about 300 photoreceptors with an average size of the outer segment of only 3 mm3 (Garm et al., 2008). As these eyes do not have a lens they have very poor spatial resolution, if any at all, so visual acuity cannot explain the high number of receptors. One explanation could be that these multiple parallel visual channels are averaged to remove noise and that the pit ocelli are used to make precise intensity measurements. Another explanation could be that groups of photoreceptors perceive different visual information such as different parts of the color spectrum. The pit ocelli of C. alata differ from those of the other examined species, and may give a clue to their function. In this species the photoreceptors contain very little screening pigment and therefore the eyes are almost colorless (Fig. 2.6D). This means that in C. alata the pit ocelli have no spatial resolution since light from any direction can access all of the receptors. In T. cystophora the pit ocelli look upwards and 25 laterally, and with their visual field having a half-width of 60 (Garm et al., 2008) they receive most of their photons from Snell’s window, like the upper lens eye. It is not known what visual information the pit ocelli respond to, or what behavioral control they are engaged in. Besides their morphology, the only evidence of their light sensitivity is their light-induced influence on the pacemaker system (Garm and Mori, 2009). Since this influence depends on the light intensity, it offers a possibility to examine the spectral sensitivity of the receptors and thereby to infer what type of photopigment they use. Concerning the photopigment it is noteworthy that molecular and immunocytochemical studies strongly suggest that it differs from that of the lens eyes (Ekstro¨m et al., 2008; Koyanagi et al., 2008; O’Connor et al., 2010a). The slit ocelli are surprisingly complex in at least some box jellyfish species. In T. cystophora, they consist of four cell types of which two are photoreceptors (Garm et al., 2008; see Fig. 2.6C). Most of the retina is composed of pigmented photoreceptors that are ultrastructurally identical to those of the pit ocelli. However, in the central part of the eye, a group of unpigmented receptors are situated on one side of the pigment slit. Even though we know nothing about their physiology, this distinct arrangement of morphologically different photoreceptor types strongly suggests that each receptor type constitutes a separate photosystem. In total, each slit ocellus of T. cystophora comprises about 250 photoreceptors, which again is a surprisingly large number considering the small size of these ocelli (Garm et al., 2008). As in the pit ocelli, a large number of photoreceptors could average out noise or make possible several visual channels. The two other cells types are nonsensory pigment cells that make up the upper part of the pigment screen, and vitreous cells that fill the slit between the epithelial cells and the receptors (Garm et al., 2008). The vitreous cells are filled by aggregations of 0.5–2 mm large vitreous vesicles with varying

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refractive index. Our measurements show that they have little optical power, which only cause a weak scattering of the light (Garm et al., 2008). We do not know their function, but it is possible that the vitreous cells perform spectral filtering and remove UV light. Interestingly, vitreous cells are not present in the slit ocelli of all cubozoan species. They are found also in C. alata (Fig. 2.6E) and in C. marsupialis (Satterlie, 2002) but are absent in C. bronzie (O’Connor et al., 2009). In T. cystophora, the outer segments of the photoreceptors in the slit ocelli are restricted to the innermost part of the slit and arranged in layers. Each of the layers is shaded differently by the pigment screen, which leaves the possibility of spatial resolution but only along the vertical plane (Garm et al., 2008). The maximum possible spatial resolution is 15–20 , which is similar to that of the lens eyes. Still, whether the slit ocelli convey such spatial information is unknown. In transverse section the pigment screen is asymmetric, which has the effect that the visual field is directed downwards. As for the pit ocelli we have tried to obtain ERG recordings from the slit ocelli but without much success. When suction electrodes are attached to the slit ocelli the recorded signals are highly variable and do not correlate with the light stimuli in a reproducible way. However, one interesting result has emerged from these recordings. Responses to stimulations of the lower lens eye can be recorded at the slit ocelli, indicating a neural connection between these two eye types (A. Garm, unpublished results).

5.3. Presence of different photoreceptor types? The differentiated photoreceptor cells in the rhopalia of cubomedusae are of the ciliary type, as judged from their ultrastructural morphology. The surface of the ciliary outer segment is enlarged by numerous microvilli that emanate from the ciliary sheath. The microvilli are not regularly arranged as in the rhabdome of a compound eye, although small sets of adjacent microvilli may form groups with parallel organization (Laska and Hu¨ndgen, 1982; Yamasu and Yoshida, 1976). Martin’s (2002) claim that the surface enlargement of the outer segment is in the form of stacks of lamellae ‘‘similar to the stacks of membranes found in vertebrate rods’’ is not supported by her electron micrographs (Fig. 2.5C and D) or by any other available data. Retinal ultrastructure has been investigated in four species, and different conclusions have been drawn with respect to the number of photoreceptor (sub)types. In their study of T. bursaria, Yamasu and Yoshida (1976) described one type of photoreceptor in the lower lens eye, the so-called pigmented sensory cells. These are interspersed with long pigment cells, which have surface enlargements by microvilli but bear no ciliary specializations. Similarly, only a single photoreceptor type was identified in C. marsupialis (Martin, 2002) and C. bronzie (O’Connor et al., 2009). In contrast, Laska and Hu¨ndgen (1982) described no less than three types of photoreceptors in the

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lens eyes of T. cystophora on the basis of differences in the morphology of their sensory cilium and microvillar organization. All three types were observed in the lower lens eye: long pigment cells, pyramid cells, and prism cells, whereas the upper lens eye contained only pyramid cells and prism cells. In addition, the pit ocelli and slit ocelli contained a fourth sensory cell type. Laska and Hu¨ndgen did not recognize any specialized pigment cells; their ‘‘long pigment cells’’ were ciliary and considered photosensory. So far, the ultrastructural studies have not provided strong evidence for clearly defined morphological photoreceptor (sub)types in box jellyfish. The four types described by Laska and Hu¨ndgen (1982) may well represent photoreceptors with easily distinguished characters in a continuum of size and morphological characters that are restricted by different biophysical constraints in the different eyes. Our own data support the interpretation that there is one basic morphological type of photoreceptors in the lens eyes and pit ocelli of box jellyfish (Fig. 2.8). This type is found also in the slit ocelli, which contain an additional type of photoreceptor (Garm et al., 2008). This does not discredit the possibility that there are photoreceptor subtypes with different physiological characteristics, which we in fact have shown to be the case in T. cystophora (O’Connor et al., 2010b). We have already described that there are photoreceptors without pigment granules, and photoreceptor cell types can also be distinguished by their chemical signature. The most compelling evidence for different types would be a selective expression of different photopigments. Again, available data are contradictory. The first attempts to identify multiple photoreceptor types by immunological methods, using antibodies against specific (zebrafish) c-opsins and (Drosophila) r-opsins, showed crossreactions with putative opsin molecules in the eyes of C. marsupialis. Antibodies against zebrafish rhodopsin, green opsin, blue opsin, and UV opsin labeled photoreceptor outer segments and/or cell bodies in the upper and lower lens eyes (Martin, 2004). There was no apparent differential distribution of immunolabeling in the retina, which would have indicated the presence of different photoreceptor subtypes within an eye. However, the morphology of the UV opsin-immunoreactive photoreceptors differed between the upper and lower lens eyes (Martin, 2004), corroborating the earlier ultrastructural studies in T. cystophora (Laska and Hu¨ndgen, 1982). On the basis of these results, Martin (2004) suggested that box jellyfish might have color vision. In contrast to Martin’s interpretation, subsequent studies strongly suggest that box jellyfish lens eyes operate by a single opsin only, and, hence, are color blind. The physiological data are reviewed above, and these are supported by immunofluorescence and molecular biological studies that indicate that the photoreceptors of the lens eyes contain only one photopigment (Ekstro¨m et al., 2008; Koyanagi et al., 2008; Kozmic et al., 2008a; O’Connor et al., 2010a). Intriguingly, though, the data are conflicting as to the type of opsin-based phototransduction. Koyanagi et al. (2008) report an

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A

B

PG

CR M

CR

N N M

Figure 2.8 Photoreceptor types in cubomedusae. All the photoreceptors in the lens eyes and the pit ocelli are of the same morphological type even though they may vary in size, especially the outer segments. They are ciliary with the ciliary membrane extending in numerous microvilli. The apical part of the cell is filled with pigment granules (PG) functioning as the shielding pigment of the eyes. Between the pigment granules and the basal nucleus (N), there is a layer of densely packed mitochondria (M). The ciliary rootlet (CR) is normally strong and extends all the way to the nucleus (A). Most of the receptors in the slit ocelli are of the just described type but there is an additional type. This type of photoreceptor has no pigment and only few mitochondria. The cilium retains the normal ciliary structure including the 9  2 ring formation of microtubules for a long distance before it produces microvilli. Their cell bodies lie in a cluster separate from the other photoreceptors of the slit ocelli (B).

opsin–Gs–cAMP phototransduction cascade in Carybdea rastonii, whereas Kozmic et al. (2008a) show evidence for a ciliary opsin–transducin–cGMP pathway in T. cystophora. Even though the lens eyes may utilize only one photopigment, it has been consistently observed that the pit and slit ocelli do not express this photopigment. Indeed, there is presently no indication as to what photopigments are utilized by these ocelli. Thus, the rhopalium contains more than one type of photoreceptor cell. The pit ocelli have visual fields that overlap that of the upper lens eye and the slit ocelli have visual fields that overlap that of the lower lens eye. Since the lens eyes and pigment cup eyes also obviously utilize different photopigments, we postulate that each eye type constitute at least one separate photosystem. The pit ocelli and the upper lens eye, and the slit ocelli and the lower lens eye, respectively, may extract different visual information from a common visual field. The minimum number of photoreceptor types required for this is two: one in the pit and slit ocelli, another in the lens eyes.

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5.4. Neuronal organization It was early realized that the rhopalium of cubomedusae contains the most complex neuronal system in cnidarians (Claus, 1878). However, it is only recently that the different neuronal components of this visual organ have begun to be systematically characterized. On the basis of ultrastructural studies and classical histological descriptions, Laska and Hu¨ndgen (1982, 1984) presented a first model of the signaling pathway from photoreceptor cells to epitheliomuscular cells in T. cystophora. In this model, photoreceptor cells would extend long neurites to a rhopalial ganglion in the upper part of the rhopalium. The rhopalial ganglion cells, in their turn, would emit neurites into the rhopalial stalk where they gather in a rhopalial nerve. The rhopalial nerve neurites would terminate on neurons in the ring nerve, and all synaptic connections would be bidirectional (Laska and Hu¨ndgen, 1984). This was a plausible model, but subsequent studies have not corroborated their interpretation. Immunochemical studies of neurons and photoreceptors, as well as electron microscopic analyses, have given a completely different view of the neuronal circuitry of the rhopalium (Fig. 2.9). The most obvious characteristic is its bilaterally symmetric nature, which likely reflects the bilaterally symmetric arrangement of the paired ocelli (Parkefelt et al., 2005). Two major subsystems connect the pit ocelli and the slit ocelli, respectively, with a major neuropil region in the posterior wall (the backside of the rhopalium) below the stalk base (Parkefelt et al., 2005). A prominent system of RFamide-immunoreactive neurons forms a horseshoe-shaped population around the base of the rhopalial stalk in the posterior wall (Parkefelt and Ekstro¨m, 2009; Satterlie, 2002; Skogh et al., 2006). LWamide-immunoreactive (Plickert and Schneider, 2004) and arginine vasotocin (AVT)-immunoreactive (Parkefelt and Ekstro¨m, 2010) neurons are found within this population. Another group of AVT-immunoreactive neurons gives rise to a dense neurite plexus around the lower lens eye; these neurons are likely postsynaptic to lower lens eye photoreceptors (Parkefelt and Ekstro¨m, 2010). RFamide-immunoreactive neurites also surround the lower lens eye retina, but the cell bodies that give rise to this innervation have not been located (Parkefelt and Ekstro¨m, 2010). In addition, the posterior and lateral walls of the rhopalium contain a large number of neurons with different chemical signatures (P. Ekstro¨m, unpublished observations), which possess an apical neurite with a putative sensory cilium that penetrate the ectoderm, and a basal neurite that branches in a common neuropil region. Finally, immunofluorescence labeling with antibodies against tubulins reveals additional neuronal populations, notably large neurons associated with the pit ocelli and neurites that surround the two lens eyes (Fig. 2.9). A homogeneous population of large neuronal cell bodies—the giant neurons (Skogh et al., 2006)—is easily distinguished in histological sections of the upper part of the rhopalium. However, this population is

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A Stalk

C

POc

ULE

Melanopsin Syntaxin PCNA

LLE

SOc Crystal B

Figure 2.9 The rhopalial nervous system. The six eyes and ocelli found on the cubomedusan rhopalium are supported by a complex nervous system, the rhopalial nervous system (RNS). One of the characteristic features of the RNS is that it is bilaterally symmetric. Using different antibodies several subsystems have been found in the RNS. (A) Top and side view of a rhopalium from Tripedalia cystophora showing subsystems immunoreactive with antibodies against syntaxin (green) and proliferating cell nuclear antigen (PCNA, purple/red). (B) Top and side view of a rhopalium from T. cystophora showing subsystems immunoreactive with antibodies against mouse melanopsin. (C) All three subsystems together. Spheres and capsules indicate clusters of cell bodies and tubes indicate bundles of neurites. Abbreviations: POc, pit ocellus; SOc, slit ocellus; LLE, lower lens eye; ULE, upper lens eye. See on-line version for color figure.

heterogeneous with respect to chemical signature. It contains RFamidepositive as well as RFamide-negative neurons, alongside tubulin-positive and tubulin-negative neurons. The tubulin-positive neurons extend neurites toward the base of the rhopalial stalk. Thus, the rhopalium contains a surprisingly complex neuronal apparatus. Are all neurons involved in processing of visual signals? We believe not. We have so far not identified any direct connections between neurons in the lateral and posterior walls and any of the eyes. Rather, these neurons—which possess apical neurites that penetrate the ectoderm and may represent dendrites with sensory specializations—may be mechanosensory and/or chemosensory neurons (Parkefelt and Ekstro¨m, 2010; Skogh et al., 2006). Still, this leaves us with an impressive set of neurons associated with the different eyes, and with one population of RFamide-immunoreactive neurons that do not have the morphological signature of putative sensory neurons. So far, we have

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identified two neuronal groups directly associated with the pit ocelli, two with the slit ocelli, two associated with the upper lens eye, and one associated with the lower lens eye. However, our data clearly indicate that there are more systems involved, and we have so far not identified any good candidates for second-order neurons for the upper lens eye. We discussed above that the upper lens eye and the pit ocelli, and the lower lens eye and the slit ocelli, respectively, collect information from vastly overlapping visual fields. Further, we hypothesized that each eye type constitutes a photosystem, filtering specific information from its visual field. For each visual field, the ocelli could register luminosity (in a given direction relative the animal’s position) whereas the lens eyes could respond to pattern. Is this hypothesis supported by the neuronal organization of the rhopalia? To address this it is important first to note that the pacemaker signal that drives the medusa’s swimming contractions is one of the major neural outputs of the rhopalium (Satterlie, 2002). Laska and Hu¨ndgen (1984) suggested that the neurons of the rhopalial ganglion constitute the output neurons of the rhopalium, that is, the pacemaker neurons. However, the pacemaker neurons have never been unequivocally identified. Ablation experiments have shown that they are located in the upper part of the rhopalium (Yatsu, 1917), and the location of the giant neurons suggest that they may constitute the pacemaker. It is not clear how the complex signals generated by the pacemaker neurons (Garm and Bielecki, 2008) are transmitted to the rhopalial nerve. We have so far no ultrastructural evidence that would verify the observation of Laska and Hu¨ndgen (1984) that neurites from giant neurons—or any other neurons in the upper part of the rhopalium—continue into the rhopalial nerve (A. Garm, unpublished observations). Our hypothesis is that signals from the pacemaker neurons are transmitted to the ring nerve via synapses in the rhopalium, by neurons close to the stalk base and in the rhopalial neuropil, or in the rhopalial nerve (Garm et al., 2006). The neurons with synaptic contacts with the rhopalial nerve are located in a region that corresponds to that of the giant neurons, but also to part of the RFamide-immunoreactive population and the AVT-immunoreactive neurons close to the stalk (A. Garm, unpublished observations). Interestingly, neither of these cell groups has been shown to possess direct contacts with any of the eyes. This suggests that all eyes transmit signals to the pacemaker via second-order interneurons, although subpopulations of photoreceptors in the pit and slit ocelli have long axons that project directly to the neuropil surrounding the stalk base (Parkefelt et al., 2005), and some of the visual signals are transmitted directly to the ring nerve, bypassing the pacemaker neurons (Garm et al., 2006). The secondorder interneurons could serve the purpose of integrating signals from the upper lens eye and pit ocelli, and lower lens eye and slit ocelli, respectively. So far, our data suggest that each eye type uses a separate channel to the pacemaker and that both eyes and the pit ocelli transmit signals to the

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pacemaker via second-order interneurons. However, there may be extensive crosstalk between channels in the extensive neuropil region, and some photoreceptors in the pit ocelli may connect directly to the pacemaker (Parkefelt et al., 2005). Thus, the definite characterization of the neural information channels in the visual system of the rhopalium requires ultrastructural studies of the synaptology of individual identified photoreceptors and neurons.

6. Multiple Opsins in Cnidarians—Multiple Photosystems? Except from the cryptochrome-based light response in some anthozoans all data suggest that photosensitivity in cnidarians, ocular as well as extraocular, is opsin-based. The data come from electrophysiological, immunocytochemical, and molecular studies. Some of the first evidence stems from ERGs from the ocelli of the hydromedusae S. tubulosa and P. penicillatus (Weber, 1982a,b). Using color filters the spectral sensitivity of the ocelli was examined and the results are in good concordance with the existing opsin templates (Govardovskii et al., 2000; Stavenga et al., 1993). Also using ERGs we have shown that the lens eyes of cubomedusae probably also utilize opsins (Coates et al., 2006; Ekstro¨m et al., 2008; Garm et al., 2007a) and this has been supported in the Australian species C. bronzie by direct measurements of the absorbance spectrum of the photoreceptors using microspectrophotometry (O’Connor et al., 2010a). As mentioned above (see Section 5.3), strong indications for the presence of opsins have also been obtained in immunofluorescence studies with antibodies against zebrafish and squid opsins (Ekstro¨m et al., 2008; Martin, 2002; Musio et al., 2001). In the last years, a number of opsin and opsin-like genes have been identified in different cnidarians. Notably, the hydromedusa C. radiatum express no less than 18 opsin genes, some of which are eye-specific. Podocoryne carnea lacks eyes but expresses two opsin genes. Hydra magnipapillata, a sessile hydropolyp with solely epidermal photosensitivity, and the sea anemone Nematostella vectensis, expresses 63 and 31 opsin genes, respectively. It is not known if all (or which) opsins form functional photopigments, but all of the Nematostella opsins and 58 of the Hydra opsins have the conserved lysine for retinal binding (Suga et al., 2008). Functional photopigments have been identified, by heterologous expression, from the cubozoans T. cystophora (Kozmic et al., 2008a) and C. rastonii (Koyanagi et al., 2008) but not from other cnidarians. Available opsin sequences indicate that cnidarians may possess at least two types of opsins: one that is phylogenetically ancient (cnidops; Plachetzki et al., 2007) and one ciliary opsin type that forms a sister group to the c-opsins. Plachetzki et al. (2007) suggested that the ancestral opsin split in two lineages:

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one that gave rise to all c-opsins and one where cnidops is the sister group to r-opsins and RGR/Go-coupled opsins. Soon after, Suga et al. (2008) corroborated these findings; they described a group of basal opsins found only in the anthozoan N. vectensis that may belong to the cnidops family. They also found a vast number of ciliary opsin genes in hydrozoans, and three Nematostella opsin genes, which together form a sister group to the c-opsins. Then, Kozmic et al. (2008a) reported the presence of a ciliary opsin in the box jellyfish T. cystophora, as well as evidence for components of the canonical opsin–transducin–cGMP phototransduction pathway of vertebrate c-opsins. Immediately afterward, Koyanagi et al. (2008) described a new phototransduction pathway in another box jellyfish species, C. rastonii. The opsin grouped with the cnidops, and utilized a previously unknown opsin–Gs–cAMP signaling cascade. In both species, the opsin was strictly localized to the camera-type lens eyes, although the distribution pattern in the eyes differed between the species. The data suggest that box jellyfish may possess both cnidops and ciliary opsin(s), and raise the intriguing possibility that both types may be found in the same retinas. Many opsin genes are widely expressed in the different body parts of hydromedusae, and only a subset are specifically expressed in the eyes (Suga et al., 2008). This raises the question whether all represent functional photopigments and what physiological mechanisms they influence. Photosensitivity in non-neural tissue is well known from many bilaterians; compelling examples are the peripheral circadian oscillators in Drosophila (Glossop and Hardin, 2002) and zebrafish (Kaneko et al., 2006), light-sensitive dermal pigment cells (Isoldi et al., 2005), and the intrinsically photosensitive iris (Tu et al., 2004). Cryptochromes are the functional photopigments in the peripheral oscillators of Drosophila (Glossop and Hardin, 2002), and possibly in the chick iris (Tu et al., 2004). Nonvisual opsins like encephalopsin, teleost multiple tissue opsin, and melanopsin may be expressed in non-neural tissue in vertebrates (Peirson et al., 2009). The large number of cnidarian opsin genes present in a given species (Suga et al., 2008) may reflect that, in a small and transparent animal, many physiological functions are directly regulated by light without any need for neural processing in a visual system. The most obvious example is the entrainment of peripheral circadian oscillators (see above), but changes in irradiance—or light within specific wavelength ranges—may also elicit direct physiological responses like triggering of changes in ciliary or muscular activity ( Je´kely et al., 2008; Kargacin and Detwiler, 1985). Metazoan evolution can be characterized as an increase in number of cell types, and an increase in cell specialization concomitant with a decrease in cell complexity (Arendt et al., 2009). Hence, it is reasonable to assume that cnidarians, which have relatively few cell types, have retained ancestral multifunctionality (encompassing, e.g., photosensitivity) in various cells and tissues. In the absence of structural photoreceptor specializations and/ or neuronal processing of the light responses, such photosensory cells can

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only respond to changes in irradiance within the wavelength range dictated by its photopigment and possible shielding pigments. As discussed above, there is still no evidence that cnidarians have multiple opsins with different spectral sensitivities. However, even expression of the same opsin in different effector cells gives these cells a certain degree of autonomy; they can respond directly to light and independently regulate multiple physiological functions. On the other hand, if opsins with different transduction mechanisms— and possibly different spectral sensitivities—independently regulate multiple physiological functions, they have to be specifically expressed in the different multifunctional photosensory cells. The regulation of restricted expression of opsin genes would then introduce a new level of complexity—or diversity—that relates to the photopigment and phototransduction mechanism employed by the cells. It has been suggested that after the evolutionary divergence of Porifera, there was a wave of gene duplications in the eumetazoan ancestor (Putnam et al., 2007). If so, this might explain the large number of opsin genes found in some cnidarians. We speculate that the duplicated opsin gene copies were ‘‘used’’ for directed expression in different cell types—a type of subfunctionalization (Force et al., 1999; Lynch et al., 2001) and an alternative to genesharing where a duplicated gene obtains a new function (Piatigorsky, 1998). In this way, several cell types could harbor functionally equivalent opsin genes, and respond independently to light. The presence of two basically different types of opsins (Gs–cAMPcoupled cnidops and transducin–cGMP-coupled ciliary opsin) in at least some cnidarians suggests that cnidarians in general do possess multiple photosystems. However, only cubomedusae appear to have multiple visual photosystems. In the other cnidarian classes, photosensitivity and/or photopigments are found in non-neural tissue or in photosensory neurons, that is, in nonvisual photosystems.

7. Conclusion In general, cnidarians do not possess multiple photosystems. Photosensory behavior in anthozoans is conveyed by diffuse non-neuronal photosensitivity. In hydroid polyps, also a diffuse—possibly neuronal— photosensory system drives rhythmic body movements. Among cnidarian medusae—jellyfish—some hydromedusae may possess more than one photosensory system, but photic stimulation of the ocelli elicit the same responses as stimulation of the intrinsically photosensory nervous system (Arkett and Spencer, 1986a,b; Satterlie, 1985a). Scyphomedusae may be regarded as possessing multiple photosystems. In some species, each

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rhopalium carries two ocelli that perceive light from different directions. These ocelli may direct vertical phototaxis and horizontal migration. Cubomedusae provide the best evidence for the evolution of multiple photosystems in cnidarians. Their visual organs, the rhopalia, contain different eye types. Two eye types—the upper lens eye and the pit ocelli— look upwards with overlapping visual fields, whereas two types—the lower lens eye and the slit ocelli—look downward with overlapping visual fields. The lens eyes have the capacity for spatial resolution and are placed along the rhopalial midline, whereas the paired pit ocelli and slit ocelli probably function as luminosity detectors. Although the lens eyes seem unable of conveying color vision, they may contain more than one type of photoreceptor cell, and the slit and pit ocelli clearly express other photopigment(s) than the lens eyes. Finally, the most compelling evidence for the existence of multiple photosystems is our observations that the signals from the different eyes have differential effects on the activity of the rhopalial pacemaker neurons, and may elicit differential swimming behavior. The multitude of opsin genes expressed in different tissues awaits experimental assessment of their functions; presently, they serve as an indication of yet more numerous photosystems. They also suggest that cnidarians have retained the ancestral animal photoreceptor types, or rather photoreceptors with ‘‘mixed characters’’ in the sense that various effector cells with different functions may be intrinsically photosensitive. Taken together with our evidence for the presence of multiple photosystems in cubomedusae, available data suggest that multiple photosystems had evolved already in early eumetazoans and that their original level of organization was that found in extant cnidarians, that is, discrete sets of special-purpose eyes and/or photosensory cells, rather than combinations of photosystems in general-purpose eyes like those found in, for example, chordates and arthropods.

ACKNOWLEDGMENTS The authors greatly appreciate the superb technical assistance offered by Carina Rasmussen, Rita Walle´n, and Eva Landgren (Lund University). We would also like to thank Poul Bennekou (University of Copenhagen) for the generosity with his equipment. The study was supported by the Swedish Research Council (grant to D.-E. Nilsson, No. 621-2005-2909) and the Danish Research Council, FNU (grant to A. Garm, No. 272-07-0163).

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Membrane Trafficking in Protozoa: SNARE Proteins, H+-ATPase, Actin, and Other Key Players in Ciliates Helmut Plattner Contents 1. Introduction 1.1. State of discussion with higher eukaryotes 1.2. State of research with ciliates 1.3. Paramecium and Tetrahymena as model systems for membrane trafficking 2. Factors Involved in the Regulation of Vesicle Trafficking 2.1. Identifying SNAREs—Criteria and methodology 2.2. Small GTP-binding proteins/GTPases and their modulators 2.3. Actin 2.4. H+-ATPase 3. Features of SNAREs 3.1. Characteristics of Paramecium SNAREs 3.2. Role of the SNARE-specific chaperone, NSF 3.3. ‘‘SNAREs and Co’’—targeting of vesicle traffic from the ER to the Golgi apparatus and beyond 4. Exocytosis and Endocytosis 4.1. Exo- and endocytosis in general 4.2. Constitutive endocytosis and exocytosis in ciliates 4.3. Stimulated exocytosis and exocytosis-coupled endocytosis in ciliates 5. Possible SNARE Arrangement in Microdomains and Membrane Fusion 5.1. General aspects 5.2. Aspects concerning ciliates 6. Phagocytosis 6.1. Phagocytosis in ciliates 6.2. Involvement of actin in phagocytotic cycle of ciliates

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Department of Biology, University of Konstanz, Konstanz, Germany International Review of Cell and Molecular Biology, Volume 280 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)80003-6

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2010 Elsevier Inc. All rights reserved.

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6.3. Role of H+-ATPase, SNAREs, and G-proteins in phagocytotic cycle of ciliates 6.4. Autophagy 7. Calcium-Binding Proteins and Calcium Sensors 7.1. Comparison of Ca2þ-signaling in ciliates with other cells 7.2. Synaptotagmin as a Ca2+-sensor 7.3. Calcium and calcium sensors in ciliates 8. Additional Aspects of Vesicle Trafficking 8.1. Guidance and support by microtubules 8.2. Additional potential key players 8.3. Pharmacology of vesicle trafficking 9. Emerging Aspects of Vesicle Trafficking in Ciliates 9.1. Contractile vacuole complex 9.2. SNAREs and ciliary function 9.3. Cytokinesis 10. Concluding Remarks Acknowledgments References

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Abstract Due to their well-defined pathways of vesicle trafficking and manyfold mutants ciliates have served as good model systems. Further studies required the development of databases, now available for Paramecium and Tetrahymena. A variety of key players have been identified and characterized based on BLAST search, domain analysis, localization, and gene-silencing studies. They include NSF (N-ethylmaleimide sensitive factor), SNAREs (soluble NSF attachment protein [SNAP] receptors), the Hþ-ATPase (V-ATPase) and actin, while Arf (ADP-ribosylation factor) and Rab-type small GTPases, COPs (coatamer proteins) and many others remain to be elucidated. The number of SNAREs, Hþ-ATPase subunits, and actins ever found within one cell type are unexpectedly high and most of the manifold vesicle types seem to be endowed with specific molecular components pertinent to trafficking. As in higher eukaryotes, multifactorial targeting likely occurs. It appears that, in parallel to higher organisms, ciliates have evolved a similar structural and molecular complexity of vesicle trafficking. Key Words: Actin, Ciliate, Hþ-ATPase, Membrane, Paramecium, SNAREs Tetrahymena, Trafficking. ß 2010 Elsevier Inc.

1. Introduction In 1997, Hutton (1997) stated ‘‘it will be intriguing to learn whether homologues exist in these organisms [the ciliates] of the syntaxin, SNAPs, synaptobrevin, synaptotagmin, or other molecules, which have been

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implicated in synaptic vesicle docking and exocytosis. . .’’ Now, detailed answers to many of these questions, and to some additional ones, can be presented.

1.1. State of discussion with higher eukaryotes Intracellular vesicle trafficking is governed by multiple molecular components and presumably the latest common eukaryotic ancestor was already endowed with a multitude of them (Dacks and Field, 2007). Key players are SNAREs, that is, soluble N-ethylmaleimide attachment protein (SNAP) receptors, small monomeric GTP-binding proteins (GTPases, G-proteins), the vacuolar type Hþ-ATPase (V-ATPase), COP (coatamer or coat protein), and clathrin-type cytosolic membrane coats as well as elements of the cytoskeleton, including microtubules and filamentous actin (F-actin, microfilaments). In addition, many more proteins, some with modulatory or auxiliary function contribute to vesicle trafficking. Specific small GTPases of types Rab and Arf (ADP-ribosylation factor) can be assigned to specific sites which also contain specific phosphoinositides (Behnia and Munro, 2005). Components are exchanged on the way through the cell. This 4D-puzzle has been repeatedly reviewed (Behnia and Munro, 2005; Jackson and Chapman, 2006; Jahn et al., 2003; Malsam et al., 2008; Pfeffer, 2007). Figure 3.1 outlines the interactions of some of the principal molecules engaged in vesicle trafficking, as to be discussed in subsequent sections. SNAREs are crucial for membrane-to-membrane interactions, that is, for docking of a vesicle to a target membrane (v- and t-SNAREs) and for final fusion ( Jackson and Chapman, 2006; Jahn and Scheller, 2006; Jahn et al., 2003; Martens and McMahon, 2008). This became increasingly evident since the pioneer work of J. Rothman’s group from the early 1990s on (Nickel et al., 1999; Rothman, 1994; Rothman and Warren, 1994; So¨llner et al., 1993a,b). Until the early 1990s, other hypotheses, specifically for membrane fusion, have been preferably envisaged and it has been largely questioned whether membrane proteins may play any role at all in membrane interactions leading to fusion. For instance, fusion was explained by Ca2þ-mediated local lipid-phase transitions. In contrast, work with the ciliated protozoan cell, Paramecium tetraurelia had suggested at that early time already a decisive role for membrane-integrated and -associated proteins (Plattner, 1981, 1987, 1989; Vilmart and Plattner, 1983). This concept had been endorsed by numerous mutations in the sequence of the secretory pathway specifically in the ciliate, P. tetraurelia (Beisson et al., 1976, 1980; Bonnemain et al., 1992; Lefort-Tran et al., 1981; Pouphile et al., 1986; Vayssie´ et al., 2000, 2001). It should also be appreciated that such work from the Beisson group, with Jean Cohen and Linda Sperling (CNRS,

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H+-ATPase

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Figure 3.1 Principal mechanisms cooperating during vesicle trafficking, as exemplified by compartments endowed with SNAREs and Hþ-ATPase as well as with interacting F-actin. These components, analyzed mainly in P. tetraurelia and to a smaller extent in T. thermophila, are in the focus of the present review on trafficking in ciliates. The right side of the scheme refers specifically to exocytosis. Sequence from left to right. Acidification: Vesicles possess a set of v- (R-)SNAREs and a Ca2þ-sensor (not yet identified in ciliates) as well as an Hþ-ATPase (bright blue) undergoing conformational change as a consequence of lumenal acidification (as shown in other cell types). Activation: The conformational change of the Hþ-ATPase allows for binding of an Arf-type small GTPase (dark blue) and its activator (red ball)—as shown in other cells, thus allowing for targeting. Tethering: Targeting to an appropriate compartment includes tethering. So far there is only evidence of some tethering effect of F-actin in ciliates, while exocyst (for constitutive exocytosis) and any other potential tethering components have not been clearly identified as yet. Docking: After tethering, docking ensues, involving pairing of the v- (R-) SNARE with the t- (Q-) SNAREs of which for simplicity only one type has been drawn. In Paramecium, we identified R-SNAREs of the type synaptobrevin (yet mainly as longin forms) and Q-SNAREs of the type syntaxin and SNAP-25-LP. As in other systems, in Paramecium only the (majority of the) first two possess a transmembrane domain which is a prerequisite to subsequent membrane fusion. Ca2þ release and influx: This occurs during stimulated exocytosis in response to a stimulus. As found with Paramecium, activation of cortical stores (alveolar sacs, green) causes Ca2þ release which precedes and entails a superimposed Ca2þ-influx (‘‘SOC mechanism’’). Membrane fusion: Increase of the local cortical cytosolic [Ca2þ] activates the system for membrane fusion, provided SNARE zippering has preceded. It produces a membrane continuum, with mixture of the contents (inside the cell) or their release (exocytosis). Regrettably little is known on other key players, such as small G-proteins/ GTPases and their regulators as well as of a Ca2þ sensor in ciliates.

Gif-sur-Yvette, France), served as a nucleation center for the development of the Paramecium genome project. While cytoskeletal elements had been acknowledged early on as important components of intracellular trafficking in many systems, the significance of

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SNAREs, of different vesicle coats, of small GTPases and, most recently, that of the Hþ-ATPase have been recognized only with some delay. Many such details are meanwhile known also from ciliates, mainly Paramecium.

1.2. State of research with ciliates While basic concepts have been detected in other cells, from yeast to mammals, work with ciliates still does not yet cover all these fields. To mention just a few of the regrettable gaps in ciliate cell biology: Apart from their presence in Paramecium (Suchard et al., 1989) and Tetrahymena (Kersting et al., 2003; Leonaritis et al., 2005; Ryals and Kersting, 1999), almost nothing is known from ciliates, for example, on the distribution and turnover of phosphoinositides—another regulation principle known from higher eukaryotes (Behnia and Munro, 2005). Moreover, since the early recognition of a complex family of small GTPases in Paramecium (Fraga and Hinrichsen, 1994; Peterson, 1991) little detailed insight has been achieved. Molecular analysis of COPs is another gap to be filled. In the years since the last review on vesicle trafficking in ciliates (Plattner and Kissmehl, 2003a) many new tools have become available and, thus, enabled the identification of important molecular aspects. This includes the cloning of the macronuclear genome of Paramecium and Tetrahymena, paralleled by key publications (P. tetraurelia: Arnaiz et al., 2007; Aury et al., 2006; Dessen et al., 2001; Zagulski et al., 2004; Tetrahymena thermophila: Coyne et al., 2008; Eisen et al., 2006; Orias, 1998). Databases are accessible as follows: http://www.genoscope.cns.fr/paramecium and http:// paramecium.cgm.cnrs-gif.fr for P. tetraurelia and http://www.ciliate.org/ for T. thermophila, respectively. For Tetrahymena, see also protein database, http://www.tigr.org/tdb/e2k1/ttg/. A database for the fish-pathogenic ciliate species Ichthyophthirius multifilliis is being elaborated (see internet). See also http://www.genenames.org for aspects of gene/protein designation, databases for protein types, and for specific protein domains. In 1987, the first transformation of a Paramecium cell has been performed by microinjection of a cloned gene (Godiska et al., 1987). This was followed by complementation cloning (Haynes et al., 1996; Skouri and Cohen, 1997) and establishment of indexed genomic libraries (P. tetraurelia: Keller and Cohen, 2000; T. thermophila: Hamilton et al., 2006). Posttranscriptional homology-dependent gene silencing (siRNA technology) is possible (P. tetraurelia: Bastin et al., 2001; Galvani and Sperling, 2002; Ruiz et al., 1998; T. thermophila: Chilcoat et al., 2001; Howard-Till and Yao, 2006; Shang et al., 2002). In Paramecium, the mechanism behind may reflect the same principle that mediates faithful elimination of the IES (internal eliminated sequences) when, in an epigenetically controlled process, micronuclear genes are edited for storage in the macronucleus by comparison with the old macronuclear genome (Garnier et al., 2004; Meyer and Cohen, 1999).

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With Tetrahymena, the efficient mass transformation achieved by electroporation (Gaertig et al., 1994) or by DNA-loaded particle bombardment (Cassidy-Hanley et al., 1997), eventually allowing also the production of germline transformants, is of big advantage. With Paramecium transformation of postautogamous cells by macronuclear injection is the rule, but from electroporation and particle bombardment also good results have been reported (Boileau et al., 1999). After adaptation to the specific code, proteins can be expressed as green fluorescent protein (GFP)-fusion proteins (Hauser et al., 2000a). Wherever available, the exploitation of special databases, for example, for SNARE proteins, with the inclusion of all organisms analyzed, proved helpful in the molecular analysis of vesicle trafficking. Every time when we try to assign proteins, identified by molecular biology, to certain subcellular components we realize the importance of previous ultrastructural and functional analyses some of which have been conducted in admirable detail. Examples that were particularly helpful to us along these lines are the painstaking analyses by Richard Allen and his associates on the phagolysosomal and the osmoregulatory system in Paramecium (Allen, RD). http:// www5.pbrc.hawaii.edu/allen/ Ciliates deserve special interest also for practical reasons as they are closely related to important protist groups which in part are animal and plant pathogens. An evolutionary relationship between ciliates and the apicomplexan parasites (Plasmodium, Toxoplasma) becomes increasingly robust in the literature, while this is somewhat less pronounced for heteroconts, such as plant pathogenic oomycetes and the large, nonpathogenic group of brown algae (phaeophyceae, kelp) (Baldauf et al., 2000). Figure 3.1 can serve as a section summary as it presents the most important interaction partners during vesicle trafficking in eukaryotic cells, including SNAREs, Hþ-ATPase, actin, and GTPases, of which the first three have been elucidated to some extent in Paramecium, while information on G-proteins in ciliates is restricted.

1.3. Paramecium and Tetrahymena as model systems for membrane trafficking Ciliates are highly organized cells, particularly with regard to vesicle trafficking, Paramecium being the best analyzed example for the time being (Allen, 1988; Allen and Fok, 2000; Fok and Allen, 1988), followed by Tetrahymena (Frankel, 2000). An outline of the main trafficking pathways is presented in Fig. 3.2. Widely different approaches have been applied particularly to Paramecium. It possesses not only numerous regularly arranged sites for the exocytosis of dense core-secretory vesicles (trichocysts) (Beisson et al., 1976; Plattner and Kissmehl, 2003a, b; Plattner et al., 1973), where exocytosis-coupled endocytosis also takes place (Allen and Fok, 1984a;

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Plattner et al., 1985a), but also for clathrin-mediated constitutive endocytosis via parasomal sacs (Allen et al., 1992). Furthermore, it disposes off welldefined sites for formation of phagosomes (oral cavity, with cytostome and cytopharynx) which, after their transcellular transport (cyclosis), release indigestable materials at the cytoproct (Allen and Wolf, 1974). Tetrahymena cells, displaying a very similar design as its larger counterpart (Frankel, 2000), also have served as a powerful model for some aspects of membrane trafficking (Turkewitz, 2004; Turkewitz et al., 1991, 2000, 2002). Its endophagosomal system (Nilsson and Van Deurs, 1983) appears similar to that in Paramecium, but the latter has been studied in much more depth. A variety of secretory mutants have also been collected from T. thermophila (Bowman and Turkewitz, 2001; Gutie´rrez and Orias, 1992; Melia et al., 1998; Orias et al., 1983; Sauer and Kelly, 1995) with similar disturbances as had been established for P. tetraurelia (Beisson et al., 1976, 1980; Bonnemain et al., 1992; Froissard et al., 2004; Gogendeau et al., 2005; Lefort-Tran et al., 1981; Pouphile et al., 1986; Vayssie´ et al., 2000, 2001). Recently, endocytosis via parasomal sacs has been analyzed in much more depth in Tetrahymena (Elde et al., 2005) than in any other ciliate. Early on, a hypothesis of protein-regulated membrane interactions was derived from work with Paramecium. It was based on a clear-cut ultrastructure of exocytosis sites (Beisson et al., 1976; Plattner et al., 1973), with proteasesensitive freeze-fracture particle aggregates (‘‘rosettes’’) in the membrane (Vilmart and Plattner, 1983) whose assembly is under genetic control (Beisson et al., 1976) and that are dispersed during synchronous exocytosis induction (Knoll et al., 1991a; Plattner, 1974). In Paramecium and Tetrahymena, some other fusion processes are also rather clearly defined, though not to the same extent as exocytosis sites.

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Figure 3.2 Main trafficking pathways in ciliates. (A) Three main vesicle trafficking pathways in ciliates, as analyzed mainly with Paramecium (to which the scheme refers), but to a considerable extent also with Tetrahymena. Green: exo-endocytotic pathways, mainly based on cited work with Paramecium (by J. Beisson and her then associates and by the present author and his coworkers) as well as with Tetrahymena (by A. Turkewitz). The general trafficking scheme is based on a figure by Kissmehl et al. (2007); therein the part concerning phago-lysosomal components (red) is based mainly on cited work with Paramecium (by R. Allen and A.K. Fok and their collaborators). Yellow: Unexpectedly, in the cited work on SNAREs, we found evidence of vivid trafficking in the contractile vacuole/osmoregulatory system of Paramecium. Abbreviations: a, ampulla; as, acidosomes; ci, cilia; cp, cytoproct; cv, contractile vacuole; ds, decorated spongiome; dv, discoidal vesicles; ee, early endosomes; er, endoplasmic reticulum; fv, food vacuole; ga, Golgi apparatus; gh, ‘‘ghosts’’ (from trichocyst release); oc, oral cavity; pm, plasmamembrane; ps, parasomal sacs; rv, recycling vesicles; sm, smooth spongiome; tr, trichocyst; trpc, trichocyst precursors. (B) The three main trafficking pathways depicted in Fig. 3.2A are shown here in more detail, each pathway with a remarkable number of membrane interactions by fusion and fission. Based on the scheme by Plattner and Kissmehl (2003a).

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During cyclosis of a phagosome—the ‘‘food vacuole’’ serving digestion of food bacteria—several defined fusion/fission processes occur (Allen and Fok, 2000; Fok and Allen, 1988, 1990). In Paramecium, it has been shown that, after a nascent phagosome has pinched off, the first step is acidification by fusion with acidosomes (Allen and Fok, 1983a), followed by fusion with lysosomes and endocytotic vesicles (Allen and Fok, 2000; Fok and Allen, 1988, 1990). In addition, lysosomal membranes and enzymes are recycled (Allen and Fok, 1984b). Furthermore, two other sets of vesicles are recycled back to the nascent phagosome. First, pieces of membrane are detached as ‘‘discoidal vesicles’’ from the phagosome once it has achieved some degree of maturation (Allen and Fok, 1983b; Allen et al., 1995). Second, membranes from old phagolysosome are recycled from the cytoproct, the site of release of spent materials, also as discoidal vesicles (Schroeder et al., 1990). Additional small round vesicles occur along the oral cavity, particularly in zones with regular arrangement of cilia (‘‘quadrulus’’ and ‘‘peniculus’’) and some vesicles slide along the ‘‘oral fibers,’’ probably to the nascent food vacuole (Ishida et al., 2001). Constitutive endocytosis by bristle coated pits/vesicles takes place by ‘‘parasomal sacs’’ that are stereotypically arranged on one side of the basis of cilia on the cell surface outside the oral cavity (Allen, 1988). Until now, one has generally assumed that constitutive endocytosis vesicles can assemble only at the sites of parasomal sacs. In this restricted area, the cell surface is not occupied by ciliary basal bodies or by alveolar sacs—the cortical Ca2þ-stores (Hardt and Plattner, 2000; Stelly et al., 1991). We now have evidence that there are potential sites for docking and detachment of small vesicles also outside this narrow region (Schilde et al., 2010), as outlined in Section 3.2. After pinching off vesicles travel to the ‘‘terminal cisternae’’ (Patterson, 1978), now considered as early endosomes that fuse with Golgi-derived vesicles to form late endosomes (Allen, 1988). The contractile vacuole complex of Paramecium (Allen, 2000; Allen and Naitoh, 2002) mainly serves osmoregulation. It has been shown to perform cyclic membrane fusions, not only at the outlet of the contractile vacuole (the ‘‘porus’’), that is, at the level of the cell membrane, but also at the sites where radial/connecting canals emanate from the vacuole (Tominaga et al., 1998a,b). Recently, we have found the unexpected occurrence of SNAREs in the contractile vacuole complex also outside these sites of periodic membrane fusions (Kissmehl et al., 2007; Schilde et al., 2006, 2008). Therefore, the contractile vacuole complex may contain many more fusion sites than previously assumed for this organelle. Remarkably, some trafficking steps can take place in Paramecium in a highly synchronous manner (Plattner et al., 1993). In particular, exocytosis (Plattner et al., 1984, 1985b) and exocytosis-coupled endocytosis (Plattner et al., 1985a, 1992) can be massively triggered and, thus, studied under highly synchronous conditions. Many parameters, ultrastructural, biochemical, and biophysical, can then be analyzed correlatively within a subsecond

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time range (Plattner and Hentschel, 2006). An example is the documentation of point fusion at a time when patch-clamp analysis could not yet approach that problem (Plattner et al., 1992). As a summary of this subsection, Fig. 3.2 highlights the main vesicle trafficking routes in Paramecium—specifically one along the endoplasmic reticulum (ER), the Golgi apparatus, and secretory vesicles; another one going along the endo-/phago-/lysosomal system; and a third one including the contractile vacuole system. Vesicle trafficking includes a multiplicity of fusion/fission steps in a P. tetraurelia cell and will be similar in other ciliates. From the multitude of membrane interaction sites, one had to expect an abundance of specific molecular key players on the different membranes involved in the respective trafficking steps, as it has actually been found.

2. Factors Involved in the Regulation of Vesicle Trafficking 2.1. Identifying SNAREs—Criteria and methodology More easily than most other proteins envisaged in this review, SNAREs can be generally identified by domain structure analysis. Criteria now to be outlined are illustrated in Fig. 3.3 and expanded to Paramecium SNAREs in Section 3.1. 2.1.1. General properties of SNAREs in other systems Whenever a BLAST search of the P. tetraurelia database has revealed high similarity, sequences were completed and subject to detailed domain analysis including the following criteria (as used, for instance, to identify plant SNAREs; Lipka et al., 2007). (i) Most SNAREs are single-span transmembrane proteins with a C-terminal transmembrane domain. (ii) This is followed by a SNARE domain,  60–70 aminoacids long, with ‘‘heptad repeats’’ centered around a ‘‘zero-layer.’’ The latter contains either an R- or a Q-residue—though with a few exceptions (Fasshauer et al., 1998; Sutton et al., 1998). The a-helical SNARE domain is able to coassemble with partner SNAREs to a quarternary transcomplex (SNAREs from opposite membranes). This is a prerequisite for membrane fusion ( Jahn and Scheller, 2006). (iii) More distally, in the case of the Qa-SNARE syntaxin, a Habc domain of  47–71 aminoacids follows; this domain allows consecutive binding of a-SNAP (unrelated to SNAP-25 and similar proteins) and consecutively of the SNARE-specific chaperone, NSF (N-ethylmaleimide sensitive factor) (Bock and Scheller, 1996; Rizo and Su¨dhof, 2002; Xu et al., 1999). (iv) In R-SNAREs a longin domain of  100–140 aminoacids may follow (‘‘longins’’; Filippini et al., 2001), for example, in most plant (Lipka et al., 2007) and ciliate (Schilde et al., 2006, 2010) R-SNAREs.

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The longin domain is absent from ‘‘brevins’’ (e.g., synaptobrevin ¼ VAMP [vesicle-associated membrane protein]), that is, in most animal R-SNAREs (Jahn and Scheller, 2006). (v) Cysteine residues in a specific C-terminal context may allow for fatty acylation (Magee and Seabra, 2005); this is the case with the SNAP-25-like proteins (SNAP-25-LPs) and with the Qbc SNARE proper, SNAP-25 (Gonzalo and Linder, 1998; Veit et al., 1996) as well as with the Qb SNAREs, Sec9 and Spo20, in yeast (Burri and Lithgow, 2004). The molecular size of SNAP-25-LPs, however, may deviate more or less from 25 kDa, as has been found in many species, from ciliates to mammals. Its Qb- and Qc-part each contain a SNARE domain and, by

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Figure 3.3 Some molecular characteristics of P. tetraurelia syntaxins (A–C), their evolutionary connection (D) and intracellular localization (E) by different methods, based on the work by Kissmehl et al. (2007). (A) The 15 types of syntaxins found in Paramecium by sequence analysis and domain structure analysis show some diversification with regard to the presence of a syntaxin domain (green), but all forms contain a SNARE domain (red), and a transmembrane region (blue). Their molecular size also varies. From Kissmehl et al. (2007). (B) Molecular modeling of PtSyx3-1 and PtSyx3-2 in comparison to 1DN1 (syntaxin 1), from R. norvegicus (C. Danzer, Diploma work, University of Konstanz) reveals striking similarities with regard to the arrangement of a-helical structure in the SNARE domain (green), with the Q-residue in the zero-layer indicated, and the structure of the Habc domain (yellow); red—linker. Unpublished images from the series by Kissmehl et al. (2007). (C) Core structure of the SNARE domain of PtSyx paralogs. Note the zero-layer with the Q residue typical of syntaxins and an exceptional A in PtSyx11-1. Also note the heptad repeats (repetitive aminoacids, yellow, in positions 3/4/7 upstream and downstream from the zero-layer), with some exceptional aminoacids set in green. A series of such heptad repeats in each of the SNAREs would align to a quarternary complex (‘‘SNARE complex’’). From Kissmehl et al. (2007). (D) Relationships between the different PtSyx

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backfolding, SNAP-25-type SNAREs can contribute the third and fourth a-helical SNARE domain to the quarternary SNARE complex (Fukuda et al., 2000; Jahn and Scheller, 2006; Malsam et al., 2008; Sutton et al., 1998). (vi) However, Qb- and Qc-domains may occur as independent proteins which, in that case, are membrane anchored by a C-terminal hydrophobic stretch (Lipka et al., 2007). Since the assignment to v- or t-type membranes may be ambiguous, SNAREs are now generally subdivided more stringently according to the aminoacid in the center (zero-layer) of the SNARE-domain which either contains an Arg/R- or a Gln/Q-residue, flanked by the periodic heptad repeats (Sutton et al., 1998). Thus, SNAREs are subdivided into R-SNAREs (synaptobrevin and related forms, including longins, v-SNAREs members), Qa(syntaxin), Qb-, Qc-, and Qb/c-SNAREs. In sum, with the exception of SNAP-25 and SNAP-25-LPs, SNAREs are normally, though not always, membrane-anchored by a C-terminal single membrane-spanning a-helical domain ( Jahn and Scheller, 2006; Jahn et al., 2003; Lipka et al., 2007; Malsam et al., 2008; Melia et al., 2002). Vesicle docking and subsequent membrane fusion requires pairing and zippering of SNAREs, whereby at least one SNARE on each side has to have a transmembrane domain (Section 3.1.2). Zippering means the formation of a quarternary coiled-coil transcomplex proceeding from the peripheral (N-terminal) to the proximal (C-terminal) part of the SNARE molecules (Lin and Scheller, 1997; Melia et al., 2002; Pobbati et al., 2006; Sorensen et al., 2006). Arg/Gln in the zero-layer support stabilization by hydrogen bonding. This has also been found by in vitro studies with reconstituted recombinant SNAREs. Again detailed analyses are required to establish the consequences of the deviating SNAREs found in P. tetraurelia. As we shall see, specificity of membrane interactions is not, or not solely determined by the organelle-specific SNAREs (see below). Rather, specific small GTPases are of crucial importance (Section 2.2) and possibly also subunits (SUs) of the Hþ-ATPase (Sections 2.4 and 3.3). Some ‘‘auxiliary’’ proteins are known to contribute, for example, a-SNAP for transient paralogs (neighbor joining tree), with probability values indicated, can be interpreted rather clearly as representing waves of whole genome duplication (pink, green, blue) discussed in the text. Composed from material contained in Kissmehl et al. (2007). (E) Intracellular distribution of PtSyx species, as determined by expression as GFPfusion proteins and by antibody labeling at the light and electron microscope level. For trafficking scheme, see Fig. 3.2A. Note association of PtSyx 9 and PtSyx10 with different vesicles probably interacting with food vacuoles; similarly uncertain is the assignment of PtSyx14 and PtSyx15 to the contractile vacuole system. Also note the presence of PtSyx1 all over the cell surface, including the oral cavity. Many other syntaxin isoforms can be clearly assigned to specific structures; they may be exchanged during trafficking, as is the case, for example, with syntaxins associated with early and later stages of the food vacuole. cph, cytopharynx; cs, cytostome; trp, trichocyst precursors; for additional abbreviations, see Fig. 3.2A. From Kissmehl et al. (2007).

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binding of NSF to the SNARE complex, as well as Munc18 (Bethani et al., 2007), Munc13, complexin, aRim, CAPS, etc., for some fine-tuning effects. In neuronal cells, they may also contribute to priming for subsequent fusion/exocytosis of neurotransmitter vesicles (De´ak et al., 2009; Wojcik and Brose, 2007). In Paramecium, a-SNAP and Munc 18 occur, whereas some of these proteins, such as complexin, do not occur in the database. 2.1.2. SNAREs in Paramecium On the basis of criteria just outlined, SNARE genes have been identified in P. tetraurelia (the only ciliate analyzed so far with this regard), followed by control of expression and intron verification (Kissmehl et al., 2007; Schilde et al., 2006, 2008, 2010). However, as in other systems, there occur also the following exceptions to the rules of SNARE characteristics. (i) A C-terminal hydrophobic aminoacid stretch may be absent (Kloepper et al., 2008); examples in P. tetraurelia are PtSyb7 and PtSyb12 as well as PtSNAP-25-LP (Tables 3.1 and 3.2). (ii) The zero-layer of the SNARE domain may contain an aminoacid other than R or Q (Fasshauer et al., 1998); for examples in Paramecium, consult Tables 3.1 and 3.2. (iii) A motif for potential fatty acylation (CAAX or other motifs) may be present, but in thorough analyses we could not verify fatty acylation where it would be expected, for example, in PtSNAP-25-LP (Schilde et al., 2008). These serious deviations made it even more important to include some additional, independent—though equally ambiguous—criteria for the identification of SNAREs in P. tetraurelia: (i) Control by Northern and/or Western blots, to verify transcription and translation, allowing for the recognition of potential pseudogenes. (ii) Expression as GFP-fusion proteins which in turn (iii) should be controlled by immunolocalization using antibodies against the endogenous protein. This may also require immunogold electron microscope (EM) analysis. Here, increased sensitivity can be achieved when GFP-labeling is combined with anti-GFP antibody labeling. (iv) Gene silencing will frequently disclose specific transport pathways although one has to bear in mind that some SNAREs can travel on rather different routes within one cell (Burri and Lithgow, 2004; Kloepper et al., 2008) and that they may have to use such routes to reach their final destination. In our work with Paramecium, these approaches require mutual control for the following reasons. (i) GFP-fusion proteins may become mistargeted. (ii) Antigenicity or copy numbers of endogenous proteins may be too low for detection and (iii) discrimination between closely related paralogs by polyclonal antibodies may not be possible—in contrast to GFP expressions. (iv) Gene silencing may not discriminate between closely related genes, particularly with the most recently generated subfamily paralogs (also called ‘‘ohnologs’’ according to an author’s name). The difference in nucleotide sequence has to be >15% as a rule to achieve selective silencing (Ruiz et al., 1998).

Table 3.1 R-SNAREs and related forms found in P. tetraurelia

SNARE-domain

Longin domain

amino acid 0-layer

þ þ þ þ þ d

þ þ þ   þ

þ þ þ (þ) (þ) þ

R R R   R

PtSyb7 PtSyb8

d þ

þ þ

þ þ

R N

PtSyb9

þ

þ

þ

H, Ne

PtSyb10

þ

þ



N

PtSyb11

þ

þ



N

PtSyb12 f Sec22

 þ

 þ

 þ

 R

Type of SNAREa)

Transmembrane domain

PtSyb1 PtSyb2 PtSyb3-1 PtSyb4 PtSyb5 PtSyb6-1

Localizationb

Endoplasmic reticulum Contractile vacuole complex Endoplasmic reticulum Small vesicles in cyclosisc Trichocyst precursors? Cytopharynx, nascent food vacuole (acidosomes), cytoproct, parasomal sacs, endoplasmic reticulum, early endosome No localization achieved Acidosomes?, cytopharynx, nascent and early stage of food vacuoles Acidosomes?, cytopharynx (domain of food vacuole formation) Ciliary basis, cell membrane/alveolar sacs complex One side of cytostome, occasionally on food vacuoles, terminal cisternae Cytosolicf Endoplasmic reticulum/Golgi apparatus

Notes: Data from Schilde et al. (2006, 2010); Sec22: Kissmehl et al. (2007). For more details on ohnologs, see Schilde et al. (2010). a PtSyb1, 2, 4, 7, and 9 are represented each by two paralogs and PtSyb 3, 5, and 8 each by one, while PtSyb6-2 is a fragment. b Parasomal sacs ¼ clathrin-coated pits, teminal cisternae ¼ early endosomes, acidosomes ¼ late endosomes studded with Hþ-ATPase for delivery to food vacuoles. For terminology, see also Section 1.3. c ‘‘Small vesicles’’ are 1 mm in size and travel with the cyclosis stream. d With a C-terminal CCXXF/Y motif. e H in PtSyb9-1, N in PtSyb9-2. f Prognosticated by sequence analysis, but questionable as a SNARE.

Table 3.2

Q-SNAREs and related forms found in P. tetraurelia

Type of SNAREa

Transmembrane domain

SNAREdomain

Syntaxin domain

Amino acid 0-layer

Qa group PtSyx1

+

+

+

Q

PtSyx2 PtSyx3 PtSyx4

+ + +

+ + +

+ + +

Q Q Q

PtSyx5 PtSyx6 PtSyx7 PtSyx8 PtSyx9 PtSyx10 PtSyx11 PtSyx121 PtSyx13d

+ + + + + + + +c

+ + + + + + + +

 +     + 

Q Q Q Q Q Q A Q

Putative pseudogene

Localizationb

Cell membrane, cytoproct, discoidal vesicles and additional recycling vesicles, nascent and early food vacuole Contractile vacuole complex Terminal cisternae, one side of cytostome Discoidal vesicles, oral cavity (small vesicles), for nascent food vacuole formation? Golgi apparatus No result achieved Food vacuoles Endoplasmic Reticulum Food vacuoles and interacting vesicles Cyclosis vesicles Food vacuoles Food vacuoles

Qc group PtSyx14 + PtSyx15 + Qb/c group PtSNAP 25-LP

+ +

 

Q Q

Contractile vacuole complexe Contractile vacuole complexe

+

Duplicate heptad repeat

Q/Qf

Contractile vacuole complex, Endoplasmic Reticulum, food vacuoles (except early stages), oral cavity, parasomal sacs, cell membrane g

Notes: From Kissmehl et al. (2007); PtSNAP-25-LP (Schilde et al., 2008). a PtSyx1, 2, 3, 4, 5, 7, 8, 9, 10, and 14 are represented by two paralogs each, in contrast to PtSyx6, 11, 12, and 15 as well as PtSNAP-25-LP (each with one paralog only). b For terminology, see Table 1 and Section 1.3. c With an extra-long C-terminal part beyond the transmembrane domain. d Ptsyx13 is a pseudogene. e Seen only after overexpression. f With two SNARE domains, each one with Q in 0-layer. g With many diffusely labeled sites beyond those indicated.

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With ciliates, additional complications may be expected from the following experience with higher eukaryotes. Specifically, the promiscuous Qa-SNARE, syntaxin 6, can interact with other Qa-, Qb-, or QbcSNAREs or with R-SNAREs (Wendler and Tooze, 2001). While SNAREs frequently occur in specific membranes, they may also pair ‘‘illegitimately’’ with noncognate counterparts when reconstituted in liposomes (Brandhorst et al., 2006; McNew et al., 2000). From this one can conclude that SNAREs possess only limited intrinsic specificity. Proofreading by some of the ‘‘auxiliary’’ proteins on the way through the cell may contribute to enhance organelle-specificity (Bethani et al., 2007). No SNARE specificity has been found for homotypic early endosome fusion (Brandhorst et al., 2006), in contrast to late endosomes where the SNARE domain is thought to be responsible for specificity (Paumet et al., 2004). In contrast, in the Golgi apparatus of yeast a combination of appropriate SNAREs mediates a high degree of specific interaction (‘‘combinatorial specificity’’) (Parlati et al., 2002). In sum, a mutual balance between the respective chances and pitfalls is mandatory to achieve reliable data on SNAREs. A cross-check of the data with those contained in a global SNARE database reachable under http://www.mpibpc.mpg.de/english/ service/bioinformatics/index.html and design of corresponding evolutionary trees is advisable. This has been included in our work with P. tetraurelia SNAREs (Kissmehl et al., 2007; Schilde et al., 2006) in an attempt to round up the identification of PtSNAREs. Thus, such data can be put in line with >3000 SNARE sequences (complete or fragmentary) that are globally available at this time (Kloepper et al., 2008). The currently available Paramecium database contains many data on SNAREs based on two sources, that is, manual annotations mainly by our group (Kissmehl et al. 2007; Schilde et al., 2006, 2008, 2010) and comparative computer search in numerous genome databases (Kloepper et al., 2007, 2008). Note on the nomenclature used in P. tetraurelia: To give an example, the v-SNARE synaptobrevin is designated as Ptsyb for the coding gene and PtSyb for the protein, respectively. This is followed by the subfamily and the ohnolog number, for example, PtSyb1-2. Results from plant molecular biology suggest that increasing diversification of secretory activity during evolution is accompanied by increased numbers of SNAREs (Rojo and Denecke, 2008; Sanderfoot, 2007). This may be expanded to the degree of complexity of the entire morphologically seizable trafficking system which in Paramecium is very high. The total number of PtSNARE genes currently estimated on the basis of results from our group is well comparable to multicellular organisms up to man. However, such comparison requires clear definition which molecules are considered a SNARE and to what extent ohnologs are considered separately. This is discussed in more detail in Section 3.1.1. When compared with Fig. 3.2B, in Paramecium the number of SNAREs may even surpass the

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number of specific membrane interaction sites currently known from structural studies. This suggests further functional (and unnoticeable structural) diversification, for example, of small vesicles associated with the digestive cycle (Schilde et al., 2010). One explanation for this wide diversification may be repeated whole genome duplications with subsequent differentiation. Only the last duplication appears to have created closely related subfamily members (ohnologs) which may serve gene amplification rather than neofunctionalization (Aury et al., 2006; Duret et al., 2008). In practice, we have identified PtSNAREs applying the following methodical arsenal. First, we performed BLAST searches in the Paramecium database. Then the putative genes were cloned and the corresponding cDNA was prepared to identify introns. The deduced aminoacid sequence served to specify in detail domains characteristic of the different SNAREs (Section 3.1) and, by molecular modeling, to check similarities with established SNAREs from other systems. Prognostication of immunogenic stretches of the protein served production of antibodies for immunolocalization at the light and EM level as well as for Western blot analyses from subcellular fractions, as far as available. This was complemented by overexpression as GFP-fusion proteins which also augmented chances for EM localization. Finally, posttranscriptional homology-dependent gene silencing was performed by feeding transformed bacteria (Section 1.2) or by microinjection of appropriate constructs into the macronucleus. A most elegant method is the ‘‘antisense ribosome technology.’’ It was developed by Chilcoat et al. (2001) for posttranscriptional gene silencing in Tetrahymena. This method involves the generation of cells transfected with the genes to be analyzed by insertion into the 26S rRNA (‘‘ribosome library’’). Among ciliates, however, its use has largely remained restricted to Tetrahymena. We finish this section by recommending for a short overall background information on the identification of SNAREs the review by Sorensen (2005). Though SNAREs are well defined by their insertion in membranes by a single C-terminal hydrophobic stretch and by specific domains, including a SNARE-domain with a defined zero-layer, etc., there are exceptions to most identification rules. Therefore, a combination of several molecular properties has to be considered, paralleled by in situ analysis (localization, gene silencing), to identify functional SNAREs also in ciliates.

2.2. Small GTP-binding proteins/GTPases and their modulators As found with higher eukaryotic systems, from yeast to mammals, small GTPases of the Arf- and Rho-type may exert, independently from SNAREs, a dominant function in determining the specificity of membrane interactions (Behnia and Munro, 2005; Cai et al., 2007; Grosshans et al.,

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2006; Novick and Zerial, 1997; Schwartz et al., 2007a,b; Zerial and McBride, 2001). In fact, specificity cannot be explained in full merely by SNAREs, as outlined in Sections 2.1 and 9. The Arf-type GTPases (in complex with their activators; see below) may be exceptional, as they may interact with SNARE complexes and also with COP-type coat proteins (Poon and Spang, 2008). One of the biggest gaps in the analysis of membrane trafficking in ciliates concerns small GTPases and their modulators, including GAPs (guanine nucleotide activation proteins) and GEFs (guanine nucleotide exchange factors), etc. Arfs are a group of larger monomeric G-proteins that are involved in budding of COP-coated vesicles in the ER and the Golgi apparatus (Anders and Ju¨rgens, 2008; Bonifacino and Glick, 2004; Pfeffer, 2007; Section 3.3). Monomeric G-proteins can also contribute to the recruitment of motor proteins ( Jordens et al., 2005) and, thereby, to the motility from the early endosome on (Nielsen et al., 1999). Specifically, Arfs also interfere with the kinetics of the F-actin system (Doherty and McMahon, 2008; D’Souza-Schorey and Chavrier, 2006), including remodeling of cortical F-actin in dense core-secretory vesicle systems (Vitale et al., 2002). Therefore, monomeric G-proteins/GTPases may exert several functions along the secretory pathway, from vesicle budding till targeting and docking. Since small G-proteins particularly of the Rab and Arf type are considered most essential determinants of vesicle targeting (Grosshans et al., 2006; Novick and Zerial, 1997), many of them serve as markers, for example, for specific stages of the endo-/lysosomal system: Rab5 is associated with early endosomes, Rab7 with late endosomes/lysosomes, and Rab11 with recycling endosomes (Behnia and Munro, 2005; Haas, 2007; Novick and Zerial, 1997). Considering the particular importance of Arf molecules in vesicle targeting our ignorance with regard to ciliates is a highly regrettable gap. Apart from some GTP-overlay studies (Section 3.3.3) only a few genes have been partially cloned and their translation products tentatively characterized and localized (Surmacz et al., 2006). An exception is putatively Arf-specific GEFs in P. tetraurelia, with homologs in other ciliates. One form related to the mammalian type, ARNO, has been cloned (Nair et al., 1999), before a list of them has been derived from homology search (Mouratou et al., 2005). This, together with the number of small GTPases and GAPs to be expected, suggests considerable diversification in ciliates. All this may contribute substantially to the impressive differentiation of vesicle trafficking, also in ciliates. Since minute diversifications may have taken place during evolution, any premature assignment to specific localization and function should be avoided as long as any detailed analyses are missing. In the T. thermophila genome (Elde et al., 2005) 69 different Rab protein genes, in addition to 8 dynamin-related genes, are found (Zweifel et al., 2009).

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Also for T. thermophila, at a FASEB meeting (2009) Aaron Turkewitz and his team (L. Bright) presented evidence of a similar number of Rabs, and intracellular localization may hopefully soon be documented. Some of them are very conserved and some other ciliate specific, each group encompassing about one-fourth of the total number. This is a remarkable number, considering that somewhat over 60 Rabs have been identified in mammalian cells and 11 in yeast (Grosshans et al., 2006). No such precise estimates are available for Paramecium as yet (or for any other ciliate species), but the number may be even higher than in other ciliate and nonciliate species due to the most recent whole genome duplication. Unfortunately, G-proteins associated with food vacuoles are not known as yet. Only at later stages food vacuoles in Paramecium are reported to acquire Rab7 (Surmacz et al., 2006) and, in analogy to mammalian cells, a Rabinteracting protein, together with the lysosomal marker, LAMP-2 (Wyroba et al., 2007). The Cda12p and Cda13p proteins that were found relevant for cytokinesis and conjugation in T. thermophila (Zweifel et al., 2009) are without any identified homolog in higher eukaryotes. From their function and localization (Section 9.3) they are considered functionally related to a Rab11interacting protein—Rab11 being a determinant and marker for recycling endosomes (Ullrich et al., 1996). To sum up this section one may state that information about small G-proteins/GTPases in ciliates, though of paramount importance for vesicle trafficking, is only rather fragmentary. The number of Rabs in Tetrahymena is known to exceed that in man. In Paramecium, examples of our fragmentary knowledge are Rab7, some Arf-related modulators, GEFand GAP-proteins. It now appears mandatory to fully clone and to characterize these components functionally and to map them topologically. Partial sequences can be retrieved from the databases and used as a starting point, before any definitive identification and appreciation of G-protein subfamilies and their modulators can be achieved.

2.3. Actin 2.3.1. General considerations on actin participation in vesicle trafficking The role of cortical F-actin in secretory vesicle docking has long been debated, from merely inhibitory (Aunis, 1998) to facilitation. Only quite recently the involvement of actin in the secretory cycle, from the Golgi apparatus (Cao et al., 2005) to vesicle docking (Vitale et al., 2002), release (Mitchell et al., 2008), pore closure (Larina et al., 2007) and ‘‘ghost’’ retrieval (Galletta and Cooper, 2009; Giner et al., 2007; Kaksonen et al., 2006; May and Machesky, 2001; Soldati and Schliwa, 2006) has become increasingly evident. To achieve such dynamics, actin filaments can associate with myosin (Bhat and Thorn, 2009). F-actin is essential in detachment

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of endocytotic vesicles, not only for exocytosis-coupled endocytosis but also for other types, such as clathrin-coated and noncoated vesicle endocytosis (Galletta and Cooper, 2009; Miaczynska and Stenmark, 2008). For a more detailed discussion of what is known about the contribution of actin to phagocytosis in higher eukaryotes (May and Machesky, 2001; Soldati and Schliwa, 2006), see Section 6. What has to be expected along these lines for ciliates? 2.3.2. Actin in ciliates The multitude of actin isoforms in P. tetraurelia is surprising (Table 3.3). Within ciliates, the highest number, up to 31, occurs in species with extensive macronuclear genome fragmentation during development (Zufall et al., 2006). We found nine subfamilies, subfamily PtAct1 with nine paralogs, PtAct5 with three, subfamilies PtAct2, 3, 4, 6, and 7 each with two isoforms, and subfamilies PtAct 8 and 9 with one form each (Sehring et al., 2007a,b, 2010). Even though a few of the numerous actin forms may also be classified as actin-related and actin-like proteins, they clearly outnumber the four actin genes reported from T. thermophila (Kuribara et al., 2006; Williams et al., 2006) and six from man (Pollard, 2001). From the abundant actin isoforms, members of seven subfamilies were investigated by immunofluorescence, by immuno-EM analysis, and as GFP-fusion proteins and nine subfamilies by gene silencing (Sehring et al., 2007a,b, 2010). These studies also yielded clues to the drug (in)sensitivity and to polymerization properties (Table 3.3) (Sehring et al., 2007b). This may be the reason why we have noticed in phalloidin-affinity labeling studies (Kersken et al., 1986) the questionable absence of phalloidin fluorescence label from some ‘‘classical’’ sites where actin would definitely have been expected. Concomitantly, using antibodies against common sequences mainly from PtAct1 paralogs we have recognized many more actin-containing sites by immuno-EM localization studies. This included the occurrence of actin at some established crossroads of vesicle trafficking (Kissmehl et al., 2004). However, in Paramecium the distribution of more widely different actin isoforms varies considerably (Table 3.3). This is in line with the involvement of actin in many phenomena. In higher eukaryotes this includes the arrangement of Golgi elements (Lin et al., 2005) and the formation of Golgi vesicles (Cao et al., 2005) as well as the endo-/phago-/lysosomal system (Kjeken et al., 2004) and thereby particularly the formation of (Yam and The´riot, 2004), and recycling vesicle formation from phagosomes (Damiani and Colombo, 2003) as well as delivery of the Hþ-ATPase via lysosomal extensions (Sun-Wada et al., 2009). Again in higher eukaryotes, actin also contributes to targeting of some SNAREs and of some SUs of the HþATPase (Section 3.3). In fact, in Paramecium many of these sites are endowed with actin with more or less pronounced selectivity.

Table 3.3 Characteristics of actin isoforms in P. tetraurelia

Actin type

Amino acid identitya,b %

ATP-binding site identitya,b %

Myosin binding site identitya,b %

PtAct1d PtAct1-1

100

100

100

PtAct1-2 PtAct1-3 PtAct1-4 PtAct1-5 PtAct1-6 PtAct1-7 PtAct1-8 PtAct1-9 PtAct2-1 PtAct2-2 PtAct3-1

100 100 90 90 60 75 70 65 60 60 45

100 100 100 100 80 95 80 45 85 i.c.p.e 55

100 100 100 100 65 70 45 65 85 i.c.p. 50

PtAct3-2

45

i.c.p.

50

Localizationc

Cytoproct Cortex, cilia, cytoproct, cytostome, oral cavity, food vacuoles Food vacuoles Cytosolic compartment Cytosolic compartment

Food vacuoles Cilia, cytosolic compartment Cilia, cortex, food vacuoles, cytosolic compartment (continued)

Table 3.3 (continued) Actin type

Amino acid identitya,b %

ATP-binding site identitya,b %

Myosin binding site identitya,b %

PtAct4-1

30

25

70

PtAct4-2 PtAct5-1f

30 40

i.c.p. 75

i.c.p. 60

PtAct5-2 PtAct5-3 PtAct6-1 PtAct6-2 PtAct7-1g PtAct7-2 PtAct8-1

40 40 30 30 25 25 35

i.c.p. 65 60 i.c.p. 20 i.c.p. 55

i.c.p. 55 55 i.c.p. 35 35 45

PtAct9-1h

20

35

20

Localizationc

Cortex, cilia, cytostome, oral cavity, nascent food vacuole Cortex, cytostome, oral cavity, food vacuoles, postoral fibers, cilia

Cytosolic compartment

Cortex, cytostome, cytopharyngeal fibers, ER/Golgi, food vacuoles, parasomal sacs

Notes: Results from Sehring et al. (2007a,b, 2010). a Amino acid sequence derived from macronuclear DNA; numbers refer to aminoacid sequence of PtAct1-1. b Rounded values (þ/ 5%). c For terminology, see Section 1.3. d Antibody labeling, without discrimination between PtAct1 subtypes. e i.c.p., identical conservation pattern within subfamily. f Also designated ARP-1. g Also designated ARP2/4. h Also designated ARP10.

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In Paramecium, surprisingly numerous actin isoforms are associated with the cell cortex (Table 3.3). Specifically PtAct8-1 is the only actin associated with parasomal sacs (Sehring et al., 2007b). Silencing of the genes of another cortical form, PtAct4, distorts the endocytotic organelles derived from them (Sehring et al., 2010). This indicates mutual dependency of these isoforms, rather than complementation. Five of the PtAct subfamilies (PtAct1-1, 1-2, 1-9, 3-1, 5-1, and 8-1) are associated with food vacuoles (Table 3.3) and, thus, may interfere with vesicle budding and/or fusion. Isoforms are exchanged during cyclosis; for instance, PtAct4-1 is restricted to nascent food vacuoles. Silencing only of some of these PtAct forms affects phagocytosis, while some of them (e.g., PtAct1-1 and PtAct1-9) may be compensated for by other forms (Sehring et al., 2007b). Propulsion of food vacuoles in the cyclosis stream by an unilateral comet-tail seen with GFP-PtAct1-2 and PtAct1-9 (Sehring et al., 2007b) is another aspect pertinent to trafficking—a hypothesis suggested by unilateral arrangement (Section 6). The presence of actin isoform 1 at the cytoproct of Paramecium, as determined by antibody staining (Sehring et al., 2007b), is in agreement with the following physiological findings. In Paramecium, cytochalasin B impedes closure of the cytoproct after defecation (Allen and Fok, 1985). In Tetrahymena another actin disruptive drug, latrunculin B, inhibits egestion of spent food vacuole contents (Sugita et al., 2009). The question which PtAct1 isoform decorates the cytoproct requires elucidation. Table 3.3 also lists the conservation of ATP- and putative myosin-binding residues in P. tetraurelia actins (Sehring et al., 2007a) which both are relevant for dynamic actin functions. In different forms of PtAct both these properties fluctuate considerably and independently from each other. We may summarize the situation in ciliates, notably in Paramecium as follows. Widely deviating actin isoforms can be associated with one specific type of vesicular organelle (e.g., the food vacuole), but the opposite also occurs, for example, PtAct8-1, associates with different organelles. Some of the actin layers, made of different types of actin, appear more dynamic than others; for instance, there occurs a coordinated exchange of actin isoforms during the phago(lyso)somal cycle in Paramecium. More aspects concerning the digestive cycle are discussed in Section 6.2. Clearly silencing of some of the actin genes affects specific vesicle trafficking steps.

2.4. H+-ATPase To some extent, this Hþ-transport-ATPase is comparable to the mitochondrial ATP synthase (Dimroth et al., 2006) although—in contrast to the mitochondrial molecule—the V-ATPase hydrolyses ATP to induce rotation of the V0 part inserted in the membrane and, thus, to translocate protons.

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2.4.1. General aspects The Hþ-ATPase (proton pump) is a hetero-oligomeric protein assembly consisting of a proteolipid (V0) and a catalytic part (V1) with exchangeable SUs. The V0 and V1 part are connected by a stalk containing an a-SU, but the V1 part can dissociate, thus leaving behind the V0 channel part (Boesen and Nissen, 2009) to which a role in membrane fusion has been assigned (Section 5.2). This complex molecule is classified as a V (vesicle)-type ATPase whose structure and modus operandi have been repeatedly reviewed (Beyenbach and Wieczorek, 2006; Forgac, 2007; Marshansky and Futai, 2008). These molecular assemblies are distributed over a variety of vesicular organelles undergoing trafficking, such as early and late endosomes as well as phago(lyso)somes (Hinton et al., 2009). Hþ-ATPases do not form a phosphointermediate, in contrast, for example, to the monomeric Ca2þ-ATPases (calcium pumps, P-type ATPases) (Carafoli, 2005). 2.4.2. Aspects pertinent to Paramecium In P. tetraurelia (the only protozoan species analyzed), the genes for the several SUs of the Hþ-ATPase have been cloned and the SUs localized by combined GFP- and antibody-techniques (Wassmer et al., 2005, 2006), as summarized by Wassmer et al. (2009) as well as in Table 3.4. Previously only the B-SU had been identified in Paramecium multimicronucleatum (Fok et al., 2002). A salient feature of our own work is the unsurpassed number of a-SUs, 17 versus 2 in yeast and 4 in the mouse (Wassmer et al., 2009). This multiplicity may be crucial for composing different holo-enzymes with different pumping kinetics (and related functions) in the multitude of organelles endowed with the HþATPase in Paramecium (Wassmer et al., 2009). This aspect is particularly intriguing as the lumenal pH achieved can transduce a signal to the cytosolic side and, thus, determine trafficking specificity, as outlined in Section 3.3. Biogenesis and targeting of the Hþ-ATPase and of its V0-SUs is a particular problem, as no signal peptide could be detected, also in Paramecium (Wassmer et al., 2005, 2006). Current views on proteins helping insertion into the ER membrane and escorting the V0 part and possibly parts of the ‘‘stalk’’ from the ER to the Golgi apparatus and beyond are discussed in Section 3.3. The many a-SU isoforms contain an ill-defined (conformational?) targeting motif in the C-terminal half of the molecule (Wassmer et al., 2006), rather than in the N-terminal half as reported for yeast (Kawasaki-Nishi et al., 2001). The a-SU isoforms are delivered selectively to different organelles in the Paramecium cell (Wassmer et al., 2005, 2006). If they were, in fact, to determine organellespecific targeting, the problem arises, how the respective isoforms are instructed to go which way. Considering the interactions with other molecules, notably SNAREs, G-proteins and actin (Section 3.3), this may illustrate the complexity of membrane traffic regulation even in single-cell organisms, such as ciliates.

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Table 3.4 Survey of compartments of P. tetraurelia cells endowed with an Hþ-ATPase and the SUs experimentally localized to the respective organelle

Organelle

Aciditya

SUs found in compartmentb

ER region Golgi apparatus Terminal cisternaec Cytostomal aread Nascent food vacuole Acidosomes Pinched off food vacuole (after fusion with acidosomes) Vesicles (lysosomes?) possibly contributing to food vacuole formation Matured food vacuole (intermediary stage) Late food vacuole Discoidal vesicles Trichocyst precursorse Mature trichocystse,f Contractile vacuole system (decorated spongiome)

No No Yes ? No Yes Yes

a7-1, c1, c4, c5 a8-1 a1-1 a1-1, a4-1 – a4-1 a4-1

Yes

a5-1, a6-1, a9-1

Yes

a5-1, 6-1, 9-1, c1, c4, c5 – – a3-1 a3-1, c1, c4, c5 a2-1, c1, c4, c5, F2

No No No No No

Notes: From Wassmer et al. (2005, 2006, 2009). a Determined by acridine orange. ‘‘No acidity’’ despite of the presence of Hþ-ATPase may be due to Hþ-binding by acidic contents (trichocysts and their precursors), presence of an H+-exchanger, or to expulsion of contents within short time periods (contractile vacuole system); see text. b Incomplete list enumerating only SUs actually localized experimentally by GFP fusion and/or antibody staining at the light and/or EM level. c Equivalent to early endosomes. d Containing ill-defined vesicles; see text. e Lack of acidity determined by electron microscopy, using antibodies against the hapten, dinitrophenol, after trapping a structural analog of this compound in vivo (Garreau De Loubresse et al., 1994). f Lack of acidity also registered by acridine orange (Lumpert et al., 1992).

In sum, the overall involvement of the Hþ-ATPase in vesicle trafficking is as follows. As known from higher eukaryotes this complex molecule and its SUs can—directly or indirectly—specifically contribute to vesicle trafficking. As discussed in detail in Section 3.3, this implies direct binding of SNAREs or of actin as well as binding of some monomeric G-proteins (Arf) and regulators that secondarily mediate specific organelle docking. In Paramecium, we have cloned and localized Hþ-ATPase SUs to specific vesicles (as discussed in the respective sections) and gene silencing experiments have been successful. Nevertheless the contribution to any specific vesicle interactions is still poorly understood in ciliates.

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3. Features of SNAREs 3.1. Characteristics of Paramecium SNAREs As we shall see, we are sometimes at the limits of identifying a molecule as a SNARE. This is so not only with ciliates where Paramecium is the only species analyzed with this regard up to now. 3.1.1. Overview of SNAREs in Paramecium as compared to other organisms Tables 3.1 and 3.2 summarize the SNAREs identified and, to a large extent, localized in P. tetraurelia (the only ciliate species analyzed so far). Figure 3.3 presents some characteristics of PtSyb species and their localization. In Paramecium, the number of SNARE genes amounts close to the highest numbers known, as we shall see. Such numbers, however, require some comments. Most syntaxins and synaptobrevin(-like) PtSNAREs are represented by subfamilies, mostly with two members each (Fig. 3.3, Tables 3.1 and 3.2). On the aminoacid level, all of the SNAREs belonging to one subfamily differ from each other in the extreme by in between >90% (PtSyx; Kissmehl et al., 2007) and 85% (PtSyb; Schilde et al., 2006). One may now assume that subfamily members would have to differ to a sufficient extent to be differentially localized and to exert differential functions—an experience which we made with various examples. If their nucleotide sequence differs by only <15%, for instance, the aminoacid sequence will generally differ even less due to the degenerated genetic code. From a practical point of view, a difference of  15 between the open reading frames of two genes allows one to achieve simultaneous gene silencing (Ruiz et al., 1998). From our experience, this generally concerns ohnolog pairs. One may, thus, assume that rather similar isoforms may merely serve gene amplification for the same function (Aury et al., 2006). On this basis, one can estimate the number of ‘‘functionally distinct’’ SNAREs in P. tetraurelia as outlined below. Some uncertainty comes from missing information on the precise difference between some isoforms and whether lack of a transmembrane domain or of a SNARE domain, etc., would restrict function as a SNARE. In detail, among Q-SNAREs we found only one gene each encoding PtSNAP-25-LP (Schilde et al., 2008), PtSyx6, 11, 12, and 15 (Kissmehl et al., 2007). For the other Q-SNAREs, we found two ohnologs in type PtSyx1–5, PtSyx7–10, as well as in PtSyx14 (Kissmehl et al., 2007). PtSyx13 may be disregarded as a putative pseudogene. The 10 twin ohnologs together with the 5 singular forms (‘‘singletons’’) may result in 15 ‘‘functionally distinct’’ Q-SNAREs in P. tetraurelia if one assumes diversification

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in function and localization, as discussed below. Similarly, we found 19 R-SNAREs (disregarding PtSyb6-2 and PtSyb12 because of fragmentation and/or of lacking clear domain structure), five of them being singletons (PtSec22 [Kissmehl et al., 2007], PtSyb3, 5, 6, 8) and seven twin ohnologs, that is, PtSyb1, 2, 4, 7, 9, 10, 11 (Schilde et al., 2006, 2010). The number of ‘‘functionally distinct’’ (¼ sufficiently diversified in function and localization) R-SNAREs may, thus, be 12. In sum, out of a total of 44 PtSNARE genes—the ones we have identified and characterized in any sufficient detail—only 27 may encode diversified ‘‘functionally distinct’’ SNARE proteins, or more likely close to 40, considering less similar ohnologs. The number of genes encoding PtSNAREs and PtSNARE-like sequences may be still higher, as concluded from database search (Kloepper et al., 2007), but detailed specification is still missing. In total, the estimated number of SNAREs in Paramecium is comparable to the number determined for Homo sapiens that is, 41 (Kloepper et al., 2007), and plants (angiosperms) where similar estimates amount to 42 in Arabidopsis thaliana or to 47 in a poplar tree species (Lipka et al., 2007). These numbers exceed those in Saccharomyces cerevisiae where 26 SNAREs (7 Qa-, 6 Qb-, 8 Qc-, and 5 R-SNAREs) are established (Burri and Lithgow, 2004). An absolutely reliable comparison of diversification is difficult to achieve because not in all systems are functional and structural criteria sufficiently known. For specific SNARE types fully identified so far in Paramecium, refer Tables 3.1 and 3.2. In other protists, the number of SNAREs is said to be lower than in higher organisms, but this conclusion has been reached from a restricted number of genomes from parasitic species (Yoshizawa et al., 2006) whose size is very likely reduced due to life style. In fact, only 17 SNAREs have been found in Giardia lamblia (Elias et al., 2008) and 18 SNARE-like proteins in Plasmodium falciparum (Ayong et al., 2007). In a phylogenetic analysis, it was possible to extrapolate a set of 20 primordial SNARE types in eukaryotes and 30 in urmetazoans (Kloepper et al., 2008). For the latter, it has been assumed that evolution of a diversified endo-/phagosomal system has enforced the acquirement of additional SNAREs. When these data are compared with the ‘‘functionally distinct’’ SNARE types in P. tetraurelia, one can conclude that such an increase in the number of SNAREs may have been anticipated by ciliates, thus supporting the particular role of the evolution of an intricate exo-/ endo-/phagosomal system as a driving force in evolution (Cavalier-Smith, 2002). For example, among Q-SNAREs we have several ones relevant for phago-lysosomal trafficking (PtSyx7, 9, 10, and 11) even without direct counterpart in metazoans; some other ones (PtSyx4-1 and 4-2 and perhaps PtSyx6-1) are involved in processes resembling transcytosis (Kissmehl et al., 2007). Also among synaptobrevin-like PtSNAREs several ones are dedicated to vesicle flow directly or indirectly bound to phagocytosis and recycling (Schilde et al., 2010).

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The type of R-SNAREs also requires a comment. Most R-SNAREs are of the ‘‘brevin’’-type in animal cells (Jahn and Scheller, 2006) and of the ‘‘longin’’-type in higher plants (Filippini et al., 2001; Rossi et al., 2004); Section 2.1. Only some animal R-SNAREs are longins, for example, the TeNT-insensitive VAMP7 (TI-VAMP, Syb-LP1) occurring in some neuronal and nonneuronal cells (Galli et al., 2006). In A. thaliana targeting of VAMP7 is regulated by the longin domain (Uemura et al., 2005). In addition to TI-VAMP/VAMP7, the longins Ykt6 and Sec22 are widely distributed, from fungi to plants and man (Rossi et al., 2004). In mammalian cells it has been found that the longin domain of VAMP7 folds back and, thus, blocks its SNARE domain when not engaged in a SNARE complex. Only in the open form—as occurring in a cis-SNARE complex after membrane fusion—can its longin domain bind the Arf-GAP, Hrb, which mediates clathrin binding and endocytosis (Pryor et al., 2008). In higher eukaryotes, the folding state of the longin domain, for example, of Sec22 (with a homolog in P. tetraurelia [Kissmehl et al., 2007]), is known to be important also for vesicle release from the ER and further targeting (Mancias and Goldberg, 2007). This may explain why N-terminal GFP labeling of another Paramecium longin, PtSyb10, inhibits exit from the ER and delivery to the cell membrane (Schilde et al., 2010). This is clearly an example calling for mutual control of trafficking pathway analysis by different methodologies. Membrane fusion depends on the occurrence of one transmembrane domain in an R- (v-) and one in a Q- (t-) SNAREs: One type of SNARE each has to be anchored in one of the two membranes (Grote et al., 2000; McNew et al., 2000). Nevertheless, SNAP-25, in spite of a lacking transmembrane segment, also contributes to the fusion process as a whole. This has been demonstrated by application of antibodies and by truncation by the SNAP-25-specific Clostridium toxin, BoNT/E (Schuette et al., 2004), as outlined in Section 8.3. 3.1.2. Specific aspects of SNAREs in Paramecium The aspects just discussed are particularly interesting if one considers the longin character of R-SNAREs in P. tetraurelia, including members of the PtSyb1, 2, 3, and 6–9 subfamilies (Schilde et al., 2006, 2010) as well as PtSec22 (Kissmehl et al., 2007). The question arises as to similar effects of the longin domain (Section 3.1.1), including targeting, in plants and in Paramecium—a question to be analyzed in future work. In Fig. 3.3, we give an example what Paramecium SNAREs look like and where they are localized. As Tables 3.1 and 3.2 show, some of the Paramecium SNAREs display various aberrant features. This may include absence of a transmembrane domain (PtSyb6 and 7), substitution of the R-residue in some PtSyb forms (PtSyb8–11) or of the Q-residue in one of the PtSyx molecules (PtSyx12). (Note that such aberrations also occur in established higher eukaryotic

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systems [Fasshauer et al., 1998]). Even a SNARE domain may not be identifiable in some of the Paramecium R-SNAREs, such as PtSyb4 and 5 which are prognosticated as SNAREs by overall homology (Table 3.1). Although they display distinct subcellular localization there may be functional implications yet to be analyzed. The expected lipidic anchor seems to be absent from PtSNAP-25-LP (Schilde et al., 2008). On the one hand, its abundance in the cytosol (apart from association with many membranes of trafficking compartments) and on the other hand the presence of other characteristic features may justify the inclusion of these molecules in the list of PtSNAREs. In fact, there are comparable examples in other cells. However, what may the absence of important features imply in functional terms—may such SNAREs be functional? For the following reasons, it appears premature at this time to appreciate any role for the truncated SNAREs we found in Paramecium. (i) In yeast, the non-NSF type cochaperone, Sec 17 (different from the NSF homolog Sec 18) can complete fusion with normally nonfusogenic trans-SNARE complexes (Schwartz and Merz, 2009). (ii) Fragments of synaptobrevin can reduce the formation of dead-end syntaxin/SNAP-25 complexes (Pobbati et al., 2006). (iii) Soluble SNAREs can associate, in yeast, with a SNARE complex and thus drive vacuole interaction and fusion (Thorngren et al., 2004). Right away one would rather envisage some inhibitory effect in the latter two cases. (iv) In contrast to these situations, inhibitory SNAREs have been identified in mammalian cells as a set of t-SNAREs endowed with a transmembrane domain and occurring in addition to ‘‘normal’’ t-SNAREs; therefore, they may serve fine-tuning (Varlamov et al., 2004). Although no such analyses have been executed with ciliates, the examples clearly indicate that absence of a transmembrane domain would not necessarily entail an inhibitory/competitive role for SNAREs lacking a transmembrane segment. Substitution of Q for R in the 0-layer of SNAREs in yeast reduces cell growth and protein secretion, but can be restored by an inverse substitution in a partner SNARE (Graf et al., 2005; Ossig et al., 2000). Similarly, a Q ! R substitution in Syb of synaptic vesicles has no dramatic effect in hippocampal neurons (De´ak et al., 2006). In fact, deviations from the orthodox 0-layer have been detected even in normal cells (Fasshauer et al., 1998; Sutton et al., 1998). However, the effect of deviating 0-layer aminoacids other than R and Q in P. tetraurelia is difficult to anticipate without detailed analysis. Deviations from the orthodox heptad repeat structure in quite a few PtSNAREs (Kissmehl et al., 2007; Schilde et al., 2006, 2010; Tables 3.1 and 3.2) may reduce SNARE specificity (Fasshauer et al., 1998; Graf et al., 2005; Paumet et al., 2004) and zippering, and, thus, fusogenicity. In conclusion, we have identified numerous SNAREs, type R, Qa, Qc, Qb/c, in P. tetraurelia, cloned the respective genes, localized the proteins, and largely probed their function by gene silencing (as discussed in

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subsequent sections). These are the only data available on SNAREs in ciliates. Their number is about twice that assumed for the ur-eukaryote particularly when one also considers the twin isoforms originating from a recent whole genome duplication (‘‘ohnologs’’). These may now mainly serve gene amplification, in order to match the requirements for intense vesicle trafficking. A substantial number of PtSNAREs contributes to the extensive endo-/phago-/lysosomal system (Sections 4.2 and 6). Clearly, ciliates have increased their SNARE repertoire independently of, but in parallel to the evolution of multicellular organization.

3.2. Role of the SNARE-specific chaperone, NSF 3.2.1. General role of NSF NSF is a hexameric AAA-type ATPase with characteristic domain structure (Hanson and Whiteheart, 2005; Whiteheart et al., 2001). NSF is generally believed to be engaged in disentangling SNARE complexes after fusion, so SNAREs can become amenable to reuse (Littleton et al., 2001). Another possibility, though less considered in the literature, is the establishment of SNARE complexes during membrane-to-membrane attachment (Ungermann and Langosch, 2005; own data in Section 3.2.2). To appreciate the significance of NSF one has to bear in mind the following details. Fusion capacity in vivo depends on the assembly of a quarternary complex of a-helices from the SNARE domains. This includes a v (R)-SNARE and two or three t (Q)-SNAREs (Sections 2.1, 3.1, and 5), among them Qa-, Qb-, Qc-, and Qb/c-SNAREs (Fasshauer et al., 1998). Normally, a Qb/c with two pin-shaped (antiparallel) a-helical SNARE domains or, more rarely, separate Qb and Qc SNAREs are superimposed to a minimal SNARE pin of two membrane-anchored SNAREs (Fukuda et al., 2000). Thus, the minimum required for fusion is an R- and a QaSNARE (Grote et al., 2000; McNew et al., 2000). Specifically, in vitro studies have demonstrated that one type of v-/R-SNARE and one type of t-/Q-SNARE suffice to mediate fusion as long as they are inserted (by their carboxy terminus) in opposing membranes, so they can form ‘‘SNARE pins’’ (Fasshauer et al., 1998; Graf et al., 2005; Malsam et al., 2008; Melia et al., 2002; Sorensen et al., 2006). Beyond the fact that in vivo one SNARE complex is made of one v-/R- and three SNARE domains from two or three t-/Q-SNAREs (Section 2.1.1) (Fukuda et al., 2000; Jahn and Scheller, 2006; Malsam et al., 2008) it then has been found, moreover, that several such complexes are radially arranged around a potential fusion site (Section 5). All this arrangement depends on the SNARE-specific chaperone, NSF. NSF starts binding after preceding binding of a-SNAP to the Habc domain of syntaxin (Bock and Scheller, 1996; Rizo and Su¨dhof, 2002; Xu et al., 1999) (Section 2.1.1).

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3.2.2. NSF in Paramecium We could for the first time identify NSF in Paramecium on the basis of its distinct domain structure (Kissmehl et al., 2002). This includes a conserved AAA domain, Walker A and B as well as SRH domains and some other features (Patel and Latterich, 1998; Whiteheart et al., 2001). Furtheron, we have demonstrated disturbance of vesicle trafficking by PtNSF gene silencing (Kissmehl et al., 2002). Then we showed that specifically the assembly of ultrastructurally defined, functional trichocyst exocytosis sites requires the activity of NSF (Froissard et al., 2002). This was demonstrated with the temperature-sensitive mutant, nd9, that does not differentiate such sites, even though trichocysts are docked at the cell periphery, when cultivated at a nonpermissive temperature of 28  C (Beisson et al., 1980). During a 28  C ! 18  C shift, functional exocytosis sites are normally assembled within 2 h, but not when cells were silenced in the NSF genes (Froissard et al., 2002). Thus, in our system, NSF serves primarily establishing SNARE complexes for subsequent membrane fusion upon stimulation. (In addition, it may also serve dismantling SNAREs after fusion). Recently, in contrast to the mainstream hypothesis, a function of NSF as a chaperone before (!) fusion was also ascertained for neurotransmitter release (Kuner et al., 2008). This analysis relied on photolytic activation of a caged NSF peptide with adequate time resolution. Normally NSF would hydrolyze ATP, paralleled by rapid release from its sites of action (Whiteheart et al., 2001). Therefore, to localize NSF at its potential sites of action, that is, fusion sites, in Paramecium we have elaborated a suitable protocol. We applied careful cell permeabilization and infiltration with the inhibitor, N-ethylmaleimide, and nonhydrolyzable ATP-g-S (Kissmehl et al., 2002), followed by staining with antibodies against NSF. This resulted in hot spots indicating sites of repetitive fusion activity, including the cytoproct, the porus of the contractile vacuoles, and their connections with the radial canals—established sites of repetitive membrane fusion (Sections 6 and 9.1). NSF gene silencing also allowed us to virtually see, in the EM, sites of vesicle interactions. Vesicles looked as if ‘‘frozen’’ at many sites where this process normally cannot be realized because of the high speed/low frequency. Figures 3.4–3.7 present some examples of this approach (H. Plattner, B. Scho¨nemann, and C. Schilde, unpublished observation). In detail, we observed unusually extensive vesicle aggregates and stacks of rough ER, intermingled with aggregates of ribosomes (as described in fungi [Garrison and Boyd, 1974], chicken embryos [Birks and Weldon, 1971], or in crystals after isolation from bacteria [Avila-Sakar et al., 1994]), together with a substantial number of autophagosomes (Figs. 3.4 and 3.6). These features, including increased autophagy (Kuma et al., 2004), are clearly indications of inhibited metabolic activity, as one might expect from vesicle

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A ar

va

app

rER

ar

ar C

B n

rER

ER sER

ar

Figure 3.4 Examples of effects of NSF silencing in P. tetraurelia cells, as analyzed by standard ultrathin section EM analysis. Ultrastructural changes in the region containing rough ER (rER), aggregates of polysaccharides particles (app) and of ribosomes (ar). (A) Unusually extensive vesicle aggregates (va) in between rough ER stacks and aggregates of ribosomes and of polysaccharide particles. Magnification 13,000. (B) Considerable dilation of ER cisternae. ‘‘n’’ labels the nucleus. Magnification 8,500. (C) Transition between rough and smooth ER (rER, sER) and an aggregate of ribosomes (ar). Magnification 13,000. Unpublished data (see text).

trafficking impairment. The rough ER lumen may be dilated in some cells (Fig. 3.4B) or in continuity with smooth membrane aggregates, also in a form otherwise not seen (Figs. 3.5 and 3.6), as if ER ! Golgi or reverse

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asv

asv

asv

Figure 3.5 Examples of effects of NSF silencing in P. tetraurelia cells, as analyzed by standard ultrathin section EM analysis. rER stacks end in an aggregate of smooth vesicles (asv), as in the framed area which is enlarged in the insert. Magnification 11,500 (insert 22,000). Unpublished data (see text).

trafficking was affected. Such situations in Fig. 3.5 display continuity of a mass of smooth vesicular/tubular membranes with emanating cisternae of rough ER. This is interesting, considering the occurrence of specific physical contact sites between the two ER forms and the Golgi apparatus in higher eukaryotes (Sparkes et al., 2009) and the assumption that a Golgi dictyosome forms at specific sites along the rough ER (Foresti and Denecke, 2008; ViveroSalmero´n et al., 2008). In the ER, experimental disturbance of the interaction between SNARE, COP (coatamer), and tethering molecules causes accumulation of vesicles at the ER boundaries (Zink et al., 2009). Similarly, in our case, NSF silencing may have inhibited in one way or another vesicle trafficking from the ER on, or it may have frozen vesicles recycling to the ER. Figure 3.6 presents varying aspects of the Golgi apparatus, from normal to barely identifiable, as well as increased autophagy (Fig. 3.6E). In fact, in higher eukaryotes, SNAREs are responsible not only for vesicle transport from the ER to subsequent organelles (mainly the Golgi apparatus) and back. In compatibility

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A

B

C

D

E

app

aps

aps

Figure 3.6 Examples of effects of NSF silencing in P. tetraurelia cells, as analyzed by standard ultrathin section EM analysis. (A–D) show variations of the aspects of the Golgi apparatus and accumulations of small electron clear vesicles, and (E) shows large electron dense vesicle, in part representing autophagosomes (aps). app, aggregates of polysaccharide particles. Magnifications: 24,000 (A, D), 29,000 (B), 18,000 (C). Unpublished data (see text).

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ci as

as as as

as app

as

app

Figure 3.7 Change of the surface membrane complex (cortex). Note numerous vesicles of variable size, including very small vesicles (arrows) between the cell membrane and the outer membrane of the alveolar sacs (as) and even at a basis of a cilium (ci) at the top right. app, aggregates of polysaccharide particles. Magnification 8,000. Examples of effects of NSF silencing in P. tetraurelia cells, as analyzed by standard ultrathin section EM analysis. Unpublished data (see text).

with our observations, in HeLa cells a SNARE protein, syntaxin 18, has been shown to mediate the organization of ER subdomains including smooth/ rough ER and ER exit sites (Iinuma et al., 2009). In Fig. 3.7, numerous minute to medium-sized vesicles are attached to the cell membrane, to alveolar sacs and to some other membranes. At the cell membrane, this frequently occurs outside parasomal sacs so that additional sites now also have to be considered for constitutive exo- and endocytosis (Section 4.2). Vesicles arrested in contact with alveolar sacs may trace their biogenesis (Kissmehl et al., 2002). This is substantiated by the transfer of the SERCA-type Ca2þ-ATPase (pump) from the ER to alveolar sacs when expressed as a GFP-fusion protein in normal Paramecium cells (Hauser et al., 2000b). The chaperone function of NSF, together with any other chaperones which may emerge in future studies, involves ATP requirement. Whether ATP is required for membrane fusion has been debated over a long time. Experiments with Paramecium cells and cortex fragments isolated from them have suggested that ATP may be required for vesicle priming, but not for fusion (VilmartSeuwen et al., 1986). Later on this has been shown more clearly in much more detailed work, including electrophysiological studies with chromaffin cells (Xu et al., 1999). However, it is not known whether such function has to be attributed solely to chaperones. In the case of NSF, ATP-dependent priming would presuppose that it becomes active before, rather than after fusion.

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In sum, NSF can be characterized as a SNARE-specific chaperone that contributes to vesicle trafficking also in ciliates. However, NSF may exert its function not only after membrane fusion, to disentangle SNAREs from a cis-arrangement resulting from fusion (as most widely assumed), but in ciliates NSF operates also before fusion. Concomitantly, NSF is required to mediate exocytosis-competence to trichocyst docking sites. According to EM analysis, NSF silencing causes abolition of vesicle trafficking and thus results in the accumulation of vesicles at unexpected sites. Only ‘‘freezing’’ such interaction sites allows their visualization because they are normally too short-lived to be recognized.

3.3. ‘‘SNAREs and Co’’—targeting of vesicle traffic from the ER to the Golgi apparatus and beyond 3.3.1. Basic background from higher eukaryotes A set of potential key players participates in vesicle trafficking from the ER on: SNAREs, Arf-, and Rab-type small GTPases (G-proteins), COPs, the Hþ-ATPase as well as actin. Among them, in ciliates, COPs as well as small GTPases and their regulators have not yet been analyzed in any detail comparable to the other molecules. In mammalian cells, right at the beginning of their travel, some longintype SNAREs can bind to the signal recognition particle receptor, that is, a heterodimeric GTPase related to Arf which exerts a GAP activity (Schlenker et al., 2006) and mediates this first step of targeting. An ArfGAP also has to bind to the longin domain of a VAMP7-type R-SNARE in normal rat kidney cells in order to mediate vesicle cycling between the Golgi apparatus and the ER (Pryor et al., 2008). Vesicle budding within the ER requires COPs (Cai et al., 2007; Rothman, 1994). This generally occurs at specific sites (Foresti and Denecke, 2008; Vivero-Salmero´n et al., 2008) frequently seen in juxtaposition to the Golgi apparatus. We have followed up this aspect by PtNSF silencing (Section 3.2.2). There are hardly any specific motifs relevant for specific vesicle targeting known from any of the systems analyzed so far (Pfeffer, 2007). Although orthologs of syntaxin display rather similar intracellular localizations in widely different cell types, even in that case no specific targeting motifs are known. However, substituting lipid anchors for transmembrane segments in the yeast Qa-SNAREs, Sso1 and 2, and the R-SNAREs, Snc1 and 2 (or their mammalian equivalents), does not affect targeting, though it blocks membrane fusion (Grote et al., 2000; McNew et al., 2000). In mammalian cells the transmembrane domain (plus flanking positively charged aminoacids) is essential for trafficking from the ER on, as is binding of SNAP-25 (Yang et al., 2006). The length of the transmembrane domain has also been reported as crucial for targeting of some syntaxins, for example, types 3, 4, and 5 in some mammalian systems (Watson and Pessin, 2001).

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More specific membrane interaction may be mediated by complex molecular interactions, including not only SNAREs, SUs of the Hþ-ATPase (see below), and monomeric G-proteins (Grosshans et al., 2006), but probably also components that are currently only partially known. For instance, ‘‘accessory/auxiliary proteins’’ may interact with SNAREs (Bethani et al., 2007; Medine et al., 2007; Rizo et al., 2006; Weninger et al., 2008). It is also unknown whether posttranslational modifications, such as phosphorylation of SNAREs, might contribute to targeting. Specifically for syntaxin 6, in mammalian cells, a motif within the middle part of the cytoplasmic domain has been found relevant for sorting (Watson and Pessin, 2000). Overall, the mode of targeting by which specific membrane interactions are achieved is assumed to be complex. Very likely it requires the interaction of several different components, and is, therefore, poorly understood for the time being. Clearly microtubule rails with associated motor proteins may contribute to efficient delivery (Soldati and Schliwa, 2006), but hardly to specific targeting proper (Hirschberg et al., 1998). The Hþ-ATPase has recently been attributed a key role in targeting vesicle flow (Maranda et al., 2001; Marshansky and Futai, 2008; Recchi and Chavrier, 2006). In mammalian (AtT-20, anterior pituitary) cells, it was shown by inhibitor studies that the Hþ-ATPase directs separate pathways for lysosomal and secretory proteins at the level of the trans-Golgi network (Sobota et al., 2009). How is specific protein targeting and subsequently specific membrane interaction possible, considering the absence of a signal peptide sequence, for instance, in SNAREs and in the SUs of V0 (proteolipid) part of the HþATPase? In fact, this is also the case in Paramecium (Kissmehl et al., 2007; Schilde et al., 2006, 2008, 2010; Wassmer et al., 2005, 2006). Some information along these lines is available from other eukaryotes, mainly yeast, where V0-SUs are inserted into the ER membrane (Forgac, 2007) by interaction with several assembly factors including the ‘‘vacuolar ATPase assembly integral membrane protein,’’ VMA21 (http://www.uniprot.org/uniprot/Q3ZAQ7), and also quite recently identified factor, Voa1p. This is a double membrane-spanning glycoprotein with a signal peptide of 24 aminoacids and a dileucyl motiv for ER retention (Ryan et al., 2008). Hþ-ATPase components can escape from this strong interaction only after full assembly. This is enabled by another assembly factor, Vma21p, which binds to COPII-type coats and thus mediates budding for delivery to the Golgi apparatus (Malkus et al., 2004). ER/Golgi SNAREs of the types Sec22 and syntaxin may be included in such complexes (Mossessova et al., 2003; Springer and Schekman, 1998). This is facilitated by the longin domain of Sec22 (Liu et al., 2004) by means of a conformational motif (Mancias and Goldberg, 2007). From the Golgi apparatus on, the V1 part of the Hþ-ATPase associates with syntaxin 1 which in kidney cells is, thus, delivered to the cell surface (Schwartz et al., 2007b). Association of the Hþ-ATPase B- (Zuo et al., 2008) and C-SU with F-actin is another regulatory aspect (Beyenbach and

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Wieczorek, 2006). Note that syntaxins, though conventionally designated as t-SNAREs, also have to travel in vesicles from the ER on for final delivery to the respective target membranes. An additional interaction partner leading to site-specific membrane delivery is clathrin, for example, for vesicle targeting to specific domains of the cell membrane (Deborde et al., 2008). It remains to be established, also with ciliates, whether there is any crosstalk to other components determining vesicle targeting. Another aspect of increasing complexity is the budding of different secretory vesicle types from different sites of the Golgi apparatus (Muniz et al., 2001; Spang, 2009). This also concerns glycosylphosphatidyl inositol (GPI)-anchored proteins, such as variant surface antigens (Section 4.2). In yeast, GPI-anchored proteins are suggested to be transported, from the ER on, in separate vesicles to the Golgi apparatus and from there to the cell membrane; this may be mediated by specific COP interactions (Mayor and Riezman, 2004; Muniz et al., 2001). Whether this would hold true for protozoan variant surface antigens and which subset of SNAREs, GTPases, etc., would be involved is not known. 3.3.2. Aspects pertinent to ER/Golgi trafficking in ciliates Only parts of this puzzle are known from the currently best analyzed ciliate, P. tetraurelia. The Hþ-ATPase can be recognized from the ER on (Wassmer et al., 2005, 2006). A longin-type Sec22 ortholog occurs (Kissmehl et al., 2007), probably also somehow intermingled between the ER and the numerous Golgi units. Unfortunately, Paramecium’s Golgi apparatus is scattered in so minute dictyosomes (Este`ve, 1972), as it is in Tetrahymena (Kurz and Tiedtke, 1993), that localization of specific components to such elements is difficult to achieve. By EM work, smooth coats comparable to COP-type vesicles also occur at the ER/Golgi interface and on the Golgi apparatus of Paramecium (Allen and Fok, 1993; Garreau De Loubresse, 1993). Similar situations are visible also in micrographs from Tetrahymena (Kurz and Tiedtke, 1993). However, molecular identification of COP SUs as well as of specific components of the clathrin-type coats, both at the trans-side of the Golgi apparatus, is still missing. Such coats could mediate formation of secretory organelles (trichocysts) and of lysosomal vesicles, respectively, as known from higher eukaryotes. During NSF gene silencing, in Paramecium, we observed that the rough ER, at some sites, is continuous with a tightly entangled mass of smooth, tubulo-vesicular membranes without any visible association with a Golgi apparatus (Fig. 3.5; Section 3.2.2). This could represent sites where normally vesicle budding and fusion would occur (Section 3.3.1). At other sites, the rough ER is considerably swollen, probably because transport of lumenal proteins fails after NSF silencing. There are several trivial, though basic open questions concerning passage through the Golgi apparatus in ciliates. Which are the molecular components of the two types of membrane coats? Do lysosomal enzymes go through the

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Golgi apparatus? To what extent is the Golgi apparatus involved in trichocyst biogenesis? Is PtAct8, a resident of the Golgi apparatus (Sehring et al., 2007a), involved in Golgi trafficking, in analogy to higher eukaryotes (Cao et al., 2005; Carreno et al., 2004)? Again we are confronted with fundamental questions that have been settled for higher eukaryotes already quite some time ago (Section 3.3.1), yet only in part for ciliates. Several reports have dealt with the release of acid hydrolases, probably of lysosomal origin, from Tetrahymena cells (Banno et al., 1993; Taniguchi et al., 1985). More detailed analysis of a cysteine protease revealed expression as a preproprotein, that is, with the potential of a passage through the Golgi apparatus (Herrmann et al., 2006). As known from higher eukaryotes, on the way through the cell, SUs of the V1 part of the Hþ-ATPase molecule can be exchanged (Marshansky and Futai, 2008), depending on the local ‘‘cellular environment’’ (Qi and Forgac, 2007). Thus, different lumenal pH values can be produced in the different compartments. In the Paramecium cell, SUs of the catalytic V1 part are also exchanged on the way through ER, Golgi, and beyond (Wassmer et al., 2005, 2006, 2009). The unsurpassed number of 17 genes encoding in Paramecium the a-SU (Wassmer et al., 2006) that forms part of the V0/V1 connecting stalk may, by numerous combinations with other SUs, endow the holo-enzyme with locally different Hþ-transport kinetics. (This aspect has not yet been analyzed in any detail.) Based on results from higher eukaryotic cells one now assumes that this can entail a transmembrane signal by conformational change of the holoenzyme depending on the lumenal pH. In detail, a conformational change can allow for the docking of specific Arf proteins and their activators which in turn can mediate specificity of membrane interaction—or at least part of the specificity (Maranda et al., 2001; Recchi and Chavrier, 2006). Although this has been shown primarily for delivery of cargo from early to late endocytotic vesicles in mammalian cells it may be a mechanism common to many trafficking steps (Brown et al., 2009; Hurtado-Lorenzo et al., 2006) including phagosome processing (Steinberg et al., 2007). In conclusion, complex interactions may represent a basic scenario to explain in part the specificity of transport steps from the ER, via Golgi apparatus, to the secretory pathway as well as to phago- and lysosomes. In fact, in Paramecium, Hþ-ATPase components occur in different combinations from the ER on as well as along the secretory pathway and in the many compartments of the endo-/lysosomal apparatus (Wassmer et al., 2005, 2006, 2009), as summarized in Table 3.4. 3.3.3. Dense core-secretory vesicle biogenesis and trafficking in Paramecium and Tetrahymena As usual with dense core-secretory vesicles, the biogenesis of ciliate ‘‘extrusomes’’ starts in the ER. As to the passage of trichocyst precursor elements through the Golgi apparatus, there is different evidence available. First,

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immuno-EM localization of the main contents proteins suggests transit through the trans-Golgi in Paramecium (Garreau De Loubresse, 1993; Vayssie´ et al., 2001) and in Tetrahymena (Turkewitz et al., 1991). Second, an antigen connecting the trichocyst matrix with the organelle membrane can also be localized to the Golgi apparatus (Momayezi et al., 1993). Third, the glycosylation pattern derived from lectin binding studies (Glas-Albrecht et al., 1990), particularly the inclusion of fucosylation sites (Allen et al., 1988), suggests such a pathway. In Paramecium, the trichocyst membrane is probably endowed with a v (R)-SNAREs, type PtSyb5 (Schilde et al., 2010), as discussed below. Small G-proteins have also been addressed by GTP-overlays and differences between secreting and nonsecreting cells have been reported (Peterson, 1991). Any molecular details, however, await exploration. Along these lines one has to consider not only docking and fusion capacity required for exocytosis, but also the multiple fusions of trichocyst precursor vesicles (Garreau De Loubresse, 1993; Gautier et al., 1994). Strikingly, trichocysts contain an Hþ-ATPase (Wassmer et al., 2005, 2006) although they are not remarkably acidic compartments (Garreau De Loubresse et al., 1994; Lumpert et al., 1992). Also precursor vesicles are not acidic (Garreau De Loubresse et al., 1994). Here, protons may contribute to the assembly of trichocyst matrix proteins (tmx, trichynins) in crystalline form, and this may be linked to posttranslational processing which in turn enables docking at the cell membrane (Gautier et al., 1994). In fact, the Meþ/Hþ exchanger, monensin, blocks maturation and transport of trichocysts (Garreau De Loubresse et al., 1994). Whenever in mutants, or by experimental manipulation proteolytic protrichynin cleavage fails, EM morphology of trichocysts is aberrant, and no delivery to the cell membrane takes place (Gautier et al., 1994; Pollack, 1974; Pouphile et al., 1986). The mostly highly acidic trichynins (Tindall, 1986) may trap protons and, thus, obscure their presence in color assays. Exchange of protons for other ions may also be relevant for trichocyst matrix assembly. With dense core-secretory vesicles of Tetrahymena (‘‘mucocysts’’) one would expect a similar situation, as they also contain acidic proteins (Bowman et al., 2005a; Chilcoat et al., 1996) and maturation by proteolytic cleavage also occurs (Turkewitz et al., 1991; Verbsky and Turkewitz, 1998). In fact, several aspects relevant for Paramecium are also relevant for Tetrahymena (Bowman et al., 2005a; Bradshaw et al., 2003; Turkewitz et al., 1991). Again different mutations are available (Bowman and Turkewitz, 2001; Orias et al., 1983). On this background, it is surprising that the assembly of the mucocyst contents core (Grl ¼ granule lattice) proteins can undergo crystallization still in the ER (Cowan et al., 2005). It would be interesting to see whether mucocysts dispose of a similar endowment with an Hþ-ATPase as trichocysts and how this contributes to processing and condensation of contents. In their tip region, Tetrahymena mucocysts accumulate an independent, soluble group of proteins (Grt ¼ granule tip) defined by a C-terminal

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b/g-crystallin domain (Bowman et al., 2005b; Rahaman et al., 2009). Homolog proteins also occur in Paramecium (unpublished observation). Whereas crystalline matrix proteins serve rapid contents decondensation, thus expelling trichocysts upon stimulation in a Ca2þ-dependent manner (Section 7), the function of crystallin-like soluble proteins remains to be determined. It is worth noting that Grt proteins are described as sticky (Rahaman et al., 2009) and that trichocyst tips contain soluble, inherently sticky secretory lectins (Haacke-Bell and Plattner, 1987) whose trafficking pathway has not been analyzed as yet. In Tetrahymena, Grl and Grt proteins, respectively, are assumed to be sorted along independent pathways (Rahaman et al., 2009). Acidification or pH-dependent maturation of secretory matrix components—which one determines trafficking? To decide this question one would have to manipulate components of the Hþ-ATPase and the endopeptidase (Collins and Wilhelm, 1981) responsible for secretory protein processing. In fact, inhibition of the subtilisin or cathepsin family proteases in T. thermophila causes a delay in the processing of the mucocyst matrix proteins (Bradshaw et al., 2003). (Note, however, that Elde et al., 2007, by contrast, assume the absence of a ‘‘convertase’’ in T. thermophila and P. tetraurelia, based on database search.) Subtilisin-type endoproteases are established enzymes for secretory proprotein processing in mammalian cells (Rouille´ et al., 1995). Remarkably, in Tetrahymena, cathepsin follows the secretory pathway (Section 4.2). Also the peptide cleaved off from the Grl proteins in Tetrahymena go the constitutive exocytotic pathway (Bowman et al., 2005a), just as in mammalian (pancreatic b-) cells (Arvan et al., 1991). Also in this cell-type acidification by the secretory organelle Hþ-ATPase is mandatory for secretory proprotein ! protein conversion by acid endoprotease activity (Sun-Wada et al., 2006). Thus, the answer to the question on top says that, also in ciliates, both factors are relevant as they condition each other. Biogenesis of mature chromaffin granules by homotypic fusion of precursor organelles requires syntaxin 6 (Urbe´ et al., 1998). For Paramecium we have suggestive evidence for the relevance of PtSyb5, based on the fact that overexpression as a GFP-fusion protein alters trichocyst processing (Schilde et al., 2010) in a way reminiscent of mutants described in Paramecium (Vayssie´ et al., 2001) as well as in Tetrahymena mucocysts (Bowman and Turkewitz, 2001). We have evidence for a crosstalk between the lumenal and the cytoplasmic side of trichocysts, some details being presented in Section 3.3.3. What may the signals be? In mammalian gland cells interaction of the lumenal with the cytoplasmic side may be mediated by syncollin and a lumenal GPIanchored protein (Kalus et al., 2002) or by the transmembrane protein, phogrin, as well as by different lumenal scaffolding proteins anchored by an a-helix (Dikeakos and Reudelhuber, 2007). In Paramecium, some monoclonal

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antibodies recognize, after fast freezing and freeze-substitution, fragile periodic connections situated between the trichocyst matrix and the membrane (Momayezi et al., 1993) so that a kind of transmembrane crosstalk appears possible. When trichynin precursors are not cleaved, as in the tl (trichless) mutant (Pollack, 1974), these are secreted by numerous small trichocyst precursor vesicles along the constitutive pathway (Madeddu et al., 1994). Looking back at the transport from the ER on, via the Golgi apparatus and beyond we emphasize the following essential findings. At least some of the SNAREs, together with some of the Hþ-ATPase SUs are inserted into the ER membrane and escorted to the Golgi apparatus by very intriguing molecular interactions (not yet known in detail from ciliates). From there, molecules can be passed over by vesicle flow to other parts of the trafficking pathway, including secretory components. Although there are reasons to assume a similar scenario for ciliates, these aspects have not yet been analyzed in detail. Specifically from ciliates we know that proper secretory contents processing and assembly is required for delivery further on along the secretory pathway. In Paramecium, this depends on the activity of the organellar Hþ-ATPase. Up to now, there is only circumstantial evidence for the occurrence of an R-SNARE in the trichocyst membrane.

4. Exocytosis and Endocytosis 4.1. Exo- and endocytosis in general From yeast to man, membrane interactions leading to exocytotic membrane fusion require a teamwork of a multitude of proteins including SNAREs and a variety of auxiliary proteins. These include SM proteins (Sec1/ Munc18 complex) as well as Rab-type GTPases and their modulators and effectors, etc. (Cai et al., 2007; Spiliotis and Nelson, 2003; Su¨dhof, 2007). However, in the details requirements differ between stimulated and constitutive exocytosis inasmuch as the latter uses an octameric protein complex for tethering vesicles to the cell membrane whose biogenesis it controls. This ‘‘exocyst’’ complex, detected in yeast (Terbush et al., 1996), has homologs up to mammals (Grindstaff et al., 1998, Yeaman et al., 2001), whereas no information is available so far for ciliates. In the course of stimulated exocytosis, ‘‘clear’’ and ‘‘dense core’’ secretory vesicles liberate their secretory contents, that is, neurotransmitters and proteins, respectively. The time period required is much shorter for clear vesicles. Ca2þ requirements and sensors are also different for the different types of stimulated exocytosis (Section 7), as are other parts of the molecular machinery engaged in membrane interaction. This is also the case with the subsequent retrieval of vesicle membranes by exocytosis-coupled endocytosis. The steady-state delivery of membrane components by ‘‘unstimulated’’ (constitutive) exocytosis

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needs no Ca2þ signal (Burgoyne and Clague, 2003; Jaiswal et al., 2009; Schwartz and Merz, 2009). For constitutive endocytosis, in the past few years a variety of mechanisms—with different cytoplasmic coats or without a coat visible in the EM—have been established, although many details are still unsettled (Kirkham and Parton, 2005; Mayor and Pagano, 2007). In human cells, some of these processes are modulated by dynamin, clathrin, and actin, but reportedly no Ca2þ is required ( Jaiswal et al., 2009). A precisely timed study with chromaffin (PC12) cells, molecules emerge at sites of constitutive endocytosis in the following sequence: clathrin, dynein, and actin (Felmy, 2009). This is complemented by an elegant recent study with higher eukaryotic systems where molecules proposed to be required for endocytosis have been subject to a detailed analysis (Ohya et al., 2009). Thereby SNAREs, Rab GTPases, and regulators of their nucleotide exchange/activation cycle, in addition to Rab effector molecules, have been probed individually and collectively in an in vitro assay The data support the concept that, Rab proteins, more than SNAREs, determine organelle-specific membrane interactions. In ciliates, only a selection of these mechanisms is ascertained; for details, see the respective sections. Exo-/endocytosis encompasses (i) stimulated dense core-vesicle exocytosis (‘‘extrusomes’’; trichocysts in Paramecium, mucocysts in Tetrahymena) and (ii) rapidly ensuing detachment of empty membranes (‘‘ghosts’’) from the cell membrane and their internalization (exocytosis-coupled endocytosis). This is independent of endocytosis by bona fide clathrin-coated vesicles (parasomal sacs) near ciliary bases. This section can be summed up as follows. The clear distinction between stimulated and constitutive exo-/endocytosis becomes evident also in ciliates. The respective sites are endowed with very different molecular components relevant for vesicle trafficking, as will be specified in the subsequent Sections 4.2 and 4.3.

4.2. Constitutive endocytosis and exocytosis in ciliates 4.2.1. Parasomal sacs as sites of constitutive exo-endocytosis Parasomal sacs are bristle-coated omega-shaped profiles stereotypically associated unilaterally with ciliary bases in Paramecium (Allen, 1988) and in Tetrahymena (Elde et al., 2005). They serve constitutive endocytosis. In Paramecium, parasomal sacs contain SUs of the Hþ-ATPase (Wassmer et al., 2006). The coat is most likely made of clathrin (though molecular proof is scant), which would be in line with its presence in the Paramecium and in the Tetrahymena databases. Involvement of clathrin-associated adaptor protein, type AP2, in Tetrahymena (Elde et al., 2005) strongly supports a clathrin-mediated mechanism. In Paramecium the situation may be similar, probably with the involvement of dynamin (Wiejak et al., 2004). (However, dynamin can equally support some other internalization modes, as

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known from higher metazoans [Mayor and Pagano, 2007; Miaczynska and Stenmark, 2008]). Accordingly, the dynamin activating protein phosphatase, type 2B (calcineurin), as specified in P. tetraurelia by Fraga et al. (2010), is enriched at parasomal sacs of P. tetraurelia (Momayezi et al., 2000). In an unrooted phylogenetic tree dynamin of the ciliates, P. tetraurelia and T. thermophila, cluster only to a small extent with each other; however, they do not at all cluster with any other group, from mammals down to the apicomplexan relatives (Breinich et al., 2009). This is just one out of many examples of independent diversification in protozoa in general and in ciliates in particular. Occurrence of SNAREs in parasomal sacs of P. tetraurelia had to be expected from proteomic analysis of clathrin-coated vesicles isolated from human cells. This has revealed NSF, different syntaxins (types 6, 7, and 8), and Rab-type monomeric G-proteins (Borner et al., 2006). In polar epithelia, specific SNAREs (TI-VAMP) are dedicated to the delivery of GPIanchored proteins to the apical surface (Pocard et al., 2007). Manipulation of SNAREs occurring in parasomal sacs membranes of P. tetraurelia, type SNAP-25-LP (Schilde et al., 2008) and PtSyb6 (Schilde et al., 2006), may contribute in the future to unraveling the poorly understood pathway of variant surface antigens and of other GPI-anchored proteins in ciliates. The association of actin, type PtAct8 with parasomal sacs (Sehring et al., 2007a) fits well the situation in coated pits/vesicles of higher eukaryotes (Giner et al., 2007; Kaksonen et al., 2006). Constitutive exocytosis delivers glycoproteins to the cell surface, among them the ‘‘variant surface antigens’’ which, in ciliates, are also called ‘‘immobilization antigens,’’ as antibodies generated against them can immobilize the respective ‘‘serotype’’ (Beale and Preer, 2008). As analyzed in more detail in Paramecium, they undergo permanent turnover (Klo¨ppel et al., 2009) since they are internalized via parasomal sacs (Flo¨tenmeyer et al., 1999), probably for degradation. On the biogenetic pathway, in Paramecium, native variant surface antigen molecules may be transported to the same sites for insertion into the cell membrane by constitutive exocytosis (Capdeville, 2000; Flo¨tenmeyer et al., 1999). Only recently, we have found evidence of additional potential sites for constitutive exocytosis and endocytosis, some also near the ciliary bases, but separate from parasomal sacs (Schilde et al., 2010). This evidence comes mainly from NSF silencing experiments (Section 3.2) that can disclose cryptic sites of membrane interactions by ‘‘freezing’’ their otherwise very fast dynamics. Variant surface antigens of the Paramecium surface are fixed by a GPIanchor (Azzouz et al., 1995, 2001) which, in contrast to other systems, is attached to the phospholipid bilayer by a ceramide residue (Benwakrim et al., 1998). Clathrin-mediated endocytosis would be one of several currently heavily discussed internalization pathways of GPI-anchored proteins (Campana et al., 2005; Frick et al., 2007; Linden et al., 2008). This pathway

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also holds for (mammalian) prion protein (Langhorst et al., 2008)—a molecule smaller than variant surface antigens. Since chances for transport via clathrin-coated vesicles increases with the bulkiness of the GPI-attached protein (Bhagatji et al., 2009), this may well explain the pathway we found for Paramecium variant surface antigens (larger than prions) by immunogold EM analysis (Flo¨tenmeyer et al., 1999). Circumstantial evidence also suggests that newly synthesized variant surface antigens are delivered to the same sites by constitutive exocytosis (Flo¨tenmeyer et al., 1999). This would not easily be recognizable on EM micrographs when both, exo- and endocytosis, alternate in a cycle because assembly of a clathrin coat requires a much longer time period, up to 1 min (Ehrlich et al., 2004), than exocytosis. Interestingly, in Trypanosoma, unequivocal evidence for the internalization of variant surface antigens, also GPI-anchored, via clathrin-mediated endocytosis has been obtained (Overath and Engstler, 2004). In protozoa, this internalization pathway may be used because—to our current knowledge—some other pathways known from mammalian cells (Kirkham and Parton, 2005; Mayor and Pagano, 2007) appear to be absent (or they are not yet identified). 4.2.2. Early and late endosomes In P. primaurelia soluble fluorescent wheat germ agglutinin, a lectin, is first accumulated below the plasmamembrane, followed by delivery to food vacuoles (Ramoino et al., 2001). Applying exogenous proxidase and EM analysis in P. multimicronucleatum reveals the transfer to disk-shaped vesicles below the basis of cilia (terminal cisternae ¼ early endosomes) and transfer to late endosomes (Allen et al., 1992). The ‘‘early’’ vesicles possess an antigen of the cell membrane. Molecular biology clearly provides an even more distinct picture as we find proteins specific for the different stages of vesicle internalization (Tables 3.1–3.4 and below). How is the molecular equipment of the terminal cisternae, that is, the early endosomes, in Paramecium (Allen, 1988)? We could localize PtSyb11 (Schilde et al., 2010), PtSyx3 (Kissmehl et al., 2007), and Hþ-ATPase SUa1 (Wassmer et al., 2005, 2006) to this organelle. No specific SNAREs for homotypic fusion between early endosomes have been found in higher eukaryotes (Brandhorst et al., 2006). This may be even more valid for Paramecium where the stereotype arrangement of these well-defined structures would not suggest occurrence of homotypic fusion. If so, the dual endowment with R- and Q-type SNAREs, PtSyx3 and PtSyb11, may indicate additional vesicle input, for example, from the Golgi apparatus (Allen, 1988). From NSF gene silencing experiments, we have recently obtained evidence of trafficking of smooth-surfaced vesicles to/from other sites of the cell membrane, even within the normally very narrow cleft between the cell membrane and alveolar sacs (Schilde et al., 2010; H. Plattner,

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B. Scho¨nemann, and C. Schilde, unpublished observation). Near the ciliary basis there may occur some particular sites predetermined for vesicle trafficking, as PtSyb10 is enriched there in patches (Schilde et al., 2010). This aspect is discussed in Section 9.2. The most crucial points of this section can be summarized as follows. Specific SNAREs, Hþ-ATPase, and actin isoforms are clearly engaged in constitutive endocytosis in ciliates, as we find specific paralogs associated with the established sites, the parasomal sacs. From NSF silencing experiments with Paramecium cells, we also obtained evidence for the relevance of SNAREs for constitutive exocytosis and/or endocytosis outside the established sites.

4.3. Stimulated exocytosis and exocytosis-coupled endocytosis in ciliates 4.3.1. Assembly of exocytosis sites After delivery to the cell surface, an estimated period of several minutes may suffice to acquire exocytotic fusion capacity of trichocysts (Plattner et al., 1993). When Paramecium cells of the temperature-sensitive mutant, nd9 (Beisson et al., 1980; Froissard et al., 2001), are transferred from a nonpermissive to a permissive temperature (28  C ! 18  C), almost all sites achieve exocytosis competence, paralleled by assembly of ‘‘rosettes’’ (aggregates of intramembraneous particles/integrated proteins seen in freezefractures; Section 5.2) within hours (Froissard et al., 2002). This does not contradict our previous estimation of much shorter times for the individual process (Plattner et al., 1993), as this seeming discrepancy is observed with all steps of the exo-endocytosis cycle due to a certain degree of asynchrony (Knoll et al., 1991a; Plattner and Hentschel, 2006). Interestingly, NSF gene silencing during the 28  C ! 18  C transfer of nd9 cells can suppress rosette assembly and acquirement of fusion competence (Froissard et al., 2002); Section 5. This implies that NSF, in this case, is required to establish SNARE complexes, rather than to disassemble them after exocytosis. The latter has been found with some other systems and then tacitly generalized (Littleton et al., 2001). In fact, the sequence we described has also been found with bovine chromaffin cells (Xu et al., 1999). Even more strikingly, recent electrophysiological studies with neuronal cells analyzed under conditions of sufficient time resolution concludes an ongoing interaction of NSF with SNAREs to maintain them in an assembled state ready for fusion (Kuner et al., 2008). Since trichocyst docking sites are newly formed over many cell divisions independently of any exocytosis, this also implies a primary role of NSF for SNARE assembly, rather than disassembly after fusion (although NSF may also support SNARE rearrangement after trichocyst exocytosis).

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4.3.2. Dynamics of exocytosis in Paramecium In Paramecium, an appropriate stimulus provokes the immediate release of trichocysts. Such a stimulus can be the contact with a predatory ciliate, such as Dileptus, whose attacks are survived selectively by exocytosis-competent cells, as detected by Harumoto and Miyake (1991). Based on this work, subsequent studies by Knoll et al. (1991b) have shown that local trichocyst release keeps the predator at a distance, thus allowing the Paramecium cell to escape. While the actual chemical stimulus is not known, a mechanical stimulus does not produce this phenomenon. In contrast, it can be perfectly mimicked by polyamines such as aminoethyldextran, AED (Plattner et al., 1984, 1985b). Meanwhile, AED has been accepted as a standard secretagogue for Paramecium. The dynamics of synchronous trichocyst exocytosis and exocytosis-coupled endocytosis has been thoroughly analyzed by quantitative quenched flow/cryofixation/freeze-fracture EM analysis (Knoll et al., 1991a; Plattner and Hentschel, 2006; Plattner et al., 1997). Synchronous trichocyst exocytosis upon stimulation with AED occurs within 80 ms, followed by 270 ms for endocytotic membrane resealing and still longer times for pinching off trichocyst ‘‘ghosts.’’ Again, the individual fusion/resealing pore has a much shorter life-time than registered for the overall phenomenon in the entire cell population analyzed. For the individual fusion event, we estimate a time requirement of 1 ms, that is, below the methodical time resolution which was available to us (Plattner et al., 1993). With liposomes, this has been substantiated in vitro by fast kinetics analysis (Kasson et al., 2006) and in vivo, by patch-clamp analysis, with mammalian cells (Breckenridge and Almers, 1987), as summarized by Sorensen, 2005). The open time of a fusion pore (opening to full width) is about in the range of 1 s. It is similar when recorded by electrophysiology with mammalian dense core-vesicle systems (Fang et al., 2008) as it is for the stimulated exo-endocytosis coupling in Paramecium. In the latter case, coupling is accelerated with increasing [Ca2þ]0 (Plattner et al., 1997), as it is in mammalian systems (Henkel and Almers, 1996; Rosenboom and Lindau, 1994), as discussed in Section 7. What is the molecular background of these processes? No ATP is required for membrane fusion per se during trichocyst exocytosis (VilmartSeuwen et al., 1986). There is currently unanimous agreement on this aspect also in other systems (Sorensen, 2005) though initially this issue had been hotly debated. In Paramecium, PtSyx1 is considered the t-type SNARE required for trichocyst exocytosis, as silencing of this gene greatly reduces the stimulated exocytotic response (Kissmehl et al., 2007). Strikingly, PtSyx1 is scattered over the entire ‘‘somatic’’ (nonciliary) cell membrane, whereas one would expect concentration at the sites with rosettes, directly above the trichocyst tips. (The aspect of potential microdomain arrangement is discussed in Section 5.) The rather ubiquitously distributed PtSNAP-25-LP is also recognized over the

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entire cell boundary (Schilde et al., 2008). Both, PtSyx1 and PtSNAP-25-LP may also mediate any other membrane fusion events occurring at the cell surface. How about other components for stimulated exo-/endocytosis and other membrane fusion processes? Unfortunately, the v-type SNARE pertinent to trichocyst exocytosis could not be identified unequivocally so far, but PtSyb5 is a realistic candidate (Section 3.3). Another open question pertinent to trichocyst exocytosis concerns the nature of the Ca2þ-sensor which has to be expected at such sites, as outlined in Section 7. Finally, the relevance of calmodulin occurring at trichocyst exocytosis sites (Momayezi et al., 1986; Plattner, 1987) expects elucidation. Calmodulin is known to be mandatory for the assembly of a functional trichocyst exocytosis site (Kerboeuf et al., 1993). In mammalian cells, calmodulin binds to synaptobrevin (Quetlas et al., 2002) and, thus, can affect the arrangement of SNAREs. By interaction with the auxiliary protein, Munc13, calmodulin also drives priming for neurotransmission (Dimova et al., 2009). However, from its Ca2þ-binding properties and kinetics, calmodulin is generally considered inappropriate to serve as a Ca2þ-sensor for a rapid exocytotic response. Beyond this, calmodulin is a Ca2þ-sensor for the different forms of endocytosis in nerve terminals (Wu et al., 2009) and, thus could exert the same function in ciliates. In sum, calmodulin may be a multifunctional component of dense core-vesicle docking/exocytosis sites also in ciliates. Actin is another modulator of exocytotic fusion pore dynamics, for example, in pancreatic acinar cells (Larina et al., 2007). Remarkably, actin flanks trichocyst docking sites in Paramecium (Kissmehl et al., 2004) and trichocyst docking is reportedly inhibited by cytochalasin B (Beisson and Rossignol, 1975). Combining fast freezing technology during stimulated synchronous trichocyst release with EM analysis (Plattner and Hentschel, 2006) has shown that membrane fusion probably occurs within a submillisecond time scale and presents itself as a 10 nm large spot (‘‘fusion pore’’). All this corresponds to the temporal and spatial resolution achievable by the method used (Knoll et al., 1991a; Section 5). Subsequently, the pore expands and thus allows access of extracellular Ca2þ to the trichocyst contents. This entails vigorous expansion of the trichocyst matrix (Bilinski et al., 1981) by Ca2þ binding to specific matrix proteins (Klauke et al., 1998), as commented in Section 7.2. Not only trichocyst membranes (‘‘ghosts’’) are internalized by exoendocytosis coupling, but also dischargeable intact trichocysts can be caused to dedock and eventually to redock. In nondischarge strains of P. tetraurelia, trichocysts can be detached from the cell surface and brought to redocking under conditions described (Pape and Plattner, 1990). This observation is supplemented by the following experiments. Secretory contents release is blocked in a P. caudatum mutant (Watanabe and Haga, 1996) because of

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defective Ca2þ-binding to secretory matrix proteins (Klauke et al., 1998). In normal cells, contents release can also be inhibited by exocytosis stimulation under conditions unfavorable to matrix expansion, thus resulting in ‘‘frustrated exocytosis’’ (Klauke and Plattner, 2000). This has been visualized by the styrene dye FM1-43 that is spontaneously incorporated into the cell membrane and diffuses into the secretory organelle membrane upon fusion (Henkel et al., 1996). In this case, trichocyst membranes fuse with the cell membrane, just as during exocytosis, but without contents release. This ‘‘frustrated exocytosis’’ is followed by resealing, internalization, and redocking of the intact organelles which can be stimulated to perform normal exocytosis. The organelle must have a signal indicating its state (Section 3.3.3) because empty ‘‘ghosts’’ would go the degradation pathway. All this has specified for the first time membrane detachment as a distinctly regulated step. We summarize this section on exo- and endocytosis as follows. The highly efficient machinery of stimulated exocytosis is only partly understood on a molecular level, also in ciliates. Actin is found around docking sites underneath the cell membrane (Section 2.3.2). PtSyx1 is involved in stimulated exocytosis, though it is distributed all over the somatic cell surface. Quite uncertain is the type of v-SNARE, possibly PtSyb5, in the trichocyst membrane (Section 3.3.3). No evidence could be found in Paramecium for any contribution of the proteolipid part of the Hþ-ATPase to exocytotic membrane fusion, as elaborated in Section 5. Finally, we observe a specific difference between the detachment of intact trichocysts and their ‘‘ghosts’’ formed by exocytosis.

5. Possible SNARE Arrangement in Microdomains and Membrane Fusion 5.1. General aspects As mentioned, for fusion to occur, two of the SNAREs forming a SNARE pin have to be anchored by a transmembrane domain in the opposing membranes. Concomitantly, exchange of a transmembrane domain by a lipid anchor in R- or Qa-SNAREs inhibits fusion (Grote et al., 2000; McNew et al., 2000). Note that Qa/b-SNAREs, such as PtSNAP-25-LP, have no transmembrane domain. Using widely different methods, the number of SNARE molecules of any type occurring on the donor and receptor side has been estimated as about six (Han et al., 2004; Montecucco et al., 2005). This would suffice to form a kind of crown of SNARE pins for tethering, zippering, and subsequent fusion of membranes. At neurotransmitter release sites, indirect methods have suggested the occurrence of at least 3 (Lu et al., 2008), more likely 5–8, or a maximum of 10–15 SNARE pins (Montecucco et al., 2005). According to the Jackson

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group, the transmembrane domains of 5–8 syntaxins finally line a fusion pore (Han et al., 2004). SNARE complexes required for fusion can be accomodated in a  3–4 nm large area (Martens and McMahon, 2008). Some other measurements revealed formation of larger SNARE microdomains that define sites for vesicle docking and fusion (Lang et al., 2001). A cluster of 75 syntaxins, 50–60 nm in size can be assembled within 10 min (Sieber et al., 2007) by homo-oligomerization via interaction of the a-helical SNARE domains (Sieber et al., 2006). Clustering syntaxin 1 (equivalent to PtSyx1) in the cell membrane can be mimicked in reconstitution studies by adding cholesterol (Murray and Tamm, 2009) under conditions that differ from those characteristic of classical ‘‘rafts’’ or microdomains induced by scaffolding proteins. The authors assume this a potential regulatory aspect of localized exocytosis site formation. In this case, it remains unclear how interaction with SNAREs of the opposed membranes would not be sterically hindered during fusion. For further comments, see below. By contrast, synaptobrevins are reported not to be liable to oligomerization (Bowen et al., 2002). However, this is what we see with PtSyb10 in the plasmamembrane (!) surrounding the basis of cilia (Schilde et al., 2010), as outlined in Section 5.2. Are such microdomains formed by SNAREs only in the cell membrane? For the time being, this remains an open question. May SNAREs at the cell surface exert still other functions? In neuronal cells, syntaxin1, together with the scaffolding proteins reggie/flotillin clusters the (normal) Alzheimer amyloid precursor protein in the cell membrane, thus facilitating normal trafficking and processing (Sakurai et al., 2008). This would mean an alternative, indirect function of SNAREs, that is, microdomain assembly of a specific protein to prepare its piecemeal removal by endocytosis. Whether the reggie/flotillin-related scaffolding protein stomatin (occurring in the Paramecium database) can serve such a function or, alternatively, mediate formation of microdomains for intracellular signaling, as executed by reggie/ flotillin-based microdomains (Langhorst et al., 2005), remains to be analyzed with ciliates. Remarkably, in mammalian cells, in such microdomains GPIanchored proteins are enriched; among them is the prion protein, PrPc. This in turn can modulate, in neurons, [Ca2þ]i dynamics upon stimulation (Powell et al., 2008). Therefore, SNAREs can induce microdomains and these may perform, in addition to membrane-to-membrane interactions, other functions such as signaling. Currently, such aspects remain hypothetical paradigms yet to be analyzed with ciliates.

5.2. Aspects concerning ciliates In Paramecium, a microdomain-type assembly has been observed with Syb10 (Schilde et al., 2010). We have combined expression of PtSyb10 as a GFPfusion protein, monitoring by fluorescence imaging, and EM-analysis by applying anti-GFP antibodies and proteinA-gold-conjugates. Possible

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functional implications are discussed in Section 9.2. Another question concerning microdomain assemblies is the molecular identity of ‘‘rosette’’ particles seen on freeze-fractured exocytosis sites of trichocysts and mucocysts of Paramecium and Tetrahymena, respectively (Orias et al., 1983; Plattner et al., 1973), as well as in some other ciliates (Bardele, 1983). If a rosette particle would represent densely packed aggregates of single membrane-spanning domains of any type of SNAREs, one particle is estimated to accommodate 75 molecules (Plattner and Kissmehl, 2003b). This figure may just coincidentally be identical with that of plasmalemmal syntaxin 1 in the aggregates described by Sieber et al. (2007). It is noteworthy, however, that a rosette is an infallible indication of the presence of an exocytosis-competent trichocyst underneath (Beisson et al., 1976; Pouphile et al., 1986; Vayssie´ et al., 2001). Nevertheless, rosette particles still could represent integral membrane proteins unrelated to SNAREs. Work with Paramecium has gradually developed to a general view of membrane fusion (‘‘focal fusion concept’’)—importantly with the inclusion of proteins in the formation of a point-like fusion pore (Knoll and Plattner, 1989; Plattner, 1981, 1987, 1989; Plattner and Knoll, 1993), rather than an extended diaphragm (the intermediate fusion structure then commonly assumed). While point fusion had been described already by others with in vivo (Heuser et al., 1979) and in vitro systems (Verkleij et al., 1979), any essential role of membrane proteins had been envisaged for the first time in the work with Paramecium. This has been endorsed by the analysis of secretory mutants obtained by the Beisson group (Beisson et al., 1976, 1980; Lefort-Tran et al., 1981; Pouphile et al., 1986) and by the evident dependency of fusion capacity on defined protein arrangements at fusion sites, the rosettes (Plattner et al., 1973; Vilmart and Plattner, 1983). While all this currently may appear trivial it was far from being so at that time. Focal fusion (point fusion) has then unequivocally been demonstrated with improved temporal and spatial resolution by patch-clamp analysis (Breckenridge and Almers, 1987; Neher and Marty, 1982). For the time being, the discussion still goes on whether lipids (Fang et al., 2008), proteins (Han et al., 2004; Jackson and Chapman, 2006) or, alternatively, both (Chapman, 2008) may line the fusion pore—probably another trivial aspect, once settled. In Paramecium, c-SUs (proteolipid, V0 part) of the Hþ-ATPase are clearly not present at preformed exocytosis sites in Paramecium (Wassmer et al., 2005) (whereas the Hþ-ATPase is present in the trichocyst membrane [as discussed in Section 3.3]). Thus, it was not possible to expand a concept derived from homotypic (vacuolar) membrane fusion in yeast (Bayer et al., 2003; Mayer, 2002; Peters et al., 2001) to exocytosis. Interestingly, the perception of this hypothesis of membrane fusion is distinctly divided— believers cluster among Hþ-ATPase experts and doubters among most of those dedicated to the analysis of membrane fusion.

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Expulsion of trichocysts is enabled by instantaneous rearrangement of crystalline matrix components (Sperling et al., 1987; Section 7.2) before trichocyst exocytosis sites are rapidly resealed within 270 ms after exocytosis ( 80 ms), that is, 350 ms after stimulation (Knoll et al., 1991a). This membrane fusion event initiates ‘‘ghost’’ retrieval and is also of the focal type (Plattner et al., 1992). For technical reasons, it could be demonstrated only at a much later time, by patch-clamp analysis, that endocytotic membrane resealing in mammalian cells is also of the point-fusion type (‘‘fission pore’’) (Rosenboom and Lindau, 1994; Roux and Antonny, 2008). The essential points of this section are the following. The molecular identity of ‘‘rosettes,’’ the prominent freeze-fracture particle aggregates seen at preformed exocytosis sites in the cell membrane of ciliates—an indicator of exocytosis competence—has not yet been elucidated. t-SNAREs contributing to exocytosis are PtSyx1 and possibly PtSNAP-25-LP, while PtSyb5 is a candidate for a trichocyst v-SNARE. No such role could be assigned to V0-type SUs of the Hþ-ATPase as they do not occur at trichocyst exocytosis sites.

6. Phagocytosis The general background, mainly derived from macrophages, is as follows. Though phagosome formation, for instance in macrophages, is perceived as an indentation of the cell membrane, formation and detachment of a phagocytotic vacuole (phagosome) requires delivery of additional membrane involving NSF (Coppolino et al., 2001) and specific SNAREs (Braun et al., 2004; Hackam et al., 1998). Substantial membrane material is contributed by fusion of Rab11-positive recycling vesicles (Braun and Niedergang, 2006; Cox et al., 2000; Huynh et al., 2007) with the participation of synaptotagminV (Vinet et al., 2008) as a Ca2þ-sensor and VAMP3 as a v-SNARE (Bajno et al., 2000). Whereas early endosomes contribute little, if any membrane material, fusion with late endosomes/lysosomes follows at a later stage (Bajno et al., 2000), thus forming a phagolysosome. This transition can be followed by the respective Rab-type G-proteins (Haas, 2007; Novick and Zerial, 1997). In macrophages, fusion of a phagosome with late endosome/lysosome also delivers the Hþ-ATPase into the phagolysosomal membrane thus formed (Sun-Wada et al., 2009). Phagosome formation is paralleled by the assembly of an actomyosin coat (May and Machesky, 2001; Soldati and Schliwa, 2006), regulated by an Arf protein (Niedergang et al., 2003), and formation of a dynamin–amphiphysin complex for pinching off (May and Machesky, 2001), that is, for closing the vacuole and its detachment. Further on, there occurs multiple input and

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exchange of components. Concomitantly, proteomics of phagosome membranes from macrophages yields a variegated picture (Garin et al., 2001).

6.1. Phagocytosis in ciliates Phagocytosis has been analyzed in most detail by Richard Allen and Agnes Fok and their associates in P. multimicronucleatum using electron microscopy, including labeling with monoclonal antibodies (Allen, 2000; Allen and Fok, 2000; Fok and Allen, 1988, 1990). The phagocytosis cycle is sketched in Figs. 3.2 and 3.5. In their pivotal study, Allen et al. (1995) describe the expansion of the nascent phagosome membrane by membrane delivery in the form of ‘‘discoidal vesicles.’’ Pinching off is supported by F-actin (Cohen et al., 1984; Kersken et al., 1986), followed by fusion with ‘‘acidosomes’’ (Allen and Fok, 1983a) and retrieval of acidosomal constituents by discoidal vesicle membrane formation (Allen and Fok, 1983b). This membrane replacement was sensitive to cytochalasin B, but not to acidification (Fok et al., 1987). Then, lysosomal components are delivered and later on retrieved (Allen and Fok, 1984b). This is complemented by retrieval of an early population of discoidal vesicles (Allen et al., 1995), before the spent food vacuole discharges its contents at the cytoproct (Allen and Wolf, 1974), with an additional, late retrieval of a second population of discoidal vesicles (Schroeder et al., 1990). See Fig. 3.8 for a selection of molecular components involved in phago(lyso)some cycling in P. tetraurelia, as subsequently discussed. Application of cytochalasins to Tetrahymena (Gronlien et al., 2002a) and Paramecium (Allen and Fok, 1985; Cohen et al., 1984; Kersken et al., 1986; Zackroff and Hufnagel, 1998) inhibits phagocytosis. The different steps, from pinching off, acidosome and lysosome fusion as well as membrane retrieval from the cytoproct, have different cytochalasin sensitivity (Allen and Fok, 1985; Fok et al., 1985, 1987). Generally F-actin is a wellestablished contributor to phagosome formation, trafficking, and processing. In ciliates, the different sensitivity to disrupting drugs must be caused by the participation of different actin isoforms with different drug sensitivity (Sehring et al., 2007a), as specified in Section 2.3.2 below, and in Table 3.3. Phago(lyso)some biogenesis requires more detailed elucidation. In Paramecium, in addition to recycling ‘‘discoidal vesicles’’ one can see, in timeresolved microscopy, small vesicles associated with the oral cavity; they vigorously travel directionally along to local cytoskeletal elements (Ishida et al., 2001). In this region, we found vesicles with PtSyb8, PtSyb9, and PtSyb10 (Schilde et al., 2010). Also among PtSyx proteins, such as PtSyx7, PtSyx9, PtSyx10 we found some that are specifically dedicated to the phagocytotic system (Kissmehl et al., 2007). Since for these vesicles no information is available about specific markers, for example, small G-proteins, it is impossible at this time to classify them accordingly, but

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A

ss a cv

ds

fv Slightly acidic

gh tr

er

Acidic

Neutral

ga cf

pm

pof rv

ee

oc dv

ps ci

cp

as

B Constitutive exocytosis vesicles

Defecation: cytoproct (constitutive exocytosis)

Terminal cisternae (early endosomes)

Maturation stages of food vacuoles

Discoidal vesicles

Syx1, 4, 7, 9, 10, 11, 12-1, SNAP-25-LP

Lysosomes

Acidosomes

Syb6-1, 8, 9, 11 + H -ATPase: a1, a4, a5, a6, a9 c1, c4, c5 Food vacuole Actin1-1, 1-2, 1-9, 3, 4, 5, 8 Act1-2: speckles and lee-side tail Act1-9: lee-side tail Act5-1: cap etc.

Nascent food vacuole (phagocytosis)

Figure 3.8 The phago(lyso)somal cycle of Paramecium. (A) Formation and maturation of the phago(lyso)somal vacuole (‘‘food vacuole’’), with the change of lumenal pH from acidic (after fusion with numerous small acidosomes, as) to near neutral and neutral, for final discharge at the cytoproct (cp). For other abbreviations, see Fig. 3.2A. Scheme adapted from Wassmer et al. (2009). (B) SNAREs, actin, and HþATPase SU isoforms contributing to the phago-/lysosomal cycle in P. tetraurelia cells. Scheme as in Fig. 3.2B. Superimposed are data combined from Kissmehl et al. (2007), Schilde et al. (2006, 2008, 2010), Sehring et al. (2007a), and Wassmer et al. (2005, 2006). Magnification of micrographs 600. Source of the basic scheme as in Fig. 3.2A; micrographs from Sehring et al. (2007b).

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they may contribute to Paramecium’s phagosome formation in one or the other way. The organelle, commonly called the ‘‘food vacuole,’’ receives its Hþ-ATPase only after pinching off, by fusion with acidosomes. These are considered late endosomes (Allen et al., 1993). This distinction from other endosomes is compatible with several facts. (i) Acidosomes are endowed with Hþ-ATPase (Allen and Fok, 1983a, 1993), just like late endosomes. (ii) In Paramecium, they may be formed as follows: The early endosomes (terminal cisternae) although also containing an Hþ-ATPase (Wassmer et al., 2006), receive additional input from Golgi vesicles (Allen, 1988) (Sections 1.3 and 4). (iii) Recycling endosomes are generally devoid of any Hþ-ATPase in other systems (Gagescu et al., 2000; Hinton et al., 2009), but—as stated— they contribute to phagosome formation in mammalian cells, as they do in the form of discoidal vesicles in Paramecium. In sum, in Paramecium late endosomes and recycling vesicles contribute to food vacuole formation. The contribution of the small cytopharyngeal vesicles mentioned above remains to be established. For a more stringent classification of additional contributors one would have to know the orthologs of the organelle-specific Rab proteins, types 5, 7, and 11, as established in higher eukaryotic cells (Section 2.2). Food vacuoles travel through the cell, mostly steadily but locally in a saltatory manner, by cytoplasmic streaming (cyclosis). Velocities registered vary from 1 to 2 mm (Nishihara et al., 1999) or 2 to 4 mm s 1 (Sikora, 1981) in P. bursaria, whereas in P. multimicronucleatum maximally 6 mm s 1 has been measured (Ishida et al., 2001). This is fast enough to postulate active propulsion (see below).

6.2. Involvement of actin in phagocytotic cycle of ciliates Recent analyses have revealed a considerable number of actins and actinrelated proteins in T. thermophila (Kuribara et al., 2006) and an even much higher number of actins (subdivided into nine subfamilies) in P. tetraurelia, with a particularly high number of subfamily 1 members (Sehring et al., 2007a,b). Analysis of potential binding sites for drugs that disrupt (such as latrunculin A) or stabilize F-actin (phalloidin, jasplakinolide) have prognosticated widely different sensitivities, from fully sensitive to insensitive (Sehring et al., 2007a). The numerous isoforms occurring in Paramecium are specified in Table 3.3, those contributing to phagocytosis in Fig. 3.8B. In Paramecium and Tetrahymena, F-actin disrupting drugs, such as cytochalasins, do not inhibit the indentation of a phagocytotic cup, but they can slow down detachment of a phagosome (Cohen et al., 1984; Kersken et al., 1986) in a dose-dependent manner (Fok et al., 1985) and fusion with acidosomes (Allen and Fok, 1983a). It thus appears that closing of a phagocytotic vacuole requires a specific actin isoform still to be determined. Specifically, PtAct4 is involved in phagosome/food vacuole formation

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(Sehring et al., 2010)—a role it may share with Act1 in T. thermophila (Williams et al., 2006). PtAct4-1 is seen in association with nascent food vacuoles and its silencing reduces food ingestion (Sehring et al., 2010). Since PtAct4 has no prognosticated binding capacity for disrupting drugs (Sehring et al., 2007a), other actin paralogs, such as PtAct1-1 and PtAct1-2, may come into play. Altogether there seems to occur some finely tuned selectivity for actin isoforms along the phagocytotic pathway in Paramecium, whereas this is not so well known from higher eukaryotes. During cyclosis, actin isoforms are exchanged in P. tetraurelia (Sehring et al., 2007b): Actin types 1, 3, 4, 5, and 8 were seen on food vacuoles (Fig. 3.8B). PtAct1-2 and PtAct1-9 as well as PtAct5-1 can either fully cover the phagosome as a smooth layer or alternatively form a smooth, but only partial cover, or alternatively produce speckles. PtAct1-2 and PtAct1-9 can also produce a comet tail-like structure on the lee-side relative to the organelle movement (Sehring et al., 2007b) to which it probably contributes by propulsion in analogy to Listeria actin tails (Tilney and Portnoy, 1989). Any sequence of these different aspects of actin arrangement during phago(lyso)some cyclosis in ciliates has not been established as yet. For comparison, in macrophages the lifetime of such a tailed aspect of (bacteria-free) phagosomes is short (Zhang et al., 2002). In a more extensive study, also with macrophages, it was found that varying actin assemblies on phagosomes can regulate docking of, and fusion with lysosomes, while comet-tails can propel the organelle (Liebl and Griffiths, 2009). According to Nishihara et al. (1999) any contribution of actin to cyclosis of endosymbiontic algae in P. bursaria is unlikely, based on cytochalasin insensitivity. However, on the one hand, this may be explained by drug insensitivity of the relevant actin isoform in those cells. On the other hand, in analogy to plants (Shimmen and Yokota, 2004), actomyosin engagement would be expected. In fact, in Tetrahymena, requirement of the nonconventional myosin, myo 1p, for cyclosis has been established (Hosein et al., 2005). Scrutinized analysis of actin subtypes with regard to latrunculin A and myosin binding in Paramecium revealed that most of the actins associated with food vacuoles (PtAct types 1-1, 1-2, 1-9, 3, 4, 5, and 8; Table 3.3) possess full (PtAct1-1 and 1-2), partial (PtAct1-9, 4, 5), or no (PtAct3, 8) latrunculin A-binding sites (whose similarity to cytochalasin binding, however, is not established in ciliates), whereas myosin binding generally decreases from PtAct1-1 to PtAct8) (Sehring et al., 2007a). (Note that the relevance of our prognostication of myosin binding capacity would need more detailed experimental confirmation.) In sum, there exists some inherent capacity to exploit actin/actomyosin for cyclosis in ciliates. Defecation is reported to be insensitive to cytochalasin in Paramecium (Cohen et al., 1984). This contrasts with the occurrence at the cytoproct of PtAct1-1 (Sehring et al., 2007b) which possesses drug binding sites

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(Sehring et al., 2007a). The discrepancy may be resolved by assuming a role for PtAct1 isoforms in the process of pinching off discoidal vesicles (known to be supported by F-actin [Cohen et al., 1984; Kersken et al., 1986]), rather than food vacuole contents discharge proper. If so, the cytoproct could be clogged for ongoing food vacuole docking and discharge. In sum, F-actin isoforms can contribute to specific interactions of the food vacuole membrane with different components of the vesicle trafficking apparatus. To our knowledge, involvement of so widely different isoforms is not known from higher eukaryotic cells, although several aspects correspond to our observations with P. tetraurelia: (i) A regulatory function of actin during phagocytosis is generally acknowledged (Damiani and Colombo, 2003; Soldati and Schliwa, 2006). (ii) Actin has been found in phagosome membrane proteomics (Garin et al., 2001). (iii) Phagosomes undergo repeated cycles of actin assembly and disassembly (Yam and The´riot, 2004). As already mentioned, phagosomes receive multiple input from the formation stage (nascent food vacuole) on (Allen and Fok, 2000; Fok and Allen, 1990). As determined with P. multimicronucleatum, the membrane of the nascent food vacuole is formed mainly from recycling vesicles of dual origin, that is, discoidal vesicles derived from the membrane of spent phagosomes discharging at the cytoproct (Schroeder et al., 1990) and those derived from an earlier stage of phagosome development (Allen and Fok, 2000). Acidosomes,  0.8 mm in diameter and endowed with HþATPase molecules, fuse right after detachment of a nascent phagosome (Allen and Fok, 1983a; Allen et al., 1993). Although the signal for fusion is not known, control of the accessibility of interacting vesicle membranes by superficial actin could be crucial. Additional small vesicles (Ishida et al., 2001), endowed with distinct v-SNARE populations (Table 3.1) interact with oral fibers (Schilde et al., 2010). Localization coincides with specific actin isoforms (Table 3.3) (Sehring et al., 2007b, 2010) and their fusion may finally also contribute to food vacuole formation. To sum up, different stages of food vacuole formation are associated with specific SNAREs (Kissmehl et al., 2007; Schilde et al., 2006, 2008, 2010), but not all stages are fully characterized with regard to actin binding. In retrospect, specific interactions of vesicles with selective actin isoforms may contribute to vectorial vesicle trafficking by mechanisms yet to be determined. The actin assemblies of varying appearance occurring on food vacuole membranes in Paramecium (Sehring et al., 2007b; Fig. 3.8B) could interfere with vacuole–vesicle fusion, as shown in yeast (Eitzen et al., 2002). The association of P. tetraurelia phagolysosomes and associated vesicles containing different actin isoforms (Sehring et al., 2007b) is paralleled by the different cytochalasin B sensitivity in P. multimicronucleatum. Acidosome and lysosome fusion as well as membrane retrieval from the cytoproct all display different sensitivity to cytochalasin B (Allen and Fok, 1983a;

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Fok et al., 1985, 1987), while they are insensitive to abolition of lumenal acidity (Allen et al., 1995). It now would appear mandatory to discriminate between actin isoforms of different drug sensitivity (Sehring et al., 2007a). Beyond differences within one species, comparative analyses in the future will have to consider the wide divergence of sequences of actins in different ciliate groups (Kim et al., 2004; Zufall et al., 2006).

6.3. Role of H+-ATPase, SNAREs, and G-proteins in phagocytotic cycle of ciliates The changing acidity in food vacuoles of Paramecium during a throughput time of 20–40 min (depending on species and strain) is illustrated in Fig. 3.8A. The precise origin of acidosomes is not established on a molecular basis, but the dominant proposal says they may be derived from subciliary early endosomes (the terminal cisternae in Paramecium [Patterson, 1978]). However, acidosomes are believed to represent late endosomes (Allen, 1988) as they receive additional input from Golgi-derived vesicles (Allen et al., 1993). The latter would be compatible with the likely biogenetic pathway of the Hþ-ATPase molecule (Sections 2.4 and 3.3). Comparison of SNAREs and of SUs of the Hþ-ATPase in acidosomes and food vacuoles in P. tetraurelia shows that the acidosomal a4-1 SU is rapidly removed from the phagosome, while other SUs are included (Wassmer et al., 2006; Fig. 3.8B). This corresponds to the morphological observation of a retrieval of acidosomal components in P. multimicronucleatum (Allen and Fok, 1983b). In P. tetraurelia, later food vacuole stages contain SUs a5, a6, and a9. Therefore, there must be several cycles of delivery and removal of HþATPase components (Wassmer et al., 2006) whose specific pathways and functional implications remain to be elucidated. In the end, old food vacuoles retain some of their Hþ-ATPase molecules, though their contents are no more remarkably acid (Wassmer et al., 2009). Acidification, first to pH 5, then to higher values (Fok and Allen, 1988), serves—in the presumable absence, or lack of evidence of an oxidative burst in protozoa—inactivation of ingested food bacteria and digestion by lysosomal enzymes if one takes into account their more or less acidic pH optima also in Paramecium (Fok and Paeste, 1982; Fok et al., 1982). Hydrolytic enzymes are delivered by fusion with lysosomes, whereas at a later stage of phagolysosome maturation lysosomal components are retrieved (Allen and Fok, 2000). Also during cyclosis, input from constitutive endocytosis (Allen and Fok, 1980) and, after exocytosis stimulation, from trichocyst ‘‘ghosts’’ ensues in Paramecium (Lu¨the et al., 1986). As mentioned in Section 4.3.2, ‘‘frustrated exocytosis’’ involves transient membrane fusion with the cell membrane without contents release, but with membrane resealing and detachment of intact trichocysts. Interestingly, frustrated exocytosis allows trichocysts to

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undergo recycling (Klauke and Plattner, 2000). This fact implies that a specific membrane signal, normally formed or exposed after contents release, is required for delivery into the phagocytotic pathway. Though this signal is not yet known it may depend on a connection between the trichocyst matrix and the trichocyst surface (Section 3.3.3). By proteomics analysis of macrophage phagosome membranes, a plethora of constituents showed up, among them the SNARE-specific chaperone, NSF, together with its adaptor protein, a-SNAP, but surprisingly no SNAREs of the v-(R-) or t-(Q-)type (Garin et al., 2001). Actin, SUs of the Hþ-pump, Rab-type, and Arf-like G-proteins have also been detected. Apart from SNAREs this fits well with origin and activity of phagosomes. For instance, phagocytotic ingestion of bacteria does require NSF (Coppolino et al., 2001) although NSF should remain attached only transiently (Section 3.3). The phagocytic system of ciliates would be particularly suitable for time-sequence analyses of phagosome formation and maturation, as it is amenable, after magneto-bead ingestion, to magnetic separation with rather precise timing (Vossku¨hler and Tiedtke, 1993). In Tetrahymena, the exchange of small GTPases has thus been detected by [a-P32]-GTP overlays (Meyer et al., 1998) or by partial sequencing (Maicher and Tiedtke, 1999). Unfortunately, this has not been pursued in full by genomics/proteomics analysis. In mammalian systems, changing lumenal pH in phagosomes is paralleled by interaction with varying monomeric G-proteins and this can act as a timer controlling the transition between different maturation stages (Steinberg et al., 2007). G-proteins probably condition specific vesicle attachments and fusions, as outlined in Section 2.2. A more comprehensive proteome analysis of T. thermophila whole phagosomes, isolated after latex feeding, has detected—beyond lysosomal enzymes—several specific G-proteins (including Sar1 and Rab1, 7, and 13 members), a longin-type synaptobrevin, actin binding proteins, and various Hþ-ATPase SUs (Jacobs et al., 2006). This is clearly compatible with the selective localization of specific types of syntaxins (Table 3.2; Kissmehl et al., 2007), synaptobrevins—which actually are longins (Table 3.1; Schilde et al., 2006), actin (Table 3.3; Sehring et al., 2007a,b), and Hþ-ATPase SUs (Table 3.4; Wassmer et al., 2005, 2006), as analyzed in situ in P. tetraurelia cells by molecular biology. For the association of calmodulin with phago(lyso)somes/food vacuoles in ciliates and other cells as well as the potential role it may exert, see Section 7.

6.4. Autophagy In P. tetraurelia, autophagocytosis was abundant after NSF gene silencing (Kissmehl et al., 2002; H. Plattner, B. Scho¨nemann, and C. Schilde unpublished observation). This may be due to the following effects. (i) Cells may

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become energetically compromised due to disturbance of heterphagocytotic food processing; mammalian cells, for instance, react by increased autophagy when energetically compromised (Galluzzi et al., 2008; Kuma et al., 2004). (ii) Formation of autophagosomes per se does not require SNAREs (Ishihara et al., 2001; Klionsky and Emr, 2000); their double membrane envelope closes with the aid of a triple-A ATPase different from NSF (Babst et al., 1998). (iii) Autophagosomes may pile up because subsequent fusion with lysosomal elements does, in fact, require SNAREs for maturation to autophagolysosomes and intraorganellar digestion (Ishihara et al., 2001; Klionsky and Emr, 2000). In retrospect the endo-/phago-/lysosomal system in ciliates is most complex. During evolution, this may have contributed to the ‘‘invention’’ and association of a great number of isoforms of SNAREs, Hþ-ATPase SUs, and actin in this subcellular compartment system. In fact, numbers of such molecules undergoing association/dissociation with food vacuoles are equivalent to, or even higher than known from most metazoans and higher plants, as discussed in Section 3.1.1. Our findings with P. tetraurelia are illustrated in Fig. 3.8.

7. Calcium-Binding Proteins and Calcium Sensors 7.1. Comparison of Ca2þ-signaling in ciliates with other cells Upon cell stimulation, Ca2þ transmits many different signals, in a direct or indirect way, in connection with widely different cell activities (Berridge et al., 2003; Clapham, 2007; Laude and Simpson, 2009), including exo- and endocytosis (Henkel and Almers, 1996). This also holds true for ciliates, but only with Paramecium has Ca2þ signaling been studied in some detail (Plattner and Klauke, 2001). Briefly, trichocyst exocytosis requires Ca2þ mobilization from alveolar sacs, the cortical Ca2þ stores, tightly followed and superimposed by Ca2þ-influx (Hardt and Plattner, 2000; Klauke et al., 2000; Mohamed et al., 2002). Locally, [Ca2þ]i increases to 5 mM (Klauke and Plattner, 1997). The more Ca2þ is present in the outside medium during stimulation, the more are all steps of an exo-endocytosis cycle accelerated, including endocytosis, that is, the final removal of trichocyst ‘‘ghosts’’ (Plattner et al., 1997). The very recently identified Ca2þ-release channels of alveolar sacs membranes are arranged such as to mediate a very rapid local Ca2þ signal upon stimulation and, thus, an efficient exocytotic response (Ladenburger et al., 2009). All this clearly resembles dense core-secretory vesicle handling in mammalian cells in several regards. For instance, exocytosis and exo-endocytosis coupling in these systems is also accelerated by increased extracellular

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[Ca2þ] and the time periods required are about the same (Henkel and Almers, 1996; Rosenboom and Lindau, 1994). Concomitantly, a Ca2þactivated protein (Ca2þ-sensor) must accelerate exocytosis. It will also promote exocytosis-coupled endocytosis by binding the adaptor for clathrin binding, AP-2, on its C2B domain (Schwartz, 2004). Does a Ca2þ-sensor of any kind occur in Paramecium and in other ciliates?

7.2. Synaptotagmin as a Ca2+-sensor Generally, the Ca2þ signal can be transmitted by binding to a variety of proteins, for example, low-capacity/high-affinity Ca2þ-binding proteins with specific Ca2þ-binding motifs. Among them are theoretically available several proteins with EF-hand motifs, as found in calmodulin, or proteins with C2-domains, as in the established Ca2þ-sensor synaptotagmin. Particularly, the latter type is considered relevant for membrane fusion (Martens and McMahon, 2008). Such a function has been demonstrated not only for exocytosis (Chapman, 2008; Lynch et al., 2008; Paddock et al., 2008) but also in vitro by reconstitution studies (Lynch et al., 2007; Martens and McMahon, 2008; Tucker et al., 2004). Therefore, synaptotagmin is believed to mediate the final reaction of the exocytotic machinery to the Ca2þ signal which arises when mammalian cells, for example, different neuronal cell types, are stimulated (Chapman, 2008; Lynch et al., 2007, 2008; Martens and McMahon, 2008; Paddock et al., 2008). As the local Ca2þ signal ranges from slightly below 10 mM, for example, in chromaffin and some other glands (Voets, 2000), to 100 mM in some nerve terminals (Neher, 1998), different synaptotagmin-type Ca2þ-sensor isoforms may be in action (Sugita et al., 2002). The delay between Ca2þ signal formation and the actual exocytotic response reflects the Ca2þ binding kinetics (Heinemann et al., 1994). The signal we recorded in P. tetraurelia during trichocyst exocytosis (Klauke and Plattner, 1997) resembles that in chromaffin cells. Could synaptotagmin be involved? To address this question, we have to scrutinize this molecule. Synaptotagmin is inserted by an N-terminal stretch in synaptic and other vesicle membranes (Perin et al., 1991). It represents the only established Ca2þ-sensor pertinent to vesicle fusion that has been analyzed in any depth. Since its detection by Matthew et al. (1981) its mode of action is increasingly unraveled. Its two C2 domains, C2A and C2B, follow the N-terminal transmembrane stretch, and bind Ca2þ rapidly at concentrations emerging upon stimulation. Thereby Ca2þ ions bind to short loops protruding from eight-stranded b-barrels of the C2 domains (Chapman, 2008). Upon Ca2þbinding, particularly the conformational change of the C2A domain causes its partial penetration into the opposite phospholipid bilayer. This binding prefers phosphatidylserine and phosphatidyl inositol 4,5-bisphosphate (PIP2), both enriched on the cytoplasmic side, of the vesicles to be fused

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and a target membrane. Synaptotagmin also favors the interaction with SNAREs (Martens et al., 2007), notably with SNAP-25, and thus increases Ca2þ-sensitivity (Lynch et al., 2007; Nagy et al., 2008). This step actually comes into play only after SNARE-pin zippering (Section 3.1.2). Then, synaptotagmin can, in vitro and in vivo, significantly accelerate membrane fusion. Although many details have been ascertained in many systems (Chapman, 2008; Lin and Scheller, 1997; Lynch et al., 2007, 2008; Martens and McMahon, 2008; Paddock et al., 2008; Pobbati et al., 2006; Sorensen et al., 2006; Tucker et al., 2004) the actual process of membrane fusion remains elusive. The role of synaptotagmin may be to force lipids into some unstable rearrangement (‘‘perturbation’’) prone to fusion. The multifarious activity of synaptotagmin evidenced by all these studies and derived from the multiple sites of occurrence in cells (below) prompted us to look carefully for synaptotagmin in Paramecium. Synaptotagmins or related proteins are usually found in different subcellular regions (Adolfsen et al., 2004) down to the Golgi region (Ibata et al., 2000). They occur in widely different cell types (Li et al., 1995) including plant cells where they contribute to cell membrane biogenesis (Schapire et al., 2008). In neurons and related cells, they participate not only in exocytosis but also in maturation of secretory vesicles (PC12 [chromaffin] cells: Ahras et al., 2006) and endocytosis, including recycling at nerve terminals ( Jorgensen et al., 1995; Nicholson-Tomishima and Ryan, 2004; Poskanzer et al., 2003). Different paralogs—probably with different Ca2þ-binding characteristics— may be in use, for example, synaptotagmin V for delivering early endosome membrane to a forming phagosome (Vinet et al., 2008) or synaptotagmin VII for fusion of lysosomes with phagosomes (Czibener et al., 2006). Proteins similar to synaptotagmin, but with only one or with more than two C2 domains have also been found, from moss to man (Craxton, 2007), but their function has remained enigmatic so far. Multiple C2-domain proteins (related to synaptotagmin, but distinct from other protein families with C2-domain) may be restricted to some intracellular sites of vesicle trafficking (Martens and McMahon, 2008) where their role remains to be analyzed, just as the precise relevance of local [Ca2þ]i for intracellular membrane fusions (see below). The absence of synaptotagmin from yeast cells (Schwartz and Merz, 2009) suggests that constitutive fusion processes can take place in the absence of a Ca2þ-sensor, if not by supplementation by an unknown functional surrogate. Generally, constitutive exocytosis is considered a Ca2þ-independent process (Burgoyne and Clague, 2003; Jaiswal et al., 2009). Beyond that, a variety of intracellular fusion processes do not need Ca2þ (Hay, 2007). This may apply to some steps of the endo-/phagosomal pathway in some cell types. In contrast, for instance, in neutrophils phagosome–lysosome fusion requires a recordable increase in [Ca2þ]i ( Jaconi et al., 1990). On these fundamentals we can now inspect the situation in ciliates.

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7.3. Calcium and calcium sensors in ciliates May calmodulin be of interest in the present context? We have detected in Paramecium 13 genes encoding putative calmodulin isoforms (R. Kissmehl, unpublished research). Besides several other sites, calmodulin has been localized, from the light to the EM level, to trichocyst docking sites (Momayezi et al., 1986; Plattner, 1987). Association of calmodulin with docking sites reflects its requirement for the assembly of functional trichocyst exocytosis sites (Kerboeuf et al., 1993). Its association with digestive vacuoles and with various other vesicles participating in trafficking (Fok et al., 2008; Momayezi et al., 1986) clearly assigns to calmodulin a role also for other vesicle trafficking pathways. This will also include the contractile vacuole complex which is labeled by microinjected fluorescent calmodulin in vivo (Momayezi et al., 1986), particularly since we now know this organelle to participate in vesicle trafficking (Section 9.1). Any direct contribution of calmodulin to exocytosis, however, is not established (Burgoyne and Clague, 2003). Its role may be indirect, for example, by binding to specific SNAREs (Quetlas et al., 2002) (Section 4.3). Also any specific role of calmodulin in other vesicle trafficking steps remains to be established. An exception may be the requirement of Ca2þ/calmodulin for completion of the vacuole docking/fusion process, as found in yeast cells (Mayer, 2002; Peters and Mayer, 1998). This recalls findings with phagocytotic vacuoles in ciliates, as they bind calmodulin in T. thermophila (Gonda et al., 2000) and P. tetraurelia (Momayezi et al., 1986; Plattner, 1987), the latter having been confirmed recently by Fok et al. (2008) for P. multimicronucleatum. Furthermore, in neuronal cells, calmodulin in conjunction with the ‘‘auxiliary’’ protein, Munc13, can form a Ca2þ sensor/effector complex ( Junge et al., 2004). Any such effects would be worthwhile exploring in ciliates. In Paramecium, the Ca2þ-increase occurring at trichocyst exocytosis sites upon AED stimulation amounts to [Ca2þ] 5 mM (Klauke and Plattner, 1997). This is in the range of a synaptotagmin type with a Ca2þ-sensitivity as in an average gland cell. For instance, 10 mM is required to activate the readily releasable pool of chromaffin cells (Voets, 2000). At the cell membrane level, in some neuronal systems, sensitivity to Ca2þ may be enhanced by specific point mutations in the syntaxin 1A molecule (Lagow et al., 2007) or by close association of syntaxin 1A with SNAP-25 and Ca2þ-channels (Hagalili et al., 2008). This disturbingly complex situation in higher eukaryotic systems can set only a quite enigmatic frame for our expectations in ciliates. Trichocyst exocytosis is considerably faster (Knoll et al., 1991a; Plattner and Kissmehl, 2003b) than any other dense core-secretory vesicle system (Kasai, 1999). Once docked, trichocysts all belong to a ‘‘readily releasable pool’’ (>95% of all trichocysts present). While we had to expect the occurrence of a Ca2þ-sensor comparable to synaptotagmin in Paramecium, the only

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sequence with closest similarity found in the database contains eight, rather than two C2 domains (R. Kissmehl, unpublished results). In no higher eukaryotic system has the function of synaptotagmin-related proteins with more than two C2 domains been elucidated up to now. This also holds true for Paramecium. Whereas synaptotagmin is the established Ca2þ-sensor in metazoans and in plants (Schapire et al., 2008), only details of this hypothesis being currently a subject of scrutinized analysis, no equivalent molecule could be identified in ciliates as yet. The fast response of Paramecium to exocytosis stimulation is supported by the vigorous expulsion of trichocyst contents. This is due to rapid decondensation of the crystalline trichocyst matrix proteins (Section 3.3) in response to extracellular Ca2þ when it gets access through the exocytotic opening (Bilinski et al., 1981). This decondensation/expulsion process is based on the Ca2þ-binding capacity of some of the contents proteins (Klauke et al., 1998) of which several ones are probably derived from one precursor. A similar mechanism, though less vigorous, may subside the release of Tetrahymena mucocysts which also contain acidic Ca2þ-binding proteins (Chilcoat et al., 1996; Turkewitz et al., 1991; Verbsky and Turkewitz, 1998). Considering the additional involvement of synaptotagmin in exocytosiscoupled endocytosis in higher eukaryotes, by binding adaptor protein 2 (AP2), this can explain the acceleration of exo-endocytosis coupling in dependency of extracellular [Ca2þ] in a variety of systems (Section 4). We do not know whether this would also include ciliates where such coupling is equally fast (Section 4.3). In summary, there is some agreement that only some, but not all intracellular fusions may require Ca2þ (Burgoyne and Clague, 2003) and a Ca2þ-sensor (Hay, 2007). All these restrictions also concern Paramecium cells—almost the only ciliate for which we have some information available. As in other cells, calmodulin may contribute to intracellular membrane interactions. Moreover, in Paramecium calmodulin enables the assembly of functional trichocyst docking sites (Kerboeuf et al., 1993). Whereas synaptotagmin is a well-established Ca2þ-sensor for exocytosis particularly in neuronal cells, in ciliates as well as in other protozoa Ca2þ-signal transduction by a sensor protein with C2 domains remains poorly understood at this time.

8. Additional Aspects of Vesicle Trafficking 8.1. Guidance and support by microtubules In this section, we shall address the auxiliary role of microtubules as a kind of long-range targeting aid, the unsettled role of some additional molecules with potential relevance for vesicle trafficking in ciliates and finally the

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considerable restrictions of using allegedly organelle- or molecule-specific drugs as a tool for analyzing vesicle trafficking in ciliates. This problem is largely exemplified by the effects on the microtubule system. In metazoans, microtubules are considered relevant for fast and directional movement of vesicles of different kinds, rather than for their precise targeting (Hirschberg et al., 1998). In a variety of higher eukaryotic cells, docking of dense core-secretory vesicles takes place by saltatory movement along microtubule rails (Lacy, 1975) and, hence, with the involvement of motor proteins (Soldati and Schliwa, 2006). Only the recruitment, but not the release of dense core-secretory vesicles is accelerated by the presence of an intact microtubule system in pancreatic acinar cells (Schnekenburger et al., 2009). Endocytosis, at least from some distance from the cell membrane on (Nielsen et al., 1999), also involves microtubules (Bananis et al., 2000; Matteoni and Kreis, 1987). Microtubules contribute not only to trafficking from the early to the late endosome (Aniento et al., 1993), but also to phagosome formation (Harrison and Grinstein, 2002; Khandani et al., 2007) and intra-Golgi trafficking (Cai et al., 2007). 8.1.1. Microtubules in ciliates Cytoplasmic microtubules are arranged in a complex pattern in Paramecium (Adoutte et al., 1991; Allen, 1988; Allen and Fok, 2000; Fok and Allen, 1988, 1990), in Tetrahymena (Gaertig, 2000), and in other ciliates. They may contain tubulin with different posttranslational modifications in ciliates (Libuso´va and Dra´ber, 2006) such as Paramecium (Adoutte et al., 1991) and Tetrahymena (Gaertig, 2000; Penque et al., 1991). Ciliates may represent a useful model for analyzing the functional meaning of the numerous posttranslational modifications that are also found in metazoans (Westermann and Weber, 2003). Moreover, some of the numerous paralogs generated in Paramecium after gene duplication acquire specific new functions (Aury et al., 2006). This complex scenario still awaits more detailed analysis. Microtubules flanc the oral cavity from where a separate population emerges, called the (post)oral fibers. In this region, at least three types of vesicles associate with microtubules (Schroeder et al., 1990). This differentiation is supported by the differential endowment with SNAREs (Schilde et al., 2010). Additional microtubules run perpendicular to the oral cavity (Adoutte et al., 1991) and still others connect the oral cavity with the cytoproct (Allen and Wolf, 1974), as summarized by Allen and Fok (2000). This set of microtubules serves recycling of membrane materials as ‘‘discoidal vesicles’’ generated from the spent phagosome membrane after contents release, thus supporting the formation of a nascent food vacuole. This is true for Paramecium (Schroeder et al., 1990) and for Tetrahymena (Sugita et al., 2009). Another chemically defined microtubule population runs around the contractile vacuole and elongates over the radial canals of the osmoregulatory system in Paramecium (Adoutte et al., 1991) and in Tetrahymena (Gaertig,

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2000). In addition, in Paramecium, single distinct microtubules have been observed to emanate from the cell periphery, deep into the cytoplasm (Adoutte et al., 1991), and may serve trichocyst docking (Aufderheide, 1977; Plattner et al., 1982). Among oral (and intracytoplasmic) microtubules in Tetrahymena there are glycinated and glutamylated forms (Gaertig, 2000). Different microtubule subpopulations made of differently posttranslationally modified tubulin participate in phagosome processing in T. thermophila (Wloga et al., 2008). Microtubule subpopulations differ in sensitivity to depolymerizing drugs, concomitantly different steps of the digestive cycle in Paramecium have widely different drug sensitivities (Fok et al., 1985). Also their cold sensitivity differs (Adoutte et al., 1991). As mentioned, in P. tetraurelia, the different microtubule populations connected to the oral cavity are associated with vesicle types endowed with different proteins. The first population, possibly comprising two or more vesicle types (also with the additional uncertainty of discrimination from acidosomes), contains SNAREs type PtSyb6 (Schilde et al., 2006), PtSNAP-25-LP (Schilde et al., 2008), PtSyb8, PtSyb9, and PtSyb10 (Schilde et al., 2010) as well as PtSyx3 and PtSyx4 (Kissmehl et al., 2007). Furthermore, these vesicles differ in their Hþ-ATPase SUs; among others, they contain SUs type a1 and a4 (Wassmer et al., 2006) and they are associated with the oral filament system. This is made not only of microtubules, but it also contains PtAct5 and PtAct8 (Sehring et al., 2007b). Some of these vesicles are vigorously catapulted from their origin to the periphery of the respective microtubule population, just as previously described by structural video-analysis (Ishida et al., 2001). How they are associated with microtubules and by which motor proteins they are propelled remains open for the time being. In Paramecium, discoidal recycling vesicles travel along microtubules to the cytopharynx. This is less evident for the population derived from partially matured phagosomes (Allen et al., 1995). Discoidal vesicles originating from the cytoproct are more clearly connected to a specific set of microtubules by dynein (Schroeder et al., 1990). Microtubule binding also occurs with the small cytopharyngeal vesicles, also probably contributing to phagosome biogenesis, since microtubules also emanate longitudinally and perpendicularly to the oral cavity (Adoutte et al., 1991) and these small vesicles are seen to travel forcefully and unidirectionally, as described above. The motor proteins, kinesin and dynein, that drive the anterograde ( ! þ) and retrograde (þ ! ) vesicle transport, respectively, along microtubules (Hirokawa and Takemura, 2005; Vallee et al., 2004) also occur in ciliates. There is much less information about kinesin and its potential contribution to vesicle transport than about dynein. T. thermophila expresses 25 and P. tetraurelia 26 dynein heavy chains (Wilkes et al., 2008). In Tetrahymena, DYH1 encodes a cytoplasmic form required for phagocytosis (independent of oral ciliary activity) (Lee et al., 1999). In P. multimicronucleatum, dynein has been

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identified by biochemical analysis as a two-headed cytosolic form (Schroeder et al., 1990). In P. bursaria, its silencing inhibits the formation/detachment of a food vacuole, thus indicating a process operating in þ !  direction. Nishihara et al. (1999) also report cyclosis inhibition by the bona fide dynein ATPase inhibitor, erythro-9-[3(2-hydroxynonyl)] adenine (‘‘EHNA’’). Accordingly, formation and/or transport of food vacuoles through the cell may be facilitated by microtubules. In agreement with this, nocodazole, an established microtubule depolymerizing agent in ciliates (Plattner et al., 2009), greatly reduces cyclosis in Paramecium (Nishihara et al., 1999). In Paramecium, the basal bodies of oral ciliary assemblies called ‘‘quadriculus’’ and ‘‘peniculus’’ are associated with selective dynein isoforms, as these microtubules stain with specific antibodies (Asai et al., 1994). This is the site of vivid transport of small vesicles (Ishida et al., 2001)—a site also stained with GFP constructs of PtAct, PtSyx, PtSyb, and SNAP-25-LP (specified above). These vesicles are transported along with the formation of food vacuoles (Ishida et al., 2001). In Paramecium, the oral cavity microtubules and the postoral fibers stained differentially when a battery of antibodies was probed (Adoutte et al., 1991); postoral fibers also contain PtSyb9-positive structures (Schilde et al., 2010). The precise, posttranslationally modified microtubule subtype involved in the different steps mentioned is not always known as yet. In higher eukaryotes, microtubules may equally contribute to ordered vesicle trafficking from deep inside the cell to the periphery as their disruption causes disintegration of the Golgi apparatus (Pfeffer, 2007) and abolition of saltatory transport of secretory vesicles (Lacy, 1975). In Paramecium, some microtubules emerge from ciliary basal bodies and hang into the cell interior vertically to the cell surface (Glas-Albrecht et al., 1991; Plattner et al., 1982). During saltatory docking (Aufderheide, 1977), these microtubules can guide trichocysts to the cell periphery. Interestingly, docking follows an inherent polarity of trichocysts, tip first, from the plus to the minus end of microtubules. (An exception is some exocytosis-incompetent mutants, such as ptA2, that do not dock their aberrant trichocysts [Pouphile et al., 1986].) This microtubule-guided polarity in Paramecium is opposite to that in most higher eukaryotic cells (Soldati and Schliwa, 2006). However, plus to minus docking has later on been observed also in MDKC (renal) cells (Bacallao et al., 1989; Bre´ et al., 1990). This is supported by the observation that chromaffin granules, isolated from bovine adrenal medulla and injected into Paramecium cells, travel to the plus end (Glas-Albrecht et al., 1991) which, in chromaffin cells, would carry them to the cell membrane. When similar experiments were conducted with chromaffin granules injected into sea urchin egg cells, granules were docked at the cell membrane (Scheuner et al., 1992), as to be expected from the minus-toplus orientation of microtubules emanating from the cytocenter in that system.

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In summary, in ciliates microtubules relevant for exo-endocytosis do not emerge from a cytocenter, but microtubule organizing centers are associated with ciliary basal bodies. The interaction of dense core-secretory vesicles with microtubules evidently follows, or imposes, an inherent directionality—another novel finding one can derive from the cited work with ciliates. Unfortunately, analysis of kinesins has been largely neglected with ciliates. 8.1.2. Additional aspects concerning microtubules in ciliates In P. multimicronucleatum, the different steps of the phagosomal cycle, from acidosome fusion to fusion with lysosomes, probably involve rather different populations of microtubules and microfilaments. This may be inferred from the different sensitivity of the different steps to disrupting drugs (Fok et al., 1985, 1987). An intriguing interaction of cytoskeletal elements, microtubules and actin, is observed in P. tetraurelia cells by immunogold EM localization in different areas of intense vesicle trafficking (Kissmehl et al., 2004)—in agreement with the effects of some drugs aiming at microtubule and microfilament function (Beisson and Rossignol, 1975). Mechanisms and functions of these interactions remain to be elucidated in detail. Most recently, Cda12p-containing vesicles relevant for cytokinesis (Section 9.3) have been documented in Tetrahymena to travel along cortical microtubules to their site of integration into the cleavage furrow (Zweifel et al., 2009). This microtubular arrangement is considered equivalent to the cytospindle described in more detail in Paramecium where it assembles just prior to cytokinesis (Delgado et al., 1990; Iftode et al., 1989). In sum, microtubules form an unspecific long-range guidance system for vesicle trafficking. Ciliates contain several regularly arranged subpopulations of microtubules, with different posttranslational modifications and varying drug sensitivity. In Paramecium, trichocysts are docked, tip first according to their inherent polarity, along microtubules from the plus- to the minus-end. (In contrast, almost all cells of higher eukaryotes operate in the opposite direction.) In ciliates, subsets of differently modified microtubules are relevant for phagosome formation, that is, to handle the multiplicity of vesicles that are seen around the oral cavity and which all contain different membrane protein signatures.

8.2. Additional potential key players Annexins are a family of proteins interacting with phospholipids/biomembranes subsequent to Ca2þ binding (Gerke et al., 2005). In Paramecium, antibodies generated against different common sequences from mammalian annexins revealed two binding sites (Knochel et al., 1996). One antibody bound to the cytoproct and the other one to trichocyst tips. Later on, another

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family of proteins with similar binding characteristics, the copines, has been detected, first in Paramecium (Creutz et al., 1998) and then in higher eukaryotic, including mammalian cells. In none of these systems, the precise localization and function has been ascertained (Tomsig and Creutz, 2002). Only more recently it was found that binding of different target proteins, including cytoplasmic actin and SNAP-25, by copines may play a functional role (Tomsig et al., 2003). Similarly, annexin A2 favors actin polymerization during endosome biogenesis, also in mammalian cells (Morel et al., 2009). This can be appreciated if one considers the relevance of actin for endocytosis (Sections 2.3 and 4). To sum up, the function of Ca2þ-dependent phospholipid binding proteins, such as annexins and copines, awaits much more detailed exploration—not only in ciliates.

8.3. Pharmacology of vesicle trafficking Many drugs are applied in cell biology of higher eukaryotes to unravel specific functions and to pinpoint their localization. A recent thorough evaluation of data published on the effects of different drugs in ciliates warns us of lacking, unspecific or even adverse effects of important drugs that otherwise are of standard use (Plattner et al., 2009). Therefore, application of such drugs to ciliates appears highly problematic, unless the effects are strictly established with the species under investigation, with the strict support by molecular biology. In animal cells, neurotoxins derived from Clostridium botulinum and Clostridium tetani cleave certain SNAREs, by the metallo-endoprotease activity of their light chain, to inactive fragments (Montecucco and Schiavo, 1995; Niemann et al., 1994). In Paramecium, our experience is as follows. The SNAREs investigated are not sensitive to the Clostridium proteases tested, that is, Botulinum and Tetanus toxins (BoNT, TeNT) and their respective subtypes, respectively (Schilde et al., 2008). In unpublished work, we had previously injected the light chains of several Clostridium toxins and achieved inhibition of trichocyst docking by BoNT/E (D. Vetter, Diploma work, University of Konstanz). Considering its specificity (Brunger et al., 2008) we had expected cleavage of SNAP25-LP which, however, did not occur (Schilde et al., 2008). These results are in line with the insensitivity of related longintype SNAREs in higher plants (Bassham and Blatt, 2008; Foresti and Denecke, 2008). In P. tetraurelia, several actin forms are predicted insensitive to the F-actin stabilisator, phalloidin, and also to depolymerizing drugs (Sehring et al., 2007a). Thus, only a subfraction of microfilaments will be affected at specific sites of a ciliate cell. Similarly, among a variety of ‘‘antimicrotubule’’ drugs only some are active at low standard concentrations (Pape et al., 1991). However, the widely different subtypes of posttranslationally modified microtubules at specific sites have not yet been analyzed accordingly. For this aspect, see Section 8.1. Among Hþ-ATPase inhibitors, only concanamycin B (Gronlien et al., 2002b), but not

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bafilomycin, is efficient in Paramecium (Fok et al., 1995). Application of brefeldin A, a blocker of binding between the Arf-GDP and the COP coat (Anders and Ju¨rgens, 2008), would be a standard assay to identify in the light microscope the Golgi apparatus by reversible dispersal. However, unpleasantly high concentrations are required in Paramecium so that control by EM immunogold labeling was advised to assign different proteins to the Golgi apparatus (Kissmehl et al., 2007). This is not exceptional as several Arf types are brefeldin A-insensitive also in plants (Anders and Ju¨rgens, 2008; Foresti and Denecke, 2008). We now can summarize as follows. In ciliates, insensitivity to otherwise established drugs may frequently be caused by aberrant binding sites (Plattner et al., 2009). This greatly restricts the repertoire of tools available for ciliate cell biology, while it opens up a wide field for future research. Examples are the insensitivity of the ciliate Golgi apparatus (or rather its formation by vesicle trafficking from the ER) to brefeldin A and of some SNAREs to Clostridium neurotoxins, the SNARE-specific endopeptidases, etc.

9. Emerging Aspects of Vesicle Trafficking in Ciliates In this section, we shall present mainly two aspects whose future development will provide important contributions to basic cell biology and to that of ciliates. (i) Unexpectedly, the contractile vacuole complex turned out to be an organelle with rather intense vesicle trafficking. (ii) Although in principle biogenesis of cilia is known to take place, in part, by vesicle trafficking, detailed information, particularly on SNAREs, is scarce. (iii) Cytokinesis is another activity of cells with considerable contribution by vesicle trafficking. Now important new aspects emerge on these aspects which will deserve enforced investigation.

9.1. Contractile vacuole complex The contractile vacuole complex/osmoregulatory system of ciliates is made up of a contractile vacuole and emanating radial canals that are continuous with a tightly attached tubular ‘‘spongiome.’’ It disposes of two preformed sites of cyclic membrane fusion/fission. These are the ‘‘porus’’ for exocytotic fluid expulsion and the connection of the radial canals with the vacuole (Allen and Naitoh, 2002). Electrophysiology has ascertained by capacitance measurements their periodic dis-/reconnection from/to the vacuole (Tominaga et al., 1998a,b). Precisely these sites, together with the vacuole docking site at the cell membrane, are labeled by antibodies against NSF, provided dissociation of NSF is inhibited in carefully permeabilized cells by adding the NSF

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inhibitor, N-ethylmaleimide, and nonhydrolyzable ATP-g-S (Kissmehl et al., 2002). Note that normally NSF would be released from membrane docking/fusion sites with each cycle (Section 3.2). Strikingly, under such conditions the other parts of the contractile vacuole complex were also labeled with antibodies against NSF, though much more faintly (Kissmehl et al., 2002). This is compatible with the localization, at the light and EM level, of some SNAREs (as GFP fusion proteins), particularly of PtSyb2 and PtSyx2 (and possibly of some other SNAREs) (Kissmehl et al., 2007; Schilde et al., 2006). These two are both restricted to the contractile vacuole complex where they occur on the membranes of the vacuole, of the radial canals (including ampullae), and on the widely branching tubular system of the smooth spongiome (Kissmehl et al., 2007; Schilde et al., 2006). A similar distribution is found with the Qb/c-type SNARE called ‘‘SNAP-25-like protein,’’ PtSNAP-25-LP (Schilde et al., 2008) as well as with an inositol 1,4,5-trisphosphate (InsP3) receptor (Ladenburger et al., 2006). In contrast, the decorated spongiome, with its abundant Hþ-ATPase molecules (Fok et al., 1995, 2002; Wassmer et al., 2005, 2006) mediating a ‘‘decorated’’ appearance in the EM, does not display any labeling for PtSyb2 or PtSyx2. Although the biogenesis of the contractile vacuole system still remains to be elucidated, a tentative interpretation of the facts reported is as follows. Based on Section 3.3 one may assume that assembly of components of the contractile vacuole complex would begin in the ER, followed by delivery via Golgi-derived vesicles. Within the spongiome one may envisage lateral segregation, perhaps enabled by the tendency of Hþ-ATPase molecules to form dimers and higher order linear clusters (Strauss et al., 2008). This in turn could drive segregation into the decorated spongiome and this process may drive its tubularization (Allen et al., 1989). In the contractile vacuole system, SNAREs may serve unexpectedly vivid membrane trafficking to allow for intense turnover of membrane components. Alternatively or additionally, SNAREs may be permanently required in diastole to revoke membrane vesiculation or tubularization occurring during systole, as described by Allen and Fok (1988)—a hypothesis to be tested in future work. Hþ-ATPase constituents may be delivered in vesicles as outlined in Section 3.3 to any level below the decorated spongiome, before they may be trapped in these terminal branches by lateral segregation, as outlined above. In summary, the contractile vacuole system now appears as an unexpectedly dynamic system—far beyond its impressive systole/diastole cycle. PtSyb2 and PtSyx2 are SNAREs exclusively found in this complex organelle, in all of its parts except the decorated spongiome (where the Hþ-ATPase exclusively resides). There is no evidence of actin in this organelle.

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9.2. SNAREs and ciliary function Detailed knowledge on vesicle trafficking for cilia biogenesis is scarce. This will be an important field for future research. We have recently obtained some fragmentary data for Paramecium, as will be discussed below, after setting a baseline available from other systems. 9.2.1. Cilia in vertebrates As known from higher eukaryotes, many constituents of cilia, or rather of their membranes, are delivered by vesicular transport close to the ciliary basis. Here, vesicles are integrated into the cell membrane by fusion for further transport in ‘‘rafts’’ (Rosenbaum and Witman, 2002). Fusion likely requires SNAREs, though information on SNAREs responsible specifically for ciliogenesis is scanty. Indirect evidence, or rather a postulate, refers mainly to the ongoing biogenesis/turnover of the ‘‘outer segments’’ of photoreceptors (Chuang et al., 2007) and to the formation of the primary cilium of nonsensory epithelia (Follit et al., 2006; Zhang et al., 2007). Delivery of rhodopsin to the retina outer segment (sensory cilium) has been supposed for some time already to involve SNAREs although more stringent evidence came up only very recently. Biogenesis of rhodopsin transport carriers destined for the sensory cilium depends on a ciliarytargeting complex comprising Arf4 and Rab11, a Rab11/Arf effector protein and an Arf-GAP (Mazelova et al., 2009a). Targeting of these carriers to near the basis of the rod outer segment (periciliary plasmamembrane) is regulated by Rab8 and its effector Sec6/8 (exocyst; Section 3.3) as well as by syntaxin 3. Based on fluorescence images this was proposed to serve vesicle delivery to the periciliary membrane (Mazelova et al., 2009b). Rab8 (Moritz et al., 2001) and Syx3 (Chuang et al., 2007) were known already previously to participate in vesicle delivery for raft formation in rod outer segments. Syx3 stays excluded from these ciliary derivatives (Baker et al., 2008), whereas SNAP-25 is reported to be found within primary cilia (Low et al., 1998). By contrast, Mazelova et al. (2009b) localized SNAP-25 to the inner rod segment. Concomitantly, no SNAREs show up in the ciliary proteome (Blacque et al., 2005; Pazour et al., 2005). This is in agreement with the assumption that newly added membrane components are incorporated into the cell membrane below the onset of cilia, as distinct membrane domains (‘‘rafts’’), for subsequent intraciliary transport (Blacque et al., 2008; Rosenbaum and Witman, 2002). Similar discrepancies emerge with the biogenesis of the primary cilium. Rab8, together with its GDP/GTP exchange factor—both promoting ciliogenesis—are on the one hand reported to mediate vesicle transport to the primary cilium and on the other hand to enter the cilia (Nachury et al., 2007). Similarly, exocyst components (Section 3.3) were scattered all over the primary ciliary membrane after overexpression (Zuo et al., 2009). In the

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absence of any vesicle traffic inside cilia (Rosenbaum and Witman, 2002) this may be due to overexpression and/or other experimental corollaries. 9.2.2. Cilia in ciliates Specifically in Paramecium, we found a candidate SNARE for ciliary function, PtSyb10, which is enriched in patches (‘‘microdomains’’) close to the basis of cilia, but still on the somatic cell surface (Schilde et al., 2010). A similar situation is seen within the oral cavity. Our finding of PtSyb10–GFP clusters in the periciliary cell membrane is surprising insofar as synaptobrevins are v-SNAREs and not liable to clustering (Bowen et al., 2002); Section 5. Simultaneous silencing of the Ptsyb10-1 and Ptsyb10-2 genes significantly reduces rotations that normally accompany depolarization-induced backward swimming (‘‘ciliary reversal’’). However, ciliary activity is coregulated by somatic channels. The actual component causing the failure observed after Ptsyb10 silencing has not been identified as yet. Interestingly, in a P. tetraurelia pawn-B mutant (unable of ciliary reversal), the d4-662 transcript relevant for ciliary reversal—probably an activator of some voltage-dependent channels—was found to be delivered close to the cell membrane when expressed as a GFP-fusion membrane protein (Haynes et al., 2000). Among the exuberant Kþ-channel genes detected in P. tetraurelia (Haynes et al., 2003) there are some that are activated by depolarization (some with, some others without Ca2þ-dependency) (Kung and Saimi, 1982; Saimi et al., 1999). Such channels mediate a delayed rectification in the context of Ca2þ-current activation by depolarization that causes ciliary beat reversal, although they are—in contrast to the ciliary voltage-dependent Ca2þ-channels—localized exclusively to the somatic cell membrane (Kung and Saimi, 1982; Machemer, 1988). In this context SNAREs could play a dual role according to some anecdotal, but paradigmatic situations in higher eukaryotes. (i) A modulator of comparable Kþ-channels in mammalian neuronal cells and cardiac myocytes is escorted by the R-SNARE VAMP7/TI-VAMP (Section 3.1) from the Golgi apparatus to the cell membrane (Flowerdew and Burgoyne, 2009). (ii) Gating of voltage-dependent cation channels is modulated by SNAREs in widely different organisms, from plants (Grefen and Blatt, 2008) to mammals (Bezprozvanny et al., 2000; Leung et al., 2007; Ramakrishnan et al., 2009). Clearly, more data are required to fully understand the situation in Paramecium. On a speculative basis, we consider it possible that Syb10 clusters found near ciliary bases in P. tetraurelia cells may be engaged in the regulation of ciliary activity, notably of the ciliary reversal response. This presupposes delivery of the respective membrane components by vesicles. The PtSyb10 clusters we observe may also contribute to form and maintain microdomains with functionally important ion channels and signal transduction (Section 5). In full agreement with a role for these PtSyb10 clusters in vesicle delivery is the following observation by EM analysis (Fig. 3.7). After NSF silencing

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we could observe small vesicles in close association with the cell membrane, including sites near the ciliary basis, where they normally are not visible (Schilde et al., 2010). The PtSyb10 microdomain we observe reminds us of the recent description of ‘‘platforms,’’ in the nonflagellar cell membrane, that are enriched in specific proteins and destined for delivery into the flagellar membrane in Trypanosoma brucei (Emmer et al., 2009). How can an R (v)-SNARE, such as PtSyb10, ever be enriched in the cell membrane? Such a situation could indicate a steady-state situation under conditions of intense membrane delivery with only partial retrieval of components. Such a situation has been found in some nerve terminals of the nematode, C. elegans (Dittman and Kaplan, 2006). To sum up, in no cell system has the contribution of SNAREs to the biogenesis of cilia, specifically of their membrane, been explored in any satisfying detail. We have obtained some evidence of the participation of PtSyb10 in the delivery and/or clustering of components relevant for ciliary reversal in Paramecium.

9.3. Cytokinesis In an elegant study, Eric Cole and his collaborators (Zweifel et al., 2009) have identified in T. thermophila two proteins, Cda12p and Cda13p, that are relevant for vesicle trafficking during cytokinesis and conjugation. They produced cells with ribosomes endowed with antisense RNA-transfected 26S-rRNA according to Chilcoat et al. (2001) for downregulation, as well as N-terminally GFP-tagged fusion proteins for overexpression. They found the GFP-fusion proteins, complemented by anti-GFP antibody labeling for immunogold EM analysis, at distinct subcellular locations. Cda12p was localized to subcortical compartments fusing with endocytotic vesicles that were labeled by the exogenous sterene dye FM4-64 (Section 4.3.2). Cda13p was assigned to a structure considered the transGolgi network and to multivesicular bodies (late endosome/lysosome intermediates). Antisense technology with Cda12p yielded defects in processing of endocytotic vesicles and in cytokinesis, whereas Cda13p proved important for conjugant separation and subsequent cytokinesis. One can conclude from this that—out of a plethora of vesicles occurring in T. thermophila (Frankel, 2000)—particular vesicles, including some derived from early and late endosomes and possibly from the Golgi apparatus, contribute to cell membrane biogenesis during cytokinesis and conjugation in ciliates (Zweifel et al., 2009). In summary, the recent findings with Tetrahymena complement our knowledge about vesicle trafficking in higher eukaryotes. There, the Q-and R-SNAREs, syntaxin 2, and endobrevin, have been identified in mammalian cells as being relevant for cytokinesis (Low et al., 2003).

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10. Concluding Remarks In ciliates, essential components of membrane trafficking have been identified, based on work mainly with Paramecium and Tetrahymena. Most data are compatible with those from other systems, while they also reveal important new aspects. Remarkably, the secretory mutants analyzed previously at the molecular level, particularly in Paramecium may all act more or less upstream of the direct membrane interactions; some of the mutations concern integral membrane proteins of unknown function. This indicates that secretory activity is regulated by a much broader spectrum of components than envisaged here. During eukaryote evolution, among the key players regulating membrane trafficking, not only Rab GTPases (Gurkan et al., 2007), but also SNAREs have widely diversified already at the level of ciliates. The same holds true for actin and some Hþ-ATPase SUs, particularly the a-SU relevant for exchangeable binding of other SUs. One also has to bear in mind the expectation of numerous ‘‘auxiliary proteins’’ that may interact with the SNARE machinery and that still await identification in ciliates. What are unsettled questions of general relevance beyond ciliates? What determines specificity, targeting, and molecular interaction of different molecules on the way through the cell? Also inhibitory SNAREs need more careful investigation. Which SNAREs are relevant for biogenesis of cilia? What is the detailed interaction of auxiliary proteins in SNARE-mediated membrane fusion? Which function has Ca2þ-binding proteins with a number of C2-domains different from synaptotagmin? How do membranes ultimately fuse and which molecules, lipids, and/or proteins, line the fusion pore? What is the role of Ca2þ-dependent phospholipid-binding proteins, annexin-like proteins, and copines? As with other systems, this aspect remains in abeyance. This list could be expanded by many detailed questions for all of the different cell systems under investigation up to now. With ciliates, some of the most significant gaps concern our uncertainty about the identity of COP-like coats. Another aspect requiring detailed analysis is the unambiguous identification of the R (v)-SNARE for dense core-vesicle exocytosis and the Ca2þ-sensor mediating exocytosis. What is the identity of ‘‘rosette’’ particles—a still enigmatic freeze-fracture/EM feature correlated with exocytosis capacity—and which function would they play? Are rosette particles microdomain assemblies of syntaxin1 (in analogy to reports from neuronal systems)? Is the arsenal of SNAREs, actin, Hþ-ATPase principal SUs, as determined for Paramecium, already complete? As to other potential key players, an involvement of Rabproteins and their regulators in regulating membrane trafficking is very poorly elaborated in ciliates, so that small GTPases require extensive

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exploration in ciliates. Which is the role of paralogs from a most recent whole genome duplication (‘‘ohnologs’’)? Are they all merely for gene amplification (Aury et al., 2006; Duret et al., 2008), that is, for enhancement of identical effects at identical sites? What is the consequence of the finding of only one Qb/c- and of only two Qc-type SNARE genes, in the absence of any known Qb gene, in P. tetraurelia? What is the role of ‘‘pseudogenes’’ of R-/Qa-type SNARE-like sequences and of those without a membrane anchor? Gaps also exist on many details about ciliate motor proteins, notably kinesins. Though this is a long list already for future research, many more could be enumerated. Also note that up to now molecular informations come only punctually, either from Paramecium or from Tetrahymena. On the positive side of our bilance we can note some aspects exceeding, or supplementing informations from ‘‘higher’’ eukaryotic systems. Secretory organelles, such as trichocysts, need not necessarily be acidic to go to the stimulated pathway of exocytosis. Nevertheless, presence of an Hþ-ATPase is required for contents processing and docking. The latter follows an inherent organelle polarity, tip first, unexpectedly from the plus- to the minus-end of microtubules. Secretory contents release by trichocysts evidently reverts or inhibits another vectorial signal, as ‘‘ghosts’’ go to the phagocytotic pathway, while this does not occur after ‘‘frustrated exocytosis’’ (transient membrane fusion without contents release). Exocytosis and endocytosis use regularly installed sites (parasomal sacs) which are epigenetically determined, as is the formation of the contractile vacuole system. Unforeseeably, this organelle undergoes intense ‘‘silent’’ membrane trafficking, with a constantly ongoing rearrangement and/or delivery of membrane materials. NSF gene silencing in Paramecium has manifested many of such cryptic membrane interaction sites which normally would not be seen. Such putative exo-endocytosis sites await more reliable and specific identification. Ciliated protozoa also have an elaborate system of membrane recycling along distinct routes. A multitude of posttranslational modifications of tubulin and a plethora of actin isoforms in Paramecium suggest specific interactions of the cytoskeleton at different sites of vesicle trafficking. Some of the molecular components of vesicles display an unforeseen complexity of paralogs at distinct subcellular sites. In particular, the phagolysosomal system is most elaborate in ciliates, with many fusions and fissions, involving an exchange of specific SNAREs, Hþ-ATPase SUs, and of actin isoforms. The diversification of vesicle trafficking in the context of phagocytotic food recruitment and processing may be the reason why the number of SNAREs and of other key players of vesicle trafficking resembles that seen on the highest level of evolution. Definitely the number of SNAREs in Paramecium, for instance, exceeds by far that extrapolated for the ur-eukaryote, and to some extent that of the urmetazoan, thus suggesting a parallel evolution of subcellular complexity. Finally, one should not overlook the role as a paradigmatic guide provided by early work with Paramecium. This concerned the elaboration

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of protein-based membrane interactions, including the ‘‘focal fusion’’ concept. Early on, outside the mainstream, this has suggested an important role for membrane proteins in membrane interaction and fusion. The recent establishment of genome databases, in conjunction with international cooperations, revive and support the significance of ciliates as model systems, also for membrane trafficking.

ACKNOWLEDGMENTS Thanks are due to Drs. Janine Beisson, Jean Cohen, and Linda Sperling (CNRS, Gif-sur-Yvette, France) for some inspiring collaborations over the years, for initiating the Paramecium genome project as well as for making available some informations relevant for our own work at an early stage, to Dr. Patrick Wincker (Genoscope, Evry, France) for developing the Paramecium genome project to a platform for general use, to Drs. D. Fasshauer and R. Jahn (Max-Planck-Institute for Biophysical Chemistry, Go¨ttingen, Germany) for access to the server for the SNARE database, to Drs. E. May and J. Hentschel as well as Doris Bliestle and her crew (all University of Konstanz) for electronic image processing, and last but not least to Lauretta Nejedli and Sylvia Kolassa for excellent technical assistance. Particular acknowledgments also go to all previous and present coworkers who contributed to the present topic by the publications cited and beyond. Among coworkers, I thank Dr. Ivonne M. Sehring for critical comments to this manuscript. Work of the author cited herein has been constantly supported by the Deutsche Forschungsgemeinschaft.

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New Insights into the Types and Function of Proteases in Plastids Yusuke Kato and Wataru Sakamoto Contents 186 188 190 190 194 197 199 201 201 201 202 203 203 204 204 205 205 206 206 206 207 207 209

1. Introduction 2. Overview 3. Major Proteases 3.1. Clp protease 3.2. FtsH protease 3.3. Lon protease 3.4. Deg protease 4. Processing Peptidases 4.1. SPP 4.2. PreP 4.3. TPP 4.4. Plsp1 4.5. Ctp 5. Intramembrane Proteases 5.1. Rhomboid 5.2. EGY1 5.3. AraSP 6. Other Proteases 6.1. SppA 6.2. CND41 6.3. GEP 7. Concluding Remarks References

Abstract Plastids change their morphology dynamically in response to environmental conditions and developmental status. Related to these plastid dynamics, the quality and quantity controls of proteins are necessary. Therefore, proteases are important as key regulators in almost all processes during the conversion of plastid types and the maintenance of plastid homeostasis. Recent progress in Research Institute for Bioresources, Okayama University, Kurashiki, Okayama, Japan International Review of Cell and Molecular Biology, Volume 280 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)80004-8

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2010 Elsevier Inc. All rights reserved.

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this field has revealed that various proteases and peptidases act on plastids. Results of the studies indicate that the vast majority of plastid proteases are homologous to prokaryotic ones because plastids are thought to originate from endosymbiosis of ancestral cyanobacteria. Moreover, the diversification of subunits of several plastid proteases has been revealed along with new insights into the functions of these homologues. This review provides basic information related to plastid proteases and the current view of their physiological roles in plastid homeostasis and biogenesis. Key Words: Plastid, Protease, Chloroplast biogenesis, Protein degradation, Thylakoid membrane, Clp, FtsH, Deg. ß 2010 Elsevier Inc.

1. Introduction Enzymatic, structural, and regulatory functions of various proteins are necessary for the cell homeostasis. However, whether it is desired or not, each protein has its own lifetime. Therefore, to sustain life, innumerable births and deaths of proteins occur during an organism’s life span. Generally, polypeptides undergo folding, processing, modification, and proper assembly as a complex to become functional proteins. The proteins are eventually degraded. In this process, the protease functions are versatile and not limited to protein-degradation processes (Gottesman et al., 1997; Wickner et al., 1999). Accumulating evidence suggests that proteases play, respectively, important roles in the proper folding of polypeptides and N-terminal or C-terminal processing of preproteins as chaperon and processing peptidases (Glaser et al., 1998; Reumann et al., 2005). Therefore, proteases act as key regulators in the life of proteins. Protease functions are fundamentally involved in almost all activities of living cells. Plastids are among the most characteristic features of plant cells. The organelle is classifiable into several types according to its functions and morphology (Fig. 4.1) (Lopez-Juez and Pyke, 2005). The most familiar plastid type is chlorophyll-containing chloroplasts in leaves. Meanwhile, leaves that have germinated in the dark contain other type of plastid, an etioplast. Chromoplasts, which contain carotenoid pigments, and amyloplasts, which store large amounts of starch, are also known as plastids of other types. Each plastid type is interconvertible. The change of plastid types occurs dynamically in response to developmental status and environmental signals. Examples of plastid conversion are the development of etioplasts into chloroplasts immediately after leaves are exposed to light and the transition of chloroplasts into chromoplasts during fruit ripening. These plastid conversions are active developmental programs rather than

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Plastid differentiation Processing and maturation Removal of abnormal proteins Etioplast

Chloroplast

Homeostasis Processing and maturation Protein quality control

Proplastid

Chromoplast Amyloplast

Senescence, Conversion of plastid type Processive degradation

Figure 4.1 Necessity of proteases for plastid life. The left panel depicts differentiation of plastids. Proplastids in the cells of meristematic tissues change their functions and morphology dynamically in response to the developmental status and the environmental signals. In plastid differentiation, the processing and maturation of proteins is probably most important because enormous plastid proteins are imported from cytosol to develop rapidly into other plastid types. Furthermore, the removal of abnormal proteins that are occasionally generated during protein folding and so on, might be necessary in this step. Once plastids are developed, quality control systems of proteins become more crucial for their homeostasis, for example, PSII repair cycle with D1 protein degradation. ATPdependent proteases complexes mainly control protein qualities. At the senescence of leaves and conversion of plastid types, large-scale degradation of plastid proteins is necessary. Consequently, processive protein degradation by various proteases is needed.

simple morphological changes. Consequently, protease activities in plastids are necessary for various processes during the conversion of plastid types. More importantly, chloroplasts are the location at which photosynthesis occurs: they are therefore the home of the most fundamental biochemical process for all living cells. Photosynthesis comprises two processes, one of which is a light-dependent reaction by which captured light energy is converted to chemical energy in the form of ATP and NADPH. The other is a light-independent reaction that converts carbon dioxide into organic compounds using chemical energy (Allen, 2002; Blankenship, 2002). Meanwhile, the photosynthetic apparatus in chloroplasts is subjected to unavoidable photooxidative damage during light irradiation (Barber and Andersson, 1992; Tyystja¨rvi and Aro, 1996). Irreversible inactivation of photosystem II (PSII) induced by excessive light, a process called photoinhibition, engenders inhibition of the growth of photosynthetic organisms (Aro et al., 1993; Barber and Andersson, 1992). Therefore, a tightly controlled protein quality control system is physiologically necessary for chloroplasts (Baena-Gonza´lez and Aro, 2002; Kato and Sakamoto, 2009). On the other hand, to date, more than 3000 proteins that have been localized to chloroplasts are estimated using programs to predict plastid

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targeting (Timmis et al., 2004). These proteins are sustained at proper levels, although the synthesis and denaturation of proteins occur constantly. Consequently, it is easy to imagine that the proteases might contribute to the control of protein levels for plastid homeostasis. Although proteolysis in plastids has been recognized since the 1980s, early approaches for identification of plastidic proteases by biochemical methods have been largely unsuccessful. This is one reason that, in contrast to protein synthesis, which has been studied extensively, protein degradation in plastids has remained poorly understood until recently. Nevertheless, recent studies using model plants, including Arabidopsis thaliana, have revealed numerous plastidic proteases that participate in proper organellar functioning through the maturation or activation of preproteins, the proper folding of proteins, and the degradation of unassembled or damaged proteins. In this review, we describe our current knowledge related to proteases identified in plastids of Arabidopsis and other higher plants. We enumerate plastidic proteases and discuss their physiological functions.

2. Overview In general, plastids are considered to have originated as endosymbiotic ancestral cyanobacteria. Therefore, it is not surprising that most chloroplastic proteases originated from bacterial homologues (Fig. 4.2) (Timmis et al., 2004). Examples of these proteases are three ATP-dependent proteases: Clp, FtsH, and Lon, which share a conserved ATP-binding motif but possess a different catalytic domain for proteolysis, and an ATP-independent protease, Deg (Gottesman, 1996; Gottesman et al., 1997). Of these major proteases, it is considered that Clp and Lon are involved in protein degradation in the stroma; others, FtsH and Deg, participate in protein degradation in thylakoid membranes (Adam, 2000; Adam and Clarke, 2002; Adam et al., 2006; Sakamoto, 2006). Furthermore, accumulating evidence shows that Clp and FtsH play critical roles in biogenesis and homeostasis of plastids. In chloroplasts, these proteases do not act as monomers but usually form large complexes, just as bacterial proteases do. It is noteworthy that these proteases are diversified and exist as multiple copies in photosynthetic organisms, in contrast to bacterial single copy proteases (Garcia-Lorenzo et al., 2006; Sokolenko et al., 2002). Proteome analyses and green fluorescent protein (GFP) transient assay using putative N-terminal transit peptide demonstrated actual chloroplast localization of some of them (Sakamoto et al., 2003). Additional studies using mutants in Arabidopsis have demonstrated that at least Clp, FtsH, and Deg form heterocomplexes in chloroplasts (Sakamoto et al., 2003; Sjo¨gren et al., 2006; Sun et al., 2007; Yu et al., 2004). The result––that these isomers of multiple complexes coordinately

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Chloroplast

Envelope

Plsp1

SPP

Rhomboid AraSP

Stroma PreP

CND41 Clp

Lon

GEP

SppA

Stromal Deg

Thylakoid membrane Plsp1

EGY1 TPP

Lumen

CtpA

FtsH

Lumenal Deg

Figure 4.2 Type and localization of plastid proteases. Proteases that have been found in plastids to date are portrayed in this figure. Several proteases are depicted as a complex according to their structural information. The left part shows a schematic representation of the model of protein targeting system for plastid proteins with signal peptidases.

regulated the accumulation levels of one another––suggests the complexity of plastid protease formation. The details are described in Section 3. On the other hand, although plastids have their own genome, only a few proteins (ca. 80) are encoded by their genome and produced within plastids. This means that most plastid proteins (estimated as more than 3000) that are encoded by the nuclear genome are synthesized in the cytosol and are imported into plastids posttranslationally (Reumann et al., 2005; Timmis et al., 2004). These proteins are synthesized as precursors with N-terminal transit peptide, and are ultimately transported into plastids through TOC/ TIC translocon machineries (Jarvis, 2008). After importation to stroma space, the stromal targeting transit peptide is cleaved from the precursor protein by the stroma processing peptidase (SPP) (Richter et al., 2005). Following removal of the transit peptide by SPP, the cleaved transit peptide is then further degraded by another metalloprotease because these fragments are expected to have toxic properties against plastid membrane structures and functions (Richter and Lamppa, 1999, 2002). The presequence protease, PreP, has been proposed to be responsible for this step (Nilsson Cederholm et al., 2009). In chloroplasts, many imported proteins have functions in stroma space; others are further targeted to the thylakoids by

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an additional pathway. These proteins are synthesized with a bipartite transit peptide, and are transported into the thylakoid space by either the Tat or the Sec pathway (Robinson et al., 2001). Then, the thylakoid targeting signal of the proteins is cleaved by the thylakoid processing peptidase (TPP) (Shackleton and Robinson, 1991). In addition, results of a recent study implied that plastidic type I signal peptidase, Plsp1, is involved in the maturation of precursors both in the envelope and in the thylakoid membrane (Inoue et al., 2005; Shipman and Inoue, 2009). Chloroplast-encoded proteins also undergo precursor processing. A well-studied example of the processing of chloroplast-encoded protein is the maturation of D1 protein in PSII by the C-terminal processing peptidase, CtpA. Details of these peptidases are presented in Section 4. Plastids contain membranes of three different types. The inner and outer envelope membranes are the permeability barrier between plastids and cytosol; thylakoid-membrane structures, which are well developed in chloroplasts, are a necessary site for photosynthesis. Because the plastid envelope and thylakoid membranes are the sites of lipid synthesis and photosynthesis, respectively, these membranes contain various proteins along with those of the stromal and luminal space. It is therefore not surprising that plastids have several intramembrane proteases to degrade membrane proteins. To date, a homologue of rhomboid protease and two homologues of a sterol-regulatory element binding protein site 2 protease, S2P, were identified in chloroplasts (Bo¨lter et al., 2006; Chen et al., 2005; Kmiec-Wisniewska et al., 2008). These peptidases are described in Section 5 and other proteases are described in Section 6.

3. Major Proteases In this chapter, we describe the plastid proteases, Clp, FtsH, Deg, and Lon, which are homologues of known bacterial proteases. The loss of these proteases often causes serious impairment of plastid homeostasis and biogenesis, thereby impairing plant growth. Moreover, the existence of their multiple copies in plant genomes is an important feature. Taken together, these proteases are grouped as major plastid proteases.

3.1. Clp protease 3.1.1. Basic structure A highly conserved ATP-dependent serine-type protease complex, Clp, was originally identified in Escherichia coli. To date, it has been found in almost all bacteria and eukaryota except for archaea, mollicutes, and some fungi (Porankiewicz et al., 1999; Yu and Houry, 2007). The functional Clp

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complex comprises two components: a catalytic component for proteolysis and an ATP-dependent chaperone. In E. coli, the subunit ClpP of the catalytic component has the catalytic triad Ser–His–Asp, which is conserved in typical serine-type proteases (Wang et al., 1997). X-ray crystallographic analyses of the catalytic component revealed two stacked rings of seven identical ClpP subunits forming a barrel-like structure enclosing a large chamber in which catalytic sites are exposed (Wang et al., 1997). On the other hand, chaperone components comprise a homohexameric ring of the Clp/HSP100 subunits, either ClpA or ClpX (Grimaud et al., 1998). Actually, ClpA has two distinct AAAþ domains (Class I), whereas ClpX has only a single AAAþ domain (Class II). The hexameric chaperon rings connect one or both ends of the ClpP catalytic component to form ClpAP or ClpXP holoenzyme complexes (Grimaud et al., 1998; Kessel et al., 1995). The ClpA or ClpX homohexamer is believed to recognize denatured or misfolded proteins; then the substrates translocated to an unfolded state recruit into the ClpP proteolytic chamber through narrow axial pores (Yu and Houry, 2007). Consequently, the formation of ClpAP and ClpXP complex enables ClpP to degrade the large native protein. By itself, ClpP proteolytic chamber can only degrade small peptides (Gottesman et al., 1997). In addition to the well-organized proteolytic system, the small adaptor ClpS confers substrate specificity on these holoenzymes to prevent the inadvertent degradation of functional proteins. The characterization of ClpS revealed that direct binding of ClpS to the N-terminal domain of ClpA enhanced the N-terminal residues-dependent substrate degradation, which is called an N-end rule (Erbse et al., 2006; Wang et al., 2008). In eukaryote cells, it is well known that the protein degradation system is mediated by the 26S proteasome N-end rule substrate recognition, the labeling of substrates via covalent linkage to ubiquitin by E3 ligases (Mogk et al., 2007). Together with the similarity of architecture between Clp and 26S proteasome, these features show that they share common principles of the unfolding-coupled processive protein degradation, although proteolytic machineries for degradation differ between prokaryotes and eukaryotes. Consequently, it is currently thought that Clp proteolytic complexes in prokaryotes are the counterparts of eukaryotic proteasomes. 3.1.2. Features in plastids In photosynthetic organisms, genome information has revealed by far the greatest diversity of Clp subunits. Although most bacteria contain only one copy of proteolytic subunit ClpP, Arabidopsis has at least 10 individual proteolytic subunits, six of which are palalogs of ClpP (ClpP1–ClpP 6) and four of which are palalogs of a ClpP-like subunit (ClpR1–ClpR4) that does not have all three residues of the Ser–His–Asp catalytic triad (Clarke et al., 2005). Recent reports show that ClpR in cyanobacteria is proteolytically inactive and that its presence in the proteolytic core complex does not

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affect the overall proteolytic activity (Andersson et al., 2009). All Clp genes are encoded in the nuclear genome, except for ClpP1, which is in the plastid genome. The vast majority of Arabidopsis Clp subunits are localized in plastids (Peltier et al., 2001, 2004; Zheng et al., 2002). Among the proteolytic subunits, only ClpP2 is localized to mitochondria and forms a complex with its chaperone components, ClpXs (Halperin et al., 2001; Peltier et al., 2004). In addition to Clp core subunits, an ortholog of the bacterial adapter protein ClpS (ClpS) and two plant-specific subunits (ClpT1-2) with sequence similarity to the N-terminal domain of HSP100 proteins have been discovered (Peltier et al., 2001, 2004). ClpS (previously called ClpT) was renamed to be consistent with the E. coli ClpS. Consequently, ClpT1 and T2 were also renamed from ClpS1 and S2. Identification of Clp subunits using native gel electrophoresis and subsequent mass spectrometry revealed that the 325–350-kDa Clp core complex contains five ClpP (ClpP1, ClpP3–ClpP6), four ClpR (ClpR1–ClpR4), and two plant-specific ClpT (ClpT1, ClpT2) in nonphotosynthetic plastids as well as chloroplasts (Peltier et al., 2001, 2004). Additional studies by immunoblot analysis using specific antibodies on separated Clp complex by native-PAGE indicate that the stromal Clp core complex is separable into two smaller subcomplexes, presumably heptameric rings: one of 230 kDa containing ClpP1 and ClpR1–ClpR4, and the other of 180 kDa containing ClpP3– ClpP6 (Sjo¨gren et al., 2006). In addition, ClpT1 is at least thought to be peripherally associated to the axial sites of the proteolytic core complex, which binds to the ClpP3–ClpP6 subcomplex, although neither ClpT1 nor ClpT2 attaches to ClpP1/ClpR1–ClpR4 subcomplex. Although the structural arrangement of the complicated ClpP core complex is poorly understood, the result––that most knockout mutant lines of Clp proteolytic core complex show the lethal phenotype, except for partial compensation for the loss of ClpR1 by the relative increase of ClpR3––indicates that each subunit is nondisplaceable and is necessary for the functional Clp protease (Clarke et al., 2005; Koussevitzky et al., 2007; Kuroda and Maliga, 2003; Stanne et al., 2009). Among the palalogs of Clp/Hsp100 AAAþ chaperone, four Clp/ Hsp100 AAAþ chaperones (ClpB3, ClpC1, ClpC2, and ClpD) are localized in chloroplasts (Moore and Keegstra, 1993; Peltier et al., 2004; Weaver et al., 1999). All have two distinct AAAþ domains and belong to the Class I Clp/Hsp100 family, but one, ClpB3, lacks the conserved ClpP binding domains (Peltier et al., 2004). Consequently, it has been suggested that ClpB3 does not form a complex with the Clp core complex, but it functions as a chaperon to prevent aggregation of abnormal proteins during heat stress (Keeler et al., 2000). In contrast, the existence of the conserved motifs in ClpC1, ClpC2, and ClpD suggests possible binding between these chaperone subunits and the Clp core complex (Peltier et al., 2004). Although no ClpCP or ClpDP complex has been confirmed yet in vivo in Arabidopsis, a

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coimmunoprecipitation experiment using the stroma fraction from pea chloroplasts demonstrated ATP-dependent interaction between ClpC and ClpP (Desimone et al., 1997). Results also showed that only a portion of ClpC associated with the translocon Tic of the inner envelope membrane, suggesting its function in the chloroplast protein import process as a chaperon (Nielsen et al., 1997). In contrast with Clp core complex mutants, the knockout mutants of ClpC are viable; clpC1 mutant showed a distinct slowgrowth and chlorotic phenotype caused by the reduced chloroplast development, whereas clpC2 mutants were visibly indistinguishable from wild type (Constan et al., 2004; Park and Rodermel, 2004; Sjo¨gren et al., 2004). However, double-mutant clpC1 clpC2 showed embryo lethality (Kovacheva et al., 2007). This result was consistent with an early study that showed that tobacco cell lines with significantly reduced levels of ClpC could not survive (Shanklin et al., 1995). Taken together, the Clp chaperon components in chloroplast might be redundant, unlike other Clp core subunits, but they are necessary in chloroplast biogenesis. 3.1.3. Function and protein substrates Analyses of the physiological function of Clp were conducted using knockdown mutants and antisense lines because most of the Clp proteolytic core complex is necessary for their viability, as described above. The downregulation of Clp complexes in the knockdown mutants and antisense lines caused slow growth and leaf chlorosis (Kim et al., 2009; Rudella et al., 2006; Shikanai et al., 2001; Sjo¨gren et al., 2006; Zheng et al., 2006). In the clpP4 antisense line, the chlorosis was more severe in the leaf midvein region. Consequently, the mutants showed the yellow-heart variegated phenotype (Zheng et al., 2006). In the chlorotic leaves, the chloroplasts were severely compromised in their thylakoid membrane structure and function. These results show that the Clp proteolytic activity is necessary for chloroplast biogenesis. Of the substrates for Clp protease, it has been predicted that Clp protease is involved in the degradation of unassembled or abnormal proteins in chloroplast, but it was difficult to identify the in vivo protein substrates of Clp protease. Until recent years, only an early study of the green alga Chlamydomonas reinhardtii showed the cytochrome b6/f complex as a putative substrate for Clp protease during nitrogen starvation (Majeran et al., 2000). However, recent advances in the proteome approach and mass spectrometry analyses have allowed further identification of putative protein substrates of Clp protease. The identification of substrates in clp mutants showed that most protein substrates of Clp protease were involved in homeostatic functions: molecular chaperons, chloroplast protein synthesis, protein import, etc. (Kim et al., 2009; Sjo¨gren et al., 2006; Stanne et al., 2009; Zybailov et al., 2009). Although it remains largely unknown whether Clp protease functions in the degradation of these proteins directly or

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indirectly, these results support the hypothesis that the Clp protease serves an important role in chloroplast housekeeping. On the other hand, the screening for mutants that exhibits overaccumulation of chlorophyll a oxygenase (CAO) indicates that Clp protease is involved in the regulation of chlorophyll b synthesis through degradation of the CAO protein (Nakagawara et al., 2007). It is noteworthy that the N-terminal half of CAO contains a recognition sequence that is recognized by Clp protease in the presence of chlorophyll b (Kanematsu et al., 2008; Sakuraba et al., 2009). This result implies the possible regulation mechanism of protein degradation by the conformational change of substrate proteins. The recognition mechanisms of specific substrates by protease in plastid remain largely unknown. For this reason, more effort is expected to provide useful clues to elucidate it.

3.2. FtsH protease 3.2.1. Basic structure A membrane-bound ATP-dependent metalloprotease that belongs to an AAA (ATPase associated with diverse cellular activities) protease subfamily of the large AAAþ protein family, FtsH is the only protease that is necessary for bacterial growth among the ATP-dependent proteases (Tomoyasu et al., 1993). It comprises the N-terminal one or two transmembrane domain and the C-terminal ATPase domain and proteolytic domain. The latter contains the His–Glu–X–X–His motif, which is characteristic for zinc-dependent metalloprotease (Ito and Akiyama, 2005). In E. coli, because FtsH is anchored in the plasma membrane and the protease domain faces the cytoplasm, the processive protein degradation by FtsH through both Nterminal and C-terminal regions on membrane is proposed at present. Results of early crystal structure studies of the isolated ATPase domain of FtsH suggest that FtsH forms a hexameric ring structure like other members of the AAA protein family (Krzywda et al., 2002; Niwa et al., 2002). In addition, interaction between FtsH and the HflKC membrane protein complex proposed the existence of stable large complex in the E. coli membrane (Kihara et al., 1996; Saikawa et al., 2004). On the other hand, the crystal structures of the whole cytosolic region of bacterial FtsH were recently determined using an FtsH construct lacking the transmembrane domains (Bieniossek et al., 2006; Suno et al., 2006). It is noteworthy that these analyses revealed protease catalytic sites of FtsH located at the peripheral region of the hexamer ring but not at the chamber wall. This result implied that FtsH contains six independent proteolytic chambers, although only one unfolding/translocating machine exists per complex. In general, the substrates of ATP-dependent proteases are first unfolded and translocated through the narrow central pore into the central proteolytic chamber where catalytic sites are exposed (Licht and Lee, 2008). Of substrates for

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FtsH, however, the structure information suggests the possibility of the atypical pathway that the unfolded polypeptides are delivered into one of the proteolytic sites of the subunit (Bieniossek et al., 2006; Suno et al., 2006). A three-fold-symmetric open-to-closed motion of the subunits was proposed to drive the translocation of a substrate to the protease catalytic sites. 3.2.2. Features in plastids In higher plants, chloroplastic FtsH was first identified from spinach leaves through immunological analysis using an antibody against E. coli FtsH (Lindahl et al., 1996). To date, 12 ftsH genes coding for fully functional proteases are found in the Arabidopsis genome (Sokolenko et al., 2002). In addition, four other homologous sequences are identified, but these lack a zinc-binding motif. Therefore, these are not regarded as functional FtsH, although they might yet fulfill ATPase functions. Of the 12 FtsH homologues, 9 (FtsH1, -2, -5, -6, -7, -8, -9, -11, and -12) were found to be located in the chloroplast by transient-expression assays with GFP fusions, and the chloroplast-targeted pair genes––FtsH1 and FtsH5, FtsH2 and FtsH8, and FtsH7 and FtsH9––were likely duplicated (Sakamoto et al., 2003). Furthermore, the presence of FtsH1, -2, -5, and -8 in chloroplasts were confirmed using immunoblot analysis and mass spectrometry (Friso et al., 2004; Sakamoto et al., 2003; Sinvany-Villalobo et al., 2004; Yu et al., 2004). These FtsHs were localized to thylakoid membranes, with their catalytic C-terminal region facing the stromal side of the membrane (Lindahl et al., 1996; Sakamoto et al., 2003). Meanwhile, one chloroplastic FtsH, FtsH11, was suggested to be dually located in both chloroplasts and mitochondria (Urantowka et al., 2005). Proteomic analysis of isolated thylakoid membranes showed that FtsH1, -2, -5, and -8 are four major isomers of chloroplastic FtsH complexes (Yu et al., 2004). Of these isomers, FtsH2 is the most abundant isomer, followed by FtsH5, FtsH8, and FtsH1, which showed the least accumulation (Sinvany-Villalobo et al., 2004). The differential protein levels of these four isomers seem to correlate well with the severity of phenotypes. The mutant of FtsH2 and FtsH5 show severe and weak leaf-variegated phenotypes, respectively, although the loss of FtsH1 and FtsH8 does not result in an obvious phenotype (Chen et al., 2000; Sakamoto et al., 2002, 2003; Takechi et al., 2000). Furthermore, several studies have revealed oligomeric complexes of chloroplastic FtsH and the interaction of FtsH2 and FtsH5 in thylakoid membranes (Sakamoto et al., 2003; Yu et al., 2004). These results, together with the result that the loss of either FtsH2 or FtsH5 caused the concomitant decrease of the other (Sakamoto et al., 2003), suggest the heteromeric hexamer form of FtsH complexes in chloroplast, which differs from the homocomplexes in bacteria. On the other hand, leaf variegation of the mutant lacking FtsH2 was rescued by overexpression of FtsH8; the

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phenotype of the mutant lacking FtsH5 was rescued by overexpression of FtsH1 (Yu et al., 2004, 2005). In contrast, the overexpression of FtsH5 was unable to rescue the leaf variegation of the mutant lacking FtsH2. Furthermore, both ftsh1 ftsh5 and ftsh2 ftsh8 double mutants showed an albino-like phenotype (Zaltsman et al., 2005b), although the ftsh2 ftsh5 double mutant remained viable with severe leaf variegation (Sakamoto et al., 2002). Taken together, these results suggest that the major heterocomplex in chloroplast comprises isomers of two types (Type A, as represented by FtsH1 and FtsH5; and Type B, as represented by FtsH2 and FtsH8), although the stoichiometry of each isomer in the heterocomplex remains unclear. Whether other chloroplastic FtsH (FtsH6, -7, -9, -11, and -12) form heterocomplexes also remains unclear. The characterization of many variegated mutants to be allelic to ftsH2 showed that the amino acid substitutions in ATPase domain caused leaf variegation (Sakamoto et al., 2004). However, no mutation has been detected in the proteolytic domain, suggesting that not all proteolytic activities of FtsH heterocomplexes are required for their function in chloroplast. Functional analysis of the proteolytic domain of FtsH heterocomplexes was attempted recently using transgenic plants that ectopically expressed a proteolytically inactive FtsH2. Although the FtsH heterocomplex containing proteolytically inactive FtsH2 apparently showed slightly reduced protease activity, they were still functional because the protease activity was provided by other isomers (Zhang et al., manuscript in preparation). Although the structure information of chloroplastic FtsH heterocomplexes is very poor, this result demonstrated that the proteolytic chamber acts independently in the FtsH heterocomplex. In fact, FtsH can maintain protease activities when at least one isomer is active. 3.2.3. Function and protein substrates As described above, the mutant lacking FtsH2 or FtsH5 shows severe and weak leaf-variegated phenotypes. The variegated leaves contain green sectors with normal-appearing chloroplasts and white sectors formed by visible cells with undifferentiated plastids (Kato et al., 2007). Recent studies have shown that undifferentiated plastids were arrested at the early stage of differentiation of plastids into chloroplasts (Sakamoto et al., 2009). The development of normal-appearing chloroplasts also proceeded very slowly in the mutant lacking FtsH2. On the other hand, the variegated phenotype apparently correlates with the level of FtsHs, suggesting a threshold model by which the overall FtsH levels destine the cell-autonomously thylakoid development (Yu et al., 2004; Zaltsman et al., 2005a). Furthermore, genetic approaches to search suppressors of leaf variegation suggest the balance between protein synthesis and protein degradation by FtsH during chloroplast biogenesis affects leaf variegation (Miura et al., 2007; Yu et al., 2008). Taken together, although the substrates of FtsH in the chloroplast

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developmental stage remain unknown, these results indicate that FtsH plays a crucial role in the formation of thylakoid membranes. Aside from the genetic and the microscopic approach to reveal the function of FtsH in chloroplast biogenesis, in vitro analyses suggest the involvement of FtsH in the degradation of several thylakoid proteins; unassembled Rieske Fe-S protein and D1 protein of PSII reaction center (Lindahl et al., 2000; Ostersetzer and Adam, 1997). In fact, the high sensitivity to photoinhibitory light exposure in the mutant lacking FtsH2 or FtsH5 suggests the involvement of major FtsH complexes in the in vivo repair cycle of PSII (Sakamoto et al., 2002, 2004). Furthermore, the defect of degradation of D1 protein under a photoinhibitory light condition in ftsh2 mutant leaves has been reported (Bailey et al., 2002). However, the D1 degradation examined in this study was limited because the presence of green and white sectors interferes with the estimation of protein levels. The frailty of variegated leaves presents technical difficulties of inhibitor or pulse-label experiments. Recently, a study using the nonvariegated suppressor lines that overcome the difficulty of handling variegated leaves demonstrated the crucial roles of FtsH2 and FtsH5 in the PSII repair cycle to avoid photooxidative stress in chloroplasts (Kato et al., 2009). To date, it is estimated that the FtsH heterocomplexes that are constituted of Type A (FtsH1 and FtsH5) and Type B (FtsH2 and FtsH8) isomers are a major protease in D1 degradation (Fig. 4.3) (Kato and Sakamoto, 2009). However, the degradation of other thylakoid proteins by this FtsH heterocomplexes requires further investigation. Of the other chloroplastic FtsH homologues, the involvement of FtsH6 in the degradation of the light-harvesting complex II during high light acclimation and senescence is suggested, along with the contribution of FtsH11 in thermotolerance (Chen et al., 2006; Zelisko et al., 2005).

3.3. Lon protease Lon is the first ATP-dependent protease to be purified from E. coli. Mounting evidence indicates that it is responsible for the degradation of abnormal, damaged, and naturally unstable proteins in addition to protein degradation to adapt to a nutritional downshift (Rotanova et al., 2006; Van Melderen and Aertsen, 2009). Comparison of the amino acid sequences of Lon proteases suggests that it has a variable N-terminal domain, a central ATPase domain belonging to the AAAþ superfamily, and a C-terminal proteolytic domain, but no transmembrane domain. It is particularly interesting that Lon has DNA binding activity, although it remains unclear whether it has a conserved domain required for DNA binding (Fu et al., 1997). Lon forms oligomeric assemblies, but the number of subunits has been variously reported. The crystal structure of catalytic domain of Lon in E. coli shows that it forms a hexameric ring-like structure with a catalytic Ser–Lys dyad (Botos et al., 2004; Park et al., 2006),

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Stroma

Light

2. Partial disassembly of photosystem II

1. Photo damage

Functional photosystem II D1

D2

LHCII

FtsH

Stroma 5. Maturation

Nascent D1 CtpA Lumen

3. Proteolysis of damaged D1

Damaged D1 4. Synthesis of newly D1 Lumen

Deg5,8

Deg1

Figure 4.3 Schematic drawing of the D1 quality control system in the PSII repair cycle. A series of PSII repair cycle steps is presented in this figure, especially steps with proteolysis are highlighted. (1) Photodamage to functional PSII complex. Although light energy is the driving force for the photochemical reaction, it always damages the photosynthetic complexes at a certain frequency. (2) Partial disassembly of damaged PSII. Damaged PSII migrate to the stroma thylakoid; partial disassembly of PSII occurs. (3) Proteolysis of damaged D1 protein. The transmembrane helices of the D1 protein are shown. The processive proteolysis by FtsH is initiated from the N-terminal end of D1 on the stromal side. Deg proteases cleave intermembrane peptide regions that are exposed to the lumenal side. It is considered that Deg proteases would assist effective D1 degradation by increasing the number of D1 endoproteolytic intermediates that are accessible to FtsH. (4) The D1 nascent chain is inserted cotranslationally into thylakoid membranes. (5) Maturation of D1. After synthesis of new D1, the C-terminal of D1 precursor is processed by CtpA peptidase for forming a functional PSII complex. LHCII, light harvesting chlorophyll complex II; D1 and D2, subunit of the reaction center of PSII.

although a heptameric ring structure has been suggested in yeast mitochondria (Stahlberg et al., 1999). Homologues of Lon have been reported in higher plants (Barakat et al., 1998; Heazlewood et al., 2004; Ostersetzer et al., 2007; Sarria et al., 1998), in spite of its absence from basic photosynthetic organisms (Sokolenko et al., 2002). In Arabidopsis, among the enumerated 11 homologous genes (GarciaLorenzo et al., 2006), four homologues, Lon1–Lon4, are probably functional based on a homology search with E. coli Lon (Ostersetzer et al., 2007). However, Lon3 is suspected to be a pseudogene because the expression of Lon3 has not been detected (Ostersetzer et al., 2007). Of three functional Lon proteins, Lon1 is localized to mitochondria and is involved in anther-specific protein degradation during tapetum formation (Sarria et al., 1998). Actually, Lon2 has the peroxisomal C-terminal localization signal and the C-terminal sequence from Lon2 fused to GFP was localized to the peroxisome ( Janska, 2005) (A. Kato, personal communication). On the other hand, in vivo transient expression assay using GFP fused with the N-terminal region of Lon4 in Arabidopsis protoplast and tobacco suspension-cultured cells indicate dual targeting of Lon4 for both chloroplasts and mitochondria. Further immunoblot analyses have indicated that Lon4 is tightly attached to the stromal side of

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thylakoid membranes (Ostersetzer et al., 2007). Together with proteomic analysis of chloroplast (Baginsky et al., 2004), Lon4 seems to play roles in chloroplast proteolysis.

3.4. Deg protease 3.4.1. Basic structure Deg protease is a periplasmic ATP-independent serine-type protease. It comprises the N-terminal proteolytic domain, and the C-terminal one or two PDZ domains, which are important for protein–protein interactions (Clausen et al., 2002). One Deg protease, DegP, has been best characterized in E. coli, in which it is not necessary for cell viability but in which it is necessary for survival at high temperatures (Skorko-Glonek et al., 1995). Using a natural substrate for DegP in E. coli, it has been determined that DegP has the unique property of functioning both as a protease and as a chaperone to prevent the accumulation of abnormal proteins. The chaperon activity of DegP is predominant at low temperatures, although the proteolytic activity increases dramatically at elevated temperatures (Spiess et al., 1999). To date, it has been proposed that the conformation change of DegP by a temperature shift might induce a switch between protease and chaperon activity. The first X-ray structure revealed the hexameric complex of DegP, which is formed by the staggered association of two trimeric rings and which encloses a central chamber where catalytic sites are exposed (Krojer et al., 2002). Following this hexameric structure, the physiologically functional model of DegP has been proposed, but it remains unknown how substrates can be channeled into the chamber for degradation or refolding. Recent works investigating structural information of DegP have provided a clue to answering this question. The studies showed that DegP forms a massive 12-mer and 24-mer spherical cage structure in the presence of substrates and reverts to a hexameric form after the substrate degradation is completed ( Jiang et al., 2008; Krojer et al., 2008). Based on these results, it is proposed that the hexameric form is the resting form of DegP and that the actual active protease/chaperon is oligomeric cages, followed by interaction of the substrate with DegP and the destabilization of two trimeric rings in a hexameric structure (Ortega et al., 2009). On the other hand, three types of oligomeric bowl-shaped structures of DegP on membranes were also revealed recently using electron microscopy image analysis (Shen et al., 2009). These bowl-shaped structures were constructed using four, five, and six trimeric units and had higher proteolytic activity, although the chaperon activity was decreased. 3.4.2. Features in plastids Similar to other major proteases, Deg proteases comprise a protein family in photosynthetic organisms. In Arabidopsis, the Deg gene family is encoded by 16 genes; four Degs (Deg1, -2, -5, and -8) are already found in the chloroplast

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as peripherally attached to the thylakoid membranes (Haussu¨hl et al., 2001; Huesgen et al., 2005; Itzhaki et al., 1998; Peltier et al., 2002; Schubert et al., 2002). Actually, Deg2 has been located on the stromal side of thylakoid membrane and Deg1, -5, and -8 have been found in the lumen. Each chloroplast Deg contains one PDZ domain, except for Deg5, which has no apparent PDZ domain (Kieselbach and Funk, 2003). The possibility of the massive spherical cage or bowl-like structure formation like E. coli DegP is currently unclear, but the hexameric formation of Deg1, -5, and -8 were demonstrated using size-exclusion chromatography or separation in BlueNative PAGE (Chassin et al., 2002; Sun et al., 2007). In addition, the pulldown assay using His-Deg5 and His-Deg8 fusion proteins showed a putative interaction between Deg5 and Deg8 (Sun et al., 2007). In addition, recently obtained results showed the presence of Deg7 and Deg11 in chloroplast stroma. Although Deg7 was largely present in stroma, Deg7 was also detected in thylakoid membrane fractions. Additional study showed that Deg7 was peripherally attached to the stromal side of thylakoid membranes, and that the association of Deg7 with thylakoid membranes was accelerated by high light treatment (L. Zhang and X. Sun, personal communication). 3.4.3. Function and protein substrates An earlier in vitro study with recombinant protein showed that the lumenal Deg1 degraded thylakoid lumen proteins, such as in vitro-translated plastocyanin and OEC33, the extrinsic 33 kDa protein associated with the oxygen-evolving complex of PSII (Chassin et al., 2002). Moreover, another in vitro study using recombinant Deg suggested that the stromal Deg2 is involved in the initial endoproteolytic cleavage of D1 protein pretreated with heat or high light intensity (Haussu¨hl et al., 2001). This result was consistent with two-step D1 degradation model by which the photodamaged D1 protein is specifically cleaved in the stroma-exposed DE-loop in an ATP-independent manner; subsequently, D1 is processively degraded by ATP-dependent protease, such as FtsH. However, in vivo study with mutants lacking Deg2 showed that the D1 turnover in these mutants proceeded at a rate that was similar to that in wild type under high light irradiation, suggesting that the cleavage of photodamaged D1 by Deg2 is not necessary for its degradation (Huesgen et al., 2006). In contrast, in vivo study of Deg1 showed that Deg1 is important for maintenance of chloroplast homeostasis because Deg1 knockout mutants are not available. The analysis using RNAi knockdown mutants of Deg1 showed high sensibility to photoinhibition and reduced levels of several degradation intermediates derived from the C-terminal part of D1 (Kapri-Pardes et al., 2007). These RNAi lines had much reduced levels of FtsH and Deg2; the variegated FtsH mutants also showed reduced levels of Deg1, suggesting coordinated accumulation between the two proteases. Of Deg5 and Deg8, these two luminal Degs are necessary for efficient PSII repair during high light irradiation but

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they are not necessary for survival (Sun et al., 2007). Degradation products derived from the N-terminal part of D1 in a thylakoid membrane were less accumulated in mutants lacking both Deg5 and Deg8 when exposed to strong light, suggesting the specific cleavage in the luminal CD-loop of D1 by these Degs (Fig. 4.3). In addition, the recent analyses of deg7 mutants under high light conditions proposed the contribution of Deg7 to D1 degradation (L. Zhang and X. Sun, personal communication). However, to date, the contribution of Degs to any in vivo proteolytic process beyond the D1 degradation remains unknown.

4. Processing Peptidases 4.1. SPP The stromal processing peptidase, SPP, is a soluble metalloprotease involved in the cleavage of most precursor targeted to the chloroplast (Richter et al., 2005). The existence of the inverted zinc-binding motif HXXEH in its Nterminal region suggests that SPP is one member of the metallopeptidase M16 family (VanderVere et al., 1995). Although it remains unclear how SPP interacts with the transit peptide, the deletion analysis of the Nterminal and C-terminal halves of SPP indicated that the N-terminal half is necessary for binding with their substrates (Richter and Lamppa, 2003). To date, an overview of the mechanism of precursor processing reaction by SPP has been determined (Richter et al., 2005). The first step of processing reaction is the binding of the transit peptide by SPP. Following recognition by SPP, the mature protein is released by endoproteolytic cleavage by SPP, but the transit peptide remains bound to SPP. The transit peptide is then cleaved again, converted to subfragments by SPP, and released. Finally, the subfragments are degraded by another metalloprotease with low substrate specificity. The antisense SPP transformant of tobacco showed chlorosis and retarded growth (Wan et al., 1998). Furthermore, most antisense transformants in Arabidopsis caused lethality to seedlings (Zhong et al., 2003), indicating that SPP plays a crucial role in chloroplast biogenesis and plant viability. In addition, the import capacity of the precursor was suppressed by the reduction of the efficient precursor processing in the antisense transformants. These results demonstrate that the function of SPP is integrated into a series of precursor import mechanisms.

4.2. PreP In higher plants, presequence protease, PreP, was identified initially from potato tuber mitochondria as a protease involved in degradation of mitochondrial presequence (Sta˚hl et al., 2002). Two homologues exist in the

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Arabidopsis genome; further studies using Arabidopsis homologues showed that both AtPreP1 and AtPreP2 are dual-targeted to mitochondria and chloroplasts (Bhushan et al., 2003, 2005; Moberg et al., 2003). An ATP-independent metalloprotease of 110 kDa, PreP contains an inverted version of the common zinc-binding motif HXXEH (Stahl et al., 2002). The crystal structure analysis of AtPreP1 showed two bowl-shaped halves that are mutually connected by a unique hinge region enclosing a large proteolytic chamber that shields the active site (Johnson et al., 2006). This structure proposed a hinge-bending motion mechanism that causes the protease to open and close in response to substrate binding. The chamber size is suitable for the peptide substrates, but it is too small to hold larger proteins. This structure prediction is consistent with the results that PreP cleaves peptides between 10 to 65 amino acid residues without apparent sequence specificity (Sta˚hl et al., 2005). Mounting evidence suggests the involvement of PrePs in the degradation of the released transit peptides by SPP. Although single T-DNA insertion lines of AtPreP1 and AtPreP2 showed no clear phenotype, the most recent study of the Arabidopsis double-knockout mutant prep1 prep2 revealed a chlorotic phenotype in true leaves with aberrant chloroplasts and mitochondria (Nilsson Cederholm et al., 2009). This result suggests the responsibility of PrePs for in vivo proteolytic events. However, the question remains of how ATP requirements that were previously predicted are involved in the transit peptide turnover.

4.3. TPP TPP, found in the thylakoid membrane, belongs to the type I signal peptidase family. The signal peptidase is called SPase I. Its orthologues are found in bacteria archaea, fungi, plants, and animals (Paetzel et al., 2002). SPase I is a serine-type protease using a Ser–Lys or Ser–His catalytic dyad. It is typically anchored to the membrane with one or two transmembrane domains. At the transport of proteins across the membranes, SPase I functions to cleave the signal peptide from the immature protein (Tuteja, 2005). In chloroplasts, TPP uses the Ser–Lys catalytic dyad mechanism that functions in the prokaryotes and mitochondria, but not the eukaryotic endoplasmic reticulum (Chaal et al., 1998). The hydropathy profile indicated that TPP has a single transmembrane region. Its catalytic center is located at the thylakoid lumen face (Chaal et al., 1998). The substrate specificity of TPP is similar to that of other SPase I (Shackleton and Robinson, 1991). It conforms to the well-known (-3, -1) rule, which states that the residues at the -3 and -1 positions are related to the cleavage site (von Heijne, 1983). These motifs are typically located in the N-terminal region of the signal peptide of lumenal proteins, which are imported via the Tat pathway into the thylakoid lumen.

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4.4. Plsp1 Another homologue of SPase I, Plsp1, has been sought in Arabidopsis as a peptidase responsible for the complete maturation of Toc75, a component of the protein translocon in the outer envelope of chloroplasts, which is synthesized as a larger precursor and which undergoes multiple cleavages during maturation (Inoue et al., 2005). The T-DNA insertion line plsp1 showed accumulation of the intermediate form of Toc75 that lacks the first processing part cleaved by SPP in the stroma, but which retains a second processing part of the transit peptide. The plsp1 shows the seedling lethal phenotype with severe reduction of the thylakoid membrane development. It is noteworthy that the in vitro chloroplast protein import assay reveals that it localized not only to envelope membranes but also to thylakoid membranes (Shipman and Inoue, 2009). Consistent with this dual localization, the disruption of Plsp1 caused accumulation of intermediates of the thylakoid lumenal oxygen evolving complex protein of PSII, OEC33, although it remains unknown whether Plsp1 is involved directly or through proper regulation of TPP in OEC33 maturation (Shipman and Inoue, 2009). Consequently, Plsp1 apparently has multiple functions in both envelope membranes and the thylakoid membranes. Together with studies of TPP, additional characterization will help to reveal multiple functions of Plsp1 in the biogenesis of chloroplast internal membranes.

4.5. Ctp Many proteins are synthesized initially as precursor polypeptides. They are subsequently converted to mature forms by proteolytic processing at the N-terminal or C-terminal of the precursor. In most cases, N-terminal processing contributes to their translocation across membranes, as described above. In contrast, the physiological significance of C-terminal processing is poorly understood. The carboxyl-terminal processing protease, CtpA was identified initially using genetic complementation analysis of the PSII deficient mutant of Synechocystis sp. PCC 6803 (Shestakov et al., 1994). In fact, CtpA has significant homology to the E. coli tail-specific protease, Tsp, which is periplasmic endopeptidase and which is involved in selective degradation of a protein bearing a nonpolar C-terminus (Silber et al., 1992). The crystal structure of CtpA from the eukaryotic alga Scenedesmus obliquus showed CtpA as monomeric and comprises three folding domains (Liao et al., 2000). The catalytic Ser–Lys dyad is located in the center of the protein, and the PDZ motif, which plays a role in protein–protein interaction and which is expected to recognize substrates, is in the middle domain. In higher plants, the peptidase that exhibits similarities to the sequences of the CtpA of Synechocystis sp. PCC 6803 was purified from spinach thylakoids using chromatography (Inagaki et al., 1996). The higher plant

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CtpA was also identified from barley and pea (Bowyer et al., 1992; Oelmuller et al., 1996). They showed proteolytic activity for C-terminal cleavage of D1 precursor as well as that in Synechocystis sp. PCC 6803. This proteolytic removal step of the C-terminal extension of D1 precursor is necessary for construction of functional PSII reaction center because it precedes the binding of the manganese cluster and proper assembly of the extrinsic proteins into PSII (Roose and Pakrasi, 2004). In Arabidopsis, however, the T-DNA insertion mutant of CtpA showed similar growth with that of wild type; the photosynthetic protein levels and the formation of thylakoid membrane protein complexes was comparable to those of wild type under normal light conditions (Yin et al., 2008). Furthermore, accumulation of D1 precursor was not detected in the mutant. Only in stressful light conditions did the mutant show retarded growth, which was probably caused by the slower syntheses of newly D1 in the PSII repair cycle. These results suggest the functional redundancy between CtpA and the other two putative CtpA homologues that were identified in the chloroplast luminal fractions by proteome analysis (Yin et al., 2008). To date, except for the involvement in processing of D1 protein in PSII assembly, selective cleavage by CtpA in protein maturation remains unknown.

5. Intramembrane Proteases 5.1. Rhomboid Rhomboid proteases are widely distributed intramembrane serine proteases. They have a Ser–His catalytic dyad within multiple transmembrane domains (typically six or seven) that cleave their substrates within the transmembrane domain (Lee et al., 2001; Urban et al., 2001). The cleavages of transmembrane substrates by rhomboids enable the soluble domains to be released from the membrane. These regulated intramembrane proteolyses play a critical role in signal transduction cascade in Drosophila (Lee et al., 2001; Urban et al., 2001). To date, several reports have described plant rhomboids (GarciaLorenzo et al., 2006; Kanaoka et al., 2005; Koonin et al., 2003). In the Arabidopsis genome, at least 15 rhomboid-like sequences exist, although two do not possess the conserved catalytic dyad in their sequence. Among 13 possible functional homologous genes, two (AtRBL1 and AtRBL2) are localized to the Golgi apparatus (Kanaoka et al., 2005); one (AtRBL12) is targeted into mitochondria (Kmiec-Wisniewska et al., 2008). Results obtained using the yeast mitochondria-based approach suggest a potential link between a rhomboid-like protease and the plastid translocon component Tic40 (Karakasis et al., 2007). Nevertheless, to date, it is known only that AtRBL11 localizes to chloroplast, as demonstrated by the transient

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expression of the GFP fusion proteins (Kmiec-Wisniewska et al., 2008). The physiological function of the rhomboids in chloroplast remains unclear. Additional studies reveal the role of rhomboid in the biogenesis and homeostasis of chloroplasts.

5.2. EGY1 An ethylene-dependent gravitropism-deficient isolated from a genetic screening yellow-green 1, EGY1 is a nuclear-encoded ATP-independent metalloprotease (Chen et al., 2005). It is highly homologous to a sterol-regulatory element binding protein site 2 protease, termed SREBP S2P, which is involved in SREBP-dependent lipid biosynthesis pathway (Brown and Goldstein, 1997). A hydropathy profile indicates that EGY1 has eight putative transmembrane helices in its C-terminus, presumably located in chloroplast membranes. It is particularly interesting that the existence of consensus sequences, as predicted to be a zinc-binding site of metalloproteases between its second and third transmembrane helices suggests that its catalytic center is embedded inside the membrane (Chen et al., 2005). Consequently, although EGY1 belongs to the class of metalloproteases, the function of EGY1 is presumed to be similar to that of rhomboid proteases. The Arabidopsis egy1 mutant was initially isolated as a mutant that was both pigmentation-deficient and defective in ethylene-stimulated hypocotyl gravitropism response (Chen et al., 2005). In this study, reduced thylakoid membrane stacking and an undeveloped lamella structure in abnormal plastids of egy1 mutants was observed, thereby suggesting that EGY1 is necessary for thylakoid membrane biogenesis. Furthermore, a recent report described that it is necessary for the biogenesis and replication of endodermal plastids, which are necessary for ethylene-dependent gravicurvature in light-grown hypocotyls (Guo et al., 2008). The involvement of EGY1 in lipid metabolic pathways is currently speculated. On the other hand, two homologues of EGY1 exist in Arabidopsis genome, but their function remains unknown.

5.3. AraSP In addition to EGY1 and its homologues, the Arabidopsis genome contains at least three S2P-like putative metalloproteases. Results of a recent study showed that one, AraAP, is located on the chloroplast inner envelope membrane (Bo¨lter et al., 2006). In fact, AraSP has 4–5 putative transmembrane helices; its conserved metalloprotease motif is localized between the first two helices. The development of chloroplasts is severely impaired in the T-DNA insertion line and antisense plants, suggesting the crucial role of AraSP in chloroplast biogenesis (Bo¨lter et al., 2006). However, the mechanism underlying AraSP regulation of its substrates during chloroplast development remains to be investigated.

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6. Other Proteases 6.1. SppA An ATP-independent serine-type endopeptidase, SppA, was identified in eubacteria as a membrane-associated protein (Pacaud, 1982). Crystallographic analysis of E. coli SppA revealed that four identical subunits form a bowl-shaped structure that encloses a hydrophobic chamber containing four separate Ser/Lys catalytic dyads (Kim et al., 2008). Based on the structural similarity between the bowl-shaped chamber in SppA and the ring structure formed of seven identical ClpP subunits, the possibility of SppA function in the quality control of periplasmic and membrane-bound proteins was proposed recently. In Arabidopsis, the SppA gene is present as a single copy in the genome. Arabidopsis SppA is tightly associated with the stromal side of thylakoid membranes (Lensch et al., 2001). It is enriched in stroma lamellae, although that of the transmembrane domains of bacterial SppA is absent. The result is that the size of purified SppA complexes from thylakoid membranes is consistent with the SppA from E. coli suggests that chloroplast SppA is exist as a homotetramer within thylakoid membranes. Recently, a study using T-DNA inserted sppA mutants demonstrated that sppA mutants have no phenotype under nonstress conditions, but the responses changed after high light acclimation (Wetzel et al., 2009). This result agrees with its light-dependent upregulation (Giacomelli et al., 2006). Consequently, although their substrates are poorly understood, this protease might have a physiological role in regulating optimal photosynthesis during light irradiation.

6.2. CND41 Actually, CND41 is a protein isolated from the chloroplast nucleoid of photomixotrophically cultured tobacco cells (Nakano et al., 1997). The presence of the active domain of aspartic protease in the deduced amino acid sequence of CND41 suggested that it has proteolytic activity. Actually, the analysis of proteolysis activity using highly purified CND41 from cultured tobacco cells showed that it has aspartic protease activity as well as DNAbinding (Murakami et al., 2000). The characterization of transgenic tobacco with low or high CND41 suggested that posttranslational activation of CND41 plays an important role in the in vivo degradation of chloroplast protein––especially Rubisco––and translocation of nitrogen during senescence (Kato et al., 2004, 2005). In Arabidopsis, two aspartic proteases exist, which are most homologous to tobacco CND41. Recently, immunoblot analyses using tobacco CND41 antibodies in several recombinant inbred lines selected based on differential leaf senescence phenotype showed that

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the signal was higher in the early senescing lines, although the signal remained very low or almost undetectable in late senescing lines (Diaz et al., 2008). Meanwhile, the accumulation of a tomato CND41 homologue, LeCND41, was clearly increased during the fruit ripening of tomato (Y. Kato et al., unpublished results). In addition, the development of plastid was accelerated in the shoot apex tissues with reduced gibberellin levels in CND41 antisense tobaccos (Sekimoto et al., 2003). Based on these results, the possibility of involvement of CND41 in the degradation of plastid proteins during the transdifferentiation process of plastids was proposed.

6.3. GEP Glutamyl endopeptidase (GEP) is a serine protease of the S9 family (Barrett and Rawlings, 1995). It can cleave the peptide bonds on the carboxyl sides of glutamic acid residues. Actually, GEPs are purified from stroma of spinach and pea chloroplast (Forsberg et al., 2005; Laing and Christeller, 1997). A homologue is present in Arabidopsis. Proteolytic analyses using recombinant peptide corresponding to the N-terminal part of pea Lhcb1 proposed the function of GEP in the degradation of LHCII in response to stress conditions (Forsberg et al., 2005).

7. Concluding Remarks Reviews of protein-degrading mechanisms in plastids have been presented already by us and other groups (Adam, 2000; Adam and Clarke, 2002; Adam et al., 2006; Clarke et al., 2005; Huesgen et al., 2005; Kato and Sakamoto, 2009; Richter et al., 2005; Sakamoto, 2006). However, the advance of research techniques has brought new insights into the function and structure of proteases one after another. Therefore, in this review, we updated the information of plastid proteases and described our current view of their physiological roles in plastid homeostasis and biogenesis. The initial publication of the complete genome sequence of Arabidopsis has accelerated the search for comprehensive homologues, the prediction of the intracellular location of protein, and analyses of mutant collections. These approaches revealed, as described above several times, that the isomers of plastid proteases were diversified during evolution of photosynthetic organisms, although the basal features are common among plastid proteases and bacterial proteases. The complexity of plastid protease complexes engenders difficulty in understanding of their functions. However, by contrast, this means that interesting investigations for elucidating the proteolytic machineries remain in this field. Of the several isomers of plastid

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proteases, the expression levels change remarkably according to environmental conditions and stresses (Sinvany-Villalobo et al., 2004). These results imply the fine-tuning of the composition of proteases in constantly changing environmental conditions. Future studies that are expected to elucidate tuning systems of plastid proteases can help to explain the biological advantages of the protease heterocomplex in plastids. On the other hand, not the study of proteases in plastids, but the results of recent studies of cyanobacteria demonstrate that the presence of ClpR subunit in Clp protease heterocomplex, despite being proteolytically inactive, does not limit the overall degradation activity of the protease (Andersson et al., 2009). Therefore, not all catalytic domains of protease complexes are necessary for adequate protease activity. In addition to our recent results related to FtsH heterocomplexes with mutated FtsH2, which lack proteolytic activity (Zhang et al., in preparation), these findings will provide insight into proteolytic machineries. Over the past decade, research into plastidic proteases has revealed many plastidic protease functions in plastids. Especially, progress in identification of intramembrane proteases over the past few years is interesting because protein quality control in thylakoid membranes is largely unknown despite their clear importance in chloroplast homeostasis. However, several questions remain to be answered. The first question is how plastidic proteases function cooperatively. An example of coordinated proteolysis by proteases in plastids is D1 protein degradation by Deg and FtsH proteases in PSII repair cycle. Results of several studies show that both the multiple cleavages by Deg proteases in the stromal and in luminal sides, and processive degradation by FtsH contribute to D1 degradation, which is the most important step of the PSII quality control system (Fig. 4.3) (Kato and Sakamoto, 2009). It is particularly interesting that, in cyanobacteria, the loss of Deg homologues does not interfere with D1 degradation (Barker et al., 2006), although FtsH homologue also plays a crucial role in degradation of photodamaged D1 (Komenda et al., 2006; Silva et al., 2003). These observations suggest that the cleavage of D1 by Deg proteases might be incorporated into the basic degradation process of D1 by FtsH as additional steps during the evolution of photosynthetic organisms. A second question relates to the specific substrates of each protease and the recognition mechanisms of unnecessary or damaged proteins. Although recent proteome analyses are continually revealing the specific substrates of each protease (Kim et al., 2009; Sjo¨gren et al., 2006; Stanne et al., 2009; Zybailov et al., 2009), the understanding of recognition mechanisms is largely lacking. It is hoped that further developments in this field and work to resolve many of its challenges will answer these questions.

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Impact of ATP-Binding Cassette Transporters on Human Immunodeficiency Virus Therapy Johanna Weiss and Walter Emil Haefeli Contents 1. Introduction 2. Drug Therapy of HIV-1: Drug Classes and Site of Action 2.1. Entry and fusion inhibitors 2.2. Nucleoside and nucleotide reverse transcriptase inhibitors 2.3. Nonnucleoside reverse transcriptase inhibitors 2.4. Integrase inhibitors 2.5. HIV-1 protease inhibitors 3. ABC-Transporters Influencing Drug Therapy of HIV-1 Infections 3.1. P-glycoprotein 3.2. Breast cancer resistance protein 3.3. Multidrug resistance-associated proteins 3.4. Other ABC-transporters possibly involved 4. Cell Models Investigating the Impact of ABC-Transporters for HIV-1 Therapy 5. Anti-HIV-1 Drugs as Substrates, Inhibitors, and Inducers of ABC-Transporters: In Vitro and In Vivo Findings 5.1. PIs 5.2. NNRTIs 5.3. NRTIs 5.4. Fusion and entry inhibitors 5.5. Integrase inhibitors 6. Clinically Relevant Drug Interactions with Anti-HIV-1 Drugs Attributed to ABC-Transporters 7. ABC-Transporters, ‘‘Cellular’’ Resistance, and Therapeutic Success 7.1. In vitro studies demonstrating the role of ABC-ransporters for cellular resistance towards anti-HIV-1 drugs

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Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Heidelberg, Germany International Review of Cell and Molecular Biology, Volume 280 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)80005-X

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7.2. In vitro evidence for modulation of cellular resistance by using drug combinations 7.3. Clinical data demonstrating the role of cellular resistance for therapeutic success 8. ABC-Transporter Polymorphisms and HIV-1 9. Concluding Remarks References

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Abstract Even though potent antiretrovirals are available against human immunodeficiency virus (HIV)-1 infection, therapy fails in a significant fraction of patients. Among the most relevant reasons for treatment failure are drug toxicity and side effects, but also the development of viral resistance towards the drugs applied. Efflux by ATP-binding cassette (ABC-) transporters represents one major mechanism influencing the pharmacokinetics of antiretroviral drugs and particularly their distribution, thus modifiying the concentration within the infected cells, that is, at the site of action. Moreover, drug–drug interactions may occur at the level of these transporters and modulate their activity or expression thus influencing the efficacy and toxicity of the substrate drugs. This review summarizes current knowledge on the interaction of antiretrovirals used for HIV-1 therapy with ABC-transporters and highlights the impact of ABCtransporters for cellular resistance and therapeutic success. Moreover, the suitability of different cell models for studying the interaction of antiretrovirals with ABC-transporters is discussed. Key Words: HIV-1, Drug therapy, Drug interactions, ABC-transporters, Cellular resistance. ß 2010 Elsevier Inc.

1. Introduction Infections with the human immunodeficiency-1 virus (HIV-1) represent one of the major pandemics of the history of humanity. According to the world health organization, 31–36 million people are infected with HIV1, 2.4–3.0 millions were newly infected in 2008, and 1.7–2.4 million HIVrelated deaths occurred (www.unaids.org). Without treatment, about 90% of HIV-1-infected people develop the acquired immunodeficiency syndrome (AIDS) after 10–15 years of infection. AIDS patients mostly die from opportunistic infections or malignancies associated with the progressive failure of the immune system. Antiretroviral treatment introduced in the late 1980s reduced mortality and morbidity, but routine access to the drugs is still not available in all countries. Even though potent antiretrovirals are available, therapy fails in a

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significant fraction of patients. Among the most relevant reasons for treatment failure are noncompliance due to complex dosage regimens or drug toxicity/side effects, but also the development of viral resistance towards the drugs applied (Waters and Nelson, 2007). The latter is promoted by subtherapeutic drug concentrations at the site of action, which can be caused either by nonadherence, inappropriate dose selection, or by active transport processes influencing drug disposition (Owen et al., 2005). Beyond metabolism by cytochrome P450 (CYP) isozymes, efflux by ATP-binding cassette (ABC-) transporters represents one major mechanism influencing the pharmacokinetics of antiretroviral drugs and particularly their distribution, thus modifiying the concentration within the infected cells, that is, at the site of action. Moreover, drug–drug interactions may occur at the level of these transporters and modulate their activity or expression thus influencing the efficacy and toxicity of the substrate drugs. This review provides an overview on the interactions of currently available antiretroviral drugs with ABC-transporters either as substrates, inhibitors, or inducers and highlights the impact of ABC-transporters for cellular resistance and therapeutic success. Moreover, we discuss the suitability of different cell models for studying the interaction of antiretrovirals with ABC-transporters and the prediction of clinical effects on the basis of in vitro findings.

2. Drug Therapy of HIV-1: Drug Classes and Site of Action HIV-1 primarily infects vital cells of the human immune system in particular CD4þ T-helper cells, but also macrophages, and dendritic cells. HIV-1 infection causes progressive CD4þ cell depletion with subsequent loss of cell-mediated immunity and occurrence of opportunistic infections or malignancies. Different classes of anti-HIV-1 drugs target different steps of the viral life cycle. Cellular infection of CD4þ lymphocytes by HIV-1, viral reproduction, and release of the virus particles include a complex series of events, which are shown in Fig. 5.1. HIV-1 entry is mediated by the viral envelope spike which recognizes and binds to a CD4 receptor and one of two coreceptors (CXCR4 or CCR5) on the surface of the target cells. After binding, the virus fuses with the plasma membrane and releases its RNA into the host cell. Each virus contains two strands of RNA, which are converted to double-stranded DNA by the viral enzyme reverse transcriptase producing ‘‘proviral’’ DNA. The proviral DNA is transported to the cell nucleus and integrated into the host’s DNA by viral integrase. This provirus may remain inactive for several years, producing few or no new

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1. Binding

8. Maturation 7. Budding 2. Fusion

CD4

HIV-1 coreceptor (i.e., CXCR4, CCR5)

6. Assembly 3. Reverse transcription

5. Transcription/ translation

4. Integration

Figure 5.1 HIV-1 life cycle.

copies of HIV-1. Once viral genes are transcribed by the host cell, precursor proteins for the generation of new HIV-1 virions are translated. These proteins and viral RNA are assembled at the cell surface to new viral particles. Before the viruses become infectious, they undergo a maturation step comprising further cleavage of precursor viral proteins by HIV-1 protease (Klimas et al., 2008). During this process, long precursor proteins are cut by viral protease into smaller proteins being important for maturation of the viruses. After assembly, the new viral particles bud off the host cell and form new viruses.

2.1. Entry and fusion inhibitors The first two steps of the viral life cycle (binding and fusion) can be inhibited by entry and fusion inhibitors, respectively. Fusion inhibitors work by binding to the attachment sites of the HIV-1 virus thus inhibiting the fusion of HIV-1 to the host cell membranes. So far, the only licensed fusion inhibitor is enfuvirtide. This peptide is metabolized to its amino acids by peptidases, that is, independent of CYP isozymes (Dando and Perry, 2003). HIV-1 variants use either CCR5 or CXCR4 receptors for their entry into target cells of the immune system. According to their tropism they are called R5 or X4 viruses. CCR5 antagonists like maraviroc bind to and change the

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molecular shape of CCR5 thus preventing an interaction with the viral attachment site. CCR5 antagonists are only effective against R5 viruses. Therefore, individual patients have to be tested for their virus variants to confirm the presence of R5 viruses before treatment with maraviroc. Maraviroc is metabolized by CYP3A4 and dose adjustment is necessary when given concomitantly with CYP3A4 inhibitors or inducers (MacArthur and Novak, 2008). Both fusion and entry inhibitors (often both referred to as entry inhibitors) disable de novo infection and cell-to-cell virus transmission.

2.2. Nucleoside and nucleotide reverse transcriptase inhibitors The next step, that is, reverse transcription of the viral RNA, can be prevented by two classes of antiviral drugs. A first class of competitive inhibitors are the nucleoside (NRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs). NRTIs are nucleoside analogues that must undergo intracellular phosphorylation before becoming active. When the viral enzyme reverse transcriptase uses these ‘‘wrong’’ building blocks, the provirus cannot be built correctly and integration into the host genome is impaired. The only NtRTI tenofovir acts as a competitive antagonist, but requires one step less of phosphorylation to become active. NRTIs are mainly excreted renally. They are not metabolized by CYPs and there is no evidence for inhibitory or inductive effects on this enzyme system (Barry et al., 1999).

2.3. Nonnucleoside reverse transcriptase inhibitors Nonnucleoside reverse transcriptase inhibitors (NNRTIs) act as noncompetitive inhibitors of the reverse transcriptase by binding near the substrate recognition site. Treatment with NNRTIs is afflicted with a high risk for fast development of resistance (Wainberg, 2003). However, they are very efficient in combination with NRTIs. In contrast to NRTIs, all members of the NNRTI class are metabolized by CYPs and are inducers and/or inhibitors of these enzymes (Smith et al., 2001). Therefore, they are prone to drug–drug interactions.

2.4. Integrase inhibitors The next step of the HIV-1 life cycle, the integration into the host genome, can be inhibited by integrase inhibitors. Raltegravir, the only approved integrase inhibitor so far, inhibits the strand transfer by integrase, which is the third and final step of provirus integration. This compound is primarily metabolized by glucuronidation via uridine diphosphate

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glucuronosyltransferase 1A1 (Katlama et al., 2009). A second integrase inhibitor, elvitegravir, is in late clinical development.

2.5. HIV-1 protease inhibitors Viral assembly and maturation can be blocked by HIV-1 protease inhibitors (PIs). As their name implies, they impair the activity of the HIV-1 protease by binding to the active site and locking it up. This protease is responsible for the cleavage of the large viral precursor polypeptides into functional proteins. Therefore, inhibition of protease results in the release of structurally disorganized and noninfectious viral particles (Pillay et al., 1995). The introduction of PIs along with the therapeutic strategy of combining compounds with different mechanisms of action (highly active antiretroviral therapy, HAART) revolutionized HIV-1 therapy at the end of the last century. HAART led to significant declines in HIV-1-associated morbidity and mortality (Bonfanti et al., 1999; Palella et al., 1998; Shafer and Vuitton, 1999). However, the pill burden for patients on PIs is often high, toxicity represents a significant problem, and lipodystrophy occurs in a high percentage of patients on PI therapy. Moreover, due to their interaction with numerous CYPs and transporters they are prone to drug–drug interactions (Mastrolorenzo et al., 2007). The potent inhibition of CYPs by ritonavir is used for boosting the pharmacokinetics of most other PIs. This interaction leads to reduced pill burden, enables resorption independent from food intake, and increases bioavailability of most PIs considerably (Walmsley, 2007).

3. ABC-Transporters Influencing Drug Therapy of HIV-1 Infections One cause of ongoing viral replication despite HAART is suboptimal penetration of drugs into sanctuary sites like the central nervous system or into the target cells like CD4þ cells. Drug exporters such as ABC-transporters are believed to be one major cause for suboptimal penetration (Sankatsing et al., 2004). The ABC-transporter superfamily comprises 49 known members (Gillet et al., 2007), which are integral membrane proteins that are capable to extrude structurally diverse substrates across extra- and intracellular membranes under hydrolysis of ATP (Dean et al., 2001). ABC-transporters are localized in many epithelial and endothelial cells and participate in absorption and excretion of drugs. Moreover, they also form barriers against drug distribution like the blood–brain barrier, the placenta barrier, the blood– testis barrier, and barriers to other sanctuary sites like leukocytes and may thus critically modulate the pharmacokinetics of their substrates (Schinkel

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and Jonker, 2003). ABC-transporters like P-glycoprotein (Pgp/MDR1/ ABCB1), the multidrug resistance-associated proteins (MRPs/ABCCs), and the breast cancer resistance protein (BCRP/ABCG2) are involved in physiological transport processes to protect tissues from xenobiotics and endogenous toxins and are often associated with multidrug resistance (MDR) (Schinkel and Jonker, 2003). These transporters may influence antiretroviral therapy in three areas: (1) at the bioavailability level (intestine/liver), (2) at sanctuary sites (brain, fetus, testis), and (3) at the target cell level (e.g., lymphocytes). The latter does not apply to entry inhibitors, because they bind to the surface of the target cell and thus do not have to enter the lymphocyte. Figure 5.2 depicts the localization of ABC-transporters most important for HIV-1 therapy.

3.1. P-glycoprotein Pgp is the best investigated ABC-transporter. It is expressed apically at high levels in many tissues like the gastrointestinal tract, liver, kidney, heart, lung, testes, uterus, brain, and on peripheral blood mononuclear cells (PBMCs)

Figure 5.2 Localization of main ABC-transporters involved in HIV-1 therapy. For MRPs, localization (apical or basolateral) is not well defined in all organs.

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and stem cells especially of the hematopoietic system (Sankatsing et al., 2004). During HIV-1 infection, some of the expression sites are of particular importance: The blood–brain barrier, the placenta barrier, and PBMCs. Whereas it is indisputable that Pgp is highly expressed on brain capillary endothelial cells (BCECs) being the major constituent of the blood–brain barrier (Rao et al., 1998; Schinkel and Jonker, 2003) its expression at other brain sites remains a matter of controversy. Most studies demonstrated apical expression of Pgp also in choroid plexus epithelia and microglial cells, but its expression in astroglial cells is equivocal (Miller et al., 2008; Rao et al., 1999; Ronaldson et al., 2004, 2008). Microglia and astroglia play an important role in HIV-1 infection because both harbor the virus within the CNS. For a more comprehensive review on localization, expression, and function of ABC-transporters in the brain, the brain barriers, and their relevance in pharmacotherapy, the reader is referred to several excellent reviews (Lo¨scher and Potschka, 2005a,b; Miller et al., 2008; Ronaldson et al., 2008). Although expression and function of Pgp in PBMCs has extensively been studied, it is still not completely clarified which cellular subpopulations express the highest amount. Pgp expression in lymphocytes was observed in 20–80 % of B cells and 30–100% of T cells (Chaudhary et al., 1992; Drach et al., 1992; Klimecki et al., 1994; Ludescher et al., 1998) and appears to depend on the activation status of the cells (Kock et al., 2007). One study reported the highest Pgp expression in CD56þ (natural killer) cells followed by CD8þ, CD4þ, and CD19þ cells (Oselin et al., 2003b) confirming earlier findings reporting highest Pgp expression and function in CD56þ cells among all investigated PBMCs (Ludescher et al., 1998). A series of other studies demonstrated high expression levels in natural killer cells (Egashira et al., 1999; Janneh et al., 2005; Klimecki et al., 1994; Malorni et al., 1998; Vasquez et al., 2005; Wilisch et al., 1993). However, one study states that in natural killer cells instead of the common 170 kDa protein a truncated (mini) form of Pgp with a molecular weight of 70–80 kDa and a more restricted substrate profile is expressed (Trambas et al., 2001). Pgp expression on PBMCs plays a major role for therapy with PIs being substrate of this efflux pump (cf. 5.1) and it has at least in vitro been demonstrated for several PIs that their intracellular concentrations depend on Pgp function in the target cells (Ford et al., 2004b; Jones et al., 2001a; Meaden et al., 2002). Pgp expression in placental trophoblasts seems to play a significant role in limiting the fetal exposure to antiretrovirals (Camus et al., 2006). This predominantly applies to the PIs being substrates of Pgp and some MRPs. In mice, it was demonstrated that more saquinavir enters Mdr1a//1b/ fetuses than wild-type fetuses (Smit et al., 1999). Pgp inhibition in wildtype mice and human placentae increased the transplacental passage of saquinavir into the fetus (Molsa et al., 2005; Smit et al., 1999). Pgp is also expressed in mammary epithelial cells but its role in the distribution of PIs or

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other antiretrovirals into the milk is unclear. There is one study in rats demonstrating no significant role of Pgp in the distribution of nelfinavir into rat milk (Edwards et al., 2005) and the other showing only low levels of Pgp in human lactating mammary epithelial cells (Alcorn et al., 2002). For a detailed review about the role of ABC-transporters in antiretroviral therapy during pregnancy, the reader is referred to a recently published review on this topic (Gulati and Gerk, 2009). 3.1.1. Pgp and CXCR4 The chemokine receptors CXCR4 and CCR5 act as essential coreceptors for HIV-1 entry into CD4þ cells and their expression has been linked to susceptibility to HIV-1 infection and disease progression. Interestingly, in PBMCs isolated from healthy volunteers Pgp and CXCR4 expression were correlated (Owen et al., 2004) and these findings were confirmed in HIV-1-positive individuals (Chandler et al., 2007a). The authors hypothesize that the emergence of CXCR4-tropic strains of HIV-1, observed in patients with high lymphocyte CXCR4 expression, may be exacerbated by decreased antiretroviral efficacy as a consequence of high transporter coexpression. 3.1.2. Interactions between Pgp and HIV-1 Drug therapy influences HIV-1 infection and Pgp activity and expression. Data also indicate a mutual interference between Pgp and HIV-1 infections independent of antiretroviral therapy. However, the underlying mechanisms are unexplained and the data obtained so far are contradictory. Already about 20 years ago two groups found an overexpression of Pgp in HIV-1-infected drug-naı¨ve tumor cell lines suggesting that HIV-1 infection itself can induce Pgp expression (Antonelli et al., 1992; Gollapudi and Gupta, 1990). In contrast, others demonstrated a significant decrease in Pgp expression and or activity in CD4þ, CD19þ, and CD16þ NK cells of HIV-1-infected individuals, whereas they suppose that the decrease in Pgp in CD4þ cells might reflect the depletion of this subpopulation during HIV-1 infection (Lucia et al., 1995). These results are partly supported by recently published data demonstrating a slightly decreased Pgp activity in lymphocytes of HIV-positive adults (Giraud et al., 2009). Also for astrocytes it has been demonstrated that HIV-1 infection (positively) and the viral envelope protein gp120 (negatively) influence Pgp expression (Langford et al., 2004; Ronaldson and Bendayan, 2006) and in brain microvascular endothelial cells the HIV-1-Tat protein induced Pgp expression (Hayashi et al., 2005). A detailed reflection of this interrelation is given in the excellent review dealing with the regulation of ABC-transporters by HIV-1-associated pathological events (Ronaldson et al., 2008). Finally in the absence of drugs, Pgp expression and function can inhibit infectivity and replication of HIV-1. In vitro virus production was significantly decreased in a Pgp overexpressing CD4þ human T-leukemic cell

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line, whereas fusion of viral and plasma membranes and downstream events were unaffected (Lee et al., 2000). These results might suggest that high Pgp expression decreases virus load, but impairs antiretroviral efficacy at least for the PIs being Pgp substrates. These data were corroborated by a study demonstrating in vitro that Pgp expressing cells are resistant towards HIV-1 infection, whereas this resistance can be reversed by the Pgp inhibitor verapamil (Speck et al., 2002). Moreover, an inverse relationship between Pgp activity in CD4þ and CD8þ lymphocytes and plasma HIV1 RNA levels was found in HIV-positive patients independent of their medication arguing for an inhibitory effect of Pgp on viral replication and not on drug disposition (Hulgan et al., 2003). Similarly, it was demonstrated in an in vivo study that efflux function of Pgp reduces intracellular HIV-1 replication (Sankatsing et al., 2007). In conclusion, Pgp is certainly the best studied and most important ABC-transporter for HIV-1 therapy. Pgp not only influences drug disposition and thus effectiveness, but also represents an important site for drug– drug interactions with antiretrovirals. In addition, it appears to influence HIV-1 infectivity and to be influenced by HIV-1 infection.

3.2. Breast cancer resistance protein BCRP exhibits a rather similar tissue expression pattern as Pgp. It is expressed in the apical membrane of tissues with barrier function like the placenta, the small intestine, the liver, and the blood–brain barrier. BCRP regulates intestinal absorption and biliary excretion of potentially toxic compounds and protects sanctuary sites of the human body, for example, the brain, stem cells, and the fetus from xenobiotics (Krishnamurthy and Schuetz, 2006; Mao and Unadkat, 2005). Like Pgp and MRPs, BCRP is also expressed in drug-resistant tumors and tumor cell lines and is supposed to be a major determinant of the MDR phenotype (Mao and Unadkat, 2005; Szakacs et al., 2008). BCRP expression has been demonstrated in primary cultures of human BCECs and also in immortalized BCEC lines, in human astrocytes, and microglia (Ketabi-Kiyanvash et al., 2007; Ronaldson et al., 2008). Its expression on the surface of PBMCs is also well established (Albermann et al., 2005; Bousquet et al., 2008b; Moon et al., 2007). Whereas PIs are only inhibitors but not substrates of BCRP (Gupta et al., 2004; Weiss et al., 2007a), at least some NRTIs are transported by this efflux transporter (Wang et al., 2003, 2004). In summary, the role of BCRP in HIV therapy is less well defined and more research is warranted to clarify whether BCRP merits consideration as a modulator of antiretroviral therapy.

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3.3. Multidrug resistance-associated proteins MRP1, 2, 4, and 5 appear to be just as important in HIV-1 therapy as Pgp. MRPs also actively efflux drugs out of cells thereby contributing to the MDR phenotype. In contrast to Pgp, MRPs mainly transport conjugated drugs. The localization differs among the family members with MRP2 sharing the distribution of Pgp. In addition to Pgp, MRP1 expression has also been studied in lymphocyte subpopulations and it also appears to depend on the activation state of the cells (Kock et al., 2007). The highest expression of MRP1 and MRP2 was found in CD4þ cells followed by CD8þ, CD19þ, and CD56þ cells (Oselin et al., 2003a). MRP3 mRNA has been detected in monocytes and a CD4þ cell line, MRP4 also in a CD4þ cell line and in lymphocytes of HIV1-infected individuals, and MRP5 mRNA is expressed in similar amounts in all subgroups of lymphocytes (Turriziani et al., 2008; Weiss et al., 2009; Zhang et al., 2006). Many MRPs are expressed at the blood–brain barrier but there is still substantial controversy about expression levels and localization of these transporters (Ronaldson et al., 2008). MRP1 and MRP2 are important modulators of PI pharmacokinetics also regulating distribution just as reported for Pgp. In addition, some PIs have been identified as MRP substrates and interestingly PI concentrations in PBMCs are correlated with MRP1 expression (Agarwal et al., 2007; Janneh et al., 2005, 2007; Jones et al., 2001a; Meaden et al., 2002; van der Sandt et al., 2001; Zastre et al., 2009). In contrast, MRP4 and MRP5 do not transport PIs, but may export phosphorylated derivatives at least of zidovudine and stavudine (Reid et al., 2003, Schuetz et al. 1999). A recent study demonstrated an inverse correlation between the expression of MRP4 and MRP5 and CD4þ cell counts suggesting that also these export transporters may modulate the success of HIV drug therapy (Turriziani et al., 2008). MRPs might not only be important for the intracellular concentration of antiretrovirals but also as a coregulator of the intracellular concentration of antioxidant compounds. It was demonstrated that MRP1 inhibition suppresses HIV-1 replication and this inhibition of MRP1 by PIs may contribute to their antiviral activity independent from inhibition of the HIV-1 protease (Lucia et al., 2005). In line with these findings, HIV-1 replication in macrophages substantially increased MRP1 and MRP5 mRNA levels (Jorajuria et al., 2004) suggesting that HIV-1 infection promotes the efflux of antiretroviral drugs from macrophages counteracting pharmacological effects particularly in the CNS. However, another group only found a significant upregulation of MRP1 but not of MRP2–7 mRNA and protein expression in brain microvascular endothelial cells and astrocytes by HIV-1Tat protein (Hayashi et al., 2006). Taken together the influence of MRPs on HIV infection is far from being understood, but the expression of MRPs on HIV-1 target cells appears to be an important factor for intracellular PI concentrations.

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Moreover, Pgp activity appears to protect the cell from the infection whereas for MRP1 the opposite is the case.

3.4. Other ABC-transporters possibly involved There are only very few studies investigating the potential influence of other ABC-transporters on HIV-1 infection and therapy rendering it difficult to evaluate their impact on HIV-1 therapy. However, current evidence suggests that it is unlikely, that these transporters play a major role. ABCB4, a phosphatidyl choline flippase, mediates the transport of phosphatidyl choline from the inner to the outer membrane leaflet. One study demonstrated that this transporter changed the lipid composition of HIV-1 thus reducing its infectivity (van Til et al., 2008). Ritonavir, saquinavir, and efavirenz but not nevirapine inhibit the bile salt export pump (BSEP) ABCB11 (McRae et al., 2006). However, so far there are no data about possible ABCB11 substrate characteristics of these antiretrovirals or about the possible clinical impact of ABCB11 inhibition for liver damage observed under antiretroviral therapy. ABCE1 belongs to the ABC-transporters, although it has no transmembrane domain and is not involved in any membrane transport functions. This cellular ATPase was demonstrated to be involved in virion production of HIV-1 (Dooher et al., 2007).

4. Cell Models Investigating the Impact of ABC-Transporters for HIV-1 Therapy As outlined above, ABC-transporters may modulate antiretroviral therapy at several sites of the organism including not only intestine and liver but also brain and lymphocytes. For a systematic investigation of the impact of an ABC-transporter on treatment response to a specific antiretroviral drug it is recommended to start with the examination of the drug’s substrate, inhibitor, and inducer characteristics. For this purpose, ABCtransporter overexpressing cell lines in comparison to the respective parental cell lines are mostly used for the assessment of substrate and inhibitor properties. These cell systems harbor the advantage that an internal control is available as corresponding parental cell line. Differences observed between overexpressing and parental cell line can therefore quite likely be attributed to the overexpressed transporter. Overexpressing cell lines are generated in two different ways: First, overexpression may be achieved by transfection/transduction of a cell line with the cDNA of the corresponding transporter. These cell systems are frequently used and the origin of the parental cell line (tissue/species) is insignificant as long as background transporter activity for the test

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compound is low in the parental cell line and does therefore not influence the results (Fro¨hlich et al., 2004; Lindenmaier et al., 2005). Some of the most commonly used cell lines transfected with ABC-transporters for HIV research are LLC-PK1 cells (porcine kidney) (Fujimoto et al., 2009; Guo et al., 2003; Storch et al., 2007), MDCKII cells (canine kidney) (Agarwal et al., 2007; Huisman et al., 2002; Shaik et al., 2007; Weiss et al., 2007a,b), HEK293 (human kidney) (Chinn et al., 2007; Gupta et al., 2004), and CHO cells (ovarial hamster cells) (Minuesa et al., 2009). These profoundly overexpressing cell lines offer the advantage that in transport assays even poorly transported compounds can be identified, while it has the disadvantage of overestimating the impact of the transporter in vivo. Conversely, in inhibition assays the IC50 values are generally higher than in vivo (with lower ABC-transporter expression) thus potentially underestimating the inhibitory potency of the test compound. The second way to induce overexpression of an ABC-transporter is the incubation of a cell line with increasing (but sublethal) concentrations of a cytostatic drug thus selecting cells with overexpression and inducing the respective transporter, respectively. In this case, it is important to use a human cell line because of reported species differences in drug transport and transporter inhibition (Kim et al., 2008; Schwab et al., 2003; Suzuyama et al., 2007; Xia et al., 2004). In HIV research, the CD4þ lymphocyte cell line CEM is frequently used after induction with, for example, vinblastine to increase Pgp expression (Cianfriglia et al., 2007; Dupuis et al., 2003; Janneh et al., 2007, 2008; Jones et al., 2001a,b; Schuetz et al., 1999; Srinivas et al., 1998). This approach harbors the problem that the inducing cytostatic might not be specific and induces more than one transporter. Therefore, before using such a cell line, it should be carefully characterized concerning its transporter configuration. Moreover, tumor or primary cell lines with their original transporter equipment are often used. Pertinent examples are Caco-2 cells (colon carcinoma) (Fujimoto et al., 2009; Stormer et al., 2002), HepG2 cells (hepatoma) (Su et al., 2004b), brain and other capillary endothelial cells (Bousquet et al., 2008b; Eilers et al., 2008; Storch et al., 2007), and PBMCs ( Janneh et al., 2005, 2007). Whereas these cell systems are closer to the in vivo system, they always express more than one transporter and assays using these cells require very specific substrates/inhibitors to ensure that, for example, transport by only one transporter is indeed investigated. The selection of a suitable cell line for the investigation of inducing effects of an antiretroviral is much more difficult. In vitro studies investigating possible induction of ABC-transporters by anti-HIV-1 drugs have been conducted in many different cell lines. However, due to different assay conditions and different equipment of different cell lines with nuclear receptors, cofactors, and other important components of the induction machinery, data obtained so far significantly differ and render comparison between in vitro studies and extrapolation to clinial situations quite difficult

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(Ronaldson et al., 2008; Weiss et al., 2008, 2009). Therefore, many aspects have to be considered and thus far there is no ideal induction model. Every cell line offers both advantages and disadvantages and because the induction machinery substantially differs between different species it is even more important than for inhibition and transport experiments that the cell line used is of human origin ( Jones et al., 2000; Moore and Kliewer, 2000). As outlined in Chapter 7, ABC-transporters play a special role in lymphocytes, restricting the access of at least some antiretrovirals to their target. It is therefore self-evident to use isolated PBMCs for the investigation of inducing effects of antiretrovirals or the assessment of modulators of intracellular drug concentration. This approach is pursued by many researchers and offers the advantage that not only PBMCs of healthy but also of HIV infected individuals can be investigated (Bousquet et al., 2008b, 2009; Chandler et al., 2003; Chinn et al., 2007; Ford et al., 2003). The main disadvantages of using isolated PBMCs are that they cannot be kept alive for a longer period and that the relative fractions of particular cell subtypes might change during culture because their lifetime may differ. Therefore and because transporter expression varies significantly between the respective cell types, changes in transporter expression/activity may also reflect changes in PBMC composition (Kock et al., 2007). In spite of everything, PBMCs offer one advantage that no other cell model possesses: It is possible to study induction of ABC-transporters in vivo when PBMCs are isolated after exposure of healthy or HIV-1-infected individuals with the respective antiretroviral at therapeutic doses. This approach prevents the difficult discussion of whether concentrations applied in vitro are relevant for the in vivo situation. But one pitfall should be considered: Studying ABCtransporter activity only in total PBMCs might reveal changes in ABCtransporter activity resulting from alterations in native T-cell populations (Hulgan et al., 2003) because HIV-1 infection disproportionately depletes circulating naı¨ve T-cells, which have different ABC-transporter activity. Therefore, such studies should also measure transporter activity in T-cell subpopulations to assess whether the effect observed does not only reflect changes in T-cell composition. To circumvent the problems discussed for native PBMCs, many researcher use established CD4þ cell lines, in particular CEM cells (Chinn et al., 2007; Maffeo et al., 2004) or Jurkat cells (Weiss et al., 2009). Beyond PBMCs there are other tissues of interest for inducing effects of antiretrovirals. The pharmacokinetics of ABC-transporter substrates are essentially influenced by transporter activity in the intestine and the liver (Fromm, 2003). Therefore, intestinal and hepatic cell lines are the most commonly used and best characterized models for induction of ABCtransporters. LS180 cells are among the most widespread models for induction and their suitability has been demonstrated in many studies (Gupta et al., 2008; Perloff et al., 2003; Schuetz et al., 1996; Stormer et al., 2002;

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Weiss et al., 2008, 2009). However, LS180 cells apparently lack the constitutive androstane receptor (CAR) (Gupta et al., 2008) and we found indeed very low levels of this nuclear receptor in LS180 cells (Weiss et al., 2009) suggesting that this cell line is not ideal to test gene induction by CAR ligands. Moreover, induction of BCRP is difficult to quantify in these cell lines due to the very low expression level (Weiss et al., 2009). Therefore, LS180 cells are a good but as all others not a perfect model to study induction of ABC-transporters. However, among the intestinal cell lines LS180 cells are more suitable than, for example, Caco-2 cells, which possess only low levels of PXR and are not inducible by the prototype inducer rifampin (Pfrunder et al., 2003). Nevertheless, Caco-2 cells have also been used by some researchers (Vishnuvardhan et al., 2003). When interested in hepatic induction, so far mostly primary human hepatocytes were used (Dixit et al., 2007). However, although closer to the in vivo situation than established cell lines, the results obtained vary more in primary cells, the cells are far more expensive, and not readily available (Martin et al., 2008). Moreover, they exhibit significant interindividual variability in the expression of, for example, drug metabolizing enzymes (Hewitt et al., 2007). Therefore, there is a need for suitable established hepatic cells lines. Several cell lines are currently under investigation for their suitability to investigate induction of drug metabolizing enzymes (Hariparsad et al., 2008; Kanebratt and Andersson, 2008) and comparable studies for transporters will follow. In general, artificial cell models with ABC-transporter overexpression are only appropriate to detect substrate and inhibitor characteristics, but not to evaluate their impact on drug therapy in the clinical situation. When investigating the impact of an ABC-transporter on distribution, intracellular levels, or drug–drug interactions of antiretrovirals cell models with ‘‘natural’’ expression levels are to be preferred. As outlined in the following chapters, in vitro studies conducted with different cell systems often lead to conflicting results. As an example, inhibitory potencies cannot be compared between different studies, because IC50 values highly depend on the cell system used, the model substrate applied, and also on the assays conditions (Taub et al., 2005; Weiss and Haefeli, 2006). Therefore, in such studies only the potency ranking will be significant. To assess the ranking order of transporter inhibition (or induction), it is therefore important to assess different drugs under identical assay conditions. The results of different assays can only be compared if standard paradigm drugs have been studied in both assays, but this is much too rarely done in practice. One question always arising when investigating ABC-transporter induction or inhibition in vitro (except for using isolated PBMCs from humans exposed to drugs) is whether the induction observed in vitro truly reflects the in vivo situation. In most cases, therapeutic plasma concentrations observed at steady state are the basis for comparison. This may, however, both overand underestimate the relevance for the in vivo situation. Overestimation

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can occur, when total instead of free plasma concentrations are considered. As listed in Table 5.1, many antiretrovirals and especially PIs and NNRTIs are highly bound to plasma proteins rendering effective free concentrations much lower. On the other hand, plasma concentrations might only be relevant for inhibition of ABC-transporters on leukocytes and the true impact on efflux Table 5.1 Important pharmacokinetic parameters of antiretroviral drugs

Compound

Cmax steady state (mg/ml)

Cmax steady state (mM)

Plasma protein binding (%)

Reference

HPIs Saquinavir Ritonavir Indinavir Nelfinavir Amprenavir Fosamprenavir Atazanavir Lopinavir Tipranavir Darunavir

2.5 11.2 12.6 3.0 7.7 4.8–6.1 2.9–5.9 9.6 80 11.2–14.9

3.2 15.5 20.7 5.3 15.2 7.7-9.8 4.1–8.3 15.3 132 20.5–27

97 98–99 60 > 98 90 90 86 98–99 99.9 95

Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Chapman et al. (2004) Ronaldson et al. (2008) Ronaldson et al. (2008) Orman and Perry (2008) Fenton and Perry (2007)

NNRTIs Efavirenz Nevirapine Delavirdine Etravirine

4.1 2.0 16 0.47–1.39

12.9 7.5 28 1.1–3.2

96–99 60 98 99.9

Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Scholler-Gyure et al. (2009)

3 0.31–5.0 1.5 0.0252 0.41–0.54 1.7 0.93 0.326

4.5 1.3–21 6.5 0.0119 1.5–2 6.8 4.1 1.1

50 <5 < 36 neglegible < 38 >4 negligible < 0.7

Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008) Ronaldson et al. (2008)

4.49

1.02

92

Dando and Perry (2003)

NRTIs Abacavir Didanosine Lamivudine Zalcitabine Zidovudine Emtricitabine Stavudine Tenofovir Fusion inhibitors Enfuvirtide Entry inhibitors Raltegravir Maraviroc

0.142 0.29 83 0.27–0.62 0.53–1.2 76

Croxtall et al. (2008) Carter and Keating (2007)

ABC-Transporters and HIV-1 Therapy

235

transporters, for example, at the apical membrane of a hepatocyte may be different because intracellular concentrations may be several-fold higher for substrates of (basolateral) uptake transporters (e.g., human organic aniontransporting polypeptide (OATPs)). Similarly, whether a drug may induce ABC-transporter expression depends on intracellular concentrations which are often much higher as demonstrated for many PIs (Ehrhardt et al., 2007; Ford et al., 2004c) and also efavirenz (Almond et al., 2005b; Burhenne et al., unpublished data). Moreover, in most cases information on drug concentrations in tissues of interest (i.e., liver or intestine) is lacking. Intestinal concentrations can be estimated by drug dose divided by the 250 ml of solution that is usually coadministered during intake and they are obviously one or two orders of magnitude higher than plasma concentrations after distribution in the body (Fenner et al., 2009). Even if an effect on pharmacokinetics of a drug can also be observed in vivo it might not be clinically relevant in terms of drug efficiency or adverse effects. This can only be assessed in the appropriate clinical setting investigating a defined end point. Taken together, the objectives of an experiment should guide the selection of the cell model. When evaluating in vitro data and before extrapolation to the in vivo situation many aspects are to be considered as outlined in Table 5.2. It is often difficult to realistically appraise the meaning of in vitro data. However, if crucial preconditions are not met (like disregard of solubility limits) data should be interpreted with caution. Nevertheless, data can still be Table 5.2 Aspects to be considered when evaluating in vitro data

Is the cell line characterized concerning its transporter expression? Might other transporters be involved in the effect observed? Is intracellular concentration modified by several transporters and if so are they expressed in the model used? Was the solubility maximum for the investigated drug considered? Antiretrovirals are often applied at concentrations exceeding the solubility limit leading to invalid results (Weiss et al., 2002) Are possible species differences relevant and if so are they appropriately considered? Which concentration range was tested? Are these concentrations relevant for the in vivo situation? Where toxic/antiproliferative effects of the drug tested? Toxicity influences inhibition (damage of the cells with leakage of substrates) and induction assays (unspecific stress response of the cells). Where direct or indirect assays applied (e.g., transport assays vs. proliferation assays)? Where plateau effects reached (appropriate duration/concentration of exposure)? Was the localization of the respective transporter considered (important for transport experiments when interpreting the data)?

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inconsistent even if all criteria are fulfilled. The main underlying reason is the fact that there are no standard or ideal assays for a particular question. Therefore, diverse assays or cell models are applied leading to a vast amount of data that may be difficult to interpret. It would go beyond the scope of this review to evaluate or judge each study mentioned. Therefore, for this purpose the reader is referred to the primary literature cited to draw his own conclusion.

5. Anti-HIV-1 Drugs as Substrates, Inhibitors, and Inducers of ABC-Transporters: In Vitro and In Vivo Findings All data and studies discussed in this chapter are summarized in Table 5.3.

5.1. PIs 5.1.1. Saquinavir Saquinavir is certainly one of the best investigated PIs concerning its interaction with ABC-transporters. It was identified as Pgp substrate already in 1998 in both in vitro studies and animal models (Kim et al., 1998a,b; Lee et al., 1998; Srinivas et al., 1998). Many subsequent studies confirmed the substrate characteristics of saquinavir (Choo et al., 2000; Eilers et al., 2008; Janneh et al., 2005; Jones et al., 2001a; Maffeo et al., 2004; Park and Sinko, 2005; Patel et al., 2002; Profit et al., 1999b; Su et al., 2004a; Washington et al., 1998b, 2000). In addition, saquinavir is also a potent inhibitor of Pgp (Bachmeier et al., 2005; Chinn et al., 2007; Gutmann et al., 1999; Kim et al., 1998a; Lucia et al., 2001; Profit et al., 1999a; Storch et al., 2007; Washington et al., 1998a), however, with lower potency than lopinavir, ritonavir, nelfinavir, and tipranavir (Storch et al., 2007). Saquinavir is a ligand of the pregnane X receptor (PXR), which is a key nuclear factor regulating a large number of genes involved in the detoxification and excretion of toxic compounds and drugs (Kliewer, 2003). Saquinavir can therefore not only induce CYP3A4, but also Pgp (Dussault et al., 2001), as verified in LS180 cells (Gupta et al., 2006, 2008; Ko¨nig et al., 2009). In contrast, the data obtained in PBMCs in vitro and in vivo as well as in CD4þ cell models are contradictory. In the CD4þ cell line CEM, an overexpression of Pgp together with an increased function after a longer incubation period (several months) of the cells with 10–20 mM saquinavir was observed (Dupuis et al., 2003). In contrast, another study demonstrated only an increase in mRNA expression after incubation of CEM cells with 5 mM saquinavir for 96 h which did not translate into changes in protein expression and 10 mM saquinavir had no effect at all (Chinn et al., 2007).

Table 5.3 Antiretrovirals as substrates, inhibitors, and inductors of ABC-transporters Compound

PIs Saquinavir

Pgp/ABCB1

BCRP/ABCG2

MRPs/ABCCs

Other ABC-transporters

Substrate: yes (in vitro and in vivo) (Kim et al., 1998a,b; Lee et al., 1998; Srinivas et al., 1998; Profit et al., 1999b; Choo et al., 2000; Patel et al., 2002; Maffeo et al., 2004; Su et al., 2004a; Janneh et al., 2005; Park and Sinko, 2005; Eilers et al., 2008; Washington et al., 1998b; Washington et al., 2000; Jones et al., 2001a,b) Inhibitor: yes (in vitro) (Kim et al., 1998a; Washington et al., 1998a,b; Profit et al., 1999a; Drewe et al., 1999; Lucia et al., 2001; Bachmeier et al., 2005; Storch et al., 2007; Chinn et al., 2007) Inductor: yes (in vitro) contradictory (in PBMCs) (Chandler et al., 2003; Chinn et al., 2007; Dupuis et al., 2003; Dussault et al., 2001; Ford et al., 2003; Gupta et al., 2006, 2008; Ko¨nig et al., 2009)

Substrate: no (in vitro) (Gupta et al., 2004) Inhibitor: yes (in vitro) (Gupta et al., 2004; Weiss et al., 2007a) Inductor: yes (in vitro) (Ko¨nig et al., 2009)

Substrate: yes (MRP2, for MRP1 contradictory) (in vitro and in vivo) no (for MRP3, MRP5) (in vitro) (Srinivas et al., 1998; Jones et al., 2001a,b; Williams et al., 2002; Meaden et al., 2002; Huisman et al., 2002; Su et al., 2004b; Janneh et al., 2005; Park and Sinko, 2005; Eilers et al., 2008) Inhibitor: yes (in vitro) (Bachmeier et al., 2005) Inductor: yes (for MRP1, MRP2, MRP4 and MRP5) in vitro (Ko¨nig et al., 2009)

Substrate: – Inhibitor: yes (ABCB11) (in vitro) (McRae et al., 2006) Inductor: –

(continued)

Table 5.3 (continued) Compound

Pgp/ABCB1

BCRP/ABCG2

MRPs/ABCCs

Other ABC-transporters

Ritonavir

Substrate: yes (in vitro) (Kim et al., 1998b; Lee et al., 1998; Srinivas et al., 1998; Jones et al., 2001a,b; Patel et al., 2002; Meaden et al., 2002; Zastre et al., 2009) Inhibitor: yes (in vitro and in vivo) (Washington et al., 1998a,b; Drewe et al., 1999; Profit et al., 1999a; Shiraki et al., 2000; Lucia et al., 2001; Perloff et al., 2002; Ding et al., 2004; Bachmeier et al., 2005; van Heeswijk et al., 2006; Perloff et al., 2003; Storch et al., 2007) Inductor: yes (in vitro) (Dussault et al., 2001; Perloff et al., 2007; Perloff et al., 2001; Perloff et al., 2003; Stormer et al., 2002; Chandler et al., 2003; Dixit et al., 2007; Gupta et al., 2008; Zastre et al., 2009)

Substrate: no (in vitro) (Gupta et al., 2004) Inhibitor: conflicting (in vitro) (Gupta et al., 2004; Weiss et al., 2007a) Inductor: –

Substrate: yes (MRP2, conflicting for MRP1) (in vitro) no (for MRP3, MRP5) (in vitro) (Srinivas et al., 1998; Jones et al., 2001a,b; Huisman et al., 2002; Zastre et al., 2009) Inhibitor: yes (MRP1) (in vitro) (Bachmeier et al., 2005; Olson et al., 2002) Inductor: no (MRP1) (in vitro) (Zastre et al., 2009)

Substrate: – Inhibitor: yes (ABCB11) (McRae et al., 2006) Inductor: –

Indinavir

Nelfinavir

Substrate: yes (in vitro and in vivo) (Hamidi, 2006; Jorojuria et al., 2004; Kim et al., 1998b; Lee et al., 1998; Srinivas et al., 1998) Inhibitor: yes (in vitro) (Bachmeier et al., 2005; Lucia et al., 2001; Profit et al., 1999a; Storch et al., 2007; Tong et al., 2007) Inductor: no (in vitro and in vivo) (Chandler et al., 2003; Ford et al., 2003; Gupta et al., 2008; Jorojuria et al., 2004) Substrate: yes (in vitro and in vivo) (Choo et al., 2000; Edwards et al., 2005; Kaddoumi et al., 2007; Kim et al., 1998b; Salama et al., 2005; Srinivas et al., 1998) Inhibitor: yes (in vitro) (Washington et al., 1998b; Profit et al., 1999a; Shiraki et al., 2000; Lucia et al., 2001; Bachmeier et al., 2005; Storch et al., 2007)

Substrate: no (in vitro) (Gupta et al., 2004; Huisman et al., 2002; Wang et al., 2003) Inhibitor: no (in vitro) (Gupta et al., 2004; Weiss et al., 2007a) Inductor: –

Substrate: yes (MRP2, for MRP1 conflicting) (in vitro) no (MRP3, MRP5) (in vitro) (Srinivas et al., 1998; Huisman et al., 2002; Jones et al., 2001a,b; van der Sandt et al., 2001; Jorojuria et al., 2004) Inhibitor: yes (in vitro) (Bachmeier et al., 2005) Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: no (in vitro) (Gupta et al., 2004; Wang et al., 2003) Inhibitor: yes (in vitro) (Gupta et al., 2004; Weiss et al., 2007a) Inductor: –

Substrate: – Inhibitor: yes (in vitro) (Bachmeier et al., 2005) Inductor: yes (MRP2) (in vitro) (Dixit et al., 2007)

Substrate: Inhibitor: Inductor: -

(continued)

Table 5.3 (continued) Compound

Amprenavir

Lopinavir

Pgp/ABCB1

Inductor: yes (in vitro and in vivo) (Chandler et al., 2003; Dixit et al., 2007; Ford et al., 2003; Gupta et al., 2008; Huang et al., 2001) Substrate: yes (in vitro and in vivo) (Choo et al., 2000) Inhibitor: yes (in vitro) (Bachmeier et al., 2005; Storch et al., 2007; Tong et al., 2007) Inductor: yes (in vitro and in vivo) (Gupta et al., 2008; Huang et al., 2001) Substrate: yes (in vitro) (Agarwal et al., 2007; Janneh et al., 2007) Inhibitor: yes (in vitro) (Storch et al., 2007; Tong et al., 2007; Vishnuvardhan et al., 2003)

BCRP/ABCG2

MRPs/ABCCs

Other ABC-transporters

Substrate: no (in vitro) (Gupta et al., 2004) Inhibitor: weak (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: – Inhibitor: yes (MRP1) (Bachmeier et al., 2005) Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: no (for murine Bcrp1) Agarwal et al., 2007 Inhibitor: yes (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: yes (MRP1, MRP2) (in vitro) (Agarwal et al., 2007; Janneh et al., 2007) Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Atazanavir

Tipranavir

Inductor: yes (partly contradictory/ in vitro) (Chandler et al., 2003; Ford et al., 2003; Gupta et al., 2008; Vishnuvardhan et al., 2003) Substrate: yes (in vitro) (Bousquet et al., 2008b; Roucairol et al., 2007; Zastre et al., 2009) Inhibitor: yes (in vitro) (Lucia et al., 2005; Perloff et al., 2005; Chinn et al., 2007; Storch et al., 2007) Inductor: yes (in vitro) (Bousquet et al., 2008b; Chinn et al., 2007; Gupta et al., 2008; Perloff et al., 2005; Zastre et al., 2009) Substrate: yes (in vitro) (Orman and Perry, 2008)

Substrate: yes (in vitro) (Bousquet et al., 2008b) Inhibitor: yes (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: yes (MRP1 (in vitro) (Zastre et al., 2009) Inhibitor: yes (MRP1, in vitro) (Lucia et al., 2005) Inductor yes (MRP1, in vitro) (Bousquet et al., 2008b)

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor:

Substrate: – Inhibitor:

Substrate: – Inhibitor: (continued)

Table 5.3 (continued) Compound

Pgp/ABCB1

BCRP/ABCG2

MRPs/ABCCs

Other ABC-transporters

yes (in vitro) (Weiss et al., 2007a) Inductor: –

– Inductor: –

– Inductor: –

Darunavir

Inhibitor: weak (in vitro) (Orman and Perry, 2008; Storch et al., 2007) Inductor yes (in vitro) (Gupta et al., 2008; Orman and Perry, 2008) yes (in vivo) (Mukwaya et al., 2005; Vourvahis and Kashuba, 2007) Substrate: yes (in vitro) (Fujimoto et al., 2009; Ko¨nig et al., 2009; Kwan et al., 2009) Inhibitor: weak (Fujimoto et al., 2009; Ko¨nig et al., 2009; Tong et al., 2007) Inductor: yes (Ko¨nig et al., 2009)

Substrate: presumably not (indirect in vitro assay) (Ko¨nig et al., 2009) Inhibitor: weak (Ko¨nig et al., 2009) Inductor: –

Substrate: contradictory (in vitro) (Fujimoto et al., 2009; Ko¨nig et al., 2009; Kwan et al., 2009) Inhibitor: – Inductor: no (for MRP1–5) (Ko¨nig et al., 2009)

Substrate: – Inhibitor: – Inductor: –

NNRTIs Efavirenz

Nevirapine

Substrate: no (in vitro, ex vivo, in vivo) (Stormer et al., 2002; Almond et al., 2005b; Dirson et al., 2006; Weiss et al., 2009; Janneh et al., 2009) Inhibitor: yes (in vitro) (Storch et al., 2007) Inductor: yes (in vitro) no (in vivo) (Stormer et al., 2002; Chandler et al., 2003; Weiss et al., 2008, 2009; Berruet et al., 2005; Mouly et al., 2002) Substrate: no (in vitro) ( Janneh et al., 2009; Stormer et al., 2002) presumably (in vivo) (Almond et al., 2005a) Inhibitor: weak (in vitro) (Storch et al., 2007) Inductor: contradictory (in vitro) (Chandler et al., 2003; Stormer et al., 2002; Weiss et al., 2008)

Substrate: presumably not (indirect in vitro assay) (Weiss et al., 2009) Inhibitor: yes (Weiss et al., 2007a) Inductor: yes (in vitro) (Weiss et al., 2009)

Substrate: presumably not (indirect in vitro assay) (Weiss et al., 2009) Inhibitor: yes (MRP1–3) (in vitro) (Bousquet et al., 2009; Weiss et al., 2007b) Inductor: yes (MRP1–3, MRP5, MRP6) (in vitro) (Bousquet et al., 2009; Weiss et al., 2009)

Substrate: – Inhibitor: yes (ABCB11) (in vitro) (McRae et al., 2006) Inductor: –

Substrate: no (in vitro) (Wang et al., 2003) Inhibitor: weak (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: – Inhibitor: yes (for MRP3), weak (for MRP1 and MRP3) (in vitro) (Weiss et al., 2007b) Inductor: –

Substrate: – Inhibitor: no (ABCB11) (McRae et al., 2006) Inductor: –

(continued)

Table 5.3 (continued) Compound

Pgp/ABCB1

BCRP/ABCG2

MRPs/ABCCs

Other ABC-transporters

Delavirdine

Substrate: no (in vitro) (Stormer et al., 2002) Inhibitor: yes (in vitro) (Storch et al., 2007; Stormer et al., 2002) Inductor: yes (in vitro) (Stormer et al., 2002; Weiss et al., 2008) Substrate: no (in vitro) (Scholler-Gyure et al., 2009) Inhibitor: weak (in vitro) (Scholler-Gyure et al., 2009) Inductor: –

Substrate: – Inhibitor: yes (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: – Inhibitor: yes (for MRP1–3) (in vitro) (Weiss et al., 2007b) Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: contradictory (in vitro) (Antonelli et al., 1992; Jorojuria et al., 2004)

Substrate: yes (in vitro) (Pan et al., 2007; Wang et al., 2003, 2004)

Substrate: yes (MRP4), possibly (MRP1– 3,5), no (MRP8) (in vitro) (Eilers et al., 2008; Guo et al.,

Substrate: – Inhibitor: –

Etravirine

NRTIs/ NtRTI Zidovudine

Didanosine

Zalcitabine

Inhibitor: no (in vitro) (Lucia et al., 1995; Shiraki et al., 2000; Storch et al., 2007) Inductor: contradictory (in vitro) (Jorojuria et al., 2004; Lucia et al., 1995; Signoretti et al., 1997; Weiss et al., 2008) Substrate: – Inhibitor: no (in vitro) (Shiraki et al., 2000; Storch et al., 2007) Inductor: weak (in vitro) no (in vivo) (Lucia et al., 1995; Weiss et al., 2008) Substrate: – Inhibitor: no (in vitro) (Lucia et al., 1995; Shiraki et al., 2000; Storch et al., 2007) no (in vitro) (Lucia et al., 1995; Weiss et al., 2008)

Inhibitor: weak (in vitro) Weiss et al., 2007a,b Inductor: –

2003; Jorojuria et al., 2004; Schuetz et al., 1999) Inhibitor: no (in vitro) (Olson et al., 2002) Inductor: yes (MRP4, MRP5) (Jorojuria et al., 2004)

Inductor: –

Substrate: no (in vitro) (Wang et al., 2003) Inhibitor: no (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: yes (MRP4) (in vitro) (Schuetz et al., 1999) Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor: no (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: yes (MRP8) (in vitro) (Guo et al., 2003) Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

(continued)

Table 5.3 (continued) Compound

Pgp/ABCB1

BCRP/ABCG2

MRPs/ABCCs

Other ABC-transporters

Stavudine

Substrate: – Inhibitor: no (in vitro) (Storch et al., 2007) Inductor: yes (in vitro) (Weiss et al., 2008) Substrate: – Inhibitor: no (in vitro) (Storch et al., 2007) Inductor: weak (in vitro) (Weiss et al., 2008)

Substrate: conflicting (in vitro) (Wang et al., 2003, 2004) Inhibitor: no (in vitro) (Weiss et al., 2007a) Inductor: – Substrate: yes (in vitro) (Wang et al., 2003) Inhibitor: no (in vitro) (Weiss et al., 2007a) Inductor: –

Substrate: yes (MRP4) (in vitro) (Schuetz et al., 1999) Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: – Substrate: – Inhibitor: – Inductor: –

Substrate: yes (in vitro and in vivo) (Shaik et al., 2007) Inhibitor: weak (in vitro) (Storch et al., 2007) Inductor: no (in vitro) (Weiss et al., 2008)

Substrate: yes (for murine transporter, not tested for human BCRP) (in vitro) (Pan et al., 2007) Inhibitor: weak (in vitro) (Weiss et al., 2007b) Inductor: –

Substrate: yes (MRP4) (in vitro) conflicting (MRP8) (in vitro) (Guo et al., 2003; Schuetz et al., 1999; Turriziani et al., 2002) Inhibitor: weak (MRP1–3) (in vitro) (Weiss et al., 2007b) Inductor: – Substrate: most likely no MRP4 substrate (in vitro) (Turriziani et al., 2006) Inhibitor: weak for MRP1 and MRP2, no inhibitor for MRP3 (in vitro) (Weiss et al., 2007b) Inductor: –

Lamivudine

Abacavir

Substrate: – Inhibitor: – Inductor: –

Tenofovir

Substrate: no (for tenofovir) yes (for tenofovir DF) (in vitro) (Ray et al., 2006; van Gelder et al., 2002) Inhibitor: weak (in vitro) (Storch et al., 2007) Inductor: no (in vitro) (Weiss et al., 2008)

Substrate: – Inhibitor: no (in vitro) Weiss et al., 2007 Inductor: –

Emtricitabine

Substrate: – Inhibitor: no (in vitro) (Storch et al., 2007) Inductor: weak (in vitro) (Weiss et al., 2008)

Substrate: – Inhibitor: no (in vitro) (Weiss et al., 2007a) Inductor: –

Entry inhibitors Raltegravir Substrate: yes (in vitro) (Zembruski et al., 2009) Inhibitor: no (in vitro) (Zembruski et al., 2009) Inductor: yes (in vitro) (Zembruski et al., 2009)

Substrate: no (in vitro) (Zembruski et al., 2009) Inhibitor: no (in vitro) (Zembruski et al., 2009) Inductor: –

Substrate: yes (MRP4) contradictory (MRP2) (in vitro) (Imaoka et al., 2007; Mallants et al., 2005; Ray et al., 2006) Inhibitor: yes (in vitro) (Bousquet et al., 2009; Weiss et al., 2007b) Inductor: no (in vitro) (Bousquet et al., 2009) Substrate: yes (in vitro) (Bousquet et al., 2008a) Inhibitor: yes for MRP1–3 (in vitro) (Bousquet et al., 2008a, 2009; Weiss et al., 2007b) Inductor: yes (MRP5) (in vitro) (Bousquet et al., 2009) Substrate: no (in vitro) (Zembruski et al., 2009) Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

(continued)

Table 5.3 (continued) Compound

Pgp/ABCB1

BCRP/ABCG2

MRPs/ABCCs

Other ABC-transporters

Elvitegravir

Substrate: yes (in vitro) (Zembruski et al., 2009) Inhibitor: no (in vitro) (Zembruski et al., 2009) Inductor: yes (in vitro) (Zembruski et al., 2009) Substrate: yes (in vitro and in vivo) (Walker et al., 2005; Zembruski et al., 2009) Inhibitor: no (in vitro) (Zembruski et al., 2009) Inductor: weak (in vitro) (Zembruski et al., 2009)

Substrate: no (in vitro) (Zembruski et al., 2009) Inhibitor: no (in vitro) (Zembruski et al., 2009) Inductor: –

Substrate: no (in vitro) (Zembruski et al., 2009) Inhibitor: – Inductor: –

Substrate: – Inhibitor: – Inductor: –

Substrate: – Inhibitor: no (in vitro) (Zembruski et al., 2009) Inductor: –

Substrate: – Inhibitor: – Inductor: MRP3 (in vitro) (Zembruski et al., 2009)

Substrate: – Inhibitor: – Inductor: –

Maraviroc

Not included: data derived from pharmacogenetic studies. For fosamprenavir and enfuvirtide there are no data at all. –, No data available.

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The reason for this discrepancy might be attributed to the different incubation periods. In another CD4þ cell line (Jurkat cells) our group could not find any expressional differences of ABCB1 during a 4-week incubation period with 10 mM saquinavir (Ko¨nig et al., 2009). In PBMCs of healthy volunteers, 10 mM saquinavir for 72 h did not provoke any change in Pgp protein expression (Chandler et al., 2003; Ford et al., 2003) and similarly in vivo in HIV-1-infected individuals no overall changes in protein expression were observed (Chinn et al., 2007; Ford et al., 2003). Taken together, these data do not argue for an influence of saquinavir on lymphocyte Pgp expression in the clinical situation but it is likely that Pgp induction is possible in other organs like the intestine. Studies addressing the interaction of saquinavir with BCRP are sparse. In vitro saquinavir was not transported by HEK cells overexpressing BCRP (Gupta et al., 2004) and this was confirmed by our group by demonstrating that MDCKII cells with BCRP overexpression are similarly sensitive towards toxic effects of saquinavir as the parental cell line (Ko¨nig et al., 2009). Although not being a BCRP substrate, saquinavir can inhibit this transporter albeit less potently than nelfinavir and lopinavir (Gupta et al., 2004; Weiss et al., 2007a) and saquinavir is also a weak BCRP inductor in Jurkat cells (Ko¨nig et al., 2009). Whereas saquinavir is a well-established substrate of MRP2 (Huisman et al., 2002; Su et al., 2004b) data for MRP1 are inconsistent and difficult to interpret. This is mainly due to the difficulty to differentiate between the action of individual MRPs in cells expressing more than one MRP, a technical flaw caused by the lack of specific MRP substrates and inhibitors. The only study with a really specific MRP assay, which studied the transport of saquinavir in MRP transfected MDCK cells, demonstrated that saquinavir is not efficiently transported by MRP1, MRP3, and MRP5 (Huisman et al., 2002). In contrast, another study found a significantly larger accumulation of saquinavir in PBMCs of patients with lower MRP1 expression arguing at least for some transport of saquinavir by MRP1 (Meaden et al., 2002). All other studies investigating saquinavir transport by MRP1 did not use specific assays for this transporter: One study found a reduced saquinavir accumulation in CEM–MRP cells, which could partly be reversed by MK571, but because these cells were selected by epirubicin they might also express other MRPs potentially inhibited by MK571, which is not specific for MRP1 ( Jones et al., 2001a). In addition, a marginal decrease of the antiviral efficacy of saquinavir in MRP1 overexpressing cells (CEM/VM-1-5) might also be caused by expression of other MRPs (Srinivas et al., 1998). Similarly, a number of other studies did not differentiate between different MRPs and although they all demonstrated transport of saquinavir by MRPs, it cannot be concluded that MRP1 was indeed the protein involved (Eilers et al., 2008; Janneh et al., 2005; Park and Sinko, 2005).

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With regard to the inhibitory properties of saquinavir on MRPs it is also open which MRP was inhibited, because the assays applied were not selective for a specific MRP (Bachmeier et al., 2005; Srinivas et al., 1998). Saquinavir induces mRNA expression of several MRPs in vitro suggesting that saquinavir might limit the access of MRP substrates to the systemic circulation or to sanctuary sites like some of the PIs and other antiretrovirals (Ko¨nig et al., 2009). Beyond these ‘‘classical’’ ABC-transporters, saquinavir has also been demonstrated to inhibit ABCB11 (BSEP) (McRae et al., 2006). However, this inhibition is most likely not of importance for drug interactions and toxicity. Summarizing the current knowledge on saquinavir and ABC-transporters reveals that Pgp and MRP2 might limit absorption of saquinavir and its access to its target cells, whereas the impact of other MRPs and BCRP still has to be defined. 5.1.2. Ritonavir Beyond saquinavir, ritonavir is the best investigated PI with respect to the interaction with ABC-transporters. It is well established that ritonavir is transported by Pgp (Jones et al., 2001a; Lee et al., 1998; Meaden et al., 2002; Patel et al., 2002; Srinivas et al., 1998; Zastre et al., 2009) and that it is one of the most potent Pgp inhibitors both in vitro and in vivo (Bachmeier et al., 2005; Ding et al., 2004; Drewe et al., 1999; Lucia et al., 2001; Perloff et al., 2002, 2003; Profit et al., 1999a; Shiraki et al., 2000; Storch et al., 2007; van Heeswijk et al., 2006; Washington et al., 1998a). Its inducing effects on ABCB1 mRNA expression are also indisputable and due to its strong PXR ligand properties expected (Chandler et al., 2003; Dixit et al., 2007; Dussault et al., 2001; Gupta et al., 2008; Perloff et al., 2003, 2007; Zastre et al., 2009). In contrast to Pgp only very little is known about the interaction of ritonavir with BCRP. There is one study excluding substrate characteristics, but it is not clear whether ritonavir inhibits BCRP (Gupta et al., 2004). Up to its solubility limit we could not demonstrate any inhibitory effect of ritonavir on BCRP transport, whereas others reported BCRP inhibition (Gupta et al., 2004) at concentrations exceeding ritonavir’s solubility limit (Weiss et al., 2002). As saquinavir, ritonavir is a substrate of MRP2 (Huisman et al., 2002) and for the same reasons as for saquinavir the data for MRP1 are not clear (Huisman et al., 2002; Jones et al., 2001a; Srinivas et al., 1998). MRP3 and MRP5 substrate characteristics of ritonavir were excluded (Huisman et al., 2002) as well as induction of MRP1 by ritonavir (Zastre et al., 2009). Like saquinavir, ritonavir was identified as an inhibitor of ABCB11, which might be associated with hepatocellular toxicity (McRae et al., 2006). In conclusion, for ritonavir the impact of Pgp has doubtlessly been demonstrated, whereas for many other ABC-transporters data are inconclusive.

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5.1.3. Indinavir Similar to ritonavir and saquinavir indinavir is a substrate and inhibitor (albeit the weakest of all PIs) of Pgp (Bachmeier et al., 2005; Hamidi, 2006; Kim et al., 1998b; Lee et al., 1998; Lucia et al., 2001; Profit et al., 1999a; Srinivas et al., 1998; Storch et al., 2007; Tong et al., 2007). In contrast, induction of ABCB1 expression was not demonstrated neither in LS180 cells nor in PBMCs in vitro or in vivo (Chandler et al., 2003; Ford et al., 2003; Gupta et al., 2008). Indinavir is neither a substrate nor an inhibitor of BCRP (Gupta et al., 2004; Huisman et al., 2002; Wang et al., 2003; Weiss et al., 2007a). So far, there are no data about inductive effects on ABCG2 expression. As for saquinavir and ritonavir indinavir has been demonstrated to be transported by MRP2, but it is less clear, whether this also applies to MRP1 (Huisman et al., 2002; Jones et al., 2001a; Srinivas et al., 1998; van der Sandt et al., 2001). Moreover, indinavir is not a substrate of MRP3 and MRP5 (Huisman et al., 2002). One study showed inhibition of MRP function by indinavir, but it did not differentiate between individual MRPs (Bachmeier et al., 2005). Concerning inducing effects on MRP expression there are no data available. In summary, the interaction of indinavir with Pgp is well established, but the role of the other ABC-transporters is not well defined.

5.1.4. Nelfinavir Nelfinavir is a well-established substrate, inhibitor, and inducer of Pgp (Bachmeier et al., 2005; Chandler et al., 2003; Choo et al., 2000; Dixit et al., 2007; Edwards et al., 2005; Ford et al., 2003; Gupta et al., 2008; Huang et al., 2001; Kaddoumi et al., 2007; Kim et al., 1998b; Lucia et al., 2001; Profit et al., 1999a; Salama et al., 2005; Shiraki et al., 2000; Srinivas et al., 1998; Storch et al., 2007; Washington et al., 1998a). According to our data, which were obtained under identical assay conditions for all antiretrovirals, it is even the most potent inhibitor among all PIs (Storch et al., 2007). Like most other PIs nelfinavir is not a substrate of BCRP, while it potently inhibits this efflux transporter (Gupta et al., 2004; Wang et al., 2003; Weiss et al., 2007a). Induction of ABCG2 expression has not been studied yet. Even less data exist on the interaction of nelfinavir with MRPs. One study demonstrated inhibiting properties of nelfinavir but without differentiating individual MRPs. Another study showed MRP2 induction by this PI (Dixit et al., 2007). Whether nelfinavir is a substrate of MRPs has not been studied. To summarize, Pgp is the most important ABC-transporter for nelfinavir therapy. Data on other transporters are too sparse to judge their impact.

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5.1.5. Amprenavir Amprenavir was consistently shown to be a substrate, inhibitor, and inducer of Pgp (Bachmeier et al., 2005; Choo et al., 2000; Gupta et al., 2008; Huang et al., 2001; Storch et al., 2007; Tong et al., 2007). Moreover, it is a weak inhibitor of BCRP without substrate properties (Gupta et al., 2004; Weiss et al., 2007a). For MRPs there is only one study demonstrating an inhibitory effect but without discriminating between individual MRPs (Bachmeier et al., 2005). 5.1.6. Fosamprenavir Fosamprenavir is a prodrug of amprenavir with lower lipophilicity and improved absorption. As an ester it is hydrolysed during the absorption process to amprenavir. So far, there are no studies addressing its interaction with ABC-transporters. 5.1.7. Lopinavir Without doubt, lopinavir is a substrate and one of the most potent inhibitors of Pgp among the PIs (Agarwal et al., 2007; Janneh et al., 2007; Storch et al., 2007; Tong et al., 2007; Vishnuvardhan et al., 2003). Whereas it is clear that lopinavir induces Pgp expression and function in LS180 cells (Gupta et al., 2008; Vishnuvardhan et al., 2003), data in PBMCs indicate that no relevant induction is provoked in the target cells of HIV-1 therapy. In vitro at a concentration of 10 mM, lopinavir did not induce Pgp expression (Chandler et al., 2003; Ford et al., 2003). Moreover, in patients receiving lopinavir/ ritonavir no induction was observed (Ford et al., 2003). In contrast at a concentration of 100 mM a significant induction was achieved in PBMCs in vitro, but this concentration is toxic for the cells and the increase in Pgp expression might therefore represent a stress response of the cells (Chandler et al., 2003). Referring to BCRP, it has been demonstrated that lopinavir is a substrate of murine Bcrp1 (Agarwal et al., 2007) but data for human BCRP are lacking. Lopinavir is the most potent inhibitor of human BCRP among the PIs (Weiss et al., 2007a). While lopinavir is a substrate of MRP1 and MRP2 (Agarwal et al., 2007; Janneh et al., 2007), it is currently unknown whether it also inhibits or induces MRPs. In summary, Pgp appears to play the major role for lopinavir therapy, but data also indicate that BCRP and MRPs might influence its pharmacokinetics. 5.1.8. Atazanavir Similar to all other PIs, atazanavir is a substrate, inhibitor, and inducer of Pgp (Bousquet et al., 2008b; Chinn et al., 2007; Lucia et al., 2005; Perloff et al., 2005; Roucairol et al., 2007; Storch et al., 2007; Zastre et al., 2009). In contrast to other PIs, it appears to be a BCRP substrate (Bousquet et al., 2008b) and it

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also inhibits this transporter (Weiss et al., 2007a). In addition, it is transported by MRP1 and both inhibits and induces this member of the MRP family (Bousquet et al., 2008b; Lucia et al., 2005; Zastre et al., 2009). 5.1.9. Tipranavir Information on tipranavir, which has only been released in 2005, is sparse. It is a substrate, inhibitor, and inducer of Pgp (Gupta et al., 2008; Orman and Perry, 2008) and an inhibitor of BCRP (Weiss et al., 2007a). 5.1.10. Darunavir Darunavir is a very weak inhibitor of Pgp and BCRP (Fujimoto et al., 2009; Ko¨nig et al., 2009; Tong et al., 2007) and it induces Pgp but not BCRP or MRP1–5 (Ko¨nig et al., 2009). Moreover, an indirect assay (antiproliferative effect in transporter overexpressing cell lines compared to the corresponding parental cell line) indicated that darunavir is a substrate of Pgp but not of BCRP or MRP1–3 (Ko¨nig et al., 2009). In two recently published in vitro studies the authors confirmed our assumption, that darunavir is a Pgp substrate, but they also assume that it is transported by MRPs (Fujimoto et al., 2009; Kwan et al., 2009). Taken together, like for all other PIs Pgp appears to play the major role for darunavir therapy.

5.2. NNRTIs 5.2.1. Efavirenz Whereas efavirenz appears not to be transported by any ABC-transporter (Almond et al., 2005b; Dirson et al., 2006; Janneh et al., 2009; Stormer et al., 2002; Weiss et al., 2009), it exerts inhibitory and substantial inducing effects on many of them. We have demonstrated inhibition of Pgp, BCRP (Storch et al., 2007; Weiss et al., 2007a), and, confirmed by another group, inhibition of MRP1–3 (Bousquet et al., 2009; Weiss et al., 2007b). Whereas it is unequivocal that efavirenz can induce Pgp expression and function in vitro (Chandler et al., 2003; Stormer et al., 2002; Weiss et al., 2008, 2009a), three studies (two in humans and one in rats) indicate that efavirenz does not provoke changes in Pgp expression in the intestine and in PBMCs in vivo (Berruet et al., 2005; Burhenne et al., unpublished data; Mouly et al., 2002). Moreover, at least in vitro efavirenz induces BCRP and several MRPs (Bousquet et al., 2009; Weiss et al., 2009) and inhibits ABCC11 but the in vivo relevance of these findings is unclear (McRae et al., 2006). 5.2.2. Nevirapine Similar to efavirenz, nevirapine is not transported by Pgp or BCRP (Stormer et al., 2002; Wang et al., 2003) but it is a weak inhibitor of Pgp, BCRP, and MRPs (Storch et al., 2007; Weiss et al., 2007a,b) and an

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inducer of Pgp at least in vitro (Stormer et al., 2002; Weiss et al., 2008). In contrast to efavirenz it does not inhibit ABCC11 (McRae et al., 2006). 5.2.3. Delavirdine Only little information is available on delavirdine. It is not a substrate of Pgp, acts as an inhibitor of Pgp, BCRP, and MRPs, and induces Pgp expression (Storch et al., 2007; Stormer et al., 2002; Weiss et al., 2007a,b). 5.2.4. Etravirine For the recently marketed etravirine, there is only one review stating that is a mild inhibitor of Pgp lacking substrate properties (Scholler-Gyure et al., 2009). In conclusion, the great difference between NNRTIs and PIs is the lack of substrate characteristics of NNRTIs. Nevertheless, their inducing and inhibiting effects on ABC-transporters might influence pharmacokinetics of other coadministered drugs and thus also the safety and success of NNRTI therapy.

5.3. NRTIs 5.3.1. Zidovudine Information on zidovudine interactions with ABC-transporters is more comprehensive than for most other NRTIs. All studies consistently report that zidovudine does not inhibit Pgp (Lucia et al., 1995; Shiraki et al., 2000; Storch et al., 2007). There is only one in vitro study indicating that this NRTI might be a substrate of Pgp (Antonelli et al., 1992). In this study, CEM-VBL100 cells expressing high levels of Pgp were less sensitive to the antiproliferative and antiviral action of zidovudine. However, this cell line was not characterized thoroughly and other transporters may have contributed to the effect observed. In contrast, another study provides strong evidence that Pgp does not transport zidovudine, because this drug did not activate Pgp ATPase activity ( Jorojuria et al., 2004). Therefore it appears unlikely, that Pgp plays a role in the transport of zidovudine. In contrast, zidovudine is a well-established substrate of BCRP and can weakly inhibit this transporter (Pan et al., 2007; Wang et al., 2003, 2004; Weiss et al., 2007a). Referring to MRPs it has been demonstrated that zidovudine is transported by MRP4, possibly also by MRP1–3 and MRP5 but not by MRP8. It lacks inhibitor properties of MRPs and induces MRP4 and MRP5 (Eilers et al., 2008; Guo et al., 2003; Jorojuria et al., 2004; Olson et al., 2002; Schuetz et al., 1999). 5.3.2. Didanosine The interaction of didanosine with ABC-transporters has not been thoroughly studied and it is unknown, whether it is a substrate of Pgp. We and others have demonstrated, that it does not inhibit this ABC-transporter

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(Shiraki et al., 2000; Storch et al., 2007). Moreover, the weak induction of Pgp function in LS180 cells by didanosine does not appear relevant for the in vivo situation as demonstrated by Lucia and coworkers (Lucia et al., 1995; Weiss et al., 2008). Moreover, didanosine is not a BCRP substrate or inhibitor (Wang et al., 2003; Weiss et al., 2007a). It is transported by MRP4 (Schuetz et al., 1999.) 5.3.3. Zalcitabine Zalcitabine does not inhibit Pgp or BCRP and does not induce Pgp expression. It is transported by MRP8 (Guo et al., 2003; Shiraki et al., 2000; Storch et al., 2007; Weiss et al., 2007a, 2008). 5.3.4. Stavudine We have demonstrated that stavudine is an inducer of Pgp without inhibitor properties (Storch et al., 2007; Weiss et al., 2008). The data about stavudine’s BCRP substrate characteristics are inconsistent (Wang et al., 2003, 2004), while it does not inhibit this transporter (Weiss et al., 2007a). Moreover, stavudine is transported by MRP4 (Schuetz et al., 1999). 5.3.5. Lamivudine For lamivudine there is no published information, whether it is transported by Pgp or not. However, it is not an inhibitor and only a weak inducer of Pgp function (Storch et al., 2007; Weiss et al., 2008). Although it is a substrate of BCRP it does not inhibit this transporter (Wang et al., 2003; Weiss et al., 2007a). Moreover, lamivudine is transported by MRP4 and possibly by MRP8, but for MRP8 the data are controversial (Guo et al., 2003; Schuetz et al., 1999; Turriziani et al., 2002). MRP1–3 are weakly inhibited by lamivudine (Weiss et al., 2007b). 5.3.6. Abacavir In contrast to most other NRTIs abacavir is transported by Pgp (Shaik et al., 2007). Abacavir has weak inhibitory potency and does not induce Pgp (Storch et al., 2007; Weiss et al., 2008). Alternatively, it is transported by BCRP, although this has only been demonstrated for the murine protein (Giri et al., 2008; Pan et al., 2007). Moreover, it is only a weak inhibitor of BCRP and MRP1–3 and is not transported by MRP4 (Turriziani et al., 2006; Weiss et al., 2007a,b). 5.3.7. Tenofovir Whereas the prodrug tenofovir disoproxilfumarate (tenofovir DF) is a substrate of Pgp the active drug tenofovir is not (Ray et al., 2006; van Gelder et al., 2002). Moreover, tenofovir only weakly inhibits Pgp and does not induce its expression (Storch et al., 2007; Weiss et al., 2008). Tenofovir does not inhibit BCRP (Weiss et al., 2007a). It is a substrate of MRP4 and

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possibly also for MRP2, but the data are inconsistent (Imaoka et al., 2007; Mallants et al., 2005; Ray et al., 2006). Tenofovir is an inhibitor of MRPs, but not an inducer (Bousquet et al., 2009; Weiss et al., 2007b). 5.3.8. Emtricitabine Emtricitabine does not inhibit Pgp or BCRP. It weakly induces Pgp function in LS180 cells (Storch et al., 2007; Weiss et al., 2007a, 2008). In contrast, it is a substrate and an inhibitor of MRP1 and induces MRP5 (Bousquet et al., 2008a, 2009; Weiss et al., 2007b). In conclusion, for NRTIs MRPs and possibly also BCRP are more relevant than Pgp, although the clinical impact is not yet well defined.

5.4. Fusion and entry inhibitors 5.4.1. Enfuvirtide For the fusion inhibitor enfuvirtide there are no published data at all concerning possible interactions with ABC-transporters. However, due to its structure and molecular weight an interaction seems unlikely. 5.4.2. Maraviroc Both in vitro and in vivo maraviroc is a substrate but not an inhibitor of Pgp (Walker et al., 2005). It is neither a substrate nor an inhibitor of BCRP or MRPs and a week inducer of Pgp and MRP3 (Zembruski et al., 2009).

5.5. Integrase inhibitors 5.5.1. Raltegravir For raltegravir only very limited information is available. It is a substrate and an inducer of Pgp without inhibitor properties and neither a substrate nor an inhibitor of BCRP and MRPs (Zembruski et al., 2009). 5.5.2. Elvitegravir Since elvitegravir is not licensed yet, there are only limited data concerning its interaction with ABC-transporters. In vitro data of our group indicate that elvitegravir is a substrate and an inhibitor of Pgp, not a substrate but an inhibitor of BCRP, and may induce several ABC-transporters including Pgp in LS180 cells (Zembruski et al., 2009).

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6. Clinically Relevant Drug Interactions with Anti-HIV-1 Drugs Attributed to ABC-Transporters HIV-1 patients usually receive not only three or more antiretrovirals within HAART, they are also exposed to a wide variety of other drugs to treat prevalent comorbidity. Because both PIs and NNRTIs are extensively metabolized by CYPs and many of them also inhibit or induce CYPs, there is a considerable potential for pharmacokinetic drug–drug interactions in HIV-1 patients already at the level of these enzymes. For an overview over published drug–drug interactions with antiretrovirals the reader is referred to reviews dealing with this topic (Barry et al., 1999; de Maat et al., 2003; Vourvahis and Kashuba, 2007; Young, 2005). In addition, there is increasing evidence, that ABC-transporters are also involved in drug–drug interactions between antiretrovirals and with other drugs. However, because many antiretrovirals also have profound effects on CYPs or are extensively metabolized by them, it is often not clear which part of an interaction can be attributed to transporters, although it must be assumed that they contribute to numerous interactions originally ascribed solely to CYPs. As an example, the increase of the maximum plasma concentration (Cmax) and the area under the curve (AUC) of maraviroc in the presence of CYP3A and Pgp inhibitors like ketoconazole, lopinavir, ritonavir, and saquinavir might be attributed to CYP3A and/or Pgp inhibition. Similarly, the decrease in Cmax and AUC of maraviroc by rifampin and efavirenz might be provoked by induction of CYP3A and/or Pgp (MacArthur and Novak, 2008). There are only very few clinical studies clearly demonstrating an interaction of antiretrovirals at the level of ABC-transporters and such studies are complicated by the absence of selective substrates or inhibitors suitable for in vivo use. One of the best qualified drugs for the assessment of Pgp function in vivo is digoxin. Our group studied the pharmacokinetic interaction between digoxin and ritonavir and demonstrated an inhibition of renal digoxin clearance by ritonavir which can most likely be attributed to Pgp inhibition by ritonavir (Ding et al., 2004). Another group found an increase of the AUC of the Pgp substrate fexofenadine which they attributed to increased bioavailability secondary to Pgp inhibition (van Heeswijk et al., 2006). Also, ritonavir-boosted lopinavir significantly increases digoxin AUC (1.81-fold) in vivo arguing for profound Pgp inhibition by this PI combination. Similarly, the interaction between the macrolide antibiotic azithromycin and nelfinavir is also attributed to Pgp inhibition. In a crossover study in 12 healthy volunteers, nelfinavir provoked an increase of azithromycin Cmax and AUC by >100% (Amsden et al., 2000). The increase in amprenavir AUC following clarithromycin pretreatment might also at least partly be due to Pgp inhibition (Brophy et al., 2000). Moreover,

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the reduced AUCs of several antiretrovirals during intake of St. John’s Wort might be caused not only by induction of CYP isozymes but also by coinduction of Pgp (de Maat et al., 2001, 2003; Piscitelli et al., 2000). A study hypothesized that the life-threatening interaction observed between antiretroviral therapy and the Pgp substrate vinblastine might also at least partly be due to Pgp inhibition (Kotb et al., 2006). Also for the Pgp and CYP3A4 substrate tacrolimus a severe interaction with ritonavir-boosted darunavir was reported, probably not only caused by CYP but also Pgp inhibition by PIs (Mertz et al., 2009). Sometimes the ABC-transporter inducing effects of PIs seem to exceed the inhibitory effect in vivo. As an example tipranavir and ritonavir-boosted tipranavir provoked a significant decrease of the AUC of the Pgp substrate loperamide (51% and 63%, respectively) whereas ritonavir alone increased the level of exposure of loperamide (by 121%) (Mukwaya et al., 2005) suggesting that for tripranavir the inducing effects prevail. Similarly, AUC and Cmax of digoxin increased after a single dose of tipranavir/ritonavir due to Pgp inhibitory effects, but decreased under steady-state conditions arguing for an induction of Pgp by tipranavir (Vourvahis and Kashuba, 2007). There are several interactions reported between PIs and tenofovir, a compound which is largely excreted by the kidneys in unchanged form and which is therefore not subject to metabolic drug interactions. A clinical study found a decrease of tenofovir clearance under concomitant treatment with lopinavir/ritonavir. Although the exact mechanisms remained unclear, the authors hypothesized that inhibition of MRP2 by PIs might be the reason for the observed interaction ( Jullien et al., 2005). This interaction was later confirmed, but the exact mechanism remained open (Kiser et al., 2008). Tenofovir also influences the pharmacokinetics of PIs. A study in HIV-1-infected patients found a decreased AUC of atazanavir in the presence of tenofovir that might be caused by induction of intestinal Pgp by tenofovir (Taburet et al., 2004). However, our in vitro data gave no hint of ABCB1 induction by tenofovir in an intestinal cell line and the authors of the in vivo study pointed out that a physicochemical interaction between atazanavir and tenofovir DF in the gut might be an alternative explanation (Taburet et al., 2004; Weiss et al., 2008). In contrast to NNRTIs, PIs, and tenofovir, the NRTIs have far fewer drug interactions. Pharmacokinetic interactions producing changes in NRTI plasma concentrations are generally unlikely to be of clinical relevance, because their effect is mainly dependent on the rate and extent of intracellular phosphorylation (Barry et al., 1999). Taken together both in vitro and clinical data indicate that PIs reveal the highest interaction potential among all antiretrovirals at the level of ABC-transporters either by being influenced by inhibitors or inducers of ABC-transporters or by inhibiting or inducing ABC-transporters themselves thus altering the pharmacokinetics of other ABC-transporter substrates.

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7. ABC-Transporters, ‘‘Cellular’’ Resistance, and Therapeutic Success Failure of antiretroviral therapy is a complex interplay between many factors including resistance towards the compounds applied. Recent investigations suggest that not only viral resistance but also host factors (‘‘cellular resistance’’) might account for clinical resistance to antiretrovirals (Cinatl et al., 1994; Ford et al., 2004c). ABC-transporters may limit the access of antiretrovirals to the systemic circulation and infected cells and contribute to the protection of sanctuary sites for viral replication. At least for PIs a high correlation between intracellular drug concentration and antiviral activity has been demonstrated (Bilello et al., 1996; Nascimbeni et al., 1999) leading to failure of antiretroviral therapy if only subtherapeutic intracellular drug concentrations are reached.

7.1. In vitro studies demonstrating the role of ABC-ransporters for cellular resistance towards anti-HIV-1 drugs One of the first studies demonstrating reduced activity of an antiretroviral compound in ABC-transporter overexpressing cells was published as early as 1991. The study demonstrated that CEM cells overexpressing Pgp are less sensitive towards the antiproliferating effects of zidovudine (Antonelli et al., 1992). Also accumulation of PIs is reduced in CEM cells with overexpression of Pgp or MRP1 suggesting that similar results may be expected with PIs ( Jones et al., 2001a,b). Indeed, overexpression of Pgp in vitro reduced both uptake and antiviral activity corroborating the hypothesis that intracellular concentrations are determined by ABC-transporters and are critical for antiviral effectiveness (Maffeo et al., 2004). Later, also the impact of BCRP on cellular resistance towards NRTIs was demonstrated (Wang et al., 2003, 2004). However, there are also data questioning the role of ABC-transporters on lymphocytes for drug effectiveness, because saquinavir, ritonavir, nelfinavir, and indinavir, which interact with Pgp and MRP1, retained their effectiveness in multidrug-resistant T-lymphocyte cell lines indicating that cellular resistance to PIs may not be a major therapeutic concern (Srinivas et al., 1998). Whereas for PIs and NRTIs an impact of ABC-transporters on cellular resistance has been widely established, the only in vitro data so far existing for NNRTIs indicate that this class of antiretrovirals is not influenced by ABC-transporters. Using an indirect in vitro assay we have recently demonstrated that the mRNA expression level of Pgp, MRP1-3, and BCRP does not influence intracellular concentrations of the NNRTI efavirenz (Weiss et al., 2009). These data are supported by Janneh and

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coworkers who demonstrated that intracellular accumulation of efavirenz and nevirapine is independent of Pgp activity ( Janneh et al., 2009).

7.2. In vitro evidence for modulation of cellular resistance by using drug combinations A very interesting study demonstrated that ‘‘intracellular boosting’’ of PIs by inhibition of drug efflux transporters is possible and enhances suppression of viral replication thus slowing the emergence of resistance mutations (Chandler et al., 2007b). In a similar work this group demonstrated that intracellular concentrations of lopinavir (in PBMCs) increase when coincubated with ritonavir, amprenavir, and atazanavir suggesting inhibition of Pgp and/or MRPs ( Janneh et al., 2007). Another study revealed an influence of efavirenz and nevirapine on atazanavir accumulation in CEM cells. Whereas efavirenz increased baseline accumulation of atazanavir presumably by inhibition of efflux transporters such as Pgp and MRP1, nevirapine induced a marked reduction of atazanavir uptake possibly due to induction of efflux transporter expression (Roucairol et al., 2007). A recently published study in PBMCs isolated from healthy donors investigated whether dual or triple combinations of tenofovir, emtricitabine, and efavirenz lead to higher intracellular concentrations after incubation for 20 h. The authors attempted to correlate changes in ABC-transporter expression/activity with intracellular drug concentrations. Although this study had some methodological flaws by using an unspecific functional assay this approach is interesting and the authors conclude that the use of combination therapy (like Atripla) improves intracellular drug concentrations of emtricitabine and tenofovir at least partly through changes in MRP activity (Bousquet et al., 2009).

7.3. Clinical data demonstrating the role of cellular resistance for therapeutic success As a logical consequence of the impact of ABC-transporters on intracellular concentrations of antiretrovirals one would expect an association between therapeutic success and individual ABC-transporter activity. However, studies investigating this potential association are rare and data are conflicting. Moreover, only very few studies investigated ABC-transporter function in addition to mRNA or protein expression. However, ultimately only transporter function under chronic antiretroviral therapy in the target population (HIV-1-infected patients) is relevant. Ideally, the function measured should also detect potential inhibitory effects of applied drugs, because an increase in transporter expression might offset concurrent inhibition. Most functional assays in PBMCs are likely not measuring the ‘‘real’’ in vivo activity of ABC-transporters on these cells, because modulating

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influences of drugs present in plasma are washed out during experimental preparation. This might be circumvented by using activity assays in whole blood instead of isolated PBMCs (Witherspoon et al., 1996). Therefore, a conclusive appraisal on the role of ABC-transporters in mediating cellular resistance and modulating therapeutic success is currently not possible. Although saquinavir, ritonavir, and nelfinavir are substrates of Pgp, accumulation of these PIs did not correlate with lymphocyte Pgp protein expression in HIV-1-infected individuals (Ford et al., 2004a,b). Similarly, saquinavir, atazanavir, or ritonavir accumulation in lymphocytes was independent of Pgp, MRP1, or BCRP protein expression (Ford et al., 2006). In addition, differences in Pgp protein expression on PBMCs do not determine virological responses to antiretroviral therapy (Agrati et al., 2003). A more recent clinical study also concludes that antiviral activity of nelfinavir and indinavir is not influenced by Pgp activity on CD4þ cells (Sankatsing et al., 2007). In contrast, another study demonstrated an inverse relationship between mRNA overexpression of Pgp and lower intracellular concentration of PIs (Chaillou et al., 2002) and other studies also observed a relation between Pgp or MRP1 protein expression in lymphocytes and accumulation of ritonavir, saquinavir, and lopinavir (Hoggard et al., 2002; Meaden et al., 2002). In HIV-1-infected patients (with and without virological failure) who were treated with PIs Pgp protein expression and activity in CD4þ cells was quantified and compared to a control group. Pgp expression in circulating CD4þ lymphocytes was not enhanced by PI treatment and was not linked to virological failure (Bossi et al., 2003). These facts do not preclude an effect of Pgp on PI absorption or efficacy in other compartments of the body such as gut, lymph nodes, or brain in patients treated with PIs. In line with in vitro data there are two in vivo studies supporting the hypothesis that efavirenz is not a substrate of ABC-transporters and therefore not influenced in its intracellular concentrations by these efflux pumps. In PBMCs of patients, intracellular AUC and Pgp protein expression on PBMCs were not correlated (Almond et al., 2005b). Similarly, we have recently demonstrated that there is no correlation between mRNA expression levels of ABCB1, ABCC1, and ABCC2 on PBMCs and intracellular efavirenz concentrations (Burhenne et al., unpublished data). In contrast to in vitro data indicating that nevirapine is not a Pgp substrate (Stormer et al., 2002) and should therefore not be influenced by Pgp protein expression, there is one in vivo study demonstrating that intracellular nevirapine exposure decreased with high Pgp expression on PBMCs in HIV-1-infected patients. The authors concluded that either nevirapine is a substrate of Pgp or a substrate of another efflux transporter highly coregulated with Pgp (Almond et al., 2005a). A recently published interesting, albeit retrospective, study investigated the mRNA expression levels of ABCB1, ABCC1, ABCC4, and ABCC5 in

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PBMCs of HIV-1-infected patients with failing antiretroviral therapy. In the HIV-1-infected group mRNA expression of all transporters investigated was significantly higher than in the control group of healthy volunteers. Moreover, ABCC4 and ABCC5 expression was significantly higher in patients who were both PI and NNRTI experienced than in PI experienced patients who were NNRTI naı¨ve. However, they did not observe a correlation between mRNA expression for all transporters and viral load and conclude that ABC-transporters are not the only factor contributing to treatment failure ( Turriziani et al., 2008). The discrepancies observed between clinical studies and between in vitro and clinical data indicate that combinations of multiple efflux transporters are more likely to be responsible for therapeutic success (Ford et al., 2004c). Moreover, there is increasing evidence that uptake transporters (not reviewed here) also influence intracellular concentrations of antiretrovirals. To give some examples, saquinavir has been demonstrated to be a substrate of OATP1A2 (SLCO1A2) (Su et al., 2004b), lopinavir is also transported by OATPs ( Janneh et al., 2008), and lamivudine is transported by human organic cation transporters (hOCTs) (Minuesa et al., 2009). Another possible reason for the discrepancies between in vitro/ex vivo and in vivo data might be the fact that the former are normally performed with only one compound neglecting the potential contribution of combination partners. Moreover, as outlined above HIV infection itself might influence ABC-transporter activity, which is not considered in in vitro studies.

8. ABC-Transporter Polymorphisms and HIV-1 Expression and function of ABC-transporters can also be influenced by genetic variation. The numerous studies investigating the impact of polymorphisms in the ABCB1 and other ABC-transporter genes produced inconsistent results. There is not only a controversy about the clinical relevance of the polymorphisms in general, but also about their relevance for antiretroviral therapy. As discussed for ABCB1 in an excellent review inconsistent results of existing pharmacogenetic studies are often caused by methodological limitations leading to invalid or hardly comparable data (Leschziner et al., 2007). Thus, studies on ABC-transporter polymorphisms and antiretroviral drug response are also heterogeneous and difficult to judge. Some of the most important studies conducted in this field are discussed in the following sections. PIs as known substrates of Pgp and some MRPs would be the first suspects of being influenced by polymorphisms in ABC-transporter genes. One study found an association of the C3435T polymorphisms in the ABCB1 gene and pharmacokinetic data of indinavir (Solas et al., 2007), but others demonstrated the contrary (Owen et al., 2005; Verstuyft et al., 2005).

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For saquinavir, atazanavir, and lopinavir no associations were found between the C3435T and G2677T polymorphisms of ABCB1 and the pharmacokinetics of the corresponding PI (la Porte et al., 2007; Ma et al., 2007; Mouly et al., 2005; Rahi et al., 2008; Winzer et al., 2003). Similarly, a recently published study demonstrated that ABCB1 C1236T, G2677T, and C3435T genotypes and their haplotypes do not predict lopinavir and ritonavir concentrations in plasma, semen, or saliva of HIV-1-infected men under stable HAART treatment (Estrela et al., 2009). For nelfinavir an association between cellular nelfinavir exposure and the C3435T variant and two other SNPs of ABCB1 (A61G and TIVS26 þ 80C) was demonstrated (Colombo et al., 2005). In contrast, no association was demonstrated for the C3435T and G2677T variants and CD4þ cell count in patients (Zhu et al., 2004). Although efavirenz is not a Pgp substrate (Chandler et al., 2003; Stormer et al., 2002; Weiss et al., 2008) an association between ABCB1 C3435T and efavirenz exposure (Fellay et al., 2002) and between this polymorphism and improved virological outcomes has been reported (Haas et al., 2005) while other studies found no influence of ABCB1 C3435T and efavirenz plasma concentrations (Winzer et al., 2003). Hence, if this polymorphism is indeed relevant it will affect different drug compartments differently. A few years ago, an association between ABCC2 gene haplotypes and tenofovir-induced proximal tubulopathy was reported (Izzedine et al., 2006); however, the results of this study were disputed later (Izzedine et al., 2007). Another group found an association between ABCC2 C-24T polymorphism and tenofovir-induced kidney tubular dysfunction but not for other SNPs investigated such as ABCC2 (G1249A, T3563A, C3972T, G4544A), ABCC4 (C669T, A3463G, T4131G), and ABCB1 (C3435T, C1236T) (Rodriguez-Novoa et al., 2009). Also the ABCC4 A3463G SNP was associated with 35% higher intracellular tenofovir diphosphate concentrations in HIV-1-infected patients (Kiser et al., 2008). For intracellular zidovudine and lamivudine triphosphate concentrations in HIV-1-infected patients no association was demonstrated for ABCG2 or ABCC2 polymorphisms, but for ABCC4 T4141G (20% increase in lamivudine triphosphate concentration) (Anderson et al., 2006). Similar to studies with single antiretrovirals, studies investigating ABCtransporter polymorphisms and response to combination therapy (HAART) also found no association between ABCB1 C3435T polymorphisms and HAART efficacy (Haas et al., 2003; Hendrickson et al., 2008; Nasi et al., 2003). In contrast, one study reported an association between the ABCB1 C3435T SNP and virological efficacy in HIV-1-infected patients with nonboosted PI-containing regimens, but not with those receiving boosted PIs (de la Tribonniere et al., 2008). Taken together the impact of ABC-transporter polymorphisms on pharmacokinetics of antiretrovirals and on therapy outcome is far from being elucidated. For a deeper insight into the pharmacogenetics of

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antiretroviral therapy the reader is referred to excellent reviews dealing with this topic (Cressey and Lallemant, 2007; Owen and Khoo, 2008; Owen et al., 2005, 2006; Rodriguez-Novoa et al., 2006).

9. Concluding Remarks Success of HIV-1 therapy is determined by many factors including subtherapeutic drug concentrations at the site of action or adverse drug reactions. About 20 years of research have identified important mechanisms how efflux by ABC-transporters may influence pharmacokinetics and consequently concentrations of antiretrovirals in HIV-1-infected cells. Numerous in vitro, ex vivo, and clinical studies have demonstrated that Pgp and several MRPs restrict the access of PIs to the systemic circulation and sanctuary sites like lymphocytes or brain, and promote renal and hepatic excretion. In contrast, NNRTIs may influence ABC-transporter expression and function, but do not appear to be influenced themselves by the activity of these transporters. However, compared to the substantial influence of CYP-mediated metabolism the impact of ABC-transporters on PI and NNRTI pharmacokinetics, drug interactions, and efficacy appears to be smaller but of potential clinical importance. In contrast, NRTIs are not influenced by CYPs and only marginally by transporters (except for tenofovir) leading to an only small interaction potential of this drug class. For entry inhibitors acting extracellularly ABC-transporters expressed at the surface of lymphocytes are likely irrelevant but their absorption and excretion can be influenced by drug transport and in vitro data indicate that some of them are inhibitors and/or inducers of ABC-transporters. Although the impact of ABC-transporters for the pharmacokinetics and thus therapeutic success of many antiretrovirals is proven, the relevance of ABC-transporter activity on the surface of target cells is less clear. Clinical data published so far are contradictory. Most inconsistencies might be attributed to the use of different methodologies, investigation of only one transporter at a time, and the interplay between different efflux and uptake transporters, which was generally ignored. Moreover, the interrelationship between ABC-transporters and expression of HIV-1 relevant proteins in HIV-1 target cells is not well defined yet. There are still in vitro studies needed to define the interaction between antiretrovirals and drug transporters. However, future studies should also consider the whole set of drug transporters and investigate both pharmacological and virological aspects. In addition, studies are needed on how modulation of ABC-transporter activity might increase therapeutic success. The ultimate goal is and will be to improve HAART and to maximize therapeutic benefit while minimizing drug toxicity.

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Rao, V.V., Dahlheimer, J.L., Snyder, A.Z., Crankshaw, C.L., Piwnica-Worms, D., 1998. MDR1 P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP) mediate the blood–cerebrospinal fluid permeability barrier of Tc-99m sestamibi in choroid plexus epithelium. J. Nucl. Med. 39, 13P. Rao, V.V., Dahlheimer, J.L., Bardgett, M.E., Snyder, A.Z., Finch, R.A., Sartorelli, A.C., et al., 1999. Choroid plexus epithelial expression of MDR1 P glycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug-permeability barrier. Proc. Natl. Acad. Sci. USA 96, 3900–3905. Ray, A.S., Cihlar, T., Robinson, K.L., Tong, L., Vela, J.E., Fuller, M.D., et al., 2006. Mechanism of active renal tubular efflux of tenofovir. Antimicrob. Agents Chemother. 50, 3297–3304. Reid, G., Wielinga, P., Zelcer, N., De Haas, M., Van Deemter, L., Wijnholds, J., et al., 2003. Characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins MRP4 and MRP5. Mol. Pharmacol. 63(5), 1094–1103. Rodriguez-Novoa, S., Barreiro, P., Jimenez-Nacher, I., Soriano, V., 2006. Overview of the pharmacogenetics of HIV therapy. Pharmacogenomics J. 6, 234–245. Rodriguez-Novoa, S., Labarga, P., Soriano, V., Egan, D., Albalater, M., Morello, J., et al., 2009. Predictors of kidney tubular dysfunction in HIV-infected patients treated with tenofovir: a pharmacogenetic study. Clin. Infect. Dis. 48, E108–E116. Ronaldson, P.T., Bendayan, R., 2006. HIV-1 viral envelope glycoprotein gp120 triggers an inflammatory response in cultured rat Astrocytes and regulates the functional expression of P-glycoprotein. Mol. Pharmacol. 70, 1087–1098. Ronaldson, P.T., Lee, G., Dallas, S., Bendayan, R., 2004. Involvement of P-glycoprotein in the transport of saquinavir and indinavir in rat brain microvessel endothelial and microglia cell lines. Pharm. Res. 21, 811–818. Ronaldson, P.T., Persidsky, Y., Bendayan, R., 2008. Regulation of ABC membrane transporters in glial cells: relevance to the pharmacotherapy of brain HIV-1 infection. Glia 56, 1711–1735. Roucairol, C., Azoulay, S., Nevers, M.C., Creminon, C., Lavrut, T., Garraffo, R., et al., 2007. Quantitative immunoassay to measure plasma and intracellular atazanavir levels: analysis of drug accumulation in cultured T cells. Antimicrob. Agents Chemother. 51, 405–411. Salama, N.N., Kelly, E.J., Bui, T., Ho, R.J.Y., 2005. The impact of pharmacologic and genetic knockout of P-glycoprotein on nelfinavir levels in the brain and other tissues in mice. J. Pharm. Sci. 94, 1216–1225. Sankatsing, S.U.C., Beijnen, J.H., Schinkel, A.H., Lange, J.M.A., Prins, J.M., 2004. P glycoprotein in human immunodeficiency virus type 1 infection and therapy. Antimicrob. Agents Chemother. 48, 1073–1081. Sankatsing, S.U.C., Cornelissen, M., Kloosterboer, N., Crommentuyn, K.M.L., Bosch, T.M., Mul, F.P., et al., 2007. Antiviral activity of HIV type 1 protease inhibitors nelfinavir and indinavir in vivo is not influenced by P-glycoprotein activity on CD4(þ) T cells. AIDS Res. Hum. Retroviruses 23, 19–27. Schinkel, A.H., Jonker, J.W., 2003. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv. Drug Deliv. Rev. 55, 3–29. Scholler-Gyure, M., Kakuda, T.N., Raoof, A., de Smedt, G., Hoetelmans, R.M.W., 2009. Clinical pharmacokinetics and pharmacodynamics of etravirine. Clin. Pharmacokinet. 48, 561–574. Schuetz, E.G., Beck, W.T., Schuetz, J.D., 1996. Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells. Mol. Pharmacol. 49, 311–318. Schuetz, J.D., Connelly, M.C., Sun, D.X., Paibir, S.G., Flynn, P.M., Srinivas, R.V., et al., 1999. MRP4: a previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nat. Med. 5, 1048–1051.

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C H A P T E R

S I X

New Insights into the Circadian Clock in Chlamydomonas Takuya Matsuo*,† and Masahiro Ishiura*,† Contents 1. Introduction 2. Behavioral and Physiological Circadian Rhythms in Chlamydomonas 2.1. Phototaxis 2.2. Chemotaxis 2.3. Cell division cycle and UV sensitivity 2.4. Stickiness to glass 2.5. Starch content 2.6. ‘‘Artificial’’ bioluminescence 3. Circadian Oscillator in Chlamydomonas 3.1. Searching for clock components 3.2. Possible molecular mechanisms of the circadian oscillator in Chlamydomonas 4. Input Pathways to the Circadian Oscillator in Chlamydomonas 4.1. Light information 4.2. Temperature information 5. Output Pathways from the Circadian Oscillator in Chlamydomonas 5.1. Transcriptional regulation of nuclear gene expression 5.2. Posttranscriptional regulation of nuclear gene expression 5.3. Transcriptional regulation of chloroplast gene expression 5.4. Posttranscriptional regulation of chloroplast gene expression 6. Concluding Remarks Acknowledgments References

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* Center for Gene Research, Nagoya University, Nagoya, Japan Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan

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International Review of Cell and Molecular Biology, Volume 280 ISSN 1937-6448, DOI: 10.1016/S1937-6448(10)80006-1

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2010 Elsevier Inc. All rights reserved.

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Abstract The unicellular green alga Chlamydomonas reinhardtii has long been used in research on circadian rhythm. Various circadian rhythms in behavior and physiology including phototaxis, chemotaxis, the cell division cycle, UV sensitivity, stickiness to glass (changes in properties of the cell surface), and starch content are observed in this alga. Soon after the isolation of clock mutants in Drosophila melanogaster and Neurospora crassa, clock mutants were also isolated in Chlamydomonas. Whereas genes responsible (clock genes) in Drosophila and Neurospora mutants have been identified and these organisms have become important models for understanding the circadian clock, Chlamydomonas clock genes have not been identified and the molecular basis of the algal clock was unclear until a few years ago. Recently, reverse and forward genetic studies revealed several clock genes in Chlamydomonas. These studies unveiled that the Chlamydomonas clock consists of both original and plant-like components. Further study of the Chlamydomonas circadian clock should provide new insights into the evolution of plant clocks. Furthermore, the identification of its clock genes makes Chlamydomonas a new model for molecular studies of the circadian oscillatory mechanisms in eukaryotes. Its simple cellular architecture may provide significant advantages for a comprehensive understanding of the intracellular temporal organization, especially between the nucleus and chloroplast. Key Words: Circadian rhythm, Chlamydomonas reinhardtii, Model organism, Clock gene, Plant clock. ß 2010 Elsevier Inc.

1. Introduction The rotation of the earth around its axis leads to environmental changes in light and temperature that define the 24-h day/night cycle. Most organisms have evolved circadian clocks to adapt their life cycle to these environmental changes (Bu¨nning, 1973). The circadian clock consists of three parts; input pathways, an oscillator, and output pathways (Fig. 6.1). The oscillator is the core of the clock, generating
24-h rhythmicity, and the output pathways mediate the rhythmicity to physiology and behavior of the organism. The circadian oscillation is self-sustained but the period length is often not exactly 24 h, and thus, daily adjustments are needed. The input pathways receive environmental cues and adjust (reset) the phase of clock oscillations to entrain to the environmental day/night cycle. The circadian clock is a cell-based genetically determined molecular machine, and a limited number of genes called ‘‘clock genes’’ are involved in the maintenance of its functions (Dunlap, 1999; Harmer et al., 2001). In the past two decades, genetic and molecular approaches have identified clock

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Input Light

Temperature

Overt circadian rhythms

Circadian clock

Environment

Oscillator

Output

Mammals Sleep−wake cycle Blood pressure Insects Eclosion Locomotor activity Plants Photosynthesis Leaf movement Fungi Conidiation Protists Cell division Taxis

Figure 6.1 Conceptual model of the circadian clock. The input pathways include photoreceptors, temperature sensing molecules, and their signaling pathways to the circadian oscillator. The circadian oscillator consists of a set of clock genes and its protein products. They form transcriptional/translational autoregulatory feedback loops. The output pathways pass the timing information from the circadian oscillator to various biological pathways. One means of mediation is the regulation of gene expression. Representative overt circadian rhythms resulting from the circadian regulation are indicated.

genes in several model organisms including animals (D. melanogaster and Mus musculus), fungi (N. crassa), land plants (Arabidopsis thaliana), and cyanobacteria (Synechococcus sp. strain PCC7942). Most clock genes and their products are not conserved across kingdoms at least not in their primary sequences, indicating that circadian clocks have evolved independently. However, a common model, the autoregulatory feedback loops that generate circadian oscillation of gene expression, has been proposed (Dunlap, 1999; Harmer et al., 2001). This model is based on genetic feedback regulation of core clock gene expression. The essential factors for maintenance of the feedback regulation are also clock components (e.g., activators and repressors for clock gene expression, kinase, phosphatase, and ubiquitin ligase for the clock proteins, and other interacting proteins). However, except in these model organisms, clock genes have not been identified and molecular mechanisms of the circadian clock are not well understood. Chlamydomonas reinhardtii (C. reinhardtii) was one such model organism. Chlamydomonas is a unicellular eukaryotic green alga. Like animals, it has an eye (eyespot) for detecting light, and arms (flagella) for swimming and mating, but like plants, it can photosynthesize with its large single chloroplast (Fig. 6.2A). Chlamydomonas is sometimes called ‘‘green yeast’’ or ‘‘photosynthetic yeast’’ because it is a simple and experimentally tractable unicellular eukaryote like yeast (Goodenough, 1992; Rochaix, 1995). Numerous techniques of molecular genetics, cell biology, and biochemistry have been used successfully in this alga, and the whole genome sequence is

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A

Flagellum

Mitochondrion

Basal bodies

Eyespot

Chloroplast Pyrenoid

B Hatching Ammonium uptake

Nucleus

Supercoiling of chloroplast DNA Nitrogen metabolism

Chloroplast RNA synthesis

Cell division Stickiness to glass

Night

Day Phototaxis

Chemotaxis

Starch content

UV sensitivity

Figure 6.2 Behavioral and physiological circadian rhythms observed in Chlamydomonas. (A) The morphology of Chlamydomonas. The cell is
10 mm in diameter. Chlamydomonas cells have a single nucleus surrounded by a cup-shaped chloroplast containing a pyrenoid. Anterior flagella are rooted in basal bodies. An eyespot is positioned just inside the chloroplast membrane. (B) Diagrammatic representation of the circadian cycle of Chlamydomonas. The arrows represent the active time of cellular processes indicated.

now available (Harris, 2001; Merchant et al., 2007). Although yeast is an outstanding model for studies on eukaryotic cellular processes, Chlamydomonas has significant advantages over yeast in studies of the chloroplast and flagella which yeast does not have. Since yeast does not exhibit obvious circadian rhythms, Chlamydomonas has the potential to be an outstanding simple model eukaryote for studying molecular mechanisms of the circadian clock. Recently, two groups identified clock genes in Chlamydomonas (Iliev et al., 2006; Matsuo et al., 2008; Schmidt et al., 2006). This alga has

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therefore become a new model for studying the molecular basis of the circadian clock. This chapter provides an overview of research into circadian rhythm in Chlamydomonas.

2. Behavioral and Physiological Circadian Rhythms in Chlamydomonas 2.1. Phototaxis Chlamydomonas displays circadian rhythms in many cellular processes (Fig. 6.2B). A representative is the phototaxis rhythm. Chlamydomonas can detect the direction of a light source with its eyespot, and swims toward or escapes from it, depending on the strength of the light (Foster and Smyth, 1980). In 1970, Bruce reported that the accumulation of Chlamydomonas cells at a light source (positive phototaxis) was stronger during the day than night under light/dark (LD) cycles, and this daily rhythm is maintained even in the absence of an external time cue (e.g., continuous light [LL] condition, constant darkness [DD]) (Bruce, 1970). One striking feature of the circadian clock is the temperature compensation of period length (Bu¨nning, 1973). In contrast to general biological processes, the frequency of circadian oscillations (period lengths) is relatively constant under different temperature conditions. Bruce measured photoaccumulation rhythms at 18, 22, and 28  C, and found period lengths of the photoaccumulation rhythms to be almost constant (Bruce, 1970). These findings might be enough to conclude the existence of an endogenous circadian clock. However, if one takes into account external daily fluctuations in gravity, cosmic radiation, and magnetic fields in laboratories, there is some doubt as to the endogenous control of the rhythmicity. One promising way to confirm endogenous control would be to conduct an experiment in space. Mergenhagen and colleagues measured photoaccumulation rhythms in a space-lab aboard the space shuttle, and demonstrated that the phototaxis rhythm is regulated by an endogenous circadian clock (Mergenhagen, 1986; Mergenhagen and Mergenhagen, 1987, 1989). Since the phototaxis rhythm is robust and can be recorded automatically, it is still a very important technique for analyzing circadian rhythm in Chlamydomonas.

2.2. Chemotaxis Another taxis observed in Chlamydomonas is chemotaxis. Chlamydomonas cells exhibit positive chemotaxis toward ammonium (Sjoblad and Frederikse, 1981) and some sugars (Ermilova et al., 1993). The chemotaxis activity toward ammonium fluctuates in a circadian manner (Byrne et al., 1992). In contrast to phototaxis, chemotaxis activity peaks in the subjective night (Fig. 6.2B). This peak is followed by a peak of ammonium uptake

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rhythm at the subjective dawn and peak of activity of NII, a key enzyme of nitrogen metabolism, in the early subjective morning (Fig. 6.2B). The phototaxis and chemotaxis rhythms are thought to confer adaptive advantages to Chlamydomonas cells. The positive phototaxis will lead the cells to light-rich environments, and enable them to achieve efficient photosynthesis during the day. In turn, the chemotaxis during the night would enable them to find a nitrogen-rich environment before the peak of nitrogen uptake and metabolism in the morning (Byrne et al., 1992).

2.3. Cell division cycle and UV sensitivity The cell division cycle is under the control of the circadian clock in most organisms (Edmunds, 1984). These two intracellular oscillatory systems are linked at the molecular level by the interactions of core molecules of the cell cycle and circadian clock (Fu et al., 2002; Hunt and Sassone-corsi, 2007; Matsuo et al., 2003; Unsal-Kac¸maz et al., 2005). Circadian control of the cell division cycle is also observed in Chlamydomonas. Chlamydomonas cells divide by a noncanonical mechanism termed multiple fission (Harris, 1989). Nuclear division and cytokinesis occur several times in the mother cell wall, and daughter cells are released synchronously by enzymatic digestion of the wall (called hatching). Under LD conditions, nuclear division and cytokinesis occur in the dark phase, and hatching occurs at the transition from dark to light. Bruce (1970) found that the daily hatching rhythm continues even under LL conditions (Fig. 6.2B). Straley and Bruce (1979) reported that a circadian rhythm was observed not only in hatching but also in cell division (nuclear division and cytokinesis). Goto and Johnson (1995) demonstrated conclusively that the hatching rhythm met three criteria of circadian rhythms: (i) persistence of about 24-h rhythmicity under constant conditions, (ii) entrainment to LD cycles, and (iii) temperature compensation of period length. Nikaido and Johnson (2000) found that Chlamydomonas cells exhibit daily rhythmicity in sensitivity to UV-radiation. Maximum and minimum sensitivity were observed in the early night phase and late night to mid-day phase, respectively (Fig. 6.2B). The rhythmicity was also observed under circadian conditions (DD). The daily/circadian rhythmic variation of UV sensitivity was thought to be due to circadianly controlled cell cycle progression (Nikaido and Johnson, 2000). UV sensitivity is known to be maximum during the G1 and early S-phase of the cell cycle in yeast (Chanet et al., 1973) and cultured mammalian cells (Burg et al., 1977). The circadian phase of maximum sensitivity to UV-radiation in Chlamydomonas corresponds well to the G1 or S-phase of its cell cycle progression (Nikaido and Johnson, 2000).

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2.4. Stickiness to glass Straley and Bruce (1979) found that Chlamydomonas exhibits a circadian change in the properties of its cell surface that can be measured as an ability to stick to glass. Maximum stickiness occurs during the subjective night under DD conditions (Fig. 6.2B). Since a larger fraction of the cells were involved in sticking than in the cell cycle under their experimental conditions, the rhythm would be independent of any surface changes accompanying cell cycle progression. They also showed that the flagella as well as the cell body are involved in the sticking, and that nonmotile cells whose flagella had been amputated exhibit a similar sticking rhythm. Importantly, no cooperative effect between the sticking and nonsticking cells was found. This finding raised the possibility that circadian rhythm mutants having an altered circadian period or phase could be enriched. To test the possibility, they carried out an experiment with a mixed culture containing 10% previously isolated long-period mutant per-4 cells (Section 3.1.1) and 90% wild-type cells. If the per-4 and WT ticked independently with their own period length in the mixture, phases of their rhythms would dissociate gradually. They recovered sticking cells when the maximum phase of the per-4 would be 180 out-of-phase with that of WT (i.e., WT was in the minimum phase). The sticking cell fraction contained 3.4-fold more mutant cells than the original mixture, thus they succeeded in enriching the mutant fraction. Unfortunately, however, they did not isolate any new clock mutants using this procedure. On the other hand, this result suggested that there is no mutual entrainment between the circadian clocks of per-4 and WT cells.

2.5. Starch content Chlamydomonas displays a robust diurnal rhythm of starch content that peaks during the night (Klein, 1987; Zabawinski et al., 2001) (Fig. 6.2B). Ball and colleagues demonstrated that the rhythm in starch content persists over 10 days even in constant conditions, and the period length is temperaturecompensated between 15 and 30  C (Ral et al., 2006). The starch content rhythm was tightly correlated to the activity of ADP-glucose pyrophosphorylase (Ral et al., 2006). Analyses of transcript and protein abundances, and enzymatic activity suggested that the circadian clock exerts its control at the regulation of mRNA abundance of the small subunit of ADP-glucose pyrophosphorylase (Ral et al., 2006; Zabawinski et al., 2001). The starch content rhythm was strongly affected in a mutant of granule-bound starch synthase I (GBSSI), and the rhythm was completely abolished in a double mutant with soluble starch synthase III (Ral et al., 2006).

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2.6. ‘‘Artificial’’ bioluminescence The artificial bioluminescence derived from luciferase reporters fused to clock-regulated promoters has been used to monitor circadian rhythms in real-time in several organisms including Arabidopsis, cyanobacteria, Drosophila, mammals, Neurospora, moss, and zebrafish (Aoki et al., 2004; Brandes et al., 1996; Kondo et al., 1993; Millar et al., 1992; Morgan et al., 2003; Vallone et al., 2004; Yamazaki et al., 2000). There are two luciferase reporters used for the monitoring of circadian rhythms in Chlamydomonas. One of them is the Renilla reniformis luciferase gene whose codons were adapted to the GC-rich nuclear genome of C. reinhardtii (cRluc) (Fuhrmann et al., 2004; Kiaulehn et al., 2007). Since the reaction of cRLUC needs only the substrate (coelenterazine) and O2 to emit light (Wilson and Hastings, 1998), it is suitable for an in vivo reporter of gene expression. Indeed, the bioluminescence of cRLUC seems to well reflect the circadian rhythms in transcriptional and posttranscriptional regulation (Kiaulehn et al., 2007; Voytsekh et al., 2008). However, preparations of samples at each time point are needed to measure cRLUC activity long term (several circadian cycles), because coelenterazine is not stable in culture media (Fuhrmann et al., 2004). Another bioluminescent reporter is the firefly luciferase gene whose codons were optimized to the AT-rich chloroplast genome of C. reinhardtii (lucCP) (Matsuo et al., 2006). Firefly luciferases use intracellular O2, Mg2þ, and ATP for light-emitting reactions (Wilson and Hastings, 1998), but the substrate luciferin is stable for at least several weeks in the media. Once luciferin is added to cultures, bioluminescence can be monitored continuously. Thus, it is suitable for a long-term and automatic high-throughput real-time monitoring of the circadian bioluminescence rhythms. Bioluminescence derived from the lucCP reporter fused to the chloroplast psbD promoter showed a circadian rhythm whose period length was temperature-compensated and whose phase could be entrained by LD cycles (Matsuo et al., 2006). Furthermore, per mutants having a mutation in the nuclear genome showed a long-period phenotype of chloroplast bioluminescence rhythms, suggesting the dominant regulation of circadian rhythmicity of the chloroplast by nuclear-encoded circadian clock components (Matsuo et al., 2006).

3. Circadian Oscillator in Chlamydomonas 3.1. Searching for clock components 3.1.1. per mutants Chlamydomonas was one of the most important models, comparable to Drosophila and Neurospora, in early molecular genetic studies of the circadian clock components. Soon after the isolation of circadian rhythm mutants

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in Drosophila and Neurospora (Feldman and Hoyle, 1973; Feldman and Waser, 1971; Konopka and Benzer, 1971), Bruce (1972) isolated circadian rhythm mutants in Chlamydomonas that exhibit altered circadian periods and phases in the phototaxis rhythm. A spontaneous short-period mutant having a 21-h period was isolated from the six wild-type strains tested. He further isolated a long-period mutant and an atypical phase mutant from nitrosoguanidinetreated populations. Genetic cross experiments of the mutants revealed that a single gene confers the mutant phenotype in the long-period mutant termed per-1 (per stands for period). Although per has also been given to the clock genes in Drosophila and mammals, the Chlamydomonas per is thought to be unrelated to those because no putative homolog of the Drosophila or mammalian per has been found in the genome of C. reinhardtii (Mittag et al., 2005). He further isolated three phototactic rhythm mutants, per-2, per-3, and per-4, from nitrosoguanidine-treated populations (Bruce, 1974). The per-2 mutant shows a long-period rhythm with an approximately 27-h period. The per-3 and per-4 were obtained by using a procedure designed to enrich rhythm mutants. He randomly picked several hundred clones mutagenized by exposure to nitrosoguanidine to prepare a mixed culture in which most cells had a normal period but presumably a small fraction had an abnormal one. The cells in the mixture were allowed to tick independently in the mixed culture for 3 or 4 circadian cycles, and then he recovered phototactic cells at the time the phototactic response of WT cells was expected to be minimum, and if there were longperiod mutant cells having approximately 27–28-h periodicity, their phototactic response would be maximum. Then, he succeeded in the isolation of circadian rhythm mutants, per-3 and per-4. Actually, these mutants had a longperiod phenotype of 27–28-h as expected. Genetic cross experiments of these per mutants revealed that single genes at different loci are responsible for their long-period phenotypes. The period lengthening effect of the per mutations was additive, that is, the period length of double, triple, and quadruple mutants was lengthened by the sum of the period lengthening of the single mutants. Bruce and colleagues analyzed vegetative diploids of per-1, per-2, and per-4 mutants, and revealed that per-1 is dominant, per-2 is recessive, and per-4 is probably incompletely dominant to their respective WT alleles (Bruce and Bruce, 1978). In addition, they found that the per-4 mutation is genetically linked to the arginine-requiring markers of linkage group I. The long-period phenotypes of the per mutants have been observed not only in the phototactic rhythm but also in other biological processes. To date, the per-1 and per-4 mutants have been subjected to assays of rhythms in sticking to glass, cell division/hatching, and artificial bioluminescence derived from the nuclear cRluc and the chloroplast lucCP (Goto and Johnson, 1995; Matsuo et al., 2006; Straley and Bruce, 1979; Voytsekh et al., 2008). In all of these assays, the per mutants exhibited long-period phenotypes similar to those observed in the phototactic rhythm. This indicates that per-1 and per-4 mutations affect the core mechanisms of the Chlamydomonas circadian clock.

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Mergenhagen (1984) isolated a spontaneous mutant exhibiting an extremely short-period (18-h) rhythm in a phototaxis assay. A genetic cross with the WT strain yielded progenies with a normal period, the short period, and arrhythmicity. The arrhythmicity was thought to be due to a new combination of genes. Unfortunately, the genes responsible in Bruce’s mutants and Mergenhagen’s mutant remain to be determined. Thereafter, until recent reverse and forward genetic studies (see below), little progress has been made in understanding the oscillator of the Chlamydomonas circadian clock. 3.1.2. Casein kinase A casein kinase gene (CK1) encoding a Ser/Thr protein kinase has been characterized as a circadian clock component in Chlamydomonas (Table 6.1; Fig. 6.3) (Schmidt et al., 2006). Casein kinases are involved in circadian oscillators in all eukaryotic models for the circadian clock (Fig. 6.5) (Gallego and Virshup, 2007; Mizoguchi et al., 2006). In Chlamydomonas, CK1 was identified as an eyespot protein, and its function was analyzed by RNA interference (RNAi)-mediated knockdown (Schmidt et al., 2006). A knockdown strain expressing CK1 at 25–40% of the WT level showed defects in the formation of flagella and hatching. The former defect was apparent in mixotrophic cultures where only 31.7% of the cells had normal flagella. On the other hand, 80.2% of the cells had normal flagella in autotrophic cultures. The phototaxis rhythm of the CK1 knockdown strain has been measured, and found to have a slightly short-period phenotype with a tendency to arrhythmicity after several circadian cycles. 3.1.3. CHLAMY1 The RNA-binding protein CHLAMY1 was found and characterized by Mittag and colleagues (Section 5.2 for details). CHLAMY1 consists of two subunits, termed C1 and C3. C1 is composed of three lysine homology (KH) domains and a protein–protein interaction domain (WW), and C3 is a CUG-BP-ETR-3-like factor (CELF) family protein bearing three RNA recognition motifs (RRM) (Table 6.1; Fig. 6.3) (Zhao et al., 2004). Both the knockdown and overexpression of C3 shifted the phase of circadian rhythms in phototaxis, while that of C1 abolished the rhythmicity (Iliev et al., 2006). Since similar defects were found in the circadian rhythm of nitrite reductase activity measured as another indicator of circadian rhythms, CHLAMY1 is involved in the core oscillatory mechanisms of the Chlamydomonas circadian clock (Iliev et al., 2006). In addition, the knockdown and overexpression of C1 caused a parallel decrease and increase in the level of C3 expression, indicating the existence of regulatory mechanisms interconnecting these clock protein expressions (Iliev et al., 2006). Interestingly, the C3 subunit shows significant homology to the rat CUG-binding protein 2, and anti-C3 antibody can recognize the rat homolog in various brain regions

Table 6.1 The Chlamydomonas circadian rhythm mutants and responsible genes Mutants

Mutagen

Phenotype

Circadian rhythms analyzed

per-1

Nitrosoguanidien

Long period; short period after treatment with a temperature cycle

per-2 per-3 per-4

Nitrosoguanidien Nitrosoguanidien Nitrosoguanidien

Long period Long period Long period

s Ck1-sil

Spontaneous RNA interference

Short period Short period

C1-sil

RNA interference

C1-ox

C3-sil

Gene affected

Gene product

Function

Reference

Phototaxis, hatching, bioluminescence (lucCP, cRluc)

Not determined

–

–

Phototaxis Phototaxis Phototaxis; stickiness to glass; cell division; hatching; bioluminescence (lucCP) Phototaxis Phototaxis

Not determined Not determined Not determined

– – –

– – –

Bruce (1972, 1974), Goto and Johnson (1995),Matsuo et al. (2006), Voytsekh et al. (2008) Bruce (1974) Bruce (1974) Bruce (1974), Straley and Bruce (1979), Matsuo et al. (2006)

Not determined CK1

– Casein kinase 1

Arrhythmic

Phototaxis; nitrite reductase activity

C1

KH and WW domain containing protein

Overexpression

Arrhythmic

Phototaxis

C1

KH and WW domain containing protein

RNA interference

Phase advance

Phototaxis; nitrite reductase activity

C3

CELF family RNAbinding protein

– Ser/Thr protein kinase Subunit of CHLAMY1 RNA-binding protein complex Subunit of CHLAMY1 RNA-binding protein complex Subunit of CHLAMY1 RNA-binding protein complex

Mergenhagen (1984) Schmidt et al. (2006) Iliev et al. (2006)

Iliev et al. (2006)

Iliev et al. (2006)

(continued)

Table 6.1 (continued) Mutants

Mutagen

Phenotype

Circadian rhythms analyzed

Gene affected

Gene product

Function

Reference

C3-ox

Overexpression

Phase advance

Phototaxis

C3

CELF family RNAbinding protein

Iliev et al. (2006)

roc15

Insertional mutagenesis

Short period

Bioluminescence (lucCP); growth

ROC15

GARP domain containing protein

roc40

Insertional mutagenesis

Long period in LL

Bioluminescence (lucCP); growth

ROC40

Single-MYB domain containing protein

roc55

Insertional mutagenesis Insertional mutagenesis

Short period

Bioluminescence (lucCP); growth Bioluminescence (lucCP); growth

ROC55

Insertional mutagenesis

Arrhythmic

Bioluminescence (lucCP); growth

ROC75

roc114

Insertional mutagenesis

Arrhythmic

Bioluminescence (lucCP); growth

ROC114

Putative transcription factor Putative transcription factor Putative subunit of E3 ubiquitin ligase

Matsuo et al. (2008)

roc75

Leucine-rich-repeat containing protein B-box zinc-finger and CCT domain containing protein GARP domain containing protein

Subunit of CHLAMY1 RNA-binding protein complex Putative transcription factor Putative transcription factor Unknown

roc66

Long period in DD

ROC66

F-box domain containing protein

Matsuo et al. (2008)

Matsuo et al. (2008)

Matsuo et al. (2008)

Matsuo et al. (2008)

Matsuo et al. (2008)

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CK1

333 a.a. PK 488 a.a.

C1 WW

KH

374 a.a.

C3 RRM

631 a.a.

ROC15 GARP

1556 a.a.

ROC40 Single MYB ROC55

1276 a.a. LRR

LRR

LRR

ROC66

2398 a.a. B-box

CCT

ROC75

1705 a.a. GARP

ROC114

1344 a.a. F-box

Figure 6.3 Schematic view of Chlamydomonas clock-related proteins. The putative functional domains are highlighted. Numbers of amino acid residues (a. a.) are indicated on the right.

including the suprachiasmatic nuclei which is the site of a central pacemaker of mammalian circadian rhythms (Klein et al., 1991; Zhao et al., 2004). 3.1.4. RHYTHM OF CHLOROPLAST The luciferase-based real-time bioluminescence reporter system is one of the strongest tools for the forward genetic screening of circadian rhythm mutants, because it enables a high-throughput assay of circadian rhythms. In cyanobacteria and higher plants, this system has been used to isolate circadian rhythm mutants (Kondo et al., 1994; Millar et al., 1995; Onai et al., 2004). We used the chloroplast bioluminescent reporter as described above (Section 2.6) for a forward genetic approach to clock genes in Chlamydomonas (Matsuo et al., 2008). To maximize the efficiency of rhythm assays, a new strain CBR34 exhibiting a more robust rhythmicity (i.e., high amplitude) in bioluminescence, was chosen from progenies of crosses between the original reporter strain and WT strains of different genetic backgrounds. Figure 6.4 shows the outline of the forward genetic analysis: (1) Insertional mutagenesis: The bioluminescent strain is transformed by a

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1) Insertional mutagenesis

2) Phenotypic screening

Hyg

105 utants rhythm mutants

3) Genetic linkage analysiss Mutant

WT

Bioluminescence

~16,000 transformants

Short period Long period Low amplitude Arrhythmic Time

4) Mapping of disrupted gene

WT 50 tagged mutants Hyg

Mutant rhythm Hyg-resistance

WT rhythm Hyg-sensitive

Mutant genome

Database searches 37 mapped mutants 30 responsible genes (or gene loci)

Figure 6.4 Illustrations of the forward genetic approach to the ROC genes. This analysis consists of insertional mutagenesis (1), phenotypic screening of rhythm mutants (2), genetic linkage analysis (3), and mapping of disrupted genes (4). Hyg represents the hygromycin-resistance marker gene. The red and green arrows indicate the specific primers and black arrows indicate the random primer used for the TAIL-PCR. See text for details.

selectable marker gene. The marker gene is integrated into random genomic loci and disrupts endogenous genes at the site of insertion. (2) Phenotypic screening: Rhythm mutants are screened by a high-throughput bioluminescence monitoring. (3) Genetic linkage analysis: The mutants are subjected to a genetic cross with the WT strain, and cosegregation of the mutant phenotype and the marker gene (antibiotic resistance) in the progeny is confirmed. Cosegregation strongly suggests that the gene responsible is disrupted by the insertion of the marker gene. (4) Mapping of disrupted genes: The flanking sequences of the inserted marker gene are identified by thermal asymmetric interlaced (TAIL)-PCR. The genes

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responsible are identified by database searches of the whole genome sequence ( JGI C. reinhardtii v3.0). The CBR34 strain was subjected to the serial analysis described above (Matsuo et al., 2008). By screening about 16,000 mutants with a random insertion of the hygromycin-resistance gene in the nuclear genome, we found 105 mutants that show defects in the chloroplast bioluminescence rhythm (Fig. 6.4-1 and 6.4-2). There were various types of mutants including those with short and long periods, advanced and delayed phase angles, and several types of low-amplitude rhythms, but the majority (78%) of the mutants were classified into the low-amplitude group. Cosegregation analysis revealed that the causative genes of the 50 mutants were ‘‘tagged’’ by the marker gene (Fig. 6.4-3). TAIL-PCR and database searches identified 30 causative genes (or gene loci) of these mutants (Fig. 6.4-4). These genes have been termed RHYTHM OF CHLOROPLAST (ROC), and could be classified by their predicted functions: flagella function, ubiquitin-proteasome, transcription and transcript metabolism, gene silencing, membrane trafficking and transport, signal transduction, DNA damage response, and apoptosis. Therefore, it has been revealed that many biological processes are associated with the maintenance of circadian rhythms of chloroplast bioluminescence in Chlamydomonas. A recent genome-wide RNAi screening for clock-related genes in mammalian cells also showed that knockdown of a large fraction of genes reduced the amplitude of circadian rhythms, and that the circadian clock is interconnected with many aspects of cellular functions (Zhang et al., 2009). Which ROC genes are the core components of the Chlamydomonas circadian clock? At least 6 mutants, roc15, roc40, roc55, roc66, roc75, and roc114, are thought to have defects in the mechanisms closely related to the core of the clock, since they show severe defects not only in the chloroplast bioluminescence rhythm but also in circadian rhythms of growth thought to reflect circadianly controlled cell cycle progression (Table 6.1) (Matsuo et al., 2008). Interestingly, roc40 and roc66 are conditional mutants in that their defects are obvious only under LL and DD conditions, respectively. They might be involved not only in the oscillator but also in the input pathways of the circadian clock. The genes responsible in the roc15, roc40, roc66, and roc75 mutants encode putative transcription factors. ROC15 and ROC75 have the GARP domain, a subclass of MYB-related DNA-binding domain (Fig. 6.3). ROC40 has a single-MYB domain (Fig. 6.3). ROC66 has a B-box zinc-finger DNA-binding domain in its N-terminal region and a CCT (CO, COL, and TOC1) domain in its C-terminal region (Fig. 6.3). On the other hand, ROC114 encodes a putative F-box protein that is thought to be a subunit of the Skp1-Cullin-F-box protein (SCF) E3 ubiquitin ligase, and ROC55 encodes a leucine-rich-repeat (LRR) containing protein (Fig. 6.3).

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3.2. Possible molecular mechanisms of the circadian oscillator in Chlamydomonas 3.2.1. Basic mechanisms of the circadian oscillator in other model organisms The core mechanisms of the circadian oscillator have been studied in animals (M. musculus and D. melanogaster), fungi (N. crassa), land plants (A. thaliana), and cyanobacteria (Synechococcus sp. strain PCC7942) (Dunlap, 1999; Harmer et al., 2001). The majority of clock genes are not conserved, however, some common features can be found in the eukaryotic circadian systems. DNA-binding proteins and their interacting proteins, kinases, phosphatases, and F-box proteins are commonly included in the eukaryotic systems. In the animal and fungal systems, the transcription of core clock genes (e.g., per, cry, tim, and frq) is activated by DNA-binding proteins (e.g., CLOCK/BMAL1 [CYCLE in Drosophila], WC-1/WC-2) (Fig. 6.5A). The mRNA and protein products of the core clock genes are subjected to posttranscriptional/posttranslational modifications by RNA-binding proteins, kinases, phosphatases, and ubiquitin ligases (Fig. 6.5A) (Gallego and Virshup, 2007; Garbarino-Pico and Green, 2007; Mizoguchi et al., 2006). The protein products, in turn, repress their own transcription by interacting with activators (Fig. 6.5A). This cycle of gene expression takes
24 h, and is thought to be important for generation of circadian oscillations. On the other hand, in higher plants, the present model of the circadian transcriptional circuit is slightly different from those of fungi and animals. In the currently accepted model, reciprocal genetic interactions between ‘‘morning genes’’ (LHY, CCA1) and ‘‘evening genes’’ (TOC1, LUX [PCL1]) are thought to form a negative feedback loop for the generation of circadian oscillations (Fig. 6.5B). But, also in this case, posttranslational regulators (e.g., CKII, ZTL) are involved in the oscillation (Fig. 6.5B). Our group proposed the existence of an autoregulatory negative feedback regulation of the evening gene PCL1 (Onai and Ishiura, 2005). 3.2.2. Evolution of plant circadian clocks The Chlorophytes (green algae including Chlamydomonas) diverged from the Streptophytes (land plants including Arabidopsis) over a billion years ago. These lineages are part of the green plant lineage (Viridiplantae), which diverged from opisthokonts (including mammals, Drosophila, and Neurospora). An endosymbiosis of an ancestral cyanobacterium is believed to be the origin of the chloroplast of the green plant lineage (Reyes-Prieto et al., 2007; Yoon et al., 2004). It is now apparent that transcription factors, RNA-binding proteins, kinases, and components of the ubiquitin ligase are involved in the clockwork of Chlamydomonas. Thus, the Chlamydomonas clock is thought to consist of similar mechanisms to other eukaryotic clock models. Particularly, the putative transcription factors involved in the

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A F-box prot proteins

DNA binding proteins

b-TrCP SLM SLMB FWD FBXL3

CLOCK CLOCK WC-1 BMAL1 CYCLE WC-2

Kinases CKId DBT CKIa CKIe CKII CKII CKII SGG CAMKII GSK3

Ub P

Core clock genes

Phosphatases

Per1 per frq Per2 tim Cry1 Cry2

PP1 PP2A PP1 PP2A PP2A PP5

RNA binding proteins LARK

Kinases

B

CKII

P

Morning genes Morni LHY CCA1

Evening genes TOC1 PCL1(LUX)

X Ub

F-box proteins F ZTL

Figure 6.5 Simplified illustrations of the genetic negative feedback models of eukaryotes. (A) A model of mouse, Drosophila, and Neurospora circadian oscillators. Representative clock-related genes/proteins in mice, Drosophila, and Neurospora are indicated in red, blue, and green, respectively. Ub and P represent ubiquitination and phosphorylation, respectively. (B) A model of the Arabidopsis circadian oscillator. Representative clock-related genes/proteins are indicated. It is likely that there are other factors (X) involved in activation of the morning genes.

Chlamydomonas clock have remarkable similarities in their DNA-binding domains to those of Arabidopsis clock proteins (Matsuo et al., 2008). The DNA-binding GARP motifs of ROC15 and ROC75 are similar to that of the Arabidopsis clock protein PCL1 (LUX) (Hazen et al., 2005; Onai and Ishiura, 2005). The DNA-binding domain of ROC40 (single MYB) is

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similar to that of LHY and CCA1 (Schaffer et al., 1998; Wang et al., 1997). The LHY homologs in Lemna plants also have a role in their circadian clocks (Miwa et al., 2006; Serikawa et al., 2008). In addition, it has recently been shown that LHY/CCA1 homologs have a similar role in the circadian oscillatory mechanisms of the moss Physcomitrella patens, distantly placed from Arabidopsis in the land plant phylogeny (Okada et al., 2009). The identification of ROC15/ROC75 and ROC40 as the clock genes in Chlamydomonas suggests that circadian clocks of green algae and land plants evolved from a common ancestral clock. On the other hand, no proteins showing strong similarity to ROC55 and ROC114 have been found in sequence databases. These would be original components of the Chlamydomonas clock. The present circadian clock of Chlamydomonas is thought to have diverged to some extent from that of the land plants. ROC66 has some similarity to the higher plant CO/COL proteins (Matsuo et al., 2008). All members of the family encode putative transcription factors having both the B-box zinc-finger DNA-binding domain at the N-terminal and the CCT domain involved in nuclear localization at the Cterminal (Griffiths et al., 2003; Robson et al., 2001). CO is a key regulator of the photoperiodic flowering pathway (Putterill et al., 1995). COL3, COL5, and COL9 also affect flowering to some extent (Cheng and Wang, 2005; Datta et al., 2006; Hassidim et al., 2009), but a large number of COLs do not. COL1 and COL2 have no significant influence on flowering time. However, interestingly, overexpression of COL1 shortened Arabidopsis circadian rhythms (Ledger et al., 2001). Recently, CrCO, another Chlamydomonas protein belonging to the CO/COL family, has been characterized (Serrano et al., 2009). Knockdown and overexpression of CrCO in Chlamydomonas induced various defects in growth, the diurnal rhythmicity of starch content, and the synchrony of cell cycle-related gene expression. Moreover, it is noteworthy that CrCO can complement the Arabidopsis co mutation and affect in a similar manner the timing of flowering and expression of the FLOWERING LOCUS T (FT) gene when expressed under different promoters in Arabidopsis. When these findings are taken together with the involvement of ROC66 in the circadian clock, coordination of the photoperiodic pathway and the circadian clock by CO and some COL proteins may be an evolutionarily conserved feature in the plant lineage. 3.2.3. Transcription factors As described in the previous section, the putative transcription factors involved in the Chlamydomonas clock have some similarities to the Arabidopsis clock proteins. Are the core transcriptional loops common to these organisms? CCA1/LHY and PCL1 (LUX) exhibit distinctive antiphase expression profiles in Arabidopsis. CCA1 and LHY are morning genes whose mRNAs accumulate in the morning in contrast to PCL1 (LUX), an evening gene whose mRNA accumulates in the evening (Fig. 6.6A)

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Circadian Clock in Chlamydomonas

A

mRNA level

Subjective night Subjective day Subjective night

Arabidopsis CCA1 LHY PCL1(LUX) TOC1

Time B

mRNA level

Subjective night Subjective day Subjective night

Chlamydomonas

ROC40 ROC15 ROC75 ROC66 Time

Figure 6.6 Schematic view of expression patterns of clock genes in Arabidopsis (A) and Chlamydomonas (B). LHY/CCA1 and ROC40 are indicated in yellow. PCL1 (LUX) and ROC15/ROC75 are indicated in blue.

(Hazen et al., 2005; Onai and Ishiura, 2005; Schaffer et al., 1998; Wang and Tobin, 1998). These expression profiles are conserved in the CCA1 and PCL1 (LUX) homologs in Oryza sativa (Murakami et al., 2007), in the LHY homologs in Lemna (Miwa et al., 2006), and in the CCA1 homologs in P. patens (Okada et al., 2009). Since ROC40 mRNA accumulates in the midsubjective night to subjective dawn (Fig. 6.6B) (Matsuo et al., 2008), the morning expression profile seems to be conserved among the clock-related single-MYB genes in Chlamydomonas and land plants. On the other hand, ROC15 shows an expression pattern similar to that of ROC40 in spite of its similarity in amino acid sequence to the GARP domain of PCL1 (LUX) (Fig. 6.6B) (Matsuo et al., 2008). Thus, it is difficult to draw the same scheme as that of Arabidopsis. On the other hand, the mRNA accumulation of ROC75, another PCL1 (LUX)-like GARP domain-encoding gene, peaks in the early to mid-subjective day, 4–8 h after ROC15/ROC40 expression (Fig. 6.6B) (Matsuo et al., 2008). Thus, it is possible that reciprocal regulation occurs between ROC75 and ROC15/ROC40. Further investigation of the genetic interactions among these ROC genes will provide new insights into the core transcriptional loops of the Chlamydomonas clock. Some other ROC genes encoding putative transcription factors, ROC56, ROC59, and ROC76, may also be involved in the

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transcriptional loops. Since phenotypes of their corresponding mutants are not so severe (Matsuo et al., 2008), they are good candidates that might form interlocked loops in the circadian oscillatory mechanisms (Brunner and Merrow, 2008). 3.2.4. RNA-binding proteins A remarkable feature of the Chlamydomonas clock is that an RNA-binding protein strongly affects core oscillations. Knockdown and overexpression of the C1 subunit of the RNA-binding protein CHLAMY1 abolishes circadian rhythms (Iliev et al., 2006). Also in other model organisms, RNAbinding proteins contribute to the circadian system. CCTR in Lingulodinium polyedrum, AtGRP7 in Arabidopsis, NOCTURNIN in Xenopus and mice, hnRNPs in rats, and LARK in Drosophila and mice are known to be posttranscriptional circadian regulators involved in circadian regulation of splicing, mRNA decay, and translation (Garbarino-Pico and Green, 2007). Although most of them have roles in specific output processes of the circadian clock, mouse LARK (mLARK) is known to influence the core clock mechanism (Fig. 6.5A) (Kojima et al., 2007). mLARK binds to the 30 UTR of mRNA of the core clock gene Per1 and positively regulates Per1 expression. Knockdown and overexpression of mLARK shortens and lengthens circadian oscillation in mammalian cells, respectively. Interestingly, ROC40 mRNA bears the CHLAMY1-binding sequence (UGrepeat) in its 30 -UTR region (Matsuo et al., 2008). Participation of CHLAMY1 in the circadian clockwork may occur through the UG-repeat of ROC40 as well as LARK and Per1 in mammals. The involvement of XRN1 (50 –30 -exoribonuclease gene), the causative gene of the roc86 mutant, also highlights the importance of the circadian regulation of RNA in the Chlamydomonas circadian system (Matsuo et al., 2008). 3.2.5. Kinases, phosphatases, and ubiquitin ligases Posttranslational regulation of clock proteins has important roles in circadian oscillatory mechanisms (Gallego and Virshup, 2007; Mizoguchi et al., 2006). Kinases and phosphatases regulate the phosphorylation status of core clock proteins, and the phosphorylation of clock proteins triggers binding to ubiquitin ligases, which mediates the polyubiquitination and the subsequent degradation of the proteins by the proteasome (Fig. 6.5). For example, in the mammalian system, casein kinase I phosphorylates core clock proteins PER1 and PER2. Phosphorylation of the PERs triggers binding of the F-box protein bTrCP, a substrate recognition subunit of E3 ubiquitin ligase, and leads to polyubiquitination and degradation by the proteasome (Fig. 6.5A) (Gallego and Virshup, 2007). In Chlamydomonas, some kinases (CK1, MUT-9 [ROC94], ATR1 [ROC69], MAPKKKK [ROC78]) and components of ubiquitin ligase (ROC114, SKP1 [ROC80]) are revealed to be involved in the circadian system (Matsuo et al., 2008; Schmidt et al., 2006). Identification

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of their target proteins will be important for understanding the Chlamydomonas clock. ROC proteins and CHLAMY1 are prospects. Biochemical analysis of these proteins in the mutants of the kinases and the ubiquitin ligase components should shed light on the posttranslational regulation of the Chlamydomonas clock. On the other hand, protein phosphatases involved in circadian oscillatory mechanisms have not yet been identified in Chlamydomonas. 3.2.6. Genetic oscillator or biochemical oscillator? Although genetic feedback loops must be an important part of the core of the circadian oscillator, circadian oscillation that is independent of the genetic feedback regulations has been found in the bacterial circadian clock system. Cyanobacterial circadian clock proteins KaiA, KaiB, and KaiC interact with each other and generated circadian rhythmicity in KaiC autophosphorylation levels in an in vitro reconstitution experiment (Nakajima et al., 2005), indicating that the Kai protein complex itself, without genetic feedback regulation, has clock oscillatory functions. One interesting question is whether the new concept found in bacteria is applicable to eukaryotes. Suggestive evidence for circadian oscillation without genetic regulation has been obtained in the green alga Acetabularia. This macroalga shows photosynthetic rhythms even in cells whose nuclei are surgically removed (Sweeney and Haxo, 1961). Since Chlamydomonas is simple and evolutionarily closer to Acetabularia than other model organisms for the circadian clock, the former might be a good model to test the reconstitution of the eukaryotic circadian clock in vitro. 3.2.7. Other factors Surprisingly, a lot of genes associated with flagella functions are included among the ROC genes (Matsuo et al., 2008). How do the flagella participate in the circadian system? Most mutants of these genes show low-amplitude phenotypes in bioluminescence rhythms. Because defects in flagella might have an impact on the physiological status of the cell, it is possible that the reduced metabolism affects the circadian rhythmicity of the clock or chloroplast bioluminescence itself (Brunner and Merrow, 2008). Many genes related to membrane (vesicle) trafficking are included among the ROC genes (Matsuo et al., 2008). Although some transcriptome studies in mammals mentioned mRNA cycling of several vesicle trafficking-related genes (Akhtar et al., 2002; Panda et al., 2002), direct functional evidence for the involvement of these processes in the circadian clock has not yet been obtained. How are these genes involved in the circadian system in Chlamydomonas? In the case of ROC81, a specific output process for the chloroplast bioluminescence rhythm would be affected. The roc81 mutant showed a normal circadian rhythm in growth rate, even though it showed arrhythmicity in chloroplast bioluminescence (Matsuo et al., 2008). Therefore, roc81 mutation affects specific output processes for the chloroplast bioluminescence rhythm. Recently, a mechanism of protein transport to the chloroplast through

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secretory vesicles, which is independent of the canonical Tic/Toc (translocation at the outer/inner envelope membrane of chloroplast) machinery, has been demonstrated (Nanjo et al., 2006; Villarejo et al., 2005). Interestingly, ROC81 encodes a protein with similarity to the N-terminal region of VTC4, a member of the vacuolar transporter chaperone complex involved in several aspects of membrane transport and vesicular trafficking (Cohen et al., 1999; Muller et al., 2002, 2003; Murray and Johnson, 2000, 2001; Nelson et al., 2000; Uttenweiler et al., 2007). The vesicular trafficking might have a role in the circadian output pathway regulating the chloroplast bioluminescence rhythm.

4. Input Pathways to the Circadian Oscillator in Chlamydomonas 4.1. Light information It is important for life on earth to keep precise phase relationships between the internal circadian clock and the external daily cycle. The input pathway adjusts (resets) the phase of the circadian clock to a precise phase angle (Fig. 6.1). One of the most important cues that resets the clock is light. Action spectra for resetting the phototaxis rhythm have been examined extensively (Johnson et al., 1991; Kondo et al., 1991). The action spectrum differed depending on the culture conditions: (i) Cells kept in darkness showed an action spectrum having two prominent peaks at 520 and 660 nm (green and red, respectively), whereas cells adapted to dim light conditions showed peaks at 450–480 and 660 nm (blue and red, respectively). (ii) The effective dose of light was distinctly different between the cells adapted to darkness and illumination, and it needed a 2000 times higher dose to gain the same effect on the illuminated cells than on the cells in darkness. (iii) The effects of the light stimuli were blocked by specific inhibitors of photosynthetic electron transport (DCMU and atrazine) only in the illuminated cells. Therefore, it is postulated that there are at least two light input pathways (DCMU-sensitive and insensitive) in the Chlamydomonas circadian clock. The DCMU-sensitive pathway would include the photosynthetic apparatus and might be an indirect pathway mediated by cellular metabolic changes depending on photosynthesis. No photoreceptors corresponding to clock resetting have been identified to date. The C. reinhardtii genome sequence has some photoreceptor genes encoding cryptochromes, phototoropins, and Chlamydomonas opsins (Chlamyopsins and Channelrhodopsins) (Grossman et al., 2004; Hegemann, 2008; Mittag et al., 2005). They are candidates for circadian blue and green light receptors. On the other hand, there are no obvious candidates for the red-light receptor. Although there are some genes encoding phytochrome-like proteins, their similarity to the Arabidopsis PHYs is very weak (Grossman et al., 2004;

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Mittag et al., 2005). In addition, the resetting by red light was not diminished by subsequent administration of far-red light ( Johnson et al., 1991; Kondo et al., 1991). These results suggest that the PHYs are not likely to be involved in phase resetting by red light. Even if the phy-like proteins are involved, they would be atypical PHYs that have diverged extensively between Chlamydomonas and Arabidopsis.

4.2. Temperature information Integration of temperature information is an important process for the circadian clock. Temperature, as well as light, is a major cue to reset the phase of the circadian clock, but, in contrast, the period length of the circadian oscillation is relatively constant at different ambient temperatures. A recent study by Mittag and colleagues shed light on the molecular mechanisms of the temperature sensing of the circadian clock in Chlamydomonas (Voytsekh et al., 2008). They analyzed the subunits of the RNA-binding protein CHLAMY at low (18  C) and high (28  C) temperatures, and found that the C1 subunit is hyperphosphorylated, and accumulation of the C3 subunit is upregulated at low temperature. The upregulation of c3 occurs at the transcriptional level through regulatory cis-elements (E-box and DREB1A-box) in the c3 upstream region. Interestingly, in Bruce’s per-1 mutant, the hyperphosphorylation of C1 subunits at low temperature was abolished and the C3 protein level was elevated at all the temperatures examined. These results indicate that a temperaturecontrolled network of C1, C3, and PER1 exists in the Chlamydomonas circadian system (Voytsekh et al., 2008).

5. Output Pathways from the Circadian Oscillator in Chlamydomonas 5.1. Transcriptional regulation of nuclear gene expression The timing information generated by the circadian oscillator is converted into peripheral behavior and physiology via the output pathways (Fig. 6.1). One important pathway is the regulation of gene expression. There are many reports showing circadian rhythms in gene expression at the mRNA level (Carter et al., 2004; Fujiwara et al., 1996; Hwang and Herrin, 1994; Jacobshagen and Johnson, 1994; Jacobshagen et al., 1996, 2001; Lemaire et al., 1999; Memon et al., 1995; Savard et al., 1996). A genome-wide analysis of the transcriptome in Chlamydomonas revealed that
2.6% of genes in the nuclear genome is under the control of the circadian clock (Kucho et al., 2005). The genes are involved in various biological processes including photosynthesis, respiration, biosynthesis, metabolism, protein export, and degradation. The accumulation of mRNA is determined by the transcription rate and mRNA

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stability. Some genes are known to be regulated by the circadian clock at the transcriptional level. The mRNA level of a light-harvesting chlorophyll binding protein LHCA1 shows a robust circadian rhythm peaking in the subjective day (Hwang and Herrin, 1994). The transcriptional rate of LHCA1 shows a robust circadian rhythm, whereas the circadian rhythm of LHCA1 mRNA stability is relatively weak, indicating that transcription is a major point of regulation by the circadian clock for this gene (Hwang and Herrin, 1994). The other gene encoding a light-harvesting chlorophyll binding protein LHCB1 also shows a robust circadian rhythm in its transcription rate, and chimeric genes that have the LHCB1 promoter region and a reporter gene NIT1 or ARS2 show circadian rhythms in the accumulation of the reporter mRNAs, suggesting circadian transcriptional regulation of the LHCB1 promoter (Jacobshagen and Johnson, 1994; Jacobshagen et al., 1996). The mRNA level of the C3 subunit of CHLAMY1 is also circadianly regulated (Kucho et al., 2005). Although the circadian rhythm of c3 transcriptional activity has not been investigated, the bioluminescence derived from the cRluc reporter fused to the c3 promoter shows circadian rhythms (Voytsekh et al., 2008). Since the bioluminescence rhythm is in phase with the circadian rhythm of c3 mRNA accumulation (Kucho et al., 2005), it seems that c3 mRNA is mainly regulated at the transcriptional level. Interestingly, the bioluminescence rhythm of the c3 reporter is abolished by a mutation of the E-box element in the promoter (Voytsekh et al., 2008). The E-box is a core cis-element for the circadian regulation of transcription in the circadian systems of Drosophila and mammals (Dunlap, 1999; Harmer et al., 2001). Basic helix-loop-helix (bHLH) transcription factors (CLOCK/BMAL1 [CYCLE in Drosophila]) bind to not only the E-box elements of the core clock genes but also those of clock controlled output genes, and activate the transcription of these genes (Dunlap, 1999; Harmer et al., 2001). E-box-mediated circadian regulation might be common to Chlamydomonas and animals. However, the transcription factor that binds to the E-box element of c3 has not yet been identified. No homologs of CLOCK and BMAL1 (CYCLE) were found in the C. reinhardtii genome (Mittag et al., 2005), and no bHLH transcription factor genes were included among the ROC genes (Matsuo et al., 2008).

5.2. Posttranscriptional regulation of nuclear gene expression The posttranscriptional regulation contributes to the circadian gene expression. A recent systematic analysis of the mammalian hepatic proteome revealed that
20% of soluble proteins show circadian expression and almost half of these proteins lack a corresponding cycling transcript (Reddy et al., 2006). A canonical example of posttranscriptional regulation is the luciferin binding protein (LBP) of L. polyedrum (Mittag, 2001). Its translational activity and protein level shows robust circadian rhythmicity,

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whereas its mRNA level does not fluctuate throughout the circadian cycle (Morse et al., 1989). The circadian controlled translational regulator (CCTR) binds to the UG-repeat sequence in the 30 -UTR of the lbp mRNA and the binding activity is negatively correlated to the LBP protein level, thus CCTR is thought to repress LBP translation in a circadian manner (Mittag et al., 1994). CHLAMY1 was originally identified as an analog of CCTR in Chlamydomonas (Mittag, 1996). The RNA-binding activity of CHLAMY1 shows a circadian rhythm peaking in the early subjective night phase (Mittag, 1996). CHLAMY1 binds to the UGrepeat-bearing 30 -UTR of mRNAs, especially mRNAs related to nitrogen and carbon metabolism (Waltenberger et al., 2001). nii1 mRNA encoding a key enzyme of nitrogen metabolism (NII) is one of the targets of CHLAMY1. The activity of NII peaks in the early day or subjective day phase under LD or LL conditions, respectively (Fig. 6.2B) (Iliev et al., 2006; Pajuelo et al., 1995). Since NII activity’s rhythm is antiphase to the binding activity of CHLAMY1 (Mittag, 1996), CHLAMY1 might act as a repressor of nii1 gene expression at the posttranscriptional level (Waltenberger et al., 2001). Posttranscriptional regulation of UG-repeat-bearing mRNAs was also suggested in experiments using a luciferase reporter gene (Kiaulehn et al., 2007). A cRluc reporter gene having HSP70A/RBCS2 fusion promoter and RBCS2 30 -UTR did not show circadian rhythm in bioluminescence. However, when the RBCS2 30 -UTR was replaced with the UGrepeat-bearing 30 -UTR of arg7, nii1, and gs2, these reporters showed circadian rhythms in bioluminescence. In addition, the phases of the rhythms were different. The peak phase of bioluminescence of nii1 and arg7 reporters was the early subjective day, whereas that of the gs2 reporter shifted to the mid-subjective night. Thus, the UG-repeat sequence in the 30 -UTR of mRNA mediates circadian expression and can determine circadian phase (Kiaulehn et al., 2007).

5.3. Transcriptional regulation of chloroplast gene expression Chlamydomonas has distinct advantages in the study of chloroplasts (Rochaix, 1995). Chlamydomonas cells contain a single chloroplast that can be easily transformed, and the cells are viable without photosynthesis because they can grow heterotrophically when supplemented with acetate (Harris, 2001). Chlamydomonas is the first organism in which the circadian regulation of chloroplast gene expression has been described. Endogenous fluctuations in the levels of chloroplast tufA, atpA, and atpB mRNAs were observed on the first day under constant conditions (Leu et al., 1990, Salvador et al., 1993). Hwang and coworkers demonstrated conclusively that tufA mRNA levels oscillated robustly for 2–3 days under constant conditions (Hwang et al., 1996). Since the transcription rate but not mRNA half-life showed circadian rhythmicity, it is obvious that tufA

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mRNA expression is mainly regulated by the circadian clock at the transcriptional level. They also showed that the transcription rates of other chloroplast genes including atpB, psbA, psaA, and rrn (ribosomal RNA) are regulated by the circadian clock. The gene expression system of chloroplasts is different from that of the eukaryotic nucleus, but rather similar compared to that of prokaryotes (Stern et al., 1997). How does the circadian clock exert its control to the chloroplast? Since per-1, per-4, and roc mutants having a mutation in the nuclear genome showed mutated circadian rhythmicity in bioluminescence of the chloroplast luciferase reporter (Matsuo et al., 2006, 2008), the chloroplast circadian rhythmicity is likely to be under the control of the nucleus. Kawazoe and coworkers showed that the circadian peaking of tufA transcription was blocked by cycloheximide treatment administered immediately before the circadian peak. Thus, a nucleus-encoded protein(s) having a relatively short-life is thought to be needed to evoke the circadian rhythm of tufA transcription (Kawazoe et al., 2000). A sigma factor would be such a protein conveying timing information to the chloroplast. The nuclear genome of C. reinhardtii carries a single copy gene for sigma-like factor termed RPOD that has a chloroplast transit peptide in its N-terminal (Bohne et al., 2006; Carter et al., 2004). RPOD protein is actually transported to the chloroplast in vivo, and initiated transcription of chloroplast genes in an in vitro reconstitution experiment using Escherichia coli RNA polymerase core enzymes (Bohne et al., 2006). Interestingly, the RPOD mRNA level is regulated by the circadian clock. The mRNA level shows a peak in mid- to late subjective night just prior to the peak of transcription of chloroplast genes, suggesting a contribution of the sigma factor to the chloroplast transcriptional circadian rhythmicity (Carter et al., 2004). The circadian control of sigma factor genes is also known in higher plants (Morikawa et al., 1999) and moss (Ichikawa et al., 2004). Another possible mechanism for the circadian regulation of chloroplast transcription is the modulation of topology of the chloroplast genomic DNA. Salvador et al. showed that the supercoiling status of chloroplast DNA in Chlamydomonas is regulated by the circadian clock. The chloroplast DNA was supercoiled during the subjective day and relaxed during the subjective night, and the peak of the superhelicities was consistent with that of transcription (Salvador et al., 1998). DNA gyrase, the type II topoisomerase catalyzing ATPdependent DNA supercoiling, is one of the candidates for the regulator of DNA superhelicity (Champoux, 2001). Chlamydomonas contains an ATPdependent topoisomerase activity that can supercoil DNA in vitro, and gyrase-specific inhibitors affect the transcription of chloroplast genes (Thompson and Mosig, 1985, 1987). In higher plants, homologs of E. coli GyrA and GyrB that are subunits of DNA gyrase have been identified. They are nucleus-encoded genes and their products are targeted to chloroplasts and mitochondria (Cho et al., 2004; Wall et al., 2004). The nuclear genome

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of C. reinhardtii contains genes that encode proteins homologous to the higher plant GyrA and GyrB (Protein ID [JGI C. reinhardtii v4.0]: 115934 [GyrA-like] and 114600 [GyrB-like]). These proteins may be involved in the circadian regulation for the supercoiling of chloroplast DNA.

5.4. Posttranscriptional regulation of chloroplast gene expression Gene expression in the chloroplast highly depends on the posttranscriptional steps (Eberhard et al., 2002; Klinkert et al., 2005). Herrin and colleagues examined daily the circadian rhythms in the translational activities of chloroplast genes by performing pulse-chase labeling experiments. Under LD conditions, the translation rate of major chloroplast proteins fluctuated robustly (10–20-fold) peaking during the subjective day (Herrin et al., 1986; Michaels and Herrin, 1990). Since such robust diurnal rhythms were observed even in the proteins encoded by relatively long-lived mRNAs (e.g., rbcL, psbA, and psbD) with constant levels throughout the daily cycle, the translational rhythmicities were not due to the amounts of mRNA (Herrin et al., 1986). On the other hand, the translational rhythmicities were relatively weak (
6. Concluding Remarks The identification of clock genes will make Chlamydomonas a new model for understanding the circadian clock. The next step is the identification of genetic and biochemical interactions of clock genes and proteins. Particularly, Chlamydomonas would have advantages in proteome analyses, because it grows easily and quickly in a large-scale culture (Harris, 2001; Wagner et al., 2004). Further molecular dissection of the Chlamydomonas clock will provide new insights into the evolution of the plant circadian clock and may highlight the core mechanisms conserved during the plant clock’s evolution.

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Although Chlamydomonas is a simple unicellular organism, various circadian rhythms can be monitored in it (Fig. 6.2B). The output pathways for these rhythms would crosstalk with each other and/or feedback to the circadian oscillator or the input pathway. One of our goals is the comprehensive understanding of such a complex circadian system in the cell. Particularly, Chlamydomonas will be ideally suited to analyze the output pathway from the circadian clock to chloroplast. Bioluminescent reporters for chloroplast and nucleus (Section 2.6), or dual reporters open a new avenue to study the nucleus–chloroplast communication via the circadian clock.

ACKNOWLEDGMENTS The authors thank all members of Ishiura’s laboratory for discussions, M. Mittag (Friedrich Schiller University Jena) for comments on the manuscript, and D. Mrozek (Medical English Service) for professional editing. Research in the authors’ laboratory is supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to M.I. and T.M. The Division of Biological Science, Graduate School of Science, Nagoya University, was supported by the Global COE grant from MEXT.

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Index

A Abacavir, 255 Actin ciliates, 102–105 general considerations, 101–102 Adenosine triphosphate (ATP) ATP-dependent proteases, 188 Clp protease, 190–194 FtsH protease, 194–197 Lon protease, 197–199 ATP-independent proteases, 188 Deg protease, 199–201 EGY1, 205 presequence protease, 201–202 Adrenal cortex atrial natriuretic peptides aldosterone inhibition, 18 binding sites, 10 cortisol secretion, 19 glucocorticoid secretion, 18 immunoreactivity, 10 phosphorylation inhibition, 18 water deprivation, 11 zona glomerulosa., 10–11, 18–19 brain natriuretic peptides aldosterone inhibition, 18–19 binding sites, 11 cGMP synthesis, 18–19 cortisol inhibition, 19 C-type natriuretic peptides, 10 natriuretic peptide receptors, 11 Adrenal medulla atrial natriuretic peptides binding sites, 12 catecholamine inhibition, 20 immunoreactivity, 11 brain natriuretic peptides binding sites, 12 tyrosine hydroxylase activity, 20 C-type natriuretic peptides binding sites, 12 catecholamine inhibition, 21 natriuretic peptide receptors, 12 Adrenocortical adenoma and carcinoma, 21–22 Amprenavir, 252 Antibiotic resistance, 294 Arabidopsis thaliana ClpP protease, 191–192

CND41, 206 CtpA, 204 Deg protease, 199 EGY1 proteases, 205 FtsH protease, 195 genome sequence, 207 GEP, 207 Lon protease, 198 Plsp1, 203 PreP, 202 rhomboid proteases, 204 SPP, 201 SppA gene, 206 Artificial bioluminescence, 288–289 Atazanavir, 252–253 ATP-binding cassette (ABC) transporters, HIV therapy ABCB4 and ABCB11 protein, 230 ABCE1 protein, 230 ABC-transporter polymorphisms and HIV-1, 262–264 antiretrovirals pharmacokinetic parameters, 234 substrates, inhibitors, and inductors, 237–248 breast cancer resistance protein (BCRP), 228 cell lines incubation, 231 LS180 cells, 232–233 overexpression, 230–231 PBMC cells, 232 primary human hepatocytes, 233 selection, inducing effects, 231–232 transfection/transduction, 230–231 tumor/primary cell lines, 231 in vitro studies, 233–236 cellular resistance anti-HIV-1 drugs, 259–260 modulation, drug combinations, 260 therapeutics, 260–262 drug-drug interactions, 257–258 elvitegravir, 256 enfuvirtide, 256 fusion and entry inhibitors, 256 integrase inhibitors, 256 localization, 224–225 maraviroc, 256 multidrug resistance-associated proteins (MRPs), 229–230

315

316

Index

ATP-binding cassette (ABC) transporters, HIV therapy (cont.) NNRTIs delavirdine, 254 efavirenz, 253 etravirine, 254 nevirapine, 253–254 NRTIs abacavir, 255 didanosine, 254–255 emtricitabine, 256 lamivudine, 255 stavudine, 255 tenofovir, 255–256 zalcitabine, 255 zidovudine, 254 P-glycoprotein (Pgp) CXCR4, 227 expression site, 225–227 HIV-1 interaction, 227–228 pharmacokinetics, 264 PIs amprenavir, 252 atazanavir, 252–253 darunavir, 253 fosamprenavir, 252 indinavir, 251 lopinavir, 252 nelfinavir, 251 ritonavir, 250 saquinavir, 236, 249–250 tipranavir, 253 raltegravir, 256 ATP-independent serine-type endopeptidase (SppA), 206 Atrial natriuretic peptides (ANP) adrenal gland (see Adrenal cortex; Adrenal medulla) hypothalamus (see Hypothalamus) pituitary gland (see Pituitary gland) Aurelia aurita ocelli types, 56 rhopalia, 54–55 Autophagy, 141–142 B Bacterial proteases, 188, 190, 207 Bioluminescence, 288, 293–295, 301–302, 304–306 Bioluminescent reporters, 288, 293, 308 Box jellyfish. See also Cubomedusae Carybdea sivickisi, 43 Chiropsella bronzie cubozoan lens eyes, 60–61 cubozoan ocelli, 62, 64 ERGs, 70 obstacle avoidance response, 48 photoreceptor type, 64

Tripedalia cystophora (see Tripedalia cystophora) Brain natriuretic peptides (BNP) adrenal gland (see Adrenal cortex; Adrenal medulla) hypothalamus (see Hypothalamus) pituitary gland (see Pituitary gland) Breast cancer resistance protein (BCRP), 228 C Casein kinase, 290 Cellular resistance, ABC transporter anti-HIV-1 drugs, 259–260 modulation, drug combinations, 260 therapeutics, 260–262 Chemotaxis, 285–286 Chiropsella bronzie cubozoan lens eyes, 60–61 cubozoan ocelli, 62, 64 ERGs, 70 obstacle avoidance response, 48 photoreceptor type, 64 CHLAMY1, 290–293, 300–301, 304–305 Chlamydomonas reinhardtii, 193. See also Circadian clock, Chlamydomonas cell division cycle, 286 chloroplast gene expression posttranscriptional regulation, 307 transcriptional regulation, 305–307 nuclear gene expression posttranscriptional regulation, 304–305 transcriptional regulation, 303–304 UV sensitivity, 286 Chlorophyll a oxygenase (CAO), 194 Chlorosis, 193, 201 Ciliated protozoa, vesicle trafficking pathway. See Vesicle trafficking pathway, ciliates Circadian clock, Chlamydomonas behavioral and physiological circadian rhythms, 283–285 artificial bioluminescence, 288 cell division cycle, 286 chemotaxis, 285–286 phototaxis, 285 starch content, 287 stickiness to glass, 287 UV sensitivity, 286 clock components casein kinase, 290 CHLAMY1, 290–293 per mutants, 288–290 rhythm of chloroplast (ROC), 293–295 clock-related proteins, 293 conceptual model, 282–283 identification, 307 input pathways light information, 302–303 temperature information, 303 molecular mechanisms

317

Index

eukaryotes, 296–297 genetic/biochemical oscillator, 301 kinases, phosphatases, and ubiquitin ligases, 300–301 plant circadian clock evolution, 296–298 RNA-binding proteins, 300 ROC genes, 301–302 transcription factors, 298–300 mutants and responsible gene, 291–292 nucleus–chloroplast communication, 308 output pathways posttranscriptional regulation, 304–305, 307 transcriptional regulation, 303–307 Clock genes, 283–284, 304, 307 definition, 282 expression pattern, 299 forward genetic approach, 293 per mutant, 289 ROC, 298 transcription, 296 Clp protease features, 191–193 function and protein substrate, 193–194 structure, 190–191 CND41, 206–207 Cnidarians cubomedusae (see Cubomedusae) hydromedusae (see Hydromedusae) multiple opsins ancestral opsin, 70–71 ciliary opsin, 70–71 gene duplications, 72 multifunctionality, 71–72 nonvisual opsins, 71 opsin genes, 70 opsin–transducin–cGMP phototransduction pathway, 71 multiple photosystem (see Multiple photosystem, cnidarians) photosensitive structures extraocular photoreception, 49–50 eyes, 51 ocelli, 50–51 photosensitivity extraocular photoreception, 49–50 photoreception, behavioral evidence, 46–49 phylogenetic relationships, 43 scyphomedusae (see Scyphomedusae) cRluc reporter gene, 288–289, 304–305 Cryptochromes, 44, 71 C-terminal processing peptidase (CtpA), 203–204 C-type natriuretic peptides (CNP) adrenal gland (see Adrenal cortex; Adrenal medulla) hypothalamus (see Hypothalamus) pituitary gland (see Pituitary gland) Cubomedusae. See also Box jellyfish lens eyes

behavioral control system, 62 C. alata, largest lower lens eye, 61 image quality, 60–61 pacemaker system, 62 spectral sensitivity, 61 temporal sensitivity, 61 unpaired lens eyes, 60 neuronal organization, 67–70 ocelli types photoreceptors, 62–63 screening pigment, 63 slit ocelli , T. cystophora, 63–64 visual information, 63 photoreceptor cells box jellyfish, 65 ciliary type, 64 lower lens eye, 64–65, 73 opsin-based phototransduction, 65–66 upper lens eye, 65, 73 rhopalium eyes and ocelli, 57–58 monociliated epithelium, 58–59 pacemaker activity, visual control, 59 pedalia and stalk, 57 vs. scyphomedusae, 56 statocyst, 57 D Darunavir, 253 Deg protease features, 199–200 function and protein substrates, 200–201 structure, 199 Delavirdine, 254 Didanosine, 254–255 E Efavirenz, 253 Elvitegravir, 256 Emtricitabine, 256 Enfuvirtide, 256 Ethylene-dependent gravitropism-deficient isolated froma genetic screening yellowgreen 1 (EGY1), 205 Etravirine, 254 Exocytosis and endocytosis ciliates, exo-/endocytosis, 125 constitutive endocytosis, 125 constitutive exo-endocytosis, ciliates early and late endosomes, 127–128 parasomal sacs, 125–127 exocyst complex, 124 stimulated exocytosis, 124 stimulated exocytosis-coupled endocytosis, ciliates dynamics, Paramecium, 129–131 exocytosis site assembly, 128

318

Index

Exocytosis and endocytosis (cont.) unstimulated exocytosis, 124–125 F Food vacuoles, 137 Fosamprenavir, 252 FtsH protease D1 quality control system, PSII repair cycle, 198 features, 195–196 function and protein substrate, 196–197 structure, 194–195

G Gene mapping, 294–295 Genetic linkage analysis, 294 Glutamyl endopeptidase (GEP), 207 Granule-bound starch synthase I (GBSSI), 287 H Human immunodeficiency-1 virus (HIV-1). See also ATP-binding cassette (ABC) transporters, HIV therapy antiretroviral drugs, 220–221 drug classes and site of action entry and fusion inhibitors, 222–223 HIV-1 protease inhibitors, 224 integrase inhibitors, 223–224 nonnucleoside reverse transcriptase inhibitors, 223 nucleoside and nucleotide reverse transcriptase inhibitors, 223 HIV-1 life cycle, 221–222 Hydractinia echinata, extraocular photosensitivity, 47 Hydromedusae. See also Sarsia tubulosa nonsensory pigment cells, 53 ocelli electroretinograms (ERGs), 53 outer segments, 53 Sarsia tubulosa, 51–52 sensitivity, 52–53 shape, 52 size, 51–52 Hypophysis, 8–10, 23 Hypothalamic–pituitary–adrenal (HPA) axis, natriuretic peptides. See Natriuretic peptides, HPA axis Hypothalamus atrial natriuretic peptides binding sites, 7 central cardiovascular regulation, 12–13 corticotrophin-releasing factor, 14 immunoreactivity, 4, 6 LHRH release, 15 neuronal norepinephrine uptake, 13

oxytocin (OT) release, 13 prolactin release, 14 vasopressin inhibitor, 13–14 brain natriuretic peptides ACTH response, 15 dopamine, 14 immunoreactivity, 6–7 neuronal norepinephrine uptake, 13 C-type natriuretic peptides ACTH response, 15 immunoreactivity, 7 LHRH release, 15 neuronal norepinephrine uptake, 13 prolactin release, 14 natriuretic peptide receptor, 8 I Immobilization antigens, 126 Indinavir, 251 Insertional mutagenesis, 293–294 Integrase inhibitors, 256 Intramembrane proteases AraSP, 205 EGY1, 205 rhomboid, 204–205 J Jellyfish, multiple photosystem. See Cnidarians L Lamivudine, 255 Laser-capture microdissection, 23–24 Leaf variegation, 195–196 Light/dark (LD) cycles, 285–286, 288, 307 Light-dependent and independent reaction, 187 Lon protease function, 198–199 structure, 197–198 Lopinavir, 252 lucCP reporter gene, 288–289 Luteinizing hormone-releasing hormone (LHRH) release, 15 M Maraviroc, 256 Membrane trafficking, protozoa. See Vesicle trafficking pathway, ciliates Microtubules, vesicle trafficking pathway cytokinesis, 150 cytopharynx, 148 disrupting drugs, 150 dynein, 148–149 kinesin, 148 metazoans, 147 oral cavity, 147–148

319

Index

saltatory docking, 149 Multidrug resistance-associated proteins (MRPs), 229–230 Multiple photosystem, cnidarians general vs. special-purpose eyes ocelli, 42 pigment cup eyes, 42 pineal organs, 42 special-purpose eye, 42, 44 photopigments color vision, 44–45 morphological types, 44 opsins, 44–45 visual channels cone pathway, 45 convergence and divergence, 46 processing capacity, 46 rod pathway, 45 N Natriuretic peptides, HPA axis effects adrenal cortex, 18–20 adrenal medulla, 20–21 hypothalamus, 12–15 pituitary gland, 15–18 expression adrenal cortex, 10–11 adrenal medulla, 11–12 hypothalamus, 4, 6–8 pituitary gland, 8–10 interaction, 5 pathophysiology adrenocortical adenoma and carcinoma, 21–22 pheochromocytoma, 22 receptors, 4 sequence, 2–3 signaling mechanism, 4 Nelfinavir, 251 N-end rule, 191 N-ethylmaleimide sensitive factor (NSF) ATP requirement, 117 characteristics, 118 fusion capacity, 112 Paramecium domain structure, 113 golgi apparatus, 115–116 inhibited metabolic activity, 113–114 NSF gene silencing, 113–117 rough ER lumen, 114–115 surface membrane complex, 117 Nevirapine, 253–254 Nonnucleoside reverse transcriptase inhibitors (NNRTIs), 253–254 Nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs), 254–256

O Ohnologs, 94 P Paramecium SNAREs aminoacid level, 108 vs. Arabidopsis thaliana, 109–110 vs. higher eukaryotes, 110 vs. Homo sapiens, 109 vs. mammalian cells, 110 vs. parasitic species, 109 Q-SNAREs, 108–109 R-SNAREs, 109 vs. Saccharomyces cerevisiae, 109 specific aspects aberrations, 110 Q substitution, O-layer, 111 subcellular localization, 111 truncated SNAREs, 111 specific protein targeting, 119 transmembrane domain, 110 Paramecium tetraurelia. See also Vesicle trafficking pathway, ciliates actin isoforms, 102–105 calcium-binding proteins and sensors calmodulin, 145 Ca2þ-signaling, 142–143 synaptotagmin, 143–144 trichocyst exocytosis, 145–146 clathrin-mediated constitutive endocytosis, 85 constitutive exo-endocytosis, 87 early and late endosomes, 127–128 parasomal sacs, 125–127 contractile vacuole complex, 87 dense core-secretory vesicle biogenesis, trichocysts Hþ-ATPase, 122 homolog proteins, 123 small G-proteins, 122 transmembrane crosstalk, 123–124 trichocyst precursor passage, 121–122 ER/golgi trafficking, 120–121 exocytosis, 84 fusion process, 85–88 G-proteins/GTPases, 101 Hþ-ATPase, 106–107 macronuclear genome cloning, 83–84 microdomains and membrane fusion focal fusion concept, 133 Hþ-ATPase, 133 N-ethylmaleimide sensitive factor (NSF) domain structure, 113 golgi apparatus, 115–116 inhibited metabolic activity, 113–114 NSF gene silencing, 113–117 rough ER lumen, 114–115 surface membrane complex, 117

320 Paramecium tetraurelia. See also Vesicle trafficking pathway, ciliates (cont.) phagocytosis, 135–137 (see also Phagocytosis) phagosome cyclosis, 87 protein-regulated membrane interactions, 85 R-SNAREs and related forms, 95–97 SNAREs (See Paramecium SNAREs) stimulated exocytosis dynamics, 129–131 site assembly, 128 trafficking routes, 88 transformation, 83–84 per mutants, 288–290 P-glycoprotein (Pgp), HIV therapy CXCR4, 227 expression brain capillary endothelial cells (BCECs), 226 mammary epithelial cells, 226–227 peripheral blood mononuclear cells (PBMCs), 226 placental trophoblasts, 226 tissues, 225–226 HIV-1 interaction, 227–228 Phagocytosis autophagy, 141–142 ciliates acidosomes, 137 actin, 137–140 cytochalasin sensitivity, 135 food vacuoles, 137 G-proteins, 141 Hþ-ATPase, 140–141 phagocytosis cycle, 135–136 phago(lyso)some biogenesis, 135, 137 SNAREs, 141 macrophages, 134 phagosome formation, 134–135 Phenotypic screening, 294 Pheochromocytoma, 12, 22 Photoinhibition, 187, 200 Photosensory organs cubomedusae (see also Cubomedusae) lens eyes, 60–62 neuronal organization, 67–70 ocelli types, 62–64 photoreceptor cells, 64–66 rhopalium, 56–59 hydromedusae (see also Hydromedusae) electroretinograms (ERGs), 53 nonsensory pigment cells, 53 ocelli, 51–53 scyphomedusae, 54–56 (see also Scyphomedusae) Photosynthesis, 187, 190, 206 Phototaxis, 285 Pituitary gland atrial natriuretic peptides

Index

ACTH secretion, 15–16 binding sites, 9 GH release, 16–17 immunoreactivity, 8–9 PRL release, 17 proopiomelanocortin, 17–18 brain natriuretic peptides cGMP accumulation, 17 immunoreactivity, 9 C-type natriuretic peptides cGMP accumulation, 17 immunoreactivity, 9 natriuretic peptide receptor, 9–10 Plant circadian clock, 296–298 Plastidic type I signal peptidase (Plsp1), 203 Plastid proteases activity, plastid conversion, 186–187 biogenesis, 188 chloroplasts, 186–187 Clp protease features, 191–193 function and protein substrate, 193–194 structure, 190–191 CND41, 206–207 Deg protease features, 199–200 function and protein substrates, 200–201 structure, 199 degradation process, 208 FtsH protease features, 195–196 function and protein substrate, 196–197 PSII repair cycle, 198 structure, 194–195 GEP, 207 identification, 188 intramembrane proteases AraSP, 205 EGY1, 205 rhomboid, 204–205 isomers, 207–208 Lon protease function, 198–199 structure, 197–198 plastid homeostasis, 187–188 plastid membranes, 190 processing peptidases CtpA, 203–204 Plsp1, 203 PreP, 201–202 SPP, 201 TPP, 202 SppA, 206 transit peptide, 189–190 type and localization, 188–189 Presequence protease (PreP), 201–202 Protease inhibitors (PIs), 236, 249–253

321

Index Proteases. See Plastid proteases Protozoa, membrane trafficking. See Vesicle trafficking pathway, ciliates R Raltegravir, 256 Rhythm of chloroplast (ROC) DNA-binding domain, 297–298 forward genetic approach, 293–295 genes, membrane/vesicle trafficking, 301–302 genetic interactions, 299 mutants, 295 Ritonavir, 250 S Saquinavir, 236, 249–250 BCRP, 249 MRP, 249–250 Pgp, 236, 249 Sarsia tubulosa neurophysiological studies, 53, 70 ocellus, ultrastructure, 51–52 positive phototaxis, 47 Scyphomedusae morphological and physiological data, 56 neurophysiological data, 54–56 pigment cells, 54 rhopalia, 54–55 Signal peptidase (SPase I), 202–203 SNAP-25-like proteins (SNAP-25-LPs), 89, 93 Soluble NSF attachment protein (SNAP) receptor (SNAREs) domain analysis criteria, 88–93 R-SNAREs, 88–89 SNAP-25-like proteins (SNAP-25-LPs), 89, 93 v/t-type membranes, 93 microdomains and membrane fusion ciliates, 132–134 reggie/flotillin-related scaffolding protein, 132 SNARE pins, 131 synaptobrevins, 132 syntaxin, 132 N-ethylmaleimide sensitive factor (NSF) (see N-ethylmaleimide sensitive factor (NSF)) Paramecium (see also Paramecium SNAREs) antisense ribosome technology, 99 BLAST search, 99 database, 98 diversification, 98–99 exceptions, 94 identification criteria, 94 mutual control, 94

SNARE specificity, 98 types, 93 Stavudine, 255 Stromal processing peptidase (SPP), 189, 201 Synaptotagmin Ca2þ-sensor, 143–144 phagocytosis, 134 Synechocystis sp. PCC 6803, 203–204 T Tenofovir, 255–256 Tetrahymena thermophila, membrane trafficking actin isoform, 105 antisense ribosome technology, 99 Ca2þ-binding proteins, 146 constitutive exo-endocytosis, 125 cytokinesis, 150, 156 dense core-secretory vesicle biogenesis mucocysts, 122–123 secretory matrix component maturation, Hþ-ATPase, 123 ER/Golgi trafficking, 120–121 fusion processes, 85 macronuclear genome cloning, 83–84 microdomains and membrane fusion, 133 microtubules, 147–148 phagocytosis, 135, 137–138, 141 Rabs protien, 101 trafficking pathways, 86 Thylakoid processing peptidase (TPP), 190, 202–203 Tipranavir, 253 Tripedalia cystophora cubozoan ocelli, 62–64 lens eyes image quality, 60 morphology, 60 pacemaker system, 62 spectral sensitivity, 61 temporal resolution, 61 upper lens eye, 61 morphological photoreceptor, 65 obstacle avoidance response, 48 ocellus, 50–51 opsin, 70–71 opsin-based phototransduction, 65–66 rhopalia, 54–55 rhopalial nervous system, 67–68 V Vesicle trafficking pathway, ciliates actin ciliates, 102–105 general considerations, 101–102 annexins, 150–151 apicomplexan parasites and, 84 calcium-binding proteins and sensors

322 Vesicle trafficking pathway, ciliates (cont.) calmodulin, 145 Ca2þ-signaling, 142–143 synaptotagmin, 143–144 Tetrahymena mucocysts, 146 trichocyst exocytosis, 145–146 ciliary function, SNAREs ciliates, 155–156 vertebrates, 154–155 contractile vacuole complex, 152–153, 158 copines, 151 cytokinesis, 156 ER to golgi apparatus ciliates, 120–121 molecular interactions, 119 motifs, 118 secretory vesicle budding, 120 specific protein targeting, 119–120 exocytosis and endocytosis (see Exocytosis and endocytosis) Hþ-ATPase general aspects, 106 Paramecium, 106–107 macronuclear genome cloning, 83–84 microtubules cytokinesis, 150 cytopharynx, 148 disrupting drugs, 150 dynein, 148–149 kinesin, 148 metazoans, 147 oral cavity, 147–148 saltatory docking, 149

Index

molecules engaged, 81–82 Paramecium (see also Paramecium tetraurelia) clathrin-mediated constitutive endocytosis, 85 constitutive endocytosis, 87 contractile vacuole complex, 87 exocytosis, 84 fusion process, 85–88 phagosome cyclosis, 87 protein-regulated membrane interactions, 85 trafficking routes, 88 transformation, 83–84 phagocytosis (see Phagocytosis) pharmacology, 151–152 principal mechanisms, 81–82 secretory mutants, 157 small GTP binding protiens/GTPases Arf-type GTPases, 99–100 monomeric G-proteins, 100 T. thermophila, 100–101 SNAREs (see also Soluble NSF attachment protein (SNAP) receptor (SNAREs)) domain analysis, 88–93 Paramecium, 94, 98–99 types, 93 Tetrahymena thermophila (see Tetrahymena thermophila, membrane trafficking) Z Zalcitabine, 255 Zidovudine, 254

H+-ATPase

Membrane fusion

v-SNARE H+

H H

H+

Ca2+

H+

H+ H+

Vesicle

Acidification

-sensor Arf (GTPase) + activator

target

H+

H+ H+ H+

H+

H+ H+

t-SNARE

Ca2+ -release +/- influx Ca2+-store

Activation

H+

H H+

H+

H+

+

H+ H+

Actin binding

Tethering

H+ H+ H+

H+

H+ H+

Docking

Helmut Plattner, Figure 3.1 Principal mechanisms cooperating during vesicle trafficking, as exemplified by compartments endowed with SNAREs and Hþ-ATPase as well as with interacting F-actin. These components, analyzed mainly in P. tetraurelia and to a smaller extent in T. thermophila, are in the focus of the present review on trafficking in ciliates. The right side of the scheme refers specifically to exocytosis. Sequence from left to right. Acidification: Vesicles possess a set of v- (R-)SNAREs and a Ca2þ-sensor (not yet identified in ciliates) as well as an Hþ-ATPase (bright blue) undergoing conformational change as a consequence of lumenal acidification (as shown in other cell types). Activation: The conformational change of the Hþ-ATPase allows for binding of an Arf-type small GTPase (dark blue) and its activator (red ball)—as shown in other cells, thus allowing for targeting. Tethering: Targeting to an appropriate compartment includes tethering. So far there is only evidence of some tethering effect of F-actin in ciliates, while exocyst (for constitutive exocytosis) and any other potential tethering components have not been clearly identified as yet. Docking: After tethering, docking ensues, involving pairing of the v- (R-) SNARE with the t- (Q-) SNAREs of which for simplicity only one type has been drawn. In Paramecium, we identified R-SNAREs of the type synaptobrevin (yet mainly as longin forms) and Q-SNAREs of the type syntaxin and SNAP-25-LP. As in other systems, in Paramecium only the (majority of the) first two possess a transmembrane domain which is a prerequisite to subsequent membrane fusion. Ca2þ release and influx: This occurs during stimulated exocytosis in response to a stimulus. As found with Paramecium, activation of cortical stores (alveolar sacs, green) causes Ca2þ release which precedes and entails a superimposed Ca2þ-influx (‘‘SOC mechanism’’). Membrane fusion: Increase of the local cortical cytosolic [Ca2þ] activates the system for membrane fusion, provided SNARE zippering has preceded. It produces a membrane continuum, with mixture of the contents (inside the cell) or their release (exocytosis). Regrettably little is known on other key players, such as small G-proteins/GTPases and their regulators as well as of a Ca2þ sensor in ciliates.

A Osmoregulatory system cv

gh

trpc tr

er

ss a

fv

ds

Phagocytotic pathway

ga pm

Exo-endocytic pathway

ee

rv oc dv

ps ci

as

cp

B Stimulated exocytosis

Constitutive exocytosis vesicles

Parasomal sacs (+ clathrin)

Stimulus Cell membrane Defecation: cytoproct (constitutive exocytosis)

Terminal cisternae (early endosomes)

Experimental de−/redocking Mature trichocyst

Maturation stages of food vacuoles

Ghosts

Discoidal vesicles

Precursor secretory vesicles Lysosomes Food vacuole

Golgi Radial canals Reversible fusion/fision

+

Contractile vacuole

?

? Rough ER

Constitutive exocytosis

Acidosomes Nascent food vacuole (phagocytosis)

Helmut Plattner, Figure 3.2 Main trafficking pathways in ciliates. (A) Three main vesicle trafficking pathways in ciliates, as analyzed mainly with Paramecium (to which the scheme refers), but to a considerable extent also with Tetrahymena. Green: exo-endocytotic pathways, mainly based on cited work with Paramecium (by J. Beisson and her then associates and by the present author and his coworkers) as well as with Tetrahymena (by A. Turkewitz). The general trafficking scheme is based on a figure by Kissmehl et al. (2007); therein the part concerning phago-lysosomal components (red) is based mainly on cited work with Paramecium (by R. Allen and A.K. Fok and their collaborators). Yellow: Unexpectedly, in the cited work on SNAREs, we found evidence of vivid trafficking in the contractile vacuole/osmoregulatory system of Paramecium. Abbreviations: a, ampulla; as, acidosomes; ci, cilia; cp, cytoproct; cv, contractile vacuole; ds, decorated spongiome; dv, discoidal vesicles; ee, early endosomes; er, endoplasmic reticulum; fv, food vacuole; ga, Golgi apparatus; gh, ‘‘ghosts’’ (from trichocyst release); oc, oral cavity; pm, plasmamembrane; ps, parasomal sacs; rv, recycling vesicles; sm, smooth spongiome; tr, trichocyst; trpc, trichocyst precursors. (B) The three main trafficking pathways depicted in Fig. 3.2A are shown here in more detail, each pathway with a remarkable number of membrane interactions by fusion and fission. Based on the scheme by Plattner and Kissmehl (2003a).

A 205

274 291 267 295

202

271 264

213

275

282 300 304

223

285

292 309 313

183

249 266 245 270

169

231

239 256 261

199

261

271 286 288

1

183

252 267 245 270

1

183

245

253 269 271

157

219

226 242 246

147

209

220 235 238

163

225

142

207 223 204 226

1

31

135

PtSyx1-1 130

32

1

290 297

PtSyx2-1 1

40

148

PtSyx3-1 1

67

167

PtSyx4-1 1 PtSyx5-1 1

36

131

PtSyx6-1 1 PtSyx7-1

PtSyx8-1

PtSyx9-1 1 PtSyx10-1 1 PtSyx11-1 1

232 249

PtSyx12-1 1 PtSyx14-1 1

179

246 261 241 265

PtSyx15-1

Syntaxin domain

SNARE domain

TMR

288

B 1DN1

Q226

PtSyx3-1

PtSyx3-2

Q242

Q242

C

Figure 3.3 (continued)

D 100 100

PtSyx14-1 PtSyx14-2 PtSyx15-1

34 100

PtSyx5-1 PtSyx5-2 PtSyx8-1

77 100

PtSyx8-2

100

PtSyx1-1 PtSyx1-2

99

PtSyx3-1 100 100

PtSyx3-2 PtSyx2-1

100

PtSyx2-2

100

PtSyx7-1

50

79

PtSyx7-2

42

PtSyx12-1 PtSyx9-1

33 100

PtSyx9-2 PtSyx10-1

96

100

PtSyx10-2 PtSyx11-1

100

PtSyx4-1 PtSyx4-2

89

PtSyx6-1

Helmut Plattner, Figure 3.3 Some molecular characteristics of P. tetraurelia syntaxins (A–C), their evolutionary connection (D) and intracellular localization (E) by different methods, based on the work by Kissmehl et al. (2007). (A) The 15 types of syntaxins found in Paramecium by sequence analysis and domain structure analysis show some diversification with regard to the presence of a syntaxin domain (green), but all forms contain a SNARE domain (red), and a transmembrane region (blue). Their molecular size also varies. From Kissmehl et al. (2007). (B) Molecular modeling of PtSyx3-1 and PtSyx3-2 in comparison to 1DN1 (syntaxin 1), from R. norvegicus (C. Danzer, Diploma work, University of Konstanz) reveals striking similarities with regard to the arrangement of a-helical structure in the SNARE domain (green), with the Q-residue in the zero-layer indicated, and the structure of the Habc domain (yellow); red—linker. Unpublished images from the series by Kissmehl et al. (2007). (C) Core structure of the SNARE domain of PtSyx paralogs. Note the zero-layer with the Q residue typical of syntaxins and an exceptional A in PtSyx11-1. Also note the heptad repeats (repetitive

aminoacids, yellow, in positions 3/4/7 upstream and downstream from the zero-layer), with some exceptional aminoacids set in green. A series of such heptad repeats in each of the SNAREs would align to a quarternary complex (‘‘SNARE complex’’). From Kissmehl et al. (2007). (D) Relationships between the different PtSyx paralogs (neighbor joining tree), with probability values indicated, can be interpreted rather clearly as representing waves of whole genome duplication (pink, green, blue) discussed in the text. Composed from material contained in Kissmehl et al. (2007). (E) Intracellular distribution of PtSyx species, as determined by expression as GFP-fusion proteins and by antibody labeling at the light and electron microscope level. For trafficking scheme, see Fig. 3.2A. Note association of PtSyx 9 and PtSyx10 with different vesicles probably interacting with food vacuoles; similarly uncertain is the assignment of PtSyx14 and PtSyx15 to the contractile vacuole system. Also note the presence of PtSyx1 all over the cell surface, including the oral cavity. Many other syntaxin isoforms can be clearly assigned to specific structures; they may be exchanged during trafficking, as is the case, for example, with syntaxins associated with early and later stages of the food vacuole. cph, cytopharynx; cs, cytostome; trp, trichocyst precursors; for additional abbreviations, see Fig. 3.2A. From Kissmehl et al. (2007).

A

ss a cv

ds

fv Slightly acidic

gh tr

er

Acidic

Neutral

ga cf

pm

pof rv

ee

oc dv

ps ci

cp

as

B Constitutive exocytosis vesicles

Defecation: cytoproct (constitutive exocytosis)

Terminal cisternae (early endosomes)

Maturation stages of food vacuoles

Discoidal vesicles

Syx1, 4, 7, 9, 10, 11, 12-1, SNAP-25-LP

Lysosomes

Acidosomes

Syb6-1, 8, 9, 11 + H -ATPase: a1, a4, a5, a6, a9 c1, c4, c5 Food vacuole Actin1-1, 1-2, 1-9, 3, 4, 5, 8 Act1-2: speckles and lee-side tail Act1-9: lee-side tail Act5-1: cap etc.

Nascent food vacuole (phagocytosis)

Helmut Plattner, Figure 3.8 The phago(lyso)somal cycle of Paramecium. (A) Formation and maturation of the phago(lyso)somal vacuole (‘‘food vacuole’’), with the change of lumenal pH from acidic (after fusion with numerous small acidosomes, as) to near neutral and neutral, for final discharge at the cytoproct (cp). For other abbreviations, see Fig. 3.2A. Scheme adapted from Wassmer et al. (2009). (B) SNAREs, actin, and Hþ-ATPase SU isoforms contributing to the phago-/lysosomal cycle in P. tetraurelia cells. Scheme as in Fig. 3.2B. Superimposed are data combined from Kissmehl et al. (2007), Schilde et al. (2006, 2008, 2010), Sehring et al. (2007a), and Wassmer et al. (2005, 2006). Magnification of micrographs  600. Source of the basic scheme as in Fig. 3.2A; micrographs from Sehring et al. (2007b).