International Review of Cell and Molecular Biology, Volume 271

V O LU M E T WO S E V E N T Y O N E INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY INTERNATIONAL REVIEW OF C...

Author: Kwang W. Jeon

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V O LU M E

T WO

S E V E N T Y

O N E

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

1949–1988 1949–1984 1967– 1984–1992 1993–1995

Editorial Advisory Board

ISAIAH ARKIN EVE IDA BARAK PETER L. BEECH HOWARD A. BERN ROBERT A. BLOODGOOD DEAN BOK HIROO FUKUDA RAY H. GAVIN MAY GRIFFITH WILLIAM R. JEFFERY KEITH LATHAM

WALLACE F. MARSHALL BRUCE D. MCKEE MICHAEL MELKONIAN KEITH E. MOSTOV ANDREAS OKSCHE THORU PEDERSON MANFRED SCHLIWA TERUO SHIMMEN ROBERT A. SMITH NIKOLAI TOMILIN

V O LU M E

T WO

S E V E N T Y

O N E

INTERNATIONAL REVIEW OF

CELL AND MOLECULAR BIOLOGY

EDITED BY

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

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

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ISBN: 978-0-12-374728-0

PRINTED IN THE UNITED STATES OF AMERICA 08 09 10 11 12 8 7 6 5 4 3 2 1

CONTENTS

Contributors

ix

1. Genetic Models of Cancer in Zebrafish

1

James F. Amatruda and E. Elizabeth Patton

1. Introduction 2. Molecular Relevance of Zebrafish Cancer to Human Disease 3. Forward Genetic Screens 4. Targeted Cancer Models in Zebrafish 5. Xenotransplantation 6. Chemical Genetics: Small-Molecule Screening in Zebrafish 7. Summary References

2. Cellular and Molecular Biological Aspects of Cervical Intraepithelial Neoplasia Fjodor Kisseljov, Olga Sakharova, and Tatjana Kondratjeva

2 5 6 10 23 26 27 28

35

1. Introduction 2. Cervical Cancer as a Unique Model of Human Carcinogenesis 3. Biology of Cervical Neoplasias 4. Cellular Aspects of Cervical Tumors Progression 5. Molecular Aspects of Cervical Carcinogenesis 6. Conclusions References

36 37 40 44 51 75 76

3. Vesicle, Mitochondrial, and Plastid Division Machineries with Emphasis on Dynamin and Electron-Dense Rings

97

T. Kuroiwa, O. Misumi, K. Nishida, F. Yagisawa, Y. Yoshida, T. Fujiwara, and H. Kuroiwa

1. Introduction 2. Structural Similarities Among Mitochondrial, Plastid, and Vesicle Division Machineries 3. Vesicle Division Machinery 4. Bacterial, Microbody (Peroxisome), Mitosome, and Hydrogenosome Division Machineries 5. Mitochondrial Division Machinery

98 101 107 111 118 v

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Contents

6. Plastid (Chloroplast) Division Machinery 7. Isolation of Organelle Division Machinery and Its Significance 8. Concluding Remarks References

4. Retromer: Multipurpose Sorting and Specialization in Polarized Transport Marcel Verge´s

1. Introduction: Basic Concepts on Coat-Mediated Vesicular Transport 2. Retromer’s Assembly and Functioning 3. Multiple Roles of Retromer: Models of Study 4. Polarized Transport Mediated by Retromer 5. Implication of Retromer and Sorting Nexins in Other Aspects of Polarity 6. Concluding Remarks References

5. Translational Control of Gene Expression: From Transcripts to Transcriptomes ¨rg Ba¨hler Daniel H. Lackner and Ju

1. 2. 3. 4. 5.

Introduction Preparation for Translation: RNA Processing and Export Regulation of Translation Emerging Concepts in Translational Regulation Global Approaches to Identify Targets of Posttranscriptional Gene Regulation 6. Concluding Remarks References

6. Phagocytosis and Host–Pathogen Interactions in Dictyostelium with a Look at Macrophages Salvatore Bozzaro, Cecilia Bucci, and Michael Steinert

1. Introduction 2. The Dynamics of Phagocytosis 3. Cellular Mechanisms of Phagocytosis 4. Regulatory Pathways Controlling Phagocytosis 5. Host–Pathogen Interactions: A Versatile New Model Host 6. Concluding Remarks References

128 137 143 144

153 154 155 160 167 179 184 185

199 200 202 204 219 227 237 238

253 254 257 260 268 281 288 289

Contents

7. Mechanobiology of Adult and Stem Cells James H.-C. Wang and Bhavani P. Thampatty

1. Introduction 2. Application of External Mechanical Forces to Cells 3. Cell-Generated Mechanical Forces 4. Mechanobiological Responses of Cells 5. Mechanotransduction Mechanisms 6. Concluding Remarks References Index

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301 303 304 306 311 329 332 334 347

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CONTRIBUTORS

James F. Amatruda Departments of Pediatrics, Molecular Biology, and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390 ¨rg Ba ¨hler Ju Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, United Kingdom Salvatore Bozzaro Department of Clinical and Biological Sciences, University of Turin, Ospedale S. Luigi, 10043 Orbassano, Italy Cecilia Bucci Department of Biological and Environmental Sciences and Technologies, University of Lecce, via Prov. Monteroni, 73100 Lecce, Italy T. Fujiwara Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan Fjodor Kisseljov N. N. Blochin Cancer Research Center, Kashirskoe shoesse 24, Moscow 115478, Russia Tatjana Kondratjeva N. N. Blochin Cancer Research Center, Kashirskoe shoesse 24, Moscow 115478, Russia H. Kuroiwa Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan T. Kuroiwa Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan Daniel H. Lackner Cancer Research UK Fission Yeast Functional Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom

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Contributors

O. Misumi Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan K. Nishida Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan E. Elizabeth Patton Edinburgh Cancer Research Centre & MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, United Kingdom Olga Sakharova N. N. Blochin Cancer Research Center, Kashirskoe shoesse 24, Moscow 115478, Russia Michael Steinert Institute for Microbiology, Technical University of Braunschweig, Spielmannstr. 7, D-38106, Germany Bhavani P. Thampatty Departments of Orthopaedic Surgery, Bioengineering, Mechanical Engineering and Materials Science, and Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Marcel Verge´s Laboratory of Epithelial Cell Biology, Centro de Investigacio´n Prı´ncipe Felipe, C/E.P. Avda. Autopista del Saler, 16-3 (junto Oceanogra´fico), 46012 Valencia, Spain James H.-C. Wang Departments of Orthopaedic Surgery, Bioengineering, Mechanical Engineering and Materials Science, and Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 F. Yagisawa Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan Y. Yoshida Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan

C H A P T E R

O N E

Genetic Models of Cancer in Zebrafish James F. Amatruda* and E. Elizabeth Patton† Contents 1. Introduction 1.1. Early evidence for cancer in fish: Carcinogenesis and genetics 1.2. The zebrafish system 2. Molecular Relevance of Zebrafish Cancer to Human Disease 3. Forward Genetic Screens 4. Targeted Cancer Models in Zebrafish 4.1. Hematologic malignancies 4.2. Pancreatic cancer 4.3. Rhabdomyosarcoma 4.4. Transgenic technical developments for cancer studies 4.5. p53 pathways in development and cancer 4.6. Melanoma 4.7. The APC-Wnt pathway 4.8. PTEN in development and cancer 5. Xenotransplantation 5.1. Zebrafish as a biological readout of signaling pathways in human cancer 5.2. Studying human tumors in zebrafish 6. Chemical Genetics: Small-Molecule Screening in Zebrafish 7. Summary Acknowledgments References

2 2 3 5 6 10 10 13 14 14 15 18 20 21 23 23 24 26 27 27 28

Abstract Firmly established as a model system for development, the zebrafish is now emerging as an effective system for the study of the fundamental aspects of tumorigenesis. In keeping with the striking anatomical and physiological similarity between fish and mammals, zebrafish develop a wide spectrum of cancers resembling human malignancies. The potential for zebrafish as a cancer model * {

Departments of Pediatrics, Molecular Biology, and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Edinburgh Cancer Research Centre & MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, United Kingdom

International Review of Cell and Molecular Biology, Volume 271 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01201-X

#

2008 Elsevier Inc. All rights reserved.

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derives from its strengths as an experimental system for developmental biology. Despite 450 million years of evolutionary distance, the pathways that govern vertebrate development including signaling, proliferation, cell movements, differentiation, and apoptosis—indeed, the same pathways that are often misregulated in tumorigenesis—are highly conserved between humans and zebrafish. This, together with a complete genome sequence and an array of tools for gene manipulation, makes the construction of robust, physiological zebrafish cancer models increasingly possible. Key Words: Zebrafish, Cancer, Development, Genetics, Carcinogen, Tumorigenesis. ß 2008 Elsevier Inc.

1. Introduction 1.1. Early evidence for cancer in fish: Carcinogenesis and genetics Tumors in fish have been recorded for decades. As in other fishes, such as Xiphophorus, medaka, and trout, tumors of diverse cellular origin can be induced in zebrafish by carcinogens. During the 1960s, Stanton (1965) used zebrafish as the first fish species to study the effects of experimental carcinogens. Exposed as young fry (aged 3 weeks) to diethylnitrosamine, zebrafish developed liver neoplasms similar to those seen in rodent models. Such observations led Streisinger (1984), one of the founders of the zebrafish system, to put forth the use of zebrafish as a sentinel for toxic and carcinogenic substances. More recently, Spitsbergen and coworkers led a comprehensive study of the effects of exposing zebrafish to carcinogens at specific stages of the life cycle (Spitsbergen and Kent, 2003; Spitsbergen et al., 2000a,b). They found that while zebrafish rarely develop spontaneous tumors, carcinogen exposure causes zebrafish to develop neoplasms in almost every tissue. Administered in the water or in the diet, the carcinogens DMBA, MNNG, MAMA, N-nitrosodiethylamine (DEN), and Aflatoxin B promoted a diverse range of neoplasms including liver, gill, blood, blood vessel, intestine, testis, heart, eye, nerve sheath, brain, pancreas, bone, and skin tumors. Spitsbergen’s experiments highlight two key points for the field: that the cellular response to carcinogens to promote tumorigenesis is the same in zebrafish as in mammals, and that zebrafish do indeed develop tumors with relevant histopathology to humans. In addition to tumor development induced by carcinogens, historical use of fish models with genetic predisposition to cancer provided a platform for initial genetic cancer studies in zebrafish. Of particular importance, genetic crosses of Xiphophorus species, X. maculatus (sword tails) and X. helleri

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(platyfish), done in the 1920s by Gordon and Kosswig showed that hybrid breeding programs produced fish with lesions ranging from preneoplastic melanocytic lesions to invasive cancer (Anders, 1991; Meierjohann and Schartl, 2006; Walter and Kazianis, 2001). The genetic cause of melanoma in such crosses is the overexpression of an X-linked dominant locus, called Tu, encoding the Xiphophorus melanoma receptor kinase (Xmrk), a gene related to the epidermal growth factor receptor (Schartl et al., 1999; Wittbrodt et al., 1989, 1992). Oncogenic Xmrk constitutively activates the mitogenactivated protein kinase (MAPK) pathway, a signaling cascade required for melanocyte development and frequently misexpressed in human melanomas. In wild-type X. maculatus, oncogenic Xmrk is prevented from causing neoplastic transformation by a tumor suppressor, R. Although the identity of which is unknown, a promising candidate strongly linked to the R locus is a CDKN2-like gene (Kazianis et al., 1998; Nairn et al., 1996). In humans, CDKN2A encodes a cell cycle inhibitor protein p16, loss of which is a frequent somatic and germ line genetic cause of melanoma in humans (Chin et al., 2006). Through serial backcrossing of X. maculatus with X. helleri, the oncogenic Tu locus becomes unlinked from R, allowing Xmrk activity to escape tumor suppression, permitting melanoma development. Like human melanomas, Xiphophorus melanomas can also be induced by environmental carcinogens, in particular ultraviolet light, providing an important example of a gene–environment interaction (Nairn et al., 1996; Setlow et al., 1989). The carcinogen, genetic, and environmental cancer studies in these fishes provided compelling evidence that the zebrafish could be used to study the fundamental genetic events of tumorigenesis, and become a model system for cancer induction and suppression studies.

1.2. The zebrafish system Key studies in the 1980s by Kimmel and Streisinger proved that zebrafish could reveal insight into significant biological questions in vertebrate development (Grunwald and Eisen, 2002). Streisinger’s selection of the zebrafish for laboratory use was based on a number of key features (NuessleinVolhard and Dahm, 2002; Westerfield, 2000), some of which are also shared by medaka and Xiphophorus model systems. Small teleost (bony) fish, adult zebrafish are 3–4 cm long, the males differentiated from females by subtle changes in color and shape. Their natural habitat, including the Ganges river and rice paddies of India, provide them with varied conditions, possibly helping them to adapt quickly to home and laboratory aquariums (Engeszer et al., 2007). About 40 animals can inhabit a 9-l tank, thus enabling large numbers of adults to be housed for relatively low cost. Responding to their environmental conditions, zebrafish sleep in the night, and breed after exhibiting courting and mating behavior at dawn. In the laboratory, most fish are kept on 14-h light and 10-h dark cycles, and more commonly,

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researchers are incorporating a dawn and dusk cycle to the environment. Females deposit eggs, which are externally fertilized by males and easily collected in breeding chambers. Healthy fish can supply over 200 embryos in one morning, and can be bred weekly. Within 3 months, the embryos are fully developed adults, ready to begin a weekly breeding regime. The ability to collect single-cell fertilized eggs coupled with the transparency of the embryo allows for vertebrate organogenesis, angiogenesis, and overall development to be viewed from conception (Fig. 1.1). One of Streisinger’s goals was to develop a vertebrate system that could be used in genetic screens. Just as genetic mutations and screening in Drosphophila and Caenorhabditis elegans had revealed the genetic basis of A

D

C

B

E

F

Figure 1.1 Embryonic development in zebrafish after fertilization. Cytoplasmic streaming creates a polarized embryo, with a clear pool of blastodisc at the top of the embryo and yolk to the bottom.Within 20 min, the first division occurs to create a twocell stage embryo (A), and the next three hours is defined by rapid synchronous cleavagesçwithout addition of volume to the embryoçto generate a cap of approximately 1000 cells poised at the top of the yolk (B). Gastrulation follows with cellular rearrangements that by 10 h postfertilization create an embryo with clearly recognizable anterior^posterior and dorsal^ventral axes (C). By 12^15 h postfertilization, somites and eyes are clearly visible (D), and by 24 h of development, the foundations of organogenesis have been established (E). At 2 days postfertilization (F), the digestive tract, heart, blood, eyes, pigment pattern, and brain are clearly patterned and visible.

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metazoan developmental biology, zebrafish provided a system in which vertebrate developmental biology could be revealed. For genetic studies two important features of the zebrafish make it unique among vertebrates: embryos can survive as haploids until day 5, and early haploid embryos can be induced to become homozygous diploid animals (Streisinger et al., 1981). Haploidy and homozygous diploidy allow for the phenotypic effect of a genetic mutation to be revealed within a single generation, an important tool in reducing the physical space and time requirement for genetic screening. Classical large-scale genetic screens led by Marc Fishman, Wolgfang Driever, Christiane Nusslein-Volhard, and Nancy Hopkins and their colleagues have revealed hundreds of mutations that cause specific developmental phenotypes (Amsterdam et al., 1999, 2004; Driever et al., 1996; Haffter et al., 1996). As discussed in more detail below, new cancer genes have been identified in zebrafish through genetic screening, and coupled with the transgenic and xenotransplantation models this method has the potential to reveal novel genetic suppressors and enhancers of a cancer phenotype (Amatruda et al., 2002; Berghmans et al., 2005a; Goessling et al., 2007a; Stern and Zon, 2003). For cancer studies, zebrafish can be monitored through larval and juvenile stages, to adulthood. As in other animal systems, tumor growth can be visualized by histology, or in live animals by examination of growths on the skin (i.e., melanoma), protrusions from the head (i.e., nerve sheath tumors), or abdominal expansion (i.e., germ-cell and hepatic tumors). Fluorescence or luminescence visualization of tumors in live fish, such as a GFP-labeled tumor cells, can identify dissemination of tumor cells in the body of the fish and permit accurate sorting of tumor cells. More recently, micro-ultrasound machines have been used to give functional and anatomical imaging of tumors in living zebrafish (Goessling et al., 2007b). Zebrafish tumors can be transplanted; zebrafish are not isogenic but serial transplantation of tumors can be achieved in fish in which the T cells have been ablated by g-irradiation (Langenau et al., 2003; Mizgireuv and Revskoy, 2006; Patton et al., 2005; Traver et al., 2003). In clonal fish lines, tumors generated by DEN—with a histological spectrum of hepatocellular carcinoma, hepatoblastomas, hepatoma, cholangiocarcinoma, and pancreatic carcinoma— could be transplanted from 3 to 35 passages (Mizgireuv and Revskoy, 2006).

2. Molecular Relevance of Zebrafish Cancer to Human Disease The pathological features shared between humans and zebrafish coupled with the conservation of cancer gene sequences between the species suggests, but does not prove, that the same molecular mechanisms are employed during tumorigenesis in both zebrafish and people. Recent studies

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from the Gong laboratory show that human and zebrafish liver tumors do indeed share a molecular framework that becomes disregulated during tumorigenesis (Lam et al., 2006). DMBA is a carcinogen that can be added to the water of young fish to induce a range of neoplastic lesions (Spitsbergen et al., 2000a). Metabolized in the liver, DMBA treatment results in a spectrum of lesions that can be correlated to clinical grade or stage of neoplastic progression in humans. Using oligonucleotide microarrays with almost 16,000 unique zebrafish gene sequences, gene expression signatures of zebrafish liver tumors were compared to normal zebrafish liver (Grabher and Look, 2006; Lam and Gong, 2006; Lam et al., 2006). As in human liver tumors, analysis revealed a noted disregulation in hallmark cancer genes, including cell cycle, apoptosis, DNA repair and replication, metastasis and cytoskeletal organization, and protein synthesis. Complied into a Zebrafish Liver Tumor Differentially Expressed Gene Set (ZLTDEGS; 1,861 genes), the expression patterns of the ZLTDEGS were compared with human liver, gastric, prostrate, and lung cancer types; zebrafish liver tumors were found to specifically resemble human liver tumors. The molecular pathways specific to liver tumors included Wnt-bcatenin and RAS-MAPK pathways—pathways frequently mutated in human liver cancers—as well as novel genes, an intriguing prospect for future studies in fish and mammals. Finally, human and zebrafish liver tumor gene expression patterns were analyzed during tumor progression: overlapping gene sets from human precancerous nodules and zebrafish low grade tumors were distinguished from high grade human liver tumors and highly anaplastic zebrafish cancers. In all, three key themes emerged from this work for the zebrafish cancer field: human and zebrafish tumors share a common molecular premise, liver cancers between species more closely resemble each other than do different tumor types within a species, and comparative oncology can reveal novel molecular mechanisms of carcinogenesis. Common molecular themes and pathways found in tumors from human and zebrafish are likely to provide important insight into tumorigenesis, as they are conserved despite evolutionarily divergence between the species. Such comparative functional genomics is essential for creating accurate models of human disease, and for the interpretation of novel genetic and chemical suppressors or enhancers of the cancer phenotype.

3. Forward Genetic Screens Potentially the most powerful use of zebrafish in cancer gene discovery is the use of forward genetic screens. In this method, random mutations are made in the genome using chemical mutagens, irradiation, or insertional mutagens such as retroviruses or transposons. The progeny of mutagenized

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animals are screened to detect a particular phenotype, and the underlying genetic mutation is identified. The spectacular success of forward genetic screens in yeast, worms, flies, and other model organisms inspired efforts to apply this technique to gene discovery in zebrafish (Patton and Zon, 2001) (Fig. 1.2). Using retroviral insertional mutagenesis in a forward genetic screen, Nancy Hopkins’ group at MIT identified 12 heterozygous genetic mutant lines of zebrafish, which reproducibly developed malignant peripheral nerve sheath tumors (MPNSTs). One of these lines carried a retroviral insertion into the neurofibromatosis type 2 (nf2) gene, a known tumor suppressor. Mutagenesis Chemical Insertional Zinc finger nucleases

Morpholinos

Transgenics

Small molecules

Xenografts

Functional genomics

Highthroughput drug screens

Host/tumor interactions

Phenotypebased screens

Cancer models

Figure 1.2 Zebrafish as a tool for cancer gene discovery. A large number of experimental techniques have been developed for the zebrafish system, enhancing its flexibility and usefulness. Mutations can be introduced into the germ line by exposure of adults to chemicals such as ethylnitrosourea or by retroviral infection of cells in developing embryos. Microinjection into one- or two-cell stage embryos can be used to introduce transgenes or to prevent translation of specific mRNAs with morpholinos. Smallmolecule screens are simple to carry out by adding compounds to the water (shown here by exposure at the 1000-cell stage, but exposure at any stage is possible). To assess the effects of these interventionsçmutagenesis, transgene expression, morpholino knockdown, or small moleculesçembryos can be assessed for morphological phenotypes, or in situ hybridization can be used to visualize gene expression (functional genomics). Transgenic animals can be grown to adulthood to assess tumor incidence and establish new cancer models. Finally, transplantation of human or mouse tumor cells into embryos as xenografts creates a platform for drug testing and allows genetic manipulation of thetumor microenvironment.

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Remarkably, the other 11 lines all carried insertions in ribosomal protein genes—the first example in which ribosomal protein mutations have been definitively linked to a solid tumor predisposition. In humans, mutations in ribosomal protein S19 cause Diamond-Blackfan anemia, a pediatric bone marrow failure syndrome in which children also have susceptibility to leukemias. Further exploration of these zebrafish ribosomal protein mutants is sure to yield important insights into the complex relationship between ribosome biogenesis and cancer (Cook and Tyers, 2007; Ruggero and Pandolfi, 2003). While conceptually simple, the approach of discovering cancer genes by identifying adult fish lines with increased tumor incidence requires large numbers of tanks and considerable infrastructure. An alternative approach instead identifies cancer-relevant phenotypes during the first 5–6 days of development, then directly tests for cancer predisposition in adults of selected genotypes. Embryos are simply maintained in a petri dish prior to screening, and only the small fraction of genetic mutant lines showing a phenotype of interest need be raised to adulthood, thereby significantly reducing the number of adult animals. Furthermore, via this approach, direct insight into cancer biology may come from screens for phenotypes of altered cell proliferation, apoptosis, and angiogenesis, as well as developmental phenotypes including cell fate determination and organogenesis. Indeed, we stress that one of the key strengths of the zebrafish system is its ability to yield insight into the mechanisms of normal developmental pathways that are themselves frequently misregulated in cancer. In a forward genetic screen to identify cancer-susceptibility genes, the Zon laboratory screened for mutations that alter cell proliferation in embryos, using the mitotic marker antiphosphohistone H3 (Shepard et al., 2005, 2007). One mutant line, crash&burn (crb), carries a mutation in the Bmyb transcription factor, and crb homozygotes have defects in genomic stability and cell cycle progression. Another mutant line, cease&desist (cds), carries a mutation in zebrafish separase (espl1), a protease that normally cleaves the cohesin complex of proteins at the metaphase–anaphase transition, allowing separation of sister chromatids. Accordingly, in separasedeficient homozygous cds mutant embryos, nearly half of the cells have an aneuploid chromosome number. To gain insight into the mechanistic pathways through which Bmyb suppresses tumorigenesis, microarray analysis of crb mutant embryos was used to generate a gene expression signature of Bmyb loss. This signature was found to be specifically conserved in a subset of nonsmall cell lung cancers lacking p53 mutations. From this, it is speculated that Bmyb loss might represent a p53-independent pathway to genomic instability, thereby contributing to tumorigenesis. Importantly, directly linking the embryonic phenotypes to a causative role in cancer carries its own set of challenges, as tumor formation almost

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always involves multiple genetic alterations resulting a long latent period. To circumvent this problem, Zon and coworkers used the Spitsbergen carcinogenesis protocols (Spitsbergen et al., 2000) to promote accelerated tumor formation. Compared with wild-type siblings, heterozygous adult carriers of the B-myb and separase mutations display increased numbers of tumors after carcinogen treatment, thus identifying these genes as novel haploinsufficient tumor suppressors. In the case of the separase mutation, a dramatic eightfold increase in epithelial tumors was observed. This finding is particularly significant because of the strong association of genomic instability with epithelial cancers in humans. Thus, the zebrafish separase mutant line represents a new platform to investigate the role of genomic instability and interacting genetic mutations in lung, breast, prostate, and other epithelial cancers. Knowing the strong association between genome instability and cancer, Cheng and coworkers used zebrafish embryos in a screen to directly detect mutations causing loss of heterozygosity (Moore et al., 2006). Employing a zebrafish line heterozygous for a pigmentation mutation, golden, and the ‘‘mosaic-eye’’ assay developed by Streisinger (Streisinger et al., 1989), they screened for embryos with patches of unpigmented cells in the eye, which were reasoned to be due to loss of heterozygosity at the golden locus. Twelve genome instability (gin) mutants were identified; trans-heterozygous gin mutants show enhanced mosaic pigment loss, suggesting that these genes may interact in a complex or in parallel pathways that are essential to maintain genome stability. gin-10 heterozygotes showed enhanced rates of spontaneous tumor formation in a wide variety of tissues. The identification of the gin genes will add to our understanding of the complex mechanisms maintaining genome stability. Two additional screens illustrate the use of zebrafish to identify genes that play a role in cell proliferation and DNA damage pathways, although not yet directly linked to cancer in zebrafish. van Eeden and coworkers used RNA in situ hybridization for the proliferation marker PCNA to isolate two alleles of a gene required for normal cell proliferation in embryos (Koudijs et al., 2005). In addition to the proliferation phenotype, the mutations also conferred distinctive developmental defects reminiscent of the previously identified zebrafish mutants, dre, uki, and lep (Haffter et al., 1996). A combination of positional and candidate cloning approaches was used to show that dre, uki, and lep encode negative regulators of the hedgehog signaling pathway (Su(fu), Hip, and ptc2, respectively), which is aberrantly activated in a range of human cancers including basal cell carcinoma and medulloblastoma (Marino, 2005; Rubin and Rowitch, 2002). A second screen from the Lees’ laboratory used the Hopkins collection of retroviral insertion mutant zebrafish (Amsterdam et al., 2004) to identify a dtl/cdt2 mutation that abrogates the G2/M DNA damage checkpoint (Sansam et al., 2006). DTL/CDT2 functions at the G2/M checkpoint, and also with the

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CUL4/DDB1 E3 ubiquitin ligase to regulate the stability of CDT1, a replication licensing factor that prevents rereplication of DNA.

4. Targeted Cancer Models in Zebrafish While forward genetic screens allow for the identification of novel genes, a reverse genetic approach allows for the direct testing of known genes in cancer development. In zebrafish, transgenes can be made to randomly integrate into the genome as DNA insertions, especially useful for expressing an oncogenic form of a gene under the control of a tissuespecific promoter. While generating transgenic zebrafish has been greatly facilitated by transposon technology (Kawakami, 2005; Kawakami et al., 2004; Kwan et al., 2007), the zebrafish field has historically been limited by our current inability to mediate gene targeting. Several avenues are being pursued to achieve gene targeting in zebrafish, including homologous recombination in cultured embryonic stem cells (Fan et al., 2006), as well as sequence-specific chimeric nucleases (Beumer et al., 2006; Porteus and Baltimore, 2003; Urnov et al., 2005). Recently, two groups demonstrated successful use of these zinc finger nucleases to induce mutations in a target zebrafish gene of interest (Doyon et al., 2008; Meng et al., 2008). Another alternative is screening large libraries of ENU-mutagenized genomes for mutations within a specific gene of interest (Wienholds et al., 2003). This technique, known as targeting-induced local lesions in genomes (TILLING), can facilitate the identification of hypomorphic, gain, or loss of function alleles in any selected gene. The Zebrafish Mutation Resource at the Wellcome Trust Sanger Institute (United Kingdom) provides a valuable community resource in the identification and free distribution of a large number of mutant zebrafish lines identified through TILLING methods (http://www.sanger.ac.uk/Projects/D_rerio/mutres).

4.1. Hematologic malignancies Hematopoiesis in fish is similar to that seen in humans, and hematologic malignancies can develop in adult zebrafish (Davidson and Zon, 2004; Trede et al., 2004). The visualization of the blood and cardiovascular systems in the developing zebrafish embryo has driven forward an understanding of their genetic programming and insight into associated human disease (North and Zon, 2003). T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematologic malignancy, can result from the inhibition of normal apoptosis of developing thymocytes. Chromosomal translocations that place T-cell oncogenes under the control of strong promoter elements have been identified in many T-ALLs. Upregulation of T-cell

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oncogenes is sufficient to cause stage-specific arrest of T-cell maturation, but additional mutations are required to promote leukemia. Tom Look and colleagues have found that overexpression of T-cell oncogene transcription factors can drive five multistep molecular pathways, of which four subgroups have increased levels of the transcription factor MYC (Ferrando et al., 2002). MYC is a central regulator of proliferation and genomic instability, and coupled with mutations in the retinoblastoma (Rb) and p53 pathway (such as INK4Ap16 and ARFp14), can drive T-ALL development. To generate a model of T-ALL, transgenic zebrafish expressing the mouse c-Myc cancer gene (mMyc) under the T-cell-specific rag2 promoter were generated (Langenau et al., 2003). As the first example of transgenic cancer in zebrafish, this study provided a way forward for future cancer model design. Tumors developed within 2 months, were genomically unstable, and could be serially transplanted. Found in the developing thymus, almost all of the T-ALLs were clonal, as measured by T-cell receptor and immunoglobulin gene rearrangements and DNA flow cytometry. While in humans, upregulation of MYC can be found in a range of T-ALL subtypes (as determined by misexpression of specific markers such as HOX11), only one subtype of T-ALL that coexpressed scl2 and lmo2 could be identified in the zebrafish T-ALL (Langenau et al., 2005a). The cause for these differences is unknown, but one possibility is that timing of the mMyc gene expression from the rag2 promoter may partly explain the differences seen in humans and zebrafish. Future models of T-ALL are needed to determine how MYC expression can lead to different subgroups of T-ALL, as seen in people. An essential component of T-ALL in humans is the activation of MYC coupled with the suppression of apoptosis. While in humans, T-ALL often contains loss of the CDKN2A locus (and thus the Rb and p53 pathways), or a direct mutation in p53, the zebrafish T-ALL display neither variable expression of mdm2, bcl-xl, bcl2, p53, or bax nor mutations in p16 or p53. Apoptosis is a highly conserved process during evolution, and is also essential for normal hematopoiesis, by maintaining hematopoietic stem cell (HSC) numbers and T-cell development. In zebrafish, for example, expression of Bcl2, working downstream of p53, throughout the embryo can suppress normal developmental and irradiation-induced apoptosis (Langenau et al., 2005b). To understand if the zebrafish model of T-ALL develops from the same basic mechanisms as in humans, a transgenic line expressing Bcl2 fused to EGFP under the Rag2 promoter was developed to determine if suppression of apoptosis is sufficient for transformation of T cells in zebrafish. Live animal imaging has been a valuable tool for the study of T-cell ontogeny and leukemogenesis, using thymocyte-specific lck and rag2 promoters to drive GFP expression (Langenau et al., 2003, 2004). As in embryos, Bcl2 can block irradiation- and dexamethasone-induced apoptosis in the larval and adult lymphocytes (Langenau et al., 2005b). In fact,

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expression of the EGFP-Bcl2 construct alone increased the number of both thymocytes and B cells sufficiently to produce thymic enlargement. In addition to increased cell number, T cells appeared abnormal in their development, and the kidney marrow displayed B-cell hyperplasia. To directly test the role of apoptosis in zebrafish T-ALL, the EGFP-Bcl2 line was crossed with the rag2-EGFP-mMyc zebrafish. While zebrafish T-ALL can be ablated with irradiation, coexpression of Bcl2 confers resistance to irradiation-induced leukemic cell death, demonstrating the importance of competent apoptosis in treatment of T-ALL. Similar findings were revealed when a truncated form of human NOTCH1 was expressed from the rag promoter (Chen et al., 2007). Mosaic expression of NOTCH1 in the thymus resulted in adult onset of T-ALL, while stable germ-line transmission of NOTCH resulted in only very late onset of tumorigenesis. When combined with the expression of Bcl2, the rate of apoptosis in the thymus was reduced and incidence of leukemia was dramatically increased. These combined works from the Look laboratory provide an important example of translating clinical findings to an animal system, forming a foundation for chemical and genetic modifier screens that may reveal insights to be translated back to the clinic in the form of more specific and effective treatments. Indeed, the ability of Bcl2 to enhance leukemogenesis in the mMyc and NOTCH transgenic models provides a sound proof of principle for future genetic enhancer screens. Recurrent chromosomal translocations that create novel fusion proteins play an important role in human cancers, especially the leukemias. To model the effects of these translocations, several transgenic zebrafish lines that express the fusion proteins have been created. The Crosier group expressed the RUNX1–CBF2T1 fusion (the product of the t(8;21) found in a subset of human acute myeloid leukemia) via RNA injection into single-cell embryos (Kalev-Zylinska et al., 2002). This transient, ubiquitous expression led to disturbances in circulation, hemorrhages, and accumulation of immature blood cell precursors. These phenotypes were similar to those obtained when morpholinos were used to knock down endogenous runx1 expression, and were similar to those described in knock-in mice overexpressing RUNX1-CBF2T1. In another model, the hematologic effects of a TEL-JAK2 fusion, found in cases of ALL, as well as atypical chronic myeloid leukemia, were modeled using zebrafish orthologues of TEL and JAK2 (Onnebo et al., 2005). Transient expression from the control of the myeloid-specific spi1 promoter produced visible hematopoietic perturbations, including expansion of the intermediate cell mass (the zebrafish equivalent of yolk sac hematopoiesis) and increased numbers of cells expressing myeloid markers such as L-plastin. The Hickstein laboratory took this approach a step further to probe the role of the TEL-AML1 fusion in the development of ALL (Sabaawy et al., 2006). TEL-AML1, the product of the

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t(12;21) translocation, is the most common genetic aberration in childhood leukemia. Previous attempts to model the effects of TEL-AML1 in transgenic mouse models have produced either no hematologic perturbation or a preleukemic hematopoietic expansion, suggesting that cooperating mutations are necessary for TEL-AML1 to induce leukemia. Ubiquitous expression of the human TEL-AML1 fusion produced fatal lymphoid hyperplasias in 6% of young animals; after longer latency (8–12 months), 3% of the transgenic zebrafish developed oligoclonal B-cell ALL, consistent with the requirement for second-site mutations in the development of leukemias. Notably, lymphoid-specific expression of TEL-AML1 from the rag2 promoter failed to cause lymphoid hyperplasia, indicating that the fusion protein acts prior to the committed lymphoid progenitor stage. Taken together, these models highlight the similarity of zebrafish and mammalian hematopoiesis and demonstrate the activity of fusion oncogenes in zebrafish leukemia models.

4.2. Pancreatic cancer People with pancreatic neuroendocrine cancers often suffer from the high expression levels of active hormones due to endocrine cell differentiation within the tumor. N-MYC dysregulation may be an early event in human b-cell tumor progression because it is expressed at higher levels in b-cell hyperplastic islets and very high levels in benign and malignant tumors. To generate a model of zebrafish pancreatic cancer, MYCN was expressed from both a zebrafish and human–zebrafish hybrid MyoD promoter, which was able to promote expression in muscle, hindbrain, spinal cord, and pancreatic islet cells (Yang et al., 2004). Importantly, expression of MYCN from both the zebrafish myod promoter and the human–zebrafish chimeric MyoD promoter caused development of neuroendocrine tumors in the cranial cavity and pancreas. Despite the importance of MyoD in muscle development and rhabdomyosarcoma, and strong expression from the humanzebrafish chimeric promoter in developing somites, rhabdomyosarcoma was not observed in these transgenic lines, causing speculation that MYCN may promote apoptosis in muscle cells or additional mutational events are required to drive carcinogenesis. To model pancreatic exocrine carcinoma, Leach and coworkers used BAC recombineering to express G12V mutant KRas, fused to GFP and under the control of the ptf1a promoter (Park et al., 2008). In developing embryos, the oncogenic KRas inhibited exocrine differentiation in pancreatic progenitor cells. Silencing of the transgene led to loss of GFP fluorescence in juvenile fish. However, the eventual growth of pancreatic carcinomas was evident as an expansion of visible GFP-positive cells, occurring in two thirds of the transgenic fish by 9 months of age. The tumor histology was similar to that of pancreatic cancer in humans, and the

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tumors showed activation of the hedgehog signaling pathway, further confirming the similarity of the fish model to the human tumor.

4.3. Rhabdomyosarcoma Rhabdomyosarcoma is the most common childhood soft-tissue tumor. Two forms of this malignancy are described: alveolar rhabdomyosarcoma (ARMS), which is driven by fusion of the PAX and Forkhead genes, and embryonal rhabdomyosarcoma (ERMS), of which the molecular basis is unknown. Recent work in zebrafish has uncovered a previously unsuspected role for the small GTPase RAS in ERMS (Langenau et al., 2007). The Zon laboratory expressed a mutant, constitutively active kRASG12D mutant from the zebrafish rag2 promoter, expecting to model a role for RAS in leukemia. Surprisingly, due to a MyoD element in the rag2 promoter, Rag2-kRASG12D fish developed early, aggressive muscle tumors resembling RMS by both histological and gene expression criteria. Further, through a gene expression microarray comparative cross-species approach, a new RAS ‘‘signature’’ in the human ERMS was revealed despite the absence of frequent RAS mutations in this tumor type. A dual-labeling strategy together with serial transplantation of limiting dilutions of tumor cells was used to illuminate the putative ERMS cell of origin. In transplant recipients, tumors were reconstituted most readily from a cell population transcriptionally, resembling activated muscle satellite cells (specifically, cMetþ, m-cadherinþ, myf5, and myoD). This work suggests that the self-renewal properties of myogenic cells are conserved, or recreated, in the RMS stem cells, and give new insight into the origin of these tumors (Langenau et al., 2007). In related work, to gain temporal control of kRASG12D expression, a transgenic line expressing a floxed kRASG12D allele driven by a ubiquitous promoter was crossed to a line expressing heat-shock inducible CRE (Le et al., 2007). This strategy resulted not only in RMS but also malignant peripheral nerve sheath tumors, intestinal cell hyperplasia, and a myeloproliferative disorder. This last condition could be induced in recipient animals by ex vivo heat shock and transplantation of kidney marrow, neatly circumventing the early lethality due to RMS and the potent effect of RAS on development. This kind of sophisticated manipulation of transgene expression and tumor transplantation will no doubt prove invaluable as other transgenic zebrafish cancer models are made.

4.4. Transgenic technical developments for cancer studies An important technical development from these studies was the coinjection and resulting cosegregation and cointegration of transgenes in the mosaic transgene tumors (Langenau et al., 2008). Building on this further, Langenau

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et al. assessed whether this was a property that could be extended to other tumor types, as well as increasing the numbers of transgenes per injection. For both RMS and T-ALL, three transgenes could be simultaneously injected at the one-cell stage and become coexpressed in the developing tumor. Like the stable transgenic experimental methods, coinjection strategies recapitulated the T-ALL radiation sensitivity, as well as bcl-2 suppression of irradiation-induced cell death. As well, transgenic coinjections were successfully used to test for genetic modifiers of tumor progression, both by coexpression in genetic mutant lines (i.e., p53 loss of function lines) as well as by coinjection of transgenic modifiers (i.e. the P53 transcriptional target gene, noxa). Considering the large number of injections into single-cell embryos that can be achieved in a day (e.g., about 1000 embryos), this technology provides a rapid platform to identify new cancer pathways, as well as test for collaborating genetic events in tumor progression. Finally, with temporal control of gene expression, such as the heat-shock inducible transgenic approaches, pathways can also be assessed for their involvement in tumor maintenance.

4.5. p53 pathways in development and cancer TP53 is the most frequently mutated gene in human cancer, with almost half of all human tumors exhibiting abnormal p53 function. In response to cellular stress, p53 acts as a tumor-suppressor protein by activating a program for cell cycle arrest, including transcriptional expression of the cell cycle inhibitor p21, DNA repair, apoptosis, and the induction of senescence. The p53-dependent cell cycle arrest and apoptotic response pathways are critical for allowing time to repair DNA damage, or for the promotion of apoptosis, such as after irradiation. The inability to properly respond to DNA damage allows further accumulation of mutations during oncogenesis (Bourdon, 2007; Fuster et al., 2007; Hussain and Harris, 2006; Vousden and Lane, 2007). While sporadic loss-of-function mutations in tumors are highly prevalent, more rarely, heterozygous TP53 germ-line mutations in people cause LiFraumeni syndrome. Patients with P53 germ-line mutations have a 50-fold increase in cancer incidence, and often suffer a broad tumor spectrum— sarcomas, leukemias, breast cancers, and others—by the age of 30 years (Malkin et al., 1990). In mice, most p53 mutant embryos have normal development, with some mice strains demonstrating an essential p53 requirement for development of craniofacial features, and while loss of p53 function can permit normal proliferation during development, it cannot suppress the formation of lymphomas and sarcomas in mice (Armstrong et al., 1995; Jacks, 1996; Jacks et al., 1994). In zebrafish, loss of p53 transcriptional activity does not appear to affect normal development, but control of the p53 pathway is important in zebrafish organogenesis. In zebrafish, mutation in the endoderm factor, digestive-organ expansion factor (def ), causes a dramatic increase in the

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p53 isoform D113p53, promoting cell cycle arrest specifically within the developing digestive organs, and providing evidence that p53 isoforms function during normal development (Chen et al., 2005). Invertebrates do not appear to have the same range of TP53 responses, and TP53 mutations do not cause tumor phenotypes in worms and flies (Lu and Abrams, 2006). Demonstrating that the p53 pathway is more closely aligned with that of the mouse and human pathways, morpholino oligonucleotide studies resulting in knockdown of zebrafish p53 revealed an essential role of p53 for apoptosis induced by DNA damage (Langheinrich et al., 2002). The p53 protein can be regulated posttranslationally via phosphorylation and targeted for degradation by E3 ubiquitin ligases such as MDM2. MDM2 is often overexpressed in tumors, promoting the loss of p53 signaling, and is a key drug target. Overexpression of MDM2 in zebrafish twocell stage embryos results in partial division of the embryonic axis (Thisse et al., 2000), and transgenic expression of MDM2 in the liver causes adult liver degeneration (Chen et al., 2008). Mice deficient in Mdm2 die in embryogenesis; this is related to elevated levels of p53, as Mdm2/;p53/ animals are viable ( Jones et al., 1995; Montes de Oca Luna et al., 1995). Knockdown of mdm2 in zebrafish causes severe embryological abnormalities (Langheinrich et al., 2002). As in mouse, simultaneous knockdown of p53 and mdm2 restores normal development suggesting that the Mdm2-p53 pathway is conserved between mammals and zebrafish (Langheinrich et al., 2002). However, caution is warranted in the interpretation of morpholino knockdown studies, as the Ekker laboratory has recently described a phenomenon of ‘‘morpholino toxicity.’’ This toxicity, which results in severe, characteristic embryonic defects, is linked to transcriptional activation of a specific isoform of p53, and the toxic effects of certain morpholinos in zebrafish can be blocked by simultaneous knockdown of p53 (Robu et al., 2007). Indeed, Robu et al. found that the embryological abnormalities induced by mdm2 knockdown were not rescued by coexpression of mRNA encoding mdm2, implying that the developmental defects may result from morpholino off-target effects. Thus, definitive definition of the role of mdm2 and its interactions with p53 in zebrafish may require the generation of an mdm2 null allele. To activate transcription, four identical TP53 molecules assemble as a homotetramer to bind DNA. As such, most frequently (greater than 95% of the time), mutations are found in the DNA-binding domain, and p53 mutations can interfere with wild-type TP53 function to generate a dominant-negative or dominant-interfering function (Blagosklonny, 2000; Gannon et al., 1990). An important aspect of relating zebrafish mutations to human mutations is both the conservation of the gene and protein sequence and analogous cancer mutations in zebrafish p53 sequences. This was found when Berghmans et al. (2005b) sequenced 2679 individual males fish, the F1 progeny of ENU-mutagenized male

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founders. Five mutations were found (one silent), and of the remaining four, three were recovered, with two orthologous to TP53 mutations found in human tumors. Both human-relevant mutations, TP53N168K and TP53M214K, were in the DNA-binding domain, and as such interfered with wild-type TP53 in vitro transactivation assays. As TP53 is a critical regulator of apoptosis, embryos from both lines were subjected to irradiation and analyzed for apoptosis in the whole embryo. Both mutations prevented proper apoptosis in response to DNA damage, and as also found with human TP53 mutations, TP53N168K displayed temperature sensitivity. The TP53M214K line has defects in the irradiation-induced G1checkpoint—reflected by the lack of p21 transcriptional activation and by flow cytometry—and carries on through the cell cycle rather than delaying approximately 2 h to repair DNA damage. Interestingly, both wild type and p53 mutants still arrested at the G2-checkpoint. This range of phenotypes associated with the TP53 mutations is an important finding for the zebrafish cancer field: it not only directly relates the function of a tumor-suppressor between fish and humans but it also directly links the types of mutation alleles identified in humans to those found in zebrafish. With the TP53 alleles well related to human cancer, coupled with the morpholino studies indicating the same molecular pathways function in human and zebrafish, the TP53 lines were followed for tumor development. Between the ages of 8 months and 17.5 months, almost 30% of the adult zebrafish developed tumors in the eye or abdominal cavity (Berghmans et al., 2005b). Histological examination revealed spindle and epithelioid cells most similar to human MPNST. By electron microscopy, tumor cells were shown to have elongated, interdigitating, cytoplasmic processes with reduplicated external lamina, features consistent with nerve sheath differentiation. Tumor cells analyzed by flow cytometry showed clonal aneuploidy with both chromosome gains and loss, indicating genome instability. Although nerve sheath tumors are observed in patients with Li-Fraumeni syndrome, the restricted tumor spectrum in zebrafish may reflect speciesspecific differences in p53 function, or perhaps a need to acquire secondary genetic mutations; the single melanoma identified in over 150 tumors in the p53 mutant fish does indeed suggest that p53 can function in promote tumorigenesis within the context of additional tissues (see below) (Fig. 1.3). Antibodies to tumor-suppressors and oncogenes, and the pathways they regulate, are key to cross-species comparative molecular oncology. David Lane and colleagues have addressed the lack of antibody reagents in zebrafish by generating a panel of monoclonal p53 antibodies that show temporal and spatial specific detection of p53 on histological sections of developing embryos after treatment with chemotherapeutic or ionizing radiation (Lee et al., 2008). Importantly, these reagents begin to bring our understanding of p53 in zebrafish in line with studies in mammals, with similarities in tissuespecific expression that is dependent on the nature of the p53-activating agent.

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A

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Figure 1.3 A diverse tumor spectrum exhibited by zebrafish. Selected examples of tumor types from adult zebrafish are shown. (A) Acute lymphocytic leukemia. The small, blue tumor cells fill the kidney marrow, surrounding the normal kidney tubules. (B) Osteosarcoma invading muscle and producing ectopic bone. (C) Hepatocellular carcinoma. (D) Intestinal adenoma. Normal intestinal epithelium is visible at lower left. (E) Spindle cell tumor, often found associated with vascular structures. (F) Pancreatic adenocarcinoma. A dense desmoplastic reaction is present. At the periphery of the tumor, pancreatic exocrine cells are seen, containing bright red cytoplasmic granules (Amatruda, Shepard, and Zon, unpublished data).

Differences do exist, however, and unlike mammals, p53 itself appears to be a p53 transcriptional target in zebrafish (Lee et al., 2008). During embryogenesis of the cancer-prone p53M214K line, mutant p53 is unable to activate a p53 transcriptional program, but remains immunoreactive. Indeed, while in normal cells, p53 protein levels are tightly controlled by Mdm2, higher mutant levels of p53 protein are detected in embryos both untreated and treated with ionizing irradiation. Here, Lane and colleagues make an important link with mouse p53 cancer models, in which mutant p53 is stabilized specifically in the tumor, and a direct examination of how and if mutant p53 stabilization contributes to tumorigenesis can now be tested in the zebrafish cancer models. Finally, such p53 monoclonal antibodies can now be used for future anticancer screening for small molecules and chemotherapeutics that activate the p53 response in wild-type and specific genetic backgrounds.

4.6. Melanoma Melanoma is the deadliest form of skin cancer, accounting for 80% of the deaths from skin cancer, and incidence continues to rise rapidly. Aggressive and resistant to most therapies, individuals with metastatic melanoma often have a life expectancy of less than 1 year. Progression from melanocyte to

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melanoma can be histopathologically defined as a series of steps (The Clark model): The first step begins as a proliferation of normal melanocytes to form a nevus, followed by atypical growth and the development of a dysplastic nevus (step 2), which then begins unlimited hyperplasia and radial growth (step 3), followed by a vertical growth phase, crossing the basement membrane and forming a tumor (step 4), and finally, successfully spreading throughout the body as metastatic tumors (step 5) (Clark et al., 1991; Mihm et al., 1971). Defining the genetic changes that promote the transition from one step to the next is a significant challenge in the melanoma field. While some genes are known (importantly, MITF and the INK4a/ARF locus controlling the ‘‘Rb-pathway’’ and ‘‘p53-pathway’’), a stepwise mole-tomelanoma molecular signature, and an understanding of how environmental factors influence transitions between steps, is far from complete. Molecular pathology of melanoma has been significantly advanced by the finding that over 60% of human melanoma and nevi have a mutation in the kinase BRAF, of which over 80% are a single activating mutation, BRAFV600E (Davies et al., 2002; Pollock et al., 2003; Stratton et al., 2004). Activated in most cancers, the RAS-RAF-MEK-ERK MAPK signaling cascade can promote a range of intracellular effects including gene expression, cell cycle progression, cell motility, and cell survival (Dhillon et al., 2007). Tumor cells can activate this pathway through diverse mechanisms, including gain-of function mutations of the components themselves, (e.g., BRAFV600E). The ‘‘addiction’’ of a broad spectrum of tumors to the continued activation of MAPK signaling makes it a prime target for pharmacological intervention, and clinical trials are ongoing with inhibitors of RAS, RAF, and MEK signaling (Gray-Schopfer et al., 2007; SeboltLeopold, 2004). The high proportion of BRAF mutations in nevi and melanoma, coupled with the knowledge that nevi with BRAF mutations can remain arrested without progressing to melanoma, suggests that BRAF can promote nevi but that additional factors are required for melanoma development (Dong et al., 2003; Pollock et al., 2003). Transgenic zebrafish expressing human BRAFV600E from the mitfa promoter (to express BRAF in melanocytes of the fish) provided causal evidence for the observations in humans (Patton et al., 2005). Fish expressing BRAFV600E developed large black lesions, histologically comparable to human nevi. The fish-nevi progressed to melanoma only when fish lacked the tumor-suppressor p53. In humans, p53 is only rarely mutated in melanoma; instead, mutations in ARF (encoding p14) prevent inhibition of the Mdm2 pathway and effectively shut down p53 activity (Sharpless and Chin, 2003). In addition, mouse genetics show that when genetic integrity of ARF is maintained, p53 mutations are found in melanomas, again providing evidence that the p53 pathway is crucial to melanoma suppression (Bardeesy et al., 2001; Walker and Hayward, 2002). The zebrafish melanomas could be serially

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transplanted, demonstrating tumorigenicity of the melanoma, and like human melanoma, the tumors had dramatic genome instability (Patton et al., 2005). This model is important for demonstrating that BRAF has a causal role in promoting nevi development, and that further alterations are required to promote further steps toward melanoma.

4.7. The APC-Wnt pathway Colon cancer incidence continues to rise in Western populations, in part due to dietary conditions, and by the age of 70 years, almost half of the people in these populations have low numbers of noninvasive, premalignant polpys in the intestinal tract. Polyps themselves are not maligant, but are considered a primary transforming event in digestive tract neoplasia (Kim and Lance, 1997). The intestinal cells of the intestine are arranged in villi, with the differentiated epithelial cells at the top of these villi to absorb nutrients and water or secrete aprotective mucus-like lining. Differentied epithelial cells soon undergo programmed apoptosis and are shed from the villi, to be replaced by younger cells derived and migrated from the lower regions of the crypt. In the lower regions of the colonic crypts, b-catenin activates the T-cell factor (TCF/LEF ) transcription factors to actively maintain stem cells and the proliferating undifferentiated enterocyte progenitors of the colonic crypts. In the upper regions of the crypts, the adenomatous polyposis coli (APC) protein inhibits the b-catenin protein, inducing cell cycle arrest differentiation (Rattis et al., 2004; Reya and Clevers, 2005). While most polyps and cases of colon cancer are sporadic, the small percentage of people carrying germ-line mutations in the APC gene develops familial adenomatous polyposis (FAP) syndrome in which polyps carpet the intestinal wall, and are highly predisposed to frank carcinoma (Fodde, 2002). The intestinal wall of the zebrafish intestine is lined with continuous, irregularly shaped, epithelial folds. Larger than the microvilli seen in mammals, their cellular organization within these folds mirrors mammalian cellular organization, with proliferation concentrated at the base of the intestinal folds, differentiation along the crypt-villi axis and in the upper regions of the fold, and self-renewal of the epithelial cells. Like the mammalian intestine, zebrafish intestinal cells include columnar-shaped absorptive enterocytes, goblet cells, and enteroendocrine cells; but unlike mammals, the zebrafish lack the Paneth cells and may have a fourth epithelial cell type analogous to mammalian M-cells (Wallace et al., 2005). Mice homozygous for APC mutations do not complete gastrulation, and heterozyotes develop tumors in the small intestine. To understand how the APC functions within the zebrafish, Hans Clevers and colleagues explored the developmental and cancer phenotypes generated by mutations in the

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APC gene (Hurlstone et al., 2003). Screening an ENU-mutagenized library for mutations in APC (via TILLING), they identified a premature stop codon corresponding to amino acid 1318 of human APC (allele apcmcr ), predicted to result in constitutive Wnt/b-catenin signaling. Genetic crosses of apcmcr heterozygous fish resulted in apcmcr homozygous embryos that died by day 3 with multiple phenotypes. Of the range of phenotypes associated with activated Wnt signaling, the heart defect observed could be rescued by RNA expressing part of the human APC gene. Detailed microscopy and gene expression signatures (with in situ hybridization markers) revealed that the apcmcr mutant embryos had excessive endocardial cushion formation; b-catenin, TCF-responsive genes, and cell cycle markers were expressed throughout the apcmcr mutant endocardium proliferation. Thus, APC in the heart has a specific role in restricting Wnt/b-catenein signaling to control proliferation and the epithelial to mesenchymal transition of the cardiac valve. The loss of the normal tight control of b-catenin and TCF gene expression in the developing apcmcr embryos strongly suggests that zebrafish APC functions in the same molecular pathways as in humans. These pathways are also similar in the adult intestine, as apcmcr heterozygous adult fish developed mammalian-like intestinal polys with increased PCNA proliferation in the intervillus pockets and high levels of b-catenin along the villus axis (Haramis et al., 2006). As with FAP patients, hepatic adenomas also develop, with increased b-catenin and PCNA expression, coupled with increased apoptotic bodies. Further characterization showed the TCF4 target genes, cmyc and axin2, are highly expressed in the intestinal and hepatic adenomas of apcmcr heterozygous adult fish. DMBA treatment increased the onset, frequency, size, and dysplasia of both the intestinal and liver tumors, and broadened the tumor spectrum to include the pancreas and bile duct. These studies show that the APC protein in zebrafish performs as a tumor suppressor, as seen in mammals—the tumor spectrum resembles that seen in people with FAP syndrome, the tumor histology is similar, and the same molecular pathways are dysregulated. The promise of these studies is the ability to now push this system toward the discovery, to search for genetic mutations or small molecules that can suppress the APC mutant phenotype in embryos, and that can then be directly tested against the adult cancer phenotype (Fig. 1.4).

4.8. PTEN in development and cancer The tumor-suppressor gene PTEN functions to control AKT signaling, and is frequently mutated in many human cancers (Di Cristofano and Pandolfi, 2000; Zbuk and Eng, 2007). Zebrafish have two PTEN orthologues, ptena and ptenb, related to the mammalian PTEN gene. Two approaches have

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A

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Figure 1.4 Embryonic developmental phenotypes and adult cancer predisposition. Mutations in cancer-relevant genes can cause developmental phenotypes in the embryos that are distinct from the cancer phenotypes found in adults. Two examples are shown: apc (A^D) and separase/espl1(E^H). (A) normal cardiac morphology. (B) apcmcr homozygous embryos display defective cardiac valve formation, resulting in trapping of blood within the atrium. (C) normal adult intestine. (D) intestinal adenoma in an apcmcr heterozygote. Brown staining indicates high level expression of the proliferation marker, PCNA. (E) close-up of the eye of a 36-hpf zebrafish embryo. Proliferating cells are stained with an antibody to phosphohistone H3. (F) Similar view of a separase/espl1 homozygous mutant embryo. Many fewer cells are proliferating, and the nuclei are polyploid and much larger than normal. (G) Normal liver. (H) Cholangiocarcinoma in the liver of a separase/espl1 heterozygous adult after exposure to the carcinogen, MNNG. (A,B, and D courtesy of Ana-Pavlina Haramis. E,F, G, and H from Shepard, Amatruda, and Zon, unpublished data.)

been used in zebrafish to examine the role of PTEN in development and disease: morpholino knockdown and germ-line ENU point mutations identified by TILLING (Croushore et al., 2005; Faucherre et al., 2007). Loss of either ptena or ptenb revealed an increase in phospho-AKT, indicating that both PTEN genes regulate AKT signaling in developing embryos. While loss of either gene alters AKT signaling, ptena and ptenb have distinct roles in development: ptena morphants have irregularities in notochord, vasculogenesis, head shape, and ear development, while ptenb morphants also have head, tail, and yolk phenotypes (Croushore et al., 2005). Such morphant phenotypes, in particular the specific changes associated with intersegmental blood vessel formation, complement mammalian and zebrafish studies showing that VEGF signals through AKT during blood vessel development (Liang et al., 2007; Tong et al., 2006). However, in mice and flies, PTEN regulates cell and organ size, as well as proliferation, phenotypes not closely analyzed in zebrafish pten morphants. Thus, while such morphant studies may point toward important roles in development for PTEN, how this relates to the cell size and proliferation remain unknown.

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In contrast to the morphant embryonic lethal phenotypes, mutants with nonsense mutations in ptena and ptenb are viable (perhaps due to maternal RNA contribution to allow embryogenesis), only becoming essential when combined as double pten a/b/ mutant lines. Importantly, for comparison to human biology, zebrafish lines with mutations in ptenb/ develop tumors at 7 months of age (Faucherre et al., 2008). Close analysis of these tumors, as well as the developmental phenotypes, will be an important step to understanding how closely aligned zebrafish PTEN function is to human gene function.

5. Xenotransplantation 5.1. Zebrafish as a biological readout of signaling pathways in human cancer The ability to easily inject zebrafish embryos with human tumor cells provides the opportunity to converge embryonic development with tumorigenesis. Cancer cells, like embryonic cells, engage in bidirectional communication with surrounding cells via molecular signaling pathways. It is this idea—communication between cancer cells and their environment—which stimulated Topczewska et al. (2006) to inject melanoma cells into the blastula-stage zebrafish and use the developing embryo as a biological readout for cancer cell signaling pathways. Earlier studies by this team had shown that human tumor cells can be injected into the developing zebrafish embryo and survive through adulthood (Lee et al., 2005). It was only with injection of highly aggressive melanoma cells, however, that embryonic changes begin to take place. Depending on the site of melanoma cell engraftment, embryos developed an outgrowth on the head, additional axial mesoderm, or an almost complete extraembryonic axis (Topczewska et al., 2006). Noting the similarity between their novel assay with the classical developmental transplantation experiments—specifically, transplantation of the dorsal organizer expressing the morphogen Nodal can induce a secondary axis—Topczewska et al. discovered that Nodal expression from the melanoma cells was able to transform the fate of the surrounding normal embryonic cells. Relating these findings to human cancer, normal human skin and melanoma samples at increasing histopathological stages of aggressiveness were examined for Nodal expression. Absent in normal skin, and most primary melanomas (except for small clusters of tumor cells in the vertical growth phase), Nodal was strongly expressed in metastatic melanoma samples. Inhibition of Nodal released the highly aggressive melanoma cellular phenotype (i.e., amelanotic, transdifferentiated), restoring the pigmentation of the melanocytes. In addition, when Nodal was reduced in aggressive melanoma cells, the cancer cells were reduced in their ability to form

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colonies by over half. Extrapolating from fish to human and mouse cells, reduction of Nodal in aggressive human melanoma cells reduced tumor incidence in the transplanted mouse by 30%. This study is especially important because of the new information it provides to the melanoma field: aggressive melanoma cells express embryonic morphogens, thereby reprogramming neighboring cells to create a prime cancer microenvironmental niche, and Nodal expression correlates with melanoma tumor progression suggesting that Nodal may be a new diagnostic marker and target for drug therapy design (Abbott et al., 2007; Hendrix et al., 2007).

5.2. Studying human tumors in zebrafish Cancer cell lines in culture allow for effective assessment of gene function on proliferation, contact inhibition, and anchorage-independent growth. However, it is difficult to assess the physiological impact of the tumor microenvironment in culture. Recently, xenotransplantation studies in zebrafish embryos and larvae have found that cell lines from a variety of human and mouse cancers, including metastatic melanoma, pancreatic, ovarian, breast, and colorectal cell lines, are capable of proliferating, invading, and forming tumor masses in the zebrafish embryo. In contrast, nonaggressive cancer cells, nontumorigenic mouse embryonic fibroblasts, or nontumorigenic human foreskin cells do not form tumor masses or induce a specific phenotype when injected into zebrafish (Lee and Herlyn, 2006; Nicoli and Presta, 2007; Nicoli et al., 2007; Stoletov et al., 2007). As the embryo is transparent, currently available genetic tools (such as fli1:egfp transgenic lines) can be used to visualize angiogenesis and single cell migration in the living tumor. The mammalian tumor xenografts induce vessel neovascularization and remodeling by recruiting zebrafish vascular endothelial cells to form vessels (Nicoli et al., 2007). Using high-resolution confocal microscopy, Richard Klemke and colleagues have revealed interactions between invading tumor cells interacting with and integrating into the vasulature by physically docking at vascular openings, or ‘‘portholes’’ caused by the secretion of VEGF from the cancer cells (Stoletov et al., 2007). Using MDA-435 breast adenocarcinoma cells engineered to overexpress human RhoC—a small GTPase that controls actin/myosin cytoskeleton, and is implicated in cancer progression—the breast cancer cells more aggressively invade surrounding tissues, and alter morphology to a rounded, amoeboid-like shape, migrating by extending small membrane protrusions and blebs. When RhoC expression was combined with expression of VEGF, the cancer cells redirected their invasion to the remodeled vessels, and enhanced intravasation into the vasculature by protruding large membrane projections into the lumen. Xenotransplantation into the zebrafish embryo avoids time-consuming mouse transplantation studies which require large numbers of cancer cells,

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and only small volumes of small molecules are needed to test effective compounds. For example, the FGF receptor-1 tyrosine kinase inhibitor SU5402 or the VEGFR inhibitor SU5416 specifically inhibited the host response to tumor neovascularization, restored integrity to highly permeable vessels, and reduced tumor cell size in the mammalian xenograft models (Nicoli et al., 2007; Stoletov et al., 2007). In addition, the Presta laboratory has shown that morpholino oligonucleotide-targeted gene inactivation of VE-cadherin induces a specific loss in tumor vasculature, providing a foundation for using the xenotransplantation model to study and identify genes that affect tumor angiogenesis (Nicoli and Presta, 2007; Nicoli et al., 2007). Geiger et al. established xenografts of human glioblastoma cells in zebrafish embryos, and demonstrated that the tumors could be successfully treated with radiation, as addition of the radiosensitizer temozolamide increased the beneficial effect of radiation. Addition of temozolamide did not adversely affect embryonic development, thus adding additional information about the safety of the drug during embryogenesis (Geiger et al., 2008). Like wise, Lally et al. identified 40 -bromo-30 -nitropropiophenone in a chemical screen as a radiosensitizer of glioma cells, and applying this to their xenografts of human glioma cells in zebrafish embryos and mice, the compound enhanced radiosensitization of the tumors without significant toxicity to other tissues (Lally et al., 2007). These studies highlight the potential for high-throughput screening in zebrafish for compounds that selectively target tumor tissue and cells. The differences observed on the host embryo between differing cancer cell lines (induction of developmental phenotype vs tumor growth in zebrafish embryos) may be in part due to differences in technical manipulation. It seems more likely, however, that different cancer cell express signals that allow for either developmental changes or tumor development. These xenotransplanation assays are unique properties of the zebrafish system, and provide an important, novel, and readily feasible system to study cancer cell–environment interaction. The potential to screen for chemical compounds that specifically affect tumor vascularization offers advantages when compared with other animal models; although small molecules are easily absorbed into the embryo and can be used for screening of small molecules at physiologically relevant doses, it is unknown how the pharmacokinetics of a given compound would compare between fish and humans. The appeal of creating human tumor xenografts in fish embryos lies in the small size of the host organism, and also in the optical transparency of the embryos, which greatly facilitates imaging studies. In contrast, the robust pigmentation of adult zebrafish can make in vivo imaging of tumor behavior difficult. The recent description of the ‘‘casper’’ fish, which lacks both melanophores and iridophores, promises to greatly facilitate the analysis of transplanted and endogenous tumors in adult zebrafish (White et al., 2008).

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6. Chemical Genetics: Small-Molecule Screening in Zebrafish A major goal of creating zebrafish cancer models—via forward or reverse-genetic strategies, as outlined above—is the creation of a platform useful for drug discovery. One way in which this might occur is through classical suppressor-enhancer screens. Imagine, for example, a second-site mutation that, when present, abrogated the tumor predisposition of a p53deficient or BRAFV600E-transgenic zebrafish. Such a mutation would identify a gene whose product is an excellent target for an anticancer pharmaceutical. Another exciting, and more direct, way of identifying targeted agents is to screen zebrafish directly with the pharmacological agents themselves. Zebrafish, particularly the embryos, offer many advantages for this kind of screen. The small size of zebrafish embryos means that several embryos can be assayed in each well of a 96- well plate holding 100–200 ml of water, minimizing the amount of drug needed. Embryos readily absorb most compounds, meaning that the small molecules can simply be added to the water (Zon and Peterson, 2005). Small molecules may thus be identified that affect cancer-relevant processes, such as angiogenesis (Chan et al., 2002, and as described above), or those that suppress the deleterious effects of a given mutation or transgene, providing lead compounds that can be developed into novel treatments, such as the rescue of the pten-deficient embryonic lethality with a phosphatidy linositol-3-kinase inhibitor (Faucherre et al., 2008). The Zon laboratory has taken a small molecule screening approach using a library of 16,000 compounds to identify a single compound that specifically rescued the crash&burn cell proliferation mutant (Stern et al., 2005). Applying the same library of chemicals to wild-type zebrafish embryos and assaying effects on cell proliferation, they then isolated 14 compounds with novel antiproliferative activity (Murphey et al., 2006). These compounds, which had not been detected in previous cell line-based screens of the library, are potential anticancer agents. Because zebrafish embryos do not suffer from the drawbacks of cell lines—such as the accumulation of mutations necessary to allow indefinite growth in vitro—it is to be expected that small molecule screens in zebrafish will continue to uncover novel biology and to contribute importantly to cancer drug development. In a small-molecule screen for new modulators of the HSC homeostasis, North et al. (2007) in the Zon laboratory identified prostaglandin (PG) E2 synthesis as a key pathway for HSC development during zebrafish embryonic development (North et al., 2007). Using the HSC markers runx1 and cmyb—required for both zebrafish and mammalian stem cell development— more than 2000 small molecules were screened for compounds that enhanced or suppressed RNA in situ hybridization patterns of runx1 and cmyb in the ventral wall of the dorsal aorta (the region analogous to the

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aorta-gonad-mesonephros in mammals). With minor affects to the vasculature, substances that affected the PG pathway, such as linoleic acid or a stable derivative of PGE2 (dmPGE2), promoted an increase in zebrafish HSCs, while inhibition of the pathway, such as with a COX2 inhibitor, caused a decrease in HSCS. In adult zebrafish, administration of dmPGE2 enhanced hematopoietic recovery after sublethal irradiation. Translating to the mammalian system, administration of dmPGE2 following 5-FU bone marrow injury enhanced bone marrow recovery in the mouse, while COX inhibitors inhibited recovery. As COX inhibitors are administered to provide relief from inflammation and pain (e.g., aspirin and other NSAIDs), these studies suggest that administration of COX inhibitors after human bone marrow transplantation may weaken HSC engraftment. On the other hand, administration of PGE2 or its derivatives may benefit patients with bone marrow failure or enhance recovery subsequent to bone marrow transplantation, and preparations are under way for a clinical trial to test this idea (L. I. Zon, personal communication).

7. Summary This is an exciting time for the field of cancer biology and genetics in the zebrafish system. With the first forward- and reverse-genetic cancer models firmly established, we can look to a rapid expansion in the repertoire of available mutants and transgenics. Continued innovations will provide for the zebrafish system the same types of powerful genome manipulations that are currently routine for yeast, worms, flies, and mice. In addition, the valuable, ongoing work from many investigators will push forward the frontiers of developmental biology, imaging, and high-throughput screening techniques in zebrafish, which will have tangible benefits for making improved cancer models. For the zebrafish system to reach its full potential, it is critical that the full genetic power of the system be applied to these cancer models. We look forward to large-scale genetic or chemical screens carried out using robust, physiological zebrafish cancer models as a platform for the identification of novel cancer genes, and promising antineoplastic compounds.

ACKNOWLEDGMENTS We thank members of the Patton laboratory for critical reading of the manuscript, Dr. Nathalie Sphyris for help with the text, and Drs. Ana-Pavlina Haramis and Hans Clevers for images for Figure 3. E.E.P. is supported by an MRC Career Development Award, and by grants from the Wellcome Trust and the Association for International Cancer Research. J.F.A. is supported by the Horchow Family Endowed Scholarship in Pediatrics and by grants from the Lance Armstrong Foundation and NIH/NCI.

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Cellular and Molecular Biological Aspects of Cervical Intraepithelial Neoplasia Fjodor Kisseljov, Olga Sakharova, and Tatjana Kondratjeva Contents 1. Introduction 2. Cervical Cancer as a Unique Model of Human Carcinogenesis 2.1. Association with HPV infection 2.2. Steps of cervical carcinogenesis 2.3. Effective prophylactic vaccines 3. Biology of Cervical Neoplasias 3.1. Cervical cancer incidence and mortality in different populations 3.2. Cervical cancer types and staging 3.3. Precancerous lesions (CINs) and their diagnosis 3.4. HPV as a causal factor of CIN and cervical carcinomas 3.5. HPV testing and typing for clinical practice 4. Cellular Aspects of Cervical Tumors Progression 4.1. Morphological terminology used in cervical pathology 4.2. Morphological classification of precancerous lesions and cancer 4.3. Cytomorphological criteria of precancerous lesions 4.4. Dynamics of precancer lesion conversion 5. Molecular Aspects of Cervical Carcinogenesis 5.1. Basic characteristics of papillomaviruses 5.2. Regulatory region of the genome 5.3. Early genes of HPV 5.4. Viral DNA in tumors and precancer lesions 5.5. Epigenetic changes in tumors and CIN 6. Conclusions References

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N. N. Blochin Cancer Research Center, Kashirskoe shoesse 24, Moscow 115478, Russia International Review of Cell and Molecular Biology, Volume 271 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01202-1

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2008 Elsevier Inc. All rights reserved.

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Abstract Cervical cancer is one of the most common cancers in women. The development of this disease involves reversible changes in the cervical tissue leading to various cellular abnormalities and ultimately to cervical cancer. Several welldefined stages of cervical neoplasia are described, namely, precancer lesions and cancer. Squamous cell carcinomas and adenocarcinomas are most frequent among them, the former being much more common. Each stage is characterized by specific morphological changes. These changes were analyzed in the context of recent molecular biology data. Cervical carcinogenesis associated with infection with high-risk human papillomaviruses (HPVs) contains several early genes that are necessary for viral replication and among them two genes (E6 and E7) play a key role in the induction of cervical carcinogenesis. The main targets of their products are tumor-suppressor genes p53 and retinoblastoma, and their function is inhibited by E6 and E7 proteins. Both E6 and E7 are multifunctional and participate in many cellular functions associated with cell proliferation. The viral genome persists in transformed cells in episomal or integrated form (or both), and possible role of such type of persistence in tumor progression is discussed. Progression of the disease also involves many epigenetic changes. These include methylation of the genes relevant to cell proliferation and differentiation, activation of telomerase, and global changes in cellular gene expression. The cervical cancer is the first cancer that can be effectively prevented by vaccination. Key Words: Cervix, Epigenetic changes, Papillomaviruses, Tumors, Tumor transforming genes. ß 2008 Elsevier Inc.

1. Introduction Several human neoplasias are known, whose association with viral infection has been confirmed. These include adult T-cell leukemia associated with human T-cell lymphotropic virus, hepatocellular carcinoma and hepatitis B and C viruses, Burkitt’s lymphoma and Epstein-Barr virus, Kaposi’s sarcoma and human herpesvirus 8, and cervical cancer and human papillomaviruses (HPV) (Zur Hausen, 1996, 2006b). These neoplasias account for 20% of malignancies arising in humans. Virus-associated human tumors have been extensively reviewed by Zur Hausen (2006b). In this chapter, various aspects of the best-studied human cancer, cervical cancer, associated with papillomaviruses are considered. We tried to integrate the molecular, biological, and cytological aspects of this disease. At the same time, many problems of its pathogenesis remain controversial and we focused only on some of them.

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2. Cervical Cancer as a Unique Model of Human Carcinogenesis 2.1. Association with HPV infection HPV infections are the most commonly diagnosed sexually transmitted (viral or oncogenic) disease today. HPV DNA testing of asymptomatic women in the general population estimated the prevalence of HPV infection to be in the range of 2–44%. This wide variation is largely explained by age differences among population samples studied and by different molecular sensitivity of assays used to detect HPV DNA. More than a hundred HPV genotypes have been identified (De Villiers, 2001). HPVs infect the skin and mucous membranes and initially cause benign epithelial lesions. Some HPVs can be oncogenic and their infection gives rise to malignant tumors. The HPV family is very heterogeneous; different high-risk HPV types can cause anogenital carcinomas as well as nonanogenital cancers (Zur Hausen, 1977, 2006a,b). Thirty-four HPV types are associated with anogenital neoplasias. Large case-control and prospective studies as well as experimental data demonstrate the dominant role of HPV-16 and, to a lesser extent, HPV-18 in carcinogenesis. Table 2.1 summarizes the data on HPV role in anogenital and nonanogenital cancers. Sexually transmitted HPVs are the major etiological factor of cervical intraepithelial lesions and cancer, that is why cervical intraepithelial neoplasia (CIN) and cervical cancer are a unique model of human carcinogenesis. Table 2.1 Roles of HPV in anogenital and nonanogenital cancers (adapted from Zur Hausen, 2006b)

Cancer type

HPV types involved

Anogenital Cervical, vulval, vaginal, 16, 18, 31, 33, 35, 39, 45, penile, and anal cancers 51, 52, 56, 58, 59, 66 Nonanogenital Head and neck Oral, tonsillar, 16, 18, 33 and oropharyngeal cancers Other Breast, prostate, 5, 8, 6, 11, 14, 16, 17, 18, 20, 23, 30, 31, 33, 47, 57 lung, colon, rectal, ovarian, bladder, nasal, sinonasal, conjunctival, laryngeal, esophageal, and skin cancers

HPVpositive (%)

90–50

25 3–10

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HPV has been recognized as an essential cause of cervical cancer, and the association applies equally to squamous cell carcinoma and adenocarcinoma. Two of the oncogenic types, 16 and 18, are jointly responsible for 70% of the world’s cervical cancer cases. Three lines of evidence confirm the etiological role of HPV in cervical carcinogenesis: (1) the identification of HPV DNA in most cervical cancer biopsy specimens worldwide (Arends et al., 1991), (2) relative risk for cervical squamous cell carcinoma and adenocarcinoma of more than 70% for several high-risk HPV types in case-control studies, and (3) relative risk of 10% for women with HPV infection in cohort studies (IARC, 1995; WHO classification, 2003; Zur Hausen, 2006a). In addition to the viral factors, genetic and immune factors play an important role in the cervical cancer initiation. Long-term use of hormonal contraception, high parity, tobacco smoking, and coinfection with HIV increase the risk of cervical cancer, which may also apply to coinfection with Chlamydia trachomatis, herpes simplex virus type 2 (HSV-2), certain dietary deficiencies, and so on. All these factors are important for the progression from cervical HPV infection to cervical intraepithelial lesions and cancer (Munoz et al., 2006). Prospective studies demonstrated a prolonged HPV infection and a low rate of clearance in smoking women, which significantly increased the rate of persistent HPV infections. Longterm hormonal contraception use may activate the genes of persistent HPVs and, probably, increase the duration of HPV persistence. The risk of cervical cancer is elevated in multiparous women. The possible role of HSV-2 in cervical cancer is difficult to interpret; HSV infections can induce selective DNA amplification of persistent HPVs and suppress the cell immune response. The effect of HIV in cervical neoplasias is mediated by the virus-induced immunosuppression and coactivation of the HPV promoter. Bacterial vaginosis, Neisseria gonorrhoeae, Treponema pallidum, and Candida albicans infections have also been considered as cofactors in cervical carcinogenesis (Zur Hausen, 2006b). The available experimental data indicate that viral gene expression of latently infected cervical cells is a prerequisite for uncontrolled growth properties and malignant phenotype. The epidemiological studies in 1990s supported this concept and identified HPV infections as a major risk factor for CIN and cervical cancer. The risk associated with various HPV types is different. Table 2.2 classifies the HPV types by their carcinogenic effect (Munoz et al., 2003).

2.2. Steps of cervical carcinogenesis The natural development of cervical cancer involves reversible changes in the cervical tissue from a normal state to various cellular abnormalities that ultimately lead to cervical cancer. The development of CIN includes a

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Table 2.2 Epidemiological classification of HPV types Group

HPV type

High-risk Probable high-risk Low-risk

16, 18, 31, 33, 39, 45, 51, 52, 56, 58, 59 26, 53, 66, 68, 73, 82 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81

sequence of events that underlie the cytological screening for cervical cancer (Mitchel et al., 1994). The HPV infection can remain latent for long periods (up to 20 years); most HPV infections are subclinical and transient with more than 90% clearance within 2 years; they spontaneously regress due to the cellular immune response. In about 10% of individuals, HPV infection may produce benign and low-grade cervical lesions. Certain HPV types can persist and eventually progress to high-grade precancerous lesions and invasive cervical cancer. Low-risk HPV types cause low-grade cervical intraepithelial lesions (CIN1 or LSIL) and high-risk HPV types cause high-grade cervical intraepithelial lesions (CIN2/3 or HSIL). HPV-6/11 cause 25% of CIN1 lesions; 75% of CIN1 lesions are caused by high-risk HPV types (25% by HPV-16/18). About 10–20% of HPV patients have persistent HPV infection. Long-term viral persistence is required to develop cervical precancer and invasive cervical carcinoma. The interaction between high-risk HPV types and host or environmental factors appears to play a role in the disease progression (Pagliusi and Aguado, 2004).

2.3. Effective prophylactic vaccines The identification of oncogenic viral infections led to the development of preventive vaccines against high-risk HPV, which were based on the expression of HPV structural proteins in recombinant vectors. Similar vaccines were developed against HPV types 16 and 18 as well as against types 6 and 11 (that cause genital warts and low-grade cervical lesions). Large-scale clinical trials have shown the preventive potential of these vaccines (Harper et al., 2004; Koutsky et al., 2002; Pagliusi and Aguado, 2004; Villa et al., 2005). The current vaccines, Glaxo Smith Kline and Merck Gardasil, cover two highly oncogenic types, HPV-16 and HPV-18. The US Food and Drug Administration (FDA) approved Merck quadrivalent vaccine that covers four viral types: high-risk HPV-16 and HPV-18 and low-risk HPV-6 and HPV-11. The approval of the Glaxo Smith Kline bivalent vaccine is expected soon. These vaccines were developed using virus-like particles containing the L1 capsid proteins of the above-mentioned HPV types.

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These vaccines are effective against the viral infection and disease; their efficacy substantially decreases in women already infected with HPV; and they are not therapeutically effective. Attempts are being made to develop therapeutic vaccines, and one of such attempts can become successful in the near future. In this case, the virus-like particle vaccine is based on a recombinant protein containing fragments of the L1 capsid protein and the major HPV oncoprotein E7 (Kaufmann et al., 2007).

3. Biology of Cervical Neoplasias 3.1. Cervical cancer incidence and mortality in different populations In 1990, cervical cancer accounted for 10% of all cancers in women worldwide (471,000 cases in total). Cervical cancer represents the third most common cancer in females and the most common cancer in subSaharan Africa, Central America, South Central Asia, and Melanesia. Approximately 233,000 women died of cervical cancer in the year 2000 and more than 190,000 of them were from developing countries. Zimbabwe and India stand out for the high incidence and high mortality from cervical cancer. In 2002, more than 273,000 deaths from cervical cancer were recorded, which accounted for 9% of female cancer deaths (Ferlay et al., 2001, 2003). The incidence and mortality rates varied 17-fold between different regions in the world; these rates were 83,437 and 39,512 in more developed regions and 409,404 and 233,776 in less developed regions, respectively; the crude and world age-standardized rates were 13.6 and 10.3 for the incidence and 6.4 and 4 for the mortality in more developed regions, whereas the respective indices were 16.6 and 19.1 for the incidence and 9.5 and 11.2 for the mortality in less developed regions (adapted from the GLOBOCAN 2002 database). About 80% of cases occurred in developing countries that do not have adequate screening programs.

3.2. Cervical cancer types and staging Approximately 70–75% of cervical cancers arise from flattened or ‘‘squamous’’ cells covering the cervix (exocervix), and 15–25% of cervical cancers are adenocarcinomas. The cervical cancer pathology is shown in Table 2.3. Squamous cell carcinoma is commonly associated with HPV-16 and adenocarcinomas with HPV-18. The tumor node metastasis staging system for cervical cancer is based predominantly on the extent of the primary tumor (Table 2.4). The lymphatic system is the most common site for metastatic spread.

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Table 2.3 Pathology of cervical cancers (Hunter, 2002) Cervical cancer type (main)

Frequency

Squamous cell carcinomas (main types): keratinizing, nonkeratinizing, non-neuroendocrine, and verrucous Adenocarcinomas (main types): adenoma malignum, mucinous, papillary, endometrioid, clear cell, and adenoid cystic Adenosquamous carcinoma Glassy cell carcinoma Neuroendocrine small cell carcinoma

75–80% 15–20% Rare Rare Rare

Table 2.4 The tumor node metastasis staging system for cervical cancers (AJCC, 2002)

T Tx T0 T1 T1a

Primary tumor Tumor cannot be assessed No evidence of primary tumor Tumor confined to corpus utery Invasive tumor diagnosed only by microscopy; stromal invasion with a maximum depth of 5 mm measured from the base of the epithelium and a horizontal spread of 7 mm T1b Clinically visible tumor confined to the cervix lesions or microscopic lesion greater than T1A T2 Tumor invades beyond uterus but not to pelvic wall or lower third of vagina T2a Tumor without parametrial invasion T2b Tumor with parametrial invasion T3 Tumor extends to pelvic wall involves lower third of vagina causes hydronephrosis or nonfunctioning kidney T3a Tumor involves lower third of vagina, no extension to pelvic wall T3b Tumor extends to pelvic wall causes hydronephrosis or nonfunctioning kidney T4 Tumor invades bladder mucosa or rectum extends beyond true pelvis N Regional lymph nodes Nx Regional nodes cannot be assessed N0 No regional nodes involved N1 Regional lymph node metastasis M Distant metastasis Mx Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis

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3.3. Precancerous lesions (CINs) and their diagnosis One of the earliest steps in cervical cancer development is CIN (or LSIL). Its progression depends on many factors. The risk of CIN is closely linked to the number of sexual partners and HPV exposure. It peaks in early reproductive life when HPV can be detected in as many as 39% of adolescents, and 20% of women under age 19 in a sexually transmitted disease clinic developed CIN2 or -3. The strong association between HPV-16 and highgrade CIN demonstrated in follow-up studies suggests that HPV infections induce high-grade lesions within a relatively short period. The risk of CIN drops substantially in the forth or fifth decades of life, which coincides with a sharp reduction in the frequency of HPV (attributed to the development of immunity to HPV) and elimination of the virus from the genital tract in most women. Precursor lesions of the cervix persist longer and progress more quickly in women with oncogenic HPV infections than in women with nononcogenic infections or without HPV. Testing cervical lesions for oncogenic HPVs may help in the identification of candidates for rapid progression (Koutsky et al., 1992; Rosenfeld et al., 1992). Cervical cytology screening is the current method for early detection of precancerous lesions and cancer. The Pap smear, named for pathologist George Papanicolaou, was introduced in the middle of the last century (Papanicolaou and Traut, 1941). For the Pap smear, cells are removed from the cervix; an optimal cervical specimen includes sampling of the squamous and columnar epithelium, encompassing in particular the transformational zone. Cervical cytology screening techniques include conventional Pap smears or liquid-based cytology. Published sensitivity estimates for Pap smears ranged from 11% to 99% with an average sensitivity between 55% and 80% and specificity between 14% and 97% (Wiley et al., 2004). Pap smears are a highly effective tool to screen for patients at risk of dysplasia or cancer but not a diagnostic tool. The purpose and benefit of the Pap smear is the early detection of abnormal cervical cells, cervical precancer, and cancer. Colposcopy using a long focal-length dissecting-type microscope (10–16 magnification) along with colposcopy-directed biopsies have become a primary method to distinguish women with abnormal cervical cytology. The colposcopic biopsy exposure to 4% acetic acid makes it possible to rule out invasive diseases and to determine the extent of preinvasive disease. Women should begin screening 3 years after the onset of vaginal intercourse or no later than 21 years of age. After initiation, cervical screening should be performed annually with conventional Pap smears or every 2 years using liquid-based cytology (IARC, 2005; Wright et al., 2002). Pap smear screening effectiveness in developing countries is discussed by specialists; the point of those who advocate this technique in developing countries is both cost effectiveness and reduction of mortality

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rates from cervical cancer, while others contend that the strategy of ‘‘see and treat’’ by the use of colposcopy would be more effective in developing countries (Goldhabe-Fiebert, 2005; Goldie et al., 2005; Sherris et al.,2007; Suba and Raab, 2004; Suba et al., 2006a,b). HPV DNA testing for primary cervical cancer has been approved by the FDA. HPV DNA test for high-risk virus types can also be used as a component of both primary screening and workup of abnormal cytology results (ACOG Practice Bulletin, 2005; NHS, 2004). However, HPV DNA testing is not recommended for women younger than 21 years; it is also not useful to test for low-risk virus types in primary and secondary screening. A standardized system for reporting the results of Pap testing was needed to ensure that the results are interpreted properly and consistently. The Bethesda System, established in 1988 and revised in 1991 and 2001, classifies the terminology used in reporting the results of cervical cytology (see below).

3.4. HPV as a causal factor of CIN and cervical carcinomas The causal role of HPV in all intraepithelial neoplasias of the uterine cervix has been established biologically and epidemiologically. HPV DNA was detected in 99.7–100% of cervical cancer cases [3000 cases of cervical carcinomas were analyzed by polymerase chain reaction (PCR)-based assays]. These results lead to the conclusion that HPV is a necessary cause of cervical cancer development. HPV types 16, 18, 31, 33, 35, 45, 52, and 58 are responsible for about 90% of all cancers of cervix. HPV-16 and -18 types are the two most common types in both squamous cell carcinomas and adenocarcinomas (Munoz et al., 2004). A few studies have reported increased risks of developing CINs grade 2/3 associated with presence of HPV-16 and -18 types and of HPV types related phylogenetically to HPV-16 and -18 (Khan et al., 2005). HPV-6 and -11 types usually cause benign cervical lesions such as exophytic genital warts (condilomata acuminata) and CINs grade 1 and are rarely associated with CINs grade 2/3 or carcinomas (Stoler, 2003).

3.5. HPV testing and typing for clinical practice HPV detection and typing in the cervical epithelium is an inherent part of the primary screening for cervical precancer and cancer (Herrington, 1995). While different techniques are used for HPV detection and typing, PCR using species- and type-specific primers is the main HPV diagnostic tool. The identification of the high-risk HPV types most common in human population, HPV-16 and -18, is significant in clinical practice; the test for HPV-16 and -18 can be performed during or after a Pap smear. The test for high-risk HPVs makes it possible to identify women that require thorough

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observation or treatment. Four conventional groups are formed after two tests: (1) negative test for high-risk HPV and no cytological changes in the cervical epithelium; examinations after every 2–3 years are recommended; (2) positive test for high-risk HPV and no cytological changes; regular examinations (at least once a year) are recommended; (3) negative test for high-risk HPV and cytological changes; regular examinations (at least once a year) are recommended; and (4) positive test for high-risk HPV and cytological changes; treatment and meticulous follow up are recommended. Invasive cervical cancer is commonly observed in never-screened women. Testing for high-risk HPV coupled with the standard Pap smear test makes it possible to predict individual prognoses and to develop individual observation and treatment programs. HPV typing is important both for the primary diagnosis (primary screening) and for monitoring women after radical treatment of precancer as well as preinvasive and microinvasive cervical carcinoma (secondary screening). Clearly, testing for high-risk HPVs will be used to identify women for prophylactic vaccination (ACOG Practice Bulletin, 2005; IARC, 1995, 2005, 2007; NHS, 2004; Wright et al., 2002).

4. Cellular Aspects of Cervical Tumors Progression 4.1. Morphological terminology used in cervical pathology Precancerous and early cancerous lesions are of clear interest. Clinical experience indicates that any malignant growth is preceded by specific changes. However, there is no common view of ‘‘precancer’’; some propose a narrow definition, while others tend to broaden it. Finally, in addition to the concept of cancer progressive development, the concept of its de novo development exists (Koutsky et al., 1992). Dysplasia became one of the key morphological criteria of this concept. This term became widespread in both practical morphology and therapy. At the same time, the limits of the term are getting more and more fuzzy, which necessitates the definition of ‘‘dysplasia.’’ Metaplasia is the replacement of one distinctive tissue with another one that differs morphologically and functionally. During metaplasia, the epithelium loses the organotypic form and function, while the histotypic type and function are preserved. These changes in tissue differentiation rely on pluripotent basal cells, which are sources of any epithelium development. Morphological evaluation of metaplastic changes should take into account not only the histotypic and structural properties but also the cytological properties of tissue elements.

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Dysplasia is an abnormal differentiation giving rise to cells with pathological properties. Metaplasia and dysplasia can develop independently; however, it is very important that dysplasia can develop on the background of metaplasia. The relationships between metaplasia, atypical hyperplasia, and dysplasia remain controversial, which leads to terminological confusion and complicates the interpretation of data. Dysplasia should be considered only as controlled and reversible precancerous abnormalities of epithelial differentiation resulting from the proliferation of cambial elements (undifferentiated pluripotent cells of the basal layer) to atypical cells with no polarity and affected histological structure ¨ sto¨r and Rome, 1994; without the membrane invasion (Anderson, 1995; O Richart, 1973; WHO, 2003). In the cervical epithelium, dysplasia is characterized by abnormal cell composition and architectonics. The cells become heteromorphic and demonstrate wide variation in the size and shape. The nuclei become hyperchromatic and oversized relative to the normal nuclei. This phenomenon is called dyskaryosis. The number of mitotic figures increases and they are found in unusual sites of the epithelial layer. In cervical dysplasia, mitoses can be detected in any (including surface) layer of the multilayered epithelium as against basal cells only in the norm. However, atypical mitoses are not commonly observed. Dysplasia is also characterized by abnormal architectonics as a loss of the normal epithelial structure, polarity, and sometimes histotypic or organotypic pattern: the vertical anisomorphy of cells is lost in the multilayered squamous epithelium and the layer is replaced with basal cells instead of progressive differentiation of the basal elements into squamous cells. Dysplasia of the cervical multilayered squamous epithelium features limited numbers of proliferation foci with affected vertical anisomorphy of cells in the layer, basal cell hyperplasia, nuclear polymorphism and hyperchromatism, enlarged nuclei, higher nuclear/cytoplasmic ratio, hyper- and parakeratotic lesions, and high mitotic activity. At the same time, the pathological elements to different extents replace the epithelial layer usually not reaching the surface layers. Different stages (grades) of dysplasia are recognized according to the degree of epithelial proliferation and structural and cellular atypia, affecting the cell organization (Arends et al., 1998; Woodman et al., 2007). The most significant morphological features of dysplasia include nuclei polymorphism and abnormal mitoses. The limits between different grades of dysplasia and preinvasive cancer or sometimes invasive cancer are not always clearly defined by morphological analysis (Al-Nafussi and Colquhoun, 1990; Al-Nafussi and Hughes, 1994; Anderson et al., 1991; Creagh et al., 1995; Heatly, 2002; Lowe et al., 1997; McCluggage et al., 1996). Adequate identification of dysplasia and its grade is of principal clinical significance and largely determines the risk of malignant transformation and treatment approach. The probability of malignant transformation of the regenerating, hyperplastic, or metaplastic epithelium is low. The risk of

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transformation increases in the case of dysplasia, and severe dysplasia demonstrating cellular changes similar to cancerous ones appears to correspond to the highest risk. The progress of the recent years in studying cervical carcinogenesis is primarily due to the elucidation of the role of papillomaviruses. Their role in dysplastic pathogenesis is considered below.

4.2. Morphological classification of precancerous lesions and cancer The morphological diversity of cervical lesions introduced several cervical lesion classifications that are briefly described below. The Papanicolaou classification This cytological classification was widely recognized worldwide until 1990s. It is based on the evaluation of cellular atypia: class I, absence of atypical cells; class II, epithelial atypias mediated by inflammation; class III, atypical cells but no diagnosis (doubtful smear); class IV, suggestive of malignancy; and class V, conclusive for malignancy. Now it is clear that many cases interpreted as false negatives were precancerous lesions (DeMay, 1996; Kobelin et al., 1998; Nanda et al., 2000; Papanicolaou and Traut, 1941; Renshaw et al., 2001; Renshaw, 1997). The WHO classification for cervical cancer screening (Anderson, ¨ sto¨r and 1995; Buckley, 1995; Lowe et al., 1997; Mitchell et al., 1996; O Rome, 1994; Richart, 1973; WHO, 2003): 1. Squamous epithelial abnormalities, benign changes in squamous epithelium. 2. Koilocytes, no changes suggestive of CIN. 3. Squamous cells, changes of undetermined significance. 4. CIN1, mild dysplasia. 5. CIN2, moderate dysplasia. 6. CIN3, severe dysplasia. 7. Carcinoma, suggestive of invasion. 8. Invasive squamous cell carcinoma. Cervical intraepithelial neoplasia. The term CIN was introduced by Richart (1973) as a disease with precancerous changes in the epithelium (Fig. 2.1A). CIN1 features the differentiation with maintained stratification and vertical anisomorphy of two-third of the epithelial layer. Surface cells can have mild atypia including the presence of koilocytes. The nuclear abnormalities (commonly mild) can be observed in different epithelial layers. Not frequent mitoses can be found in the basal layers. Atypical mitoses are very rare (Fig. 2.1B). CIN2 features the differentiation in a half of the epithelial layer; nuclear abnormalities (of different grades) are observed in both upper and basal/ parabasal epithelial layers. The number of mitoses increases, particularly, in

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Figure 2.1 Histology of cervical intraepithelial neoplasias: (A) zone of transformation, (B) CIN1, (C) CIN2, and (D) CIN3.

the deep layers, and abnormal mitotic figures appear (amitosis) (Fig. 2.1C). CIN3 features abnormal differentiation usually extended to all epithelial layers; significant nuclear abnormalities are also observed in all layers; the number of mitoses and amitoses increases (Fig. 2.1D). The histological and, particularly, cytological markers of cervical precancerous conditions vary between different authors and can provide for subjective estimates of the nuclear and cellular properties corresponding to each CIN stage. This necessitates the identification of more contrast conditions of precancerous epithelium, which was realized in the Bethesda classification. The Bethesda Nomenclature System (National Cancer Institute Workshop, 1998; Schenk et al., 1998; Sherman et al., 1992; Solomon and Nayar, 2003): The National Cancer Institute of United States has developed and proposed the Bethesda Nomenclature System (1989, 1991, 2003) for practical use. This classification recognizes the following abnormalities: 1. Atypical squamous cells of undetermined significance (ASCUS). 2. Squamous intraperitoneal lesion features the abnormal growth of cervical squamous cells and is cytologically divided into low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL), distinguished by the lesion volume and the number of atypical cells. 3. Squamous cell carcinoma.

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The analysis of published data on the correlation between the cytological and histological criteria demonstrates that the differentiation into HSIL and LSIL is more consistent than the CIN1, CIN2, and CIN3 grades (Ismail et al., 1990; Massad et al., 2001; Robertson et al., 1989; Sherman et al., 1992, 2003).

4.3. Cytomorphological criteria of precancerous lesions In terms of morphology, CIN1 and LSIL often resemble dysregenerative and metaplastic changes. Many cytologists claim that the mild dysplasia diagnosis is poorly reproducible since the main changes take place deep in the multilayered squamous epithelium. Atypical cells are not numerous and isolated in different, largely surface layers. Mild dysplasia often correlates with HPV markers: koilocytes, dyskeratocytes, and binucleated cells (Al-Nafussi and Colquhoun et al., 1990; Kurman et al., 1991; Lee et al., 1997; Noller, 2005; Stoler, 2003; Stoler and Schiffman, 2001; Ziol et al., 1998). 4.3.1. Koilocytosis Koilocytosis is the main marker of HPV infection. Koilocyte is a cervical squamous cell producing HPV particles with the owl’s eye appearance resulting from the nucleus compression and halo formation around it. Morphological examination demonstrates cells with a wide semitransparent perinuclear zone (halo) and vacuolated cytoplasm (Fig. 2.2). The significance of detecting koilocytes in cervical smears is discussed; however, its relationship with viral activity and risk of dysplasia progression remains unclear (Dudding et al., 1996; Elit et al., 1999; Herrington, 1995; Kruse et al., 2003a,b).

Figure 2.2 Koilocytes.

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The cytological markers of the virus include nuclear polymorphism in the basal layer cells coupled with koilocyte detection in the surface layers, a combination of dyskaryosis in some cells and koilocytosis in others, and the appearance of atypical immature cells both isolated and in clusters. The ‘‘blurry’’ chromatin structure with dense structures in the nucleus as well as two- to four-nucleate cells with the overlapping nuclei that adhere to one another can also be considered as a specific marker. 4.3.2. CIN1 (LSIL) Most pathologists tend to consider CIN1 as a result of HPV infection, which can spontaneously regress, particularly, if only a single virus type is detected (Dudding et al., 1996; Elit et al., 1999; The ALTS-LSIL triage group, 2003; Woodman et al., 2007; Zur Hausen, 2002). Hence, koilocytosis is a manifestation of squamous intraepithelial changes. Overall, these changes in the cervical epithelium range from the norm to carcinoma. The discussion about the significance of koilocytosis in cervical smears still continues (IARC, 1995, 2007; Jeffers et al., 1994; Lee et al., 1997; Londesborough et al., 1996; Martin-Hirsch et al., 2002), although most specialists consider it as the most reliable pathognomonic indication of active HPV infection and replication (Baak et al., 2006; Herrington et al., 1995; Lowe et al., 1997; Zur Hausen, 2002). At the same time, koilocytic atypia is revealed in less than a half smears in cervical abnormalities (Kruse et al., 2003a,b; Lee et al., 1997; Stoler and Schiffman, 2001), while nuclear atypia is much more important for the adequate screening. The assessment of most nuclear changes is also subjective. Note that both nuclear and cytoplasmic atypias are observed in cytological preparations from not only neoplastic but also benign, reactive, and metaplastic tissues. 4.3.3. CIN2 þ CIN3 (HSIL) CIN2 is the soft spot in terms of diagnosis and clinical approach. In most European countries, mild and moderate dysplasias are grouped together and distinguished from severe dysplasia and carcinoma in situ, while the Bethesda classification conversely includes CIN2 into HSIL. CIN2 proved to be associated with the persistence of the high-risk virus in many cases, and hence with its transformation to CIN3 and cancer is of high risk (Arends et al., 1991; Cuzick et al., 1994). Accordingly, the most efficient screening programs are aimed to identify and ablate CIN2 and CIN3. It was also proposed that CIN2 is closer to CIN1 and CIN3 in terms of the risk of malignant progression (IARC, 1995, 2007; Mitchell et al., 1996; Sherman et al., 2003; Zur Hausen, 2002). In other words, the risk of CIN2 progression to invasive cancer observed in a minor fraction of women remains poorly predictable and obscure.

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As dysplasia progresses, the number of dyskaryotic cells increases, atypical characters appear in cells of the intermediate and parabasal layers, the nuclear/cytoplasmic ratio decreases, the proportion of cells with irregular outline increases, and clusters of abnormal cells appear. The limits between different grades of dysplasia and preinvasive or sometimes invasive cancer are not always clear in morphological study. Dysplasia features scattered arrangement of atypical cells, while cancer demonstrates more pronounced cellular and nuclear polymorphism, high number of cells with homogeneous granular chromatin, and clusters of clearly atypical cells.

4.4. Dynamics of precancer lesion conversion As mentioned above, mild dysplasia (CIN1 and LSIL) represents heterogeneous pathologies. Clearly, most of them are of polyclonal origin and can limit the proliferation of cells infected with low-risk HPV. However, a minor fraction of dysplasias simultaneously or successively convert to CIN3, carcinoma in situ, or invasive cancer. This can be attributed to two mechanisms: clonal selection of cells with the least differentiated phenotype or independent development of different morphological variants of CIN. The proportion between these mechanisms and their role in precancerous changes remain unclear. High-risk HPVs (types 16 or 18) are more commonly associated with severe dysplasia and carcinoma in situ. These virus types can favor the clonal progression or induce cancer de novo. CIN3 as well as in situ and invasive carcinoma always represent a monoclonal lesion (both independent and successive). Recent genetic data indicate possible progression of dysplasia by clonal selection; however, the malignant transformation of target cells (cells in the transformation zone) substantially depends on other mechanisms apart from HPV infection. Target cells in the transformation zone can differentiate in two directions, squamous or glandular epithelium; and HPV type was proposed to determine the differentiation pathway of cells infected or transformed by the virus. Low-risk viruses and HPV-16 are commonly associated with squamous cell neoplasia, while HPV-18 is associated with the glandular differentiation (cervical glandular intraepithelial neoplasia). Overall, this is reflected in the cytological and histological pattern of different variants of dysplasia: mild dysplastic proliferation foci feature a more diverse cell composition compared with severe dysplasia and carcinoma in situ (Hopman et al., 2005). LSIL and ASCUS identified by cytological screening proved to persist longer and progress faster in women infected with oncogenic HPV compared with patients infected with nononcogenic HPV or uninfected ones (IARC, 1995, 2007; Kruse et al., 2003a,b; The ALTS-LSIL triage group, 2003). Cytological screening supplemented with HPV typing will likely improve the diagnosis of the risk of CIN1 and SIL transformation to CIN3

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(Arends et al., 1991; Cox, 1995; Cuzic et al., 2003; Cuzick et al., 1994; Garrenstroom et al., 1994; Herrington et al., 1995; Ziol et al., 1998). HPV sequences in the abnormal cervical epithelium is a molecular marker of CIN and cancer; however, it provides no information about the progression stage, degree of cell differentiation, and disease prognosis. Complex evaluation of molecular and cytomorphological markers of dysplasia can shed light on the CIN pathology and diagnosis and improve the selection of individual treatment and monitoring strategy of CIN patients as well as the results of the primary and secondary screening of cervical cancer. HPV infection was proposed to induce only minimum transitory changes in the epithelium (Grenko et al., 2000; Stoler and Schiffman, 2001; Woodman et al., 2003); however, in the absence of therapy, the infection can persist and transform to CIN3 or, in a fraction of women, to cancer (after 10 or more years). Published data indicate that about 10% of CIN1 cases and 20% of CIN2 progress to CIN3, and at least 12% of CIN3 cases transform to cancer (Arends et al., 1998; McIndoe et al., 1984; Woodman et al., 2003; Zur Hausen, 2002). Clearly, the prediction of the risk of CIN progression requires, in addition to the test for high- and low-risk HPV, the analysis of morphological properties and different markers such as p16, Ki-67, p53, retinoblastoma, cytokeratins 14 and 13, as well as DNA ploidy test. At the same time, such tests should be quantitative and separate surface, middle, and deep epithelial layers for the accurate identification of CIN and evaluation of its biological potential (Baak et al., 2006; Dehn et al., 2007; Kruse et al., 2002, 2003a,b; Lane and Wells, 1994).

5. Molecular Aspects of Cervical Carcinogenesis 5.1. Basic characteristics of papillomaviruses Papillomaviruses contain double-stranded circular DNA from 7.2 to 8 kb. In contrast to polyomaviruses (SV40 is one of them), only one DNA strand is transcriptionally active (Chow and Broker, 1994; De Villiers, 2001). Nine open reading frames (ORFs) were identified in the genome of papillomaviruses, which code for two groups of proteins: late genes encoding two structural proteins, major capsid protein L1 and minor capsid protein L2, and early genes (E1–E7) with the key role in DNA replication control, viral replication, and initiation and maintenance of the transformed phenotype of the cells. Both groups of genes are controlled by the upstream regulatory region (URR), also called the long control region (LCR), with binding sites for different transcription factors of viral or cellular origin (Chen et al., 1982; Danos et al., 1982) (Fig. 2.3).

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E2 E5 E4 L2

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Viral assembly (L1 and L2)

• •

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• •

•

•

•

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••

Viral DNA replication (E6 and E7)

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Episomal viral DNA

.•• •

.•

.•

(E1 and E2, E6 and E7) Infection of basal cells (E1 and E2)

Basal (stem) cells Basement membrane Normal epithelium

Infected epithelium

Figure 2.3 Structure of human papillomavirus and cycle of its reproduction in epithelium.

The life cycle of HPV is associated with different layers of epithelium (Fig. 2.3). More than 20 RNA types can be transcribed from the full-length viral genome. Their qualitative and quantitative composition can vary with cell types and persistence of the viral genome (episomal or integrated; see below). At the same time, the constitutive expression of two early oncogenes E6 and E7 is the key event in the transformation (Hawley-Nelson

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et al., 1989; Munger et al., 1989; Von Knebel Doeberitz et al., 1992; Yee et al., 1985). The E6 gene transcript may present in several splice forms. Splicing is typical of other mRNA species largely encoded by the early proteins (Fauquet et al., 2005). This chapter largely focuses on the E6 and E7, which are the main oncogenes in papillomaviruses, and their expression is required to both initiate malignant transformation and maintain the transformed phenotype (Mantovani and Banks, 2001; Munger et al., 2001).

5.2. Regulatory region of the genome The nonstructural region of the viral genome is represented by the URR, also called the LCR, comprising about 12% of the genome. The main function of this region is to control transcription of epithelial-specific genes as well as viral genes during cell differentiation. The URR contains many binding sites for transcription factors of both viral and cellular origin (Hawley-Nelson, 1989; May et al., 1994; Munger et al., 1989; Schwarz et al., 1985; Von Knebel Doeberitz et al., 1992; Yee et al., 1985). The regulation of viral gene expression and its coordination with the expression of cellular genes is quite complex, and most regulatory functions are mediated by the URR sequences. Additional regulatory mechanisms include the utilization of different promoters, alternative splicing (largely, in the E1 gene), differential transcription termination, and different stability of viral RNAs. cis-active elements within the URR control the transcription of the major viral oncogenes E6 and E7. Most transcription factor-binding sites identified in the URR are common for many HPV types; however, type-specific ones have also been found. The common sites include the TFID binding to TATA box 30 bp upstream of the early transcription start site as well as the binding sites for transcription factors Sp-1 and Ap-1 downstream of these sequences. The binding sites for transcription factors NF-1, TEF-1, TEF-2, Oct-1, Ar-2, KRF-1, and YY1 were found in many HPV types (Bauknecht et al., 1992; Butz and Hoppe-Seyler, 1993; Ishiji et al., 1992; Mack and Laimins, 1991; O’Connor and Bernard, 1995; O’Connor et al., 1996, 2000). The keratinocyte-specific enhancers provide for the epithelial tropism. Anogenital papillomaviruses in addition feature glucocorticoid-responsive sequences (Gloss et al., 1987).

5.3. Early genes of HPV 5.3.1. E1 gene This ORF is transcribed into polycistronic mRNA and no individual mRNA has been found for this gene. The encoded protein plays the key role in viral DNA replication. E1 binds and hydrolyzes ATP to exert a helicase activity and interacts with the cellular DNA polymerase a. This protein binds to the proximal URR, where the viral origin of replication is

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located. The E1 primary structure is the most conserved among papillomaviruses. This helicase is required for efficient DNA replication. E1 is also involved in HPV replication, which requires its phosphorylation by the cyclin/cdk complex, and this process promotes the cytoplasmic localization of E1 (Bonne-Andrea et al., 1995; Deng et al., 2004; Holt et al., 1994; Li et al., 1993; Ustav and Stenlund, 1991; Ustav et al., 1991; Yang et al., 1993). 5.3.2. E2 gene This gene codes for at least two proteins functioning as transcription factors. These gene products are the main viral factors of the genome transcriptional activity (Androphy et al., 1987; Bouvard et al., 1994; Chiang et al., 1992; Demeret et al., 1997; Hegde, 2002; Ustav and Stenlund, 1991). The E2 products contain two critical domains, the C-terminal DNA-binding domain and the N-terminal transactivation domain, which provide E2 dimerization on specific binding sites (Dostatni et al., 1991). In human keratinocytes, E2 functions as a transcriptional activator. The URR in high-risk HPV contain four E2-binding sites, three of which are essential for the viral life cycle, that is, for the regulation of DNA replication and promoter repression (Stubenrauch et al., 1998). E2 overexpression suppresses proliferation, arrests the cell cycle, and triggers apoptosis (Blachone and Demeret, 2003). The E2 ORF often contains deletions in cervical cancer since the circular viral DNA is commonly linearized in this gene region during the viral DNA integration into the genome (Schwarz et al., 1985). Usually this leads to uncontrolled expression of the E6 and E7 genes and eventually to cancer progression (Romanczuk and Howley, 1992). Mutations in the E2 ORF, particularly, in the URR-binding sites, increase the immortalizing potential of HPV-16 DNA. In cervical carcinogenesis, a break in the E2 ORF and the subsequent integration of viral DNA into the cell genome are relatively late events that are not observed before the CIN3 stage (Durst et al., 1992; Klaes et al., 1999; Matsukura et al., 1989). E2 protein can interact with E1 to stimulate viral DNA replication and promote E1 binding to the origin of replication (Seo et al., 1993). 5.3.3. E4 gene The E4 protein is synthesized from mRNA representing one of early spliced RNA species starting in the E1 ORF and including the whole E4 ORF. This is the major viral transcript in HPV-infected cells. The gene product is localized in differentiating epithelial cells. The involvement of E4 in transforming cells or maintaining the viral DNA as a circular episome has not been confirmed. Apparently, the main E4 function is related to the productive infection since this protein affects normal differentiation, and thus favors the viral particle maturation. E4 can interact and induce the collapse of the cytokeratin network in the cell (Chow et al., 1987; Doorbar et al.,

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1991; Roberts et al., 1993). In addition, the recruitment of this protein to the active cyclin B/cdk complex releases it from the nucleus and arrests the cell cycle in G2. This arrest allows the viral DNA amplification (Nakahara et al., 2002; Rai et al., 2004). 5.3.4. E5 gene The product of this gene is the major oncoprotein in bovine papillomavirus (Schiller et al., 1986). During HPV infection, E5 has a very low transforming potential. This 80-amino acid highly hydrophobic protein is largely localized to the endoplasmic reticulum membranes, Golgi system, and to a lesser extent to cytoplasmic membranes (Bubb et al., 1988; Conrad et al., 1993; Halbert and Halloway, 1988). E5 complexes with EGF receptor, although no growth factor receptors are activated in this case. E5 binds to the membrane-bound ATPase, which is a part of the gap junction complex. E5 in high- and low-risk viruses activates the mitogen-activated protein kinase (MAPK) pathway and decreases major histocompatibility complex (MHC) class I synthesis. In addition, E5 induces the perturbation of MHC class II maturation (Cartin and Alonso, 2003). E5 can cooperate with E6 to induce the proliferation of the primary cells and mouse NIH 3T3 cells, which results in the mouse cell transformation. E5 contributes to human keratinocyte transformation after the transfection with the E6 and E7 genes (Stoppler et al., 1996). Thus, E5 is an important factor during early infection. Regular losses of this gene after viral DNA integration also confirm that E5 is hardly essential during late viral oncogenesis. 5.3.5. Transforming gene E6 and its functions 5.3.5.1. Biological effects The product of this major HPV oncogene is a 151-amino acid protein. It includes four Cys-X-X-Cys motifs, which form two zinc finger structures (Barbosa et al., 1989; Cole and Danos, 1987) (Fig. 2.4). The E6 protein is detected both in the cytoplasm and in the nucleus of infected cells and can interact with many cellular proteins. Three nuclear localization signals were identified in E6, and the mutations in these regions prevent E6 relocation from the cytoplasm to the nucleus, p53 degradation, and cell immortalization. In contrast to E6 in high-risk HPV, this protein in low-risk HPV-6 is localized to the cytoplasm (Cooper et al., 2003). The expression of this protein alone immortalizes several human cell types. E6 can cooperate with the Ras oncoprotein to immortalize rodent primary cells and with E7 of high-risk HPV to efficiently immortalize human cells. It also stimulates substrate-independent growth of mouse NIH 3T3 cells (Garner-Hamrick et al., 2004; Hawley-Nelson et al., 1989; Lowe et al., 1994; Munger et al., 1989). Transfection of the E6 gene under the control of the keratin K14 promoter induces cell hyperproliferation and epidermal hyperplasia as well as benign skin tumors in adult mice. Hence,

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Bak p300 p53

p53 C

N

C

Zn2+

C

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C

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PKA site C Zn2+

ETQV

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hDIg MAGI-1 MUPP-1 hScrib

Figure 2.4

Structure of high-risk human papillomavirus E6 gene.

E6 is involved in early carcinogenesis resulting in benign tumors, whereas E7 plays the key role in late carcinogenesis leading to malignant transformation (see below) (Hall and Alexander, 2003; Song et al., 1999). E6 can activate the vascular endothelial growth factor promoter and, thus, mediate angiogenesis (Lopez-Ocejo et al., 2000). E6 upregulates the expression of the fibroblast growth factor-binding protein, an angiogenic switch molecule, in human keratinocytes. The expression of this factor increases in early carcinogenesis and this process is independent of immortalization and transformation ( Vikhanskaya et al., 2002). At the background of high angiogenic activity, the induction of hypoxia-inducible factor 1a was observed in cervical cells transformed with E6 and E7 (Tang et al., 2007). E6 overcomes the antiproliferative signals and induces serumdeprived cells that overexpress inhibitors of cyclin-dependent kinases 16ink4a or p27kip1 to enter the S phase. This activity is conserved in high-risk HPVs (Malanchi et al., 2004). The progression of cervical carcinomas features chromosomal rearrangements resulting from the fusion of broken chromosomal ends. The frequency of anaphase bridges depends on telomerase activity in clones expressing E6/E7, and a correlation has been demonstrated between the E6 expression, telomerase activity, and chromosomal instability. Anaphase bridges also correlate with the presence of micronuclei (Duensing and Munger, 2003). E6 controls the cytoskeletal dynamics in keratinocytes by targeted degradation of p53 and can contribute to the invasive phenotype maintenance (Cooper et al., 2007). E6 is a multifunctional protein, and its interaction with different cellular target proteins as well as viral E7 plays the key role in the initiation and progression of genital tumors (Mantovani and Banks, 2001; Zur Hausen, 2006b).

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5.3.5.2. Interaction with tumor-suppressor p53 The main E6 target is the tumor-suppressor p53. In normal cells, this protein is induced in response to DNA damage to promote cell cycle arrest or apoptosis (ElDeiry et al., 1993; Lane and Wells, 1994). In HPV-infected cells, E6 of high-risk HPV inhibits the oncosuppressive function of p53 by its degradation via the ubiquitin–proteasome pathway (Pim et al., 1994; Scheffner et al., 1990), which lowers the level of this protein. This prevents cell cycle arrest or apoptosis induction (Matlachewski et al., 1986). In normal cells exposed to stress in the absence of HPV, p53 can be bound by ubiquitin ligase Mdm2 and degraded in proteasomes (Honda et al., 1997). In the case of HPV infection, this degradation pathway is inhibited and p53 degradation becomes entirely dependent on E6. E6 realizes the p53 degradation by recruiting another ubiquitin ligase, E6-AP. The N-terminal domain of E6 binds to this enzyme, which results in a stable complex that is later degraded in proteasomes (Schwarz et al., 1998). E6-AP can bind to several kinases of the SRC family including the Blk kinase, which is degraded via the ubiquitin pathway (Oda et al., 1999). E6, E6-AP, and p53 can form a primary complex, and the domains involved in the protein interaction and complexing have been identified (Cooper et al., 2003). In addition, the HECT domain of the ubiquitin ligase E6-AP in the complex with E6 targets and degrades the tyrosine phosphatase PTPN3 in vivo and in vitro ( Jing et al., 2007). This enzyme is a membrane-associated tyrosine phosphatase involved in the regulation of tyrosine phosphorylation of growth factor receptors and the 97-kDa valosin-containing protein (known as CBC-48 in yeast), which is mutated in colon cancer. Hence, E6 can regulate phosphotyrosine metabolism by targeting the degradation of tyrosine phosphatase. Pals1-associated tight junction (PATJ) protein is a novel target for degradation mediated by E6 of HPV-18 and its isoform E6*. PATJ protein is required for the assembly of such aggregates and is critical for tight junction formation in polarized cells. The E6* protein has lost the PDZ domain (see below) but not the capacity to degrade this complex. This is the first example of two protein isoforms with the same function (Stors and Silverstein, 2007). In addition, there are several more E6-mediated pathways to abolish the inhibition of p53-suppressor activity (Mantovani and Banks, 1999). For instance, E6 can prevent the in vivo repression of p53 transcription by binding the C-terminus of p53. This can be realized by several mechanisms: (1) E6 can compete with p53 for its DNA-binding site, (2) high-risk HPV can interact with the transcriptional activator p300/CBP and repress p53responsive promoters (Lechner and Laimins, 1994), and (3) p53 translocation into the nucleus can be inhibited. The cytoplasmic localization of p53 can be due to masking the nuclear localization signal by binding the p53 C-terminus to E6 (Tao et al., 2003).

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Note that no complete p53 degradation is observed in HPV-infected cells. It is not improbable that the presence of minor p53 quantities is due to the E6* protein translated from one of spliced mRNAs, which interferes with the full-length E6 for binding to p53 (Mantovani and Banks, 2001). Thus, the interaction between E6 and p53 is a key event in cell malignization since long-term degradation of p53 can accumulate genetic mutations in infected cells (Reznikoff et al., 1996; White et al., 1994). 5.3.5.3. E6 activities not associated with p53 Deregulation of transcription and DNA replication by HPV E6. E6 of both high- and low-risk viruses can modulate transcription from many viral and cellular promoters. The targets of such deregulation include p300/CBP, a transcriptional coactivator of many genes involved in the regulation of the cell cycle and immune response. The E6 interaction with p300/CBP can downregulate the transcriptional activity in cooperation with viral E2 (Patel et al., 1999). E6 can interact with the bifunctional TRIP-Br factor that functions as a transcriptional integrator of the E2F1/DP1/RB cell cycle regulatory pathway (then named TRIP-Br1) and as an antagonist of the cyclin-dependent kinase suppression of p16INK4a (then named p34SEI-1). TRIP-Br1 is efficiently induced by E6 of high- and low-risk HPV without degradation of the complex (Cooper et al., 2003). The E6 protein disturbs cellular DNA replication. Normal cells have limited period of proliferative activity due to continuous shortening of terminal DNA fragments (telomeres) after each cell cycle. In immortal cells, this process is stopped by switching on telomerase, an enzyme that elongates telomeres. In HPV-infected cells, E6 activates the gene coding for the telomerase structural subunit (hTERT) (Klingelhutz et al., 1996) and induces telomerase erosion and chromosomal instability (Plug-DeMagio et al., 2004). The complex of E6 with c-Myc can activate the telomerase promoter, although this can be done only by E6 of high-risk HPV. On the other hand, it was shown that the p53 degradation rather than telomerase induction by E6 is required to bypass the cell crisis and immortalize cells under the influence of E6/E7 (McMurray and McCance, 2004). The interaction between E6 and E6-AP is not essential for the telomerase activation (Sekaric et al., 2008). E6 of both high- and low-risk HPV can interact with hMcm7, a component of the DNA replication complex. At the same time, hMcm7 is a substrate for E6-AP ubiquitin ligase, and E6 can trigger the degradation of this complex in vivo (Kukimoto et al., 1998; Kuhne and Banks, 1998). Mitogenic activity of E6. E6 induces cell hyperproliferation and epidermal hyperplasia, and these events do not depend on p53 (Song et al., 1999). E6TP1 can be involved in this process. This protein is highly homologous with the family of GTPase-activating proteins, which are negative

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regulators in the MAPK cascade. E6TP1 protein is involved in the inhibition of RAP-mediated mitogenic signaling (Gao et al., 1999). Inhibition of apoptosis. Several proapoptotic factors have been identified as E6 targets. One of them, Bak, is expressed in the upper epithelial layers and is a target for both high- and low-risk HPV. E6 stimulates ubiquitindependent degradation of Bak catalyzed by E6-AP (Thomas and Banks, 1999). The transcription factor c-Myc is degraded by proteasomes in normal cells, and high-risk HPV can sharply increase this degradation (Gross-Mesilaty et al., 1998). The interference with the p53/PUMA/Bax cascade also contributes to the antiapoptotic function of E6 (Vogt et al., 2006). The suppression of caspases 3 and 8 by E6 inhibits apoptosis (Filippova et al., 2004). E6 activates NF-kB-inducible activity of cIAP (an antiapoptotic protein) and protects against apoptosis in a PDZ-binding motif-dependent manner ( James et al., 2006). Interference with epithelial organization and differentiation. E6 activities include the inhibition of the terminal differentiation of epithelial cells, which normally leads to keratinization and cell death. E6 increases the resistance of human keratinocytes to serum- and calcium-induced differentiation via the p53-independent pathway (Sherman et al., 1997). E6 can also interact with E6BP/ERC-55, which is a calcium-binding protein localized in the endoplasmic reticulum. Apparently, the E6 complex with this protein can mediate the inhibition of p53-independent apoptosis (Chen et al., 1995). E6 targets also include paxillin, a protein-mediating signaling from the plasma membrane to focal adhesions and to the actin cytoskeleton, and its activity is regulated by tyrosine phosphorylation in response to various stimuli including integrins and growth factors. The E6 interaction with paxillin does not lead to its degradation (Tong and Howley, 1997). Interactions with PDZ proteins. A conserved C-terminal domain, which is not involved in p53 binding and degradation, is an important feature of E6 from high-risk HPV. This domain is biologically important since mutations in it impair the ability of E6 to transform rodent cells and immortalize keratinocytes. This region contains a PDZ-binding motif (XT/SXV), which mediates binding proteins with PDZ domains (Crook et al., 1991; Pim et al., 2000).To date, sequences of 80–90 amino acids containing this motif have been found in different proteins involved in the functioning of ion channels, signaling enzymes, and adhesion molecules (Kim, 1997). The first protein identified as an E6 target was hDlg, a human homologue of the Drosophila tumor-suppressor Dlg, required for the formation of adherens junctions, regulation of cell adhesion, apicobasal polarity, and proliferation in epithelial tissues. The loss of this gene function causes aberrant morphology and embryonic lethality (Kijono et al., 1998). hDlg colocalizes with E-cadherin at adherens junctions of epithelial cells and interacts with several proteins including the APC tumor suppressor. The hDlg/APC complex formation blocks cell cycle progression. HPV E6 can target hDlg and

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promote its ubiquitin-mediated degradation (Gargiol et al., 1999). Dlg domains feature variations in high-risk HPVs. HPV-18 E6 has a canonical PDZ-binding motif (ETQV), while HPV-16 E6 has a suboptimal consensus site (ETQL). Accordingly, HPV-18 E6 binds and degrades hDlg more efficiently. The proteins binding E6 through their PDZ domains include hScrib, a protein expressed at epithelial tight junctions, which are a substrate for ubiquitination by the E6/E6-AP complex in vitro and for proteasomal degradation mediated by E6 in vivo (Kijono et al., 1998). The degradation of proteins with PDZ domains mediated by E6 or E6-AP complexes modulates the Notch1 signaling. Notch ligands DELTA1 and DELTA4 interact with Dlg1, which directly binds the E6/E6-AP complex. JAGGED1, a cellular Notch ligand, also contains a PDZ motif (Six et al., 2004; Ascano et al., 2003). LNX, a member of PDZ-containing motives in E3 ubiquitin ligases, can induce Notch signaling. Both JAGGEDI and Notch signaling upregulated in H-SIL and carcinomas (Nie et al., 2002). E6 targets localized to cell junctions include the MAGI-1 protein, which binds to the first of five PDZ domains. This protein complexes with bcatenin, which is known to be deregulated in many human cancers (Glausinger et al., 2000). E6 can also bind to MUPP1, a protein containing several PDZ domains and involved in the signaling (Grm and Banks, 2004). CAL, the cystic fibrosis transmembrane regulator-associated ligand is associated with the Golgi apparatus. This protein interacts with the PDZ motif of E6 that results in the ubiquitin–proteasome degradation, that is, E6 can mediate cellular trafficking processes ( Jeong et al., 2007). 5.3.5.4. Other E6 targets Several more E6-binding proteins have been identified. These include interferon regulatory factor (IRF)-3 and calciumbinding protein E6-BP (Chen et al., 1995; Elston et al., 1998; Ronco et al., 1998). E6 also interferes with insulin signaling (Chen et al., 1995) and promote retinoblastoma protein phosphorylation (Malanchi et al., 2002), while the transcription activator Gps2 and E6 inhibit this activity (Degenhardt and Silverstein, 2001). HPV-18 E6 expression inhibits the Jak-STAT pathway activation by interferon-a but not by interferon-g; in this case, E6 directly interacts with the Tyk2 kinase to modulate its activity (Li et al., 1999). A novel E6-targeted protein, E6TP1, has been identified. There is a direct correlation between its capacity to bind and target E6 for ubiquitinmediated degradation and the transforming activity. It proved homologous to GAP-activating Rap proteins (small G-proteins) (Singh et al., 2003). Methyl guanine methyl transferase (MGMT), a DNA repair enzyme, is a proteolytic E6 target (Malanchi et al., 2002). E6 proved to be involved in the p63 pathway. This pathway has been recently revealed by microarray analysis of 22,000 genes, where HPV

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E2-activated genes and 38 new mitotic genes repressed by E2 were identified. The p63 target genes are activated through silencing of the E6/E6-AP pathway, while the mitotic genes are largely repressed through E7 silencing (Teissier et al., 2007). 5.3.6. Oncogene E7 and its role in carcinogenesis 5.3.6.1. Biological properties E7 is detected in 98% of cervical cancers with no correlation between the expression level and tumor progression (Garner-Hamrick et al., 2004; Song et al., 2000). The E7 gene product is a 98-amino acid phosphoprotein that can bind zinc and contains two Cys-XX-Cys domains. The zinc-binding and two Cys-X-X-Cys domains are similar to those in E6. The N-terminal region of E7 contains two domains, CR1 and CR2 with conserved sequences also shared by adenoviral E1A and large T antigen of SV40. Both domains are crucial for the cell transformation by adenovirus and SV40 (Fig. 2.5). E7 is phosphorylated by casein kinase II (Munger et al., 2001). Its phosphorylation varies during the cell cycle. In the early cycle, E7 is phosphorylated by casein kinase II; however, its activity decreases toward the S phase and E7 is phosphorylated by an unidentified phosphorylase in S phase (Massimi and Banks, 2000). E7 can immortalize primary keratinocytes and transform immortalized NIH 3T3 and, at a low-frequency, human keratinocytes ( Jewers et al., 1992). Myc and E7 cooperate to immortalize human keratinocytes after a pronounced crisis period (Liu et al., 2007). The E7 protein is localized to the nucleus; however, its minor quantities are detectable in the cytoplasm. It is imported to the nucleus via a nonclassical pathway. E7 lacks the classical nuclear localization signal and its

LXCXE CKII 1

15 CR1

37 CR2

C-X-X-C

98 C-X-X-C pRB,p107,p130(binding) pRB,p107,p130(degradation) pRB,p107,p130(E2F complex disruption) IRF-1 F-actin p48 (ISGF3) p21cip1 TBP TAF110 p27kip1 AP-1 hTid α-Glucosidase Mi2β (HDAC) IGFBP-3 M2 PK MPP2 (Forkhead TF) S4 (26S Proteasome)

Figure 2.5

Structure of high-risk human papillomavirus E7 gene.

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transport does not depend on the major import receptors (Angeline et al., 2003). E7 targets to the centrosome fraction, where it associates with the centrosomal regulator g-tubulin in a pocket protein-independent manner. E7 can induce different cell types for apoptosis (Webster et al., 2001). In immortal urothelial cells, E7 induces p53-dependent apoptosis after X-ray exposure (Puthenveettil et al., 1996). E7 sensitizes cervical keratinocytes for apoptosis and interleukin-1a release (Iglesias et al., 1998). E7-induced apoptosis destabilizes pRb, stabilizes p53 ( Jones et al., 1997), and induces the S phase. These processes are independent events in immortalized rodent cells (Alunni-Fabbroni et al., 2000). Apoptosis can be induced by simultaneous expression of HPV-16 E7 and inhibitor of cyclin-dependent kinases (cdk/p21), which is accompanied by the activation of cathepsin B (Kaznelson et al., 2004). E7 expression abrogates certain negative growth factors, which triggers p53-directed G1 arrest, and also inhibition of proliferation mediated by TGF-b in resting suprabasal keratinocyres. Decelerated cell differentiation and high cdk2 activity are observed in E7-expressing keratinocytes ( Jones et al., 1997). E7 of HPV types 6, 16, and 18 mediate transcriptional control of MHC class I heavy chains, antigen processing subunit 1, and proteasome subunit low molecular mass protein 2, which represses these activities (Georgopoulos et al., 2000). Essentially, E7 can inhibit antiviral and antiproliferative activity of interferon-a (Barnard et al., 2000). 5.3.6.2. Interaction with tumor growth suppressors The amino acid sequence of E7 contains a canonical site CXCXE of binding with 105 kDa retinoblastoma protein (pRb), a major tumor suppressor in the cell. It belongs to the group of pocket proteins, which also includes p130 and p107 (Zur Hausen, 1996). The latter proteins have the same E7-binding sites as pRb, but their synthesis differs during the cell cycle: pRb is synthesized throughout the cycle, p107 largely in S phase, and p105 in G0 phase (Berezutskaya et al., 1997; Classon and Dyson, 2001). In normal cells, pRb complexes with transcription factor E2F. E7 binding to this complex destabilizes it and promotes its phosphorylation by cyclin-dependent kinases and E2F release (Edmonds and Vousden, 1989; Weintraub et al., 1985). Consequently, this transcription factor induces genes that advance the cell cycle to S phase or apoptosis. These include genes required for DNA synthesis, such as DNA polymerase and thymidine kinase. Noteworthily, the pRb/E7 complex formation is not limited to high-risk HPV, and low-risk HPV E7 can complex with pRb, but unlike high-risk E7, low-risk E7 cannot activate E2F-inducible genes or degradation of pRb (Piboonnjom et al., 2003; Weintraub et al., 1995). Similar to other pocket proteins, pRb is degraded via the ubiquitin–proteasome pathway (Berezutskaya and Baghi, 1997). E7 associates with an enzymatically active cullin 2 ubiquitin ligase

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complex and the E7/pRB complex contains cullin (Hu et al., 2007; McCaffrey et al., 1999). E7-mediated degradation of pRb is essential for the efficient reversal of cell cycle arrest in G1 mediated by p16ink4. Thus, the E7 capacity to neutralize the inhibiting effect of p16ink4 correlates with its capacity to promote pRb degradation (Giarre et al., 1999). HPV-16 reprograms suprabasal cells for DNA synthesis in correlation with the capacity of E7 to bind pocket proteins but independently of its capacity to promote their degradation. In contrast, the capacity to differentiation perturbation correlates with the E7 capacity to both bind and degrade pocket proteins (Ledt et al., 2005). E7 directly interacts with the coactivator protein p300 (similar to adenoviral E1a type 6 and 16). Such interaction can mediate transcriptional control by E2 (Bernat et al., 2003). The recently described 600 kDa protein is associated with pRb and can interact with E7, which leads to the degradation. This interaction does not depend on pocket proteins and is mediated by the E7 N-terminal domain, which is involved in transformation irrespective of binding to pRb (Huh et al., 2005). However, the destabilization of the pRb tumor suppressor and pocket proteins by E7 is not sufficient to reverse the cell cycle arrest in human keratinocytes (Helt and Galloway, 2001) or to induce cervical dysplasia or neoplasia (Wu et al., 2006). 5.3.6.3. Interaction with cyclin-dependent kinases and their inhibitors E7 can also interact with some proteins crucial for the promotion of cell proliferative activity. These include p21 and p27, protein inhibitors of cyclin-dependent kinases. High-risk HPV E7 directly binds to the cyclin A/cdk2 complex and indirectly (via p107) binds to the cyclin E/cdk2 complex; in this case, the kinase activity associated with cdk2 is maintained (Arroyo et al., 1993; Dyson et al., 1992) and E7 directly activates cdk2 (He et al., 2003). In contrast, low-risk E7 increases the level of cyclins A and E (Martin et al., 1998). Thus, the inhibition of pRb and p21 by HPV E7 efficiently reverses the cell cycle arrest, which consequently causes genomic instability in cells. The interaction with the ARF locus is an important part in the E7 functional activity. The ARF gene encodes a tumor-suppressor protein that inhibits cell cycle progression (Quelle et al., 1995). It functions upstream of p21 by stabilizing and activating p53, which is mediated by the ARF gene product, p14arf, binding to MDM2, an inhibitor of p53 activity. In addition, p14arf can bypass the p53-dependent pathway and regulate the cell cycle arrest and p21-dependent apoptosis (Hemmati et al., 2005). The ARF protein can also interact with some proteins of the E2F family to induce their degradation via the ubiquitin–proteasome pathway (Herdman et al., 2006). Precancer lesions and cervical cancers demonstrate elevated expression of both p14arf and p16ink4a. Paradoxically, HPV E6

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inactivates the p16ink4a gene in immortal cells expressing E6 alone, but p16ink4a (and inhibitor of cyclin-dependent kinases) is overexpressed in E6/E7-transformed cells (Volgareva et al., 2004). In the latter case, the overexpression of p16ink4a is due to the inactivation of pRb by E7 and the subsequent transcriptional activation of the p16ink4a gene by E2F. Since p16ink4a blocks the cyclin D/cdk4/6 complexes, its proliferationinhibitory effect is abolished by E7 via a direct stimulation of cyclins E and A. The overexpression of p14arf can have a similar effect if its inhibitory action on E7 is suppressed by unknown factors; in this case, ARF overexpression does not interfere with the E7 functions (Reznikoff et al., 1996; Wang et al., 2004). 5.3.6.4. Interaction with histone deacetylases The group of these proteins can inhibit E2F-inducible promoters by binding to pRb. E7 binds to histone deacetylase (HDAC) independently of its binding to pRb. These enzymes directly inactivate E2F proteins through deacetylation. Essentially, mutations in the HDAC-binding domain in E7 destabilize the episomal viral genome and increase the life span of cells in culture (Brehm et al., 1998; Weintraub et al., 1995). The activation of tyrosine phosphatase Cdc25 depends on the E7–HDAC binding. This interaction silences IRF-1, an important factor of interferon signaling and immune surveillance in persistent HPV infection. Note that this effect applies only to interferona-induced genes but not to interferon-g-induced ones (Park et al., 2000). In human keratinocytes, E7 induces histone acetylation, which can contribute to the formation of transcriptionally active chromatin, an essential factor of expression of genes important for cell cycle progression (Zhang et al., 2004). E7 of HPV types 6, 16, and 18 interacts with acetyltransferase pCAF, a coactivator of different transcription factors including p53 (Avvakumov et al., 2003). 5.3.6.5. Induction of chromosomal instability Both E6 and E7 can independently induce chromosomal instability; however, their combined action generates mitotic abnormalities and aneuploidy. The latter is largely due to structural centrosome abnormalities (Duensing et al., 2004; Munger et al., 2004). E7 expression in normal diploid cells induces duplication of centrosomes and centrioles, whose number increases as viral infection progresses and viral DNA accumulates in the genome. This process does not depend on E7 interaction with pRb or cell cycle checkpoints (Pett et al., 2004). Other abnormalities in cells expressing E7 include anaphase bridges and lagging chromosomes, which indicate errors in mitotic spindle attachment and double-stranded DNA breaks (Duensing and Munger, 2002a). E7 induces abnormal centrosomes, which corresponds to early malignant transformation (Duensing and Munger, 2002b; Duensing et al., 2001) and also tetrasomy in keratinocytes culture (Southern et al., 2001) as well as

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abnormal duplication through a pocket protein-independent mechanism, which leads to abnormal centrosome duplication (Duensing and Munger, 2002b). HPV-16 E7 activates the Fanconi anemia pathway and, consequently, accelerates chromosomal instability in Fanconi anemia cells (Spardy et al., 2007). 5.3.6.6. Other protein targets of E7

Gene of B-myb protein is regulated by E2F and is transcriptionally activated by high-risk HPV E7. In cells expressing HPV E7, B-myb is constitutively overexpressed (Lam et al., 1994). Promyelocytic leukemia protein (PML) induces senescence being overexpressed in human primary fibroblasts. This process is abolished by E7. The inhibition of PML-induced senescence requires both pRb-dependent and pRb-independent processes (Bishop et al., 2005). Proto-oncoprotein DEK binds nucleic acids and positively regulates high-risk HPV E7. Elevated DEK expression can be a common event in human carcinogenesis reflecting its ability to inhibit senescence (WisaDraper et al., 2005). Transcription factors (TATA-binding protein including TBP, members of the AP-1 family, fork head domain factor MMP 2) (Antimore et al., 1996; Lusher-Fitlaff et al., 1999; Massimi et al., 1997; Phillips and Vousden, 1997). Ca-binding protein S100P and mitochondrial ADP/ATP carrier protein (Schonning et al., 2000). S4 subunit of the 16S proteasome (Berezutskaya and Baghi, 1997). Mi2-b protein, a component of the HDAC complex (Brehm et al., 1998). Insulin-like growth factor (IGF)-binding protein 3 (Mannhardt et al., 2000). Pyruvate kinase type M2. This glycolytic enzyme can be dimeric or tetrameric, and E7 shifts the balance toward the dimeric form (Zwerscke et al., 1999). IGF-binding protein-3 (Mannhardt et al., 2000). Phoshorylated F-actin (Rey et al., 2000). Tyrosine phosphatase Cdc25 that is involved in the G1/S transition through the activation of the cyclin E/cdk2 and cyclin A/cdk2 complexes. The Cdc25A promoter is activated by E7 and its levels sharply increase in E7-expressing cells (Katich et al., 2001). E7 maintains high levels of Cdc25A during deregulation of the cell cycle arrest through the dissociation of the pRb/E2F/HDAC-1 repressor complex (Nguen et al., 2002). Skip protein that interacts with the C-terminus of E7, which inhibits Skip transcriptional activity (Prathapam et al., 2001).

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17 b-estradiol receptor; it complexes with E7 in the cytoplasm but translocates to the nucleus after the hormone is added to promote S-phase entry or accumulation of p21cip1 in differentiated keratinocytes (Balsitis et al., 2006). Serine/threonine phosphatase 2A (PP2A). E7 interaction with PP2A activates the protein kinase B/Akt pathway. Being activated after cell attachment and growth factor signaling, this secondary messenger transmits signals to the cell nucleus to inhibit apoptosis and increase cell survival during proliferation. E7 binds to both the 35 kDa catalytic and 65 kDa structural subunits of PP2A (Pim et al., 2005).

5.4. Viral DNA in tumors and precancer lesions 5.4.1. HPV DNA persistence at different stages of tumor progression Analysis of viral DNA status in cervical precancer lesions and carcinomas meets some technical problems. The early views on the existence of viral DNA as an episome at the early stages of tumor progression and integration into the cellular genome at later stages are still largely accepted; however, recent studies have unveiled many interesting facts, clarifying the mechanisms of tumor progression and the significance of DNA persistence type in this process. Note that different methodical approaches can be used to attack this problem, which is important for the result interpretation. The interpretation of the results may depend on many factors, including: first, the multilayered structure of the epithelium and the localization of the DNA forms to specific layers (see below); second, the heterogeneity of all early lesions, and hence the analysis of different lesion stages at the same time; third, the heterogeneity of cervical cancer resulting from the contamination by stromal and normal cells; and fourth, different methods to analyze the DNA status. These methods can be divided into two groups: (1) amplification of papillomavirus oncogene transcripts (Klaes et al., 1999) and RNA in situ hybridization (Van Tine et al., 2004), and (2) detection of the integrated DNA irrespective of its transcriptional status (Southern blot hybridization, real-time PCR, restriction site PCR, and FISH (Adler et al., 1997; Cooper et al., 1991; Cullen et al., 1991; Durst et al., 1985; Thorland et al., 2003, 2007). Simultaneous application of these techniques in combination with laser dissection could provide significant information about the effect of cellular factors on the type of viral DNA persistence; however, publications involving all these techniques are very rare. As shown above, cervical precancer lesions can be divided into low and high grades (LSIL and HSIL). LSIL is a productive HPV infection with a low risk of progression to invasive disease, while HSIL is an abortive infection, in which the late life cycle of HPV is not supported, and

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consequently no viral particles are produced. To date, we cannot distinguish two major LSIL variants: (1) LSIL capable of progression and containing viral DNA and (2) LSIL that will regress (constituting the bulk of lesions) (Bosch et al., 2002; Remmink et al., 1995). Cervical carcinogenesis is largely associated with squamous epithelium, the structure of which can significantly modulate the HPV replication cycle as well as malignant transformation. The normal epithelium includes four main layers: basal layer (stem cells), parabasal layer, squamous layer, and mature squamous layer. HPV infection is initiated in the basal layer; episomal viral DNA persists in the parabasal cells; viral DNA is replicated in squamous cells; and mature squamous cells produce and release viral particles (Durst et al., 1992; Stoler et al., 1992) (Fig. 2.3). In the normal replication cycle, basal cells of squamous epithelium contain 50–100 copies of circular (episomal) HPV DNA per cell. The expression of viral oncogenes E6 and E7 from episomal DNA is strictly controlled and high expression levels are observed only in suprabasal mitotic cells. In these cells, viral oncogenes induce unprogrammed S-phase induction, which triggers the host replication system required to amplify viral genes and produce virions (Cheng et al., 1995). In this productive system, the expression of viral oncogenes has no carcinogenic effect since it occurs in cells abandoned from squamous epithelium due to their continuous renewal. Hence, the SIL progression requires both the spatial (expression in different layers) and quantitative (expression level) deregulation of this transcriptional control, which eventually leads to high and predominant expression of viral oncogenes in the epithelium. Basal epithelial cells with high levels of viral oncogene expression are maintained over long periods in the cervical epithelium and are targets for the main oncogenic events such as differentiation inhibition and induction of high chromosomal instability. These events drive the process toward the malignant phenotype (Daniel et al., 1995; Jeon et al., 1995). 5.4.2. Integration of viral DNA In most cervical carcinomas, the induction of oncogene expression (and mutations in the host genome) occurs in cells with HPV DNA integrated into the cellular genome (Daniel et al., 1995). In this case, truncated copies of viral DNA integrate into the genome; however, they obligatorily include E6 and E7 genes. Such cells have proliferative advantages, which provide for their growth in mixed populations. The integration requires a doublestranded break in both viral and cellular DNA. The integration is not an essential stage in the productive virus cycle and is accompanied by the deletion of genes not required for the infectious virus production (largely, L1 and L2). Although various parts of the viral genome can be lost, the DNA breakage is most commonly observed either in the 30 -terminal E1 ORF or in the E2 ORF, which leads to a complete or partial loss of the E2

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gene encoding a potential inhibitor of transcription from viral promoters (Bernard et al., 1989; Hwang et al., 1993; Romanczuk et al., 1990). The integration leads to elevated expression of E6 and E7 and high stability of the transcripts, while the protein products bind and inactivate the major cellular tumor suppressors, p53 and pRb. These effects are limited to highrisk HPV (Luft et al., 2001; Munger et al., 2004). Since the expression of the telomerase catalytic subunit (hTERT) is inhibited by HPV E2, the viral integration considerably activates telomerase, one of the key factors of immortalization (Kijono et al., 1998; Klingelhutz et al., 1996; Lee et al., 2002; Veldman et al., 2001). Hence, HPV integration is required for cellular immortalization, deregulation of proliferation, and high genomic instability, which are direct markers of cells involved in malignant transformation (Hanahan and Weinberg, 2000; Hafner et al., 2007; Kraus et al., 2008; Vinokourova et al., 2008). The occurrence of integrated papillomavirus forms (IPFs) in the carcinomas reaches 100% and 80% in HPV-18- and HPV-16-infected stage, respectively. The rate of HPV integration considerably varies from almost no IPFs in LSIL to about 15% transcriptionally active IPFs in HSIL (Ziegert et al., 2003). Transcriptionally silent IPFs with disrupted E2 genes have been reported (Kisseljov et al., 2001). Such differences can be attributed to the methods used for IPF identification. Under some (not yet identified) conditions, these IPFs can be induced, which leads to the selection of cells with active viral genes, and such cells are a potential source for the selection of oncogenic variants. The integration sites are localized by either FISH or integration site sequencing. No specific cellular sequences have been revealed and the integration into the same genomic region occurs in different nucleotide sequences (Wentzensen et al., 2004). Apparently, the preference for the integration into common fragile sites exists. Such sites have been revealed in 38% of 192 integrated copies analyzed (Thorland et al., 2000, 2007; Wentzensen et al., 2002). It is possible that integration into such sites offers a selective advantage or introduces cellular DNA breaks. Several reports have described the integration into transcriptionally active genomic regions (Durst et al., 1987). Such insertional mutagenesis has been shown for HPV-18 integrating into the MYC locus at 8q24 (Ferber et al., 2003a; Peter et al., 2006; Wentzensen et al., 2002). Rare integration events were mapped to the TERT and FANC loci at 5p15 and 9q22, respectively (Ferber et al., 2003b; Wentzensen et al., 2004). HPV integration into the MYC locus activates the gene expression; however, the significance of these data is not obvious (Peter et al., 2006). Thus, the viral DNA in HPV-induced lesions can persist in several forms: full-length episomal and transcriptionally active; integrated truncated and expressing oncogenes E6 and E7; and integrated, presumably truncated, but silent. Accordingly, different cancers and precancer lesions can have

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different combinations of these viral DNA forms, and there is a clear trend to the selection of cells with integrated transcriptionally active viral DNA (Evans et al., 2002; Evans and Cooper, 2004). The clones of such cells are beneficial for uncontrolled proliferation. The presence of several types of viral DNA persistence points to the corresponding systems of transcriptional control, and such systems should include both viral and cellular factors since the viral regulatory region URR is present in all forms. Clearly, the regulation of episomal DNA activity is mediated by E2 (see above), while it can hardly contribute to IPF transcriptional control (being deleted) and cellular factors should play the key role in this process. 5.4.3. Transcriptional regulation of integrated and episomal DNA Since malignant cells can contain IPFs that were silent in SIL, there is a negative control of IPF transcription. As demonstrated previously, E6 and E7 are largely expressed in the upper spinous and granular layers of the epithelium, while much lower expression can be detected in proliferating basal cells, that is, the transcriptional repression of IPF takes place in basal cells. The CCAAT/enhancer-binding protein is critical for the repression, and its binding sites have been found in URR types 16 and 18 (Zhao et al., 1997). Other tissue transcriptional factors such as CCAAT displacement protein can contribute to the repression, and their binding sites have been found in the URR of many HPV types (Apt et al., 1996; O’Connor and Bernard, 1995). One more possible transcription factor is chromatin remodeling by histone deacetylation (Zhao et al., 1999). The transcriptional inhibition has been demonstrated in vivo too. Grafting of immortalized (but not tumorigenic) HPV-infected keratinocytes to nude mice inhibited the early viral genes in epithelial cells, while no silencing was observed after grafting of tumorigenic ones (Bosch et al., 1990; Durst et al., 1991; Zur Hausen, 1986). This introduced the concept of cellular interfering factor (Zur Hausen, 2000). These data allow to propose a possible role of macrophage-derived cytokines (TNF-a, TGF-b, and interleukin-1) (Braun et al., 1990; Kyo et al., 1994; Malejczyk et al., 1994; Rosl et al., 1994; Woodworth et al., 1990), and the resistance to these factors can be due to in vivo tumorigenicity. No such data have been obtained for the episomal forms. Hence, the inhibition of expression of viral oncogenes from IPF requires their transcriptional block in basal cells and resistance to the inhibitory cytokines in high-risk HPV-immortalized keratinocytes. The differences in the transcriptional control of the episomal and IPF DNA can be due to several factors including the activation of the neighboring host sequences that can be modulated by IPF but not episomes, differences in the URR chromatin structure, or differences in the functions of URR regions, in particular, the matrix attachment region (Stunkel et al., 2000).

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5.4.4. Interaction between episomal and integrated HPV DNA This type of interaction was discussed in details by Pett and Coleman (2007). It is well known that the viral DNA integration should take place in cells containing considerable episome quantities. This is the key moment since the E2 regulatory activity depends on the DNA physical state (integrated or episomal). At the same time, E2 should have no effect on episomal DNA transcription but can substantially inhibit IPF DNA transcription. It is possible that the E2 protein expressed from the episome can inhibit expression from IPF through the interaction with the IPF’s URR and underlies the IPF silencing. Overcoming this inhibition can represent a mechanism to select cells with integrated viral DNA (Dowhanick et al., 1995; Hwang et al., 1993). Cells with oncogenes actively expressing from IPF are selected in W12 in vitro culture. This selection is preceded by spontaneous and rapid loss of E2-expressing episomes (Pett et al., 2006). This rapid episomal loss is associated with the activation of antiviral response genes induced by interferon type I (which can induce episomal clearance per se and likely is the primary trigger) (Herdmann et al., 2006). In addition, W12 cells can contain silent IPFs in the population containing almost exclusively episomal DNA, and the selection for cells with active IPFs starts only after episomal inhibitory activities are eliminated. It is difficult to say to which extent this selection can be analyzed in vivo; at least, we have demonstrated silent IPFs at the background of expressing ones in cervical carcinomas (Kisseljov et al., 2004). The expression from IPFs should be relatively active since there are usually one or two integration events, which necessitates the amplification of the viral fragments to gain a stronger growth advantage. This has been directly demonstrated on several cell lines with up to 100 IPF copies (Bechold et al., 2003). Among cells containing multiple copies of IPF DNA, those containing one or several transcription centers (which can result from the URR methylation) are selected (Van Tine et al., 2001, 2004). In particular, in the case of two head-to-tail copies, the selection can be mediated by E6/E7 silencing, resulting from the E2 gene degradation. The level of transcription from IPF can vary in the course of the disease. High expression levels can be required at the early stages to favor additional genetic changes. After such changes were made, high-level expression and numerous transcription centers are not needed any more (Van Tine et al., 2004). 5.4.5. HPV DNA status and progression of the disease To what extent the disease progression depends on the proportion between different DNA forms? According to the FISH data, the spatial expression pattern of the viral genes E6 and E7 is the main distinction between LSIL

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and HSIL: their expression in the basal epithelium is inhibited in LSIL and found throughout the epithelium in HSIL (Durst et al., 1992; Higgins et al., 1992; Stoler et al., 1992). Since only a minor fraction of cells in HSIL contain transcriptionally active IPFs, the spatial deregulation, that is, the inhibition of episomal expression of the oncogenes in a different epithelial layer, should precede complete selection for IPF in HSIL. In addition, such deregulation of expression from episomes can favor complete selection for IPF in HSIL as well as new integration events (Kessis et al., 1996). In this case, IPFs can remain latent until the transcriptional inhibition is eliminated. It is possible that the loss of E2-expressing episomes is the key event in the selection for full-length IPFs, which takes place before and independently of the spatial deregulation of episomal expression of the oncogenes. This means that transcriptionally active viral IPFs contribute to the cervical carcinogenesis by the quantitative deregulation (upregulation) of E6 and E7 at the stage of HSIL with the subsequent induction of genomic instability and mutagenesis required to trigger malignant progression. If episomal clearance is induced in a population of cells with spatially deregulated episomal expression of viral oncogenes and the expression of E6 and E7 is not maintained, the loss of antiapoptotic effects of viral oncogenes induces their apoptosis (Herdman et al., 2006). As a result, such cells are eliminated from population. According to this scenario, only cells that can potentially maintain the expression of E6 and E7, that is, containing latent IPFs, are derepressed after episomal loss. Hence, the main factor of selection for IPFs is their capacity to maintain the expression of E6 and E7 and to inhibit apoptosis. The subsequent clonal selection relies on cells maintaining high levels of E6 and E7 expression from IPFs. Thus, in cancers released from episomes, the integration is a critical prerequisite for neoplastic progression since the absence of latent IPFs leads to lesion regression. However, there may be exceptions from this sequence of events. In particular, it can occur if the neighboring cellular sequences can modulate the effects of cellular factors on viral IPFs, that is, host DNA sequences can modulate host transrepressors or viral E2. In the latter case, the integration events are resistant to the transcriptional inhibition by E2 and require no episomal loss for selection. Indeed, some cervical carcinoma cell lines containing high-risk HPV IPFs are relatively resistant to E2 (Von Knebel Doeberitz et al., 1991). This phenomenon can also explain several clinically detected cases of rapid lesion progression (Peitsaro et al., 2002). At the same time, 12.5% of cervical carcinomas contain episomal transcripts only, which assumes an alternative carcinogenic pathway. In such cases, the transcription of the viral oncogenes from high-risk HPV episomes is quantitatively deregulated by alternative mechanisms that also confer resistance to the inherited mechanisms of episomal clearance such as mutations in the URR that bind transcriptional repressors such as YY1 (May et al., 1994).

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5.5. Epigenetic changes in tumors and CIN Above, we described the genetic changes associated with cervical cancer progression. These include mutations in the cellular genome or persistence of viral DNA as a source of additional genetic confirmation, and the cellular genes and their products interacting with viral oncogenes were described. However, all cancers always feature epigenetic changes. Such changes leave the gene structure unaltered and are independent of mutations but rely on the changes in its transcriptional activity. Such changes directly linked to cancer progression include methylation, telomerase induction, and changes in the specific expression pattern of other cellular genes largely controlling cell proliferation and differentiation. Epigenetic changes are typical of nearly all cancer types and provide for individual patterns of cancer. 5.5.1. DNA methylation DNA Methylation is prominent among epigenetic changes. Most genes contain CpG islands that contain CG stretches of different length within their promoter regions and/or first exons. Cytosine in these dinucleotides can be methylated by cellular methyltransferases (Esteller, 2006). Under normal conditions, these islands are demethylated in transcriptionally active genes and have no effect on transcription. However, in many types of cancer, malignant transformation is accompanied by hypermethylation of CpG islands in many genes, which results in their transcriptional inactivation (Sova et al., 2006; Woodman et al., 2007). Hypermethylation can be reversed by some agents such as 5-azadeoxycytidine (Kisseljova and Kisseljov, 2005). Cervical cancers are no exception to this pattern, and numerous genes subject to methylation have been identified in cervical cancer (Ivanova et al., 2007; Woodman et al., 2007). It should be noted that methylation targets include genes that control differentiation and proliferation, in particular, those encoding suppressors of tumor growth. Hence, methylation can be one of the main epigenetic events in tumor progression and an individual cancer marker, that is, the pattern of hypermethylated genes is specific for each cancer type irrespective of progression stage. The effect of HPV infection on the methylation pattern remains unclear; however, the infection is most likely not the governing factor of this process since methylation is typical of other cancer types too. The phenomenon of aberrant methylation, its early manifestation, and highly sensitive assays for it allow DNA methylation to be used as an early and sensitive marker of cancer (Badal et al., 2003). Extensive data on hypermethylated genes in cervical precancer lesions and carcinomas can be found in the review by Woodman et al. (2007). The number of hypermethylated genes and their methylation level clearly tend to increase as the tumor progresses (from precancer lesion to carcinoma).

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At the same time, the level and frequency of methylated genes can also differ between progression stages. The genes tested for methylation can be divided into several groups: Group 1: Eleven genes hypermethylated in HSIL, while their methylation level in LSIL is relatively low. These include the genes encoding APC (adenomatosis polyposis coli), CCNA (cyclinA1), CDH1 (E-kadherin), CDKN2A (cyclin-dependent kinase inhibitor 2A), DAPK1 (death-associated protein kinase), HIC1 (hypermethylated in cancer 1), IGF4 (immunoglobulin superfamily F4), RARb (retinoic acid receptor b), ROBO1 (roundabout, axon guidance receptor, homologue 1), SLIT1, and SLIT2 (slit homologues 1 and 2). Group 2: Few genes demonstrating no increase in DNA methylation in HSIL relative to LSIL. These include FANCF (Fanconi anemia, complementation group F), FHIT (fragile histidine triad gene), MGMT (O6-methylguanine-DNA methyltransferase), PTEN (phosphatase and tensin homologue, mutated in multiple advanced cancer 1), RASSF1 (Rasassociation [RakGDS/AF6] domain family), SLIT 3 (slit homologue 3), TERT (telomerase reverse transcriptase), and TIMP 3 (tissue metalloproteinase inhibitor 3). Group 3: Several genes whose methylation was not tested in SIL but proved to be high in carcinomas. These include C15ORF48 (chromosome 15 open reading frame 48), MT1G (metallothionein 1G), POU2F3 (POU domain, class 2, transcription factor 3), SFRP 1 (secreted frizzled-related protein 1), SPARC (secreted protein, acidic, rich in cysteine [osteonectin]), TFPI 2 (tissue factor protein inhibitor 2), and TNFRSF10C (tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain). Group 4: Several genes with no or low-level methylation in LSIL relative to carcinomas. These include HSPA2 (heat-shock 70-kDa protein 2), SOCS1, SOCS2 (suppressors of cytokine signaling 1 and 2), TWIST1 (twist homologue 1), and CDH13 (H-cadherin). Note that the methylation data were obtained using different methods, some of which can produce false-positive or false-negative results. Accordingly, some of these genes can be assigned to different groups later. Methylation studies in cervical carcinomas have demonstrated interesting patterns. For instance, the hTERT gene is unmethylated in normal cells, while in 82% carcinomas, it is methylated at specific sites unrelated to transcription control regions (Zinn et al., 2007). Hence, the methylation of this gene is an early event in cervical carcinogenesis. A different pattern is observed for the inhibitor of cyclin-dependent kinases 16ink4a; in contrast to other cancers, its gene is only marginally methylated, which has little effect on the number of cells with elevated expression of 16ink4a (Ivanova et al., 2007; Klaes et al., 2001).

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5.5.2. Activation of telomerase It is common knowledge that normal cells are limited to 30–50 divisions, after which they die. This is due to end underreplication of chromosomes, which shortens the terminal repeats TTAGGG called telomeres. After reaching the critical telomere length, the cell loses the capacity to proliferate and dies. A minor proportion of cells in the total population can persist and progress to the immortal stage, when they continue to proliferate without being malignant. This progression is due to the activation of telomerase, a ribonucleoprotein complex with reverse transcriptase activity. The major components of this complex are the RNA template and the hTERT protein. Its gene is localized to chromosome 5 (5p15.33) (Harley et al., 1994). No telomerase activity is observed in normal cells and benign tumors but is induced in immortal and malignant cells (Skvortsov et al., 2006; Snijders et al., 1998; Sprague et al., 2002; Takahura et al., 1998; Van Duin et al., 2003; Wissman et al., 1998, 2000; Yashima et al., 1998; Zheng et al., 1997). In cervical carcinomas, this enzyme is induced by high-risk HPV E6 so that these cancers have an additional factor favoring active proliferation of transformed cells (Klingelhutz et al., 1997; Renaud et al., 2007). Apart from the theoretical significance, studies of this enzyme can shed light on the early stages of carcinogenesis since many early lesions (LSIL) can regress, and telomerase can become an extra diagnostic marker. 5.5.3. Pattern of gene expression A decreased or increased gene expression without mutations in it is a classical example of epigenetic changes. An extensive analysis of this phenomenon was made possible by the advent of expression microarrays, which allowed nearly all genes to be analyzed for the expression. This technique relies on comparative analysis of expression in two biological systems, which offered the possibility to study the following cell culture combinations: normal and HPV-transformed keratinocytes, normal keratinocytes and cells transfected with HPV E6 or E7 (i.e., immortalized cells), and keratinocytes containing only integrated or episomal viral DNA. Similar variants can be used to study tumors; however, interpretation of the obtained data is preliminary considering the tumor population heterogeneity. Such combinations include normal cells and cells in lesions at different stages (LSIL, HSIL, and carcinoma), precancerous and cancerous lesions, carcinomas from different patients and the same histological picture, or individual cell populations in the same carcinoma. This approach provided extensive data (Alazawi et al., 2002; Chang and Laimins, 2000; Cheng et al., 2002; Nees et al., 1998; Nishizuka et al., 2001; Rosty et al., 2005; Santin et al., 2005; Sopov et al., 2004; Wilde et al., 2007; Wong et al., 2003) for in-depth analysis; however, several clear conclusions emerge. First, normal cells always differ from tumor cells by upregulation of some genes

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(more than 100) and downregulation of others, while the expression level of the bulk of genes remains unaltered; second, the gene expression pattern varies as cancer progresses, although no features or individual gene patterns associated with the disease progression have been revealed; third, HPVinfected cells demonstrate a higher total expression level compared with HPV-free cells; fourth, the pattern of gene expression is specific for each carcinoma; fifth, the gene expression pattern can be used to predict sensitivity to radiation therapy; and sixth, several groups of genes have been identified, whose expression is typical of cervical cancers: interferonresponsive genes, genes induced by the transcription factor NF-kB, and genes regulated during the cell cycle and DNA synthesis. The expression of 14,500 known genes in 11 primary HPV-16 and -18-infected stage I–II cervical cancers was studied in comparison with 5 normal keratinocyte cultures (Santin et al., 2005). In total, 240 and 265 genes were identified that were upregulated or downregulated, respectively, in tumors in comparison to normal keratinocytes. The genes upregulated more than twice included cyclin-dependent kinase inhibitor 2A (CDKN2A/ p16), mesoderm-specific transcript, forkhead box M1, v-myb myeloblastosis viral oncogene homologue (avian)-like2 (v-Myb), minichromosome maintenance proteins 2, 4, and 5, cyclin B1, prostaglandin E synthase (PTGES), topoisomerase II-a (TOP2A), ubiquitin-conjugating enzyme E2C, CD97 antigen, E2F transcription factor 1, and dUTP pyrophosphatase. The list of downregulated genes included transforming growth factor-b1, transforming growth factor-a, CFLAR, serine proteinase inhibitors (SERPING1 and SERPINF1), cadherin 13, protease inhibitor 3, keratin 16, and tissue factor pathway inhibitor-2 (TFPI-2). These results were double-checked by realtime PCR and immunocytochemistry for some of these genes. The presence of numerous genes whose expression varies in cervical carcinogenesis and the presence of different experimental models (different HPV-infected cell cultures, different stages of the disease, and individual variation of cervical carcinomas) seriously complicate interpretation of the obtained data; however, such studies are clearly promising for the identification of both new diagnostic markers and targets for cancer therapy (Zhai et al., 2007).

6. Conclusions The data presented in this chapter indicate that cervical carcinomas are an extremely interesting model of cancer. (1) The first effective vaccine has been developed to prevent papillomavirus infection and cervical cancer development. (2) Cervical cancer is one of several tumors of confirmed papillomavirus etiology. (3) Two viral genes crucial for tumor initiation and

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transformed phenotype maintenance were identified. (4) The suppression of genes encoding the main tumor growth suppressors, p53 and retinoblastoma protein, by viral oncoproteins E6 and E7 proved to be critical for cell malignant transformation. (5) Each carcinoma demonstrates a range of epigenetic changes underlying individual properties of carcinomas. (6) The gradual tumor progression, precancer stages, and relative simplicity of biological sample isolation from patients make it possible to trace the dynamics of molecular processes in tumor progression. (7) Further deciphering of the molecular mechanisms of cervical cancer development should lead to the development of therapeutic vaccines and drugs for targeted therapy.

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C H A P T E R

T H R E E

Vesicle, Mitochondrial, and Plastid Division Machineries with Emphasis on Dynamin and Electron-Dense Rings T. Kuroiwa, O. Misumi, K. Nishida, F. Yagisawa, Y. Yoshida, T. Fujiwara, and H. Kuroiwa Contents 1. Introduction 2. Structural Similarities Among Mitochondrial, Plastid, and Vesicle Division Machineries 3. Vesicle Division Machinery 3.1. Endocytosis, origin, and evolution of vesicle division machinery 3.2. Structure and function of vesicle division machinery 4. Bacterial, Microbody (Peroxisome), Mitosome, and Hydrogenosome Division Machineries 4.1. Origin, structure, and function of bacterial, microbody, mitosome, and hydrogenosome division machineries 5. Mitochondrial Division Machinery 5.1. Diversity in the evolution of modes of mitochondrial division 5.2. Origin and evolution of mitochondrial division machinery 5.3. Structure, function, and constriction process of the mitochondrial division machinery 6. Plastid (Chloroplast) Division Machinery 6.1. Evolutionary diversity in modes of plastid division 6.2. Origin and evolution of plastid division machinery 6.3. Structure, function, and constriction process of plastid division machinery 7. Isolation of Organelle Division Machinery and Its Significance 7.1. Isolation of organelle division machinery 7.2. Proteins and genes involved in the organization of organelle division machinery

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Research Information Center of Extremophile, Rikkyo (St. Paul’s) University, Tokyo 171-8501, Japan International Review of Cell and Molecular Biology, Volume 271 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01203-3

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Abstract The original eukaryotic cells contained at least one set of double-membranebounded organelles (cell nucleus and mitochondria) and single-membranebounded organelles [endoplasmic reticulum, Golgi apparatus, lysosomes (vacuoles), and microbodies (peroxisomes)]. An increase in the number of organelles accompanied the evolution of these cells into Amoebozoa and Opisthokonta. Furthermore, the basic cells, containing mitochondria, engulfed photosynthetic Cyanobacteria, which were converted to plastids, and the cells thereby evolved into cells characteristic of the Bikonta. How did basic singleand double-membrane-bounded organelles originate from bacteria-like cells during early eukaryotic evolution? To answer this question, the important roles of the GTPase dynamin- and electron-dense rings in the promotion of diverse cellular activities in eukaryotes, including endocytosis, vesicular transport, mitochondrial division, and plastid division, must be considered. In this review, vesicle division, mitochondrial division, and plastid division machineries, including the dynamin- and electron-dense rings, and their roles in the origin and biogenesis of organelles in eukaryote cells are summarized. Key Words: Endocytosis, Organelles, Division machineries, Vesicles, Mitochondria, Plastids, Cyanidioschyzon merolae. ß 2008 Elsevier Inc.

1. Introduction The concept of organelle division (OD) machinery arose to explain the mechanism of organellokinesis (so-called organelle division, corresponding to cytokinesis), after mitochondria and plastid nuclear divisions. These organelles have their own DNA and are considered to be descendants of endosymbiotic prokaryotes (Gillhum, 1994; Gray, 1992). The mitochondrial DNA in the Amoebozoan, Physarum polycephalum, is associated with various basic DNA-binding proteins, including Glom, organized into an electron-dense, rod-shaped mitochondrial nucleus (nucleoid) (Fig. 3.1) (Kuroiwa, 1973, 1974, 1982; Kuroiwa et al., 2006; Sasaki et al., 2003). These mitochondrial nuclei are the largest in eukaryotes and are thus equivalent to the salivary gland chromosomes in Chironomus sp. cells, if we compare mitochondria with cells. This system also has the advantage that the mitochondria are very simple in shape and divide according to a simple sequence, including spherule-, ovoid-, and dumb-bell-shaped structures, with mitochondrial nuclear division (Fig. 3.1) (Kuroiwa et al., 1977).

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Figure 3.1 Electron micrographs showing dividing mitochondria with mitochondrial nuclear (nucleoid) division in the slime mold, Physarum polycephalum. (Aa and Ab) Serial sections show mitochondrial nuclear division (mn in Aa) and mitochondriokinesis (arrow in Ab) at the middle phase of division. An electron-dense MD ring appears at the equator (arrow in Ab). (Ac and Ad) The mitochondria have small MD rings at the final phase of mitochondrial division (arrows). Ad is the higher magnification image of Ac. (Ae) A bridge of MD ring between daughter mitochondria appears at the final phase of mitochondrial division (arrow). (Af ) When the cells were treated with cytochalasin B, the ovoid-shaped structure appeared as mitochondrial division (mitochondriokinesis) was inhibited. Scale bars represent 1 mm. Aa, from Kuroiwa (1982); Ac, Ad, from Kuroiwa et al. (1977); Ab, Af, from Kuroiwa and Kuroiwa (1980).

The process of mitochondrial division can be clearly classified into two main events: mitochondrial nuclear division and mitochondriokinesis (so-called mitochondrial division), which corresponds to cytokinesis in cells.

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In plastids, the DNA is also organized into spherical or opal necklacelike, compact DNA–protein complexes, which are referred to as plastid nuclei (Kuroiwa, 1991; Kuroiwa and Suzuki, 1981; Sakai et al., 2004). The HU-like protein in plastid nuclei has a similar role to Glom in mitochondrial nuclei (Kobayashi et al., 2002; Sasaki et al., 2003). Thus, eukaryotic cells contain three types of nuclei: cell nuclei, mitochondrial nuclei, and plastid nuclei. The organization and division of organelle nuclei have been summarized (Kuroiwa, 1982; Kuroiwa et al., 1994; Sakai et al., 2004). The concept of mitochondrial division machinery has arisen to explain mitochondriokinesis, which occurs after, or during, mitochondrial nuclear division. Since treatment with cytochalasin B, an inhibitor of actin rearrangement, inhibited mitochondriokinesis, but not mitochondrial nuclear division, in P. polycephalum, the existence of a contractile ring-like structure, as seen in cytokinesis, was suggested to occur at the mitochondrial division site (Fig. 3.1). A small, ring-like structure and a smooth bridge between daughter mitochondria were found in P. polycephalum and in the green alga, Nitella flexilis, respectively (Fig. 3.1) (Kuroiwa, 1982; Kuroiwa et al., 1977, 1994). In 1993, large electron-dense double rings, called mitochondrial division rings (outer and inner MD rings), were first found at the cytoplasmic (outer) side and matrix (inner) side of the equatorial region of dividing mitochondria in C. merolae (Kuroiwa et al., 1993). However, no actin was identified in the MD rings in C. merolae or Cyanidium caldarium RK-1, although a contractile, actin ring for cytokinesis was found in C. caldarium (23.4 Mbp genome size), using immunofluorescence and immunoelectron microscopy with antibodies to actin (Kuroiwa et al., 1998; Suzuki et al., 1995). Such an electron-dense outer MD ring can be seen at the division site in almost all eukaryotes, though these MD rings become smaller in size in higher animals and plants (Kuroiwa et al., 2006). Furthermore, in 1986, we found that plastids divide using an electrondense dividing ring, called the plastid dividing (PD) ring, situated at the cytoplasmic side of the plastid division site in C. caldarium RK1 (Mita et al., 1986). Such an electron-dense outer PD ring can be seen at the division site in almost all Bikonta. In addition, in 1989, a small electron-dense (vesicle division, VD) ring, of 40 nm in diameter, was observed around the neck of vesicle buds formed from the plasma membrane (Koenig and Ikeda, 1989; Praefcke and McMahon, 2004). MD ring, PD ring, and VD rings are 1 mm, 4 mm, and 40 nm in maximum diameter, respectively. They are different in size but have common rings such as the electron-dense ring and the dynamin ring. Recent molecular biological and genome studies (Bramhill, 1997; Erickson, 2000; Hinshaw, 2000; Miyagishima et al., 2006; Osteryoung and Nunnari, 2003; Osteryoung and Vierling, 1995; Praefcke and McMahon, 2004; Yoshida et al., 2006) allow us to reveal the common fundamental functions of these rings and to make evolutionary deductions about traits within eukaryotes. In this review, we describe the

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structure, function, and origin of the MDF (MD ring, Mda1 ring, dynamin ring, FtsZ ring, and putative ring), PDF (PD ring, dynamin ring, FtsZ ring, and putative ring) and VD machineries (VD ring, dynamin ring, and putative rings).

2. Structural Similarities Among Mitochondrial, Plastid, and Vesicle Division Machineries It has been suggested that the inner MD ring originated from bacterial division machinery through endosymbiosis. Filamentous temperaturesensitive ( fts) genes for bacterial cytokinesis were identified in Escherichia coli mutants collected by Hirota et al. (1968). These fts mutants have defective cytokinesis and, as a result, elongate to form filaments. FtsZ is found in E. coli (4.6 Mbp genome size) as a key bacterial division protein and forms a ring beneath the cytoplasmic membrane at the division site (Bi and Lutkenhaus, 1991). FtsZ is a GTPase that is structurally similar to tubulin (Lo¨we and Amos, 1998) and is conserved in most Bacteria and Archaea, except in symbiotic bacteria (Kuwahara et al., 2007). The formation of the FtsZ ring is the first event at the division site and initiates the recruitment of the other proteins that constitute the bacterial division complex. Thus, of the many proteins involved in division, FtsZ is thought to play a central role in prokaryotic cell division (Bramhill, 1997; Errington et al., 2003; Lutkenhaus, 1993). As a result, FtsZ was thought to participate in mitochondrial division. Actually, ftsZ genes are present in C. caldarium (Takahara et al., 2000) and in the Heterokontophyte, Mallomonas splendens (Beech et al., 2000), and FtsZ forms a ring on the matrix side of the membrane at the mitochondrial division site in C. merolae (Takahara et al., 2001). However, the FtsZ ring is not the inner electron-dense MD ring. The outer MD ring seemed to be made of dynamin. Dynamin was originally identified as a nucleotide-dependent microtubule-binding protein in calf brains. There, GTPase dynamin molecules appeared to function as cross-bridges that underwent microscopic movements driving the sliding of the microtubule filaments (Shpenter and Valee, 1989). It is well known that dynamins act as mechanochemical enzymes with a wide range of functions, such as the fission and fusion of vesicles and mitochondria. Among the Saccharomyces mutations that cause defects in the distribution and morphology of mitochondria (mdm mutants), which were described in the early 1990s (McConnell et al., 1990; Yaffe, 1999), mdm29 was mapped to DNM1, one of three dynamin-related yeast genes (Otsuga et al., 1998). Expression of dynamin-related Drp1 in human cultured cells resulted in aggregation of mitochondria (Smirnova et al., 2001). These results suggest

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that dynamins are related to mitochondrial morphology, distribution, and division. Analogous to the function of conventional dynamin at the plasma membrane, DRP-1 in Caenorhabditis elegans (Labrousse et al., 1999) and Dnm1p in yeast (Bleazard et al., 1999) are found on the cytosolic side of the membrane at mitochondrial division sites. The dynamin ring is different from the outer MD ring and both of these rings are essential for contraction and pinching-off, respectively, of the MDF machineries (Nishida et al., 2003). Furthermore, it was found that a novel protein, mitochondrial division protein 1 (Mda1), which is an ortholog of Mdv1 in Saccharomyces cerevisiae (Tu and Nunnari, 2000), formed a ring with dynamin and was essential for mitochondrial division (Nishida et al., 2007). Sesaki and Jensen (1999) proposed that Dnm1 mutants in yeast show defective mitochondrial division, an activity antagonistic to fusion, suggesting that mitochondrial shape is normally controlled by a balance between division and fusion which requires Dnm1p and Fzo1p, respectively. Dynamin genes are distributed universally throughout the Bikonta, the Opisthokonta, and Amoebozoa. Although mitochondrial FtsZ has been identified in primitive eukaryotes, it has apparently been lost in animals, fungi, and higher plants, as revealed by various genome sequencing projects. Some scientists have proposed that dynamin replaced the function of FtsZ in mitochondrial division during evolution. This is unlikely, because in C. merolae, FtsZ and dynamin occupy opposite positions with respect to the organelle membranes: FtsZ is located inside and dynamin outside the membrane at the mitochondrial division site, and dynamin forms a ring at the cytoplasmic side alongside the MD ring, as described (Miyagishima et al., 2003a; Takahara et al., 2000). With regard to structures related to plastid division, Suzuki and Ueda (1975) first reported the appearance of electron-dense material, which they considered to be a septum, at the narrow bridge between daughter proplastids or amyloplasts in Pisum sativum. Similar electron-dense deposits have been observed at the narrow neck of dividing chloroplasts and have been described as ‘‘bar forms’’ or ‘‘plaques’’ in the dividing chloroplasts of Phaseolus vulgaris (Whatley, 1980), Atriplex semibaccata, and Sesamum indicum (Leech et al., 1981). Subsequently, Chaly and Possingham (1981) reported an electron deposit as part of an annulus located between the inner and outer envelopes at the narrow bridge in P. sativum. In 1986, we found that plastids divide using an electron-dense dividing ring, called the plastid dividing (PD) ring, situated at the cytoplasmic side of the plastid division site in C. caldarium (Kuroiwa et al., 1989; Mita and Kuroiwa, 1988; Mita et al., 1986). An inner PD ring (Hashimoto, 1986) and a middle PD ring (Miyagishima et al., 1998a) were then identified on the inner, stromal side at the constricted division site in Avena sativa, and on the middle side, between the inner and outer membranes, in C. merolae, respectively. The inner PD

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ring initially seemed to be an artifact due to the pressure caused by contraction of the outer PD ring. However, Miyagishima et al. (1998b) elegantly proved that the inner PD ring appeared before the formation of the outer PD ring. The outer PD rings are distributed universally throughout the algae, mosses, and higher plants, but the middle PD ring is indistinct in higher plants (Kuroiwa et al., 1998; Tewinkel and Volkmann, 1987). The outer PD ring is composed of a bundle of fine filaments, 5–7 nm in diameter, which are released from small vesicles around the plastid division site (Mita and Kuroiwa, 1988; Mita et al., 1986; Miyagishima et al., 2001). It is believed that the PD ring in higher plants is small and seen only during the later stages of plastid division, whereas the PD rings in the red algae, C. caldarium and C. merolae (Kuroiwa et al., 1998; Mita and Kuroiwa, 1988), and in the green alga, Nannochloris bacillaris, are present throughout plastid division (Ogawa et al., 1995). However, in Pelargonium zonale, a single PD ring-like structure, similar to that in algae, is observed throughout plastid division (Kuroiwa et al., 2002). These results suggest that PD rings are universal structures throughout the Bikonta. The width and thickness of the outer PD ring increases with progressive contraction at the division site, whereas those of the middle and inner PD rings do not change. Thus, the outer PD ring is thought to be the core component in the motive force of the division machinery (Kuroiwa et al., 1998; Mita and Kuroiwa, 1988). Certainly, cytochalasin B treatment inhibited plastid division (plastidokinesis) (Kuroiwa et al., 1998; Mita and Kuroiwa, 1988). However, we were unable to identify actin signals in the PD ring filaments, using immunofluorescence and immunoelectron microscopy with antibodies to actin, although the contractile ring was stained with rhodamine-phalloidin (Suzuki et al., 1995). The outer PD rings probably do contain an actin-like protein. The secondary plastids in algae of the genus Mallomonas (Synurophyceae) show an electron-dense belt surrounding the plastid isthmus; this putative PD ring is related to the inner pair of the four plastid membranes, suggesting that it is homologous to those of plastids in green and red algae. The PD ring does not contain actin (indicated by lack of staining with phalloidin), and displays filaments or tubules of 5–10 nm in diameter that may be homologous to the tubules described in red algal PD rings (Kuroiwa et al., 1998; Weatherill et al., 2007). There are striking similarities between MD and PD machineries. In 1995, the homologue of the bacterial ftsZ gene was found in A. thaliana and discovery of the plant FtsZ opened a new field for the study of organelle division (Osteryoung, 2001; Osteryoung and Nunnari, 2003; Osteryoung and Vierling, 1995). The genes were divided into two families: AtftsZ1 and AtftsZ2 (AtftsZ2–1 and AtftsZ2–2) (Osteryoung et al., 1998) and currently, their products exist in plastids of most Bikonta. Strepp et al. (1998) succeeded in inducing giant plastids in the moss, Physcomitrella patens, by disrupting its

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nuclear ftsZ gene and Osteryoung et al. (1998) also produced giant plastids using anti-sense technology. Although the inhibition of chloroplast division seems to be responsible for the formation of giant plastids, it remains to be established why all cells contain giant chloroplasts, and where the FtsZ proteins act in plastids. It was shown, using immunofluorescence microscopy with antibodies to bacterial FtsZ, that many gold particles indicative of FtsZ signals were located on the stromal side of the membrane at the plastid division site in C. merolae (Kuroiwa et al., 1999) and later it was clearly shown, using immunofluorescence microscopy, that FtsZ formed a ring at the plastid division site in Lilium longiflorum (Mori et al., 2001a,b), A. thaliana (Vitha et al., 2001), and P. zonale (Kuroiwa et al., 2002). On the basis of biochemical experiments, it has been proposed that the FtsZ1 and FtsZ2 rings correspond to the inner and outer PD rings, respectively. However, immunoelectron microscopy with antibodies to plant FtsZ revealed that the FtsZ ring in L. longiflorum (Mori et al., 2001b) and the FtsZ1 and FtsZ2 rings in P. zonale (Kuroiwa et al., 2002) were located on the inner side of the inner PD ring. The various mutants of model organisms such as yeasts, A. thaliana, and mammals play important roles in the analysis of the mechanisms of organelle division. To identify the genes that regulate plastid multiplication, 12 recessive A. thaliana mutants were collected and named arc (accumulation and replication of chloroplast) mutants (Marrison et al., 1999; Pyke, 1999). These mutants contain 1–15 giant chloroplasts per cell, suggesting that chloroplast division is partially inhibited in three mutants. In the arc5 mutants, chloroplasts begin to divide but appear to stop when they become centrally constricted, suggesting that the arc5 gene product is required to complete the separation process (Robertson et al., 1996). Conversely, arc1 mutants have a larger number of smaller plastids per cell than the wild type (Pyke and Leech, 1992). In arc10 and arc11 mutants, the mesophyll cell plastids are highly heterogeneous in size within a single cell (Pyke, 1999). This size heterogeneity might be caused by the presence of a subpopulation of plastids that do not divide, or that divide by some other form of abnormal plastid division, such as asymmetric division. Although the proplastid division in meristem tissues is also perturbed in arc6 (Robertson et al., 1996) and arc12 mutants, arc3 and arc5 mutations appear to especially affect plastid division (Pyke, 1999). Some of these mutations were recently mapped. In 2003, the dynamin (MDM1) gene related to plastid division was found in the genome of C. merolae, and MDM1 formed a ring, which associated with PD rings at the plastid division site (Miyagishima et al., 2003a; Surridge, 2003). arc5 is the dynamin gene in A. thaliana (Gao et al., 2003). Thus, it was thought that mitochondria and plastids divided using organelle division (OD) machineries including the dynamic trio: MD/PD rings, dynamin ring, and FtsZ ring. Furthermore, it was pointed out that prototypes of the MDF machinery (MD ring, dynamin ring, FtsZ ring,

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Mda1 ring, and some undefined rings) and PDF machinery (PD ring, dynamin ring, FtsZ ring, and some undefined rings) were similar to the plasma membrane vesicle division (pVD) machinery (Fig. 3.2) (Kuroiwa et al., 2006). There are some arguments suggesting that the electron-dense collar (VD ring) seen around the neck of clathrin-coated pits in vivo may not actually be dynamin. We would therefore like to explain the definition of VD machineries. In 1989, a small electron-dense ring, of 40 nm in diameter, was observed around the neck of vesicle buds formed from the plasma membrane (Koenig and Ikeda, 1989; Praefcke and McMahon, 2004). Since then, it has been generally believed that this electron-dense ring or spiral consists of dynamin recruited to the vesicle necks. This idea was supported by the fact that the endocytic protein, dynamin, binds directly to liposomes, deforming them into tubules in vitro, and plays critical roles in membrane fission and curvature during clathrin-mediated endocytosis (Hinshaw, 2000; Hinshaw and Schmid, 1995; Praefcke and McMahon, 2004; Takei et al., 1999). However, there is no direct in vivo evidence that the VD rings contain dynamin: (1) Using immunoelectron microscopy with antibodies to dynamin, gold particles indicating dynamin signals were not located directly on the filaments of the VD ring in vivo, but only near the VD ring. (2) The dynamin rings in MDF and PDF machineries in C. merolae were 20 or 100 times larger in diameter and more than 1000 times greater in volume than that of VD machinery (Miyagishima and Kuroiwa, 2006; Miyagishima et al., 2003b; Nishida et al., 2003). However, even when the dynamin rings were observed clearly by immunoelectron and immunofluorescence microscopy, they were never observed directly using electron microscopy (Miyagishima et al., 2003b; Nishida et al., 2003). (3) Although fine filamentous linear structures and rings of FtsZ proteins were constructed in in vitro experiments (Erickson et al., 1996), they have never been seen as electron-dense filaments in vivo in prokaryotes (Bi and Lutkenhaus, 1991), C. merolae (Takahara et al., 2000) or P. zonale (Kuroiwa et al., 2002). As the assembled electron-dense collar is indeed a fission machinery, it might be expected that a complex of dynamin and some functional counterpart would be fundamentally required for vesicle formation. Thus, we proposed the idea that the small electron-dense ring consists of an unknown material and that dynamin plays a role in the electron-dense VD ring as a major regulator of membrane pinching-off events at the division site (Kuroiwa et al., 2006). A series of evolutionary events relates bacterial contractile rings (FtsZ rings) and eukaryotic contractile rings (VD ring, MD rings, and PD rings). When an ancestral eukaryotic cell with a cell nucleus and single-membranebounded organelles incorporated an a-Proteobacterium, it regulated mitochondrial division using the MD ring, instead of reducing FtsZ ring function, and transferred more than 80% of the genes from the purple bacteria to its

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Figure 3.2 Origin and evolution of eukaryotic cells with emphasis on organelle division (OD) machineries, including plasma membrane vesicle division (pVD), cytoplasmic vesicle division (cVD), mitochondrial division (MDF), and plastid division (PDF) machineries. (Aa) Basic eukaryotic cells are composed of double-membrane-bounded organelles (cell nucleus, mitochondrion containing mitochondrial nucleus (mt-n) and plastid containing plastid nucleus (pt-n)) and single-membrane-bounded organelles (ER, Golgi-apparatus, lysosomes (vacuoles), microbodies).With phagocytosis, microbodies, mitochondria, and plastids are generated from Eubacteria. Mitosomes and hydrogenosomes are generated from mitochondria. (Aa, Ab) The formation of

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own genome. When a Cyanobacterium was engulfed by a eukaryotic cell containing a mitochondrion and became a plastid, plastid division came to be regulated by a PD ring. Almost all cells in eukaryotes, such as Bikonts, Opisthokonts, and Amoebozoa, which evolved from one ancestral eukaryotic cell, divide by means of the MDF and PDF machineries and are diverse in organelle number, shape, and division. Therefore, we subsequently describe VD, MDF, and PDF machineries, from an evolutionary aspect.

3. Vesicle Division Machinery 3.1. Endocytosis, origin, and evolution of vesicle division machinery Endocytosis might have been the first vesicular trafficking event to evolve in the transition from prokaryote to eukaryote (Fig. 3.2). It is well known that cells in the Amoebozoa engulf some of the extracellular fluid, including nutrients and ions dissolved or suspended in it, by endocytosis. A portion of the plasma membrane is invaginated and pinched off, forming a membranebounded vesicle called an endosome. Since endocytosis is observed from Amoebozoa to Bikonta and Opisthokonta, it must originate in early protoeukaryotes (Fig. 3.2) (Kuroiwa et al., 2006). Endocytosis is divided into pinocytosis and phagocytosis, and encompasses several diverse mechanisms which control endocytic vesicle formation, such as clathrin- and caveolinindependent pinocytosis (90 nm), caveolin-mediated pinocytosis (60 nm), clathrin-mediated pinocytosis (120 nm), macropinocytosis, and phagocytosis (Conner and Schmid, 2003). VD machineries are thought to have evolved in an order of increasing complexity (Fig. 3.3). Caveolaemediated endocytosis can be blocked by either overexpressing dominant negative mutants of dynamin, or by disrupting actin assembly (Pelkmans and Helenius, 2002). Early eukaryotic cells probably gave birth to dynamin during clathrin- and caveolin-independent endocytosis and it is now globally used in caveolae-mediated endocytosis, clathrin-mediated endocytosis, and phagocytosis at plasma membranes (Figs. 3.2 and 3.3) (Hinshaw, 2000; Praefcke and McMahon, 2004; Sever et al., 2000; Stowell et al., 1999). Recent work has revealed that both invagination and scission of clathrincoated vesicles and local actin polymerization are highly coordinated, eukaryotic cell organelles consists of two phases: before and after mitochondria and plastids are generated from phagocytosis. (Ab, Ac) Primordial pVD machinery is generated by endocytosis (pinocytosis) and branches into pVD and cVD machineries. (B) Electron micrograph shows pinocytosis (arrows) of Cyanidium caldarium RK-1. Scale bar: 0.5 mm.

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Figure 3.3 Structure, origin, evolution, and mechanisms of vesicle dividing machinery. (A) Evolution of cytoplasmic vesicle division (cDV) machinery for endocytosis was suggested by fine structures. On the basis of the complexity of the structure, clathrinand caveolin-independent pinocytosis, caveolin-mediated pinocytosis, clathrinmediated pinocytosis, macropinocytosis, and phagocytosis may have evolved in that order. (B) Electron micrograph shows phagocytosis of a bacterium (long arrow) and MDF machinery (short arrow) of a dividing mitochondrion in Amoeba proteus. (C) Vesicle formation process of clathrin-mediated endocytosis. The vesicle is formed in the order of positioning of electron-dense VD ring (Ca), contraction of collar by VD ring and putative ring (Cb) and pinching-off of the vesicles by cytoplasmic vesicle division (cVD) machinery including the VD ring, putative ring, and dynamin ring (Cc^Ce). (D) A dynamin molecule contains functional domains such as GTPase, middle and PH (pleckstrin-homology) domains, respectively. The collar of a dividing vesicle is

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resulting in the efficient formation of coated vesicles in endocytosis (Merrifield et al., 2002). Tsujita et al. (2006) found that formin-binding protein (FBP) and/or CIP4 contributed to the formation of the VD machinery–protein complex, which included neural Wiskoff-Aldrich syndrome protein and dynamin, during the early stage of endocytosis in human cells. Since FBP and CIP4 exist only in Opisthokonts, clathrin-mediated VD machineries have evolved from simple structures to more complex ones. With regard to intracellular transport vesicles, mutations in the only dynamin gene in Drosophila melanogaster and C. elegans inhibit endocytic vesicle formation, but have not been reported to affect the formation of intracellular transport vesicles. Neither a dynamin-like molecule nor GTP hydrolysis is required for vesicle formation from the ER or the Golgi apparatus (Springer et al., 1999). On the other hand, Praefcke and McMahon (2004) summarized the global role of dynamins and dynaminrelated proteins as mechanochemical enzymes involved in a wide range of functions, such as the fission and fusion of vesicles and organelles, including vesicular transport and Golgi apparatus. With the development of singlemembrane-bounded organelles after invagination of the plasma membrane in eukaryotic cell evolution, the VD machinery probably evolved into two types of apparatuses: plasma membrane vesicle dividing (pVD) machinery for vesicle formation at the plasma membrane, such as synaptic vesicles, and cytoplasmic vesicle dividing (cVD) machinery for vesicle formation at organelle membranes, such as transport vesicles from the endoplasmic reticulum (ER) and secretory vesicles from the Golgi apparatus (Fig. 3.2). The pinocytosis at the plasma membrane expanded to macropinocytosis for the formation of large endocytic vesicles called macropinosomes on the plasma membrane and autophagy in the cytoplasm, accompanied by seemingly chaotic membrane ruffling. The other pinocytosis VD machinery evolved into the VD machinery for phagocytosis of bacteria (Fig. 3.3).

3.2. Structure and function of vesicle division machinery Analogy between the roles of the MD and PD machineries suggests that endocytotic vesicle fission from the membrane is a coordinated process requiring positioning of pVD machinery at the formation site, contraction

composed of VD rings, dynamin rings, and putative rings. (E) Two models for the formation of vesicles in vivo are shown. In a model, the dividing site is pinched off into vesicles by the elongating force of only the dynamin ring (Ea). In another mechanism, analogous to that of MDF machinery, the collar is divided by a biochemical reaction between the PH domain in the VD machinery and the constricted plasma membrane (Eb). Scale bar: 0.5 mm (B). A, D, modified from Conner and Schmid (2003); D, Ea, modified from Praefcke and McMahon (2004).

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of the vesicle neck by the VD machinery, and pinching-off of the vesicle neck by the VD machinery. With regard to pinocytosis, the four basic mechanisms described above occur, but the mechanism of positioning and contraction of the membrane at the division site, except in clathrinmediated endocytosis, is unclear (Fig. 3.3). The positioning of the clathrin receptor is also unclear. In the first step of clathrin-mediated endocytosis, clathrin and adaptin assemble into a polygonal lattice, which helps to deform the overlying plasma membrane into a vesicle bud (Conner and Schmid, 2003; Praefcke and McMahon, 2004). No electron-dense VD ringformation has been observed. The VD ring materials are probably recruited to form a ring, 5–7 nm in width, at the budding site on the plasma membrane and the VD ring then constricts the vesicle neck region (Fig. 3.3). Recently, Shimada et al. (2007) determined the crystal structures of the EFC domains of human FBP17 and CIP4, in which the defining feature of the PCH proteins is an evolutionarily conserved EFC/F-BAR domain required for membrane association and tubulation (contraction) at the vesicle formation site. They suggested that induction of the EFC domain was needed to drive tubulation. Mutations that impair filament formation also impair membrane tubulation and cell membrane invagination. Furthermore, FBP17 is recruited to clathrin-coated pits only in the late stages of endocytosis (Shimada et al., 2007). As the FBP gene is identified only in the Opisthokonta, it is thought that in these organisms, rings of these proteins which include the EFC domain, have developed as a cooperative structure with the VD ring. In the case of other eukaryotes, the VD machineries may be a complex of the VD ring, the dynamin ring, and the putative rings (like the primitive FBP ring) (Fig. 3.3). Two models for the formation of vesicles in vivo are shown. In one model, the dividing site is pinched off into vesicles by the elongating force of only the dynamin ring. In another mechanism, based on analogy with the MDF machinery, the dynamin proteins assemble around the neck of the bud and may pinch off together with the VD ring and putative FBP-like ring by a biochemical reaction between the PH domain in the VD machinery and the constricted plasma membrane. In the biochemical reaction, the dividing site does not elongate (Fig. 3.3). The VD ring may be thought of as a template that recruits dynamin proteins to the neck, but the mechanism of pinching-off is unknown. With regard to the VD machinery for vesicle formation at organelle membranes, such as transport vesicles from the ER and secretory vesicles from the Golgi apparatus, the mechanisms of positioning and contraction of the cVD machineries are unclear. Since dynamins function in the budding of endosomes, steps similar to those involved in vesicle formation from the plasma membrane must also be used for the formation of these transport vesicles (Fig. 3.2).

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4. Bacterial, Microbody (Peroxisome), Mitosome, and Hydrogenosome Division Machineries 4.1. Origin, structure, and function of bacterial, microbody, mitosome, and hydrogenosome division machineries 4.1.1. Bacterial division machinery Although it is thought that phagocytosis is typically restricted to specialized mammalian cells (Conner and Schmid, 2003), it occurs in all eukaryotic cells, including the amoeba, Acanthamoeba castellanii (Avery et al., 1995). The mechanisms of endocytosis have diversified considerably since its early evolutionary beginnings (Figs. 3.2 and 3.3). The VD machineries, which developed from the pVD machinery for pinocytosis at the plasma membrane, expanded to machineries for phagocytosis with cell enlargement, before endosymbiosis of a-Proteobacteria. Dynamin is not identified in prokaryotes but is found in all eukaryotes, and is thus a eukaryote-specific GTPase family. Many bacteria can form intracellular structures such as mesosomes and thylakoid-like stacks by invagination of the plasma membrane, but they do not have dynamin. The proto-host cells must catch small bacteria using pVD machinery for phagocytosis on the plasma membrane (Fig. 3.3), and then secrete digestive enzymes directly into the endocytic vacuole containing the captured bacteria. Some endosymbiotic a-Proteobacteria have evolved mechanisms to avoid destruction, even after they have been engulfed by phagocytes, whereas the host cells controlled the bacterial division using unique division machineries, which included dynamin. Bacteria provide important information related to organelle division. Bacterial division is composed of two main events: bacterial nuclear division and bacterial cytokinesis. During division, bacteria, such as E. coli and Synechocystis, change from a spherical shape (or rod shape) to ovoid shape (or elongated rod shape) and then to dumb-bell-shaped (or elongated rod-shaped, with constriction at the division site), during progression of the cell cycle, and they finally divide by cytokinesis, just after bacterial nuclear division (genome separation). Bacterial cytokinesis occurs using filamentous temperature-sensitive ( fts) (A, I, K, L, N, Q, W, Z, H) and other genes (min C, D, E, ZipA), which have been identified ubiquitously in Eubacteria (E. coli and Bacillus subtilis) (Bramhill, 1997; Errington et al., 2003) and Cyanobacteria. These genes are summarized by Miyagishima et al. (2005). Almost all of the mutants were originally collected in the 1960s (Hirota et al., 1968). Bacterial cytokinesis, as well as organelle division, is controlled

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directly by contractile ring-like division machineries. There are also indirect regulatory systems, such as the formation and positioning of the division machineries after or during bacterial nuclear division. min C-D genes are involved in the indirect regulation of bacterial division. Correct positioning of the bacterial FtsZ ring requires the proteins MinC, MinD, and MinE. MinC acts as a division inhibitor, preventing FtsZ ring formation at locations other than mid-cell, by directly interacting with FtsZ to prevent polymerization. The activity of MinC is dependent on MinD, which forms heterodimers with MinC and directs it to the cell membrane, acting as an assembly protein for MinC and MinE. The MinD/MinC protein complex has further been shown to oscillate between the two cell poles in bacteria, and MinD activity is dependent on MinE, which also oscillates between the poles. MinE probably acts as a topological specificity factor, moving the MinD/MinC division inhibitor complex away from the midcell region. In short, the Min system acts as a negative regulator, restricting separation to the mid-cell region and preventing the formation of division septa at other sites within the cell (Errington et al., 2003; Miyagishima and Kuroiwa, 2006). The ftsZ gene is one of the most conserved genes for bacterial cytokinesis and is required for direct regulation of bacterial division. It has been thought to play a central role in prokaryotic cell division. FtsZ is a GTPase that is structurally similar to tubulin (Lo¨we and Amos, 1998) and self-assembles into an initial ring structure beneath the plasma membrane at the division sites (Bi and Luthkenhaus, 1991; Lutkenhaus, 1993). FtsZ recruits other Fts proteins. In E. coli, the dependence pathway for assembly is completely linear, with the sequence FtsK!FtsQ!FtsL! FtsW!FtsI!FtsN. The bacterial dividing machinery (divisome) is composed of Fts Z, A, L, I, Q, N, K, W, and Zip A at the division site. An FtsZ ring has never been observed as an electron-dense structure at the bacterial division site but has been visualized by immuno-light and -electron microscopy. The FtsZ proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli, which has a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins, with their major domains outside the cell (Errington et al., 2003). The functions of most of these proteins are unclear, with the exception of at least one penicillinbinding protein (FtsI), which catalyzes a key step in cell wall synthesis in the division septum. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. DivIB/FtsQ is a conserved component of the divisome in bacteria with cell walls, suggesting that it plays a role in synthesis and/or remodeling of septal peptidoglycans (Robson and King, 2006). There are at least two models for contraction at the bacterial division site: contraction at the division site occurs either by sliding of FtsZ

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molecules or by destruction of the FtsZ molecules (Bramhill, 1997). The latter hypothesis is probably more likely and is supported by the fact that the FtsZ proteins decrease in volume with contraction of the ring. However, in addition to the divisome, including FtsZ and other division proteins, the roles of a linker complex (including FtsI) between the divisome and the cell wall, and the role of the cell wall (including peptidoglycans) in constriction and closure of the division septum are not yet clear. In addition, it has been revealed that, although the thioautotrophic intracellular symbiont, Calyptogena okutanii, apparently undergoes division, ftsZ and related genes are missing in this bacterium (Kuwahara et al., 2007). A lack of ftsZ has been reported in many bacteria (Chlamydiae, Ureaplasma mycetes, and Pirellula sp.) and in Crenarchaeota (Glo¨ckner et al., 2003). The mechanism of bacterial cytokinesis without FtsZ remains to be studied as a key to understanding bacterial division. 4.1.2. Microbody division Single-membrane-bounded microbodies (peroxisomes), double-membranebounded mitosomes and hydrogenosomes, as well as mitochondria, are suggested to be organelles of bacterial origin (Figs. 3.2 and 3.4). Microbodies are spherules, 0.5–2 mm in diameter, and are characterized by the presence of the enzyme, catalase, which protects the cell from oxidative stress such as that caused by peroxide produced following the exposure of cellular metabolites to large amounts of molecular oxygen, which is produced by Cyanobacteria. Microbodies also play a role in b-oxidation of fatty acids. They are present in a wide variety of eukaryotic cells, from Amoebozoa to Bikonts and Opisthokonts. The number of microbodies ranges from 1 in C. merolae, and increasing to 10–1000 per cell in Bikonts and Opisthokonts. The evolutionary origin of microbodies remains unsolved, and investigations into either a symbiogenetic or cellular membrane invagination event have been inconclusive. Microbodies have been acquired by primitive eukaryotic cells by endosymbiotic phagocytosis of a prokaryote (probably an aerobic Gram-positive bacterium, such as Listeria or Mycoplasma) (De Duve, 1982), which has eventually lost its DNA (Fig. 3.4) (Cavalier-Smith, 1987). Gold et al. (1999) reported that a ubiquitously expressed dynamin isoform had a role in phagocytosis in macrophages. Dynamin is enriched in early phagosomes, and expression of a dominant-negative mutant of dynamin significantly inhibited particle internalization at the stage of membrane extension around the particle. Thus, dynamin must play a role in the formation of phagocytic vesicles containing endosymbiotic bacteria. These results suggest that microbodies arise from the fission of preexisting organelles rather than from a de novo pathway that originates in the ER.

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By contrast, regarding the behavior of microbodies in multi-microbody cells in Opisthokonta, Van der Zand et al. (2006) proposed the idea that microbodies are not autonomously multiplying organelles, but are derived from the endoplasmic reticulum. Schlu¨ter et al. (2006) provided evidence against a prokaryotic ancestor of microbodies, using a probe for a microbody proteome: (1) the microbody membrane is composed of purely eukaryotic components and (2) the microbody matrix protein import system shares mechanistic similarities with the ER/proteasome degradation process. However, there are no clear serial electron micrographs of the microbody-less mutant cells demonstrating the process of budding of microbodies from the ER. However, it is difficult to show the correct shapes and numbers of the single-membrane-bounded microbodies lacking DNA in Opisthokont cells, as these cells contain many organelles including microbodies, mitochondria, lysosomes, Golgi apparatus, and ER which are mixed and divided randomly. There is strong evidence suggesting that microbodies arise from preexisting organelles. A C. merolae cell is small (1.5–2 mm in diameter) and contains just one spherical microbody, one cell nucleus, one mitochondrion, one chloroplast, one Golgi apparatus, and a few lysosomes per cell, divisions of which can be highly synchronized by dark/light cycles. Thus, the morphology of the microbody and its interactions with other organelles could be observed three-dimensionally by fluorescence microscopy, transmission electron microscopy, and computer-assisted three-dimensional reconstructions of serial thin sections. The results showed that the microbody increased to twice its volume during mitosis, changed from sphericalto ovoid-shaped, and finally to dumb-bell-shaped and divided by binary fission (Fig. 3.4) (Miyagishima et al., 1999a). Since the ER does not develop in C. merolae cells, there is no doubt that the microbody multiplies by division of the preexisting microbody. These results support the idea that microbodies are autonomous organelles that multiply by growth and division, like mitochondria and chloroplasts (Lazarow and Fujiki, 1985). It may multiplication cycle in C. merolae is revealed by epifluorescence microscopy (Ba^Bf ). The round microbody in interphase cells moves to the equatorial plane of the cell and chloroplast (cp) in early M-phase (arrow in Ba), elongates in this plane, then winds around the mitochondrion (Ba^Bc), and finally divides by binary fission (Bd^Bf, C, D). The microbodies bind to MTOC (short arrow in Bf ) through mitochondria. Microbodies proliferate in four steps: namely association with mitochondria, elongation, branching, and fission (C, D). (C) Electron micrograph shows two electron-dense dividing apparatuses about 30 nm in diameter (double arrowheads in Ca, Cb, D).They are identified at the point where the microbody associates with the two daughter mitochondria (mt) (C, D). (E) Hydrogenosomes also divide by binary fission. Putative hydrogenosome dividing apparatus exists at the dividing site (arrow in E). Scale bars: 1 mm (Ba), 0.5 mm (Ca), 0.2 mm (Cb). B, Ca, Cb, from Miyagishima et al. (1999a); E, from Benchimol et al. (1996).

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include two proposals for either a symbiogenetic or cellular membrane invagination event. It is considered in this review that a bacterial ancestor of a microbody was acquired by the primitive eukaryotic cell, lost its DNA and the bacterial cell membrane, and eventually bacterial matrix and interspace between outer and inner envelope were mixed in the putative premicrobody (Fig. 3.4). In the case of autonomously multiplying microbodies, a suite of genes is required to control fission of the organelles in order to maintain their numbers. The combined efforts of several groups have identified some 32PEX genes that contribute to biogenesis or maintenance of microbodies (Van der Zand et al., 2006). Generally, microbody divisions in mammalian cells, trypanosomes, yeast, and algae progress in the order of elongation, constriction, and fission, and part of the process depends on the PEX11 membrane proteins. Higher level expression of Pex11pb promotes peroxisome division in mammalian cells (Li and Gould, 2002; Schrader et al., 1998). PEX11 and two PEX11-related proteins are the predominant membrane proteins of the microbodies of Trypanosoma brucei (Voncken et al., 2003). They concluded that PEX11 family proteins played important roles in determining microbody membrane structure. Dynamin-like protein 1 (DLP1), which is essential for mitochondrial division, was recently reported to also be involved in peroxisome division (Koch et al., 2004; Li and Gould, 2002; Tanaka et al., 2006) and is recruited to peroxisomes in part by PEX11. Fis1p is a single-membrane-span protein located in the outer membrane of mitochondria, where it regulates mitochondrial fission in mammalian cells through an interaction with DLP1 (Dohm et al., 2003; Yoon et al., 2003). Fis1p has now also been found in peroxisomes. In conjunction with DLP1, it appears to support the fission not only of mitochondria, but also of microbodies (Koch et al., 2003, 2004; Van der Zand et al., 2006). DLP1 is a kind of dynamin which is related to mitochondrial outer membrane fission, and also functions in the membrane fission of microbodies (Praefcke and McMahon, 2004). In addition, a microbody division gene of bacterial origin is not yet known in eukaryotes. Microbodies are probably descended from endosymbiotic bacteria which have lost their DNA and inner membrane (Fig. 3.4). Fts1 plays important roles in peroxisome division and the maintenance of peroxisome morphology in mammalian cells, possibly in a concerted manner together with Pex11pb and DLP1 (Kobayashi et al., 2007). However, the location and function of PEX1, DLP1, and Fis1 at the microbody division site remain little understood. C. merolae cells possess PEX 1, 2, and 3 genes, but no electron-dense ring or dynamin ring has been observed between daughter microbodies. Instead of a dynamin ring, Miyagishima et al. (1999b) identified an electron-dense apparatus, 30–50 nm in diameter, between the microbody and the mitochondrion, which may play a role in segregating the microbodies (Fig. 3.4). Therefore, microbodies bind to spindle pole bodies through mitochondria

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(Nishida et al., 2005). Microbodies divide by binary fission after constriction at their equators. The putative microbody division (MBD) machinery at the division site may be responsible for dividing the microbody. Although the substance of the apparatus has not been determined, this may be an example of the diversity of organelle division machinery. 4.1.3. Mitosome and hydrogenosome division In some parasitic organisms, cells have small, double-membrane-bounded mitosomes or hydrogenosomes, instead of mitochondria. A mitosome is an organelle found in some anaerobic or microaerophilic organisms, such as the intestinal parasites of humans, Entamoeba histolytica (Ghosh et al., 2000; Tovar et al., 1999), Microsporidia, and Giardia intestinalis (Regoes et al., 2005), which do not have mitochondria. These organisms cannot gain energy from oxidation, a process which is normally performed by mitochondria. Mitosomes are almost certainly derived from mitochondria and have a double-walled membrane (Vavra 2005), but do not contain DNA (Ghosh et al., 2000; Leon-Avila and Tovar, 2004). Thus, the genes for mitosomal components are contained in the nuclear genome. The Giardia lamblia genome encodes a single dynamin-like protein. Hence the single dynamin-like proteins in Spironucleus, Giardia, and Encephalitozoon may be involved in mitosome division, but their structures, locations, and functions are not clear. Hydrogenosomes are 1 mm in diameter and are so called because they produce molecular hydrogen. Like mitochondria, they are bound by distinct double membranes and have an inner membrane with cristae-like projections. Hydrogenosomes evolved from mitochondria by the concomitant loss of classical mitochondrial features, most notably its genome (Van der Giezen and Tovar, 2005). Hydrogenosomes have been identified in several trichomonad species (Tritrichomonas foetus, Trichomonas vaginalis, Tritrichomonas suis, Trichomonas galinae, Tritrichomonas augusta, and Monocercomonas sp.) (Benchimol et al., 1996). It was shown by electron microscopy that the hydrogenosome could divide by binary fission (Benchimol et al., 1996). A hydrogenosomal genome could not be detected in Neocallimastix or T. foetus (van der Giezen and Tovar, 2005). Almost nothing is known about hydrogenosome division in anaerobic parasitic flagellated protozoans. However, Benchimol et al. (1996) showed that hydrogenosomes are elongated, showing a constriction in the central region. Microfibrillar and tubular structures which can be seen in their electron micrograph must be related to the division process (Fig. 3.4). These results suggest that microbodies, mitosomes, and hydrogenosomes do not contain their own genome but can divide using putative microbody division (MBD), mitosome division (MSD), and hydrogenosome division (HGD) machineries, respectively.

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5. Mitochondrial Division Machinery 5.1. Diversity in the evolution of modes of mitochondrial division Mitochondria are typical organelles which multiply by division of preexisting organelles and are distributed in almost all eukaryotic cells, with increasing numbers per cell and changes of shape associated with evolution (Fig. 3.5). In primitive eukaryotes, the basic shape of mitochondria is spherical and there is only one per cell, but they have changed remarkably during evolution, concomitant with increasing biodiversity (Fig. 3.5). A primitive eukaryotic cell has a single spherical mitochondrion, which multiplies by binary division, progressing through spherical-, ovoid-, and dumb-bell-shaped structures during division, like bacteria. In the primitive red algae, C. merolae and C. caldarium, cells contain one mitochondrion, which changes through spherical, ovoid, and dumb-bell shapes during the cell cycle, and finally divides by binary fission (Fig. 3.5). The primitive red alga, Galdieria sulphuraria (Cyanidium caldarium M8), has a larger genome size (45 Mbp) than C. merolae (Matsuzaki et al., 2004), C. caldarium, and the Apicomplexa, and still has only one mitochondrion per cell, but the mitochondrial shape has become irregular or net-like in structure throughout the cell and life cycles (Fig. 3.5). Mitochondria increase only by division in lower and simple eukaryotes. However, in Amoebazoa, Bikonts, and Opisthokonts, the cells have evolved mitochondrial fusion, as well as division (Fig. 3.5). In P. polycephalum, the mitochondria go through spherule, ovoid, and dumb-bell shapes during the mitochondrial division cycle, but fuse during specialized stages (sporulation and amoebae) of the life cycle with the gene (ORF640p) in mF plasmids (Kawano et al., 1991, 1993; Takano et al., 1994, 2002). Studies in yeast and C. elegans suggest that the establishment and maintenance of different mitochondrial morphologies depends, in part, on the equilibrium between opposing fission and fusion events (Shaw and Nunnari, 2002; Van der Bliek, 2000). It is therefore believed that mitochondria are dynamic structures that divide and fuse continually throughout the life of almost all eukaryotic cells. However, in Euglena gracilis, Chlamydomonas reinhardtii (Osafune et al., 1972, 1975), and S. cerevisiae (Kuroiwa 1982; Miyakawa et al., 1984), irregularly shaped mitochondria elongate or constrict frequently during the life cycle, but mitochondrial division and fission occur only at specific phases of the life cycle (Fig. 3.5) (Ehara et al., 1990; Kawano et al., 1991, 1993; Takano et al., 1994). This is probably significant: Fzo1/Mgm1, which affects mitochondrial fusion in yeast, must be studied at each stage of the life cycle. In ciliates, higher algae, higher plants, and humans, the mitochondrial shape is peanut- or stringshaped and the number of mitochondria per cell can increase remarkably to

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over 2000 in animal cells (Fig. 3.5). In addition, the mitochondria not only divide, but also fuse (Kuroiwa, 1982; Miyakawa et al., 1984; Nishibayashi and Kuroiwa 1985; Satoh and Kuroiwa, 1991). Despite the diverse changes

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in shape and number of mitochondria in eukaryotes, all mitochondria can arise from preexisting mitochondria by binary or unequal fission, after mitochondrial nuclear division, using the MDF machinery.

5.2. Origin and evolution of mitochondrial division machinery It is thought that, based on the morphological similarity between slime mold mitochondrial division and a-Proteobacterium (Rickettsia felis) division (Kuroiwa et al., 1977), eukaryotic host cells engulfed Rickettsia to form mitochondria by phagocytosis. After symbiosis and nutrient exchange between the bacteria and the host cells, as suggested in Amoeba proteus ( Jeon and Jeon, 1976), more than 80% of the bacterial genome was lost, but the ftsZ gene was transferred to the host nucleus. Thus, mitochondria became semi-autonomous. By the loss of many bacterial division genes, the host genome controlled mitochondrial division using the MD ring, the Mda1 ring, and the dynamin ring, which were formed at the cytoplasmic side of the membrane at the division site (Figs. 3.5–3.8). Thus, the MDF machineries are composed of a chimera of rings from bacteria (FtsZ) and eukaryotes (MD ring and dynamin ring). Compared with the VD machineries, which pinch off the neck of vesicles 40 nm in diameter, the MDF machineries become more complicated. In the Amoebozoan, P. polycephalum, the Bikonts, and the Opisthokonts, a small electron-dense MD ring was observed between daughter mitochondria at a late phase of division (Kuroiwa et al., 1977, 1995, 2006), whereas large MD rings, 1 mm maximum circumference, were identified at the equator of dividing mitochondria throughout division in unicellular algae, such as C. merolae (Kuroiwa et al., 1993, 1995), C. caldarium (Kuroiwa et al., 1998, 2006), and Nannochloropsis oculata (Hashimoto, 2004). Certainly, a-Proteobacterial-type FtsZ was identified from the Amoebozoans such as Dictyostelium discoideum (Gilson and Beech, 2001) and C. merolae (Takahara et al., 2000) and the heterokont alga, Mallomonas (Beech et al., 2000). Mitochondrial dynamin was also identified in Amoebozoa, Bikonts (Arimura and Tsutsumi, 2002), the Opisthokont yeast (Bleazard et al., 1999), nematodes (Labrousse et al., 1999), and mammals (Smirnova et al., 2001). Compared with the distribution of the MD and dynamin rings, sequences resembling bacterial ftsZ for mitochondrial division were not found in the genomes of the Opisthokont, S. cerevisiae, or those of C. elegans, the newly sequenced genomes of other nonphotosynthetic eukaryotes (Gilson and Beech, 2001) or A. thaliana (Osteryoung and McAndrew, 2001). This suggests that other proteins have replaced ftsZ in these other eukaryotic species (Erickson, 2000). The origins of heterokont host cells with secondary chloroplasts are unknown. However, the red algae and heterokont host cells have large MD rings, mitochondrial ftsZ, and the S-adenosylmethionine synthetase (sam)

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gene in C. merolae is very similar to that in Aureococcus sp., suggesting that a secondary endosymbiotic host organism, which had lost its chloroplast, (Nozaki et al., 2003), diverged from the red algae. The heterokont cells contain chloroplasts with ring-like nuclei (nucleoids) (Kuroiwa and Suzuki, 1981; Misumi et al., 2008). Circular chloroplast nuclei have been identified at the periphery of the chloroplast in the red alga, G. sulphuraria (Kuroiwa et al., 1981, 1989), suggesting that the symbiotic eukaryote with secondary chloroplasts was a red alga, such as the ancestor of G. sulphuraria.

5.3. Structure, function, and constriction process of the mitochondrial division machinery It should be considered that the division of mitochondria is regulated directly and indirectly by genes whose products may or may not be localized within the division machinery in the mitochondria. In this review, we have focused on the direct regulation of mitochondrial division by the MDF machinery. Synchronous cell cycling in culture is very useful for the study of the dynamics of organelles, such as mitochondrial and plastid division, and has been used in C. reinhardtii (Osafune et al., 1972), E. gracilis (Ehara et al., 1990; Osafune et al., 1975), and S. cerevisiae (Kuroiwa et al., 1984; Miyamura et al., 1986). However, these organisms have irregularly shaped mitochondria which fuse, as well as divide, during their life cycles. Therefore, as it is difficult to study the MDF machinery in detail in these organisms, highly synchronous cultures of C. merolae, which has a simple mitochondrial division system, were developed (Fig. 3.6) (Nishida et al., 2003; Terui et al., 1995). The mitochondria change through spherical, ovoid, and dumb-bell shapes and divide for 3 h during the 24 h mitochondrial division cycle (Fig. 3.6). The detailed time schedule of mitochondrial divisions in C. merolae was determined by observing living cells and

The PD and MD rings are formed simultaneously at the outside of the organelles during phase1 (Aa, Ab) and the PD rings begin to constrict constantly at the division site, while the MD ring constricts rapidly during phases 2 and 3, after no constriction for 2 h. (B) The division processes in mitochondria and chloroplasts were observed by fluorescence microscopy after staining with DiOC6 (bright in Ba^Bd).The mitochondria are simple in shape and divide as shown in Ab and Ba^Bd, Be, Bf, and Ca-Cc are electron micrographs of dividing mitochondria with PD rings. Bf is a higher magnification of Be. Mitochondrial dividing rings (arrow in Bf ) are formed at the division site of the dividing mitochondrion (mt) (Be and Bf ) and contract. (C) The mitochondrial rings are doublets of an inner ring in the matrix (long arrow in Cb) and an outer ring in the cytoplasm (short arrows in Bf, Ca^Cc).The width and thickness of the mitochondrial outer rings (upper and lower in D) increase with contraction of the division site, while those of the mitochondrial inner rings do not change.Therefore, the volume of the outer PD ring does not change during mitochondrial division (D). Hor in Ab, horizontal view; Ver in Ab, vertical view; mb, microbody. Scale bar: 1 mm (Bd), 50 nm (Cc). Aa, from Nishida et al. (2003); Cb, from Miyagishima et al. (2003b); D, from Miyagishima et al. (1999b).

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organelle divisions by light and electron microscopy (Miyagishima et al., 1999b). The MD and PD rings were formed simultaneously when the diameter of the plastid division site reached 1.3 mm. Immediately after

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the formation of the rings, contraction of the plastid division site started before that of the mitochondrial division site, and progressed constantly during phase 1. On the other hand, the MD ring did not contract immediately after its formation, but began to contract a few hours later, though its speed was four times as high as that of the PD ring (Fig. 3.6). The mitochondrial division phases are defined as phase 1 (positioning and formation of MDF machinery), phase 2 (contraction of MD machinery), and phase 3 (pinching-off of membrane by MDF machinery) (Fig. 3.6). 5.3.1. Positioning Bacterial type min C-D genes, which relate to positioning of the division site, were not identified in cells of the red alga, C. merolae, but the FtsZ ring was formed at the mitochondrial division site. An unknown signal for the separation of daughter mitochondrial nuclei probably promotes the formation of the mitochondrial (mt) FtsZ ring and the inner MD ring at the inner matrix of the mitochondrial division site. However, the coupling between mitochondrial nuclear division and mitochondriokinesis can be disrupted by the addition of the inhibitor of DNA synthesis, ethidium bromide, and thus mitochondria without mitochondrial nuclei arise (Kuroiwa, 1982). Similar events occur in the process of plastid division: one of the daughter plastids did not contain DNA while another did contain DNA after treatment with the inhibitor of gyrase, nalidixic acid (Itoh et al., 1997). These results suggest that a connection between organelle nuclear division and organellokinesis is essential, but loose. The mtFtsZ ring could not be visualized in vivo by electron microscopy, probably due to poor contrast,

control of FtsH, and the division site is determined. Dynamin is located in cytoplasmic patches. In the constriction phase (Ad^Af ), after FtsZ ring formation, the Mda1 ring and outer MD ring appear on the cytoplasmic side of the outer membrane. The inner MD, FtsZ, Mda1, and outer MD rings begin to constrict the equator of the dividing mitochondrion. Dynamin is not involved in early constriction (Ad ). When the mitochondrion has constricted at the division site, dynamin is recruited from the patches in the cytoplasm to form the MDF machinery with Mda1 and the outer MD ring (Ae, Af ) and finally migrates to a space inside the thickened outer MD ring and outside the outer membrane (Ag). In the first step of the pinching-off phase (Ag^Ai), the inner PD ring and the FtsZ ring are split to form a patch in each matrix.The dynamin ring pinches off the membrane of the bridge between the daughter mitochondria (Ag).The remnants of the MDF machinery then stick to one side of the daughter mitochondria and the dynamin ring is broken into small patches. (B) Putative molecular model of pinching-off at the bridge between daughter mitochondria. Inner MD and FtsZ rings are split to form two divided matrices and then the dynamin ring pinches off the outer and inner membranes simultaneously, at the center of the bridge, by a biochemical reaction between the PH domain and the double membranes under the control of Mda1and the outer MD ring.The inner PD ring and the FtsZ ring have been eliminated from the center.

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but was seen using immunofluorescence and immunoelectron microscopy. The mtFtsZ ring forms at the matrix side of the membrane before contraction of the division site, at an early stage of mitochondrial division. The mtFtsZ ring and the inner MD ring are conserved throughout contraction of the mitochondrial division site, but disappear before pinching-off of the double membranes between daughter mitochondria (Nishida et al., 2003). Regarding the PDF machinery, the plastid (pt) FtsZ ring formation began significantly earlier than PD ring formation in the multi-FtsZ ring system of tobacco Bright Yellow 2 (BY-2) cells (Miyazawa et al., 2001; Momoyama et al., 2003). The morphological and functional analogies between the PD ring and the MD ring suggest that the mtFtsZ ring is formed just after the inner putative ring formation, the FtsZ ring is recruited on the inner putative ring, and then the inner MD ring, the outer MD ring and the Mda1 ring are formed via unknown anchor proteins at the division site (Nishida et al., 2003; Yoshida et al., 2006). Since the mt-FtsZ ring and the inner MD ring decrease in volume with contraction of the division site (Nishida et al., 2003; Takahara et al., 2000), they may promote the positioning of the division site and aid contraction by partial destruction of their FtsZ materials. 5.3.2. Contraction Contraction of the MD machinery starts just before, and finishes after, the end of mitochondrial nuclear division. It is not known how the mtFtsZ ring and inner MD ring recruit the electron-dense outer MD ring. The MD ring may relate to the contraction of the equatorial region of the dividing mitochondria. The dimensions of the outer MD ring, which recruits on the site of the inner MD ring and FtsZ ring, are 70 nm 10 nm (width thickness), before the initiation of contraction (Fig. 3.6). The behavior of the inner MD ring is very similar to that of the mtFtsZ ring. The inner MD ring and the FtsZ ring coexist as mitochondrial divisionassociated proteins at the matrix side, mediating with the scaffolding of the outer MD ring, are contracted by self-digestion and are finally digested before the separation of the daughter mitochondria (Miyagishima et al., 1999b). On the other hand, since the increase in width and thickness of the outer MD ring parallels the decrease in diameter of the mitochondrial division site during phases 2 and 3, with the dimensions reaching 130 nm 40 nm (width thickness) just before pinching-off the bridge between daughter mitochondria, the outer MD ring appears to play a major role in generating the motive force of contraction, probably by causing sliding of fine filaments (Fig. 3.6). Recently, a new protein, CMR185C, also named Mda1, containing a predictable coiled-coil region and WD40 repeats (Nishida et al., 2007), similar to Mdv1/Fis2/Gag3 in yeasts (Fekkes et al., 2000; Mozdy et al.,

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2000; Van der Bliek, 2000) has been identified. Mdv1/Fis2/Gag3 can bind to Dnm1 in the yeast two-hybrid system, but binding is too weak to be detected by immunoprecipitation. Mda1 could be purified with Dnm1, using its binding ability. Mda1 formed a dividing ring with the MD ring and was localized on the MDF machinery throughout division (Nishida et al., 2007). These results suggest that mitochondrial division is facilitated by contraction of the MDF machinery containing the inner MD ring, the outer MD ring, the FtsZ ring, the Mda1 ring, and some undefined rings such as FtsH and the putative linker between the inner and outer rings assembled at the division site, although the role of each ring in the mechanisms responsible for the sliding of MD filaments remains to be solved. The contracted MD ring appears clearly as an electron-dense and thick bundle at the later phase of division in almost all eukaryote cells, but does not pinch off the bridge between daughter mitochondria (Fig. 3.6). 5.3.3. Pinching-off It is well known that the dynamin family is involved genetically in mitochondrial division in the Amoebozoa (Wienke et al., 1999), Bikonta (Nishida et al., 2003), and Opisthokonta (McConnell et al., 1990; Otsuga et al., 1998; Shaw and Nunnari, 2002; Smirnova et al., 2001; Yaffe, 1999). However, the role played by dynamin in the MDF machinery is relatively unknown. To elucidate the function of dynamin, it is important to determine its location, though this cannot be seen in detail in multimitochondrial cells using immunofluorescence or electron microscopy. Nishida et al. (2003) observed, using immunofluorescence microscopy, that during phase 2, soon after the contraction of the MD ring, the dynamin patches moved from the cytoplasm to the MD rings (Fig. 3.7). In in vitro experiments, dynamin binds strongly to the WD40 repeat-containing protein, Mda1, which forms at an early phase of mitochondrial division. It is likely that the Mda1 ring, which has already been formed at the division site, recruits the dynamin and forms the MDF machinery, including the FtsZ ring (Figs. 3.7 and 3.8) (Nishida et al., 2007) and dynamin then moves from the surface of the Mda1 and outer MD rings to the inside of these rings (Figs. 3.7 and 3.8). In yeast, C. elegans, and mammals, Dnm1/DRP-1/Drp1 is required to sever the mitochondrial outer membrane, and the Mdv1/ Fis2/Gap3 protein can form a link between Dnm1 and the other components of the division apparatus by means of seven WD repeats (Van der Bliek, 2000). As a result, the MDF machinery is organized in the order of putative inner ring, FtsZ ring, inner MD ring, outer MD ring, Mda1 ring, and dynamin ring (Figs. 3.7 and 3.8). An important observation has been made with regard to the pinching-off of the bridge of daughter mitochondria in C. merolae (Nishida et al., 2003): during the final phase 3 of mitochondrial division, the FtsZ ring separates into each daughter

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mitochondrion and disappears with the inner PD ring, whereas the outer electron-dense MD ring, the Mda1 ring, and the dynamin ring remain to build up a narrow, tubular bridge between the daughter mitochondria (Figs. 3.7 and 3.8). At the final stage of division, gold particles indicating Mda1 signals appeared on the entire surface of the bridge, whereas those indicating dynamin signals were located only at the center of the bridge in which the outer and inner membranes were pinched off, suggesting that the final and simultaneous fission of the double membranes of the bridges between daughter mitochondria was achieved not by the MD and Mda1 rings, but by the dynamin ring (Figs. 3.7 and 3.8). After division of the mitochondria, the MDF machineries attached to alternative daughter mitochondria. This behavior of the MDF machinery reflects the behavior of the VDF machinery between vesicles and plasma membrane. Before separation of daughter cells, the dynamin rings transformed into many dynamin patches and became distributed in the cytoplasm (Nishida et al., 2003).

6. Plastid (Chloroplast) Division Machinery 6.1. Evolutionary diversity in modes of plastid division Plastids, like mitochondria, dynamically change in size, shape, and number per cell during the evolution of the Bikonta. In single cellular algae, plastids evolved through spherical, irregular, network-like and small, peanut-like shapes (Fig. 3.5). Cells of the primitive red alga, C. merolae, and the green alga, Ostreococcus tauri, contain only one spherical- or globular-shaped plastid per cell, throughout their life cycles. Although C. caldarium, Chlorella sp., and C. reinhardtii form four endospores, they basically retain one sphericalor bell-shaped plastid (Fig. 3.9). G. sulphuraria (Kuroiwa et al., 1989) and Plasmodium falciparum (Foth and McFadden, 2003) form 16 endospores, each of which has Aˆ one plastid. The plastid dynamically changes through ovoid-, elongate-, and irregular-shaped forms, and then divides into small pieces (Fig. 3.9). In addition to division and elongation of plastids, the fusion of plastids can be identified at specific stages during the life cycle in organisms ranging from C. reinhardtii and E. gracilis to higher plants (Osafune et al., 1972, 1975). The green algae cells in the Euconjugatae and in Heterokont diatom cells contain large, irregular-shaped plastids. In E. gracilis, the plastid repeatedly divides and fuses throughout the mitotic cycle (Osafune et al., 1975). In higher multi-plastid algae and plants, the number of plastids per cell increases remarkably, and these plastids undergo not only division, with plastid nuclear division, but also fusion. The plastids multiply through a division cycle of ovoid-shaped plastids and complicated, pleomorphic plastids, according to the stage of the life cycle and the

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Figure 3.9 Scanning and transmission electron micrographs showing plastid division machineries in C. merolae. (A) The outer PD ring is observed at the equatorial region of the dividing plastid (Aa). It is seen as two small, electron-dense spots at the division site in cross-sections of C. merolae cells by transmission electron microscopy (arrows in Ab, Ac).These electron-dense spots increase in width and thickness with contraction of the division site (Ab, Ac). (B) The process of PD ring formation is studied in detail in serial sections. The inner PD ring is formed at the putative division site (short arrow in Ba^ Be) and then the outer PD ring is formed outside of the inner PD ring (long arrow in Bc^Be). (C) The outer PD ring (upper) increases remarkably in width and thickness with the contraction of the division site (Bc^Be) while the inner PD ring (lower) does not change. Accordingly, the volume of the inner PD ring decreases constantly, while that of the outer PD ring does not change, suggesting that the plastid divides by decomposition of the inner PD ring and contraction of the outer PD ring. * in B, plasma membrane. Scale bars: 500 nm (Ac), 100 nm (Be). Aa, from Miyagishima et al. (1999b); A, from Miyagishima et al. (1998a); B, from Miyagishima et al. (2003a).

particular organ (leaf, stem, root, etc.): the plastids are globular-, ovoid-, and dumb-bell-shaped in the leaf, whereas they show irregular and amoebaelike shapes in the embryo and root (Duckett and Ligrone, 1993; Kuroiwa and Kuroiwa, 1992; Nishibayashi and Kuroiwa, 1982). Despite the diversity in size, shape, and number per cell of plastids throughout plant evolution, all plastids multiply by binary or unequal division, after plastid nuclear division.

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6.2. Origin and evolution of plastid division machinery It is thought that plastids were derived from free-living Cyanobacteria, which were engulfed by mitochondria-containing eukaryotic host cells, by phagocytosis (Margulis, 1970). Cyanobacteria have an elaborate and highly organized system of internal membranes which function in photosynthesis. Cyanobacteria divide using at least 19 genes, including fts (H,E, I, K, Q, W, Z ), min(C, D, E ), SulA, ftn 6, cikA, cdv(1, 2, 3), and ylm(E, G, H ), which relate directly or indirectly to inner cell membrane systems (Miyagishima et al., 2005). During primary plastid endosymbiosis, more than 80% of bacterial division genes, including the cyanobacterial division genes, such as ftsL, ftsN, ftsQ, mukB, mukF, minC, and others, were lost. The remaining bacterial division genes were also progressively lost, and as a result, several sets of bacterial division genes remained in the nuclear genome in red algae ( ftsZ ) (Matsuzaki et al., 2004; Ohta et al., 1998, 2003), green plants ( ftsH, ftsZ, minD, minE, sulA, ylmG, ylmH ), brown plants ( ftsH, ftsW, ftsK, ftsZ, minD), and other plants, while ftsH of C. merolae (Itoh et al., 1999), and two adjacent genes homologous to minD and minE of Chlorella vulgaris, exist in the chloroplast genome (Wakasugi et al., 1997). In secondary plastid endosymbiosis, even the most conserved ftsZ gene was lost in Toxoplasma gondii (Apicomplexa) in which the apicoplast (plastid) can divide using an outer PD ring-like structure) (Matsuzaki et al., 2001), suggesting that the mechanism of apicoplast division would be very useful in helping to understand the basic mechanism of plastid division (Foth and McFadden, 2003). As min (D, E ) products exist throughout the entire stroma of plastids, and the peptideglycan layer is formed at the surface of plastids, they must indirectly control plastid division. In A. thaliana, Maple et al. (2002) showed that minE can act as an evolutionarily conserved topological specificity factor during plastid division and can act together with minD during chloroplast division. A single point mutation in minD results in altered localization patterns inside chloroplasts (Fujiwara et al., 2004). Plastid division is also controlled by genes related to peptidoglycan formation. In the evolution of plastids involving peptidoglycans, plastids arose monophyletically from a Cyanobacterium with peptidoglycan. The plastids of the moss, P. patens, have nine homologous genes related to peptidoglycan biosynthesis: Mur (A-F), Ddl, pbp, and Dac. One of the genes for an Fts mutant ( fts1) encodes a penicillin-binding protein 3 (Pbp3) that is required for formation of the peptidoglycan layer of the division septum in E. coli (Weiss et al., 1999). Gene knockout of the P. patens pbp gene inhibited chloroplast division in this moss (Machida et al., 2006). However, the peptidoglycan layer at the plastid division site has been lost in the red algae and higher plants and no pbp genes have been found in C. merolae or A. thaliana (Machida et al., 2006).

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With the loss of many bacterial division genes, the host nuclear genome controls plastid division using the outer PD ring and the dynamin rings, which form on the cytoplasmic side of the membrane at the division site (Fig. 3.9) (Kuroiwa et al., 2006; Matsuzaki et al., 2004; Mita and Kuroiwa, 1988; Ohta et al., 2003). The PD ring appears to have evolved from the basic VD machinery. The electron-dense PD ring is composed of inner, middle, and outer PD rings. The outer PD ring in C. caldarium and C. merolae is a bundle of fine filaments, 5–7 nm in diameter (Kuroiwa, 1989; Mita et al., 1986; Miyagishima et al., 2001). Plastid divisions in C. caldarium were inhibited by cytochalasin B, but not by Cremart, an inhibitor of tubulin assembly. The contractile ring for cytokinesis was stained with anti-actin antibodies, but the outer PD ring was not stained. These results suggest that the filaments of the outer PD ring are actin-like filaments (Mita and Kuroiwa, 1988). Therefore, the PDF machineries are composed of a chimera of rings from Cyanobacteria (FtsZ) and eukaryotes (PD ring, dynamin ring, and other unknown rings). The PDF machinery is thought to become larger than the MDF machinery and to develop into stronger, larger, and more complicated structures to control the division of the large chloroplasts (Figs. 3.9 and 3.10) (Kuroiwa et al., 1998, 2006). The PDF machineries of C. merolae, in particular, reach about 5000 nm maximum circumference (Kuroiwa et al., 2006) and similarly large PDF machinery is observed in the green alga, N. bacillaris (Ogawa et al., 1995). However, the middle PD ring between the double membranes is not seen in higher plants, and the inner, and small outer, PD rings are only observed in the late phase. It was once thought that the PD ring appeared only during the late phase of plastid division. However, in P. zonale, the PDF machinery could be observed throughout plastid division, although the size of the machinery in higher plants tends to become small (Kuroiwa et al., 2002). Despite the dynamic changes in plastid shape and number in diverse Bikonta, the PDF machinery, including the FtsZ ring, the inner PD ring, the outer PD ring, and the dynamin ring, remains a universal structure in plastid division.

6.3. Structure, function, and constriction process of plastid division machinery 6.3.1. Positioning The time schedule of plastid divisions was determined in living cells by observations in C. merolae (Fig. 3.6) (Miyagishima et al., 1999b). The PD ring forms when the diameter of the plastid division site reaches 1300 nm. The PDF machinery is formed at the division site in the order FtsZ ring, inner PD ring, outer PD ring, and dynamin ring, and contracts and pinches off the bridge between daughter plastids (Fig. 3.9). The coordinated process of plastid division by the PDF machineries is therefore subdivided into

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positioning (scaffolding) and constriction of PDF machineries and fission of double membranes at the division site (Figs. 3.9, 3.10, and 3.13). It is suggested that, in green plants, minD and minE play roles in positioning the formation of the FtsZ rings. Since there are no plastids without nuclei (nucleoids), the progress of plastid nuclear division must relate to the function of min (D, E) and finally to formation of the FtsZ ring. However, the red algae and Apicomplexa do not contain a homologue of min (D, E), suggesting that unknown materials may have an important role in the positioning of the PDF machinery. The pleomorphic division of plastids suggests that the formed FtsZ ring attracts the inner and outer PD rings. There is pleomorphic plastid division in addition to binary division. Plastids are divided by binary division in red and green algae and in the leaves of land plants. FtsZ is organized in a ring at the central region of the dividing plastids, and soon recruits the inner PD ring to the division site. The different timing of formation of the FtsZ and the PD rings is clearer in pleomorphic plastid division. Pleomorphic plastid division has been reported in the vascular parenchyma of the ferns, Ophioglossum, Hymenophyllum, Trichomanes, and Pteridium (Duckett and Ligrone, 1993), in the embryo of the higher plant, P. zonale (Kuroiwa and Kuroiwa, 1992), in cultured tobacco BY-2 cells (Miyazawa et al., 2001; Momoyama et al., 2003) and in the apicomplexan parasite, Plasmodium falciparum (Waller et al., 2000). A typical pleomorphic plastid develops into a complex form with many branches and divides into multiple, discrete, rod-shaped plastids. In synchronous culture of BY-2 cells, the formation and division of multiple FtsZ rings could be traced easily. FtsZ rings appear in elongated plastids, but pleomorphic plastids with multiple FtsZ rings do not divide simultaneously using PDF machineries at all the FtsZ ring sites, because the PD ring, which

(B) Immunoelectron micrograph shows that silver grains (10 nm in diameter) (indicating the FtsZ ring) appear inside of the outer PD ring (arrow) at the division site, when anti-FtsZ antibodies to bacterial FtsZ were used. (C) Immunoelectron micrographs show that silver grains (indicating FtsZ) appear inside of the outer (arrow) and inner PD rings when anti-FtsZ antibodies to C. merolae FtsZ were used (Ca). Even after the FtsZ rings were separated into two regions (Ad, Ae), electron-dense PD rings remained at the bridge between the daughter plastids (Cc, Cd), suggesting that the FtsZ ring does not relate to pinching-off of the bridge. The PD ring must pinch off the bridge. (D) Immunofluorescence micrographs show that the behavior of the FtsZ ring (arrows) with (Da^Dg) and without chloroplasts in P. zonale (Dh^Dm) is similar to that in C. merolae. (E) The dynamin ring appears at the division site during the early phase of plastid division (bright line in Ec) is observed during contraction of the site (Ed, Ee) and pinches off the bridge between the daughter plastids (Ee, Ef ). Immunoelectron microscopy shows that the dynamin ring is formed at the surface of the PD ring (Eg, Eh), and finally migrates into the inside of the PD ring (Ei). Scale bars: 1mm (Af, Dg, Dm), 100 nm (Eh, Ei). A, C; from Miyagishima et al. (2001); B, from Kuroiwa et al. (1999); D, from Kuroiwa et al. (2002); E, from Miyagishima et al. (2003a).

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drives the constriction of the plastid, is formed only at a limited FtsZ ring site for one division. These results suggest that the multiplied FtsZ rings localize in advance at the expected sites of division, and the formation of the PD rings at each FtsZ ring site occurs in a certain order. ftsH, which inhibits plastid division, is a universal gene in eukaryotes and its products are distributed in chloroplasts (Itoh et al., 1997). The gene probably plays an important role in positioning of the PDF, as well as MDF, machineries (Fig. 3.13). It is unclear how the signal of the FtsZ ring or inner PD ring formation transmits from the stroma to the cytoplasm at the plastid division site. The existence of the complex of FtsZ ring and outer PD ring, even after complete digestion of the double membranes in the isolated PDF machineries by a detergent, suggests that there is an anchor or a linking structure through the membrane between the inner (FtsZ and inner PD ring) and outer PD rings (Fig. 3.13) (Yoshida et al., 2006). This putative linking structure may recruit many somewhat electron-dense granules (SEG), 40–90 nm in diameter, as these granules are distributed along the bundle of filaments (outer PD ring) and often one fine filament can be seen to be formed from one granule (Kuroiwa et al., 1998; Mita and Kuroiwa, 1988). These results suggest that SEG assemble the linking structure and that materials in the SEG are converted to the fine filaments of the outer PD ring. This may be similar to the phenomenon whereby cellular actin rapidly cycles between the monomeric (G-actin) and polymeric (F-actin) forms. 6.3.2. Contraction The outer PD ring can be visualized by scanning and transmission electron microscopy (Figs. 3.9 and 3.11). The outer PD ring grows thicker during contraction. The process of formation of the electron-dense PD ring can be studied. The inner and middle PD rings first appear at the division site and then the outer PD rings are formed outside of them, just before the PDF machinery (FtsZ ring, PD ring, and Pdv1 ring) starts to contract (Fig. 3.9). Miyagishima et al. (2006) showed that PDV1 and PDV2 rings in A. thaliana appeared during the early phase of division and mediated recruitment of dynamin to the plastid division site at a late phase of division (Miyagishima et al., 2006). As these genes were not identified in the C. merolae genome (Matsuzaki et al., 2004; Ohta et al., 1998, 2003), the putative linking structure may play a role for the PDV. The PDF machinery grew thicker and maintained a constant volume, while the thickness of the FtsZ ring and inner PD ring did not change and their volume decreased at a constant rate with contraction, suggesting that the FtsZ and inner rings were digested (Figs. 3.9 and 3.10). By contrast, the outer PD ring’s dimensions were 15 nm 10 nm (width thickness) before the initiation of contraction, and were 180 nm 45 nm (width thickness) at the end of the process (Miyagishima et al., 1999b). Therefore, the volume of the outer PD ring

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Figure 3.11 Isolation of plastids with PDF machineries from synchronized dividing cells. (A) Scanning electron micrographs show isolated dividing chloroplasts with outer PD ring (arrows). (B) Electron micrographs of isolated dumb-bell-shaped plastids with the constricted PDF machinery, negatively stained (Ba, Bb). Ba is a higher magnification of Bb. The outer PD ring is a bundle of fine filaments, 5^7 nm in diameter (arrow in Bb). Negative images of the ring at early (Bc) and late stages (Bd) of the contraction show that the filaments are composed of particles 5 nm in diameter during contraction. (C) Isolation of PDF machineries from dividing chloroplasts in C. merolae cells. Phasecontrast (Ca, Cd) and immunofluorescence images of the FtsZ (bright in Cb, Ce) and dynamin rings in isolated chloroplasts (bright in Cc, Cf ) at the early and late phases of chloroplast division. Isolated, intact PDF-machineries show ring (Cg) and spiral structures (Ch). (D) SDS-PAGE of isolated PDF-machineries with the outer membrane (Da) and isolated PDF machinery fractions (Db). In D, * show dynamin (106.6 kDa) and ** phycobilisome linker polypeptide (96.4 kDa). (E) Immunoblotting of FtsZ and dynamin proteins at each isolation step from left to right; whole cell (Ea), isolated chloroplast (Eb), isolated PDF machinery with the outer membrane (Ec), and isolated PDF machinery fraction (Ed). * FtsZ; ** dynamin. (F) Manipulation of an intact PDF machinery (top) and a dynamin-released PDF machinery (bottom) with optical tweezers.The base line of one end of each of the PDF machineries is fixed to the cover glass (top lines), while the other end is trapped by the optical tweezers (arrowheads) and an infrared

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does not change during plastid division (Fig. 3.9). Since the decrease in diameter of the division site parallels the increase in the width and thickness of the outer PDF machinery, the PDF machinery may provide the motive force for contraction. What generates the motive force? It was thought that the fine filaments, 5–7 nm in diameter, continued to contract by sliding during plastid division, using the attachment site of the filaments and myosin-like proteins on the surface of the plastid (Kuroiwa et al., 1998). However, a recent study has suggested that the function of dynamin in the operation of PDF machineries can be explained by two steps: first, as a mediator of filament sliding at the early phase of plastid division, and then to pinch off the neck of the dividing chloroplast at the late phase (Yoshida et al., 2006). Just before contraction of the dividing chloroplast, the dynamin vesicles would move from the cytosol to the outside of the outer PD ring and release dynamin to form a PDF machinery with the PD ring (Miyagishima et al., 2003a). The sliding of the PD-ring fine filaments is caused by myosin-like proteins, but no genes encoding myosin or myosinlike proteins were found in the C. merolae genome (Matsuzaki et al., 2004; Yoshida et al., 2006). Thus, it is likely that dynamin, rather than myosin-like proteins, drives the sliding of the PD-ring fine filaments and causes the pinching-off of the bridge between daughter plastids required for plastid division. 6.3.3. Pinching-off At the final phase 3, the FtsZ ring separates into the stroma of each daughter plastid and disappears, but the outer PD ring forms the bridge between daughter plastids (Figs. 3.6, 3.9, and 3.10) (Miyagishima et al., 2003a). The behavior of FtsZ in algae bears a striking resemblance to higher plants (Fig. 3.10) (Kuroiwa et al., 2002). The dynamin ring appears at the division site during the early phase and can be observed throughout plastid division (Fig. 3.10). Even during the process of constriction of the PDF machinery in C. merolae, the dynamin molecules move dynamically from the surface to the inside of the machinery ring, and are finally associated with the outer

laser (bottom lines). (G) Immunoelectron micrographs show the distribution of dynamin and FtsZ proteins (gold particles 15nm in diameter) in isolated PDF machineries after negative staining. Most of the immunogold particles (10 nm in diameter) (indicating dynamin) form a line along the outside of the fine filaments of the PDF machinery (Ga, Gb). Gb is a higher magnification of Ga. Immunogold particles are localized on the inside and outside of the filamentous PDF machineries (Ga-Gd). Most of the small immunogold particles (indicating dynamin) are distributed on the outside of the PDF machineries, as well as in the loose region, and are localized between thin filaments. Upon contraction, the immunogold particles move from the outer peripheral area (Ga^ Gc) to the inside of the PDF machinery (Gd). (A, Cf ) scale bars: 1 mm (Ba, C), 100 nm (Gd); B, from Miyagishima et al. (1999b); C, D, E, F, Gc, and Gd, fromYoshida et al. (2006).

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lipid membrane of the plastid at the division site (Figs. 3.10 and 3.13). The mechanism of movement of dynamin in the machinery as well as in the cytoplasm is unknown. At the late phase of plastid division, almost all of the dynamin molecules assemble around the lipid bilayer and pinch off the membranes at the bridge between the daughter chloroplasts (Fig. 3.10) (Miyagishima et al., 2003a). It is generally believed that only dynamin forms a helix around the neck of a nascent vesicle formed from the plasma membrane and that cooperative GTP hydrolysis results in the lengthwise extension of this helix, breaking the vesicle neck by physical extension. Organelle division is thought to occur in a similar way. However, the analogy of function with MDF machinery suggests that the vesicles and the plastids seem to be divided by a chemical reaction by the VD and PDF machineries, respectively (Figs. 3.10 and 3.13). All dynamins contain a GTPase domain that binds and hydrolyses GTP, a middle domain, and a GTPase effector domain (GED) that are involved in oligomerization and stimulation of GTPase activity. Since dynamins contain a pleckstrinhomology (PH) domain, a transmembrane domain, or a sequence for lipid attachment in Amoebozoa, Bikonts, and Opisthokonts (Fig. 3.3) (Praefcke and McMahon, 2004; Verma and Hong, 2005), this domain is supposed to react directly with the lipid bilayer of the plastid outer membrane by a chemical reaction, pinching it off. However, in order to divide plastids, the fission of the outer and inner membranes must occur simultaneously. After division of the plastids, the PDF machinery, like the MDF machinery, attaches to only one daughter plastid. This may reflect the response of the VDF machinery to the unequal structure of the vesicles and the plasma membrane.

7. Isolation of Organelle Division Machinery and Its Significance 7.1. Isolation of organelle division machinery Although FtsZ and dynamin are ubiquitous, key players in division, models for organelle division will ultimately have to incorporate the functions of the various other division proteins that have been identified. To elucidate the structure and function of the OD machinery, cell biological and morphological approaches, as well as genetic, genomic, and biophysical approaches, are essential. To fill the gap between in vivo and in vitro results relating to MDF and PDF machineries, to reveal components such as the linker and unknown MD and PD ring proteins, and to understand the mechanisms whereby the inner and outer membranes are simultaneously pinched off by the MDF and PDF machineries, isolation of intact OD machineries is required.

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We found that P. polycephalum mitochondria are simple shapes and contain the largest electron-dense, rod-shaped nuclei (nucleoids), similar to the situation in the salivary gland chromosomes of Chironomus dorsalis cells However, the cell contains many mitochondria, which do not divide synchronously and have small obscure MD machineries (Kuroiwa et al., 1977). Since then, we have searched for a suitable model organism for light and electron microscopic studies of organelle divisions and have found C. merolae to be such an organism suitable for organelle research (Misumi et al., 2005, 2008). The advantages can be summarized as follows: (1) mass culture is very easy. (2) The cells are small in size and contain the minimum set of doublemembrane-bounded and single-membrane-bounded organelles, which can be made to divide synchronously by light/dark cycles (Fig. 3.6). (3) The PD and the MD machineries are the largest ones in eukaryotes and the PDF machinery is 10 104 times larger in volume than that of the VD machinery. (4) 100% of the nuclear (16,546,747 bp) (Matsuzaki et al., 2004; Nozaki et al., 2007), mitochondrial (32,211 bp) (Ohta et al., 1998), and plastid genomes (149,987 bp) (Ohta et al., 2003) have been sequenced (Fig. 3.12). Almost all of the genes have no introns and the gene number is the smallest of the eukaryotes and thus proteome analysis is very easy. All genes related to organelle divisions seem to be evenly distributed (Fig. 3.12). (5) The intact OD machineries can be isolated from the cells at each stage during the cell cycle, and (6) nuclear transformation by homologous recombination is developed (Minoda et al., 2004). To isolate PDF machineries from C. merolae, dividing chloroplasts were obtained from highly synchronized cells treated with Nonidet P-40 (NP-40) to dissolve the thylakoid and inner membranes. The purity of the isolated PDF machineries was examined by fluorescence microscopy, scanning and transmission electron microscopy (Fig. 3.11). The isolated chloroplasts contain PD rings, which clearly show a bundle of fine filaments, 5–7 nm in diameter (Fig. 3.11). The plastids were treated with n-octyl-b-Dglucopyranoside (OG) to dissolve their outer envelopes, after NP-40 treatment (Fig. 3.11). As a result, the membrane-free, PDF machineries form super-twisted rings, circular rings, and spirals. In addition, when isolated PDF-machinery fractions, with or without the outer membrane, were analyzed by SDS-PAGE and MALDI-TOF-MS, dynamin was not detected in the outer membrane fraction (NP-40 treatment), but was present in the fraction without the outer membrane (NP-40 and OG treatments). Immunoblotting indicated that dynamin and FtsZ were concentrated in the isolated PDF-machinery fraction (Figs. 3.11 and 3.12) (Yoshida et al., 2006). Thus, the isolated PDF machinery was intact, and was composed of an FtsZ ring (inside) and PD and dynamin rings (outside), connected through the membranes, suggesting that there is the linking structure through the membrane between the inner and outer rings (Fig. 3.13).

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Figure 3.12 Distribution of genes related to mitochondrial and plastid division machineries in the complete nuclear, mitochondrial and plastid genome sequences, and MALDI-TOF-MS analysis. (A) The eukaryotic genome of C. merolae is composed of 16,727,945 nucleotides, consisting of mitochondrial (32,211 bp) (Aa), plastid (148,987 bp) (Ab), and 20 linear unambiguous DNA molecules (cell nuclear; 16,546,747 bp) (Ac). All genes related to mitochondrial and plastid division are distributed throughout the chromosomes. (B) SDS-PAGE of isolated PDF machineries (Ba) compared with interphase fraction (Bb). (Bc) The unique spot in PDF machineries has been proteome-analyzed by MALDI-TOF-MS, suggesting that about 20 more genes are related to plastid and mitochondrial division. Aa, from Ohta et al. (1998); Ab, from Ohta et al. (2003); Ac, from Matsuzaki et al. (2004).

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Figure 3.13 Model for plastid division. (A) The outer and inner membranes of the plastid are shown in dark gray and green, respectively. The location of the inner PD ring, the FtsZ ring, the outer PD ring, the dynamin ring, and dynamin patches are indicated. Images of dynamin are represented with red broken lines. In the positioning phase (Aa^Ac), the inner PD and FtsZ rings form from the matrix side with the help of FtsH proteins, and the division site is determined. Dynamin is located in cytoplasmic patches. In the constriction phase (Ad^Af), after FtsZ ring formation, the outer PD and then the dynamin rings appear on the cytoplasmic side of the outer membrane (Ad). The inner PD, FtsZ, outer PD, and dynamin rings begin to constrict at the plastid division site (Ae, Af ). In the first step of the pinching-off phase (Ag), when the plastid has

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7.2. Proteins and genes involved in the organization of organelle division machinery All protein samples, namely the NP-40-insoluble fraction and the isolated PDF-machinery fraction, were analyzed by one- or two-dimensional SDSPAGE. Fifty spots and bands were analyzed with MALDI-TOF-MS. Since 100% of the C. merolae genome has been sequenced, and almost all their genes have no introns, it was easy to search the genes. The database of the C. merolae genome (5014 sequences) was compared with other databases, using the Mascot (Matrix Science Ltd.) software program. The maximum number of missed cleavages was set at one. The following criteria were used to assign an identification: a probability score calculated by Mascot of higher than 100. Probability scores of Coomassie blue-stained bands were 133 (CmDnm2/DRP5) and 304 (phycobilisome linker polypeptide), respectively. After analysis by SDS-PAGE, the Coomassie blue-stained bands of the predicted dynamin (CmDnm2/DRP5; 106.6 kDa) and phycobilisome linker polypeptide (96.4 kDa) were analyzed by peptide mass fingerprinting (PMF) measurements using MALDI-TOF-MS. The existence of supertwists and spirals showing isolated PDF machineries suggested that the PDF machineries were responsible for generating the dividing force for contraction during the early phase of plastid division, thereby posing the question of which rings and/or factors within the PDF machineries generate the force. The presence of conformational changes could not be confirmed by the GTPase assay. The FtsZ-released and intact PDF machineries showed spiral

constricted sufficiently to become tubular at the division site, the dynamin ring migrates to a space inside the thickened outer PD ring and outside the outer membrane (Ag).The FtsZ ring splits to form two fragments, one in each daughter plastid stroma, and then disappears, and the PDF machinery simultaneously pinches off the outer and inner membranes (Ag, Ah). PDF machinery remnants then stick to one side of the daughter plastids and are reduced to patches (Ah). (B) Model showing two steps for the functioning of dynamin molecules in the PDF machinery. In the first step, the GTPase dynamin molecules in the PDF machinery function as cross-bridges that undergo microscopic movements to drive the sliding of the 5^7 nm filaments in the PD ring during the early phase of chloroplast division.The dynamin ring generates the driving force for contraction with the aid of the other rings. In the second step, the dynamin molecules move from the surface of the PDF machinery to the inside of the machinery, and play a role in pinching off the narrow bridge between daughter plastids during the late phase of chloroplast division. (C) Putative molecular structure of PDF machinery at the late phase of chloroplast division is imaged based on isolated machineries. After the inner PD and FtsZ rings are split to the stroma of the daughter plastids, the dynamin ring then simultaneously pinches off the outer and inner membranes at the center of the bridge, by a biochemical reaction between the PH domain and the double membranes, under the control of the outer MD ring. A, modified fromYoshida et al. (2006).

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structures, whereas the dynamin-released PDF machineries were straight. The results were confirmed by optical tweezer experiments. When individual spiral PDF machineries were stretched to four times their original lengths by optical laser trapping, they returned to their original sizes upon release. In contrast, dynamin-released straightened PDF machineries were unable to recover from stretching (Fig. 3.11). To reveal the dynamics of the PD, dynamin, and FtsZ rings in the PDF machineries, we examined more intact, isolated PDF machineries by immunoelectron microscopy. Immunogold particles, indicating dynamin signals, were located in a spiral line along the periphery of the supertwisted and spiral PDF machineries (Fig. 3.11). However, the dynamin-released region became disorganized. After treatment with OG for 20 min, the disassembled PDF machineries included a smooth PD ring as a bundle of fine filaments, with dynamin colocalized between the filaments, supporting the notion that the dynamin ring generates the motive force for contraction within the PD ring. As the contraction of the PDF machineries progressed, the supertwisted PDF machineries changed into compact circles. Immunogold particles, indicating the FtsZ signals, were located on the inside of the filamentous circular PD ring, where the membrane was dissolved. In chloroplasts in vivo, these rings seemed to be linked to each other through holes that appeared at the groove of the division site, as revealed by scanning electron microscopy (Yoshida et al., 2006). In contrast, the immunogold particles indicating dynamin signals were found on the outsides of the filamentous circular PD ring, and some of the particles could be seen between the thin filaments. When contraction occurred during the late phase of chloroplast division, the dynamin signals moved from the outside to the inside of the constricted PDF machineries. This does not conflict with the in vivo observations using immunoelectron microscopy (Figs. 3.10 and 3.11). We suggest that the function of dynamin in the operation of the PDF machineries can be explained by two steps: first, as a mediator of filament sliding at the early phase of chloroplast division, and then to pinch off the neck of the dividing chloroplast at the late phase. It is thought that the dynamin molecules finally pinch off the membranes in the bridge between daughter plastids, suggesting that the mechanism of pinching-off of the plasma membrane by the PDF machinery is very similar to that by MDF machinery and VD machinery, despite the difference in size. Primitive VD machinery probably evolved into VD machinery, MDF machinery, and PDF machinery, and thus common mechanisms must operate for the division of these organelles. It is possible to isolate the MDF machineries by using improved techniques, indicating that the proteome of large isolated PDF and MDF machineries provides the key to understanding the basic components, structure, and function of the different OD machineries (VD, MD, and PD machineries).

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8. Concluding Remarks In early proto-eukaryotes, VD machinery including dynamin first occurred as a small apparatus for endocytosis (pinocytosis) on the cell membrane. It diverged into two types of machineries for pinocytosis on the plasma membrane: the formation of synaptic vesicles and the pinchingoff of buds from transport vesicles of ER and secretory vesicles of Golgi apparatus. VD machinery consists of a small ring, 120 nm in circumference, because for pinocytosis and budding, the drop engulfed and transport vesicles formed are relatively small, and thus a strong motive force for their formation is not required. With the enlargement of host proto-eukaryotic cells, the cells needed to engulf small a-Proteobacteria, like Rickettsia felis, using the VD machinery for phagocytosis on the plasma membrane, which therefore evolved from the VD machinery for pinocytosis. The VD machinery for phagocytosis diverged into mitochondrial division (MD) machinery following the symbiotic incorporation of a-Proteobacteria into the host cells. As almost all bacteria, except thioautotrophic intracellular symbiotic bacteria, divide using the FtsZ ring, MDF machineries include a chimera of the FtsZ ring from bacteria and the MD ring, the dynamin ring, the mitochondrial division protein 1 (Mda1) ring, and some undefined rings from host eukaryotes. The MDF machinery developed strong structures 1 mm maximum circumference because host cells needed to control the division and multiplication of mitochondria, which evolved into diverse shapes and numbers per cell. In the Opisthokonta, the FtsZ rings were lost from the MDF machinery. In the evolutionary process to Bikonta (plants), the host cells containing mitochondria may have engulfed ancestral photosynthetic Cyanobacteria, probably using VD machinery for phagocytosis of the Cyanobacteria. With the change from Cyanobacteria to plastids, VD machinery also changed into plastid division (PDF) machineries, which included the FtsZ ring, dynamin ring, and PD ring. Thus, the process of formation of the PDF machineries is similar to that of the MD machineries. However, the PDF machineries become large (4 mm maximum circumference) in C. merolae as plastids are larger than mitochondria. The PDF machinery, like the MD machinery, is also a chimera of the rings from bacteria and eukaryotes. Hydrogenosomes, mitosomes, and microbodies, which have no DNA, also multiply by binary division of the preexisting organelle and thus these divisions also need to be studied. Organelle division (OD) machineries, including VD, MDF, and PDF machineries, represent a dynamic front of interaction between the endosymbiont and the host cell nuclear genome. The mechanism of action of OD machineries in the constriction and division (fission) of membranes of vesicles, mitochondria, plastids, and microbodies has been studied in mutants, by in vivo observation

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and by in vitro experiments. To fill the gap between in vivo and in vitro experiments and to prove directly the structure, role, and function of each ring of the OD machineries, the isolation and biochemical analyses of OD machineries are essential. The cells of C. merolae offer unique advantages for the study of OD machineries, because they contain a minimum set of organelles, the division of which can be highly synchronized by the light/ dark cycle. C. merolae is an alga in which 100% of the three genomes— nucleus (16,546,747 bp), mitochondrion (32,211 bp), and plastid (149,987 bp)—have been sequenced and it has the largest MDF and PDF machineries of the eukaryotes, which will be isolated almost completely in the near future. All of this information will, in turn, help to elucidate the origin, evolution, and fundamental mechanisms of both single- and doublemembrane-bounded organelles, using microarray and proteome analyses.

ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research (No. 19207004 to T.K.), and by grants from the Japan Society for Frontier Project ‘‘Adaptation and Evolution of Extremophile’’ (to T.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and from the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN, to T.K.).

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Retromer: Multipurpose Sorting and Specialization in Polarized Transport Marcel Verge´s Contents 1. Introduction: Basic Concepts on Coat-Mediated Vesicular Transport 2. Retromer’s Assembly and Functioning 3. Multiple Roles of Retromer: Models of Study 3.1. Yeast 3.2. Protozoa 3.3. Plants 3.4. Nematodes 3.5. Arthropods 3.6. Mammalian cells 3.7. Mouse 4. Polarized Transport Mediated by Retromer 4.1. Polarity of PIN proteins in plant development 4.2. Secretion of Wnt proteins in animal embryo development 4.3. Transcytosis of the polymeric immunoglobulin receptor 5. Implication of Retromer and Sorting Nexins in Other Aspects of Polarity 5.1. Polarity establishment in yeast 5.2. Membrane remodeling 5.3. Altered retromer-mediated transport and Alzheimer’s disease 6. Concluding Remarks Acknowledgments References

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Abstract Retromer is an evolutionary conserved protein complex required for endosometo-Golgi retrieval of lysosomal hydrolases’ receptors. A dimer of two sorting nexins—typically, SNX1 and/or SNX2—deforms the membrane and thus cooperates with retromer to ensure cargo sorting. Research in various model Laboratory of Epithelial Cell Biology, Centro de Investigacio´n Prı´ncipe Felipe, C/E.P. Avda. Autopista del Saler, 16-3 (junto Oceanogra´fico), 46012 Valencia, Spain International Review of Cell and Molecular Biology, Volume 271 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01204-5

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2008 Elsevier Inc. All rights reserved.

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organisms indicates that retromer participates in sorting of additional molecules whose proper transport has important repercussions in development and disease. The role of retromer as well as SNXs in endosomal protein (re)cycling and protein targeting to specialized plasma membrane domains in polarized cells adds further complexity and has implications in growth control, the establishment of developmental patterns, cell adhesion, and migration. This chapter will discuss the functions of retromer described in various model systems and will focus on relevant aspects in polarized transport. Key Words: Retromer, Sorting nexin, Vps26, Vps35, Vps29, Traffic, Polarity, Development. ß 2008 Elsevier Inc.

1. Introduction: Basic Concepts on Coat-Mediated Vesicular Transport Communication of cells with the external environment is a key aspect in multicellular organisms. An elaborated internal membrane system formed by compartments or organelles with a well-defined composition of resident molecules allows such communication. By this membrane system, the cell exports material to the external milieu in a secretory or biosynthetic (exocytic) pathway and, at the same time, imports or internalizes molecules into intracellular compartments in an endocytic pathway. These two pathways are in close connection in such a way that, while many components are continuously moving along each pathway, other components are exchanged between them. To move molecules between intracellular compartments, a donor compartment undergoes a budding process involving membrane deformation, tubulation, and scission of a vesicle containing the material to be transported, with the consequent fusion of this vesicle with a target compartment. As a requirement for proper cell functioning and homeostasis, a whole set of molecules in each compartment maintains a strict control of the transport processes. Among these molecules, there are certain cytosolic proteins that assemble on the membrane forming coat complexes. Functioning as a single entity, these complexes perform a dual role in vesicle transport. Thus, the coat has a structural function in shaping the membrane into regions of high curvature. In addition, it has a sorting function in capturing specific cargo into these deformed membrane areas, which involves specific interactions between sorting motifs on the cargo molecules and the coat components that recognize these motifs (Gu¨rkan et al., 2006; Kirchhausen et al., 1997; Rothman, 1994; Schekman and Orci, 1996). A classic coat protein is clathrin, whose selectivity in subcellular localization and cargo selection is often determined by clathrin adaptor protein (AP) complexes and by additional accessory factors. These coats function in

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transport events between plasma membrane, endosomes, the trans-Golgi network (TGN), and lysosomes. Four heterotetrameric AP complexes are known to act at various subcellular locations. At the plasma membrane, clathrin coats contain AP-2 in addition to various accessory proteins that can also operate as monomeric adaptors and thus have been defined as alternate adaptors (Robinson, 2004; Traub, 2003). At the TGN and at the endosome, clathrin coats contain AP-1 and/or monomeric Golgi-localized, g-earcontaining, ADP ribosylation factor (ARF)-binding (GGA) proteins. Another endosomal complex is AP-3, which can function with or without clathrin. Also on endosomes, clathrin coats can be found associated with Hrs—the hepatocyte growth factor-regulated tyrosine kinase substrate— instead of with AP complexes. Finally, at the TGN, AP-4 appears to mediate sorting in a clathrin-independent manner (Bonifacino and Traub, 2003; Evans and Owen, 2002; Hicke and Dunn, 2003). Other coats not involving clathrin are the components of the coatomer protein complex I (COPI) or II (COPII), which mediate transport in the secretory pathway. COPI functions in transport through the Golgi apparatus and in retrograde transport between the Golgi and the endoplasmic reticulum (ER), whereas COPII delivers proteins from the ER to the Golgi (anterograde pathway) (Lee et al., 2004). Research in the yeast model system has unequivocally aided to the discovery and knowledge of the diverse membrane coat complexes functioning in membrane traffic. Among them, we will focus on a multimeric protein complex found required for endosomal retrieval to the Golgi apparatus of a restricted group of transmembrane proteins (Horazdovsky et al., 1997; Nothwehr and Hindes, 1997; Seaman et al., 1997). Because of its role in protein retrieval, it was named retromer (Seaman et al., 1998).

2. Retromer’s Assembly and Functioning The vacuolar protein sorting (Vps) group comprises a large number of proteins required for protein targeting to the yeast vacuole, the organelle equivalent to the mammalian lysosome but also involved in storage and homeostasis (Bowers and Stevens, 2005; Bryant and Stevens, 1998; Horazdovsky et al., 1995). The retromer complex is constituted by five of these Vps subunits. In yeast, as in mammalian cells, soluble enzyme precursors are sorted by specific receptors at the late-Golgi/TGN membrane to be delivered to the vacuole/lysosome. The carboxypeptidase Y receptor Vps10p is a major sorting receptor for hydrolases in yeast, while the mannose-6-phosphate receptors (MPRs) take over this function in mammalian cells. GGAs are recruited at the late-Golgi/TGN membrane through

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interaction with ARF GTPases, and subsequently cooperate with AP-1 and additional accessory factors to package Vps10p or MPRs into clathrincoated vesicles, which will be released and delivered to the endosomal system. Under the acidic environment of the yeast prevacuolar compartment (PVC), or in the mammalian endosome, the hydrolase precursors dissociate from their receptors and follow their fate into the lytic compartment, where they are cleaved to produce their active forms. The empty receptors do not get degraded and, instead, they recycle back to the Golgi for reuse, undergoing new rounds of enzymes delivery. This is the transport step mediated by the retromer complex and in which various components of a retrograde transport machinery also participate (Bonifacino and Rojas, 2006; Seaman, 2005; Verges, 2007). Retromer subunits are organized into two subcomplexes. The Vps26– Vps35–Vps29 subcomplex is in charge of cargo-recognition, whereas a dimer of two sorting nexins—in general, formed by SNX1 and/or SNX2—constitutes a second subcomplex that deforms the membrane to ensure efficient cargo sorting. Remarkably, the genes encoding for retromer’s subunits, retromer’s assembly (Haft et al., 2000), and its function are conserved throughout evolution. Thus, while yeast retromer retrieves Vps10p from endosomes-to-Golgi, the mammalian retromer is required for recycling in a similar manner one type of MPR, the cation-independent MPR (CI-MPR) (Arighi et al., 2004; Seaman, 2004). Numerous studies in yeast or in mammalian cells have contributed to elaborate working models of retromer’s assembly and functioning (Bonifacino and Rojas, 2006; Carlton et al., 2005; Seaman, 2005; Seet and Hong, 2006; Verges, 2007; Worby and Dixon, 2002). However, newer structural and biochemical data focusing on Vps35 (Gokool et al., 2007a; Hierro et al., 2007; Restrepo et al., 2007; Zhao et al., 2007), the key subunit that interacts directly with cargo (Arighi et al., 2004; Nothwehr et al., 2000), should be incorporated in the context of studies on the subunits performing a more secondary role in cargo-recognition, that is, Vps26 (Collins et al., 2008; Shi et al., 2006) and Vps29 (Collins et al., 2005; Wang et al., 2005). Indeed, since this chapter was submitted, new reviews dealing with the biology of retromer and SNXs have been released (Collins, 2008; Cullen, 2008), including one that discusses retromer’s requirement for various physiological and developmental processes that depend on retrograde transport (Bonifacino and Hurley, 2008; see Section 3). A first level of study of retromer’s biology has been addressing the regulation of the complex’s assembly and association with membranes. From early experiments in the yeast model and later in higher eukaryotes, it became clear that assembly of retromer involves the cooperation of its two subcomplexes. Compelling evidence from various laboratories points to a leading participation of phosphoinositide 3-kinases (PI3Ks) on determining the precise membrane association of the SNXs dimer. The SNXs

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consequently provide a site for proper membrane location of the cargorecognition subcomplex, thus ensuring efficient cargo sorting (Arighi et al., 2004; Burda et al., 2002; Gullapalli et al., 2004; Oliviusson et al., 2006; Rojas et al., 2007; Seaman and Williams, 2002; Verges et al., 2007). These data agree with the conception that SNXs bind preferentially to membrane regions rich in phosphatidylinositol 3-phosphate [PtdIns(3)P] or other 3-phosphoinositides (PIs) via their common N-terminal phox homology (PX) domain. In addition, SNXs dimerize through their C-terminal coiledcoil regions forming a functional banana-shaped Bin/amphiphysin/Rvs (BAR) domain, which contributes to their association with tubular endosome microdomains rich in 3-PIs and may provide, by itself, the sufficient energy for membrane deformation and tubule formation (Carlton et al., 2004; Zhong et al., 2005). Indeed, SNX1 itself has now been found to define a specialized sorting exit from early endosomes toward the TGN (Mari et al., 2008), which develops into uncoated vesicles and short, nonbranched tubules that appear distinct from the previously described tubular endosomal network that takes cargo to multiple destinations (Bonifacino and Rojas, 2006). How do the SNXs dimer and the cargo-recognition subcomplex cooperate? At least in the mammalian retromer, it has been observed that the three subunits of the cargo-recognition subcomplex assemble forming a stable heterotrimer that interacts weakly or transiently with the SNXs dimer, suggesting that assembly and association of the two subcomplexes with membranes is independently regulated (Haft et al., 2000; Rojas et al., 2007; Verges et al., 2007). The structure of the cargo-recognition subcomplex has been elucidated and conserved motifs determining interaction between each subunit have been identified. From this research, an overall very similar architecture between the yeast retromer and its ortholog complex in higher eukaryotes is proposed (Fig. 4.1). In this conception, Vps26 regulates membrane association of Vps35 (Gokool et al., 2007a; Restrepo et al., 2007; Zhao et al., 2007), while Vps29 is required for interaction of the Vps26–Vps35–Vps29 subcomplex with the SNXs dimer (Collins et al., 2005). Vps35, whose flexible a-solenoid structure contains multiple putative binding sites to cargo and to SNXs, can extend along the deformed membrane interacting with Vps26 and Vps29 at each end (Hierro et al., 2007). For Vps29, which has a phosphoesterase fold, it has not been conclusive whether it shows phosphatase activity, and most likely the main function of its metallophosphoesterase site is to provide a scaffold for the C-terminal half of Vps35 (Collins et al., 2005; Damen et al., 2006; Hierro et al., 2007; Wang et al., 2005). Vps26, which binds to the N-terminal end of Vps35 through a loop and contiguous residues at its C-terminal domain, has an arrestin fold (Collins et al., 2008; Shi et al., 2006). As arrestins bind to seven-membrane-spanning G-protein-coupled receptors (GPCR) to mediate their endocytosis and inhibit receptor’s

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I235

[C−]

yI172 yS173 yK174

[N−]

Figure 4.1 Functional residues/domains in retromer’s cargo-recognition subcomplex. The mammalian subcomplex is displayed, with data obtained for the yeast counterpart also incorporated.Vps35 interacts with Vps26 at its N-terminus through the conserved PRLYL motif, being R107/L108 essential for the interaction. Residues I235, P247/R249, and perhaps adjacent residues in a loop at the C-terminal lobe of Vps26, form a contiguous surface for interaction withVps35 and are required for Vps26 integration in the subcomplex.This largely applies to bothVps26 paralogs, although inVps26B, L197 and R199 (in the drawing, the aminoacid numbering forVps26A is shown), located on an adjacent

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signaling (Lefkowitz and Whalen, 2004), it has been speculated that Vps26 may analogously undergo an arrestin-like conformational change. Remaining integrated in the subcomplex, this process would allow Vps26 binding to a putative transmembrane protein, thus explaining its regulatory role in membrane association (Shi et al., 2006). This has not been demonstrated, and in fact additional biochemical studies have shown that surface residues involved in arrestin function do not appear to be conserved in Vps26 (Collins et al., 2008). While this most recent study makes it unlikely, the idea would fit with data showing that, when Vps35 is mutated in residues of the conserved PRLYL region required for its interaction with Vps26, Vps35 does not associate with endosomal membranes. These data also suggest that initial recruitment of Vps26 to the membrane is a requirement for proper membrane association of the whole cargo-recognition subcomplex (Gokool et al., 2007a). Yet, it is not clear whether assembly of this subcomplex takes place sequentially on the endosomal membrane, or whether it is recruited to the membrane after previous assembly as a unit in the cytosol (Collins et al., 2008; Gokool et al., 2007a; Hierro et al., 2007). In the process of cargo selection, the subcomplex is predicted to align more or less parallel to the membrane, allowing its multiple SNX and surface, are also involved in the interaction.Vps26 may also undergo a conformational change and interact with a putative transmembrane (TM) protein perhaps through exposed residues at its N-terminal half. Within this region, residues 172-174 confer a dominant negative phenotype to yeast Vps26p; of these, I172 and K174 are conserved, but none are relevant for mammalian Vps26 integration in the complex or for retromer’s function.Vps35 interacts withVps29 along its C-terminus and several residues are implicated (see Hierro et al., 2007 for specific residues). Segments 534-541 and 579-589 of Vps35 include residues blocking Vps29 metallophosphoesterase site, whereas segments 629-637,672-675,722-729 and 769-776 comprise other residues contactingVps29. Segment 488-499 encompasses other residues involved. In Vps29, residues V90 and I91, located next to its metal binding site, are essential for binding toVps35. Segment 92-104 includes residues surrounding the metallophosphoesterase site and residues 139-142 also interact withVps35. An intact metal-binding site inVps29, included within its first 115 residues, is required for the subunit’s stability.The proposed Zn2þ -dependent enzymatic activity of Vps29 appears dependent on residues D8, N39, D62, H86 and H117 located at its presumed catalytic site. L25 and L152, located at the hydrophobic region opposite toVps35 binding, are also important for retromer’s functioning and are probably involved in interaction with SNXs. As depicted, at least two regions of Vps35 interaction with the N-terminus of SNX1 exist. Interaction with cargo may take place in any of the five hydrophobic grooves located between a-helices at the outer surface of Vps35. The region extending from 500-693 is important for interaction with the CI-MPR. In yeast, residues D528, L563 and L568 of Vps35p are putative binding sites of Vps10p, but they are not conserved in higher eukaryotes.The conserved yeast D123 (D132 in humans) is required for DPAP A retrieval, but it is not found in the hydrophobic grooves region. Finally, Y791 was found as a possible Src phosphorylation site on Vps35. Selected refs: Arighi et al., 2004; Collins et al., 2008; Collins et al., 2005; Damen et al., 2006; Gokool et al., 2007a; Gullapalli et al., 2004; Haft et al., 2000; Hierro et al., 2007; Lock et al., 1998; Nothwehr et al., 1999; Reddy and Seaman, 2001; Seaman and Williams, 2002; Shi et al., 2006;Wang et al., 2005.

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cargo-binding sites to interact in a cooperative way. This way, Vps35 would interact with cargo embedded in the curved membrane (Hierro et al., 2007). How interaction of retromer with cargo is regulated deserves detailed examination, and perhaps it involves phosphorylation of SNXs or other posttranslational modification on some of the subunits or on associated proteins (Gokool et al., 2007b; Seaman, 2005).

3. Multiple Roles of Retromer: Models of Study Research in model organisms has shown that retromer participates in endosomal sorting of various cargos (Bonifacino and Rojas, 2006; Seaman, 2005; Verges, 2007). Retromer can cooperate with other SNX family members besides SNX1 or SNX2 (Kama et al., 2007; Strochlic et al., 2007; Wassmer et al., 2007). Some SNXs can also act independently, in cooperation with other Vps proteins, AP complexes, or perhaps with other adaptors (Chin et al., 2001; Gullapalli et al., 2006; Hettema et al., 2003). Establishing links when possible, the following Subsections deal with the participation of retromer in multiple sorting events.

3.1. Yeast The basic mechanisms of protein delivery to the vacuole, including the retromer’s machinery, are conserved between the two most studied yeast models, that is, the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe (Iwaki et al., 2006; Takegawa et al., 2003). Most of the studies on yeast retromer have been performed in S. cerevisiae. Binding sites to S. cerevisiae Vps35p have been revealed for Vps10p and also for the model protein A-ALP (Fig. 4.1), the latter, a TGN-membrane protein consisting of the N-terminal cytosolic domain of dipeptidyl aminopeptidase A (DPAP A) fused to the transmembrane and luminal domains of alkaline phosphatase (ALP) (Nothwehr et al., 1999, 2000). However, yeast retromer also recycles from endosomes Kex2p which, like DPAP A, is also a Golgi-resident endopeptidase involved in proteolytic processing of the secreted mating pheromone a-factor (Nothwehr and Hindes, 1997; Seaman et al., 1997). These sorting processes require of the SNXs Vps5p and Vps17p; Vps5p is ortholog of SNX1 and SNX2, whereas Vps17p exists only in fungi. Another SNX required for Kex2p and DPAP A retrieval is Grd19p, the SNX3 ortholog (Voos and Stevens, 1998). Grd19p, along with Vps5p and Vps17p, are also required for late endosome retrieval of Pep12p—a target-soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (t-SNARE)—but Grd19p is not involved in Vps10p retrieval (Hettema et al., 2003; Voos and Stevens,

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1998). Endocytic recycling of the Fet3p-Ftr1p iron transporter complex is also dependent on Grd19p. Here, Grd19p functions as a cargo-specific accessory component of retromer, perhaps as a coincidence sensor detecting Fet3p-Ftr1p on PtdIns(3)P-containing endosomes, or as an adaptor linking retromer to cargo (Strochlic et al., 2007). Retromer components have also been found in a complex with Snx4p and the Batten diseaserelated protein Btn2p functioning in endosome-to-Golgi retrieval of the yeast Golgi integral membrane protein (Yip1p)-interacting factor (Yif1p) (Kama et al., 2007), although the involvement of Snx4p in retromer transport is controversial (Hettema et al., 2003). In addition to endosome-to-Golgi retrieval, Vps35p has recently been implicated in the late endosome-to-vacuole pathway and proposed to function in vacuole biogenesis (Takahashi et al., 2008). From a different perspective, human secretory proteins exogenously expressed in yeast can also be recycled from endosome-to-Golgi supposedly through a Vps10p/ retromer-mediated process (Agaphonov et al., 2005). Finally, yeast retromer has also been linked to broader aspects of cell function unrelated to membrane traffic control. In this regard, Vps35p has been involved in the regulation of gene expression, but the mechanism, which may implicate the PI3K Vps34p and downstream kinases, is not clear (Voronkova et al., 2006).

3.2. Protozoa When studying the virulence mechanisms of the protozoan parasite Entamoeba histolytica, a research group came up with the implication of retromer’s cargo-recognition subcomplex in transport of cysteine proteases to phagosomes (Nakada-Tsukui et al., 2005). During pathogenesis of invasive amoebiasis, these enzymes contribute to overcoming the protective mucus barrier by degrading the extracellular matrix, and to avoiding the host immune response by cleavage of secreted immunoglobulins (Que and Reed, 2000). The amoeba’s Vps26–Vps35–Vps29 subcomplex interacts with the small GTPase EhRab7A only when bound to GTP, suggesting that it acts, as a Rab7 effector, in retrograde transport of putative protease receptor(s) from PVCs or phagosomes to the Golgi (Nakada-Tsukui et al., 2005). However, no evidence of such receptor(s) has been reported so far. EhVps26 and EhRab7A directly interact by the EhVps26 C-terminal 117 residues, a region rich in charged amino acids and absent in yeast Vps26p, of variable length and poor conservation in different orthologs and without significant homology to any known domain (Collins et al., 2008; NakadaTsukui et al., 2005; Reddy and Seaman, 2001). Since amoeba lacks orthologs of Vps5p and Vps17p, or other BAR domain-containing SNXs, a different picture in retromer’s action probably takes place in this microorganism, perhaps involving other PtdIns(3)P-binding proteins (Nozaki and

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Nakada-Tsukui, 2006). If we assume that EhRab7A recruits retromer to prephagosomal and phagosome membranes in E. histolytica, and an equivalent mechanism actually exists in higher eukaryotes, a possible requirement of GTPase activity to provide the energy for retromer’s coat formation should be considered. Concerning this aspect, a retromer-interacting protein containing a putative phosphate-binding loop (P-loop) motif has been identified in mammalian cells; EHD1—the Eps15 homology (EH) domaincontaining protein 1—presents this glycine-rich sequence capable of ATP/ GTP-binding, suggesting that it may recruit downstream proteins needed for membrane tubulation (Gokool et al., 2007b). An EH domain protein sharing 45% identity with human EHD1 is in fact present in the E. histolytica genome (XP_655680). EHD1, however, is not present in yeast, although EH domain members are found highly conserved in yeast and other unicellular organisms (Naslavsky and Caplan, 2005), suggesting that these could perform a similar role in retromer’s action throughout evolution.

3.3. Plants Orthologs of all retromer subunits are present in the plant genome (Vanoosthuyse et al., 2003). In tobacco cells, the cargo-recognition subcomplex was immunodetected at the periphery of a PVC (referred to as multivesicular body) colocalizing with BP-80 (Oliviusson et al., 2006), a receptor equivalent to yeast Vps10p and also known as vacuolar sorting receptor (VSR) in Arabidopsis thaliana (Humair et al., 2001). AtVSR interacts with AtVps35 (Oliviusson et al., 2006). Endosomal retrograde transport of AtVSR1, one of the seven Arabidopsis VSRs, was first found to require AtVps29. This recycling step ensures efficient delivery by AtVSR1 of precursors of seed storage proteins to protein sorting vacuoles (PSVs), and therefore it is key for seed maturation (Fuji et al., 2007; Shimada et al., 2006). More recently, the implication of AtVps35 in this process was investigated. In cooperation with AtVps29, and therefore as constituents of retromer, AtVps35 recycles AtVSR1 from PSVs to the Golgi, ensuring protein storage in seeds (Yamazaki et al., 2008). While redundancy was found among the three AtVps35 genes existing in plants, AtVps35b appeared the most essential isoform for plant viability (Yamazaki et al., 2008); intriguingly, this was the only isoform that others did not detect in the assembled retromer ( Jaillais et al., 2007). VPS35 mutations turn into dwarfism, as in VPS29 mutants, but unlike in these or in VSR1 mutants, they also cause early leaf senescence (Shimada et al., 2006; Yamazaki et al., 2008). These data implicate retromer in growth and developmental processes in plants and also imply that AtVps35 binds to other cargos. Indeed, retromer subunits in Arabidopsis have been found required for polarized endosomal traffic of the phytohormone auxin ( Jaillais et al., 2006, 2007). This topic will be discussed in Section 4.1.

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3.4. Nematodes The use of a simple and well-studied model organism, the worm Caenorhabditis elegans, has shed light on many aspects of embryonic development, including the understanding of the highly conserved Wnt signaling pathway. The recent focus on identifying components regulating synthesis, secretory transport and release of Wnt proteins out of the cell has led to the demonstration that retromer is required for specialized Wnt secretion; this was first shown by two independent studies (Coudreuse et al., 2006; Prasad and Clark, 2006). Essentially, these studies provided evidence of retromer’s role in Wnt-producing cells for the establishment of anteroposterior (A/P) neuronal polarity (Prasad and Clark, 2006) and for ensuring the formation of a Wnt gradient along the A/P body axis, an essential function of retromer found conserved in vertebrate development (Coudreuse et al., 2006). As will be discussed in Section 4.2., more recent findings (Pan et al., 2008; Yang et al., 2008), also corroborated in cultured mammalian cells (Yang et al., 2008), establish that retromer carries out its role in Wnt secretion by mediating transport of the conserved transmembrane protein MIG-14; in Drosophila, its ortholog is Wntless (Wls), name that will be used throughout this chapter.

3.5. Arthropods Researchers working in the fruit fly model have also explored the participation of retromer in animal development. Wls—also named Evenness interrupted (Evi) or Sprinter (Srt)—was first found required for delivery of the Wnt protein Wingless (Wg) to the plasma membrane (Ba¨nziger et al., 2006; Bartscherer et al., 2006; Goodman et al., 2006). Newer data, also confirmed in cultured mammalian cells, imply that Wls is subsequently endocytosed and then recycled by retromer to the TGN, where it will be reutilized for further rounds of Wg secretion (Belenkaya et al., 2008; Franch-Marro et al., 2008; Port et al., 2008). The model proposed in Drosophila melanogaster is therefore consistent with that outlined from research in C. elegans (Ching and Nusse, 2006; Eaton, 2008). In a different context, a study mainly performed in Drosophila S2 cells demonstrates the importance of DmVps35 in endocytic traffic, actin polymerization and signaling by controlling levels or activity of the Rho family GTPase Rac1. Here, reduction in Rac1-dependent actin polymerization would facilitate endocytosis of signaling receptors (Korolchuk et al., 2007). How DmVps35 regulates Rac1 activity is unknown, but the authors remind us of situations in which Rac1 upregulation has been associated with alterations in traffic, such as in polymeric immunoglobulin A (pIgA) transcytosis ( Jou et al., 2000), a pathway also affected upon Vps35 depletion in polarized mammalian cells (Verge´s et al., 2004). Perhaps, these

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findings might be linked to the reported interaction of retromer with EHD1 (Gokool et al., 2007b). EHD1 may interact with epsin through the EH domain (Naslavsky and Caplan, 2005). Interestingly, epsins stabilize cellular sites for activation of Cdc42, another Rho family member and key regulator of actin cytoskeleton dynamics and cell polarity, thus allowing selection of effectors to activate the appropriate signaling pathway (Aguilar et al., 2006). The mentioned study also provides evidence of DmVps35 requirement in neuromuscular junction (NMJ) development (Korolchuk et al., 2007). In this regard, it is intriguing that Vps26 expression is elevated in isolated mouse NMJs in comparison to adjacent extrajunctional material, which was interpreted as an involvement of retromer in NMJ stability perhaps by maintaining or retrieving NMJ-bound receptors (Nazarian et al., 2005). Finally, the study raises the possibility that DmVps35 has tumor suppressor properties because its loss leads to cell overproliferation, which could derive from upregulation of various signaling pathways (Korolchuk et al., 2007). Loss of function of Vps subunits of the endosomal sorting complex required for transport (ESCRT) machinery leads also to overproliferation, an aspect in fact well studied in Drosophila (Leibfried and Bellaiche, 2007). While ESCRT mutations lead to accumulation of signaling receptors in the endosomal compartment owed to their impaired downregulation, it seems that VPS35 loss induces tumorigenesis through signaling deriving directly from the plasma membrane (Korolchuk et al., 2007). Contrary to this idea, cell proliferation in colon cancer associated with reduced SNX1 levels has been explained instead by increased endosomal signaling (Nguyen et al., 2006). Tumorigenesis upon VPS35 loss seems, however, paradoxical if we consider the importance of retromer in pre- and early embryonic (highly proliferative) stages of mammalian development (Griffin et al., 2005; Hwang et al., 1996; Lee et al., 1992; Verges, 2007).

3.6. Mammalian cells Albeit without sequence homology, Yeast Vps10p and mammalian MPRs follow equivalent routes and both perform essentially an identical function in enzyme delivery into the lytic compartment (Bonifacino and Rojas, 2006; Bonifacino and Traub, 2003; Bowers and Stevens, 2005; Ghosh et al., 2003). It was not surprising, then, that studies in cultured mammalian cells—mostly, HeLa cells and fibroblasts, but also mouse embryonic stem cells—demonstrated that retromer performs an equivalent function in higher eukaryotes as in yeast, retrieving from endosomes-to-Golgi the CI-MPR (Arighi et al., 2004; Seaman, 2004, 2005). This finding agrees with the concept that protein sorting and traffic operate under the regulation of well-conserved mechanisms.

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Retromer has been implicated in mediating sorting of additional cargos in mammalian cells. These include the pIgA receptor (pIgR), which transcytoses pIgA in polarized epithelial cells. The cargo-recognition subcomplex of retromer associates with pIgR in a postendocytic step and promotes transcytosis of pIgR–pIgA in Madin-Darby canine kidney (MDCK) cells (Verge´s et al., 2004). While the mechanism of cargo sorting by retromer has not yet been elucidated, more molecules transported by retromer, either directly or indirectly, have been added to the list. One is the intriguing case of the b-site amyloid precursor protein (APP) cleaving enzyme (BACE) and its substrate b-APP, proteins implicated in Alzheimer’s disease (AD) pathogenesis and whose endosome retrieval was shown to be mediated by GGAs and retromer in transfected HeLa cells (He et al., 2005; Small et al., 2005). Further studies in human subjects and in fibroblast cell lines implicated a member of the Vps10p-domain family, the sortilin-related receptor (SorLA)—also known as LDL receptor (LDLR) relative with 11 ligandbinding class A repeats (LR11)—in endosomal retrieval of BACE and bAPP in a retromer-dependent pathway, thus preventing generation of the neurotoxic amyloid-b (Ab) peptide (Rogaeva et al., 2007). The role of retromer in SorLA transport is perhaps not surprising considering traffic similarities between the CI-MPR and Vps10p-domain family members, such as the proneurotrophin receptor sortilin (Bronfman and Fainzilber, 2004; Jansen et al., 2007; Nielsen et al., 2001), which endosome-to-Golgi retrieval is also mediated by retromer through an interaction requiring a conserved motif also necessary for CI-MPR retrieval (Canuel et al., 2008; Seaman, 2004, 2007). Indeed, the brain is the organ with the highest expression of Vps10-domain family proteins (Hampe et al., 2001). Another receptor family with high expression in brain and with demonstrated importance in neurodegeneration is that of the LDLR, to which SorLA also belongs (Cam and Bu, 2006; Jaeger and Pietrzik, 2008). Another member of this family, the LDLR-related protein-6 (LRP6), may also associate with Vps35. LRP6 is a Wnt coreceptor and its interaction with Vps35 was found important for Wnt signal transduction in pheochromocytoma-derived PC12 cells transfected with Wnt-1, although a possible effect on Wnt secretion could not be analyzed in this system (George et al., 2007). In another context, retromer has also been implicated in recycling of secretory granule membrane proteins; this was observed during regulated secretion of insulin granules in rat insulinoma cells (Zhao et al., 2007). Research in cultured mammalian cells has also provided evidence that retromer performs additional roles apart from endosomal protein sorting. In particular, a study using the lung carcinoma-derived A549 cells, which present a well-defined lamellipodia, showed that retromer is also found at the plasma membrane (Kerr et al., 2005). These studies were aimed at exploring differences between the two Vps26 paralogs, Vps26A and

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Vps26B; Vps26B is present only in chordates and shares more than 80% homology with Vps26A, although they display some differences at their carboxy terminus. Further research confirmed that both paralogs interact with equal affinity with Vps35 and therefore are expected to form two distinct retromer complexes. Besides endosomes, both complexes also associate with actin-rich regions at the plasma membrane. The cell surface pool of retromer may therefore function in cell migration or adhesion (Collins et al., 2008; Kerr et al., 2005), which perhaps helps understanding Vps35 action on actin polymerization through Rac1 as seen in Drosophila (Korolchuk et al., 2007). For relevance in polarized traffic, some of these aspects will be discussed further in Sections 4 and 5. Finally, retromer’s retrograde pathway can be exploited by extracellular toxins, such as bacterial Shiga and cholera toxins, or the plant toxin ricin. These need to undergo retrograde transport from endosomes-to-TGN, and subsequently follow in reverse the secretory pathway till the ER to reach the cytosol, where they carry out their toxic activity (Sandvig et al., 2004). Endosome-to-Golgi transport of these toxins shares some regulatory components with the CI-MPR pathway (Falguieres et al., 2001; Lauvrak et al., 2004; Saint-Pol et al., 2004). Recent work performed in HeLa cells has now established that SNX1 is also required for efficient endosome-to-TGN transport of Shiga toxin. While SNX2 appears less involved, neither SNX1 nor SNX2 are, however, necessary for retrograde transport of cholera toxin (Bujny et al., 2007). Further analysis led to a model in which sorting of Shiga toxin is initiated at early endosomes in a clathrindependent step, which subsequently requires retromer to process endosome tubules for retrograde transport (Popoff et al., 2007). Contrasting with the negligible participation of SNX2 seen here, another study showed that both SNX1 and SNX2 are required for efficient Shiga toxin traffic to the Golgi in kidney epithelial-derived Vero cells (Utskarpen et al., 2007). Analogously, retrograde transport of the plant toxin ricin was also found dependent on SNXs. Here, knocking down SNX2 or SNX4 inhibited the pathway to a similar extent as by perturbation of the PI3K Vps34, suggesting implication of retromer as well as additional SNXs (Skanland et al., 2007).

3.7. Mouse The essential role of retromer in animal development was first demonstrated by studies in mouse models showing that the knockout for VPS26 and the double knockout SNX1/SNX2 share a nearly identical phenotype and are both embryonic lethal (Griffin et al., 2005; Lee et al., 1992; Radice et al., 1991; Schwarz et al., 2002). Data obtained in tissue and fibroblasts derived from these genetically modified mice suggest that retromer, in cooperation with SNXs, performs an essential contribution during embryogenesis

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unrelated to controlling sorting of the CI-MPR (Griffin et al., 2005); these studies have been previously reviewed (Verges, 2007).

4. Polarized Transport Mediated by Retromer We understand cell polarity by the asymmetrical distribution of cellular components within a cell or group of cells. This specialized distribution forms biochemically and functionally distinct cell surface domains, allowing their effective and specific interaction with different extracellular environments. Different types of cell polarity exist in multicellular organisms. That is, apical–basal polarity of an epithelial monolayer; A/P polarity, seen, for instance, in asymmetric cell division, cell migration, and axon specification; and planar cell polarity (PCP), generally understood as the process of orienting fields of cells along a common axis. In all cases, generation of cell polarity requires active remodeling of microtubule and actin cytoskeletons and depends on polarized vesicle traffic to different cellular domains. Most of the work in deciphering the mechanisms for establishment of polarity has been carried out in polarized epithelial cells (Dow and Humbert, 2007). The study of membrane transport in polarized cells has an additional layer of complexity owed to the increased number of choices that an itinerant molecule can follow. Thus, a long-standing question in cell biology has been how traffic of endocytosed proteins is regulated in polarized epithelial cells. In these cells, apical and basolateral plasma membrane domains are separated by specialized cell–cell junctions. Newly synthesized plasma membrane proteins are first sorted in the TGN for delivery to the apical or basolateral surface. Proteins can then be endocytosed from one surface and either be recycled back to that surface or get degraded in the lysosome. Alternatively, they can be transcytosed to the opposite plasma membrane domain (Mostov et al., 2003; Rodriguez-Boulan et al., 2005; Shin et al., 2006). Sorting to the plasma membrane, endocytosis, recycling, and transcytosis of cell-surface molecules are key processes for maintaining cell polarity. Altered traffic and localization of molecules can dramatically affect epithelial barrier and transport functions, thereby causing disease (Stein et al., 2002). Next, data implicating retromer as well as SNXs in polarized traffic will be reviewed (Table 4.1).

4.1. Polarity of PIN proteins in plant development Auxin (indole-3-acetic acid) is a key element in many plant growth and developmental processes, such as embryogenesis, photo- and gravitropism, apical dominance (dominance of the main central stem), and phyllotaxis

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Table 4.1 Retromer and sorting nexins in polarized transport Protein(s) implicateda

Described function

Model system or organism

References

Vps17p (retromer)

Late endosome-to-Golgi retrieval of Snc1p for its transport to the spindle pole body in sporulation Early endosome-to-plasma membrane recycling of Ste6p in polarized secretion of the mating pheromone a-factor Endosome-to-plasma membrane recycling of PIN proteins to ensure auxin polarity in development Generation of a Wnt signal for neuron migration and for establishment of A/P neuronal polarityb Formation of a Wnt gradient along the A/P body axis Polarized surface distribution of Wls (endosome-to-Golgi recycling)c

Yeast

Morishita et al. (2007)

Yeast

Krsmanovic et al. (2005)

Arabidopsis thaliana

Jaillais et al. (2006, 2007)

Caenorhabditis elegans

Coudreuse et al. (2006); Prasad and Clark (2006) Coudreuse et al. (2006) Franch-Marro et al. (2008); Port et al. (2008) Verge´s et al. (2004, 2007) Bryant et al. (2007)

Snx4p

Vps29–SNX1 (retromer) Vps35–Vps26–Vps29 (retromer) Vps35–Vps26–Vps29 (retromer) Retromer

C. elegans Drosophila

Retromer

Transcytosis of pIgA by the pIgR

MDCK cells

SNX1

E-cadherin recycling from macropinosomes and endosomes in reestablishment of cell–cell adhesion and polarity Regulation of tight junction architecture, which is affected by enteropathogenic bacteria

Semipolarized cell line from human breast adenocarcinoma Polarized epithelial cell lines from human intestine

SNX9

Alto et al. (2007)

Ab productiond

HeLa cells

Small et al. (2005)

HeLa cells HepG2 cells

He et al. (2005) Nielsen et al. (2007)

Retromer

Endosome-to-Golgi recycling of BACEd Endosome-to-Golgi recycling of SorLA and sortilind,e Endosome-to-Golgi recycling of SorLAd

Human fibroblasts

Retromer

Endosome-to-Golgi retrieval of sortilind

HeLa cells, monkey COS-7 cells, and mouse ESC

Rogaeva et al. (2007) Canuel et al. (2008); Seaman (2004, 2007)

Vps35–Vps26 (retromer) Vps26 (retromer) SNX1 (retromer?)

A/P, anteroposterior; BACE, b-site amyloid precursor protein-cleaving enzyme; ESC, embryonic stem cells; MDCK, Madin-Darby canine kidney; PIN, plant-specific pin-formed; pIgA, polymeric immunoglobulin A; pIgR, pIgA receptor; SNX, sorting nexin; SorLA, sortilin-related receptor; Vps, vacuolar protein sorting. a Retromer subunit(s) or SNX. b Vps29 was not found required for establishment of A/P neuronal polarity (Prasad and Clark, 2006)). c Wls was localized at post-Golgi subapical structures (Port et al. (2008)) or at the basolateral cell surface (Franch-Marro et al. (2008)). d All these studies imply the role of retromer in endosome-to-Golgi recycling of b-APP, or the b-APP–BACE complex through SorLA, or perhaps through sortilin, thereby regulating generation of Ab. Although polarized traffic of these proteins may be relevant for neurotoxicity and AD pathogenesis, aspects of polarity were not addressed. e Unlike SNX1, Vps35 appeared only involved in SorLA traffic.

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(leaf arrangement) (Leyser, 2006). It acts as a morphogen, and thus it spreads from a localized source forming a concentration gradient that provides spatial information throughout a developing tissue. From its site of synthesis, different concentration thresholds that will influence the expression of target genes in the site of action are established. This governs the pattern of tissue development, in particular, the positions of specialized cell types within a tissue. Auxin translocation from cell to cell takes place in a polar way, a process known as polar auxin transport (PAT) (Muday and Murphy, 2002). For its controlled directionality, PAT is a unique mechanism for transmitting spatial and temporal signals during plant development. Auxin entry into cells is facilitated by an auxin influx carrier (AUX1) and by additional related proteins. Auxin moves out of cells through an efflux carrier machinery involving, among other proteins, an integral plasma membrane transporter of the plant-specific pin-formed (PIN) protein family. Both AUX1 and PIN proteins display an asymmetric distribution in the plasma membrane, consistent with their role in controlling the polarity of auxin movement (Blakeslee et al., 2005; Vieten et al., 2007). During plant development, PIN family members are dynamically relocated within each auxin transporting cell. Their polarized distribution in a tissue-specific way is likely determined by specific protein targeting pathways ( Jurgens and Geldner, 2007). How the polar localization of influx and efflux carriers is related to controlling the directionality of auxin flow is still unclear, but it is assumed that the asymmetric localization patterns of these components has a functional meaning (Vieten et al., 2007). Recent studies using the root meristem model of Arabidopsis have contributed to the understanding of PIN protein subcellular distribution and traffic. Previously, PIN proteins were proposed as likely substrates of the protein Ser/Thr kinase PINOID (PID), found responsible for targeting these from the basal to the apical plasma membrane (Friml et al., 2004; Kaplinsky and Barton, 2004). Protein phosphatase 2A (PP2A) has now been found to reversibly antagonize the effects of PID by dephosphorylating PIN proteins and targeting these to the basal surface (Michniewicz et al., 2007). Relevant for this chapter are the achievements by Gaude and collaborators that implicate the retromer subunits AtSNX1 and AtVps29 in PIN protein traffic (Table 4.1). Their studies address the role of retromer on endosomal (re)cycling of two well-studied PIN protein family members, PIN1 and PIN2, as a requisite for their suitable distribution at the plasma membrane. In roots, whereas PIN1 is basally located, PIN2 is apical in epidermal cells but basal in cortical cells ( Jaillais et al., 2006, 2007). Only three SNX genes are found in A. thaliana, encoding for AtSNX1, AtSNX2a, and a closely related AtSNX2b (Vanoosthuyse et al., 2003), which contrasts with 30 SNX members presumably expressed in humans, and even already 10 in yeast (Carlton et al., 2005; Seet and Hong, 2006). Such a genome simplification is intriguing, but it can also be observed by a

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database search in simple animals, such as in C. elegans, suggesting that SNXs in these have to cover wider roles in protein traffic than their counterparts in other organisms. Plant SNX1 mutants display short primary roots, small number of secondary roots, and altered root gravitropic response. This phenotype resembles that of a GNOM mutant, which encodes for a membrane-associated guanine-nucleotide exchange factor (GEF) for ARF GTPases that is sensitive to the fungal drug brefeldin A (BFA). While such a weak and pleiotropic phenotype suggests redundancy among Arabidopsis SNXs, the postembryonic lethality often seen in double SNX1/GNOM mutants indicates that they perform partially overlapping roles but participate in distinct pathways ( Jaillais et al., 2006). Based on colocalization with GFP-tagged Rab proteins and costaining with the membrane-selective fluorescent dye FM4–64, AtSNX1 localizes in endosomes that enlarge or aggregate upon BFA treatment ( Jaillais et al., 2006). Another widely used pharmacological tool exploited in this study was wortmannin. Wortmannin treatment led to enlargement of AtSNX1-labeled endosomes but it did not affect PIN1 localization, suggesting that PIN1 recycles through a more direct GNOM-dependent pathway, sensitive to BFA but insensitive to wortmannin. Instead, traffic of endocytosed PIN2, but not its surface polarity, was affected by wortmannin, markedly in root epidermal cells and less in cortical cells. This indicates differences in PIN protein pathways and suggests that PIN2 traffic is differentially regulated in these cell types ( Jaillais et al., 2006). The rather subtle SNX1 null phenotype ( Jaillais et al., 2006) differs from the striking dwarf phenotype of the VPS29 null mutant (Shimada et al., 2006). However both these mutants appeared with cotyledon and embryo defects similar to those seen in some combinations of PIN null mutants, suggesting that AtVps29 and AtSNX1 operate, in both shoot and root, in common developmental pathways. In addition, Arabidopsis SNX1/VPS29 double mutants are lethal ( Jaillais et al., 2007), reminiscent of what has been seen in mouse models (Griffin et al., 2005). Indeed, the existence of a plant retromer essential for PIN protein polarity, and thus important for plant growth, was confirmed by further studies in the same laboratory ( Jaillais et al., 2007). AtVps29 perfectly overlaps with AtSNX1 in endosomes sensitive to both BFA and wortmannin (see above). When VPS29 was depleted, AtSNX1 remained in an enlarged endosomal compartment not containing internalized FM4-64, whereas GNOM distribution was unaffected. This suggested to the authors that AtVps29 specifically controls morphology and therefore homeostasis of AtSNX1-containing endosomes, probably by mediating an endosomal sorting pathway ( Jaillais et al., 2007). In summary, these pharmacological tricks allowed the distinction of two endosomal populations. One would be a GNOM-regulated early endosome, which fuses with the TGN under BFA treatment even in the absence of functional retromer, and appears equivalent to a mammalian endosome involved in

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direct recycling to the plasma membrane. Another endosome, containing plant retromer, is positive for Arabidopsis Rab GTPases RabF1 and RabF2 ( Jaillais et al., 2006, 2007), related to mammalian Rab22 and Rab5, respectively, and therefore likely associated with early endosomal traffic (Molendijk et al., 2004; Rutherford and Moore, 2002). However, this second compartment remains separated of the BFA-induced compartment in the VPS29 mutant and appears downstream in PIN protein recycling ( Jaillais et al., 2007). It would be interesting to better characterize such compartment, which might be also positive for members of the RabA group, some of them related to mammalian Rab11 or Rab25, or the RabC group, related to mammalian Rab18, and thus putatively associated with endosomal/postGolgi recycling or polarized endosomal traffic (Cole and Fowler, 2006; Molendijk et al., 2004; Rutherford and Moore, 2002; Samaj et al., 2006). A link between retromer and specialized Rab-mediated sorting pathways would be consistent with their cooperative action at the ‘‘tubular endosomal network’’ as a sorting station for specialized endosome transport to various destinations, including recycling or transcytosis (Bonifacino and Rojas, 2006). Gaude and collaborators clearly demonstrated that both PIN1 and PIN2 traffic is specifically affected in VPS29 mutants ( Jaillais et al., 2007), whereas only PIN2 is transported through the AtSNX1 endosome ( Jaillais et al., 2006). This discrepancy is intriguing, perhaps reflecting the implication of other SNXs in PIN protein traffic. Hara-Nishimura and collaborators reported that AtVps35 levels are reduced in VPS29 mutants (Shimada et al., 2006), in agreement with the effect seen in mammalian cells by knocking down or knocking out any subunit of the cargo-recognition subcomplex (Arighi et al., 2004; Griffin et al., 2005; Gullapalli et al., 2006; Rojas et al., 2007; Seaman, 2004; Verge´s et al., 2004). We can therefore assume that retromer function is clearly compromised in VPS29 mutants, although retromer may still be partially operative in SNX1 mutants. Another puzzling issue observed in roots of VPS29 mutants is that both PIN1 and PIN2, although accumulating in enlarged endosomes, localize at the correct plasma membrane domain at steady state ( Jaillais et al., 2007). This phenotype is compared to the GNOM mutant phenotype, in which PIN1 recycling, but not polarity, is also altered. However, while PIN1 polarity at the cellular level is maintained, coordinated polar localization of PIN1 is defective in GNOM mutant embryos (Steinmann et al., 1999), suggesting that endocytic recycling through a GNOM-retromer pathway is essential for establishing coordinated PIN1 polarity among cells ( Jaillais et al., 2007). Therefore, AtVps29 acts on establishing PIN protein polarity (particularly, PIN1) during development of both embryo and root. Finally, signaling by a Rho-of-Plant (ROP) Rho-related GTPase (Xu and Scheres, 2005a) in lateral root appearance was found to require AtVps29 ( Jaillais et al., 2007). It is not well understood how ROP proteins distribute to the apex of the root growing tip (Samaj et al., 2006). ROP proteins also localize

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in intracellular spots and their polar localization is sensitive to BFA (Molendijk et al., 2001). Such is the case of ROP2, which partially colocalizes with internalized FM4-64 in BFA-treated roots, implying an ARFdependent intracellular traffic (Xu and Scheres, 2005b) and perhaps an endocytic recycling to the apical surface (Samaj et al., 2006) mediated by AtVps29/retromer. How does plant retromer work? As suggested, it should be considered the possibility that it actually retrieves PIN proteins from endosomes-toTGN ( Jurgens and Geldner, 2007), in analogy with retromer’s evolutionary conserved pathway (Seaman, 2005) and in contrast to a proposed direct recycling to the plasma membrane ( Jaillais et al., 2007). Indeed, the TGN in root cells has been found to function as an early endosome connecting endocytic and secretory traffic (Dettmer et al., 2006). Therefore, it is possible that transport to the plasma membrane in retromer mutants becomes altered indirectly ( Jurgens and Geldner, 2007). In studies of seed storage protein transport, an endosomal-to-TGN ‘‘recycling’’ pathway was indeed proposed to be affected in VPS29 or VPS35 mutants and to be responsible for missorting the storage protein receptor AtVSR1 (Shimada et al., 2003, 2006; Yamazaki et al., 2008). Retromer may interact directly with PIN1 or PIN2. Probably falling in a cytosolic loop between the second and third proposed N-terminal transmembrane domains (Muller et al., 1998), a putative sequence involved is F70LA, which to some extent agrees with the proposed three-residue W/F-L-M/V consensus motif required in CI-MPR and sortilin for endosome-to-Golgi retrieval (Seaman, 2007). Alternatively, interaction may also take place through the YSSL sequence present in a loop between the sixth and seventh transmembrane domains, at the C-terminal hydrophobic region (Muller et al., 1998), which fits with the consensus domain YXXF (F: hydrophobic residue) found required for interaction of sortilin with Vps35 (Canuel et al., 2008). This latter sequence is also identical to the Tyr-based signal in Vps10p required for proper late Golgi-to-PVC cycling (Cooper and Stevens, 1996).

4.2. Secretion of Wnt proteins in animal embryo development During animal development, neurons extend their processes along the A/P or dorsoventral (D/V) body axis. Secreted guiding factors regulating D/V neural migration have been well defined and studied, but the molecules involved in A/P neural migration are only now beginning to be uncovered. Among molecules controlling neuron guidance mechanisms along the A/P axis are members of the Wnt family of secreted proteins. Wnt proteins are morphogens that participate in establishing neural polarity upon binding to Frizzled, their receptor on target cells (Silhankova and Korswagen, 2007). Thus, the Wnt signaling pathway affects embryogenesis at multiple aspects, including cell proliferation, migration, fate specification, polarity,

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differentiation, and axon outgrowth and guidance (Bovolenta et al., 2006; Cadigan and Liu, 2006; Chien and Moon, 2007; Gordon and Nusse, 2006; Mikels and Nusse, 2006; van Amerongen and Berns, 2006). By genetic and morphologic approaches that allow the visualization of individual migrating neurons, studies of Wnt protein biology in the nematode model system have added an essential contribution to these aspects. An example can be found in EGL-20, one of the five C. elegans Wnt proteins that performs both a permissive and an instructive function in providing directional cues for migrating neurons during development (Pan et al., 2006; Silhankova and Korswagen, 2007; Whangbo and Kenyon, 1999; Zinovyeva and Forrester, 2005). Some of the migrating neurons in C. elegans display a distinctive polarity, which is regulated by highly conserved guidance receptors and also by Wnt proteins (Silhankova and Korswagen, 2007). Such is the case of the anterior (ALM) and posterior (PLM) lateral microtubule neurons, two of the six touch-receptor or mechanosensory neurons that mediate touch sensitivity (Bounoutas and Chalfie, 2007). In these neurons, Wnt proteins probably act as permissive organizers of neural polarity rather than as instructive growth cone attractant or repellent signals. That is, Wnt proteins guide axon outgrowth along the A/P body axis not by attracting or repelling neuron growth cones but by inverting the overall A/P orientation of the neurons and its polarized processes (Hilliard and Bargmann, 2006). While PLM polarity is dependent on the canonical Wnt/b-catenin pathway, the process of PCP is not (Lawrence et al., 2007; Seifert and Mlodzik, 2007; Zallen, 2007). Whereas the mechanism of Wnt signal processing in target cells has been deeply studied and is now well characterized, the factors that regulate Wnt secretion are still not well understood. Wnt proteins undergo posttranslational modifications, such as N-glycosylation, which may mediate their interaction with heparan sulfate proteoglycans once at the cell surface. They are also palmitoylated by the O-acyltransferase porcupine, and both glycosylation and palmitoylation are necessary for secretion of an active Wnt ligand. Subsequent interaction of Wnt with Wls is required for Wnt secretion. Before their targeting to the plasma membrane, Wnt proteins are proposed to follow a specialized secretory pathway that involves their association with cholesterol-sphingolipid-rich raft microdomains and with lipoproteins in an endosomal-like compartment. Finally, Wnt polarized secretion, as well as their further reinternalization and recycling, may influence the subsequent processing of the Wnt signaling in target cells (Coudreuse and Korswagen, 2007; Hausmann et al., 2007; Mikels and Nusse, 2006; Miura and Treisman, 2006). The demonstration in C. elegans that retromer is required for long-range diffusion and efficient signaling of secreted Wnt proteins (Coudreuse et al., 2006; Prasad and Clark, 2006) quickly became matter of speculation and led to debate. Indeed, these two independent studies are relevant for various reasons. First, they provided an

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additional piece of knowledge in the important process of Wnt making, of which little data existed until then. Second, at least from evidence provided by one of these studies, retromer’s role in Wnt-producing cells emerged evolutionary conserved, as seen reproduced in frog embryos and in cultured human fibroblasts (Coudreuse et al., 2006). Third, they revealed that retromer performs a specialized function during animal embryogenesis, thus solving an earlier debate arising from data in mouse models on whether retromer has a housekeeping or a rather specific function (Hwang et al., 1996; Lee et al., 1992; Radice et al., 1991). Fourth, neural polarity was shown to depend on retromer (Table 4.1). And fifth, these studies also suggested that retromer may be implicated in sorting and processing of additional signaling molecules. Various possible models were proposed to explain how Wnt proteins may be assisted by retromer to the cell surface and how retromer may influence Wnt signaling (Coudreuse and Korswagen, 2007; Hausmann et al., 2007; He and Axelrod, 2006; Mikels and Nusse, 2006; Verges, 2007). Retromer might promote association of Wnt proteins in endosomes with components required for specialized ligand transport, such as lipoprotein particles. This fits with the observation that reduced lipoprotein levels in Drosophila’s larvae produces a similar phenotype as retromer depletion in C. elegans development (Pana´kova´ et al., 2005). In this model, retromer would directly transport the secreting ligand to the cell surface (Mikels and Nusse, 2006). It was also proposed that Wls could act as a Wnt receptor driving Wnt proteins from the TGN to the endosome, thus facilitating their association with lipoproteins. Either by an anterograde (TGN-to-endosome) or retrograde pathway, in analogy to its conserved role, retromer may guide a putative Wnt receptor to ensure proper delivery of Wnt proteins to a specialized secretory pathway, away from constitutive secretion (Coudreuse and Korswagen, 2007; Hausmann et al., 2007). Like Frizzled, Wls was predicted to be a member of the GPCR family (Wistrand et al., 2006), agreeing with the idea that a cargo-activated GPCR mediates fission at the TGN for delivery of vesicle carriers to the plasma membrane (Bard and Malhotra, 2006; Hausmann et al., 2007). Wls could well be the hypothetical transmembrane receptor interacting with Vps26 (Shi et al., 2006), although technically Vps26 does not function as an arrestin (Collins et al., 2008; see Section 2). This was an attractive model, but it did not fit with data seen in retromer mutant worms, in which intracellular levels of Wnt (Prasad and Clark, 2006) or Wnt secretion (Coudreuse et al., 2006) are not affected, in contrast with the strong evidence of Wls requirement for secretion of Drosophila’s Wg (Ba¨nziger et al., 2006; Bartscherer et al., 2006; Goodman et al., 2006). Alternatively, retromer could even play a more indirect task, recycling a receptor for enzymes or other components needed for posttranslational modifications of secreting Wnt proteins (Verges, 2007).

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Five different laboratories worked hard and quickly to solve this puzzle, and clearly established a solid link between Wls and retromer. Using different model systems, that is, C. elegans, Drosophila, and cultured mammalian cells, such as HeLa cells and 293 fibroblasts, they reached a general consensus demonstrating that retromer-mediated transport of Wls is conserved in Wnt-producing cells (Belenkaya et al., 2008; Franch-Marro et al., 2008; Pan et al., 2008; Port et al., 2008; Yang et al., 2008). An aspect analyzed by some of these studies was the polarity of ALM and PLM neurons in C. elegans, which is frequently altered in single and multiple WNT mutants and in mutants of retromer subunits (Prasad and Clark, 2006). Similarly, this altered polarity is seen in WLS mutants, as well as in mutants for DPY-23, the nematode m-subunit of clathrin’s AP-2 complex. With enhanced defects in VPS35/DPY-23 double mutants, Garriga and collaborators demonstrated the genetic interaction among WLS, DPY-23, and retromer (Pan et al., 2008). Like CeVps35 (Coudreuse et al., 2006), EGL-20, Wls, and DPY-23 (Pan et al., 2008) are also required for neuronal migration. Observing that DPY-23 or VPS35 mutations change the distribution and levels of ectopically expressed Wls in EGL-20 secreting cells, Garriga and collaborators concluded that DPY-23 is required for reinternalization of Wls, and retromer is required for its subsequent recycling from endosomes-to-TGN. Both steps are necessary for new rounds of Wnt protein secretion to the plasma membrane assisted by Wls, which therefore functions as a Wnt sorting receptor (Pan et al., 2008). This simple model disagrees with the more elaborated idea previously proposed by Korswagen and collaborators. In their earlier model, these authors suggested that retromer is not necessary for Wnt secretion, but instead it is needed for obtaining Wnt proteins competent for long-range signaling, while short-range signaling could still proceed in the absence of functional retromer (Coudreuse et al., 2006). However, the data in their subsequent work essentially agree with that from Garriga’s laboratory and in addition they provide a possible explanation on the signaling range issue. Thus, they provide evidence of an AP-2-mediated internalization of Wls and also demonstrate that retromer prevents Wls lysosomal degradation (Yang et al., 2008). They now propose that the previously observed effect on signaling range could have been caused also by decreased Wls levels, which lead to reduced Wnt secretion. As Wls is not completely eliminated in the absence of retromer’s function, Wnt secretion becomes only partially affected. As a result, short-range signaling may still proceed (Coudreuse et al., 2006; Yang et al., 2008). Studies in Drosophila’s wing imaginal disc, the tissue from which the wing derives, also provide evidence that DmVps35 is required for Wg secretion by acting on Wls recycling (Belenkaya et al., 2008; Franch-Marro et al., 2008; Port et al., 2008). The results by these groups basically agree that DmVps35 has a role in both short- and long-range signaling, with a more noticeable participation even at short range. Their conclusions are supported by analysis

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in Drosophila’s embryo-derived S2 cells and in mammalian cells. Lin and collaborators suggest that discrepancies in action range depend on the target gene analyzed and thus can be attributed to their different sensitivity to extracellular Wnt levels (Belenkaya et al., 2008). It has also been proposed that the maternal contribution of DmVps35 allows residual Wg secretion sufficient to affect gene transcription even at distal cells (Eaton, 2008). Longrange signaling in Drosophila can indeed be promoted by a mechanism unrelated to retromer, as seen for the spreading of both Wg and Hedgehog (Hh). Such is the case of reggie-1 (flotilin-2), a major component of lipid raft microdomains that stimulates apical secretion of Wg and allows packing of Wg and Hh for their long-range spreading (Katanaev et al., 2008). There seems to be a consensus that retromer is not implicated neither in Hh (Port et al., 2008) nor in Notch signaling (Belenkaya et al., 2008; FranchMarro et al., 2008), suggesting specificity for Wnt signaling. On the influence of retromer in canonical versus noncanonical Wnt signaling, Lin’s laboratory demonstrated its requirement for secretion of Wnt proteins headed for either pathway (Belenkaya et al., 2008). These studies also provided a detailed analysis of the subcellular distribution of Wls and retromer, often of exogenously expressed proteins. Upon reaching the plasma membrane, Wls undergoes clathrin-dependent endocytosis, and AP-2 (Pan et al., 2008; Yang et al., 2008), shibire/dynamin (Port et al., 2008), as well as Rab5 (Belenkaya et al., 2008) were found implicated. Importantly, two of the studies gave information, although somewhat contradictory, of the polarized distribution of Drosophila’s Wls (Table 4.1). Thus, Basler and collaborators showed a predominantly apical distribution of Wls in Wg-producing cells of wing discs, perhaps accumulating in a post-Golgi apical compartment (Port et al., 2008). This agrees with Wg’s apical localization (Strigini and Cohen, 2000), previously found altered upon WLS mutation, which already implicated Wls in Wg’s apical sorting (Bartscherer et al., 2006). On the contrary, the electron microscopy analysis performed by Vincent and colleagues revealed that Wls is instead more predominantly found at the basolateral membrane, although DmVps35 deficiency also turned into loss of Wg’s apical localization at the disc epithelium (Franch-Marro et al., 2008). Finally, the possible interaction between Wls and retromer was also addressed, often using exogenously expressed proteins. While without success in one case (Yang et al., 2008), two different teams demonstrated a reciprocal association between Wls and Vps35 by coimmunoprecipitation (Belenkaya et al., 2008; FranchMarro et al., 2008). However, Wls does not have a W/F-L-M/V consensus motif (Seaman, 2007) conserved throughout species and exposed away from proposed transmembrane domains (Ba¨nziger et al., 2006; Goodman et al., 2006). The same applies to the other proposed retromer-interacting sequence, YXXF (Canuel et al., 2008). Nevertheless, it is plausible that the interaction takes place directly through an as-yet-unidentified motif

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and, in line of all this evidence, the important contribution of retromer’s interaction with Wls for Wnt production cannot be questioned. Since this chapter was submitted, a new study revealed that retromer not only functions in Wnt-producing cells but also in Wnt target cells. Importantly, in this latter case retromer-mediated transport affects the polarity of b-catenin WRM-1 before the terminal asymmetric divisions of the stem cell-like epithelial seam cells (Kanamori et al., 2008).

4.3. Transcytosis of the polymeric immunoglobulin receptor Transcytosis of the pIgR, which transports pIgA across epithelia, has been extensively studied in hepatocytes, enterocytes, and pIgR-expressing MDCK cells. In these cell types, pIgR binds pIgA at the basolateral plasma membrane and is transcytosed through various intracellular compartments to the apical surface. There, the ligand-binding portion of pIgR is cleaved off and released together with the ligand into the apical lumen, where it will perform antigen exclusion. Since an epithelial cell has to transport cargo selectively by transcytosis across two different environments, proper sorting of proteins along this pathway is essential for maintaining cell polarity (Rojas and Apodaca, 2002; Tuma and Hubbard, 2003). The MDCK cells constitute the most studied epithelial cell model. As in vivo, pIgR efficiently transcytoses pIgA in MDCK cells, with almost no degradation. Transcytosis of pIgR–pIgA has been an area of considerable study in MDCK cells and this pathway has been found regulated by multiple components (Rojas and Apodaca, 2002; Tuma and Hubbard, 2003). Retromer interacts with pIgR in endosomes isolated from rat liver and in MDCK cells (Verge´s et al., 2004). Unpredictably, it was found that retromer does not protect pIgR from degradation, but rather prevents its basolateral recycling and stimulates its transcytosis, thus resulting in a more efficient basolateral-to-apical pIgR–pIgA transport across the cell (Table 4.1). These studies also implied that retromer must have a more general role in endosomal retrieval pathways. In agreement with previous data implicating PI3Ks in the regulation of transcytosis (Cardone and Mostov, 1995; Hansen et al., 1995), and with the dependence on the PI3K Vps34p seen in yeast retromer function (Burda et al., 2002), PI3Ks were also found required for an efficient pIgR–pIgA transcytosis mediated by retromer (Verges et al., 2007). SNXs mislocalize when PI3Ks are inhibited, and the cargo-recognition subcomplex, still efficiently recruited to the membrane, is unable to cluster cargo for efficient sorting from appropriate locations (Verges et al., 2007; see Section 2). Additional molecules likely play a role in clustering receptor–ligand complexes, perhaps establishing a sorting platform at certain membrane microdomains where clustering will take place (Wallrabe et al., 2007).

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How retromer mediates pIgR–pIgA transport remains to be elucidated. By coimmunoprecipitation, the interaction with Vps35 was mapped at the last 30 amino acids of the receptor’s tail (Verge´s et al., 2004). Earlier data attributed this region to guarantee a rapid pIgR–pIgA endocytosis (Breitfeld et al., 1990), but additional research demonstrated that it is required for ligand stimulation of pIgR transcytosis (Luton et al., 1998). It contains the sequence FLF (F: hydrophobic residue), which largely agrees with the W/ F-L-M/V conserved motif (Seaman, 2007), and a putative YXXF consensus motif (Canuel et al., 2008), both proposed as a requirement for retromer’s-mediated transport. Importantly, these last 30 residues are required for coupling pIgR transcytosis to a signaling pathway (Luton et al., 1998) involving activation of the Src family member p62Yes, a tyrosine kinase associated with pIgR (Luton et al., 1999). Recent findings suggest a cross talk between pIgR and the epidermal growth factor receptor (EGFR); upon pIgA binding to pIgR, EGFR gets phosphorylated by p62Yes in rat liver endosomes and in MDCK cells. EGFR may couple pIgR to the PI3K/Akt and the mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK) signaling cascades that would modulate pIgR transcytosis (Tao Su and Keith E. Mostov, unpublished data). Given the complexity of possible elements that may interconnect pIgR transcytosis with the PI3K (Lindmo and Stenmark, 2006) or with the MAPK/ERK pathways (Anderson, 2006), clearly more work is needed to clarify this aspect. However, from the current evidence a simplified model can be envisaged. That is, EGFR activation by pIgA binding to pIgR directs pIgR–pIgA through endosomes in a PI3K/retromer-dependent pathway, maintained by MAPK/ERK signaling and likely assisted by specific Rab proteins (van IJzendoorn et al., 2002), adaptor complexes (Anderson et al., 2005), raft-associated molecules (de Marco et al., 2002), and even the exocyst complex (Oztan et al., 2007).

5. Implication of Retromer and Sorting Nexins in Other Aspects of Polarity 5.1. Polarity establishment in yeast The budding yeast S. cerevisiae is a model system to study establishment of cell polarity and polarized exocytosis. Yeast cells are highly polarized during most of their life, but more strikingly during asymmetric enlargement of the bud that will pinch off to form a daughter cell. In the budding process, a small area of the mother cell surface specializes, involving polarized location of many components to this area. This promotes growth of the bud. Many genes have been found implicated in this process and a working model for polarity establishment in yeast has been elaborated (Irazoqui and Lew, 2004). An

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evident conclusion is that the overall mechanisms of vesicle transport for the establishment of cell polarity are well conserved from yeast to animal cells (Brennwald and Rossi, 2007). An example of conservation is seen in the participation of endosomal Rab11 in targeting post-Golgi or endocytic vesicles to the plasma membrane of mammalian cells in an analogous way as the yeast Rab GTPase Sec4p functions through interaction with the Sec15p component of the evolutionary conserved exocyst complex (Wu et al., 2005; Zhang et al., 2004). To date, the direct contribution of retromer or SNXs in such mechanism has not been reported. Nevertheless, two related cases are worth mentioning (Table 4.1). First, the implication of Snx4p, but not retromer, in early endosome-to-plasma membrane recycling of the ATPbinding-cassette (ABC) transporter Ste6p, which is required for polarized secretion of the mating pheromone a-factor (Krsmanovic et al., 2005). Snx4p action on Ste6p is comparable to that on the vesicle-SNARE (vSNARE) Snc1p, which is retrieved from early endosomes-to-Golgi in a retromer-independent but Snx4p-dependent pathway (Hettema et al., 2003). On the contrary, Snc1p, when synthesized during sporulation, is, however, retrieved from late endosomes-to-Golgi in a pathway implicating retromer but not Snx4p. This process takes place before spore formation and contributes to Snc1p transport to the spindle pole body for formation of a double lipid bilayer known as the prospore membrane (Morishita et al., 2007). Cell asymmetry can thus be accomplished by endocytic recycling, appropriate in situations where transient or variable polarity is required, such as in the yeast cell seeking its mating partner (Valdez-Taubas and Pelham, 2003).

5.2. Membrane remodeling SNX9, one of the best characterized SNXs, is the most evident example of a SNX involved in physically coupling actin polymerization to plasma membrane deformation or remodeling during endocytosis. The presence in SNX9 of a Src homology 3 (SH3) domain that directly interacts with dynamin and with the Wiskott-Aldrich syndrome protein (WASP) provide to this protein such exclusive properties (Seet and Hong, 2006; Shin et al., 2008; Yarar et al., 2007). The functional membrane-remodeling unit of SNX9 is constituted by the PX and BAR domains, which cooperate for tubulation of PI-containing membranes and have been crystallized (Pylypenko et al., 2007). SNX18, a member of this SNXs subfamily containing a SH3 domain, tubulates endosomes as part of an AP-1 budding machinery (Haberg et al., 2008). It has also been shown that pathogenic proteins can hijack the SNX9–WASP signaling complex to affect membrane traffic events through alteration of actin cytoskeleton dynamics. Thus, analysis of intestinal epithelial cells revealed SNX9 as the major target of EspF, an effector protein of enteropathogenic Escherichia coli, and this

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interaction was found implicated in breaking down tight junction architecture during bacterial pathogenesis (Table 4.1) (Alto et al., 2007). Another SNX somehow involved in regulating dynamics of surface molecules is the poorly studied SNX20. SNX20, constituted by a PX domain plus extra motifs of unclear function, interacts directly with the P-Selectin glycoprotein ligand-1 (PSGL-1), a ligand of adhesion molecules functioning in leukocyte tethering and activation. It was postulated that SNX20 directs PSGL-1 to the proper plasma membrane location for its subsequent delivery to ezrin/radixin/moesin (ERM) proteins and anchorage in restricted cell membrane domains (Schaff et al., 2008). Unlike for SNX1, a link between SNX9 or SNX20 and retromer has not been reported. Interestingly, SNX1 has been implicated in intracellular sorting and recycling of the adhesion receptor E-cadherin, emerging as an important component in reestablishment of cell–cell adhesion and epithelial polarity (Table 4.1) (Bryant et al., 2007). Once internalized, E-cadherin recycling constitutes a way to regulate its function at cell–cell contacts (D’Souza-Schorey, 2005). SNX1 appears to mediate recycling of E-cadherin from macropinosomes and endosomes after its EGF-induced internalization (Bryant et al., 2007). How retromer may be coupled with SNX1 in E-cadherin recycling was, however, not investigated. Another SNX associated with newly formed macropinosomes upon EGF stimulation is SNX5, which can heterodimerize with SNX1 (Kerr et al., 2006). Although SNX1–SNX5 association in vivo appears controversial, SNX5 has been presented as a potential component of retromer (Wassmer et al., 2007). The presence of a retromer pool at plasma membrane regions with active actin polymerization provides a hint to this idea, or perhaps more confusion, since there a participation of SNX1 is not clear (Collins et al., 2008; Kerr et al., 2005). A completely different mechanism may govern retromermediated traffic of cell surface/cytoskeleton-associated proteins, perhaps along the lines of the reported effects of DmVps35 in endocytosis and actin cytoskeleton through Rac1 (Korolchuk et al., 2007); see Section 3.5. Altogether, these data open up a new topic of retromer’s biology and more work is needed to decipher the mechanism involved.

5.3. Altered retromer-mediated transport and Alzheimer’s disease It was once proposed that axon and somatodendritic surfaces of hippocampal neurons were functionally equivalent, respectively, to apical and basolateral plasma membrane domains of MDCK cells. Thus, molecular mechanisms to sort surface proteins share some features in these two cell types (Dotti and Simons, 1990; Dotti et al., 1991). Although since then many exceptions have been found, comparing polarity of neurons with MDCK cells has been very useful to study polarized traffic (Horton and

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Ehlers, 2003; Winckler and Mellman, 1999) (Fig. 4.2). Differences between dendritic and basolateral targeting have been attributed to differential expression of sorting adaptors in the two cell types or to different downstream events responsible for directing proteins to different domains (Icking et al., 2007; Silverman et al., 2005). On the contrary, sorting of proteins to the axon requires signals that are not present in apical proteins ( Jareb and Banker, 1998). A major difficulty in neurons for delivering molecules to the axon terminals is the axon length, which in large mammals, including humans, can be over 1-m long. Thus, regions most active in signal transduction and intercellular communication are generally found at enormous distances from the soma, where many of the targets and effectors of these signaling events are located. It is not surprising, then, that highly specialized membrane transport machinery is needed to direct movements along neuron cytoskeleton tracks, allowing neuronal viability and differentiation. Such spatial separation therefore makes neurons potentially more vulnerable to minor impairments in endosomal transport processes (Mann and Rougon, 2007; Nixon, 2005; Stokin and Goldstein, 2006). Indeed, many lines of evidence agree that a defective axonal transport is linked to neurodegenerative diseases. In this regard, many proteins associated with AD pathogenesis have been found localized in axonal compartments, in particular, at presynaptic terminals. These include b-APP, BACE, subunits of the g-secretase complex, and Tau. Most of these proteins have in fact a role in promoting axonal growth (Stokin and Goldstein, 2006). As discussed below, recent studies have provided a connection between the pathways followed by some of these proteins and retromer. The current model proposes a link between b-APP–BACE and retromer through the multiligand receptor SorLA (Table 4.1). The reduced levels of retromer subunits (Small et al., 2005) and of SorLA observed in brains of AD patients (Offe et al., 2006) were quickly related to the genetic association reported between SORLA and AD (Rogaeva et al., 2007). The association found between SorLA, b-APP (Andersen et al., 2005; Offe et al., 2006), and BACE (Spoelgen et al., 2006) prompted the analysis of the molecular mechanism controlling SorLA traffic and its influence in b-APP processing. As seen in various cell lines in culture, endosomal sorting of b-APP or the b-APP–BACE complex (Spoelgen et al., 2006) through SorLA is mediated by GGA, although it is less clear the implication of retromer or the phosphofurin acidic cluster sorting protein-1 (PACS-1) (Nielsen et al., 2007; Schmidt et al., 2007), the latter, a connector protein also functioning in endosome-to-TGN transport of various proteins, including the MPRs (Wan et al., 1998). The role of retromer would be to ensure efficient endosomal retrieval to the TGN of SorLA bound to intracellular b-APP, thus limiting b-APP cleavage by BACE, which is maximal in acidic endosomes (LaFerla et al., 2007; Small and Gandy, 2006). On the contrary, retromer has also been suggested to provide

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plgR

plgR HA NGFR CD8a

CD44 FcRgII-B2

GPI GAT-1 ngCAM

BACE g

EGFR ngCAM APP LDLR a g plgR TfR VSV

GPI GAT-1 NGFR ngCAM APP FcR g II-B2 CD8a BACE g CD44 HA

CD44 CD8a HA FcRgII-B2 VSV

APP EPGR LDLR plgR TfR

Figure 4.2 Comparison of polarized traffic in epithelial MDCK cells and neurons. Examples of endogenous and exogenous proteins sharing or not an analogous targeting between the two cell types are shown. Proteins apically targeted in MDCK cells, or to the axon in neurons, and their major apical pathways, are shown in red.Those proteins/ pathways headed toward the basolateral plasma membrane in MDCK cells, or to the dendritic domain in neurons, are in blue. Only their preferential targeting is indicated, although if a protein has a split distribution it is shown at both surfaces.When evidence of transcytosis has been provided, proteins following this indirect route are marked in a rectangle at their final destination. The implication of retromer, highlighted in yellow arrows, is shown for pIgR transcytosis in MDCK cells and for endosome-to-Golgi retrieval in neurons; while not demonstrated in neurons, it is implicit for either SorLA or sortilin. Many pathways and names of intracellular compartments are omitted for simplicity. Abbreviations (see others in text): a: a-secretase activity (to our knowledge, no information on its polarity in neurons has been reported); CD8a: lymphocyte protein CD8a; CD44: lymphocyte hyaluronate receptor CD44; FcRgII-B2: macrophage Fc receptor gII-B2; g: g-secretase activity/complex; GAT-1: g-aminobutyric acid transporter; GPI: glycosylphosphatidylinositol anchored proteins; HA: viral hemagglutinin protein; Ng-CAM: neuron-glia cell adhesion molecule; p75/NGFR: low affinity nerve growth factor receptor; TfR: transferrin receptor; VSV: glycoprotein of the vesicular stomatitis virus. Selected refs: Anderson et al., 2005; Bonzelius et al., 1994; Brandli et al., 1991; Capell et al., 2002; de Hoop et al.,1995; De Strooper et al.,1995; Dotti et al.,1991; Dotti and Simons, 1990; Fuller and Simons, 1986; Haass et al., 1995; Hunziker et al., 1991; Hunziker and Mellman, 1989; Jareb and Banker, 1998; Kamal et al., 2001; Koo et al., 1990; Lazarov et al., 2005; Mostov and Deitcher, 1986; Pietrini et al., 1994; Schuck and Simons, 2006; Silverman et al., 2005; Siman and Salidas, 2004; Simons et al., 1995; Tienari et al., 1996;VergeŁs et al., 2004;Yamazaki et al.,1995.

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morphologic and functional complexity, perhaps through its effect in Wnt signaling, in the entorhinal cortex of the brain hippocampal formation, a region particularly vulnerable to AD that also shows reduced levels of retromer during this pathology (Small, 2008). However, evidence supporting this hypothesis is currently lacking. Alternatively, SorLA reduction in AD could be totally independent of retromer, and instead caused by genetic SORLA deficiency, which turns into increased amyloidogenic processing of endogenous b-APP and b-APP-induced neurogenesis in AD brains (Rohe et al., 2008). None of these studies addressed the possible relevance of a polarized sorting pathway in a SorLA–retromer sorting step. As seen previously in polarized MDCK cells, both b-APP and BACE behave in a polarized fashion, albeit establishing a correlation with their polarized traffic in neurons is conflictive (Capell et al., 2002; Haass et al., 1994) and, as discussed above (Fig. 4.2), different adaptors are likely implicated in intracellular delivery of these proteins in each cell type (Icking et al., 2007). Finally, the possible implication of sortilin in this process has not yet been considered. Unfortunately, SorLA does not have in its tail neither of the consensus motifs, W/F-L-M/V (Seaman, 2007) or YXXF (Canuel et al., 2008), proposed as a requirement for retromer-mediated transport. Therefore, still many pieces remain to be solved to understand the effect of retromer’s interaction with SorLA (or sortilin) on b-APP–BACE transport and Ab neurotoxicity. Since this chapter was submitted, a new study has addressed retromer’s deficiency—as observed in AD—in mouse and fly brain, and found that it causes hippocampal-dependent memory and synaptic dysfunction, neurodegeneration, and Ab accumulation (Muhammad et al., 2008).

6. Concluding Remarks Diseases can be caused by alterations in the sorting signals of certain proteins or in the trafficking machinery delivering these proteins to their proper destinations. Thus, the study of endosomal function and dysfunction can be very useful in understanding pathologies and become a starting point for developing new drugs to treat disease. As shown by research in various model systems, retromer has an important contribution in developmental processes and its function may be altered in certain pathologies. Retromer, with an evolutionary conserved role in endosomal sorting, has thus emerged as a protein complex implicated in multiple aspects of protein traffic. As a complex elaboration of its somehow restricted function in yeast, retromer’s role in endosomal transport and secretion of molecules in a polarized way has essential implications in cell organization at early stages of development in multicellular organisms. The study of retromer’s mechanism should then

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teach us about the endosomal sorting machinery that operates normally but also about pathological consequences from alterations of this machinery. Recent data have contributed to a better understanding of how retromer’s subunits assemble, how they are recruited at the endosome membrane, and how they cooperate in performing a sorting function. In cooperation with SNXs, retromer mediates transport through active segregation of cargo into endosome tubules, which is now seen as a more efficient way than the nonselective or default sorting proposed in early experiments in the field of membrane traffic. Thus, retromer must effectively contribute to proper cargo sorting within a complexity of various destinations. With the added knowledge of retromer’s machinery to cell biology, our current understanding of how traffic of endocytosed proteins is regulated in both nonpolarized and polarized cells has grown considerably. Still, we miss how the action of this multimeric protein complex is coordinated in the context of other factors implicated in tubule or vesicle formation, or even with molecules that provide specificity for membrane docking, tethering, and fusion. Clearly, research soon to come will serve to elucidate the intriguing contribution of retromer in these important aspects of cell biology.

ACKNOWLEDGMENTS I am a recipient of a ‘‘Ramo´n y Cajal’’ contract by the Ministerio de Educacio´n y Ciencia, Spain. Work in my laboratory was funded by a grant from the Ministerio de Sanidad y Consumo (PI 07/0895), Spain.

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C H A P T E R

F I V E

Translational Control of Gene Expression: From Transcripts to Transcriptomes ¨rg Ba¨hler,1 Daniel H. Lackner and Ju Contents 1. Introduction 2. Preparation for Translation: RNA Processing and Export 3. Regulation of Translation 3.1. Mechanisms of translation initiation in eukaryotes 3.2. Rationale for regulating translation 3.3. Targets for translational regulation: Initiation factors, mRNAs, and ribosomes 3.4. Classic examples of translational regulation 4. Emerging Concepts in Translational Regulation 4.1. P-bodies and translation 4.2. Regulation by small RNAs 4.3. Interplay between miRNAs and P-bodies 4.4. Translational regulation through alternative transcripts 5. Global Approaches to Identify Targets of Posttranscriptional Gene Regulation 5.1. Translational profiling 5.2. Proteomic approaches to study translational regulation 5.3. mRNA turnover 5.4. RNA-binding proteins and their target RNAs 6. Concluding Remarks Acknowledgments References

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Cancer Research UK Fission Yeast Functional Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom Current address: Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, United Kingdom

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International Review of Cell and Molecular Biology, Volume 271 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01205-7

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2008 Elsevier Inc. All rights reserved.

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Abstract The regulation of gene expression is fundamental to diverse biological processes, including cell growth and division, adaptation to environmental stress, as well as differentiation and development. Gene expression is controlled at multiple levels from transcription to protein degradation. The regulation at the level of translation, from specific transcripts to entire transcriptomes, adds considerable richness and sophistication to gene regulation. The past decade has provided much insight into the diversity of mechanisms and strategies to regulate translation in response to external or internal factors. Moreover, the increased application of different global approaches now provides a wealth of information on gene expression control from a genome-wide perspective. Here, we will (1) describe aspects of mRNA processing and translation that are most relevant to translational regulation, (2) review both well-known and emerging concepts of translational regulation, and (3) survey recent approaches to analyze translational and related posttranscriptional regulation at genome-wide levels. Key Words: Translation, Posttranscriptional control, mRNA processing, P-bodies, microRNA, microarray, ribosome, RNA-binding protein. ß 2008 Elsevier Inc.

1. Introduction The control of gene expression is a fundamental process to bring the genome to life, and misregulation is usually associated with disease. It is now well established that gene expression is regulated at multiple levels, and emerging data suggest that the diverse processes involved in this regulation are integrated with each other (Hieronymus and Silver, 2004; Maniatis and Reed, 2002; Mata et al., 2005; McKee and Silver, 2007; Moore, 2005; Orphanides and Reinberg, 2002; Proudfoot et al., 2002). Gene regulation can be divided into transcriptional and posttranscriptional control (Fig. 5.1). Furthermore, proteins themselves can be regulated by posttranslational modifications and protein degradation. Transcriptional control has received much attention, through both traditional single-gene studies (Kadonaga, 2004) and genome-wide approaches, including expression profiling (Bertone et al., 2005; Lockhart and Winzeler, 2000), transcription factor binding studies, and identification of regulatory sequence elements (Hanlon and Lieb, 2004; Sandelin et al., 2007), as well as chromatin remodeling and epigenetic analyses (Bernstein et al., 2007; Kouzarides, 2007; Li et al., 2007). In comparison, posttranscriptional control has been less extensively studied. This discrepancy is apparent when searching within the scientific literature: approximately 55,000 articles are found in PubMed for the query ‘‘transcriptional regulation,’’

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Figure 5.1 Scheme of different layers of gene regulation. The regulatory processes are listed according to their involvement in transcriptional, posttranscriptional, or posttranslational control. Adapted with permission from Mata et al. (2005).

whereas ‘‘posttranscriptional regulation’’ only returns about 5700 hits. This bias reflects historical and technical reasons: it is clear that transcription is one of the fundamental and intuitively important steps for gene regulation, and techniques to study transcription and transcriptional control are well established in the scientific community. An increasing appreciation of the importance of posttranscriptional gene regulation is emerging. Posttranscriptional regulation mechanisms comprise various processes such as mRNA processing (polyadenylation, capping, and splicing), mRNA export and localization, mRNA decay, and mRNA translation (Fig. 5.1). Despite this variety of regulatory mechanisms, they all have one thing in common: they ultimately control if, where, and how efficiently a given mRNA is translated into protein. Consequently, translation and translational control are central to posttranscriptional regulation of gene expression. We will therefore first discuss in some detail different mechanisms and strategies for the regulation of translation in eukaryotes and will then give an overview of recent efforts to study posttranscriptional regulation of translation and related mRNA processes on a genome-wide scale.

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2. Preparation for Translation: RNA Processing and Export Before a transcript can be exported from the cell nucleus to become available for the translation machinery in the cytoplasm, it has to undergo a series of processing steps: the mRNA acquires a cap structure at the 50 terminus, introns are spliced out from the pre-mRNA, and a specialized 30 end of the mRNA is generated, usually by polyadenylation. These maturation steps are cotranscriptional and can influence each other’s activities (Proudfoot et al., 2002). Only a brief overview of these processes will be given, as far as they are relevant to translational regulation, while referring to key reviews that present more detailed views of these RNA processing steps. The first processing step is the addition of the m7G cap structure to the 50 end of the nascent mRNA and takes place after 20–30 nucleotides (nt) have been synthesized (Gu and Lima, 2005; Shatkin and Manley, 2000). In a three-step reaction, the nascent transcript is hydrolyzed, the GMP moiety from GTP is added to the first nt of the pre-mRNA, and GMP is methylated at position N7. The m7G cap is important for mRNA stability and translation (see below). In the nucleus, the m7G cap is bound by the twosubunit cap-binding complex (CBC), and, after export of the mRNA to the cytoplasm, is replaced by the translation initiation factor 4E, which represents an essential step in translation initiation. As the coding sequences of most mRNAs in eukaryotes are interrupted by introns, these introns must be spliced out of the pre-mRNA to generate a functional mRNA. Splicing requires consensus sequences in the premRNA, which mark the exon–intron boundaries, and the spliceosome, the catalytic complex which carries out the enzymatic reactions to remove the introns and ligate the flanking exons (Collins and Guthrie, 2000; Jurica and Moore, 2003; Kramer, 1996; Patel and Steitz, 2003). The spliceosome consists of five small ribonucleoprotein particles (snRNPs: U1, U2, U4, U5, and U6), each of which is made of a small nuclear RNA (snRNA) and associated proteins, as well as numerous accesory proteins. In fact, well over a hundred different proteins are thought to function as splicing factors ( Jurica and Moore, 2003). The catalysis of the splicing reaction itself is dependent on RNA–protein, RNA–RNA, and protein–protein interactions. Furthermore, the alternative use of exons (alternative splicing) can contribute to protein variety by allowing one gene to produce multiple isoforms (Matlin et al., 2005). Most mRNAs also bear a specific structure in the form of a poly(A) tail at their 30 end. The only known protein-coding genes lacking poly(A) tails are metazoan histone mRNAs (Marzluff, 2005). Polyadenylation is achieved in two steps: the nascent mRNA is cleaved near the site of polyadenylation, which is followed by poly(A) synthesis (Proudfoot and O’Sullivan, 2002;

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Shatkin and Manley, 2000; Zhao et al., 1999). In analogy to splicing, formation of the poly(A) tail requires multi-subunit polyadenylation complexes and specific sequence-elements in the pre-mRNA. In mammalian cells, the site of cleavage lies mostly between an AAUAAA hexamer motif and a GU-rich downstream element (DSE) (McLauchlan et al., 1985). The AAUAAA hexamer is bound by the cleavage and polyadenylation specificity factor (CPSF), and the DSE interacts with the cleavage stimulatory factor (CstF). Cleavage factors I and II (CF I; CF II) are also required. Whereas both poly(A) polymerase (PAP) and CPSF are required for cleavage of the pre-mRNA and poly(A) addition, CstF is necessary for the endonucleolytic cleavage and, together with CPSF, for the recruitment of CF I and CF II (MacDonald et al., 1994; Murthy and Manley, 1995; Takagaki et al., 1989). The principle of poly(A) tail formation is the same in yeast and mammalian cells, and the protein complexes involved have orthologous components, but also specific accessory factors that are only found in one of the species (Proudfoot and O’Sullivan, 2002; Shatkin and Manley, 2000; Stevenson and Norbury, 2006). Furthermore, in yeast, a variable A-rich element substitutes for the AAUAAA hexamer motif, and there are three polyadenylation complexes: cleavage polyadenylation factor (CPF), which contains the PAP and several factors homologous to CPSF, cleavage factor IA (CF IA), and cleavage factor IB (CF IB). The emerging poly(A) tail is bound by nuclear and cytoplasmic poly(A)binding proteins (PABPs). PABPs are thought to influence the final length of the poly(A) tail positively by stimulating the processivity of PAP, as well as negatively by interacting with the poly(A) nuclease (PAN) (Mangus et al., 2003). Furthermore, PABPs are involved in nuclear export and are also important for the initiation of translation (Section 3.1.2). The poly(A) tail is also crucial for several other posttranscriptional regulatory mechanisms in the cytoplasm, and cytoplasmic PAPs can regulate the translational state and stability of various target mRNAs via modifying the length of the respective poly(A) tails (Read and Norbury, 2002; Stevenson and Norbury, 2006). The best-studied example is probably the translational regulation of maternal mRNAs in Xenopus oocytes, which are stock-piled in a translationally repressed state with short poly(A) tails that become polyadenylated upon activation and, as a consequence, translated (Mendez and Richter, 2001; Richter, 2007). mRNA decay by exonucleolytic mechanisms is also usually preceded by a shortening of the poly(A) tail (Parker and Song, 2004; Wilusz et al., 2001), and recently deadenylation of poly(A) tails has also been shown to occur in microRNA (miRNA)-mediated gene regulation (Giraldez et al., 2006; Wu et al., 2006). Mature mRNAs need to be exported from the nucleus to the cytoplasm for translation. Export through the nuclear pore complex (NPC) occurs in the context of messenger ribonucleoprotein particles (mRNPs) that are assembled cotranscriptionally (Cole and Scarcelli, 2006; Stewart, 2007;

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Stra¨sser et al., 2002). mRNPs contain the mRNA and associated RNAbinding proteins (RBPs) that bind to the mRNA during the processing steps (Aguilera, 2005; Moore, 2005). Apart from the aforementioned CBC or PABPs, such RBPs include SR (serine/arginine rich) and hnRNP (heterogeneous nuclear RNP) proteins, or the exon junction complex (EJC), which is a set of proteins loaded onto the mRNA upstream of exon–exon junctions as a consequence of pre-mRNA splicing. These factors are important for the association of the mRNP with the NPC and the export into the cytoplasm, and some of them stay associated with the mRNA as it is exported, whereas others are restricted to the nucleus. Furthermore, nuclear export is important for quality control, as faulty or unprocessed mRNAs are not only useless but also potentially harmful if translated in the cytoplasm; this quality control step is coupled to RNA processing and the mRNP composition. It needs to be emphasized that although we introduced mRNA transcription, capping, splicing, polyadenylation, and nuclear export as sequential events, these events seem to be tightly integrated with each other both spatially and temporally (Aguilera, 2005; Moore, 2005; Proudfoot et al., 2002).

3. Regulation of Translation Translation can be divided into three major steps: initiation, elongation, and termination. Translation initiation comprises the events that lead up to the positioning of an elongation-competent 80S ribosome at the start codon of the mRNA. Polypeptide synthesis takes place during the elongation phase. The completed polypeptide is released after the ribosome encounters a stop codon during translation termination. Several lines of evidence indicate that initiation is the rate-limiting step for translation. When cells are treated with low doses of elongation inhibitors (e.g., cycloheximide) such that total protein synthesis is only minimally affected, the translational efficiency of most mRNAs is not altered (Lodish and Jacobsen, 1972; Mathews et al., 2007; Walden et al., 1981). Furthermore, the average density of ribosomes along the mRNA is significantly lower than the maximum packing capacity of one ribosome per 30–40 nt (Arava et al., 2003; Lackner et al., 2007; Mathews et al., 2007; Wolin and Walter, 1988). This maximum capacity can be obtained by treating mRNAs with drugs that slow down elongation. The complexity and importance of translation initiation compared to elongation and termination is further underscored by the fact that only few dedicated factors are needed for the latter two processes, whereas more than 25 proteins are needed to ensure proper translation initiation (Pestova et al., 2007; Preiss and Hentze, 2003).

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Therefore, it is not surprising that most translational regulation is executed at the level of initiation (Gebauer and Hentze, 2004; Holcik and Sonenberg, 2005; Mathews et al., 2007; Preiss and Hentze, 2003). We will provide an overview of the molecular mechanisms of translation initiation as far as they are directly relevant to the regulation of translation. More detailed reviews of the molecular events of translation initiation in mammalian and yeast cells are available (Hinnebusch et al., 2007; Pestova et al., 2007). Note that much of the molecular data on translation have been acquired using either in vitro studies with purified components to reconstitute translation events or genetic studies in the budding yeast Saccharomyces cerevisiae. For descriptions of translation elongation and termination, we refer to recent reviews (Ehrenberg et al., 2007; Taylor et al., 2007).

3.1. Mechanisms of translation initiation in eukaryotes 3.1.1. Preinitiation complex formation Translation initiation starts with the formation of the 43S preinitiation complex (Fig. 5.2). As physiological conditions favor the association of small (40S) and large (60S) ribosomal subunits to form complete 80S ribosomes, but only free ribosomal subunits can initiate translation, it is important that posttermination ribosomes dissociate (Pestova et al., 2001; Preiss and Hentze, 2003). In prokaryotes, this dissociation is achieved through a ribosome-recycling factor, which shows no known eukaryotic equivalent (Kisselev and Buckingham, 2000). The eukaryotic initiation factors (eIFs) eIF3, eIF1, eIF1A, and eIF6 are thought to promote this dissociation in eukaryotes, but its mechanism is unknown. Recent data suggest that the activity of these factors is not sufficient to prevent formation of 80S ribosomes (Pestova et al., 2007; Preiss and Hentze, 2003), and it is thought that dissociation of 80S ribosomes is directly linked to 43S preinitiation complex formation (Pestova et al., 2007). The first step in 43S preinitiation complex formation is the assembly of the ternary complex (Figs. 5.2 and 5.3). The ternary complex consists of eIF2, a hetero-trimer of a, b, and g subunits, methionyl-initiator tRNA (Met-tRNAiMet) and GTP, and its assembly is regulated by the guanine nt exchange factor (GEF) eIF2B (Fig. 5.3). GTP is hydrolyzed after recognition of the AUG start codon producing eIF2 bound to GDP, which has a tenfold reduced affinity for Met-tRNAiMet (Hinnebusch et al., 2007). eIF2B promotes the GDP–GTP exchange to regenerate active eIF2 (Fig. 5.3) (Hinnebusch et al., 2007; Pestova et al., 2007; Preiss and Hentze, 2003). Binding of the active ternary complex to the 40S ribosomal subunit is aided independently by eIF1, eIF1A, and eIF3 in mammalian cells (Pestova et al., 2007; Preiss and Hentze, 2003). In budding yeast, eIF1, eIF3, eIF5, and the ternary complex can be isolated as a multifactor complex

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Met 2

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Figure 5.2 Major molecular events that lead to cap-dependent translation initiation. For a detailed description see main text. Reproduced with permission from Gebauer and Hentze (2004).

(MFC), which raises the possibility that this MFC is recruited to the 40S subunit as a preformed unit (Hinnebusch et al., 2007). The 43S preinitiation complex is then ready to bind to the 50 end of the mRNA.

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Figure 5.3 Formation of the active ternary complex. The ternary complex consists of eIF2, a hetero-trimer of a, b, and g subunits, the initiator tRNA (Met-tRNAiMet) and GTP, and its assembly is regulated by the guanine nucleotide exchange factor (GEF) eIF2B: GTP is hydrolyzed after recognition of the AUG start codon producing eIF2 bound to GDP, which has a tenfold reduced affinity for Met-tRNAiMet. eIF2B promotes the GDP^GTP exchange to regenerate active eIF2. Reproduced with permission from Gebauer and Hentze (2004).

3.1.2. Recruitment of preinitiation complex to mRNA Recognition of the m7G cap structure at the 50 end of the mRNA is mediated by eIF4F, which contains the three subunits eIF4E, eIF4G, and eIF4A (Fig. 5.2): eIF4E binds directly to the m7G cap structure, eIF4A is a DEADbox RNA helicase that is thought to unwind secondary structures in the 50 UTR (untranslated region) so that the 43S complex can scan along the mRNA, and eIF4G is thought to act as scaffold protein (Hinnebusch et al., 2007; Pestova et al., 2007; Preiss and Hentze, 2003). In mammalian cells, eIF3 from the preinitiation complex interacts with the central domain of eIF4G (Lamphear et al., 1995). This interaction has not yet been found in budding yeast, where eIF4A is also not stably associated with eIF4E and eIF4G (Goyer et al., 1989; Hinnebusch et al., 2007). Altogether, the binding of the preinitiation complex to the mRNA involves the cooperative activities of eIF4F, eIF3, eIF4B, and possibly the PABP. PABP was initially identified as a protein that associates with the poly(A) tail at the 30 UTR of the mRNA. The concerted binding of PABP and eIF4E to eIF4G is thought to pseudo-circularize the mRNA (Fig. 5.2) (Wells et al., 1998). Furthermore, PABP Pab1p is essential for translation initiation in budding yeast (Sachs, 2000). This circularization provides a possible framework by which 30 UTR-binding proteins can regulate translation initiation, as most known regulatory sequences are found in the 30 UTR, despite the fact that translation starts at 50 end of the mRNA (Gebauer and Hentze, 2004).

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3.1.3. mRNA scanning and AUG recognition After proper assembly at the 50 end of the mRNA, the preinitiation complex needs to scan along the mRNA to find the AUG start codon (Kozak, 1989, 2002). The model of scanning had originally been proposed by Kozak (1999), and despite the fact that most biochemical and genetic data are consistent with the model, direct physical intermediates of the scanning process have not been identified to date. The 43S preinitiation complex can bind to an mRNA having an unstructured 50 UTR independent of eIF4F, eIF4A, and ATP, but needs eIF1 or eIF4G to scan to the start codon. However, an mRNA with a structured 50 UTR additionally requires eIF4F, eIF4B, ATP, and eIF1A (Pestova and Kolupaeva, 2002; Pestova et al., 1998). eIF4A helicase and eIF4F are thought to promote unwinding of the secondary structure of the mRNA, while eIF1 and eIF1A are thought to promote a structural conformation of the 43S preinitiation complex, which allows scanning in 50 –30 direction. 3.1.4. Ready to go: Formation of translation-competent 80S subunit The 43S preinitiation complex recognizes the start codon through formation of base pairs between the anticodon loop of the initiator tRNA and the AUG start codon (Fig. 5.2). This stable complex is known as the 48S initiation complex. Selection of the correct start codon is dependent on eIF1 (Pestova and Kolupaeva, 2002; Pestova et al., 1998). Several events then take place in order for the 60S subunit to join the 48S complex and form the 80S ribosome. eIF5 promotes the hydrolysis of eIF2–GTP, and, as a consequence, most of the initiation factors including eIF2–GDP dissociate from the small ribosomal subunit, leaving the initiator tRNA bound to the start codon (Hinnebusch et al., 2007). Recently, it has been found that a second step of GTP hydrolysis is necessary for 60S joining and to render the resulting 80S ribosome competent for polypeptide synthesis: GTPase activity of eIF5B is stimulated by 60S subunits and even stronger by 80S ribosomes. GTP-bound eIF5B stimulates 60S subunit joining, and GTP hydrolysis occurs after 80S subunit formation and is essential for the release of eIF5B (Lee et al., 2002; Pestova et al., 2000; Shin et al., 2002). Taken together, two steps of GTP-hydrolysis are required for 80S ribosome formation, which also provide a checkpoint for proper start codon recognition. 3.1.5. Cap-independent translation initiation The cap-dependent events of translation initiation described above are most common for cellular mRNAs. However, a cap-independent way of initiating translation can happen through internal ribosomal entry sites (IRES). IRES are heavily structured sequence elements in 50 UTRs of some mRNAs with no obvious conserved consensus sequence (Baird et al., 2006). The

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structured IRES segment in 50 UTRs has an active role in the recruitment of the 40S subunit. IRES elements are found in viral mRNAs and also in certain cellular mRNAs that are involved in growth control, differentiation, apoptosis, or oncogenesis (Doudna and Sarnow, 2007; Elroy-Stein and Merrick, 2007). These mRNAs are usually only weakly translated under normal conditions, but can be more efficiently translated upon downregulation of cap-dependent translation. In-depth reviews on the topic of IRES are available (Fraser and Doudna, 2007; Hellen and Sarnow, 2001; Jackson, 2005; Spriggs et al., 2005; Stoneley and Willis, 2004).

3.2. Rationale for regulating translation Why do cells regulate translation and how do they benefit from it? There are several possible answers to this question, which are also addressed by (Mathews et al., 2007). Regulation at the translational level can happen rapidly without the necessity of going through all the upstream processes of gene expression such as transcription, mRNA processing, and mRNA export. Furthermore, translational regulation is usually reversible, as it is often mediated through reversible protein modifications such as the phosphorylation of initiation factors. The need for translational control is also apparent for systems where transcriptional control is not possible, such as reticulocytes, which lack a nucleus, oocytes, or RNA viruses. Another reason for the regulation of translation is spatial control of gene expression within the cell (Schuman et al., 2006; St Johnston, 2005). The requirement for localized protein production in neurons or during development can only be met by translational regulation, as transcriptional regulation is restricted to the cell nucleus. Translational regulation also provides flexible control of gene expression: given the complex mechanisms of translation initiation outlined above, there are many molecular targets for translational regulation, which consequently can change translational efficiencies for many or only a few mRNAs. A last but important reason for translational regulation lies in the fine tuning of gene expression, and there are numerous examples of genes that are regulated at both the transcriptional and translational levels (e.g., GADD45a or TNF-a; Lal et al., 2006; Saklatvala et al., 2003).

3.3. Targets for translational regulation: Initiation factors, mRNAs, and ribosomes Translational control can in principle be divided into global regulation of translation and mRNA-specific regulation (Gebauer and Hentze, 2004). Global regulation affects the translational efficiency of most mRNAs through a general tuning of translation, while mRNA-specific regulation only affects the translation of selected mRNAs. In some cases, however, this

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simple distinction cannot be made; for example, the general downregulation of cap-dependent translation enhances translation of a subset of IRESbearing mRNAs (Sections 3.1.5 and 5.1). What are the targets for translational control at the initiation step and what are the basic principles? A simple answer to this question would be that most translational regulation either inhibits or promotes the association of mRNAs with the translation apparatus. Given the plethora of translation initiation factors, it is not surprising that many of them are targets in translational regulation, and many are controlled posttranslationally (Dever, 2002; Raught and Gingras, 2007). A key target for many regulatory mechanisms is the cap-binding protein eIF4E, which can be bound by inhibitory proteins that subsequently hinder binding of the mRNA (see below for more details). Global regulation of translation is generally mediated through modifications of translation initiation factors. Another target for translational regulation is the mRNA itself, via cisregulatory elements that are bound by trans-acting factors. The cis-regulatory elements on the mRNA can be found anywhere along the mRNA, but for most well-characterized examples of translational regulation these elements are present in either the 50 or 30 UTRs (Fig. 5.4). mRNA-specific translational regulation happens mostly via RNA-bnding proteins that recognize cis-regulatory elements of a given mRNA. The ribosome itself can also be targeted to exert translational regulation, and several of its protein constituents can undergo posttranslational modifications. A well-studied example is the phosphorylation of ribosomal protein S6 (rpS6) by ribosomal S6 kinase (S6K), which was first shown more than 30 years ago (Gressner and Wool, 1974). A correlation of rpS6 phosphorylation with an increase in translation initiation, especially of mRNAs posessing a 50 -

Hairpin IRES 5 m7GpppN

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Figure 5.4 Cis-acting sequence elements that influence translation initiation of specific mRNAs. The m7G cap structure at the 50 end and the poly(A) tail at the 30 end of mRNAs are both essential elements for cap-dependent translation initiation. Additionally, specific sequence elements in the 50 or 30 UTRs (ovals) can influence translation initiation in combination with bound trans-acting factors. Structured elements such as hairpins can inhibit translation initiation and structured internal ribosomal entry sites (IRES) can mediate cap-independent translation initiation. Upstream open reading frames (uORFs) usually inhibit translation initiation for the downstream start codon. Reproduced with permission from Gebauer and Hentze (2004).

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terminal oligopyrimidine sequence (TOP mRNAs), prompted the hypothesis that translation of TOP mRNAs is regulated through this phosphorylation ( Jefferies et al., 1994). However, recent data contradict this model of a simple causal relationship between rpS6 phosphorylation and translational efficiency: a double knockout of both S6K homologues in mouse cells (Pende et al., 2004) or a knockin of nonphosphorylatable rpS6 (Ruvinsky et al., 2005) do not affect translational regulation of TOP mRNAs. The elucidation of the exact mechanism of rpS6 phosphorylation on translation is further aggravated by the discovery of various alternative substrates of S6K, which also include factors involved in translation initiation (Ruvinsky and Meyuhas, 2006). Ribosomal proteins are also modified through ubiquitination (Spence et al., 2000), methylation (Bachand and Silver, 2004; Swiercz et al., 2005), and a recent report identified ribosomal proteins as targets for NEDDylation (Xirodimas et al., 2008). In budding yeast and other organisms, many genes encoding ribosomal proteins are duplicated. The open reading frame (ORF) and the protein sequence of the paralogues are similar, while the UTRs and intron sequences can differ. Ribosomal gene pairs were generally considered to be functionally equivalent, and it was thought that the gene pairs were retained to keep up with the cell’s strong need to synthesize ribosomal proteins and ribosomes (Warner, 1999). However, recent genome-wide screens for genes required for various cellular processes such as telomere length homeostasis (Askree et al., 2004), centromeric cohesion (Marston et al., 2004), cellular life span (Steffen et al., 2008), or for genes that exhibit deleterious haploinsufficient interactions with actin (Haarer et al., 2007) uncovered specific effects for only one of the paralogues of the ribosomal protein, whereas deletion of the other paralogue would not affect the studied biological process. Functional specificity among duplicated ribosomal proteins was further corroborated by recent work from Komili et al. (2007): localized translation of ASH1 mRNA in S. cerevisiae is dependent on a specific subset of ribosomal proteins. Furthermore, phenotypes and transcriptomes largely differ between mutants in nearly identical paralogues. Taken together, this work is a nice example of a combination of cell biology and systems biology approaches, which reveals that paralogues of ribosomal proteins rarely behave in the same way. The biological reasons for these differences are not clear. One possibility could be that specific ribosomal proteins are involved in cellular processes other than translation. Another intriguing possibility is heterogeneity of ribosomes: the cell could construct various kinds of ribosomes, which differ in terms of paralogue composition and posttranslational modifications, and these specialized ribosomes could play roles in the regulation of translation of specific subsets of mRNAs. Further work will be needed to elucidate the exact mechanism behind this apparent ribosome specialization, especially in light of the similarity between the paralogues, some of which share the exact same protein sequence.

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3.4. Classic examples of translational regulation Translational regulation is crucial for diverse physiological processes. It is involved in the response to cellular stress (Holcik and Sonenberg, 2005), in the misregulation of gene expression during cancer (Schneider and Sonenberg, 2007), in apoptosis (Morley and Coldwell, 2007), in development (Thompson et al., 2007), and in the establishment of synaptic plasticity and, consequently, in learning and memory (Klann and Richter, 2007). Many examples of translational control have been reported both within and outside these areas. Instead of giving a broad overview of these regulatory mechanisms, we will focus below on several well-studied examples for which the underlying molecular mechanisms have been reasonably well identified. Most of the regulatory mechanisms presented here—such as the regulation of ternary complex formation, the regulation of translation via eIF4E-binding proteins, or the posttranscriptional regulation via ARE-elements—are probably conserved for most eukaryotes, although these processes have mostly been studied in budding yeast and mammalian cells. Other regulatory mechanisms—such as the translational regulation of gene expression in Drosophila or Xenopus development— probably apply specialized mechanisms to meet the specific requirements of gene regulation in different organisms. The underlying principles for these regulatory mechanisms, however, are found in diverse variations in many eukaryotic cells.

3.4.1. Regulation of ternary complex formation Exposure of cells to stress conditions (e.g., oxidative stress, nutrient limitation, hypoxia, temperature stress) results often, if not always, in a global downregulation of translation (Holcik and Sonenberg, 2005). One of the best-studied examples for this downregulation is the control of the availability of active ternary complexes (Fig. 5.5). Binding of Met-tRNAiMet to the 40S subunit through the ternary complex is an essential step in translation initiation as described in Section 3.1.1 (Figs. 5.2 and 5.3). After the exposure to stress, the a-subunit of eIF2 (eIF2a) is phosphorylated and thereby inhibits the exchange of GDP for GTP by eIF2B and, as a consequence, formation of active ternary complexes is strongly reduced, and translation is downregulated globally (Dever et al., 1992; Gebauer and Hentze, 2004; Holcik and Sonenberg, 2005; Ron and Harding, 2007). The molecular mechanism for this inhibition is based on the fact that phosphorylated eIF2a-GDP turns into a competitive inhibitor of eIF2B, as eIF2B has a much higher affinity toward phosphorylated eIF2a–GDP than toward unphosphorylated eIF2a–GDP (Rowlands et al., 1988). There are at least four kinases that have been identified to phosphorylate eIF2a at

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Figure 5.5 Inhibition of global protein synthesis in response to stress through phosphorylation of eukaryotic initiation factor-2a. Several protein kinases (GCN2, PKR, HRI, or PERK) can phosphorylate the a-subunit of eIF2 in response to various stress conditions.This phosphorylation inhibits the GTP^GDP exchange on eIF2 by reducing the dissociation rate of the guanine nucleotide exchange factor eIF2B, thus inhibiting active ternary complex formation. As a consequence, translation initiation is globally downregulated. Reproduced with permission from Holcik and Sonenberg (2005).

Ser51 in the response to various stresses (Fig. 5.5; Dever et al., 2007): the haem-regulated inhibitor (HRI) is induced by haem depletion; general control nondepressible 2 (GCN2) is mainly activated by amino acid starvation; protein kinase activated by double-stranded RNA (PKR) is stimulated in response to viral infection; PKR-like endoplasmic reticulum kinase (PERK) is activated during endoplasmatic reticulum (ER) stress and the unfolded protein response (UPR).

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3.4.2. Regulation through uORFs Whereas translation of most mRNAs is downregulated by eIF2a phosphorylation, translation of several specific mRNAs can be upregulated in response to reduced availability of ternary complex. Gcn2p kinase is upregulated in response to various starvation conditions in budding yeast, expression of GCN2 is upregulated through a mechanism that recognizes a lack of amino acids; this is mediated through binding of uncharged tRNAs to the kinase (Dong et al., 2000). Ternary complex formation and global translation are downregulated as a consequence. However, GCN4, encoding a master transcriptional regulator that activates transcription of amino acid-biosynthesis genes, is translationally upregulated under these conditions (Hinnebusch and Natarajan, 2002). This upregulation is achieved by regulatory upstream open reading frames (uORFs). Four of these uORFs can be found in the 50 UTR of the GCN4 mRNA (Hinnebusch, 2005; Hinnebusch and Natarajan, 2002): Under optimal growth conditions and availability of ternary complex, translation usually starts at uORF1 and ribosomes then resume scanning to translate uORF2, uORF3, and uORF4 (Fig. 5.6). However, ribosomes cannot reinitiate translation after

AUG5 GCN4

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Figure 5.6 Translational regulation of GCN4 by upstream open reading frames (uORFs).With low levels of eIF2a-phosphorylation and abundant active ternary complex, ribosomes initiate translation at uORF1, resume scanning, and reinitiate translation at uORF2, uORF3, or uORF4. However, they do not resume scanning to reinitiate translation at the start codon of GCN4. When cells are starved for amino acids, eIF2a becomes phosphorylated and, as a consequence, the number of active ternary complexes decreases. Under this condition, reinitiation at uORF2^uORF4 happens less frequently and scanning can resume to the actual start codon of GCN4, which is then translated. Reproduced with permission from Holcik and Sonenberg (2005).

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termination at these latter uORFs and, as a consequence; the main coding region of GCN4 mRNA is not translated. Upon eIF2a phosphorylation, when ternary complexes become limiting, ribosomes are more likely to resume scanning without reinitiating translation at the downstream uORFs, and translation is initiated at the actual start codon of GCN4 (Fig. 5.6). The response to amino acid starvation via the Gcn2p kinase seems to be an evolutionarily conserved mechanism, as it was recently shown that Gcn2p activity in the mouse brain is essential for a restricted intake of diets lacking essential amino acids (Hao et al., 2005; Maurin et al., 2005). These studies reveal that the Gcn2 pathway recognizes depressions in serum amino acid levels that occur during consumption of food with an imbalanced composition of amino acids, which results in a behavioral response that limits the consumption of imbalanced foods and favors the intake of a balanced diet. The mammalian transcription factor Atf4 is regulated in a similar way by uORFs in response to ER stress or amino acid starvation (Harding et al., 2000; Scheuner et al., 2001), and there is evidence that Gcn2 also regulates synaptic plasticity through modulation of Atf4 translation (Costa-Mattioli et al., 2005, and references therein). There are numerous other examples of mRNAs whose translation is regulated by uORFs (Dever, 2002). Recent genome-wide bioinformatics approaches in yeast and mammals suggest that the occurrence of functional uORFs is widespread and might be a common regulatory mechanism of translation (Cvijovic et al., 2007; Iacono et al., 2005).

3.4.3. Regulation by eIF4E inhibitory proteins An important step during translation initiation is the binding of the m7G cap by eIF4F (Fig. 5.2). The backbone of this complex is eIF4G, which interacts with eIF4E and the helicase eIF4A. Translation initiation can be regulated by the disruption of eIF4E–eIF4G binding through inhibitory proteins, which were originally called 4E-BP (for 4E binding proteins) (Richter and Sonenberg, 2005). These inhibitory proteins have been reported to control a variety of biological processes such as development or cell growth, and may also repress tumour formation (Richter and Sonenberg, 2005). 4E-BPs compete with eIF4G for the binding to eIF4E, and the binding affinity is regulated through phosphorylation of 4E-BPs (Gingras et al., 1999): in the hypo-phosphorylated state, 4E-BPs bind to eIF4E and prevent translation initiation, while in the hyper-phosphorylated state, 4E-BPs binding to eIF4E is blocked. In addition to 4E-BPs, several other proteins can bind eIF4E in an mRNA-specific manner to inhibit translation initiation. The mRNA specificity for these proteins comes through interactions with sequence-specific elements within the mRNA or through the interaction with RBPs.

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In Xenopus oocytes, many mRNAs remain dormant with short poly(A) tails. When the oocytes are stimulated by progesterone for maturation, these mRNAs become polyadenylated and translationally active. A cytoplasmic polyadenylation element (CPE) in the 30 UTR of the mRNA is important for both masking and translational activation of the mRNA and is bound by the cytoplasmic polyadenylation element binding protein (CPEB) (Mendez and Richter, 2001; Richter, 2007). When dormant, CPEB is bound by Maskin, which inhibits the binding between eIF4E and eIF4G (Fig. 5.7),

elF4G Masked m7G elF4E Maskin Weak

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Figure 5.7 Translational regulation by the cytoplasmic polyadenylation element (CPE). mRNAs with a CPE in their 30 UTRs are translationally repressed in developing oocytes by binding of the cytoplasmic polyadenylation element binding protein (CPEB) and Maskin. Maskin interacts directly with the cap-binding protein eIF4E and prevents its association with eIF4G, which is crucial for translation initiation. CPEB inhibits association of the cleavage and polyadenylation specificity factor (CPSF) with the AAUAAA sequence motif resulting in short poly(A) tails. Oocyte maturation leads to phosphorylation of CPEB. Consequently, Maskin dissociates from eIF4E and CPSF binds to the AAUAAA motif. Binding of CPSF recruits poly(A) polymerase that extends the poly(A) tail.These events lead to translation initiation in the previously translationally repressed mRNAs. Reproduced with permission from Kuersten and Goodwin (2003).

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acting as an mRNA-specific 4E-BP (Cao and Richter, 2002). After stimulation of the oocyte to complete meiosis, CPEB stimulates cytoplasmic polyadenylation of the mRNA; the poly(A) tail is bound by PABP, which then can bind eIF4G and displace Maskin (Fig. 5.7; Cao and Richter, 2002). In turn, this cytoplasmic polydenylation also activates the synthesis of C3H-4, which leads to deadenylation of a subset of of mRNAs in a negative feedback loop required to exit meiotic metaphase (Belloc and Me´ndez, 2008). During translational repression, the CPEB-containing complex also includes PARN, a poly(A)-specific ribonuclease that contributes to the short poly(A) tail of target mRNAs by overriding the polyadenylating activity of the PAP GLD2 (Kim and Richter, 2006). Recently, a combinatorial code of sequence motifs in 30 UTRs was uncovered that determines not only whether mRNAs will be translationally repressed by CPEB but also the pattern of polyadenylation-dependent translational activation (Pique´ et al., 2008). Another example of an mRNA-specific 4E-BPs is the homeodomain transcription factor Bicoid, which (apart from its activity as transcription factor) inhibits translation of Caudal mRNA in Drosophila (Dubnau and Struhl, 1996; Rivera-Pomar et al., 1996). Similar to Maskin, Bicoid has an eIF4E-binding motif and was initially thought to directly bind to eIF4E (Niessing et al., 2002). However, recent work showed that Bicoid interacts with d4EHP (Drosophila 4E-homologous protein), an eIF4E-like protein that can interact with the m7G cap, but not with eIF4G (Cho et al., 2005). Recent studies have also identified Cup as a translational regulator in Drosophila, which interacts with eIF4E and prevents eIF4F complex formation and translation initiation (Nakamura et al., 2004; Nelson et al., 2004; Wilhelm et al., 2003). Nanos and Oskar are examples of mRNAs regulated by Cup. 3.4.4. Other mechanisms of mRNA-specific translational regulation AU-rich elements (AREs) are present in the 30 UTR of many mRNAs and are potent sequence elements of posttranscriptional gene regulation. AREs influence the stability or translation of a given mRNA, usually through binding of ARE-specific RBPs (Barreau et al., 2005). AUF1 was the first ARE-binding protein to be identified and was shown to exist in four isoforms (Wilson et al., 1999). Association of ARE-binding proteins of the AUF1 family with AREs promotes degradation of mRNAs encoding cytokines (IL-3, GM-CSF) or cell cycle regulators (p16INK4a, p21WAF1/ CIP1, cyclin D1) (Lal et al., 2004; Raineri et al., 2004; Wang et al., 2005). AUF1 also interacts with the heat-shock proteins hsc70-hsp70, eIF4G, and PABP (Laroia et al., 2002). Despite its role in promoting mRNA decay, recent work shows that AUF1 can induce translation of MYC protooncogene mRNA (Liao et al., 2007): downregulation of AUF1 abundance by RNA-interference (RNAi) did not result in altered MYC mRNA levels,

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as expected based on earlier in vitro studies (Brewer, 1991), but significantly reduced MYC mRNA translation. In contrast, TIAR, another ARE-binding protein, was shown to suppress translation of MYC mRNA. Despite competitive binding of AUF1 and TIAR to the MYC ARE, translational upregulation through AUF1 was not simply achieved by suppression of TIAR binding, as shown in double knockdown experiments (Liao et al., 2007). Repression of translation through the ARE-binding protein TIAR has been shown for several mRNAs such as GADD45a (Lal et al., 2006) and the translation initiation factors eIF4A and eIF4E, especially in response to UV radiation (Mazan-Mamczarz et al., 2006) and to TNFa (Gueydan et al., 1999). Additional ARE-binding proteins have been identified (e.g., HuR, Myer et al., 1997; TTP, Carballo et al., 1998; or KSRP, Gherzi et al., 2004), and it is well recognized that AREs in conjunction with their ARE-binding proteins can influence gene expression through the modulation of mRNA turnover and translation. However, despite the identification of a large number of ARE-bearing mRNAs and ARE-binding proteins, the full complexity of this regulatory mechanism is far from understood. 3.4.5. Multistep regulation of translation As is evident from some of the examples given above, translational regulation can be exerted as a multistep mechanism, which means that more than one mechanism is used to ensure tight translational control for critical proteins whose misexpression would be deleterious for the cell. One good example for this kind of control is the translational regulation of malespecific-lethal (msl-2) mRNA in Drosophila. Expression of MSL-2 in females causes inappropriate assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila (Kelley et al., 1995). MSL-2 expression is inhibited by Sex-lethal (SXL), a female-specific RBP that also regulates sex determination via alternative splicing (Forch and Valcarcel, 2003). First, SXL promotes retention of a facultative intron in the 50 UTR of msl-2 and then represses its translation (Bashaw and Baker, 1997; Gebauer et al., 1998; Kelley et al., 1997). SXL binds to sites in the 30 UTR and the intronic 50 UTR of msl-2 (Fig. 5.8) and represses translation in a dual way: SXL bound to the 30 UTR inhibits recruitment of the 43S preinitiation complex, and SXL bound to the 50 UTR can inhibit scanning of the 43S preinitiation complex if it escapes the first inhibitory mechanism (Beckmann et al., 2005). Furthermore, to exert its function via the 30 UTR, SXL requires the RBP UNR (upstream of N-ras) as a corepressor (Abaza et al., 2006; Duncan et al., 2006; Grskovic et al., 2003).

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Figure 5.8 Translational regulation of male-specific-lethal (msl-2) mRNA in Drosophila through a multistep mechanism. msl-2 translation is inhibited by Sex-lethal (SXL), a female-specific RNA-binding protein (RBP). First, SXL promotes retention of a facultative intron in the 50 UTR of msl-2 and represses its translation. SXL binds to sites in the 30 UTR and the intronic 50 UTR of msl-2 and represses translation in a dual way: binding to the 30 UTR inhibits recruitment of the 43S preinitiation complex, and binding to the 50 UTR can inhibit scanning of the 43S preinitiation complex. To exert its function via the 30 UTR, SXL requires the RNA-binding protein UNR (upstream of N-ras) as a corepressor. In male cells, SXL is not expressed and msl-2is translated. Reproduced with permission from Duncan et al. (2006).

4. Emerging Concepts in Translational Regulation In the past few years, two new ways to modulate mRNA fate at the posttranscriptional level have attracted much attention. One is the discovery of cytoplasmic processing bodies (P-bodies), initially described as foci within the cell with a high concentration of mRNA decay enzymes (Bashkirov et al., 1997; Cougot et al., 2004; Ingelfinger et al., 2002; Lykke-Andersen, 2002; Sheth and Parker, 2003; van Dijk et al., 2002). The other discovery is that of small RNAs, which can regulate stability and translation of target mRNAs (Bartel, 2004; Filipowicz, 2005; ValenciaSanchez et al., 2006). Interestingly, recent work suggests a connection between P-bodies and miRNA-mediated gene regulation (Liu et al., 2005a,b; Sen and Blau, 2005). These novel concepts will be introduced below, with a focus on their involvement in translational regulation. We will also describe recent examples for how the modulation of alternative transcripts can affect translation.

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4.1. P-bodies and translation P-bodies were first visualized by various groups using microscopy to localize factors involved in mRNA decay such as DCP1, DCP2, XRN1, and LSM (Bashkirov et al., 1997; Cougot et al., 2004; Ingelfinger et al., 2002; LykkeAndersen, 2002; Sheth and Parker, 2003; van Dijk et al., 2002). In mammalian cells, GW182 protein is another marker of P-bodies, which are therefore sometimes also referred to as GW bodies (Eystathioy et al., 2002, 2003). mRNA decay in eukaryotes can be controlled in different ways via endonucleolytic or exonucleolytic pathways (Parker and Song, 2004; Wilusz et al., 2001). Exonucleolytic degradation is usually initiated by deadenylation of poly(A) tails. Transcripts are then degraded from their 50 ends by the exonuclease XRN1, following removal of the 50 cap (decapping), which is the most common route for decay. Alternatively, the exosome complex can degrade transcripts from their 30 ends before decapping. P-bodies are probably a site of mRNA decay, as intermediates in the 50 -30 degradation pathway are localized to P-bodies (Sheth and Parker, 2003). Furthermore, mutations in the decapping enzymes (DCP1, DCP2) or in the 50 -30 exonuclease XRN1 increase the size and number of P-bodies, which leads to a clogging of the system (Sheth and Parker, 2003). Factors of the nonsense-mediated decay (NMD) pathway, which is responsible for the rapid degradation of mRNAs with a premature stop codon (Conti and Izaurralde, 2005), are also found in mammalian P-bodies (Unterholzner and Izaurralde, 2004). However, it is not clear whether P-bodies are the only site of 50 -30 decay, as enzymes involved in this process are also found elsewhere in the cytoplasm of yeast (Heyer et al., 1995) or mammalian cells (Bashkirov et al., 1997). It is also unclear whether mRNAs need to be deadenylated to enter P-bodies. In yeast, the deadenylase Ccr4p does not visibly localize to P-bodies (Sheth and Parker, 2003), but the mammalian homolog does (Cougot et al., 2004). In mammalian and yeast cells, depletion of Ccr4p results in a reduction of P-bodies (Andrei et al., 2005; Sheth and Parker, 2003), which supports the model that mRNAs need to be deadenylated before entering P-bodies. What are the connections between P-bodies and translation? Several lines of evidence indicate that mRNAs exist in two states: actively translated and associated with polysomes or translationally repressed and associated with P-bodies. When yeast cells are exposed to stress translation is inhibited at the level of initiation, which is reflected by a strong decrease in polysomes (Coller and Parker, 2005). While translation gets downregulated, P-bodies increase in size (Coller and Parker, 2005). After removal of the stress, P-bodies decrease in size and polysomes re-form, even in the absence of new transcription (Fig. 5.9; Brengues et al., 2005). Therefore, P-bodies in yeast seem to serve as sites of mRNA storage, which can be released back

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Figure 5.9 Movement of mRNAs between polysomes and P-bodies. Glucose starvation leads to inhibition of translation, which is evident from diminished polysomes (A, F). Translational inhibition also results in increased numbers and sizes of P-bodies, which are visualized using the green fluorescent protein (GFP)-tagged reporters Dcp2p (G) and Dhh1p (H), whose presence in P-bodies is dependent on mRNAs. After the readdition of glucose, polysomes reappear (K) and P-bodies largely disappear (L, M). These findings are consistent with a move of mRNAs from polysomes to P-bodies after the inhibition of translation, and reentering of mRNAs into the polysome pool after translation is restored. Reproducedwithpermission from Brengues etal.(2005).

into the translating pool without actually undergoing decay. The idea that the recruitment of mRNAs to P-bodies interferes with translation initiation and that only mRNAs not yet associated with ribosomes can be localized to P-bodies is strengthened by the finding that inhibition of translation elongation causes P-bodies to disappear, whereas inhibition of translation initiation increases the size and number of P-bodies (Andrei et al., 2005; Brengues et al., 2005; Cougot et al., 2004; Sheth and Parker, 2003; Teixeira et al., 2005). In budding yeast, the decapping activators Dhh1p and Pat1p are required for translational repression (Coller and Parker, 2005). In mammalian cells, several proteins with established roles in translational repression localize to P-bodies: RCK/p54, CPEB, and the eIF4E inhibitory protein eIF4E-T (Andrei et al., 2005; Chu and Rana, 2006; Ferraiuolo et al., 2005; Kedersha et al., 2005; Wilczynska et al., 2005). However, the exact mechanism of how mRNAs shuttle into P-bodies and become translationally repressed is not known.

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Another kind of cytoplasmic foci linked to translational repression can be observed in mammalian cells after exposure to stress: stress granules (SGs) contain translationally silent mRNAs, which are associated with preinitiation complexes lacking the ternary complex and which can shuttle back into polysomes after the removal of the stress (Kedersha and Anderson, 2002). Despite the analogy to P-bodies and some shared components, SGs are distinct subcellular entities as they also contain SG-specific components such as 40S ribosomal subunits, translation initiation factors, and AREbinding proteins (Kedersha et al., 2005). Despite these differences, fusion events and close associations between SG and P-bodies are evident (Kedersha et al., 2005; Wilczynska et al., 2005).

4.2. Regulation by small RNAs Two types of small RNA molecules have emerged as regulators of mRNA stability and translation in the last decade: miRNAs and short interfering RNAs (siRNAs). Current estimates from bioinformatic analyses suggest that the human genome encodes hundreds of different miRNAs and that they could regulate up to 30% of all genes (Lewis et al., 2005). However, only a few miRNAs and their targets have been validated to date. miRNAs and siRNAs are short RNAs of 21–26 nt and are distinguished based on their biogenesis (Jackson and Standart, 2007; Kim, 2005): miRNAs are derived from longer precursors that include a 70 nt imperfectly base-paired hairpin segment; siRNAs are of similar length but are derived from perfectly complementary RNA precursors. Despite the different modes of biogenesis, processing of both siRNAs and miRNAs is dependent on Dicer, and the regulatory function for both RNAs is exerted through proteins of the Argonaute (Ago) family: miRNAs and siRNAs associate with Ago proteins to form RNA-induced silencing complexes (RISCs), through which they modulate gene expression. During RNAi, exogenously introduced siRNAs target mRNAs for endonucleolytic cleavage (Tomari and Zamore, 2005), which has now also been described for miRNAs in plants (Allen et al., 2005; Llave et al., 2002) and mammals (Yekta et al., 2004). Initially, it was thought that perfect base pairing between the miRNA/siRNA and the target mRNA favors endonucleolytic cleavage, whereas imperfect base pairing results in target repression by alternative mechanisms. However, endonucleolytic cleavage can still occur even with mismatches between the miRNA and the target mRNA (Mallory et al., 2004; Yekta et al., 2004). In animal cells, most miRNAs are only partially complementary to their target mRNAs, and the downregulation of protein levels is usually greater than the downregulation of mRNA abundance, suggesting regulation at the level of translation ( Jackson and Standart, 2007). The classic example is lin-4 miRNA regulating lin-14 mRNA in Caenorhabditis elegans through

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interaction with its 30 UTR (Arasu et al., 1991; Wightman et al., 1991). This regulation does not involve changes in mRNA levels, but protein levels are dramatically altered. As lin-14 mRNA could be found associated with polysomes in both the active and the repressed state, it was suggested that translation of the mRNA is repressed at a point after initiation (Olsen and Ambros, 1999). A recent study using an artificial CXCR4 siRNA directed against a luciferase reporter with six bulged target sites in its 30 UTR reported a similar result as described for lin-14 repression (Petersen et al., 2006): luciferase expression is strongly downregulated without large changes in mRNA abundance, and repressed mRNAs are still associated with polysomes. Furthermore, repression is also seen for IRES-initiated translation, which further suggests a repressive mechanism that acts after translation initiation (Petersen et al., 2006). The authors suggest a ribosome drop-off at various points along the ORF resulting from miRNA repression (Petersen et al., 2006). It is hard to understand, however, how the polysomal distribution under repressed conditions with continuous ribosome drop-off would be similar to the distribution in an activated state ( Jackson and Standart, 2007). In contrast to the idea that miRNAs regulate mRNAs after translation initiation, two reports point toward initiation as the regulated step (Humphreys et al., 2005; Pillai et al., 2005). Using the same CXCR4 system, Humphreys et al. (2005) show a similar strong downregulation at the protein level of a luciferase reporter mRNA bearing four partially complementary binding sites for the CXCR4 siRNA. However, this downregulation is not seen with IRES-containing mRNAs. Furthermore, the downregulation is dependent on the 50 cap and 30 poly(A) sequences. Pillai et al. (2005) have also used luciferase reporters, with either one perfectly complementary or three imperfectly complementary target sites for let-7 miRNA. Expression of the reporter is downregulated, and reporter mRNA containing imperfect let-7 target sites is found in lighter polysomal fractions upon expression of let-7 miRNA. Furthermore, using in vitro synthesized mRNAs, it has been shown that the 50 cap is necessary for miRNA-mediated repression (Pillai et al., 2005). However, in contrast to the study by Humphreys et al. (2005), repression is not markedly relieved when the poly(A) tail is absent (Pillai et al., 2005). Taken together, the two latter studies strongly support miRNA-mediated repression at the level of translation initiation. What could be the reason for the discrepancies in miRNA-mediated translational repression reported by these various groups? First, in their study, Petersen et al. (2006) used a reporter mRNA that was transcribed in the nucleus by RNA polymerase II, whereas in the other two studies by Humphreys et al. (2005) and Pillai et al. (2005), the reporter mRNAs were cotransfected with the miRNA. Second, the number, origin, specificity, and location of target sites on the reporter might influence the observed effect.

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Furthermore, in a recent paper, Thermann and Hentze (2007) describe the formation of heavy miRNPs after repression by the miR2 miRNA in Drosophila. These miRNA–mRNA assemblies, which the authors call ‘‘pseudo-polysomes’’ show the same sedimentation characteristics as polysomes, but form even under conditions of effectively blocked 60S subunit joining (Thermann and Hentze, 2007). It is not clear what these pseudopolysomes are, but it is tempting to speculate that they represent smaller RNA–protein assemblies that combine to form particles similar to P-bodies. However, no such formation of pseudo-polysomes has been observed using a mouse cell-free translation system to study miRNA-mediated translational repression in vitro (Mathonnet et al., 2007), but this system further supports the case of translational repression by miRNAs at the level of initiation: repression of a luciferase reporter is not due to mRNA degradation but due to inhibition of translation. Furthermore, two other groups who use in vitro systems for the study of miRNA-mediated translational repression come to a similar conclusion: Wakiyama et al. (2007) apply a cell-free system with extracts from HEK297F cells, in which miRNA pathway components are overexpressed to recapitulate the let-7 miRNA-mediated translational repression. In their systems, both the cap and the poly(A) tail are required for translational repression, which again points toward initiation as the regulated step (Wakiyama et al., 2007). Additionally, let-7 miRNA mediates the deadenylation of the target mRNA, and the authors conclude that this deadanylation step is not a mere consequences of translational repression as it still happens when translation is repressed by cycloheximide. Wang et al. (2006) use a rabbit reticulocyte lysate in vitro translation system in conjunction with luciferase mRNA reporters that contain imperfect complementary binding sites to the CXCR4 siRNA to study miRNA-mediated translational repression. Apart from showing again that a cap and the poly(A) tail are required for translational repression via miRNAs, they also show that increasing poly(A) tail length alone on the reporters can increase miRNA silencing (Wang et al., 2006). All these studies build a strong case in favor of a scenario, in which miRNAs repress translation at the initiation step. A recent study also shows that human Ago2, one of the effector proteins of miRNA-mediated repression, possesses a cap-binding motif, which is involved in translational repression (Kiriakidou et al., 2007). However, it is also possible that miRNAs exert their repression on translation through different mechanisms, and that repression of translation initiation is only one aspect or an early effect by miRNA-mediated repression of gene expression. As a consequence, it will be important and necessary to validate the regulatory mechanism for each miRNA-target pair individually. Furthermore, translation could also be indirectly influenced by miRNAs, for example, by acting on the adenylation status of the 30 end of mRNAs (Giraldez et al., 2006; Wakiyama et al., 2007; Wang et al., 2006; Wu et al., 2006).

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A further addition to the ever-growing number of mechanisms by which miRNAs can affect gene expression comes from the surprising finding that members of the miRNA pathway and miRNAs themselves can also function in the upregulation of translation. Under serum starvation and thus cell cycle arrest, TNFa becomes translationally upregulated, which is dependent on AREs in the mRNA (Section 3.4.4). In order to identify the ARE-binding proteins, Vasudevan et al. (2007) used a biochemical approach and found that miRNA-related proteins, fragile-X-mentalretardation-related protein 1 (FXR1) and Ago2, were both associated with the AREs under serum starvation. The authors could further demonstrate that FXR1 and Ago2 are both directly involved in the translational upregulation of TNFa mRNA (Vasudevan and Steitz, 2007). Furthermore, the same authors studied if actual miRNAs are involved in this process and they could show that miRNA 369-3 directs the association of FXR1 and Ago2 with the AREs of TNFa (Vasudevan et al., 2007). Furthermore, they also show that other miRNAs (let-7 and CXCR4) have the same stimulating effect on the translation of target transcripts upon cell cycle arrest. The authors suggest that miRNA function oscillates during the cell cycle: they repress translation of targets in proliferating cells, whereas they can mediate translation activation in a state of cell cycle arrest (Vasudevan et al., 2007). They further speculate that such a switch between repressive and activating function could be the cause for the sometimes contradictory results documented for miRNA function in different experimental systems. To date, no definitive mechanism for miRNA-dependent gene regulation has been established, which is not surprising given the recent emergence of this field. Furthermore, it seems unlikely that there is one unifying mechanisms that will explain miRNA function. Instead, it seems more likely that miRNA-mediated regulation is involved in several aspects of gene expression through a variety of diverse mechanisms, many of which remain to be identified. However, as publications in this area keep pouring in, we should soon obtain a better picture of the full extent of gene regulation by miRNAs.

4.3. Interplay between miRNAs and P-bodies Several recent reports have found connections between gene regulation via miRNAs/siRNAs and P-bodies. Pillai et al. (2005) show that mRNAs that are translationally repressed by let-7 miRNA localize to P-bodies or to cytoplasmic foci adjacent to P-bodies. Argonaute proteins, the effector of miRNA-mediated regulation, also localize to P-bodies (Liu et al., 2005b; Sen and Blau, 2005); these proteins interact with GW182, a key P-body subunit in mammalian cells, and depletion of GW182 impairs the repression of miRNA reporters ( Jakymiw et al., 2005; Liu et al., 2005a).

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A recent report shows the reversibility of miRNA-mediated repression and the involvement of P-bodies: Bhattacharyya et al. (2006) used the cationic amino acid transporter (CAT-1) mRNA or reporter mRNAs bearing the CAT-1 30 UTR, which is negatively regulated by the miR-122 miRNA. In Huh7 cells, miR-122 is endogenously expressed, CAT-1 protein levels are significantly downregulated, and both CAT-1 and miR-122 are present in P-bodies. However, after exposure to stress, CAT-1 mRNA can escape the translational repression, and this derepression and exit from P-bodies is dependent on ARE elements in its 30 UTR. Bhattacharyya et al. (2006) could further show that the ARE-binding protein HuR is necessary for the release from translational repression and P-body entrapment. The above examples strongly suggest that P-body components are important for gene regulation via miRNA/siRNA-mediated repression. However, the P-body environment or P-body components important for this interaction remain to be determined. Recent work suggests that disruption of P-bodies does not necessarily affect siRNA-mediated regulation (Chu and Rana, 2006). Therefore, concentration of miRNAs and their targets in P-bodies could be a consequence rather than a prerequisite of miRNA/siRNA-mediated gene regulation. Taken together, regulation of gene expression via small RNAs and sequestration to P-bodies, and the interplay between mRNA translation and decay adds further complexity to posttranscriptional control. As 30% of human genes are potential miRNA targets (Lewis et al., 2005), it is entirely possible that miRNAs exert their functions in a combinatorial way: a given mRNA could be regulated by several miRNAs, and a given miRNA could target several mRNAs. Clearly, further research will be needed to elucidate the molecular events behind these regulatory mechanisms.

4.4. Translational regulation through alternative transcripts As pointed out above, translational control is by no means independent of other layers of gene regulation, and virtually every step upstream of translation can influence the translational efficiency of a given mRNA (Fig. 5.1). Here, we will provide examples of recent work that describes how changes in transcript structure can ultimately effect translation. In the fission yeast Schizosaccharomyces pombe, the trancription factor Sre1p, an ortholog of the mammalian sterol regulatory element binding protein (SREBP), is essential for anaerobic growth and activates transcription under low-oxygen conditions. However, the general transcriptional activation via Sre1p does not necessarily include an upregulation of protein levels: tco1, a gene potentially involved in oxygen-regulated lipid transport and a target of Sre1p, is downregulated at the level of translation under lowoxygen conditions (Sehgal et al., 2008). This downregulation is paradoxically mediated by an upregulation of transcription: under low-oxygen

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conditions, Sre1p directs transcription of tco1 from an alternative promoter, resulting in a transcript with an extended 50 UTR compared to the transcript under normal oxygen levels. This longer transcript forms a stable structure in its 50 UTR, explaining the downregulation at the level of translation (Sehgal et al., 2008). Another study shows that changes in the 50 transcript structures can also induce translation: Law et al. (2005) examined a population of mRNAs that are only weakly translated in rapidly growing budding yeast cells. These weakly (or ‘‘undertranslated’’) mRNAs were identified based on data from genomic studies, which combine sucrose-gradient centrifugation with global measurements of transcripts using microarray technology (Section 5.1). Gene Ontology categories such as responses to stress and external stimuli were enriched within the undertranslated transcripts, and 17 transcripts chosen for detailed study showed indeed an increase in translation in response to the corresponding stimulus such as nitrogen starvation, pheromone response, or osmotic stress (Law et al., 2005). Interestingly, the majority of these transcripts also showed an altered 50 structure in response to the stimulus, which again illustrates the interconnectivity between regulation at the level of transcription and translation. The authors speculate that the altered transcript structure arises through the use of alternative promotors and that this mechanism of translational control allows low-level transcription and maintenance of open chromatin structures while avoiding protein production of the corresponding gene (Law et al., 2005).

5. Global Approaches to Identify Targets of Posttranscriptional Gene Regulation The advent of microarray technologies allowed genome-wide studies of gene expression at the level of steady-state mRNA abundance. Furthermore, microarrays combined with chromatin immunopreciptitations provided an invaluable tool to identify transcription factor binding sites and chromatin modifications on a global scale. Together, these approaches revealed global networks of transcriptional control in a variety of organisms and physiological conditions (Babu et al., 2004; Barrera and Ren, 2006; Luscombe et al., 2004; Walhout, 2006). As gene expression is often regulated at posttranscriptional levels, it is important to also gain an understanding of these regulatory processes and their targets on a genome-wide scale. In the same way, that DNA and its interactions with transcription factors and chromatin modifiers is integral to transcriptional regulation, mRNA and its association with RBPs is crucial for posttranscriptional gene regulation. Consequently, recent work of many groups has focussed on large-scale analyses of mRNA–protein interactions

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and mRNA dynamics. Many of these studies employ microarray-based approaches to unravel a variety of processes such as (1) the global association of mRNAs with specific RBPs, (2) mRNA stability, and (3) the association of mRNAs with ribosomes and thus the efficiency with which these mRNAs are translated. These large-scale approaches are especially useful to identify potential targets for each of the myriads of possible posttranscriptional regulatory mechanisms, and building on this knowledge can in turn be useful to examine the underlying molecular mechanisms of the regulatory processes. Here, some of these techniques and resulting findings will be introduced.

5.1. Translational profiling Translational efficiency can be measured on a genome-wide scale by assessing the number of ribosomes that are bound to a given mRNA. This is achieved by combining the traditional method of polysome profiling with microarray technology, referred to as translational profiling (Fig. 5.10):

Sucrose gradient

1) Resolve ribosome-bound mRNAs by density gradient centrifugation

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80 S 60 S 40 S

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Figure 5.10 Genome-wide measurement of translation is achieved by combining polysome profiling with microarray technology, referred to as translational profiling. mRNAs are resolved on a sucrose gradient by ultracentrifugation according to their density, which reflects the number of associated ribosomes.The gradient is fractionated and a polysome profile is obtained by measuring RNA abundance. From the light to the heavy fractions: free mRNAs, ribosomal 40S and 60S subunits, the monosome or 80S subunit, and the polysome fractions corresponding to mRNAs with increasing numbers of bound ribosomes. mRNAs from the different fractions are then extracted and quantified using microarrays.

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Usually, cells are treated with the elongation-inhibitor cycloheximide, which ‘‘traps’’ ribosomes on the mRNAs they are translating. Cellular lysates are then resolved according to their density on a sucrose gradient by ultracentrifugation. As the ribosome is a large macromolecular complex with a molecular mass above three megadalton (Taylor et al., 2007), the density of the mRNA–ribosome particles is determined by the number of ribosomes bound to mRNAs. The sucrose gradient is then fractionated and a polysome profile is obtained by measuring RNA abundance (Fig. 5.10; right panel). mRNAs from the different fractions can then be extracted and globally quantified using microarrays. In most studies that applied this approach to study translational regulation, the pool of mRNAs associated with polysomes was compared to the pool of untranslated (or poorly translated) mRNAs or total mRNA preparations to define translationally regulated transcripts (Bachand et al., 2006; Bushell et al., 2006; Dinkova et al., 2005; Iguchi et al., 2006; Johannes et al., 1999; Kash et al., 2002; Kuhn et al., 2001; Qin and Sarnow, 2004; Rajasekhar et al., 2003; Spence et al., 2006; Thomas and Johannes, 2007). Other studies used more than 10 fractions spaced along the polysome profile, which were all probed to microarrays to obtain higher-resolution data of ribosome association for mRNAs (Arava et al., 2003; Lackner et al., 2007; MacKay et al., 2004; Preiss et al., 2003; Qin et al., 2007). Using translational profiling, the effect on global and mRNA-specific translational regulation was examined in a variety of conditions. Examples are the exposure of cells to environmental stress such as hypoxia, treatment with rapamycin, heat shock, or change in carbon source (Grolleau et al., 2002; Kuhn et al., 2001; Preiss et al., 2003; Thomas and Johannes, 2007), the translational regulation during the mitotic cell cycle, meiosis, or during recovery from cell cycle arrest (Iguchi et al., 2006; Qin and Sarnow, 2004; Serikawa et al., 2003), the dependence of mRNAs on specific translation initiation factors (Dinkova et al., 2005; Johannes et al., 1999), and translational regulation in response to oncogenic signaling or in transformed cells (Rajasekhar et al., 2003; Spence et al., 2006). One of the first studies using translational profiling was conducted by Johannes et al. (1999): they examined the requirement for cap-dependent translation initiation by analyzing the association of mRNAs with polysomes in poliovirus-infected cells with reduced eIF4G concentrations. Most of the examined mRNAs showed the expected downregulation in translation, whereas a small percentage remained associated with polysomes or even showed increased polysome association. These mRNAs are probably translated via IRES-mediated translation initiation, and include mRNAs encoding immediate-early transcription factors and mitogen-acitvated regulators ( Johannes et al., 1999). Another study conducted in C. elegans investigated the effect of the selective knockout of one isoform of the cap-binding translation initiation factor eIF4E (Dinkova et al., 2005).

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Mutant worms show a mixture of phenotypic effects, reproduce more slowly, and exhibit egg laying defects. Using translational profiling, several mRNAs could be identified that showed changes in their polysomal association without corresponding alteration in total mRNA levels. Interestingly, these mRNAs were enriched for genes with functions related to egg laying, providing a possible explanation for the observed phenotype (Dinkova et al., 2005). Kuhn et al. (2001) measured the translational response in budding yeast cells to the transfer from a fermentable (glucose) to a nonfermentable (glycerol) carbon source. This shift resulted in a global downregulation of translation. mRNAs encoding ribosomal proteins were strongly downregulated in terms of total mRNA abundance as well as in their translational status, indicated by a diminished association with polysomal fractions. However, a few mRNAs showed increased association with polysomes, and most of these mRNAs also showed increased abundances in their total mRNA levels. A similar connection between changes in total mRNA levels and polysome association was described in a study that examined translational regulation in response to treatment with rapamycin and heat shock (Preiss et al., 2003). mRNAs that showed increased abundance in response to the treatment often showed increased translational efficiency, and a similar correlation was evident for mRNAs with decreased abundance. A similar relationship between changes in total mRNA levels and translational efficiency was observed in response to treatment with mating pheromone in budding yeast (MacKay et al., 2004). This coordination between changes in transcript levels and translation was termed ‘‘potentiation’’ (Preiss et al., 2003). Further studies will be required to determine whether potentiation happens through coordinated yet independent regulation of transcription and translation or whether it is a consequence of favored translation of mRNAs from de novo transcription over aged transcripts. For example, de novo transcription could influence mRNP composition or could simply provide ‘‘intact’’ messages with long poly(A) tails, which are then more efficiently translated (Lackner et al., 2007; Beilharz and Preiss, 2007). Translational profiling was recently applied to study translational changes in response to hypoxia (Thomas and Johannes, 2007). When PC-3 cells were grown under hypoxic conditions, translation was globally downregulated, concomitant with mammalian target of rapamycin (mTOR) inactivation and phosphorylation of eIF2a (Section 3.4.1), and mRNAs encoding ribosomal proteins were found to be most sensitive to the global translational downregulation. Again, several mRNAs were identified that escaped the translational downregulation and still were associated with polysomal fractions under hypoxic conditions (Thomas and Johannes, 2007). The authors suggested that translational regulation of these mRNAs might be initiated via cap-independent mechanisms. This is another example of how certain mRNAs can be selectively translated in response to a specific

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stimulus, while most other cellular mRNAs are translationally downregulated in this condition. These sets of mRNAs could only be identified using genome-wide, unbiased approaches, as their involvement in certain biological processes is unexpected and could not have been anticipated by more hypothesis-driven approaches. Arava et al. (2003) and Lackner et al. (2007) provide comprehensive views of translational efficiency in rapidly growing budding and fission yeast cells, respectively. mRNA extracted from 12–14 fractions across the polysomal profile were analyzed on microarrays, and the translation profiles were used to determine the average number of ribosomes associated with a given mRNA on a genome-wide scale. This approach revealed several interesting findings. For most mRNAs, 70–80% of the transcripts were associated with polysomal fractions. Among the few mRNAs not associated with polysomal fractions, several were known to be translationally regulated. Furthermore, ribosomes were spaced well below the maximum packing capacity on most mRNAs, which corroborates the fact that translation initiation is the rate-limiting step in translation. The density of associated ribosomes varied strongly between transcripts and showed an inverse correlation to the length of the transcript. Moreover, integration of high-resolution translational profiling data with other global data sets revealed that translational efficiency is aligned with mRNA half-lives, transcriptional efficiency, mRNA stability, and poly(A) tail lengths in both budding and fission yeasts (Beilharz and Preiss, 2007; Lackner et al., 2007), highlighting a substantial coordination between different layers of gene regulation. Qin et al. (2007) used a high-resolution translational profiling approach to study the extent of translational control during early Drosophila embryogenesis. Accordingly, mRNAs that were known to be spatially repressed by translational mechanisms in the early fly embryo had only a small portion of their transcripts associated with polysomal fractions.

5.2. Proteomic approaches to study translational regulation Currently, translational profiling is the method of choice to examine translational regulation on a genome-wide scale. Microarray technology has become robust, reliable and also affordable, and combined with proper and careful analysis, translational profiling is a powerful tool to screen for translationally regulated mRNAs. However, recent advances in proteomic approaches will also be useful to study translational regulation. Two studies combined the measurement of absolute protein levels using proteomics and total mRNA levels using microarrays (Lu et al., 2007; Newman et al., 2006). Newman et al. (2006) exploited a collection of yeast strains, where each protein is fused to green fluorescent protein (GFP) under the control of its own promoter; using a flow cytometry approach, GFP abundance was

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measured for each strain and mRNA levels were measured using microarrays. Lu et al. (2007) used a mass spectrometry approach together with a novel algorithm to make absolute measurements of protein levels. Both studies concluded that changes in protein levels were largely due to changes in the abundance of the corresponding mRNAs, but certain mRNAs were identified for which changes in protein level could not be attributed solely to a change in mRNA level. These mRNAs are prime candidates for regulation at the translational level or at the level of protein stability. There are disadvantages to these proteomic approaches: in the case of the GFP-tagged strain collection, the tag could interfere with translational regulation via sequence elements in the UTR or with protein targeting and turnover, and mass spectrometry approaches do not yet manage to identify every expressed protein in the cell and are biased toward abundant proteins. However, as these techniques improve, they will become increasingly important for the genome-wide study of translational control.

5.3. mRNA turnover mRNA turnover is regulated by multiple mechanisms (Parker and Song, 2004; Wilusz et al., 2001). Deadenylation of transcripts is a key step in these regulatory mechanisms, and mRNAs are then decapped and degraded via the XRN1 exonuclease or, alternatively, mRNAs can be degraded without decapping by the exosome complex. In certain cases, mRNAs are degraded via endonucleolytic mechanisms, for example, via the RNAi machinery (Tomari and Zamore, 2005). Furthermore, NMD serves as a quality control mechanism to degrade faulty mRNAs with a premature stop codon. These mRNAs are decapped and directly degraded without prior deadenylation (Fasken and Corbett, 2005). mRNAs that are lacking proper stop codons are degraded without decapping by the exosome in a process called nonstop decay (Vasudevan et al., 2002). Global mRNA stability is often measured by blocking transcription with drugs or by using mutants of RNA polymerase II. At different times after the transcription block, mRNAs are isolated and probed on microarrays (Fig. 5.11; Mata et al., 2005). Using this approach, genome-wide mRNA stability has been determined in various organisms such as yeast (Grigull et al., 2004; Wang et al., 2002), plants (Gutierrez et al., 2002), and human cell lines (Raghavan et al., 2002; Yang et al., 2003). The picture emerging from these studies is that mRNA decay is a controlled process and that decay rates vary substantially between different transcripts. mRNA decay rates often also correlate among mRNAs that encode functionally related proteins or proteins of the same macromolecular complex (Grigull et al., 2004; Wang et al., 2002). mRNAs encoding transcription factors,

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Block of transcription

Extraction of mRNA at different times after transcriptional inhibition t1

t2

t3

etc...

cDNA labeling and hybridization

Figure 5.11 Genome-wide measurements of mRNA half-lives. Transcription is blocked using drugs or mutants of RNA polymerase II. At different times after the transcriptional block, mRNAs are isolated and quantified using DNA microarrays to deduce mRNA half-lives. Reproduced with permission from Mata et al. (2005).

parts of the transcriptional machinery, proteins involved in ribosome biogenesis and the translation machinery generally show short half-lives, whereas mRNAs encoding central metabolism proteins show longer halflives (Grigull et al., 2004; McCarroll et al., 2004; Wang et al., 2002; Yang et al., 2003). Short half-lives for mRNAs involved in transcription or translation could be advantageous for rapid regulation of these central processes in response to changing environmental conditions. Note, however, that the transcriptional shutdown itself, and the use of drugs or RNA polymerase II mutants in these experiments could trigger cellular stress responses (Grigull et al., 2004). Thus, the short half-lives of mRNAs involved in transcription and translation could reflect a response to stress, and half-lives for the same mRNAs may actually be longer in unstressed cells at steady state.

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For many mRNAs, short half-lives correlate with the presence of ARE elements in their 30 UTRs, but not all rapidly decaying mRNAs have ARE elements (Raghavan et al., 2002; Yang et al., 2003). No obvious correlation between mRNA stability and mRNA features such as ORF length, transcriptional efficiency, or mRNA abundance seems to exist (Wang et al., 2002), but mRNA half-lives are globally aligned with poly(A) tail lengths and translational efficiency in fission yeasts (Lackner et al., 2007). In a recent study, Shock et al. (2007) determined the global decay rates of mRNAs at various stages during the intraerythrocytic development cycle of Plasmodium falciparum, the pathogen causing human malaria. Interestingly, as the parasite passes through the examined developmental stages, decay rates decrease globally for essentially all examined mRNAs, which suggests that posttranscriptional control is the main mechanism of gene regulation in this pathogen. Such genome-wide regulation of mRNA turnover, however, has not been described for any other organism. Insights into the global regulation of mRNA decay also comes from measuring total mRNA levels in cells deleted for factors involved in mRNA degradation. An example is the measurement of global effects in yeast or mammalian cells compromised for NMD function (He et al., 2003; Mendell et al., 2004). In addition to its known involvement in mRNA quality control, a new aspect of this pathway was detected in these global studies: hundreds of mRNAs accumulated as a consequence of NMD inactivation, and they were enriched for mRNAs with specific functions. In mammalian cells, many of the enriched mRNAs are involved in amino acid metabolism (Mendell et al., 2004). As NMD requires translation, which is inhibited by amino acid depletion, the authors suggest that the abundance of these transcripts is regulated by NMD to couple their mRNA levels to amino acid availability; inhibition of translation and NMD could increase the abundance of these transcripts to turn on amino acid biosynthesis (Mendell et al., 2004). Thus, these genome-wide studies reveal that NMD not only functions in ensuring quality control of mRNAs but also acts as a more general regulator of gene expression. In another recent genome-wide approach, Hollien and Weissman (2006) showed that the inositol-requiring enzyme 1 (IRE-1), which functions in activating the UPR due to accumulation of misfolded proteins in the ER, is involved in the specific and immediate degradation of a subset of mRNAs during the UPR. IRE-1 plays a role in the detection of unfolded proteins in the ER and subsequently activates a transcription factor, X-boxbinding protein 1 (XBP-1), through endonucleolytic cleavage of its mRNA. In this study, IRE-1 or XBP-1 were depleted by RNAi in Drosophila S2 cells in which the UPR has been induced. Global mRNA levels from these cells were then measured using DNA microarrays. A subset of mRNAs was identified, whose repression was dependent on IRE-1 but not on XBP-1, and IRE-1 mediates the degradation of these mRNAs (Hollien and Weissman, 2006).

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5.4. RNA-binding proteins and their target RNAs Central to virtually all aspects of posttranscriptional gene regulation, from mRNA processing and export to mRNA decay and translation, is the interplay between mRNAs and RBPs. Some RBPs bind most of the transcripts in the cell, whereas others bind only a small set of specific mRNAs, exerting a specialized control to those mRNAs (Hieronymus and Silver, 2004; Keene, 2007; Mata et al., 2005; Moore, 2005). Furthermore, RBPs may act in a combinatorial way, as each mRNA can be bound by several RBPs. In budding yeast, there are about 600 proteins estimated to have RNA-binding capacity, and this number is probably even higher in mammalian cells (Maris et al., 2005; Moore, 2005). Much insight into gene regulation via RBPs has come from the genome-wide identification of their targets via ‘‘RBP Immunoprecipitation followed by chip analysis’’ (RIP-chip, Fig. 5.12): RBPs are immunopurified together with their associated RNAs, via an epitope-tag or via an antibody against the RBP of interest; the RNAs are then isolated from the precipitate, purified, labeled, and hybridized onto microarrays. In one of the first studies to employ this technology, Tenenbaum et al. (2000) used cDNAfilter arrays containing 600 murine genes to identify mRNAs associated with the RBPs HuB, PABP, and eIF4E, which are all involved in the regulation of translation. Even though only a few mRNAs were analyzed, each RBP bound a different subset of mRNAs, with PABP being associated with many mRNAs and HuB associated with only few mRNAs. Furthermore, the pattern of association of mRNAs with HuB was altered after cells were induced to differentiate by treatment with retinoic acid. One of the most comprehensive studies using RIP-chip was conducted by Gerber et al. (2004) who identified targets for five members of the Pumilio family of RBPs in budding yeast (Puf1p-Puf5p). Dozens to hundreds of mRNAs were associated with each of the five Puf proteins, and the subsets of mRNAs bound to each of RBP were enriched for common functional groups or subcellular localizations. Puf1p and Puf2p associated with mRNAs encoding membrane-associated proteins; Puf3p nearly exclusively bound mRNAs that encode mitochondrial proteins; Puf4p associated with nucleolar ribosomal RNA-processing factors; and Puf5p associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. Furthermore, distinct sequence motifs were enriched in the 30 UTR of mRNAs bound by Puf3p, Puf4p, and Puf5p (Gerber et al., 2004). A related motif was identified in mRNAs that coimmunoprecipitate with the Drosophila Pumilio protein (Gerber et al., 2006). Many of the mRNAs associated with Pumilio in Drosophila also encode functionally related proteins, but these mRNAs are not related to the mRNAs associated with Puf3p in budding yeast (Gerber et al., 2006). RIP-chip approaches were also used to identify global targets of RBPs involved at other levels of posttranscriptional gene regulation such as

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Epitope RBP

RB

P

Extract of RBP-mRNA complexes

Binding to beads or antibody

RBP

Sepharose beads

Isolation and elution from beads RBP

Purification of isolated RNA

Hybridization of labelled cDNA

Figure 5.12 Genome-wide determination of mRNA targets of RNA-binding proteins (RBPs) by ‘‘RBP Immunoprecipitation followed by chip analysis’’ (RIP-chip). RBPs are immunopurified together with their associated mRNAs, followed by mRNA isolation, labeling, and hybridization onto microarrays. Reproduced with permission from Mata et al. (2005).

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splicing (Gama-Carvalho et al., 2006), nuclear mRNA export (Hieronymus and Silver, 2003; Kim Guisbert et al., 2005), mRNA decay (Duttagupta et al., 2005), and poly(A) tail length control (Beilharz and Preiss, 2007). Common to these studies is the finding that RBPs involved in a common process often share mRNA targets, but on top of that, each RBP seems to have unique targets, whereas mRNAs targeted by a certain group of RBPs often share functional specificity. Furthermore, RIP-chip studies also provided clues to unexpected functions of RBPs. An example is the identification of previously unknown mRNAs associated with the yeast La protein (Lhp1p). Lhp1p is involved in the biogenesis of noncoding RNAs transcribed by RNA polymerase III, and thus many noncoding mRNAs were identified as targets of this RBP (Inada and Guthrie, 2004). However, Lhp1p was also found to bind a subset of coding mRNAs, such as HAC1, which encodes a transcription factor required for the UPR. Follow-up experiments indicate that Lhp1p plays a role in the translational regulation of HAC1 mRNA (Inada and Guthrie, 2004, 387). Recently, RIP-chip approaches were also employed to measure translation on a global scale. In this case, the RBP is an epitope-tagged ribosomal subunit, and polyribosomal complexes corresponding to ribosome-bound mRNAs are immunopurified. The feasibility of these approaches was first shown in budding yeast (Inada et al., 2002). The ribosomal protein Rpl25p was epitope-tagged, and immunopurification via the epitope tag yielded intact polysomal fractions. Zanetti et al. (2005) used a similar approach with epitope-tagged ribosomal protein rpL18 to isolate polyribosomes in Arabidopsis. The authors also probed the mRNA from these immunopurified complexes with microarrays and compared the data to total cellular mRNA samples. Their data show that for most genes the mRNAs are associated with polysomal complexes with an average association of 62%, which is slightly below the number of ribosome association determined for yeast mRNAs by translational profiling (Arava et al., 2003). This technology could become a powerful complementary tool to study translational regulation in varying conditions or different cellular subtypes, and also to identify substrates of potential ribosomal subtypes containing different protein isoforms (Section 5.1).

6. Concluding Remarks Translation is a complex process mediated by large ribonucleoprotein machines, the ribosomes. Maintaining maximal translational output is a major effort and energetically very costly. Cells therefore globally tune the

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translation of transcripts to the physiological requirements dictated by environmental or intrinsic conditions. Besides this global translational tuning, cells also use a great variety of transcript-specific mechanisms to adjust the production of selected proteins to current needs. The elegant mechanistic studies reviewed here provide deep insights into several sophisticated processes of translational regulation, while the powerful genome-wide analyses provide overviews of the targets and global strategies for translational control, thus complementing more traditional studies. Although much has been learnt about translational control, this level of gene regulation is still relatively poorly understood compared to transcriptional regulation. More work is required to obtain a full picture on the extent and role of translational regulation in different organisms and in different conditions. Recent data on translational and other posttranscriptional regulation via small RNAs further add to the complex picture of gene expression control. The great abundance and diversity of noncoding RNAs emerging from current studies raises the possibility that more of these RNAs play important roles in translational regulation. Proteins are the readout of translational control, and future progress in proteomic approaches should further advance our understanding of translational and posttranslational regulation. Cells integrate multiple regulatory levels to fine-tune gene expression, and it is not well understood how the different processes of translational control are coordinated with each other and with additional levels of gene regulation. An ultimate goal is to obtain a systems-level understanding of multilevel gene expression programs to help predict the contribution of translational regulation for different genes and for different biological processes. It seems certain that scientists working in this fascinating field will not become bored any time soon.

ACKNOWLEDGMENTS We thank Franc¸ois Bachand, Traude Beilharz, Thomas Preiss, and Katsura Asano for comments on the manuscript. Work in our laboratory was funded by Cancer Research UK [CUK] Grant No. C9546/A6517.

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Wang, W., Martindale, J. L., Yang, X., Chrest, F. J., and Gorospe, M. (2005). Increased stability of the p16 mRNA with replicative senescence. EMBO Rep. 6, 158–164. Wang, Y., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., and Brown, P. O. (2002). Precision and functional specificity in mRNA decay. Proc. Natl. Acad. Sci. USA 99, 5860–5865. Warner, J. R. (1999). The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24, 437–440. Wells, S. E., Hillner, P. E., Vale, R. D., and Sachs, A. B. (1998). Circularization of mRNA by eukaryotic translation initiation factors. Mol. Cell 2, 135–140. Wightman, B., Burglin, T. R., Gatto, J., Arasu, P., and Ruvkun, G. (1991). Negative regulatory sequences in the lin-14 30 -untranslated region are necessary to generate a temporal switch during Caenorhabditis elegans development. Genes Dev. 5, 1813–1824. Wilczynska, A., Aigueperse, C., Kress, M., Dautry, F., and Weil, D. (2005). The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules. J. Cell Sci. 118, 981–992. Wilhelm, J. E., Hilton, M., Amos, Q., and Henzel, W. J. (2003). Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz. J. Cell Biol. 163, 1197–1204. Wilson, G. M., Sun, Y., Sellers, J., Lu, H., Penkar, N., Dillard, G., and Brewer, G. (1999). Regulation of AUF1 expression via conserved alternatively spliced elements in the 30 untranslated region. Mol. Cell. Biol. 19, 4056–4064. Wilusz, C. J., Wormington, M., and Peltz, S. W. (2001). The cap-to-tail guide to mRNA turnover. Nat. Rev. Mol. Cell Biol. 2, 237–246. Wolin, S. L., and Walter, P. (1988). Ribosome pausing and stacking during translation of a eukaryotic mRNA. EMBO J. 7, 3559–3569. Wu, L., Fan, J., and Belasco, J. G. (2006). MicroRNAs direct rapid deadenylation of mRNA. Proc. Natl. Acad. Sci. USA 103, 4034–4039. Xirodimas, D. P., Sundqvist, A., Nakamura, A., Shen, L., Botting, C., and Hay, R. T. (2008). Ribosomal proteins are targets for the NEDD8 pathway. EMBO Rep 9, 280–286. Yang, E., van Nimwegen, E., Zavolan, M., Rajewsky, N., Schroeder, M., Magnasco, M., and Darnell, J. E., Jr. (2003). Decay rates of human mRNAs: Correlation with functional characteristics and sequence attributes. Genome Res. 13, 1863–1872. Yekta, S., Shih, I. H., and Bartel, D. P. (2004). MicroRNA-directed cleavage of HOXB8 mRNA. Science 304, 594–596. Zanetti, M. E., Chang, I. F., Gong, F., Galbraith, D. W., and Bailey-Serres, J. (2005). Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiol. 138, 624–635. Zhao, J., Hyman, L., and Moore, C. (1999). Formation of mRNA 30 ends in eukaryotes: Mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63, 405–445.

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C H A P T E R

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Phagocytosis and Host–Pathogen Interactions in Dictyostelium with a Look at Macrophages Salvatore Bozzaro,* Cecilia Bucci,† and Michael Steinert‡ Contents 1. Introduction 2. The Dynamics of Phagocytosis 3. Cellular Mechanisms of Phagocytosis 3.1. Bacterial adhesion to the cell surface: The search for phagocytosis receptors 3.2. The actin cytoskeleton in phagocytosis 3.3. Phagosome fusion with endolysosomal vesicles and killing of bacteria: The other players 4. Regulatory Pathways Controlling Phagocytosis 4.1. Heterotrimeric G protein in phagocytosis 4.2. Phosphoinositides and calcium ions 4.3. Small G proteins of the Ras and Rac families and tyrosine kinases 4.4. The Rab family in intracellular phagosome maturation 5. Host–Pathogen Interactions: A Versatile New Model Host 5.1. Resistance/susceptibility genes of the host to infection by microbes 5.2. The Nramp family in Dictyostelium and Nramp1 as host defence factor 6. Concluding Remarks Acknowledgments References

* {

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Department of Clinical and Biological Sciences, University of Turin, Ospedale S. Luigi, 10043 Orbassano, Italy Department of Biological and Environmental Sciences and Technologies, University of Lecce, via Prov. Monteroni, 73100 Lecce, Italy Institute for Microbiology, Technical University of Braunschweig, Spielmannstr. 7, D-38106, Germany

International Review of Cell and Molecular Biology, Volume 271 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01206-9

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2008 Elsevier Inc. All rights reserved.

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Abstract Research into phagocytosis and host–pathogen interactions in the lower eukaryote Dictyostelium discoideum has flourished in recent years. This chapter presents a glimpse of where this research stands, with emphasis on the cell biology of the phagocytic process and on the wealth of molecular genetic data that have been gathered. The basic mechanistic machinery and most of the underlying genes appear to be evolutionarily conserved, reflecting the fact that phagocytosis arose as an efficient way to ingest food in single protozoan cells devoid of a rigid cell wall. In spite of some differences, the signal transduction pathways regulating phagosome biogenesis are also emerging as ultimately similar between Dictyostelium and macrophages. Both cell types are hosts for many pathogenic invasive bacteria, which exploit phagocytosis to grow intracellularly. We present an overwiew, based on the analysis of mutants, on how Dictyostelium contributes as a genetic model system to decipher the complexity of host–pathogen interactions. Key Words: Dictyostelium, Legionella, Mycobacteria, Klebsiella, macrophages, phagocytosis, host–pathogen interactions, cytoskeleton, signal transduction, phosphoinositides, G proteins, Nramp. ß 2008 Elsevier Inc.

1. Introduction Phagocytosis is a process initiated by binding of the particle to the cell surface, its progressive surrounding by the plasma membrane and ingestion of the newly produced vesicle, called the phagosome. The phagosome undergoes maturation by fusing with vesicles of the endocytic pathway and by gradually acquiring properties typical of the lysosome. In metazoa, with a developed immune system, phagocytosis is a feature of specialized, ‘‘professional phagocytes’’ (macrophages, neutrophils, and dendritic cells), which are capable of ingesting and killing a large variety of microorganisms (Haas, 2007; Stuart and Ezekowitz, 2005). In addition to this protective function against microbes, phagocytosis plays a crucial role in noninflamatory depletion of apoptotic cells, thus in tissue remodeling and development. A few pathogenic microbes exploit the phagocytic pathway to invade the cells and, by escaping digestion, cause different sorts of infections (Celli and Finlay, 2002; Gruenberg and van der Goot, 2006; Mueller and Pieters, 2006). Infectious diseases represent a serious health threat, being the first cause of mortality in the world, further complicated by the emergence of various resistant microbe strains associated to multidrug resistance. A thorough knowledge of the subcellular mechanisms underlying phagocytosis, and their subversion by invasive microbes, may help in designing more effective thrapeutic approaches to fight intracellular pathogens. Of great

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help for such understanding is the development of model organisms amenable to genetic manipulation that facilitate identification and analysis of genes regulating phagocytosis and modulating host resistance to pathogens. The awareness that phagocytosis is evolutionary very ancient, being already present in unicellular amoebae, has favored in recent years the establishment of a few such models, like Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster (Pradel and Ewbank, 2004; Stuart and Ezekowitz, 2008). Dictyostelium cells, like all ‘‘social amoebae’’ or ‘‘cellular slime molds,’’ live in the forest soil and feed on bacteria. As long as bacteria are available, the solitary amoebae ingest them, grow and proliferate by binary fission. When the bacteria are depleted, the cells stop growing and enter the social phase of their life cycle, by collecting into multicellular aggregates, which like all multicellular organisms undergo cell differentiation and morphogenesis. The outcome is a fruiting body, consisting of a slender stalk bearing on top a ball of resistant spores (Kessin, 2001). The phagocytic totipotentiality of growth phase cells gradually declines when cells enter the multicellular stage and form tight aggregates. When exposed to bacteria, starving preaggregating or aggregating cells fully revert to the phagocytic stage, but after aggregate compaction less than 20% of the cells in the aggregate display the ability to phagocytose and grow on bacteria (Gambino et al., 1992). At the migrating slug stage, this number is further reduced to about 1%, and evidence has been recently provided that these cells, named ‘‘sentinel cells,’’ may represent a reservoir of immune-like cells that circulate within the slug, swallow invading bacteria and toxic substances, and are left behind the slug during its migration (Chen et al., 2007). The idea of a primitive innate immunity system active during multicellular development is further supported by the occurrence in the Dictyostelium genome of potential homologues to innate immunity signaling proteins found in animals or plants, including Toll-like Interleukin Receptor (TIR)-domain containing proteins, WRKY transcription factors, potential LRR-domain receptors (Chen et al., 2007). Vegetative Dictyostelium cells are highly efficient phagocytes, and their capacity exceeds that of neutrophils severalfold, each cell being able to digest about 300 bacteria per hour. The cells are considered ‘‘professional phagocytes,’’ as they ingest a large variety of bacteria, yeast, apoptotic cells, as well as inert particles. In contrast, the various species of social amoebae usually do not swallow one another, thus displaying a high degree of selfnon-self recognition. A cannibalistic species was described, Dictyostelium caveatum, which is able to eat its neighbors, when cocultured with other species. Interestingly, D. caveatum cells do not devour each other, except for a mutant that has completely lost self-non-self recognition (Waddell and Duffy, 1986). Unfortunately, very little is known on the genetic background of D. caveatum.

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Phagocytosis has been mostly studied in the species D. discoideum, which is used here synonymously as Dictyostelium, except when otherwise indicated. For the molecular analysis of phagocytosis, a breakthrough was the isolation of laboratory axenic strains that grow in minimal or rich axenic media (see Kessin, 2001). In contrast to the wild-type strains, which are strictly dependent on bacterial phagocytosis for growth, the axenic strains have gained the function of swallowing nutrients by fluid-phase endocytosis, without loosing the ability to phagocytose. These strains are thus ideal for a genetic approach to phagocytosis, as mutants defective in phagocytosis, but able to grow by fluid-phase endocytosis, can be selected. With the establishment in the course of the last 25 years of molecular genetic techniques for transforming cells, disrupting single genes and randomly generating mutants by restriction enzyme-mediated insertion, the shortcomings of asexual Dictyostelium genetics could be overcome, making the system amenable to molecular genetic manipulation. The recent completion of genome sequencing has made available new tools for postgenomic analysis of phagocytosis (Eichinger et al., 2005; Eichinger and Rivero, 2006). The relevance of Dictyostelium as model system for phagocytosis and for immunity is highlighted by the broad spectrum of bacteria that are taken up by the cells, and by its susceptibility to infection by microbes that are pathogenic also for animals and humans. A first systematic attempt to identify pathogenic bacteria for Dictyostelium can be traced back to a paper by Depraite`re and Darmon (1978). These authors tested 45 Gram-negative and 23 Gram-positive bacterial species for their capacity to support Dictyostelium cell growth, and found that the cells were able to ingest and degrade most of them, including several species of Enterobacter, Serratia, Salmonella, Klebsiella, Yersinia, Pseudomonas, Staphylococcus, Listeria, and several Bacilli. A few species or strains, such as highly pigmented Serratia marcescens strains, Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas rubescens, Bacillus thuringiensis, Bacillus laterosporus, and Listeria monocytogenes, were found to be pathogenic. Except for P. aeruginosa and Bacillus thuringiensis, which killed the cells by secreting toxic substances, the other bacteria were phagocytosed and were toxic at different degree for Dictyostelium cells (Depraite`re and Darmon, 1978). These authors also reported that B. anthracis was not ingested by Dictyostelium cells. Recent studies have detected other pathogenic microbes and have shown that host–pathogen interactions can be studied at molecular level in Dictyostelium, by exploiting all the advantages of a lower eukaryote easily amenable to molecular genetic, biochemical, and cell biology investigations (see Section 5). Both fluid-phase endocytosis and phagocytosis in Dictyostelium have been reviewed in recent years (Duhon and Cardelli, 2002; Maniak, 2002). Host–pathogen interactions have been the subject of sectorial reviews (Solomon and Isberg, 2000; Steinert and Heuner, 2005). In this chapter,

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we will reexamine both phagocytosis and host–pathogen interactions in Dictyostelium, in the light of recent developments in both fields. When appropriate, comparisons with results on mammalian macrophages will be made. Fluid-phase endocytosis and macropinocytosis will not be discussed, and the readers are invited to look at the excellent reviews by Maniak (2002, 2001).

2. The Dynamics of Phagocytosis Several studies have made use of cells expressing GFP-fused proteins and fluorescently-labeled bacteria, latex beads or yeast particles to dissect the dynamics of phagocytosis in vivo. In these studies, cells and particles are coincubated on glass, to follow both the amoeboid movements of the cell in its attempt to hold and encapsulate the prey and the intracellular fate of the ingested phagosome. Alternatively, coincubation in shaken culture has been used in pulse-chase experiments for quantitative measurements of uptake and intracellular phagosome maturation or for isolating phagosomal populations for proteomic or transcriptomic studies. In a highly simplified scheme, it is assumed that particle binding leads to progressive actin recruitment at the site of attachment and along the rim of the membrane enveloping the particle, leading eventually to closure of the phagocytic cup around the particle. Formation of the phagocytic cup resembles, thus, pseudopod formation during chemotaxis, as in both cases the actin cytoskeleton drives a local membrane extension (Fig. 6.1). Occasionally, ‘‘sinking’’ of bacteria into the cell has been observed, a process that possibly requires a different rearrangement of the underlying cytoskeleton (Fig. 6.1). A basic difference between a chemotactic pseudopod and a phagocytic cup is that the first is better explained with a trigger model and the second with a zipper model. In the trigger model, a chemotactic signal initiates an all-or-none response, leading to pseudopod formation. In the zipper model, phagocytosis is assumed to be locally controlled by progressive, sequential binding of cell surface receptors to the particle, a process that can be interrupted at any moment (Swanson and Baer, 1995). Evidence for the zipper model has been provided in Dictyostelium, using yeast particle as prey. It has been shown that particle binding does not necessarily lead to uptake, and that about half of the attempts are aborted, though actin is locally recruited. Even formation of a leading pseudopod in front of the particle or prolonged binding is no guarantee of success (Maniak et al., 1995; Peracino et al., 1998). These early studies were done with yeasts, which are 3–5 mm in diameter. Similar studies with bacteria are difficult to perform, due to flagellar-induced or brownian motion of the bacteria.

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Figure 6.1 Phagocytosis of bacteria by Dictyostelium cells. (A^C) Scanning electron micrographs of Dictyostelium cells incubated with E. coli B/r. (A, B) Typical examples of pseudopod-like phagocytic cups with bacteria inside are indicated (arrows). (C) The arrow points to a bacterium ‘‘sinking’’ into the cell. The round particles are latex beads. (D, clockwise) Confocal image series of a living cell, expressing GFP-actin, ingesting a yeast particle. The time elapsing from actin recruitment to the phagocytic cup to its disappearance from the ingested particle takes about 1 min. (SEM in collaboration with the late M. Claviez, confocal fluorescence microscopy with B. Peracino.)

Alternative approaches with bacteria or latex beads have made use of the particle-tracking method, to reduce particle movement and favor adhesion (Ishikawa et al., 2003), or the agar overlay technique, where a thin agar sheet gently pressing the cells creates a ‘‘two-dimensional’’ condition, in which the bacteria are mostly immobile and the cell ingests them by extending large pseudopodia around (Clarke and Maddera, 2006). Also under these conditions, it has been qualitatively or semiquantitatively shown that attached bacteria or beads are often not phagocytosed. Successful phagocytosis events can be, however, very rapid. Both with yeasts and bacteria, the time elapsing from initial actin recruitment at the phagocytic cup to disassembly of the actin coat around the phagosome can take as less as 60 s (Blanc et al., 2005; Clarke and Maddera, 2006; Dormann et al., 2004; Insall et al., 2001; Loovers et al., 2007; Maniak et al., 1995; Peracino et al., 1998). The ingested phagosome undergoes successive fusion events with vesicles of the endolysosomal compartment, resulting in acidification, delivery of a large variety of hydrolases, postlysosomal maturation and eventually fusion of the ‘‘postlysosomal’’ vesicle with the plasma membrane for excreting undigested debris. From this point of view, phagocytosis in Dictyostelium is considered a linear process, starting with uptake and

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ending with excretion of undigested material, though retrieval and recycling of several membrane components has been shown to occur from the very early steps upward (Gotthardt et al., 2002; Ravanel et al., 2001). Fusion of the phagosome with acidic vesicles, visualized by using pHsensitive probes, such as neutral red or Lysotracker red, or GFP-fused subunits of the vacuolar Hþ ATPase (the major agent responsible for acidification) is a rapid event, detectable within 1–2 min after phagosome formation (Clarke and Maddera, 2006; Maniak, 2001; Peracino et al., 2006; Rupper et al., 2001a). In contrast to macrophages, acidification is a transitory event: in pulse-chase experiments, it has been shown that phagosomes, as well as macropinosomes, become acidic (pH 5.0) within 5 min, then the pH gradually increases to about 6.0 between 10 and 60 min and above 6.0 thereafter (Aubry et al., 1993; Padh et al., 1993; Rupper and Cardelli, 2001). Neutralization of the pH is linked to retrieval of the vacuolar Hþ ATPase (Aubry et al., 1993; Clarke et al., 2002a). Neutralization is also accompanied by shape changes of the phagosome. The nascent phagosome is characterized by tight apposition of the phagosomal membrane with the ingested particle. Between 15 and 60 min postuptake, there is an increase in the number of phagosomes containing multiple bacteria, suggesting homotypic fusion between phagosomes. Later on, the multiparticle phagosomes become swollen, giving rise to socalled ‘‘spacious phagosomes,’’ large vacuoles in which degraded bacteria are visible (Rauchenberger et al., 1997; Rupper et al., 2001b). At the level of single cell phagocytosing few bacteria, digestion has been calculated to be accomplished in 4 min from ingestion (Clarke and Maddera, 2006), though at population levels longer times, averaging 20 min, have been calculated (Aubry et al., 1993; Clarke et al., 2002a; Maselli et al., 2002). Release of undigested debris is instead a slow process, going on over several hours postuptake. Phagocytosis in shaken cocultures with bacteria is usually studied by using a 100- to 1000-fold excess of fresh bacteria in simple salt solution, in which the bacteria cannot grow. Under optimal conditions, cells ingest about 1000 bacteria per generation (3 h), with an uptake rate of 4–5 bacteria per minute (Bozzaro and Gerisch, 1978; Gerisch, 1959; Vogel et al., 1980). Pulse-chase experiments with bacteria or latex beads under similar conditions have been designed for isolation and proteomic characterization of purified phagosomes during their maturation (Bogdanovic et al., 2002; Gotthardt et al., 2002, 2006; Rezabek et al., 1997). These studies have confirmed very early recruitment of the V-HþATPase in the phagosome, concomitantly with shedding of the actin coat and delivery of proteins regulating vesicle fusion, such as the small G protein Rab7, the SNARE components Vti1, syntaxin 7, syntaxin 8, and the lysosomal marker LmpB. A second step in the maturation of phagosomes, starting between 3 and 15 min postuptake, is characterized by recruitment of lysosomal enzymes, such as cathepsin D and cysteine proteinases

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CP-p34, together with lysosomal membrane markers, such as the CD36/ LIMP family members lmpA and lmpC, and the SNARE component Vamp7. Slightly delayed is the acquisition of lysosomal glycosidases, such as a-mannosidase and b-glucosidase. Acquisition of postlysosomal markers, such as the vacuolin B protein, is a late event, occurring 60–90 min postinternalization (Rauchenberger et al., 1997). The maturation process is accompanied by extensive retrieval and recycling, which occur with different kinetics for different markers, confirming the occurrence of complex and successive fusion events (Gotthardt et al., 2002). Discrepancies in the reported kinetics of delivery, but in particular recycling, of proteins depend on whether living bacteria or latex beads are used as particles, very likely because bacteria are degraded while latex beads are indigestible inert particles. The timing of uptake and phagosome formation is similar for both particles, but subsequent phagosome maturation is slowed down for the latex beads. Toxic effects of the engulfed beads, particularly at high concentrations, cannot be excluded, though cell lysis is rarely observed, and the cells fully recover after latex bead excretion. In addition to the above-mentioned proteins, characterization of the phagosomal proteome has led to identification of about 180 proteins, most of which are involved in membrane traffic, metabolism, signal transduction, protein biosynthesis, and degradation (Gotthardt et al., 2006). Proteomic fingerprinting of the phagosome has confirmed substantial remodeling during the different phases of phagocytosis, with major changes occurring shortly after uptake and at the postlysosomal exocytic stage. A preliminary lipidomic characterization of early phagosomes compared to the plasma membrane has revealed a significant shift from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species toward lyso-PC and -PE and toward phospholipids with shorter acyl chains and lower unsaturations (Gotthardt et al., 2006). Enrichment of sterols in the phagosomal membrane, compared to the bulk of plasma membrane, was previously reported (Favard-Sereno et al., 1981). Consistent with these results is the recent observation in microarray studies that phagocytosis induces down- and upregulation of genes involved in phospholipid and sterol biosynthesis, respectively (Sillo et al., 2008).

3. Cellular Mechanisms of Phagocytosis 3.1. Bacterial adhesion to the cell surface: The search for phagocytosis receptors As just mentioned, Dictyostelium cells ingest bacteria very efficiently, when coincubated in shaken culture. Under these conditions, tight particle adhesion is a prerequisite for phagocytosis, and mutants have been isolated which

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are able to phagocytose when plated on a bacterial lawn but not in suspension (Ceccarelli and Bozzaro, 1992; Cohen et al., 1994; Cornillon et al., 2000). In principle, these mutants could be defective in membrane receptors that mediate particle binding, in actin cytoskeletal components that stabilize adhesion and give rise to the phagocytic cup, or signaling factors that regulate binding and/or actin rearrangement. Such a well-defined mutant is a talin-null mutant, which is strongly defective in both phagocytosis of Escherichia coli B/r in suspension and cell adhesion to plastic or glass. Only bacteria devoid of the carbohydrate moiety of cell surface lipolysaccharides, such as the rough Salmonella minnesota R595 strain, are adhesive enough to be recruited by talin-null cells and phagocytosed (Niewohner et al., 1997). The talin-null mutant illustrates the complexity of the potential receptor–cytoskeletal interactions in phagocytosis. Talin is known to mediate F-actin interaction with the membrane, though it is not essential in Dictyostelium, as macropinocytosis is not affected in the mutant (Niewohner et al., 1997). Bacterial binding, in contrast, has to be stable enough for the particle to be engulfed. It has been suggested that talin could be part of a specific complex with binding receptors for E. coli, not however for S. minnesota R595. Weakening of this complex would then reduce selectively E. coli uptake in the mutant. Alternatively, S. minnesota R595 binding may be stronger, despite talin absence, thus resisting the shear forces generated by shaking and ultimately leading to uptake (Niewohner et al., 1997). The search for membrane receptors involved in phagocytosis has been elusive in Dictyostelium for several years, though there is some indication that things may now change. The existence of different receptors for bacteria, latex beads or yeast particles was first inferred by Hellio and Ryter (1980) in inhibition experiments with lectins. Yeast uptake, in particular, correlated with Wheat Germ Agglutinin-binding membrane receptors. By using chemical mutagenesis, Vogel and his coworkers (1980) provided evidence for the existence of two types of receptors on the surface of Dictyostelium cells for E. coli B/r. A mutant was isolated that failed to ingest hydrophilic latex beads, while retaining the ability to phagocytose bacteria. Phagocytosis in the mutant, but not in the parental strain, could be inhibited by the addition of glucose, suggesting the existence of two receptors; a lectin-type receptor that could interact with the terminal glucose on the E. coli lipopolysaccharide, and a ‘‘nonspecific’’ receptor, inactive in the mutant and responsible for the uptake of hydrophilic latex beads. Shortly later, using sugar-derivatized polyacrylamide flat gels, it was shown that Dictyostelium cells possessed three surface receptors for binding to glucosides, mannosides, or N-acetylglucosaminides (Bozzaro and Roseman, 1983a). Binding to glucoside was particularly interesting, as it interfered in a subtle way with the ability of the cells to form stable aggregates, leading to the suggestion that the glucoside receptor could act as a sensor for bacteria (Bozzaro and

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Roseman, 1983b; Gambino et al., 1992). Chemical mutagenesis and selection for binding to sugar-derivatized gels resulted in mutants that failed to bind to glucosides. The mutants displayed strongly reduced phagocytosis of E. coli B/r, but not latex beads (Ceccarelli and Bozzaro, 1992), thus confirming Vogel’s hypothesis of two distinct membrane receptors. Unfortunately, chemical mutagenesis in Dictyostelium does not allow identifying the mutated genes, and biochemical attempts to purify the potential receptors have been unsuccessful. The approach of Vogel and coworkers has been recently combined with restriction enzyme-mediated insertion transformation to generate randomly mutagenized tagged cell populations and select for phagocytosis mutants (Cornillon et al., 2002). Two mutants were isolated that were defective for phagocytosis of latex beads, but not bacteria (Klebsiella pneumoniae or E. coli). The mutants displayed also reduced substrate adhesion and motility, suggesting a basic common role for the tagged genes. The candidate genes, Phg1 and Phg2, belong to a family of nine-transmembrane domain proteins present in plants, yeast and humans, but fail to meet the criteria of adhesion proteins. Phg1 definitely does not act as phagocytosis receptor, rather it has been proposed to regulate phagocytosis, possibly by modulating surface expression of other proteins involved in adhesion (Benghezal et al., 2003). Phg2 has been recently shown to be a serine/threonine kinase that regulates actin cytoskeleton dynamics, adhesion, and motility, in addition to phagocytosis (Blanc et al., 2005) (see Section 4.2). Another early candidate, gp130, also fails to meet the criteria of phagocytic receptor. Gp130 was identified as potential target of a poly-specific antiserum that inhibited phagocytosis as well as the EDTA-labile form of intercellular adhesion (Chia and Luna, 1989). The notion that a common receptor may be involved in phagocytosis and EDTA-labile cell–cell adhesion is appealing, as the same receptor may be used to sense the environment, allowing preaggregative starving cells to rapidly switch from development to growth, if bacteria turn to be available. However, a gp130 knockout mutant failed to display any defect in both functions (Chia et al., 2005). In addition, the most prominent candidate for EDTA-labile cell–cell adhesion, the 27 glycoprotein DdcadA, belonging to the cadherin family, is not required for phagocytosis (Chia et al., 2005). A promising candidate gene in this context is sadA, which encodes an integral membrane protein, and whose deletion results in cells strongly defective in binding to glass substratum, in phagocytosis and EDTA-labile contacts (Fey et al., 2002). Functional studies are required to better define SadA role in phagocytosis. A family of five proteins, distantly related to human b-integrin, have been recently identified (Cornillon et al., 2006). These proteins, named Sib (similar to integrin beta)-A to -E, display a conserved cytoplasmic region that binds the cytoskeletal protein talin. Disruption of SibA results in cells defective in latex beads, but not bacteria (Klebsiella), phagocytosis. The cells

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are also defective in cell-substratum, though not in cell–cell adhesion (Cornillon et al., 2006). SibA could thus be one of nonspecific ‘‘hydrophilic’’ receptors hypothesized by Vogel et al. (1980). Genes encoding potential phagocytosis receptors have been detected in a recent genome-wide transcriptomic study designed to identify genes relevant for phagocytosis of or growth on E. coli (Sillo et al., 2008). Transcripts from cells exposed briefly to the bacteria or in exponential growth on bacteria were compared with each other and with transcripts from axenically growing cells. Clusters of genes could be identified that were regulated by either phagocytosis or growth on bacteria. Among genes strongly upregulated by phagocytosis are genes encoding a carbohydratebinding membrane protein, a putative scavenger receptor, and a few other putative membrane proteins. The potential role of these genes in phagocytosis can now be analyzed by generating knockout mutants and by functional studies.

3.2. The actin cytoskeleton in phagocytosis The colocalization studies of actin and actin-binding proteins with the phagocytic cup and the phagosome, mentioned in the first section, pinpoint the importance of the actin cytoskeleton in the process. Phagocytosis is inhibited by drugs affecting actin polymerization such as cytochalasin or latrunculin (Hacker et al., 1997; Maniak et al., 1995). No similar reports exist on drugs affecting the microtubule cytoskeleton, but microtubules have been suggested to be involved in intracellular transport of phagosomes (Clarke and Maddera, 2006). The Dictyostelium genome harbours members of the two major actinnucleating families, the Arp2/3 complex and formin, as well as their regulators, Scar/Wave, WASP, and ENA-VASP. Scar-Wave, WASP, or ENA-VASP are responsible for the localized activation of Arp2/3 or formin in the membrane, in response to signals mediated by small G proteins and phosphoinositides. The Arp2/3 complex induces actin nucleation into a filament network, while formin is mainly responsible for actin filament elongation (Faix and Grosse, 2006; Ibarra et al., 2005). Schematically, formin is responsible for membrane structures such as filopodia, whereas the Arp2/3 complex induces pseudopodial and lamellipodial extensions. Arp2/3 and Scar/WAVE are recruited within seconds to phagocytic or macropinocytic cups, concomitantly with actin polymerization (Insall et al., 2001). Disruption of the single scar/Wave gene leads to a strong decrease in the rate of phagocytosis and macropinocytosis, concomitant with a 50% decrease in F-actin cell content (Seastone et al., 2001). There are two WASP encoding genes in Dictyostelium and the double knockout is probably lethal. Mutants having WASP1 disrupted and WASP2 knocked down display reduced chemotaxis. Whether they are also impaired in phagocytosis

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has not been reported (Myers et al., 2005). Formin, which is essential for filopodia formation, is also found beneath the phagocytic cup (Faix, personal communication), but its disruption, as well as inactivation of the regulatory Vasp gene, while affecting particle adhesion, does not hinder phagocytosis (Han et al., 2002). In addition to F-actin-nucleating factors, remodeling of the actin cortex requires the activity of a variety of actin-binding proteins, which regulate either availability of actin monomers, bundling, cross-linking, or fragmenting of actin filaments or their association with the plasma membrane. Because of the prominent role of motility processes in Dictyostelium development, several studies have been performed to examine the function of these proteins (reviewed in Noegel and Schleicher, 2000). Several actinbinding proteins are transiently recruited to the phagocytic cup and/or the phagosome, with kinetics roughly similar to actin itself, but only a few of them have been shown, by gene knockout or overexpression experiments, to control phagocytosis. Negative results may be due, in some cases, to redundancy in the cytoskeletal proteins, which can be eventually bypassed by generating double or triple mutants. Thus, single mutants for the F-actin cross-linking proteins a-actinin or gelation factor are mildly defective in phagocytosis, but a double mutant displays a 50% reduction in the phagocytosis rate (Rivero et al., 1996). A profilin I/II double mutant, in contrast to the single mutants, is characterized by enhanced phagocytic activity and a thick actin cortex, due to higher F-actin content (Temesvari et al., 2000). The thick actin cortex favors in some way phagocytosis. The mutant phenotype can be reversed by second suppression of the CD36/LIMPII homologue, LimpA, a protein that is located on endolysosomal vesicles. The interaction between these two genes is unclear, except that both profilins and LimpA bind to PI(4,5)P2. It has been reported that LimpA inactivation reduces the abnormal F-actin content in the double mutant, and this could be the immediate reason for the rescuing effect on phagocytosis (Temesvari et al., 2000). The F-actin destabilizing protein cofilin, and its regulator protein Aip1, localize to phagocytic cups and stimulate membrane ruffles (Aizawa et al., 1997; Konzok et al, 1999). Cofilin disruption is very likely lethal (Aizawa et al., 1996), but Aip1 disruption impairs phagocytosis, while overexpression stimulates it (Konzok et al., 1999). The reduction in the phagocytosis rate in the Aip1 null mutant has been correlated with a slower rate of membrane protrusion around the phagocytic cup, suggesting that Aip1 enhances via cofilin the pool of actin monomers available for new actin filaments at the phagocytic rim (Konzok et al., 1999). Cofilin overexpression, however, has no effect on phagocytosis (Aizawa et al., 1996). Coronin, an actin-associated protein found in many species, was originally shown in Dictyostelium to be enriched at the leading front of chemotaxing cells and in crown-shaped extensions of axenically growing cells (de Hostos et al., 1991).

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The protein is also transiently recruited to phagocytic cup and is released from the phagosome shortly after its closure. Knockout mutants are strongly defective in yeast particle uptake (Maniak et al., 1995). Early phagosomes are also coated with comitin, an F-actin-binding protein that is associated with Golgi and post-Golgi vesicle membranes. Comitin knockout mutants are defective in phagocytosis of yeast particles and E. coli, but not latex beads. The mutants are otherwise normal for growth, pinocytosis, secretion, chemotaxis, and motility (Schreiner et al., 2002). The reduced uptake of E. coli, in contrast to latex beads, suggests that the defect may be linked with particle binding, rather than uptake, considering that both particles are of similar size but the E. coli surface is more hydrophilic. Comitin would then resemble talin in its effects, though the mechanism of action remains obscure. Myosins serve as actin-linked motors and are expected to play a major role in regulating contraction, intracellular vesicle delivery, and thus migration, cytokinesis, maintenance of cell shape, morphogenesis, and least, but not last, phagocytosis (Yumura and Uyeda, 2003). Class I and VII myosins have been shown to be required for phagocytosis in Dictyostelium, whereas the conventional myosin II is not essential (Durrwang et al., 2006; Fukui et al., 1990; Maselli et al., 2002; Titus, 1999). Disruption of myosin VII, in particular, results in strong defect in initial particle binding, and thus in particle uptake (Titus, 1999). Intriguingly, MyoVII forms a complex with talin, and its disruption leads to an 80% decrease in cytosolic talin content, suggesting that the strong defect in the myoVII mutant may be indirectly mediated by depletion of talin (Galdeen et al., 2007). Knocking out MyoIK, MyoIA/IB, IA/IC, or MyoIE also affects phagocytosis, mainly due to reduced cortical tension and particle binding (Durrwang et al., 2006; Jung et al., 1996; Schwarz et al., 2000; Soldati, 2003). It is likely that the major function of these unconventional myosins is to deliver components that are required for membrane recycling and extension rather than providing the force for internalization. This would fit with the observations that myosin VII is found at filopodial tips, but not in phagocytic cups, and that other class I myosins reside only transiently in phagocytic cups or phagosomes (Durrwang et al., 2006; Schwarz et al., 2000; Tuxworth et al., 2001). Actomyosin interactions are regulated by myosin heavy chain (MHC) and light chain (MLC) kinases. PakB, the myosin I heavy chain kinase, when expressed in the activated form, is strongly enriched in macropinocytic and phagocytic cups, and leads to increased phagocytosis (de la Roche et al., 2005). Remarkably, MHCK (myosin II heavy chain kinase), whose activity leads to myosin II disassembly from the actin cytoskeleton, is localized to phagocytic cups (Steimle et al., 2001). MHCK activity could be responsible for myosin II exclusion from the phagocytic cup, thus locally inhibiting actomyosin contractile fiber formation and rather favoring actin filament assembly into pseudopodial-like extensions.

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3.3. Phagosome fusion with endolysosomal vesicles and killing of bacteria: The other players The ultimate outcome of phagocytosis is killing and digestion of the internalized bacteria. This is achieved by activation of several processes: production of toxic oxygen radicals (respiratory burst), acidification of the phagosomal lumen and depletion of essential divalent metals, degradation of the bacterial cell wall, and digestion by hydrolytic enzymes. In phagocytic leukocytes, a membrane-associated NADPH oxidase complex produces large quantities of superoxide radicals from molecular oxygen. Dismutation of O2 to H2O2 and formation of hydroxyl radicals (OH) and hyperchlorous acid (HOCl) generate potent microbicidal compounds (Minakami and Sumimotoa, 2006). The NADPH oxidase (cytochrome b558) is made of at least two membrane-bound subunits and two cytosolic components, is usually inactive, and is activated by a variety of stimuli associated with phagocytosis. The cytosolic components are targeted to phagosomes in a process involving the small G proteins Rac1 or Rac2, which activate the oxidase (Minakami and Sumimotoa, 2006). Dictyostelium cells possess three isoforms (noxA, noxB, noxC) of the large cytochrome b558 subunit and an isoform (p22phox) of the small subunit. A double knockout noxA/noxB displays normal phagocytosis and macropinocytosis. More importantly, no evidence has been provided so far for superoxide production in Dictyostelium in vivo or in vitro (Lardy et al., 2005). Thus, whether a respiratory burst is induced during phagocytosis in Dictyostelium remains open. As mentioned in Section 2, the phagosome undergoes extensive remodeling in its route to phagolysosomal maturation. The phagosomal vesicle looses quite rapidly its actin coat and undergoes rapid acidification (Aubry et al., 1993; Padh et al., 1993). The kinetics of uncoating is roughly similar for actin (Peracino et al., 1998), actin-binding proteins (Insall et al., 2001; Konzok et al., 1999; Lee et al., 2001; Maniak et al., 1995; Rupper et al., 2001b), as well as signal transducers and effectors, such as RacF1 (Rivero et al., 1999), the myosin I heavy chain kinase PakB (de la Roche et al., 2005), or myosin II heavy chain kinase (Gotthardt et al., 2002; Steimle et al., 2001). The V-Hþ ATPase, the major if not unique agent of acidification, is enriched in the contractile vacuole (CV) system and in acidic vesicles independent of the CV. The latter vesicles fuse indifferently with phagosomes and macropinosomes, and are considered a prelysosomal vacuolar reservoir (Clarke et al., 2002a,b; Neuhaus et al., 2002; Padh et al., 1989; Peracino et al., 2006; Souza et al., 1997). Prelysosomal nonacidic vesicles have also been described, which contain on their surface Natural Resistance Associated Membrane Protein (Nramp)1, an endolysosomal divalent metal transporter conferring resistance to invasive pathogenic bacteria, as will be discussed in Section 5.2. These vesicles

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are distributed in the cytoplasm, enriched in the trans-Golgi network, where they colocalize with comitin and the Vti1 SNARE protein, and undergo rapid fusion with phagosomes or macropinosomes, immediately after their acidification (Peracino et al., 2006). Fusion of Nramp1-decorated vesicles with phagosomes is very likely stimulated by Vt1, since this protein colocalizes with Nramp1-decorated vesicles also outside of the Golgi. As mentioned in Section 2, the Vti1 protein is part of a SNARE complex with syntaxin 7, syntaxin 8, and VAMP7 in early endosomes and mediates endosome fusion with lysosomal vesicles (Bogdanovic et al., 2002). Disruption of the Nramp1 encoding gene has only mild effects on phagocytosis and growth on E. coli, suggesting that the Nramp1 function, namely depleting the phagosome of divalent metals, in particular iron, is not essential for killing nonpathogenic bacteria (Peracino et al., 2006). Another protein, which is required for delivery of both V-Hþ-ATPaseand Nramp1-decorated vesicles to phagosomes, is the actin-binding, cyclase-associated protein (CAP) (Sultana et al., 2005). CAP binds the vatB subunit of the vacuolar ATPase and is supposed to mediate its interaction with the actin cytoskeleton. CAP-null mutants display altered distribution of both V-Hþ-ATPase- and Nramp1-decorated vesicles and higher endosomal pH (Sultana et al., 2005). Digestion of bacteria is accomplished by different types of lysosomal hydrolases, which are abundant in vegetative Dictyostelium cells, and are delivered in successive phases to phagosomes (Gotthardt et al., 2002; Rodriguez-Paris et al., 1993; Souza et al., 1997; Temesvari et al., 1994). The Dictyostelium genome also encodes a large family of pore-forming peptides, which act by perforating the membrane of bacteria. Some are synthesized as prepropeptide precursors, like the mammalian defensins, others as prepromultipeptide precursors, like the naegleriapores (Leippe et al., 2005), and may give rise to a large variety of peptides upon hydrolytic cleavage. Two of them have been shown to be potent antibacterial agents in vitro, and have been proposed to kill the bacteria in the phagosome, before these are degraded by hydrolases (Leippe, personal communication). The Dictyostelium genome harbours several lysozyme-encoding genes, some belonging to the bacteriophage T4 type and others to the C-type lysozymes. A novel unconventional class, the ALY class, including four members, has been described. The purified ALYA lysozyme displays antibacterial activity against Gram-positive, but not Gram-negative bacteria. Disruption of the alyA gene results in a 60% reduction of total lysozyme activity, and does not hinder growth on E. coli, though increased phagocytosis has been reported, possibly to compensate for defective digestion (Mueller et al., 2005). The postlysosomal vesicle, devoid of the vacuolar ATPase, is coated with coronin, actin, and vacuolin A and B. Both vacuolin A and B are postlysosomal markers (Rauchenberger et al., 1997). Vesicles decorated with both Nramp1 and vacuolin coexist with vesicles decorated with

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vacuolin only. It is possible that Nramp1- and vacuolin-positive vesicles represent an intermediate step in the postlysosomal pathway (Peracino et al., 2006). Disruption of the vacuolin A encoding gene has no effects. A vacuolin B-null mutant, instead, displays abnormally large postlysosomal vacuoles and delayed release of fluid-phase marker, though surprisingly these defects do not affect the rate of cell growth in axenic medium or bacteria ( Jenne et al., 1998). Maturation of postlysosomal vesicles is also impaired in the LvsB-null mutant (Charette and Cosson, 2007; Harris et al., 2002). LvsB is a protein most similar to LYST/Beige, the gene that is mutated in the Chediak-Higashi Syndrome, which is characterized by the presence of enlarged lysosomes. In the LvsB-null mutant, there is abnormal acidification of postlysosomal compartments, probably due to inappropriate fusion of early endosomes and postlysosomal vesicles (Kypri et al., 2007), supporting the notion that LvsB is a negative regulator of heterotypic fusion (Harris et al., 2002).

4. Regulatory Pathways Controlling Phagocytosis Phagocytic cup formation and closure, as well as postlysosomal vesicle exocytosis are mainly actin-cytoskeleton-based processes. It is therefore not surprising that signal transduction pathways regulating the actin cortex also regulate phagocytosis. Assembly and reshaping of the actin meshwork are controlled by signals originating at the site of particle attachment and transmitted to the cell interior by heterotrimeric G proteins, monomeric G proteins of the Ras and Rac family, and by enzymes regulating membrane phospholipids, in particular phosphoinositides. Cytosolic tyrosine kinases, particularly of the syk family, are important effectors in macrophage FcgRdependent phagocytosis (Greenberg, 1995). A similar role for Dictyostelium tyrosine kinases has not been reported, though a membrane tyrosine kinase has been recently involved in phagosomal maturation (see further below). Phagosomal traffic, like all intracellular traffic, is also controlled by the small G proteins of the Rab family (Zerial and McBride, 2001), which is largely represented in the Dictyostelium genome.

4.1. Heterotrimeric G protein in phagocytosis The heterotrimeric G protein is a major regulator of chemotaxis-based motility both during growth and development of Dictyostelium. It is activated by canonical seven-transmembrane receptors and, in addition to chemotaxis, regulates also pathways controlling cell development and differentiation (Manahan et al., 2004). Several Ga subunits exist in

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Dictyostelium, which are expressed at different times during growth or development and associate with one Gb and one Gg subunit (Wu and Devreotes, 1991). Indirect evidence for G protein involvement in phagocytosis was provided by using aluminum fluoride as inhibitor (Browning and O’Day, 1995), whereas direct evidence was obtained by knocking out the Gb subunit. The Gb~-null mutant displays strong reduced phagocytosis rate of bacteria, and to a lower extent latex beads, but is normal for macropinocytosis, thus providing a first evidence for signaling discriminating phagocytosis from macropinocytosis (Peracino et al., 1998). The phagocytosis defect in the mutant correlates with inefficient actin reorganization at the rim of the progressing phagocytic cup, and thus with phagosome closure, while particle binding as well as initial phagocytic cup formation at the site of adhesion are almost unaffected. This raises the possibility that actin recruitment at the site of particle binding is a stochastic process, independent of signaling. Indeed, intense and spontaneous actin remodeling in the absence of signals occurs incessantly in the actin cortex in the order of subseconds (Diez et al., 2005). This intense activity has been proposed to confer the plasticity required for rapidly forming phagocytic cups and adapting to external stimuli (Diez et al., 2005), but it can also explain why many phagocytic events are aborted also in wild-type cells. If so, then the major effect of heterotrimeric G protein activation, following particle binding, could be to superimpose local organization to a stochastic process. A possible Ga partner of the Gb subunit is Ga4. This alpha subunit is enriched in purified phagosomes together with the Gb at earliest time points, coinciding with phagosome formation, and at late maturation stages, close to exocytosis (Gotthardt et al., 2006). Since the postlysosomal vesicles are recoated with actin before excretion, these data suggest a close link between the heterotrimeric G protein and actin reorganization at different steps of the phagocytic process. As expected if Ga4 is the relevant Ga subunit associated to Gb, Ga4 gene inactivation also results in reduced phagocytosis rate (Gotthardt et al., 2006). How the heterotrimeric G protein is activated remains open. A possibility is lateral clustering of particle-binding membrane proteins with seventransmembrane receptors to form a signaling complex that may activate the G protein. A recent DNA microarray study has shown that two of the five genes encoding tetraspanins in Dictyostelium genome are upregulated in cells undergoing phagocytosis (Sillo et al., 2008). Tetraspanins are fourtransmembrane proteins, which act as scaffold to link together membrane receptors and other signaling effectors (Hemler, 2005). Tetraspanins have been recently involved in integrin-dependent phagocytosis in retinal pigment epithelia and in antigen-presenting cells (Chang and Finnemann, 2007), and tetraspanin-clustering with a G protein-coupled receptor (GPCR) has been also reported (Little et al., 2004). Tetraspanins may thus furnish the link for clustering particle-binding membrane proteins with

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GPCR’s into a signaling complex, which regulates actin recruitment and, possibly, membrane recycling and gene expression during phagocytosis. The Gb protein may act as integrator of signals arising in this signaling complex, independently of a specific ligand binding to a GPCR. Alternatively, particle binding may stimulate local secretion of cytokines that in turn activate GPCR’s and thus the hetrotrimeric G protein. Folate and other secreted bacterial metabolites are known to act as chemoattractants in Dictyostelium. Since the Gb-null mutant is, however, also partially defective in latex beads uptake, an autocrine cytokine would be a more appealing candidate. Such a potential candidate is GABA (g-aminobutyric acid), for which G protein linked GABAB-like receptors (GPCR family 3) exist in Dictyostelium (Prabhu and Eichinger, 2006). Genes encoding two such receptors have been shown to be upregulated by phagocytosis (Sillo et al., 2008). In addition, phagocytosis leads to coregulation of genes involved in glutamate and GABA biosynthesis (Sillo et al., 2008). Functional studies by generating null mutants for genes encoding tetraspanin or glutamate decarboxylase can validate these hypotheses.

4.2. Phosphoinositides and calcium ions Candidate downstream transducers of the heterotrimeric G protein and/or particle-binding membrane receptors are small G proteins of the Ras and Rac families, membrane lipids generated by the activity of phospholipases, PI kinases and phosphatases, and calcium ions. Phosphatidylinositide metabolism, in particular, plays a major role in signal transduction, actin remodeling, and membrane trafficking in eukaryotic cells, including Dictyostelium (Payrastre et al., 2001; Willard and Devreotes, 2006; Yeung and Grinstein, 2007). Phosphatidylinositides are glycerolipids containing a D-myo-inositol head group, which can be phosphorylated at positions 3, 4, and 5 by specific PI kinases. In addition to PI kinases, activation of phospoholipase C or PI phosphatases generate different classes of lipid species, which act as binding sites for effector proteins with specific recognition domains, such as PH, PX, FYVE, ENTH/ ANTH, and FERM domains (Downes et al., 2005; Lemmon, 2003, 2007). Phosphoinositides account for 11% and 9% of total lipids in Dictyostelium plasma membrane and endolysosomal membranes, respectively (Nolta et al., 1991). Phosphoinositide dynamics during particle uptake in Dictyostelium has been studied by using GFP-fused PH domains with different PI specificities (Dormann et al., 2004; Blanc et al., 2005; Loovers et al., 2007), and can be summarized as follows. PI(4,5)P2 (phosphatidylinositol, 4–5, bisphosphate) is distributed uniformly on the plasma membrane, and may be responsible for basal actin enrichment in the membrane cortex. Although the details remain controversial, it is established that PI(4,5)P2 is important in both activation and localization of actin nucleation factors and actin-binding

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proteins, such as profilin, coronin, gelsolin, DAPI1, or CAP. It is unclear whether there is a burst of PI(4,5)P2 accumulation in the phagocytic cup, but PI(4,5)P2 declines concomitantly with closure of the phagocytic cup, starting at its base, while PI(3,4,5)P3 accumulates and peaks just after phagosome closure, disappearing rapidly thereafter. PI(3,4)P2 increases concomitantly with PI(3,4,5)P3, but declines with a much slower rate. Disassembly of the actin coat from the internalized phagosome correlates timely with PI(4,5)P2 decrease, rather than with disappearance of PI(3,4,5) P3 or PI(3,4)P2 (Fig. 6.2). PI(4,5)P2 is the target of PLC or PI3K activity, yielding the second messengers diacylglycerol (DAG) and inositol(1,4,5)P3 (IP3), or PI(3,4,5) P3, respectively (Fig. 6.3). A pharmacological approach to characterize signal transduction pathways regulating phagocytosis has led to identical results in two different labs (Peracino et al., 1998; Seastone et al., 1999), namely the requirement of phospholipase C (PLC) and intracellular calcium for efficient phagocytosis. PLC inhibitors and intracellular calcium chelators inhibited significantly the extent of phagocytosis, whereas PKA, PKC, PLA2, or tyrosine kinase inhibitors as well as PI3K inhibitors did not affect bacterial uptake. Remarkably, PI3K inhibitors affect macropinocytosis (Seastone et al., 1999). In addition to pharmacological data, several lines of evidence support a central role for PI(4,5)P2 and PLC activity in phagocytosis. The bulk of PI(4,5)P2 is reduced to 40% of control in a myo-inositol auxotrophic mutant following myo-inositol starvation. Under these Phagosome closure

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Figure 6.2 Time course of phosphoinositide metabolism and recruitment of actinbinding proteins during phagocytosis.The relative abundance of different phosphoinositide types in the membrane of phagocytic cup/phagosome was measured over time by using GFP fused to specific PH domains. Relative changes in the binding of GFPcoronin and GFP-ABD probes to phagocytic cup/phagosome are also shown.The figure summarizes data from the following references: Dormann et al. (2004), Blanc et al. (2005), Loovers et al. (2007).

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D

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Figure 6.3 Pathways for interconversion of phosphatidylinositol polyphosphates involved in Dictyostelium phagocytosis. Only identified gene products, and their pathways, are shown. Phosphorylation reactions and kinases are in light grey; dephosphorylation reactions and phosphatases are in dark grey. Phospholipase C (PLC) is in black. In bold are enzymes, whose encoding genes have been disrupted and/or the activity studied with specific inhibitors; in italics are indicated gene products, which have been identified, but their activity in phagocytosis has not been studied so far. Abbreviations: PI, phosphatidylinositol; PI(X)P, phosphatidylinositol(X)phosphate; DAG, diacylglcyerol; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; PTEN, phosphatase and tensin homologue (encoded by 1 gene); PI3K-I, phosphatidylinositol 3-kinase, class I (encoded by 3 genes); MTMR2 (myotubularin related phosphatase 2); Dd5P4, OCRL-1 homologue inositol 5-phosphatase; DdPIK5, PI3 kinase specific for PI. See text for details.

conditions, bacterial uptake, but not fluid-phase endocytosis, is strongly reduced in the mutant (Fischbach et al., 2006). Particle binding and phagocytic cup formation are inhibited in the Phg2-null mutant defective in a serine/threonine kinase that is associated to the plasma membrane. This association is mediated by a PH domain that binds to PI(4,5)P2 or PI(4)P sites. Deletion of this binding region in the Phg2 kinase prevents complementation of the phagocytic defect in the null mutant (Blanc et al., 2005). As already mentioned, the Phg2-null mutant is defective in phagocytosis, but not in macropinocytosis. Whatever the function of this kinase in phagocytosis, the finding that its activity requires binding to PI(4,5)P2 or PI(4)P sites supports the idea that these PI sites are important for phagosome formation. This conclusion is further supported by another study, in which phospholipase D (PLD) activity was inhibited with butan-1-ol (Zouwail et al., 2005). Inhibition resulted in strong reduction of de novo synthesis of PI(4,5) P2, probably due to PLD-dependent stimulation of a PI(4)P5K activity acting on PI(4)P (Fig. 6.3). PLD is supposed to activate PI(4)P5K via production of the second messenger phosphatidic acid. Phagocytosis was partially inhibited in cells treated with butan-1-ol. F-actin recruitment to membrane extensions was also inhibited, with retraction of filopodia and pseudopodia, while most F-actin localized in intracellular spots. The bulk of

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PI(4,5)P2 is presumably not affected by the short treatment with butan1-ol, rather fluctuations in PI(4,5)P2 concentrations, regulated by PLD, have been suggested to be important for controlling local actin recruitment (Zouwail et al., 2005). Interestingly, ABD-GFP localized to the cell cortex, suggesting that the PI(3,4,5)P3 binding sites were mostly unaltered upon butan-1-ol treatment. These data support a model whereby biosynthesis or hydrolysis of PI (4,5)P2 regulate actin recruitment to the phagocytic cup and actin disassembly from the phagosome, respectively. Inhibiting PLC may result in inefficient disassembly of the actin coat, and thus inhibition of phagosomal membrane fusion and/or phagosome detachment from the membrane. Studies in mammalian cells have shown that PI(4,5)P2 sites are effectively removed from the plasma membrane by PLC activation, but not by PI3K (Stauffer et al., 1998). In Dictyostelium, the PTEN 3-phosphatase is associated with the membrane and its removal is a prerequisite for accumulation of PI(3,4,5)P3, independently of PI3K recruitment to the membrane (Iijima and Devreotes, 2002). PTEN binds to PI(4,5)P2 sites, thus it can be hypothesized that PLC activation leads to PTEN removal, and PTEN release in turn favors formation of PI(3,4,5)P3 by PI3K acting on the remaining PI(4,5)P2 sites. Indeed, PTEN is removed quite rapidly and selectively from the base of the progressing phagocytic cup (Dormann et al., 2004). PI3K could thus contribute to actin disassembly from the phagosome, but PI(4,5)P2 hydrolysis by PLC activity appears to be the most relevant factor, which would be consistent with the dynamics of PI sites and actin disassembly shown in Fig. 6.2 as well as the differential results with PLC and PI3K inhibitors on phagocytosis mentioned above. Dictyostelium PLC is similar to mammalian PLC-d, and is activated by calcium, like the mammalian homologue (Drayer and van Haastert, 1992). As mentioned, intracellular calcium chelators also inhibit phagocytosis (Peracino et al., 1998; Seastone et al., 1999). Calcium is stored in the endoplasmic reticulum (ER), and tubules of the ER have been shown to come into close association with phagosomes during uptake, though no fusion of ER membranes with the phagosome has been observed (Lu and Clarke, 2005; Muller-Taubenberger et al., 2001). It is possible that association of ER with the phagocytic cup is required for local release of calcium, following phagocytic stimuli. Calcium increase may stimulate PLC activity as well as activate proteins involved in phagosomal membrane tethering and fusion. In addition to the pharmacological approach with inhibitors, the involvement of PI3K and the antagonist PTEN phosphatase in phagocytosis has been studied with mutants. Disrupting the single pten gene has no effect on bacteria phagocytosis, but reduces yeast particle uptake (Dormann et al., 2004). There are six PI3K genes in the Dictyostelium genome, five belonging to the class IB group and one to class III. The class IB enzymes consist of the p110g catalitic subunit, and a regulatory subunit, the p101 protein. Double

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disruption of PI3K1/2 has no effect on bacteria and latex beads phagocytosis rate, but inhibits endocytosis (Buczynski et al., 1997b; Zhou et al., 1998), thus confirming the data with PI3K inhibitors. The double mutant is characterized by strongly reduced levels of both PI(3,4)P2 and PI(3,4,5)P3 as well as strong reduction of F-actin-enriched ruffles and crown-shaped membrane extensions, which are typical of pinocytosing cells (Zhou et al., 1998). The observation that this clear-cut effect on the actin cortex does not inhibit bacterial phagocytosis, though inhibiting macropinocytosis, suggests that the latter process is more sensitive than phagocytosis to changes in actin dynamics. This is in agreement with recent findings from DNA microarray studies, indicating that genes encoding actins, several actinbinding proteins and myosins are upregulated in cells undergoing macropinocytosis compared to phagocytosis (Sillo et al., 2008). A differential requirement for PI3K in phagocytosis is suggested by the work of Dormann et al. (2004). These authors found that PI3K inhibitors inhibited bacterial uptake, in contrast to previous reports. However, the assay used to measure uptake, namely a decrease in the optical density of a bacterial suspension over 7 h, does not allow distinguishing effects of the inhibitor on bacterial uptake from effects on phagolysosome maturation and bacterial degradation. In contrast, the assay used for yeast particle uptake in the same paper was a short-term assay and inhibition of uptake was evident. It is therefore possible that there is a differential requirement for PI3K, depending on the particle to be internalized. This conclusion is further supported by the finding that PTEN disruption has no effect on bacterial, but a partial inhibitory effect on yeast uptake (Dormann et al., 2004). It is conceivable that the combined activity of PLC and PI3K leads to a more rapid disappearance of PI(4,5)P2 sites, and this effect may be more relevant for phagocytosis of larger rather than smaller particles. In addition to PLC and PI3K, the product of the OCRL-1 homologue Dd5P4 gene, the inositol 5-phosphatase, also tags PI(4,5)P2, catalyzing its dephosphorylation into PI(4)P (Loovers et al., 2007). This phosphatase can also dephosphorylates PI(3,4,5)P3 (Fig. 6.3). Dd5P4-null mutants are defective in both yeast particle phagocytosis and macropinocytosis (Loovers et al., 2007). The defect in phagocytosis has been related to impaired progression and closure of the phagocytic cup, rather than particle binding or phagocytic cup formation. It has been proposed that lack of conversion of PI(3,4,5)P3 to PI(3,4)P2 may be responsible for this inhibition. However, the Dd5P4 phosphatase displays higher specificity for PI(4,5)P2 than PI (3,4,5)P3 (Fischbach et al., 2006), therefore other possible explanations are at hand: increased PI(4,5)P2 expression in the mutant, as a result of Dd5P4 gene disruption, would be consistent with the observed inhibition of phagocytosis as well as with the above-mentioned PLC inhibition data. PI (4,5)P2 persistence would lead, in both cases, to delayed actin disassembly and thus to defects in phagosome closure and further maturation. This idea

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fits also well with the finding that total PI(3,4)P2 and PI(3,4,5)P3 sites were unaltered in the mutant compared to parental cells (Loovers et al., 2007). A complication in interpreting data with the Dd5P4 phosphatase mutant is that this phosphatase also possesses a RhoGAP domain, which is highly specific for Rac1 (Loovers et al., 2007). As will be shown further below, constitutively active Rac1 inhibits both phagocytosis and macropinocytosis. The observation that the isolated catalytic domain of the phosphatase fails to rescue the Dd5P4-null mutant raises the possibility that the observed effects of gene disruption on both phagocytosis and macropinocytosis may be mediated by the RhoGAP domain rather than by the 5-phosphatase activity (Loovers et al., 2007). Taken together, this wealth of results pinpoints the importance of phosphoinositides, in particular changes in PI(4,5)P2 dynamics, for phagocytic uptake, though additional studies are required to better define the role of specific PI classes, the generating enzymes and downstream effectors in regulating the different steps in actin dynamics. The recent generation of a sextuple mutant, in which all PI3K’s plus PTEN have been deleted, may help in better defining the role of PLC. Interestingly, the sextuple mutant shows only a mild defect in chemotaxis, suggesting that actin dynamics in response to chemoattractants is essentially unaltered. The mutant grows slowly in axenic medium and on a bacterial lawn, but whether this is due to defective macropinocytosis and phagocytosis has not been reported (Hoeller and Kay, 2007). The emerging picture of phagocytosis control by modulation of PI(4,5)P2 sites in Dictyostelium is remarkably similar to what has been described for mammalian phagocytes (Scott et al., 2005). Phosphoinositides regulate also phagosomal intracellular traffic and phagolysosomal maturation (Rupper et al., 2001b). Transition from acidic to nonacidic postlysosomal vacuoles is delayed upon inhibition of PI3K either with drugs or by gene disruption, suggesting a role for this kinase in intracellular vesicle fusion at later stages of endocytosis. Indeed, both in the double PI3K1/2 mutant and after treatment of control cells with PI3K inhibitors formation of multiparticle spacious phagosomes is delayed and phagosomes remain acidic for longer time than in control cells (Rupper et al., 2001b) (see also Section 2). A similar phenotype has been described for Akt/PKB, a well-known effector of PI3K (Rupper et al., 2001b; Zhou et al., 1995). The mechanism of action of PI3K and Akt in stimulating vesicle maturation is unclear. An artificial increase in phagosomal pH has been shown to rescue formation of spacious phagosomes. It has thus been proposed that PI3K and Akt may favor delivery or removal of ion channels in the maturing phagosome, thus regulating counter ion conductance (Rupper et al., 2001b). A class III PI3K, homologous to yeast Vps34p, named DdPIK5, also exists in Dictyostelium, and DdPIK5 antisense expression inhibits growth on bacterial lawn (Zhou et al., 1995). Whether the growth defect is due to impaired phagocytosis has not been checked.

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4.3. Small G proteins of the Ras and Rac families and tyrosine kinases Dictyostelium cells possess several small G proteins of the Ras, Rac, and Rab families, as well as a large number of both GDP/GTP exchange factors (GEF’s) and G-activating proteins (GAP’s) (Vlahou and Rivero, 2006; Weeks and Spiegelman, 2003). Rac proteins in particular are well-known regulators of the actin cytoskeleton, by activating WASP/WAVE family proteins and formins (Ridley, 2006), whereas Rab’s are master timers of intracellular vesicle traffic and fusion (Zerial and McBride, 2001). Small G proteins of the Ras family act as molecular switches in signal transduction pathways and have been involved in regulating G proteindependent and -independent processes, including cell growth and cytokinesis, spontaneous and chemotactic motility, adenylyl cyclase activation, and actin polymerization (Sasaki and Firtel, 2006; Weeks and Spiegelman, 2003). Of the eight Ras genes present in the Dictyostelium genome, only two appear to be involved in phagocytosis, namely, RasS and the Ras-like protein Rap1. RasS-null mutants are characterized by many elongated actin protrusions and are defective in both phagocytosis and macropinocytosis, but display enhanced cell migration (Chubb et al., 2000). The small Ras-like protein Rap1 also regulates phagocytosis, as the phagocytosis rate of both bacteria and latex beads is increased in cells overexpressing a wildtype form of the protein and is decreased upon expression of a dominantnegative form (Seastone et al., 1999). A potential downstrean effector of RasS and Rap1 is the serine/threonine Phg2 kinase, which has been shown to interact in vitro with RasS, RasG, and Rap1. Phg2-null mutants display altered actin polymerization and are defective in phagocytosis and in adhesion to hydrophilic, but not hydrophobic, surfaces, similarly to talin- or myo VII-null mutants. They are not defective in macropinocytosis, further supporting a role for this kinase in particle adhesion and phagocytic cup formation (Gebbie et al., 2004). There are 18 genes encoding Rho GTPases in the Dictyostelium genome, all belonging to the Rac subfamily. No homologues of Cdc42 and the founding member Rho have been found (Eichinger et al., 2005; Vlahou and Rivero, 2006). Nine genes, namely, Rac1A to C and RacB, C, E, F1, G, and H, have been studied in detail. RacE, apparently regulates cytokinesis, but not phagocytosis or endocytosis (Larochelle et al., 1997). RacF1 accumulates transiently in phagocytic cup and phagosome, but its ablation has no effect on phagocytosis or endocytosis. Overexpressor, constitutive or dominant-negative mutants were not studied (Rivero et al., 1999). Rac1, RacB, RacC, RacG, and RacH appear to regulate phagocytosis, though with somewhat different effects. The Rac1 members, Rac1A, -B, and -C, display similar phenotypes, both in term of actin dynamics and effects on

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phagocytosis (Dumontier et al., 2000). Overexpression of the wild-type forms leads to formation of extended, very mobile filopodia, whereas expression of constitutive active forms stimulates crown-shaped extensions, typical of macropinocytosing cells. Bacterial and yeast phagocytosis are slightly stimulated or inhibited in cells overexpressing wild-type Rac1 or dominant-negative Rac1, respectively. In contrast, strong inhibition was found in cells expressing constitutively active Rac1. Macropinocytosis was also strongly inhibited in the latter cells (Dumontier et al., 2000). A decrease in phagocytosis rate was found in cells expressing constitutively active as well as dominant-negative RacB (Lee et al., 2003). Extensive actin protrusions were observed in constitutively active RacB cells, but it is open whether the inhibitory effect on phagocytosis is linked to these extensions, since they are not found in dominant-negative mutants. Overexpression of constitutively active RacC stimulated bacterial phagocytosis, but had the opposite effect on macropinocytosis (Seastone et al., 1998). RacC has been recently shown to regulate actin dynamics through WASP activation and to be also required for PI3K translocation to the membrane (Han et al., 2006). RacG is targeted to the rim of the progressing phagocytic cup and disappears from the phagosome immediately after its internalization. This GTPase favors formation of actin meshwork via Arp2/3, in a WASPindependent pathway (Somesh et al., 2006b). RacG overexpression or expression of a constitutively active form increased the rate of yeast particle uptake, a process that was counteracted by PI3K inhibitors. RacG ablation has no effect on yeast phagocytosis (Somesh et al., 2006b). Effects on bacterial phagocytosis have not been reported for RacG. A severe defect in both macropinocytosis and latex beads phagocytosis was observed in cells overexpressing wild-type RacH. RacH-deficient cells display, instead, normal phagocytosis rates, although fluid-phase uptake is reduced by 50%. In contrast to the other Dictyostelium Rac GTPases, RacH localizes mostly to ER and Golgi membranes. Thus, the mutant phenotypes are possibly related to altered intracellular vesicular traffic. Indeed formation of large vacuoles and recruitment of vacuolin, a marker of postlysosomal vacuoles, were impaired in the mutant, which also displays reduced acidification (Somesh et al., 2006a). These results establish Rac GTPases as important regulators of the actin cytoskeleton during phagocytosis, both during uptake and during phagosome maturation, but underline also a high degree of redundancy and overlapping functions, which explains the absence of phenotypes when single genes are disrupted. In contrast to macrophages, where they play a major role, tyrosine kinases have not been involved in Dictyostelium phagocytosis, except for a membrane tyrosine kinase-like protein, termed VSK3. VSK3 consists of a single-transmembrane domain, a C terminal catalytic domain and a TIG (immunoglobulin-like fold) domain, typical of the MET kinase family.

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The protein localizes to the surface of late endosomes or phagosomes, and its ablation leads to a reduction in bacterial and yeast phagocytosis, not however in macropinocytosis (Fang et al., 2007). Particle uptake is normal in VSK3 knockout mutant, but fusion of internalized phagosomes with endolysosomal vesicles is impaired, suggesting a role for this kinase in phagosomal maturation. The kinase is enzymatically active in vitro and the kinase domain is essential for the function of the protein (Fang et al., 2007).

4.4. The Rab family in intracellular phagosome maturation As mentioned, small G proteins of the Rab family regulate formation of transport vesicles from donor compartments, their traffic on cytoskeleton, and tethering to acceptor compartments (Zerial and McBride, 2001). In the Dictyostelium genome, 58 genes encode Rab proteins, most of which fail to undergo expression changes in a DNA microarray comparing cells engaged in phagocytosis versus macropinocytosis, suggesting that the Rab machinery is mostly unaltered in phagocytosis and macropinocytosis (Sillo et al., in press). Only a handful of Rab proteins have been studied at molecular genetic level. Consistent with the previous conclusion, mutations affecting specific Rab’s, such as RabD (related to mammalian Rab14) or Rab7, affect both phagocytosis and macropinocytosis (Harris and Cardelli, 2002; Rupper et al., 2001a). Rab7 is recruited to phagosomes immediately after actin uncoating and appears to regulate fusion of early endosomes/phagosomes with a subset of lysosomes-containing a-mannosidase and LmpA, not, however, the vacuolar ATPase (Buczynski et al., 1997a; Laurent et al., 1998; Rupper et al., 2001a). RabD appears, instead, to mediate phagosomal homotypic fusion into multiparticle large phagosomes (Harris and Cardelli, 2002). In contrast to these proteins, the homologue of mammalian Rab11 is located specifically in the contractile vacuole, but expression of a dominantnegative form leads to increased phagocytosis. A Rab protein specifically acting on phagocytosis, not pinocytosis, is Rab21 (previously termed RabB), belonging to the group V family. Constitutively active or dominant-negative Rab21 increases or decreases, respectively, the uptake rate of yeast particles (Khurana et al., 2005). Rab21 forms a complex with two interactors, a Lim-domain and a Calponin-Lim-domain protein, that activate or inhibit Rab21, respectively. The interactors are slightly enriched in the nascent phagosome, though no enrichment of Rab21 was found (Khurana et al., 2005). Also in macrophages Rab proteins have been recognized as key factors for phagosome formation and phagolysosome biogenesis although, in contrast to Dictyostelium, a specific Rab protein for phagocytosis has not been identified yet. In macrophages, activated Rab5a is recruited rapidly, already during actin assembly, and transiently to newly formed phagosomes. Recruitment of active Rab5a on phagosomes is required for the subsequent

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enrollment of Rab7 (Kitano et al., 2008; Roberts et al., 2000; Vieira et al., 2003). Early phagosomes acquire Rab7 from a soluble pool and/or by fusion with Rab7-containing endosomes. Acquisition of Rab7 enables phagosomes in macrophages to fuse with late endocytic compartments, such as late endosomes and lysosomes. Indeed, activated Rab7 recruits on the phagosomal membrane its effector RILP, which is responsible for dynein-dynactin recruitment. Recruitment of motors promotes movement of phagosomes toward the MTOC and formation of tubular extensions, which fuse with late endosomes and lysosomes, allowing acquisition of lysosomal content and maturation of phagosomes into phagolysosomes (Harrison et al., 2004). No RILP homologue has been found in the Dictyostelium genome (Bucci, unpublished observations). Phagosomal acquisition of Rab5 in macrophages stimulates phagocytosis of latex beads but not Fcg or C3 receptor-mediated phagocytosis, indicating a differential regulation of various kinds of phagocytosis (Duclos et al., 2000). Regulation of Rab function by cytokines has been recently demonstrated. Cytokines IL-6 and IL-12, through the activation of specific kinases regulate the expression of particular endocytic Rab proteins (Bhattacharya et al., 2006). Indeed, IL-6 induces expression of Rab5 through the activation of ERK leading to increased fusion of early endocytic compartments, whereas IL-12 induces Rab7 expression through the activation of p38 MAPK leading to increased lysosomal transport (Bhattacharya et al., 2006). Interestingly, the recruitment of both Rab5 and Rab7 to phagosomes is modulated by the phosphatidylinositol 3-kinase (PI3K) and inhibition of PI3K by wortmannin impairs phagolysosomes biogenesis despite the presence of active Rab7 on phagosomes (Vieira et al., 2003). Phagosome biogenesis depends on plasma membrane availability and therefore recycling it is thought to be important for phagocytosis. In mammalian cells, endosomal recycling is under the control of Rab4 and Rab11, which function sequentially. Rab4 regulates recycling from sorting endosomes and Rab11 controls transport through the perinuclear endosomal recycling compartment. Active Rab11 has been detected on nascent phagosomes in macrophages, and it is required for rapid recycling and retrieval to the plasma membrane of phagosomal content (Cox et al., 2000; Leiva et al., 2006). Indeed, it has been demonstrated that expression of a dominant-negative Rab11 mutant decreases the rate of transferrin efflux and, as a consequence, impairs FcgR-dependent phagocytosis, while expression of a constitutively active Rab11 mutant causes increased transferrin recycling and enhances phagocytosis (Cox et al., 2000; Leiva et al., 2006). Also the Rab coupling protein (RCP), which interacts with Rab4 and Rab11, has been detected on the early phagosomal membrane. RCP is localized within particular protein subdomains on the early phagosomal membranes and controls recycling from these membranes. Indeed,

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overexpression of RCP stimulates recycling and phagocytosis while expression of a truncated form inhibits them (Damiani et al., 2004). Proteomic studies have detected also Rab2, Rab5b, Rab5c, Rab9, Rab10, and Rab14 on phagosomes containing latex beads (Garin et al., 2001). The role of Rab2 in promoting engulfment and degradation of apoptotic cells has been investigated only in Caenorhabditis elegans, while a detailed study on Rab14 has demonstrated that this Rab protein is important for fusion of the mycobacterial phagosome with early endocytic compartments (Kyei et al., 2006; Mangahas et al., 2008). Mycobacterium tuberculosis survives in macrophages arresting phagosomal maturation. Rab14 silencing or expression of Rab14 dominant-negative mutants lead to phagolysosomal maturation of phagosomes containing live mycobacteria, whereas overexpression of Rab14 or of a constitutively active Rab14 mutant blocks maturation of phagosomes containing dead bacteria (Kyei et al., 2006). Rab21 and Rab22 have been initially localized to the early endosomal compartment in mammalian cells and it has been demonstrated that they regulate endocytosis and morphology of sorting early endosomes (Mesa et al., 2001; Simpson et al., 2004). Both proteins in macrophages were transiently recruited on latex beads containing phagosomes similarly to Rab5 (Roberts et al., 2006). Interestingly, on M. tuberculosis-containing phagosomes Rab22 was retained and enriched and, similarly to Rab14, maturation to phagolysosomes was impaired. Silencing of Rab22 increased phagosome maturation whereas overexpression of a constitutively active mutant prevented maturation of phagosomes-containing dead bacteria (Roberts et al., 2006). Therefore, the presence of active Rab14 and active Rab22 on phagosomes in macrophages seems to be important to inhibit or delay phagolysosomal biogenesis. Recently, comparison of phagosomes containing wild-type and a noninvasive mutant of Salmonella enterica serovar typhimurium in macrophages has allowed identification of 18 Rab proteins (included the ones previously shown as phagosome-associated), whose kinetics of association were recorded (Smith et al., 2007). Among the newly identified there are Rab8b, Rab13, Rab23, and Rab35 (Smith et al., 2007). Beside the numerous differences in the amount and in the kinetics of association/dissociation of certain Rab proteins in phagosomes containing the wild type or the mutant Salmonella, this study demonstrates that formation and maturation of phagosomes into phagolysosomes is a very complex event controlled by a network of Rab GTPases. Moreover, this study identified Rab23 and Rab35 as additional Rab proteins whose function is necessary for phagosome maturation (Smith et al., 2007). It is therefore now clear that biogenesis of phagosomes and phagolysosomes in macrophages is a process that involves several different Rab GTPases and their effectors and that cannot be explained only by the single transition between Rab5 and Rab7. We are, therefore, still far away from its complete comprehension although several

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players have now been identified. Because of the large number of genes encoding Rab proteins in the Dictyostelium genome, these cells would be excellent candidates for a systematic molecular genetic study of the Rab family in phagocytosis.

5. Host–Pathogen Interactions: A Versatile New Model Host Phagocytosis is exploited by invasive bacteria for entering the cell and proliferating in protected intracellular niches. Having examined the molecular mechanisms underlying phagocytosis, in this section we will review briefly studies on Dictyostelium cell–pathogen interactions that have shed light on genetic host determinants of susceptibility or resistance to infection by invasive bacteria. Although it was known from the early report of Depraite`re and Darmon (1978) that a few bacteria were pathogenic for Dictyostelium, the system has emerged as a suitable experimental model for bacterial infections only in recent years, the first two pioneering reports, both with Legionella pneumophila, being only 8 years old (Hagele et al., 2000; Solomon et al., 2000). Shortly later, research was extended to Mycobacterium avium and Mycobacterium marinum (Skriwan et al., 2002; Solomon et al., 2003), P. aeruginosa (Cosson et al., 2002; Pukatzki et al., 2002), Vibrio cholerae (Pukatzki et al., 2006), K. pneumoniae (Benghezal et al., 2006), and to nonculturable endosymbionts (Neochlamydia sp.TUME1 and Parachlamydia sp.UWE25) of Acanthamoeba (Skriwan et al., 2002). Worth mentioning is also Criptococcus neoformans, an environmental fungus that can cause meningitis and is also phagocytosed by Dictyostelium (Steenbergen et al., 2003). Very recently, evidence has been provided that Neisseria meningitidis, also a potent agent of fulminating meningitis, is phagocytosed by Dictyostelium cells and is, at least in part, pathogenic (Colucci et al., 2008).

5.1. Resistance/susceptibility genes of the host to infection by microbes As a soil amoeba, Dictyostelium can be natural host of opportunistic bacteria, and may thus have developed defence mechanisms against aggressive microbes. Unlike other amoebae, such as Acanthamoeba castellanii or Hartmanella vermiformis, Dictyostelium is amenable to genetic analysis. The availability of several knockout mutants and the possibility of easily screening for novel mutants with variable resistance to infection make these cells attractive for identifying host defence factors. Table 6.1 is a summary of

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Table 6.1 Host cell factors that affect Dictyostelium–pathogen interactions Host cell factor

Exp. Approach

Effects on infection

Pathogen

References

F-actin

Inhibitors

L.p.

a-Actinin/ ABP120 Coronin

Knockout

Balest and Bozzaro (unpublished results) and Lu and Clarke (2005) Fajardo et al. (2004)

Comitin

Knockout

Myosin1(A/B)

Knockout

Profilin I/II

Knockout

Daip1

Knockout

Villidin

Knockout

Lim C/D

Knockout

Gb subunit

Knockout

RacH

Knockout

Uptake down/ growth up Uptake down/ growth down Uptake down/ growth normal Uptake down/ growth up Uptake normal/ growth up Uptake normal/ growth up Uptake down/ growth normal Uptake down/ growth down Uptake down/ growth down Uptake down/ growth down Uptake down/ growth up

PLC Calcium level

Inhibitors Inhibitors

Knockout

Uptake down/n.t. Uptake down/n.t.

L.p.

L.p.

Fajardo et al. (2004) and Solomon et al. (2003) Schreiner et al. (2003)

L.p.

Solomon et al. (2000)

L.p. L.p.

Balest and Bozzaro (unpublished results) and Ha¨gele et al. (2000) Fajardo et al. (2004)

L.p.

Fajardo et al. (2004)

L.p.

Fajardo et al. (2004)

L.p.

Fajardo et al. (2004)

L.p., M.m.

Balest and Bozzaro (unpublished results) and Hagedorn and Soldati (2007) Fajardo et al. (2004) Fajardo et al. (2004)

L.p., M.m.

L.p. L.p.

Calnexin

Knockout

Calreticulin

Knockout

PI3K

Inhibitors

PI3K1/2

Knockout

Phg1

Knockout

Nramp1

Knockout Overexpression

Vacuolin B

Knockout

RtoA

Knockout

Kil1

Knockout Overexpression

TirA AMPK

Knockout Overexpression/ antisense

Uptake down/ growth down Uptake down/ growth down Uptake down/ growth up Uptake down/ growth up Uptake normal/ growth up Uptake normal/ growth up Uptake normal/ growth down Uptake normal/ growth down Uptake normal/ growth down Uptake normal/ growth up Uptake normal/ growth down N.t./growth up Growth up

L.p.

Fajardo et al. (2004)

L.p.

Fajardo et al. (2004)

L.p.

K.p.

Balest and Bozzaro (unpublished results) and Weber et al. (2006) Balest and Bozzaro (unpublished results) and Weber et al. (2006) Benghezal et al. (2006)

L.p., M.a., M.m. L.p., M.a.

Peracino et al. (2006) and Soldati (personal communication) Peracino et al. (2006)

M.m.

Hagedorn and Soldati (2007)

L.p.

Li et al. (2005)

K.p.

Benghezal et al. (2006)

K.p.

Benghezal et al. (2006)

L.p. L.p.

Chen et al. (2007) Francione and Fisher (personal communication)

L.p.

283

Pathogen uptake and intracellular growth in Dictyostelium mutants or upon treatment of wild-type cells with inhibitors (F-actin: cytochalasin A, latrunculin; PLC: U73122; intracellular calcium levels: BAPTA-AM, Thapsigargin; PI3K: wortmannin, LY24002). N.t., not tested; L.p., L. pneumophila; K.p., K. pneumoniae; M.a, M. avium; M.m., M. marinum.

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Dictyostelium genes, which have been found to affect resistance to infection by different pathogens. The system can also be used for mapping microbial virulence genes, as shown for Pseudomonas, virulent Vibrio strains (Alibaud et al., 2008; Cosson et al., 2002; Pukatzki et al., 2002, 2006), Legionella (Hagele et al., 2000; Weber et al., 2006), Klebsiella (Benghezal et al., 2006), and Neisseria (Colucci et al., 2008). The state of the art has been reviewed recently (Steinert and Heuner, 2005), therefore we will mainly examine some open questions and the latest developments on host defence factors identified in infection studies with Legionella, Mycobacteria, and Klebsiella. Microbial virulence genes will not be discussed. In contrast to Legionella and Mycobacteria, K. pneumoniae is not pathogenic for wild-type Dictyostelium cells. Sensitive to K. pneumoniae is, instead, the Phg1-null mutant (Benghezal et al., 2006). As mentioned in Section 3.1, the Phg-1 null mutant is defective in latex beads, but not bacteria uptake. K. pneumoniae is no exception, but the bacteria survive intracellularly. Suppression genetics has led to the identification of a second gene, kil1, whose overexpression in the Phg1-background rescues the mutant. The kil1 protein product is a sulfotransferase involved in sulfation of sugars and glycoproteins; sulfated glycoproteins are undetectable in the mutant. It has been suggested that Phg1 and Kil1 might be both involved in the delivery of sulfated lysosomal enzymes to the phagosome (Benghezal et al., 2006). These should, however, be rather specific for Klebsiella, as other bacterial species are killed by the double mutant. The dynamics of infection has been most studied with Legionella pneumophyla and Mycobacteria (M. avium or marinum). In contrast to Mycobacteria, which are phagocytosed by Dictyostelium cells also under shaking, Legionella is taken up, and to a rather limited extent, only if coincubated with cells in dishes; cocentrifugation is often used to improve the uptake (Solomon et al., 2000). There is some evidence that Legionella uptake occurs by macropinocytosis, rather than phagocytosis (Balest and Bozzaro, unpublished results). Both Legionella and Mycobacteria survive and proliferate intracellularly, following a route somewhat similar to what has been described for macrophages. In the case of Mycobacteria, early recruitment of V-Hþ ATPase to phagosome and acidification are strongly reduced, suggesting that phagosomal maturation is arrested or bypassed (Hagedorn and Soldati, 2007). Reduced acidification of the Mycobacterium-containing vacuole has also been reported for macrophages (Sturgill-Koszycki et al., 1994). At later stages of infection, the Mycobacteria proliferate in neutral postlysosomal, vacuolin-positive spacious vacuoles, which however do not fuse with the plasma membrane, but undergo rupture to deliver the bacteria in the cytosol (Hagedorn and Soldati, 2007). How the bacteria escape from the cytosol is unclear.

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Intracellular growth, but not uptake, is enhanced in Nramp1-null mutant for both M. avium (Peracino et al., 2006) and M. marinum (Soldati, personal communication), suggesting that control of iron homeostasis is important for establishing a friendly environment for Mycobacteria (see also next section). Remarkably, intracellular growth is also enhanced in a RacHnull mutant, but is inhibited in a vacuolin B-null mutant, in which acidification and normal phagosomal maturation are somewhat restored (Hagedorn and Soldati, 2007). Enhanced intracellular growth in the RacH-null mutant may be due to the defective acidification that has been reported for this mutant (Somesh et al., 2006a, and Section 4.3). The vacuolin B-null mutant is characterized by very large vacuoles and impairment in excretion of postlysosomal debris (see Section 3.3). Because of structural similarities with caveolin, it has been suggested that lack of the vacuolin coat may facilitate fusion/fission of the Mycobacteria-containing phagosome with vesicles of the endocytic pathway, thus favoring recruitment of vacuolar ATPase and lysosomal enzymes, which would be counteractive for mycobacterial survival (Hagedorn and Soldati, 2007). Legionella infection in the Dictyostelium host system has been extensively studied. The L. pneumophila genome analysis has identified several homologues of eukaryotic genes, and it has been speculated that the respective proteins may allow Legionella to communicate with eukaryotic cells ( Jules and Buchrieser, 2007). Moreover, the analysis of the Dictyostelium transcriptome upon infection with L. pneumophila has provided a better understanding of the manifold host cell responses (Farbrother et al., 2006). Functional annotation of the differential regulated genes revealed that by establishing its replicative niche Legionella not only interferes with bacterial degradation and intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. Functional studies revealed that uptake requires polymerization of the actin cytoskeleton, and is reduced in some mutants defective in actin-binding proteins or myosin I (Table 6.1 and Fajardo et al., 2004; Lu and Clarke, 2005; Steinert and Heuner, 2005). Following a short phase of rapid movement, apparently along microtubules, the Legionella-containing vesicle is rapidly decorated with calnexin, with calreticulin, and with the protein marker HDEL, suggesting close association with the ER and the ER/preGolgi intermediate compartment (Fajardo et al., 2004; Li et al., 2005; Lu and Clarke, 2005). During the following 2–3 h postinfection, the Legionella-containing vesicle transforms into a large replicative vacuole still decorated with calnexin and calreticulin (Fajardo et al., 2004; Lu and Clarke, 2005). These data are in agreement with studies in macrophages and support the notion that maturation of the Legionella replicative vacuole occurs by fusion with secretory vesicles recruited from the ER (Kagan and Roy, 2002; Swanson and Isberg, 1995). Similarly to macrophages, also in Dictyostelium the Legionella-containing vacuole recruits mitochondria within 30 min post-infection (Francione and Fisher, personal communication). In contrast

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to nonpathogenic bacteria, whose phagosomes rapidly fuse with V-Hþ ATPase, delivery of the vacuolar ATPase to the Legionella vacuole has not been observed during the first hours of infection (Balest and Bozzaro, unpublished results; Lu and Clarke, 2005). A partial recruitment has been reported if TAMRA- or TRITC-labeled Legionella are used for infection (Peracino et al., 2006). However, Legionella viability is strongly reduced upon fluorescence labeling, thus the V-Hþ-ATPase-decorated vesicles most likely contain sick or dead bacteria. In addition to the vacuolar ATPase, no fusion with lysosomal vesicles, assessed by using antibodies against lysosomal markers (DdLIMP), has been observed, supporting the notion that Legionella avoids quite rapidly the endosomal maturation pathway (Lu and Clarke, 2005). Similarly to macrophages (Sturgill-Koszycki and Swanson, 2000), however, the vacuole transforms late during infection into a ‘‘spacious’’ replicative vacuole decorated with the V-Hþ ATPase, and thus presumably acidic (Balest and Bozzaro, unpublished results). Phosphoinositide metabolism appears to play a major role for the establishment of the replicative vacuole. Intracellular growth of Legionella is strongly enhanced by PI3K inhibitors or in the PI3K1/2-null mutant (Weber et al., 2006). Initial docking and fusion of the Legionella-containing vacuole (LCV) with the ER are unaffected, but formation of spacious vacuoles is strongly reduced in cells lacking functional PI3K (Weber et al., 2006). The described effect is reminiscent of the inhibition of spacious postlysosomal vacuoles by PI3K inhibitors during phagosomal maturation (see Section 4.3), and suggests that any agent that delays or blocks the endocytic pathway favors the establishment of a replicative niche. Weber et al. (2006), however, provide some evidence that Legionella subverts the host cell PI metabolism, favoring enrichment of PI(4)P in the LCV. This phospholipid anchors specifically SidC, one of the secreted protein substrates of the type IV Intracellular multiplication/Defective organelle trafficking (Icm/Dot) type IV secretion system. The Legionella Icm/Dot type IV secretion system is a conjugation apparatus that is required for vesicle traffic and formation of the LCV, and is essential for Legionella pathogenicity, though not required for intracellular bacterial replication (Hilbi et al., 2001; Vogel et al., 1998). Icm/Dot substrates are of particular interest. SidM and LidA target the mammalian Rab1, a small GTPase regulating ER-toGolgi traffic. RalF recruits and activates ADP-ribosylation factor 1 (Arf1), a small GTPase involved in retrograde vesicle transport from Golgi apparatus to ER (Derre´ and Isberg, 2005; Nagai et al., 2002). The function of the SidC protein is unknown. In Legionella-infected Dictyostelium cells as well as macrophages, the protein localizes to the cytoplasmic surface of vacuoles. In the absence of functional PI3K, SidC recruitment to LCV is increased, suggesting that PI(4)P sites are enriched. Interestingly, PI(4)P sites are also enriched in vesicles harbouring wild-type Legionella compared to D-icmT/ Dot Legionella mutant, suggesting that the generation of PI(4)P sites is, at least in part, Icm/Dot dependent (Weber et al., 2006).

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Dictyostelium cells possess cytosolic Toll Interleukin1 Receptor (TIR)domain proteins (Chen et al., 2007). The tirA gene, encoding one such protein, is highly expressed in growth phase cells and in ‘‘sentinel cells,’’ the small cell subpopulation in tight aggregates and slugs, which has conserved the ability to phagocytose and is thought to act as specialized neutrophil-like clearer of pathogens in the multicellular organism (see Section 1). TirA gene disruption results in cells forming minute colonies on nonpathogenic bacterial lawn and displaying enhanced killing by Legionella (Chen et al., 2007). Remarkably, the viability of the mutant cells is reduced upon growth on nonpathogenic bacteria, suggesting a potential role of TirA in the cell response to grow on bacteria. Consistent with mitochondria recruitment to the replicative vacuole, mitochondrially-diseased cells are more susceptible to infection. Increased susceptibility appears to be linked to chronic AMPK activation, and can be suppressed by antisense inhibiting endogenous AMPK (Francione and Fisher, personal communication). Similarly to Mycobacteria, intracellular growth of Legionella is enhanced in Nramp1-null mutants (Peracino et al., 2006), establishing this protein as a crucial Dictyostelium resistance factor to infection against invasive bacteria.

5.2. The Nramp family in Dictyostelium and Nramp1 as host defence factor The Nramp1 has been widely studied in macrophages, following his discovery as a genetic factor responsible for macrophage innate resistance to various intracellular pathogens ( Vidal et al., 1993). It is part of a larger Nramp (or solute carrier 11, SLC11) family, whose members confer resistance to metal chelation in yeast, mediate dietary iron uptake in the apical membrane of epithelial cells of the brush border, and iron supply to erythrocytes (for reviews, see Forbes and Gros, 2001; Nevo and Nelson, 2006). Distant orthologs of Nramp exist in Gram-positive and Gramnegative bacteria, the prototype probably being proton-dependent manganese transporters (Richer et al., 2003). Based on sequence conservation of the overall membrane structure and functional studies in different model systems, it is established that Nramp proteins are Hþ-dependent divalent metal transporters (for a recent review, see Courville et al., 2006). Two Nramp parologs, Nramp1 and Nramp2, exist in mammals. Nramp1 is selectively expressed in the endosomal pathway of professional phagocytes, whereas Nramp2 is expressed on the cell surface or subcellular membrane compartments of most tissues. Mutations in Nramp2 functions have been discovered in patients suffering iron disorders, such as microcytic anemia or serum and hepatic overload. Nramp2 is essential for intenstinal iron transport in rodents (Gunshin et al., 2005; Su et al., 1998). Mutations in Nramp1 have, instead, been linked to innate susceptibility to mycobacterial diseases, Salmonella infection and to autoimmune diseases (Bellamy, 2003; Blackwell et al., 2003; Jabado et al., 2003; Malik et al., 2005). Dictyostelium

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cells possess two genes, Nramp1 and Nramp2, Nramp1 being closer to mammalian Nramp1 (Peracino et al., 2006). The Nramp2 gene is intronless, whereas the Nramp1 gene contains a single intron at the N-terminus, at a site conserved in all higher eukaryotes (Richer et al., 2003). Nramp1 is expressed exclusively on the membrane of endolysosomal vesicles and in the Golgi, and starts to be recruited to macropinosomes and phagosomes, containing nonpathogenic bacteria, within 2 min from uptake (Peracino et al., 2006). Nramp2 decorates, instead, exclusively the tubular network of the contractile vacuole (Peracino and Bozzaro, unpublished results). Nramp1-null mutants, as mentioned in the previous section, are more susceptible to infection by both Legionella and Mycobacteria than the parental wild-type strain. Phagocytosis of pathogenic as well as nonpathogenic bacteria is only slightly affected in the mutant, but intracellular growth of Legionella and Mycobacteria is enhanced. Constitutive Nramp1 expression rescues the mutants and protects effectively against Legionella infection (Peracino et al., 2006). Nramp2-null mutants have been recently generated, but they have not been tested so far for infection. Iron transport studies with isolated phagosomes have provided evidence in Dictyostelium for Hþ-dependent and -independent iron transport via Nramp1, suggesting that the protective role of Nramp1 is due to iron depletion from the Legionella- or Mycobacterium-containing vacuole (Peracino et al., 2006). Iron is an essential metal for all cells and is known that Legionella and Mycobacteria accumulate a large amount of iron. For Nramp1 to deplete the phagosomal lumen of iron, a functional V-Hþ ATPase is essential. The vacuolar ATPase fails, however, to be recruited to the LCV during the first hours of infection (Lu and Clarke, 2005; Peracino and Bozzaro, unpublished). Whether Nramp1 is normally recruited to the LCV is unknown. A constitutively expressed GFP fusion protein decorates about 40% of LCV’s from 2 to 24 hours of infection (Peracino and Bozzaro, unpublished). We have found, however, that Legionella represses expression of the endogenous Nramp1 gene, such that 12-h postinfection no RNA is detected (Peracino et al., 2006). It is thus possible that when the vacuolar ATPase is recruited to the Legionella-reproductive vacuole, the endogenous Nramp1 protein is no more expressed, and iron cannot be transported out of the vacuole. Expression of Nramp1 from a constitutive promoter circumvents the Legionella-induced repression of the endogenous promoter, favoring iron transport, and thus inhibiting Legionella growth.

6. Concluding Remarks Genetics is clearly the hallmark of Dictyostelium as model for phagocytosis and host–pathogen interactions, when compared to macrophages. The ease with which one can generate mutants and analyze them with all sorts of

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cell biological tools is one of the advantages offered by this amoeba, and explains the explosion of activity in the field of phagocytosis and host– pathogen interactions in the last 10 years. Recent proteomic and microarray studies have highlighted several new proteins/genes and biological processes somewhat linked with phagocytosis, which can be investigated in the near future. In addition, in-depth dissection of the well-established regulatory pathways discussed above will be made possible by novel mutants generated recently in different labs. Further studies with cytoskeletal proteins may help elucidating one of the black boxes in phagocytosis, namely, which factors are involved in closure of the phagocytic cup. Most Rab proteins still await genetic analysis in Dictyostelium, and the understanding of intracellular traffic control by Rabs also in mammalian cells would tremendously profit from this analysis. The repertoire of Dictyostelium defence mechanisms is largely unknown and its investigation may lead to identification of novel antibacterial peptides. The only drawback for infection studies is that cells do not survive at temperatures above 27 C, and this may be critical for pathogens encoding virulence factors expressed only at 37 C. In spite of this, it is possible to imagine that Dictyostelium cells may be used as easy and cheap experimental model for early screening of novel drugs against invasive bacteria.

ACKNOWLEDGMENTS Work in the laboratory of SB was supported by funds of the Italian Ministry of University and Research (PRIN program) and the Piedmont Region (RSF and RSA projects).

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Mechanobiology of Adult and Stem Cells James H.-C. Wang and Bhavani P. Thampatty Contents 303 304 306 306 307 308

1. Introduction 2. Application of External Mechanical Forces to Cells 3. Cell-Generated Mechanical Forces 3.1. Cell traction force 3.2. Cell traction force versus a-smooth muscle actin 3.3. Cell traction force microscopy 3.4. Roles of cell traction force in cell migration and tissue morphology 4. Mechanobiological Responses of Cells 4.1. Adult cells 4.2. Stem cells 5. Mechanotransduction Mechanisms 6. Concluding Remarks Acknowledgment References

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Abstract Mechanical forces, including gravity, tension, compression, hydrostatic pressure, and fluid shear stress, play a vital role in human physiology and pathology. They particularly influence extracellular matrix (ECM) gene expression, ECM protein synthesis, and production of inflammatory mediators of many load-sensitive adult cells such as fibroblasts, chondrocytes, smooth muscle cells, and endothelial cells. Furthermore, the mechanical forces generated by cells themselves, known as cell traction forces (CTFs), also influence many biological processes such as wound healing, angiogenesis, and metastasis. Thus, the quantitative characterization of CTFs by qualities such as magnitude and distribution is useful for understanding physiological and pathological events at the tissue and organ levels. Recently, the effects of mechanical Departments of Orthopaedic Surgery, Bioengineering, Mechanical Engineering and Materials Science, and Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 International Review of Cell and Molecular Biology, Volume 271 ISSN 1937-6448, DOI: 10.1016/S1937-6448(08)01207-0

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2008 Elsevier Inc. All rights reserved.

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loads on embryonic and adult stem cells in terms of self-renewal, differentiation, and matrix protein expression have been investigated. While it seems certain that mechanical loads applied to stem cells regulate their self-renewal and induce controlled cell lineage differentiation, the detailed molecular signaling mechanisms responsible for these mechano-effects remain to be elucidated. Challenges in the fields of both adult- and stem-cell mechanobiology include devising novel experimental and theoretical methodologies to examine mechano-responses more closely to various forms of mechanical forces and mechanotransduction mechanisms of these cells in a more physiologically accurate setting. Such novel methodologies will lead to better understanding of various pathological diseases, their management, and translational applications in the ever expanding field of tissue engineering. Key Words: Fibroblasts, Chondrocytes, Smooth muscle cells, Endothelial cells, Stem cells, ECM, Mechanotransduction, Tissue engineering. ß 2008 Elsevier Inc.

List of Abbreviations 2D, Two-dimensional; 3D, Three-dimensional; ACL, Anterior cruciate ligament; ALP, Alkaline phosphatase; a-SMA, a-smooth muscle actin; BMSC, Bone marrow stem cell; CBF, CCAAT-binding factor; Cbfa1, Cbfa1/Runx2 is a key transcription factor associated with osteoblast differentiation; CHP, Cyclic hydrostatic pressure; CNF-1, Cytotoxic necrotizing factor type 1; COX-2, Cyclooxygenase-2; cPLA2, calcium-dependent cytosolic phospholipase A2; CREB, cAMP-response element binding protein; CTF, Cell traction force; CTFM, Cell traction force microscopy; ECM, Extracellular matrix; EPCs, Endothelial progenitor cells; FAs, Focal adhesions; FAK, Focal adhesion kinase; FRET, Fluorescence resonance energy transfer; FPCGs, Fibroblast-populated collagen gels; GPCRs, G-proteincoupled receptors; ICAM1, Intercellular adhesion molecule-1; IGF-1, Insulin-like growth factor-1; IHP, Intermittent hydrostatic pressure; IL-1, Interleukin-1; MAPK, Mitogen-activated protein kinase; MCL, Medial collateral ligament; MCP1, Monocyte chemotactic protein-1; mDia1, A mammalian homolog of Drosophila diaphanous protein; MLCK, Myosin light chain kinase; MMPs, Matrix metalloproteinases; MSCs, Mesenchymal stem cells; NF-kB, Nuclear factor-kB; OCN, Osteocalcin; PDGF, Plateletderived growth factor; PDGFRb, Platelet-derived growth factor receptor, beta polypeptide; PDL, Periodontal ligament; PECAM-1, Platelet endothelial cell adhesion molecule-1; PGA, Poly(glycolic acid); PGE2, Prostaglandin E2; ROCK, Rho-associated kinase; SMAD, Small mothers against decapentaplegic, which are a class of proteins that modulate the activity of TGF-b

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ligands; SMCs, Smooth muscle cells; TGF-b, Transforming growth factorb; VCAM1, Vascular cell adhesion molecule-1; VEGF, Vascular endothelial growth factor; VEGFR2, Vascular endothelial growth factor receptor 2; VSMC, Vascular smooth muscle cell.

1. Introduction Mechanical forces that originate externally from the environment influence many aspects of human health and disease (Banes et al., 1990). Gravity, tension, compression, fluid shear stress, and hydrostatic pressure are just a few examples of the forces that constantly act on cells within organs and tissues (Davies et al., 1995; Grodzinsky et al., 2000; Kakisis et al., 2004; Lehoux et al., 2006; Silver et al., 2003; Wang, 2006). Besides these external mechanical forces, cells also generate their own mechanical forces, known as cell traction forces (CTFs). Cells use CTFs to migrate, maintain their shape, and generate mechanical signals. As such, CTFs play a fundamental role in many biological processes such as wound healing, angiogenesis, and metastasis (Wang and Lin, 2007). Different types of cells in the body are subjected to various levels of mechanical forces. Fibroblasts of the skin, lung, heart, tendons and ligaments, vascular smooth muscle cells (SMCs) and endothelial cells in blood vessels, and chondrocytes in cartilage are all types of cells that are subjected to large mechanical forces, or loads, and are referred to as mechano-responsive cells. Cellular responses to loads depend on loading conditions (e.g., the type, magnitude, duration, and frequency of loading); they also depend on cell type, cell source, developmental stage, and cell microenvironment, such as surrounding matrix proteins as well as soluble factors (Frangos, 1993; Grinnell, 2003; Vandenburgh, 1992). Cells use multiple sensing mechanisms to detect mechanical loads and transduce them into intracellular signals that lead to modulation of many vital cellular functions, such as proliferation, differentiation, migration, adhesion, apoptosis, and gene and protein expression (Bartling et al., 2000; Chien et al., 2005; Geiger and Bershadsky, 2002; Hsieh and Nguyen, 2005; Pradhan and Sumpio, 2004; Sarasa-Renedo and Chiquet, 2005; Wang et al., 2007). The mechanotransduction by which cells transduce mechanical forces into biochemical responses have been under intensive investigation for the past two decades. Highly coordinated extensive cellular components including the cytoskeleton, adhesion complexes, and ion channels have been implicated as the predominant mediators of mechanotransduction (Burridge and Chrzanowska-Wodnicka, 1996; Ingber, 1991; Sadoshima and Izumo, 1997).

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While it has been long established that mechanical forces are essential in the regulation of tissue homeostasis and remodeling, only recently has the concept that mechanical forces are also essential regulators in the development of successful tissue engineering constructs for tissue repair and replacement become evident (Akhyari et al., 2002; Garvin et al., 2003). A recent advance made in the tissue engineering field is the selective differentiation of mesenchymal stem cells (MSCs) into specific cell lineages by applying various mechanical loading conditions, along with providing the appropriate matrix environment and biochemical factors (Altman et al., 2002; Huang et al., 2004a; Park et al., 2007). Although many types of cells respond to mechanical forces much like they respond to biochemical stimuli, cellular mechanotransduction mechanisms, especially for stem cells, are far less explored and understood.

2. Application of External Mechanical Forces to Cells Over the years, various in vitro systems have been developed by applying mechanical forces to a population of cells to study cellular mechanobiological responses (Brown, 2000; Huang et al., 2004b) as well as mechanics of single cells such as cellular deformation and viscoelasticity (Bao and Suresh, 2003; Zhu et al., 2000). These systems take into account in vivo loading conditions of various types of cells. For fibroblasts, epithelial cells, and SMCs, mechanical stretching was applied to a population of cells using deformable elastic substrates (e.g., silicone membrane) that were coated with extracellular matrix (ECM) proteins for promoting cell attachment (Banes et al., 1985; Brown, 2000; Leung et al., 1977; Wang et al., 1995). For endothelial cells lining the blood vessel wall, fluid shear stresses were applied using flow chambers (Brown, 2000; Chun et al., 1997; Hermann et al., 1997), as these cells are predominantly subjected to shear stress from blood flow in vivo (Davies, 1995). On the contrary, chondrocytes in cartilage are subjected to compression as well as hydrostatic pressure in vivo. Therefore, corresponding mechanical forces were applied to these cells using various types of in vitro systems (Brown, 2000; Huang et al., 2004b). One advantage of these systems is that the loading parameters, such as loading magnitude and frequency, can be easily controlled. Another advantage of the systems is that the mechanical properties of the substrates (e.g., stiffness) and their surface chemistry can be readily modified. When stretching cells, the substrate underlying a population of cells can be stretched either uniaxially or biaxially. Under uniaxial stretching, the substrate is lengthened in its stretching direction whereas it is compressed in its perpendicular direction. This type of stretching is quite suitable for mechanical loading of cells from tendons and ligaments, such as the patellar

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tendon and anterior cruciate ligament (ACL), as these cells are aligned with their long axis along the tendons or ligaments and are therefore subjected to mainly uniaxial stretching in vivo. On the contrary, biaxial stretching can be applied to the substrate by stretching it in two mutually perpendicular directions. The stretching, however, can be either an equibiaxial stretch, where substrate strains are the same in all directions, or nonequibiaxial stretch, where substrate strains vary with respect to stretching direction (Lee et al., 1996). This type of mechanical stretching is most suitable for dermal fibroblasts, as they are randomly oriented in the ECM and are stretched in all directions in vivo. Several biaxial stretching systems have been developed to provide a mechanical environment for cultured cells with a number of advanced features, including input force quantitation, loading parameter controls, and homogenous deformation of a cell population (Lee et al., 1996; Sotoudeh et al., 1998; Waters et al., 2001). However, there are a few limitations associated with these systems. The first is that the input strain is measured on the loading system (referred to as a ‘‘clamp-to-clamp’’ strain), or on the substrate (referred to as substrate strain), not actually on the cells. Similarly, the second limitation is that only a fraction of the input strain may actually be delivered to the cells. This limitation is partially due to the differential adherence of individual cells in the population of cells: some cells may adhere to the matrix more strongly than others, subjecting them to different strain levels. Consequently, cellular responses in these in vitro systems are heterogeneous, and gene and protein expressions measured only represent the average response of a population of cells to mechanical stretching. An additional limitation associated with uniaxial stretching systems is that cells on the substrate assume a random orientation in static culture, but when being stretched, they orient away from the stretching direction and toward a direction that has minimal substrate deformation (Wang et al., 1995, 2001b). Consequently, the strains acting on the cells that have reoriented are minimal. To overcome this cell reorientation problem, cell alignment was induced using microgrooved substrates (Mata et al., 2002; Walboomers et al., 1999, 2000; Wang and Grood, 2000). Fibroblasts on microgrooved substrates were shown to align with the microgrooves and maintain an elongated shape, mimicking the cell alignment and orientation in vivo (Wang et al., 2003b) (Fig. 7.1). Moreover, application of cyclic uniaxial stretching to cells on the microgrooved surface did not change cell alignment regardless of initial cell orientation with respect to stretching direction (Loesberg et al., 2005; Wang et al., 2005b). The microgrooved substrate has also been used to show that the mechanobiological response of fibroblasts to mechanical stretching depends on their orientation with respect to stretching direction (Wang et al., 2004b). An additional advantage of using the microgrooved substrate is that it can also control the organization of collagen matrix produced by cells in culture (Wang et al., 2003a).

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A Reorientation

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Figure 7.1 The need to control cell alignment and organization in studying cellular mechanobiological response.When cells are grown on smooth culture surfaces and subjected to cyclic uniaxial stretching, they tend to reorient toward a direction with minimal substrate deformation (A). This cell reorientation during mechanical stretching makes it complicated, if not impossible, to properly interpret experimental results as substrate strains acting on cells during stretching keep changing. One approach to control such cell reorientation response is using microgrooved substrate (B). Regardless of stretching or not, cells remain aligned in microgrooves and are therefore subjected to relatively constant stretching. A more physiological experimental model is including matrix such as collagen gel (C) to surrounding cells as in vivo. In such a model, the cells deform the collagen gel matrix because of the traction forces they produce and at the same time are subjected to external mechanical stretching. One disadvantage of using collagen gel is its low mechanical strength for mechanical stretching. Therefore, many bioscaffolding materials with a higher mechanical strength are used to embed cells and apply mechanical loads to cells.This approach has been widely used in functional engineering of tissue constructs.

3. Cell-Generated Mechanical Forces 3.1. Cell traction force Cells can respond to external mechanical forces, but just as importantly, cells also generate internal forces, or cellular contraction, resulting from actin– myosin interactions. Intracellular contraction is transmitted to the underlying substrate, and the forces on the substrate are called CTFs. CTFs are essential for cells to migrate, maintain shape, and generate mechanical

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signals (Wang and Lin, 2007). As such, CTFs are implicated in many biological processes including wound healing, embryogenesis, angiogenesis, and inflammation (Li et al., 2007). Detailed knowledge of how CTFs are regulated and transmitted to the ECM is thus important for understanding physiological and pathological events at the tissue and organ levels. Several intracellular proteins, including Rho proteins, Rho-associated kinase (ROCK), and mammalian homolog of Drosophila diaphanous protein (mDia1) (Anderson et al., 2004), are known to regulate the formation of stress fibers and focal adhesions (FAs), and thus CTF generation and transmission (Watanabe et al., 1999). Moreover, mitogen-activated protein kinases (MAPKs) can phosphorylate myosin light chain kinase (MLCK) and increase MLC phosphorylation, which leads to actomyosin contraction and hence, CTF generation.

3.2. Cell traction force versus a-smooth muscle actin During wound healing, large traction forces are generated at the wound site by myofibroblasts. Myofibroblasts contain a contractile apparatus—actin filaments and nonmuscle myosin II, which is similar to that of SMCs (Gabbiani, 2003; Tomasek et al., 2002). Unlike fibroblasts, however, myofibroblasts express a-smooth muscle actin (a-SMA), form a-SMA-containing stress fibers (Grinnell, 1994), and generate greater contractile force than fibroblasts (Hinz et al., 2001). a-SMA is a prominent actin isoform in vascular SMCs and generally comprises 14–18% of total actin content (Arora and McCulloch, 1994). The actin isoform has been recognized as the underlying molecule that enhances traction forces of myofibroblasts (Herman, 1993; Serini and Gabbiani, 1999). Overexpression of a-SMA upregulates the myofibroblast traction force (Hinz et al., 2001). A recent study was able to show that while a-SMA is not required for CTF generation, its expression upregulates CTF magnitude in a nearly linear fashion (Chen et al., 2007). While appropriate traction forces of myofibroblasts are required for wound closure, ECM regeneration, and remodeling, the excessive traction force of myofibroblasts that persistently exist in the wound site is responsible for wound contracture, fibrosis, and other fibro-proliferative disorders during pathological conditions (Gabbiani, 2003). The mechanisms by which a-SMA protein expression regulates myofibroblast traction force are currently under investigation. They may be related to modification of stress fibers and FAs. In myofibroblasts, there is an abundant a-SMA in stress fibers and at FA sites (Hinz et al., 2001). The presence of a-SMA may allow the stress fibers and FAs to sustain large traction forces (Goffin et al., 2006; Wang et al., 2006a). Also, the incorporation of a-SMA into actin filaments may enhance force transmission between cortical actin filaments and FAs (Dugina et al., 2001; Hinz and Gabbiani, 2003). The N-terminal sequence AcEEED of a-SMA has been identified to

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be crucial for incorporation of a-SMA into stress fibers and for force generation (Chaponnier et al., 1995; Clement et al., 2005). Finally, high levels of a-SMA expression may increase CTF by Rho-dependent activation in addition to enhancing stress fiber formation (Bogatkevich et al., 2003; Skalli et al., 1990). It is well recognized that transforming growth factor-b (TGF-b) promotes differentiation of fibroblastic cells into myofibroblasts by upregulation of a-SMA and thus enhances CTF (Desmouliere et al., 1993; Evans et al., 2003; Kopp et al., 2005). The TGF-b-induced a-SMA expression requires the induction of the extra type III domain A (ED-A) form of the matrix protein fibronectin and signaling molecules of small mothers against decapentaplegic (SMAD) family (Kobayashi et al., 2006; Moustakas et al., 2001; Serini et al., 1998). Many soluble factors such as basic fibroblast growth factor (bFGF), prostaglandin E2 (PGE2), and g-interferon inhibit TGF-b 1-upregulated a-SMA expression and thus likely cause the downregulation of CTF (Burgess et al., 2005; Hjelmeland et al., 2004; Kawai-Kowase et al., 2004; Kolodsick et al., 2003; Yokozeki et al., 1999).

3.3. Cell traction force microscopy To determine CTFs, several methods have been developed (Wang and Lin, 2007). These include the use of thin silicone membranes (Harris et al., 1980), microfabricated cantilevers (Galbraith and Sheetz, 1997), micropost force sensor arrays (Li et al., 2007; Tan et al., 2003), and cell traction force microscopy (CTFM) (Butler et al., 2002; Dembo and Wang, 1999; Yang et al., 2006). A unique strength of CTFM is that it can quantify traction forces of both individual cells and a group of cells (Li et al., 2008; Wang et al., 2002). The current CTFM methods involve three major steps. The first step involves three parts: (a) plating cells on an elastic polyacrylamide gel (PG) embedded with fluorescent microbeads in a plastic dish; (b) taking digital images of individual cells on the PG substrate using a charge-coupled device (CCD) camera system on an inverted microscope, which yields ‘‘force-loaded’’ images; and (c) removing cells and then taking digital images at the same location, which yields a ‘‘null-force’’ image. The second step determines the substrate displacement field produced by CTFs by pairing ‘‘null-force’’ and ‘‘force-loaded’’ images using image analysis algorithms. The third and final step determines the CTFs from the substrate displacements by computation based on elasticity theory (Wang and Lin, 2007) (Fig. 7.2). A limitation of using the CTFM method to determine CTFs is that, unlike in vivo, cells reside on a two-dimensional (2D) substrate without the surrounding ECM. An alternative approach to assess CTFs is the use of collagen gels to embed cells. Fibroblast-populated collagen gels (FPCGs) is such a commonly used experimental model. Fibroblasts within the gels

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Figure 7.2 (A). Schematic of cell traction force microscopy (CTFM).The CTFM technique involves three major steps (numbered 1, 2, and 3. See also the text for detailed description). Using CTFM, traction forces of individual cells can be determined; (B). the CTFs of a fibroblast; and (C). the CTFs of a myofibroblast. Adopted from Fig. 1 in Chen et al. (2007).

generate traction forces, which deform the gels (Cukierman et al., 2002; Grinnell, 2003). The FPCG contraction may be estimated by measuring area changes in FPCG (Campbell et al., 2004) or quantified using a culture force monitor (Campbell et al., 2003). These two methods have been used to show that TGF-b1 induces a larger contraction than TGF-b3 (Campbell et al., 2004), and that ‘‘healing ligament fibroblasts,’’ even after being

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passaged a few times, generate a larger contraction than their normal counterparts (Agarwal et al., 2006), suggesting that cells after culturing retain their phenotype in vivo to certain degrees.

3.4. Roles of cell traction force in cell migration and tissue morphology CTFs are essential for enabling cell migration. There are three sequential steps in each cell migration event: (a) the protrusion force generated at the cell’s leading edge by one-directional actin polymerization drives the formation of new lamellipodia and filopodia and FAs at the front; (b) the CTFs pull the cell body forward; and (c) the cell detaches at the rear. The direction of the CTFs at the front and the rear of the cell always points inward (Galbraith and Sheetz, 1997). These centripetal CTFs work toward breaking cell’s FAs (Cramer et al., 1997). As the migrating cell forms stronger FAs at the front than at the rear (Schmidt et al., 1993), the imbalance between the adhesion force and the traction force at the front edge and at the rear is the net driving force for cell migration. In normal fibroblasts, the lamellipodia provides nearly all the forces needed for protrusion of the front edge. The pattern changes of CTF often occur ahead of changes in the cell migration direction. This suggests a front locomotion mechanism of cell migration in which the dynamic traction force at the cell’s front actively pulls the cell body forward. When fibroblasts are transformed with H-ras, spots of weak, transient CTFs are scattered among small pseudopods and arranged in random directions. These weak, randomized CTFs result in the abnormal migration behavior of H-ras-transformed fibroblasts (Munevar et al., 2001). Besides its role in cell migration, CTF also regulates tissue patterning and morphogenesis by modulating both ECM and cell growth. It has been noted that the traction force generated by a migrating cell normally ranges from tens to even hundreds of nano-Newtons (Burton et al., 1999; Lee et al., 1994; Tan et al., 2003), a force that is far greater than the net force that pulls the cell forward (Lee et al., 1994). The larger-than-needed CTFs have been postulated to be used by fibroblasts to help construct connective tissue during morphogenesis (Stopak et al., 1985). When fibroblasts are embedded in collagen gel, the CTF makes the collagen fibers aligned, creating patterns which are similar to tissue or organ capsules (Harris et al., 1981). Also, when fluorescently labeled collagen is injected into chicken embryos, CTFs are thought to responsible for the rearrangement of the collagen gel into anatomical patterns (Stopak et al., 1985). CTF regulates morphogenesis through modulation of Rho signaling. Epithelial branching is prohibited in embryonic mouse lung rudiments when cell tension is dissipated by treatment with ROCK inhibitor Y27632. On the contrary, lung branching is greatly enhanced when CTF is increased with Rho activator CNF-1 (Moore et al., 2005).

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4. Mechanobiological Responses of Cells 4.1. Adult cells Mechanical forces are potent regulators of matrix gene and protein expression in various types of cells, particularly those from connective tissues subjected to large mechanical forces in vivo, such as skin, tendon, and ligaments (Chiquet, 1999; Shimizu et al., 1998; Wang et al., 2007). Collagen is the most abundant component of the ECM in these and many other tissues and therefore, many in vivo and in vitro investigations of mechanical loading effects have focused on the relationship between mechanical loads and collagen synthesis. While it is difficult to draw general conclusions from in vitro studies due to the array of factors affecting cellular mechanobiological response, many in vitro studies have consistently shown enhanced cellular collagen expression in response to mechanical loading, which is associated with interactions with exogenous growth factors in various types of cells and/or the production of autocrine growth factors by loaded cells (Kim et al., 2002; Mouw et al., 2007; Nakatani et al., 2002; O’Callaghan and Williams, 2000). For example, when fetal rat cardiac fibroblasts were grown on an elastin substrate, cyclic biaxial stretching increased type I procollagen synthesis in the presence of serum or growth factors such as TGF-b1 and IGF-1 (Butt and Bishop, 1997). Adult rat cardiac fibroblasts on a collagen substrate responded to 10% static uniaxial stretching with elevated TGF-b1 activity as well as increased mRNA levels of collagen type III without affecting collagen type I mRNA levels (Lee et al., 1999). A similar increase in collagen type III mRNA levels was observed in neonatal rat cardiac fibroblasts on a laminin substrate under biaxial stretching (Atance et al., 2004). The speculation is that type III collagen mRNA is increased soon after the onset of mechanical loading while there is a delay in the stimulation of collagen type I mRNA. These events closely resemble those of in vivo animal models of pressure-overloaded hypertrophied myocardium, where an increase in type III collagen is often seen early on followed by a large sustained increase in type I collagen (Cleutjens et al., 1995). This phenomenon may indicate the extent of tissue damage and wound healing, since early deposition of type III collagen followed by type I collagen is characteristic of tissue repair (Woo et al., 1999). This phenomenon is further emphasized by the differential responses of ACL fibroblasts and medial collateral ligament (MCL) fibroblasts to mechanical loading. It is known that an injured ACL does not heal, whereas an injured MCL heals well (Frank et al., 1983a,b). In support of this clinical observation, in vitro experiments show that cyclic biaxial stretching of ACL fibroblasts increases only type I collagen mRNA expression whereas MCL fibroblasts under similar stretching conditions respond with an increase in

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type III mRNA expression and a decrease in collagen type I mRNA expression (Hsieh et al., 2000). While it is known that cells respond to mechanical forces by changing their expression of genes such as collagen, the cellular mechanobiological response is generally cell type-dependent. For example, the application of uniaxial stretching of ACL fibroblasts increased both collagen type I and collagen type III mRNA levels (Kim et al., 2002). However, tendon fibroblasts under uniaxial stretching increased collagen type I mRNA levels without a significant change in collagen type III mRNA, and this increase in collagen type I gene expression was stretching magnitude-dependent (Yang et al., 2004). On the contrary, tendon fibroblasts in collagen gels subjected to cyclic uniaxial stretching expressed collagen genes I, III, and XII as well as fibronectin and tenascin (Garvin et al., 2003), suggesting that the ECM influences cellular mechanobiological response. The type of loading condition also has differential effects on periodontal ligament (PDL) fibroblast response. Cyclic equibiaxial stretching increased the expression of collagen type I, whereas the same magnitude of cyclic compression decreased it (Howard et al., 1998). This phenomenon may explain in vivo tooth remodeling, as PDL in vivo is subjected to tension on one side but compression on the other side, and the tension side is characterized by bone synthesis whereas the compression side is characterized by bone resorption. Like fibroblasts, chondrocytes are also responsive to mechanical forces (Hall et al., 1991; Lammi et al., 1994). Compression, a major form of mechanical loading on cartilage, modulates cartilage-specific macromolecule biosynthesis and matrix deposition by chondrocytes (Guilak et al., 1994). The matrix molecule expression of chondrocytes depends on loading magnitude, frequency, and duration (Gray et al., 1989; Ragan et al., 1999). Two major cartilage matrix components, aggrecan and type II collagen, are independently regulated by intermittent hydrostatic pressure (IHP). For example, expression of aggrecan mRNA levels increased in response to low magnitudes and short duration of IHP (Ikenoue et al., 2003; Smith et al., 2000). Type II collagen mRNA levels, on the contrary, were not affected by short duration or low magnitude and increased only at higher magnitudes applied at intervals and longer durations. In addition to the types of cells and mechanical loading, cell–ECM interactions are also known to affect collagen expression. For example, fetal lung fibroblasts responded to cyclic biaxial stretching by enhancing collagen type I mRNA expression when the cells were cultured on a laminin or elastin substrate but not on a fibronectin substrate (Breen, 2000). Cell response also varied depending on whether stretching was applied statically or dynamically. A 5% static stretching increased ratio of collagen type III to type I by 5% as compared to unstretched controls, whereas a 5% cyclic stretching induced a 70% increase (Carver et al., 1991).

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The load-induced collagen expression in many types of cells has been related to the response of growth factors to mechanical loading. TGF-b1 is one of the most potent inducers of procollagen a1(I) gene expression (Coker et al., 1997). In two studies using tendon fibroblasts and ACL fibroblasts, cyclic uniaxial stretching increased collagen expression with a concomitant increase in expression of TGF-b1 mRNA and protein (Kim et al., 2002; Yang et al., 2004). In human dermal fibroblasts, a similar parallel increase in TGF-b1, procollagen a1(I) mRNA levels, and total collagen synthesis occurred when cells were subjected to cyclic biaxial stretching (Parsons et al., 1999). A TGF-b1-mediated increase in gene expression of collagen type I, III, and V was observed in response to cyclic biaxial stretching in human ligament cells (Nakatani et al., 2002). Vascular SMCs also responded to cyclic stretching by increasing collagen type I mRNA expression with a parallel increase in TGF-b expression ( Joki et al., 2000; Li et al., 1998). Endothelial cells increased TGF-b mRNA expression and total collagen synthesis in response to cyclic stretching as well (O’Callaghan and Williams, 2000). The presence of two potential strain response regions within the proximal promoter may be responsible for the stretching-induced collagen expression that have been identified (Lindahl et al., 2002). One contains an inverted CCAAT-box, whose binding activity of CCAAT-binding factor, CBF/NF-Y, is enhanced by both mechanical stretching and TGFb1 at this site (Lindahl et al., 2002). CBF regulates human COL1A1 promoter activity in human dermal fibroblasts, and binding activity is higher in scleroderma fibroblasts, which produce excessive collagen (Saitta et al., 2000). This observation suggests that transcription factor CBF/NF-Y may be involved in the upregulation of collagen gene expression. Furthermore, CBF has been shown to bind to SMAD proteins, the major components of TGF-b signaling pathway, and Sp1, a human transcription factor (Bishop and Lindahl, 1999; Chen et al., 1999). SMAD and Sp1 proteins cooperate to mediate TGF-b1-induced collagen expression (Poncelet and Schnaper, 2001). Also, mechanical compression of rabbit chondrocytes transfected with human COL2A1 gene increased the level of type II collagen mRNA expression by transcriptional activation, possibly through the Sp1-binding sites residing in the proximal region of COL2A1 gene promoter (Mouw et al., 2007). Besides inducing the expression of collagen and TGF-b, mechanical stretching also elicits a cellular inflammatory response. In tendon cells, application of uniaxial mechanical stretching increased the expression of calciumdependent cytosolic phospholipase A2 (cPLA2) and cyclooxygenase2 (COX-2), and the production of PGE2 at stretching magnitudes of 8% and 12% (Wang et al., 2003b, 2004a). Similarly, tendon explants subjected to cyclic compressive loading at 3 MPa and 12 MPa induced a loading magnitude and duration-dependent increase in the production of PGE2, a product of

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the inducible enzyme COX-2 (Flick et al., 2006). COX-2 is a mechanosensitive enzyme as demonstrated in the dynamic compression of cartilage explants (Gosset et al., 2006), where cartilage explants under intermittent compression and dynamic compression increased COX-2 expression and PGE2 production (Fermor et al., 2002; Gosset et al., 2006). Mechanical stretching of tendon cells also caused a synergistic effect on production of matrix metalloproteinases (MMPs) with interleukin-1b (IL-1b) (Archambault et al., 2002; Yang et al., 2005). IL-1b is a potent inflammatory cytokine, which induces expression of catabolic mediators MMPs, COX-2, and PGE2 in tendon fibroblasts (Thampatty et al., 2007; Yang et al., 2005). In PDL fibroblasts, similar catabolic effects due to mechanical stretching were also noted (Shimizu et al., 1998; Yamaguchi et al., 1994). In chondrocytes, large cyclic tensile loading (15–18% equibiaxial strains) increased proinflammatory gene expression as evidenced by enhanced iNOS (inducible nitric oxide synthase) RNA and nitric oxide (NO) production (Agarwal et al., 2004). The inflammatory process of vascular endothelial cells and SMCs are also regulated by mechanical forces in the form of shear stress (Cunningham and Gotlieb, 2005; Harrison et al., 2006). Shear stress increases intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin expression induced by TNF-a in endothelial cells (Chiu et al., 2004; Walpola et al., 1995). Nonlaminar flow can result in gene expression of proinflammatory transcription factors such as nuclear factor-kappaB (NF-kB) in the vascular wall (Brooks et al., 2004; Nagel et al., 1999). The activation of NF-kB generally leads to increased expression of proinflammatory genes including those encoding cytokines, VCAM1, ICAM-1, and tissue factor monocyte chemotactic protein-1 (MCP-1) (Harrison et al., 2006). NF-kB activation and ICAM-1 expression depend on the integrin activation pathway in vitro. Fluid shear stress triggers the conformational activation of integrins which mediate NF-kB activation. A pathway upstream of integrin activation identified is a mechanosensory complex that comprises platelet endothelial cell adhesion molecule-1 (PECAM-1), VE cadherin, and vascular endothelial growth factor receptor-2 (VEGFR-2). In PECAM-1-knockout mice, NF-kB and downstream inflammatory genes in the regions of disturbed flow are no longer activated (Tzima et al., 2005). While the in vitro studies described above provide useful insight into the role of mechanical forces in tissue homeostasis and pathophysiology, there are a number of factors that still need to be taken into account for proper interpretation of experimental data. For example, cell shape is usually not controlled in these studies in vitro, so cells are likely in different phenotypic states. Micropatterning technology has been demonstrated to be able to control for cell shape (Chen et al., 1998), and this technology may be a useful tool in precisely defining cellular mechanobiological responses in future studies. Cell orientation is another factor that needs to be considered

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in cell stretching experiments, as cells on smooth culture surfaces under cyclic uniaxial stretching are known to reorient toward a direction along which mechanical force or substrate deformation on the cells is minimal (Wang et al., 2001b). Thus, the cell reorientation must be prevented so that individual cells are all subjected to the same substrate strains during cell stretching experiments. One solution to this problem would be to use equibiaxial stretching systems, which produce isotropic strains on smooth surfaces such that cells are subjected to the same surface strains regardless of their orientation. However, equibiaxial stretching may not be physiological for those cells such as tendon and ligament fibroblasts that are under uniaxial stretching in vivo. Another solution, as noted earlier, is the use of microgrooved substrate to control cell orientation by taking advantage of cell contact guidance behavior (Wang and Grood, 2000). There are other factors that also need to be carefully considered. Surface topography is one such important factor that can influence cell shape, orientation, and adhesion on the substrate (Chou et al., 1995). The coating of a substrate with an ECM protein is another important factor that can modulate cell function (Breen, 2000; Reusch et al., 1996). The ECM proteins typically used as substrate coating for cell attachment include collagen type I, fibronectin, elastin, and laminin. It is known that cell responses to mechanical loading depend on the type of matrix protein coated. For example, collagen type I is expressed by fibroblasts cultured on laminin and elastin but not on fibronectin under mechanical loading conditions (Breen, 2000). Similarly, SMCs increase myosin expression in response to mechanical loading when cultured on laminin or collagen matrix but not on a fibronectin matrix (Reusch et al., 1996). Since heterodimeric integrin family constitutes major cellular receptors for ECM proteins, binding to different integrin types may elicit different signaling pathways. Therefore, the matrix protein selected for the coating of a substrate is crucial in determining specific cellular mechanobiological responses. Finally, while the in vitro systems used in these studies offer convenience in investigating cellular mechanobiological responses, it is considered less physiological because cells on the 2D substrates (e.g., silicone membrane) often used in cell stretching experiments lack the ECM surrounding the cells. Therefore, three-dimensional (3D) experimental models have been developed to include collagen matrices, which allow the study of cellular mechano-responses in a system that is more representative of the in vivo environment (Grinnell, 1994, 2003). The ability of cells to generate tension within a collagen matrix is crucial in determining cell fate, as fibroblasts that can generate tension in attached collagen matrices proliferate, whereas cells that cannot in floating matrices become quiescent and apoptotic (Grinnell et al., 1999; Rosenfeldt and Grinnell, 2000). The utility of collagen gels, however, is limited by their low mechanical strength to sustain repetitive mechanical loading, which is a significant drawback for the development of

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tissue constructs in the tissue engineering field. Biomaterials with a large mechanical strength were therefore used as scaffolding materials (Wang et al., 2006b). One example is the native silk fibroin fibers, which has been explored for engineering the ACL in cultures under dynamic mechanical loading (Vunjak-Novakovic et al., 2004). Another example of biomaterials includes poly(glycolic acid) (PGA) (Kim et al., 2000). Application of cyclic mechanical stretching to fibronectin-coated PGA scaffolds seeded with SMCs upregulated cellular expression of elastin and collagen and also increased the Young’s modulus and ultimate strength of the tissues (Kim et al., 1999). The findings of this study suggest that the appropriate combinations of mechanical loading and polymeric scaffolds can enhance the mechanical properties of the tissues.

4.2. Stem cells Stem cells are cells characterized by the abilities to perpetuate themselves through self-renewal and to generate mature cells of a particular tissue through differentiation. Stem cells are rare in most tissues, and it appears that each tissue arises from tissue-specific stem cells (Reya et al., 2001). Currently, most studies focus on stem cell biology, including isolation, identification, and characterization; fewer studies have investigated the effects of mechanical loading on stem cells (Table 7.1), which is vital for gaining a better understanding of the biology and pathology of load-bearing tissues. Mechanical loading is essential for the development, function, and repair of the major components of the musculoskeletal system such as bones, tendons, ligaments, and cartilage. Much work concerning the mechanobiological responses of stem cells arise from the desire to create functional tissue engineering constructs. For example, concurrent application of tensile and rotational loading to human and bovine bone marrow stem cells (BMSCs) in collagen gels resulted in several features characteristic of ligament cells, including expression of collagen types I and III, and tenascin-C, increased cell alignment and density, and the formation of oriented collagen fibers (Altman et al., 2002). The findings of this study show that mechanical loading alone can direct BMSC differentiation into a preferential ligament cell lineage without specific inducers of ligament cell differentiation. In another study, tissue engineering constructs were created for tendon repair using rabbit MSCs in type I collagen sponges ( Juncosa-Melvin et al., 2006). Compared to nonloading constructs, cyclic mechanical stretching of the tissue constructs resulted in three- and fourfold gene expressions of collagen type I and III, respectively, and 2.5 times greater stiffness of the tissue construct ( Juncosa-Melvin et al., 2006, 2007). With appropriate mechanical loading, human MSCs (hMSCs) also can differentiate along the osteogenic pathway without the need for osteogenic supplements. For example, when subjected to uniaxial cyclic stretching,

Table 7.1 Mechanobiological responses of stem cells

Type of cells

Type of loading

Loading conditions

Human and bovine BMSCs

Stretching and torsion

Translational 10%, rotational 25% in collagen gels

Rabbit MSC

Cyclic stretching

4% in collagen gels, 8 h/day for 2 weeks

Human MSCs

Cyclic uniaxial stretching

Human MSCs

Cyclic equibiaxial stretching

10% and 12%, 4 h/ day for 7 and 14 days in collagen matrices 3%, 8 h

8%, 48 h

Response of stem cells

Cell lineage commitment

Increased expression of collagen types I and III, tenascinC, increased cell alignment, density, formation of oriented collagen fibers Increase in gene expression of collagen types I and III Increase in BMP2 mRNA levels

Ligaments

Altman et al. (2002)

Tendon

Juncosa-Melvin et al. (2006, 2007)

Osteogenic

Sumanasinghe et al. (2006)

Increase in gene expression of cBfa1, OCN, and ALP Increase in gene expression of collagen types

Osteoblasts at low magnitude (3%), short duration (8 h) Tendon/ligament at high magnitude (8%),

Chen et al. (2008)

References

(continued)

Table 7.1 (continued) 318 Type of cells

Type of loading

Loading conditions

Human BMSCs

Cyclic uniaxial stretching

8%, 3 days

Rat bone marrow MSCs

Uniaxial cyclic stretching

2000 microstrains, 40 min

Mouse bone marrow stromal cell line, ST-2

Equibiaxial stretching

0.8% and 5%, 6 h

Human BMSCs

Cyclic compression

Rabbit bone marrow MSCs

Cyclic compression

5%, 10%, and 15%, 48 h 7994 Pa, 0.33 Hz, 7 days

10%, 14 days

Response of stem cells

I, III, and tenascin-C for high magnitude Increase in the gene expression of Bfa1, ALP, OCN, collagen types I and III Increase in gene expression of cBfa1, Ets-1, and ALP, and cell proliferation Increase in gene expression of cBfa1, and increase in ALP activity Downregulation of ALP activity Increase in the gene expression of collagen type II and aggrecan Increase in gene expression collagen type II and aggrecan

Cell lineage commitment

References

long duration (48 h) Osteoblasts

Jagodzinski et al. (2004)

Osteoblasts

Qi et al. (2008)

Osteoblastic

Koike et al. (2005)

Chondrogenic

Angele et al. (2004)

Chondrogenic

Huang et al. (2004a)

Bovine BMSCs

Dynamic compression

10%, 16 days

Rabbit BMSCs

Cyclic compressive loading

15%, 1 Hz, 4 h/ day, 2 days

Human MSCs

Intermittent hydrostatic pressure

0.1 MPa, 14 days

10 MPa, 14 days

Human MSCs

Cyclic compression

10%, 0.1, 0.5, and 1 Hz, 8 h/day, 2 days in fibrin gels

Human MSCs

Cyclic hydrostatic pressure

Steady at 7.5 MPa for 14 days, 4 h/ day at 1 Hz in agarose gels

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Increase in gene expression of collagen type II and aggrecan Increase in gene and protein expressions of Sox-9, c-Jun, and TGF-b Increase in gene expression of Sox-9 and aggrecan Increase in collagen type II gene expression Increase in aggrecan gene expression at all frequencies; increase in collagen type II at 1 Hz only Transient increase in Sox-9 gene expression by both types of loading

Chondrogenic

Mouw et al. (2007)

Chondrogenic

Huang et al. (2005)

Chondrogenic

Miyanishi et al. (2006a,b)

Chondrogenic

Pelaez et al. (2008)

Beginning stage of chondrogenesis

Finger et al. (2007)

(continued)

Table 7.1 (continued) 320 Type of cells

Type of loading

Loading conditions

Human MSCs

Intermittent hydrostatic pressure

Ramped at 1 MPa at day 1, 0.5 MPa increase in subsequent days, 7.5 MPa at day 14, 4 h/day at 1Hz 1 MPa, 1 Hz 4 h/ day, 10 days in collagen sponges

Human BMSCs

Cyclic uniaxial and equibiaxial stretching

10%, 1 Hz for 1–3 days on collagen or elastin membranes

Response of stem cells

Cell lineage commitment

References

No change in collagen type II, and aggrecan gene expression

Increase in gene expression of collagen type II, aggrecan, Sox-9, and collagen type I; increased accumulation of proteoglycan; no change in Runx2 mRNA levels Transient increase in a-SMA, SM22a and collagen type I gene expression by uniaxial stretching Downregulation of SM a-actin and SM-22a by equibiaxial strain

Chondrogenic

Wagner et al. (2008)

Smooth muscle

Park et al. (2004)

Murine embryonic progenitor cell line

Cyclic equibiaxial stretching

10%, 1 Hz for 6 days

Murine embryonic progenitor cell line

Steady fluid shear stress

15 dynes/cm2, 6 and 12 h

Mouse embryonic stem cells

Fluid shear stress

1.5–10 dynes/cm2, 1–3 days

321

Increase in gene and protein expressions of a-SMA and SMMHC; synergistic effect with TGF-b1 Increase in gene and protein expressions of CD31, VEcadherin, vWF; increase in gene expression of angiogenic growth factors, VEGF, VEGFR; decrease in expression of growth factors (TGF-b, PDGFb, and PDGFR) associated with SMC Increase in gene and protein expression of Flk-1, Flt-1, VEcadherin, and PECAM-1

Smooth muscle

Riha et al. (2007)

Endothelial

Wang et al. (2005a)

Endothelial

Yamamoto et al. (2005)

(continued)

Table 7.1

(continued)

Type of cells

Type of loading

Loading conditions

Mouse embryonic stem cells

Cyclic uniaxial stretching

2, 4, 8, and 12%, 1 Hz, 24 h

Mouse embryonic stem cells

Fluid shear stress

10 dynes/cm2, 24 h

Human MSCs

Cyclic uniaxial stretching

5%, 1 Hz, 2–4 days on collagen I coated micropatterned silicone

Response of stem cells

Cell lineage commitment

Increase in gene and protein expression of VSMC markers, a-SMA, and SM-MHC Increase in gene expression of aSMA, SMA 22a, PECAM-1, VEGFR-2 Increase in gene expression of SMC marker calponin 1; decrease in gene expression of cartilage matrix markers (biglycan, COMP, collagen type X1a, and collagen type XI a1

Smooth muscle

Shimizu et al. (2008)

Cardiovascular

Illi et al. (2005)

Smooth muscle

Kurpinski et al. (2006)

References

BMSC, bone marrow stem cell; MSC, mesenchymal stem cell; BMP-2, bone morphogenetic protein-2; a-SMA, asmooth muscle actin; OCN, osteocalcin; PDGFR, platelet-derived growth factor receptor; ALP, alkaline phosphatase; SMC, smooth muscle cell; TGF-b, transforming growth factor-b; PECAM-1, platelet endothelial cell adhesion molecule-1; VEGFR-2, vascular endothelial growth factor receptor-2; VSMC, vascular smooth muscle cell.

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hMSCs in 3D collagen type I matrices significantly upregulated bone morphogenetic protein-2 (BMP-2) mRNA levels (Sumanasinghe et al., 2006). Therefore, application of mechanical loads to tissue constructs may be a useful means of inducing desired stem cell differentiation and hence facilitating the fabrication of functional tissue constructs. Recently, several experimental approaches using various types of mechanical loading conditions have been developed for selective differentiation of stem cells into specialized cells. For instance, the influence of mechanical stretching on the gene expression of tendon-related and osteoblast-specific markers in hMSCs has been investigated recently (Chen et al., 2008). hMSCs subjected to cyclic equibiaxial stretching with low magnitude (3% strain) and short duration (8 h) promoted expression of genes typical of osteoblasts, or cBfa1/Runx2, a key transcription factor associated with osteoblast differentiation, osteocalcin (OCN), and alkaline phosphatase (ALP), while cyclic stretching with high magnitude (8% strain) and long duration (48 h) upregulated genes (collagen type I and III, and tenascin-C) typical of tendon/ligament cells (Chen et al., 2008). In contrast, cyclic uniaxial stretching (8% strain) of BMSC for 4 and 7 days preferentially upregulated differentiation markers typical of osteoblasts (ALP, OCN, cBfa1/Runx2) ( Jagodzinski et al., 2004). In rat bone marrow MSCs, a brief bout of cyclic uniaxial stretching induced cell proliferation, increased ALP activity, and upregulated expression of two osteogenic transcription factors (cBfa1/Runx2, Ets-1) as well as ALP (Qi et al., 2008). However, the increase in transcription returned to control levels within 12 h after mechanical loading ceased, suggesting that the effect is transient. Stretching the cells for longer time periods may drive sustained osteoblastic differentiation. The effect of mechanical strain in mouse bone marrow stromal cell line ST-2 also showed that low equibiaxial strains for a short duration (0.8% and 5%, 6 h) stimulated osteoblastic differentiation (Koike et al., 2005). In addition, application of equibiaxial strain induced changes in cell morphology, proliferation, and differentiation. At all strain magnitudes (5%, 10%, and 15%) applied, the proliferation of ST-2 cells increased in a stretching magnitude-dependent manner. The level of mRNA for Cbfa1/Runx2, a key regulator of cell growth and differentiation of mesenchymal bone cell progenitors, was upregulated at 0.8% and 5% strains at 6 h and but downregulated at 5%, 10%, and 15% at 24 and 48 h. ALP activity significantly increased at 0.8% and 5% at 24 h; however, the activity decreased at 10% and 15% at 48 h (Koike et al., 2005). The difference in type of stretching and duration thus affect the differential response of MSCs in terms of morphology, proliferation, and differentiation. TGF-b1-induced chondrogenesis of human BMSCs has been established previously (Barry et al., 2001; Johnstone et al., 1998). However, recent studies show that the application of compressive loading can direct controlled differentiation of BMSCs into mature chondrocytes equally as

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effectively as chondrogenic mediums (Schumann et al., 2006). Both cyclic mechanical compression and cyclic hydrostatic pressure (CHP) enhanced the chondrogenic phenotype of BMSCs with increased collagen type II, aggrecan, and proteoglycan contents (Angele et al., 2003, 2004). Cyclic compression of rabbit BMSCs also increased expression of chondrogenic markers, aggrecan and collagen II, as effectively as TGF-b1 (Huang et al., 2004a). In addition, combining cyclic compressive loading with TGF-b1 treatment promoted collagen type II expression more effectively than TGF-b1 alone. Significant stimulation of chondrogenic markers, collagen type II and aggrecan, was also reported in bovine BMSCs that were subjected to dynamic compression in the presence of TGF-b and dexamethasone (Mouw et al., 2007). Moreover, dynamic compression upregulated SMAD2/3 phosphorylation in samples with and without TGF-b1. The interaction between mechanical loading and TGF-b1 signaling could be due to a wide range of potential mechanisms. TGF-b1 signaling may lead to pSMAD activation of mechano-sensitive proteins, such as focal adhesion kinase (FAK) and paxillin, which might directly increase mechanosensitivity of cells. Alternatively, TGF-b signaling may indirectly amplify the effects of mechanotransduction by increasing the transcription of downstream targets (Sox-9, collagen type II, aggrecan) of mechanical stimulation in chondrocytes. On the contrary, mechanical stimulation may modulate TGF-b signaling by enhancing the production of TGF-b or expression of its receptors through upregulation of mRNA levels, efficiency in translation, or both. For example, it was shown that cyclic compressive loading promoted gene expressions of Sox-9, c-Jun, and TGF-b receptors and production of their corresponding proteins in rabbit BMSCs in 3D agarose culture (Huang et al., 2005). Combining IHP and TGF-b3 also had similar effects, showing increased gene expression of cartilage matrix proteins, collagen type II and aggrecan, and increased Sox-9 expression in hMSCs as pellet cultures (Miyanishi et al., 2006a,b). When compared to unloaded controls, IHP at 0.1 MPa increased Sox-9 and aggrecan gene expression, while collagen type II increased only at IHP of 10MPa at 14 days (Miyanishi et al., 2006a,b). The data show that the regulation of two major matrix proteins, collagen type II and aggrecan, are pressure magnitude-dependent. Similar pressure magnitude-dependent expression of collagen type II and aggrecan is reported in hMSCs in fibrin gels subjected to compression at various frequencies. Aggrecan gene expression was upregulated at all frequencies (0.1, 0.5, and 1 Hz), while only 1Hz frequency stimulated collagen type II gene expression when compared to control cells (Pelaez et al., 2008). Exposure to IHP altered the morphological organization of the ECM surrounding the hMSCs into more uniform and compact structure compared to the sponge-like, dispersed matrix in cultures in absence of IHP (Miyanishi et al., 2006b). Also, chondrogenic mRNA levels were increased with just exposure to IHP alone without

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presence of TGF-b3. Although the exact mechanisms for chondrogenic differentiation in response to growth factors and mechanical loading are unknown, selective cell adhesion, integrin expression, or macromolecule assembly in response to loading may underlie this observation. A recent study investigated the differential effects of ramped and steady applications of CHP on chondrogenic differentiation of hMSCs in absence of TGF-b (Finger et al., 2007). The findings indicate that hydrostatic pressure may induce chondrogenesis in hMSC-seeded agarose constructs without the need for TGF-b stimulation. In addition, their data also show that hSMCs are capable of withstanding high initial pressure (7.5 MPa) that may initiate chondrogenesis faster than lower pressure (1 MPa). Specifically, steady and ramped application of CHP initially increased (day 4) and then decreased (days 9 and 14) collagen I gene expression and transiently increased Sox-9 gene expression. However, aggrecan and collagen type II were not detected within the initial 14 days of loading. Although previous studies observed increased collagen type II and aggrecan gene expression at 14 days, these studies applied a combination of chondrogenic media and hydrostatic pressure to hMSCs or rabbit bone marrow MSCs (Angele et al., 2003, 2004; Huang et al., 2005) or utilized different culture methods for cells as pellet cultures (Miyanishi et al., 2006a), where close proximity of the cells may have accelerated the differentiation compared to those cultured in 3D gels. On the contrary, chondrogenic differentiation was initiated in both pellet and 3D alginate cultures of hMSCs in presence of TGF-b3 under intermittent dynamic compression (Campbell et al., 2006). With the onset of chondrogenesis, the gene expression of collagen types II and X and aggrecan all increased over the 10-day culture period, with a transient expression of Sox-9 by day 8 followed by a decline. The accumulation of collagen X would indicate differentiation toward chondrocyte hypertrophy; however, many studies reported the expression of this gene at early stages of chondrogenesis under high cell density conditions, which is the case in this study. Transient gene expression of Sox-9 in early chondrogenesis also has been reported previously. Moreover, Sox-9 expression may be sensitive to factors such as cell density fluctuations, ECM environment, and gel encapsulation causing modification in cell morphology and nutrient delivery. IHP applied to hMSCs for 10 days in collagen type I sponges in a mixed osteochondrogenic medium supplemented with TGF-b1 increased the gene expression of chondrogenic markers Sox-9, aggrecan, and collagen type-II (Wagner et al., 2008). The upregulation of these genes supports the view that chondrogenesis is regulated in part by mechanical signals. Additionally, there were noticeable increases in proteoglycan accumulation and collagen type I gene expression. The increase in collagen type I expression observed in this study contradicts with another similar study (Huang et al., 2004a), which may be likely due to a combination of scaffold, mechanical

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loading conditions, and culture conditions. There was no difference in expression of Runx2 mRNA between the constructs exposed to hydrostatic pressure and unloaded controls, which suggests that neither osteoblast differentiation nor chondrocyte maturation was modulated by the hydrostatic pressure under these conditions. Compared to the constructs that were loaded without cells or unloaded constructs, cell/scaffold constructs that were exposed to HP had a much more compact structure. This observation suggests that MSCs subjected to HP actively contracted the soft matrices. The mechanism for contraction of fibroblasts and endothelial cells has been shown to depend on actin cytoskeleton and the RhoA/ROCK pathway, which regulates tension in the actin cytoskeleton (Kolodney and Wysolmerski, 1992). The disruption of the RhoA/ROCK pathway leads to decrease in contraction. Recent studies have shown that inhibition of the RhoA/ROCK pathway in mesenchymal limb bud cells transforms cell shape to a spherical chondrocyte morphology and decreases the downstream mRNA expression of chondrogenic genes collagen type II and aggrecan (Woods and Beier, 2006; Woods et al., 2005). These observations suggest that cell shape, cytoskeletal tension, and chondrogenesis are interrelated. Despite the variability in pressure application, culture conditions, and scaffolds in stem cell commitment to chondrogenesis in vitro, the overall findings of previous studies suggest that hydrostatic/compressive pressure may play critical role in cartilage development and regeneration in vivo. The determination of how hemodynamic forces influence vascular cell differentiation represents another exciting area of mechanobiology research. Recent studies attempted to harness cyclic stretching to differentiate BMSCs to SMC lineage (Hamilton et al., 2004; Park et al., 2004). Uniaxial cyclic stretching induced differentiation of human BMSCs into SMCs, as they increased expression of SM a-actin (or a-SMA) and SM-22a, which are marker proteins for contractile SMCs (Park et al., 2004). However, cyclic equibiaxial stretching downregulated expression of SM a-actin and SM-22a (Park et al., 2004). Therefore, the study showed that different types of mechanical stretching (i.e., uniaxial vs biaxial) produce differential effects on BMSC differentiation. In addition, DNA microarray analysis revealed that uniaxial stretching for 24 h increased SM contractile markers, SM-22a and calponin, but did not significantly change the expression level of marker genes for other cell types such as osteoblasts, chondrocytes, ligaments, and endothelial cells. In contrast, 3% equibiaxial stretching promoted MSC differentiation into osteoblastic cells in osteogenic media (Simmons et al., 2003). The type of stretching seems to have great influence on stem cell differentiation. The different strain distributions from uniaxial and biaxial stretching may regulate the conformation and/or localization of mechanotransduction signaling molecules and either turn on different signaling pathways or have opposite effect on the same pathway, leading to different cellular responses.

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One potential problem with the studies, however, is that the cell populations were derived from bone marrow aspirates and hence may include various types of cells, which may complicate the interpretation of mechanical stretching effects. Therefore, the use of a homogeneous murine embryonic mesenchymal progenitor cell line (CH3/10T1/2), as used in another study, is advantageous for furthering the understanding of the effects of mechanical forces on vascular cell differentiation. After applying cyclic equibiaxial stretching to CH3/10T1/2 cells, the cells adopted an elongated, spindle-shaped morphology with parallel arrangement, suggesting a change toward a mature SMC phenotype. In addition, cyclic stretching increased the mRNA and protein levels of a-SMA and smooth muscle myosin heavy chain (Riha et al., 2007). Furthermore, when 10T1/2 cells were exposed to fluid shear stresses, the cells increased expression of endothelial specific markers such as CD31 and cadherin in addition to changes in cell morphology and alignment, suggesting that these cells differentiated into mature endothelial cells (Wang et al., 2005a). Both studies show the induction of two different phenotypes (SMCs vs endothelial cells) from one mesenchymal cell line with two different types of hemodynamic forces (vascular stretching vs fluid shear stress). Alteration of cell function by a mechanical stimulus such as shear stress can be used as a novel technique to induce stem cell differentiation. Fluid shear stress has been previously shown to affect the differentiation of endothelial progenitor cells (EPCs) (Yamamoto et al., 2005). When EPCs were subjected to fluid shear stress, the cells elongated and oriented their long axes in the direction of flow. In addition, shear stress induced proliferation and expression of VEGF receptors, ICAM and VCAM, both at mRNA and protein levels when compared to static controls. Shear-stressed EPCs formed tubelike structures and developed an extensive tubular network faster than their static controls. Therefore, the utilization of mechanical force to manipulate EPCs may be useful in the development of efficient tissue engineered constructs or the maturation of EPC cultures in vitro for cell therapy. Fluid shear stress also has been shown to selectively differentiate Flk-1-positive embryonic stem (ES) cells into vascular endothelial cells. When Flk-1-positive ES cells were subjected to shear stress, their cell density markedly increased, and a larger percentage of cells were in S and G2(M) phase than they were in static controls (Yamamoto et al., 2005). Shear stress significantly enhanced the expression of vascular endothelial cell-specific markers Flk-1, Flt-1, VE cadherin, and PECAM-1 at both mRNA and protein levels. Shear stress, however, did not have any effect on markers of epithelial or smooth muscle, namely, keratin, or a-SMA. This study also reported that shear stress activates Flk-1 in a ligand-independent manner and that activation of Flk-1 plays a critical role in endothelial differentiation of Flk-1-positive ES cells. However, when the same cells were subjected to cyclic uniaxial stretching, the results were quite different. For example, cyclic uniaxial stretching (2%, 4%, 8%, or

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12%, 1Hz) of Flk-1-positive ES cells for 24 h markedly increased VSMC markers a-SMA and smooth muscle-myosin heavy chain (SM-MHC) in a stretching magnitude-dependent manner (Shimizu et al., 2008). However, cyclic stretching (8%, 1 Hz, 24 h) significantly decreased the expression of EC marker Flk-1 but had no effect on the expression of other EC markers (Flt-1, VE cadherin, and PECAM-1), the blood cell marker CD3, or epithelial marker keratin. In addition, cell proliferation was increased, and cells oriented perpendicular to the stretching direction in response to cyclic stretching, which suggests that the cells’ response to mechanical stretching may change during stretching. Finally, the PDGFRb kinase inhibitor completely blocked cyclic stretching-induced cell proliferation and VSMC marker expression. The results suggest that activation of PDGFRb plays an important role in VSMC differentiation from ES cells. Taken together, the results of the two studies using fluid shear stress and cyclic stretching suggest that both stimuli may act by a common mechanism in which growth factor receptors are activated by mechanical forces without ligand binding. Directing stem cells into cardiovascular lineage utilizing mechanical stretching has been attempted recently. Mouse ES cells exposed to fluid shear stress (10 dynes/cm2, 24 h) induced several cardiovascular markers including a-SMA, SMA 22-a, PECAM-1, and VEGF receptor-2 (Illi et al., 2005). In addition, stress activated transcription from VEGFR-2 promoter. The study shows how laminar flow effects could be successfully utilized for cardiovascular differentiation. Besides fluid shear stress, mechanical stretching can also be used to direct ES cell differentiation into cardiovascular lineage. Equibiaxial strain (10%, 2 h) of ES cells induced HIF-1a and VEGF mRNA and protein, which are involved in cardiovascular development (Schmelter et al., 2006). Reactive oxygen species have been shown to function as transducers of mechanical strain-induced cardiovascular differentiation of ES cells (Schmelter et al., 2006). Cyclic uniaxial stretching applied to cells on elastic substrates causes cells to align perpendicular to the stretching direction, which is different from that in the vascular wall that is anisotropic and mainly in the circumferential direction. To simulate the vascular cell alignment and investigate the anisotropic mechanical sensing by MSCs, elastomeric membranes with parallel microgrooves were developed using soft lithography (Kurpinski et al., 2006). This topographic pattern kept the cells aligned parallel to the stretching direction. MSCs in such microgrooves were subjected to cyclic uniaxial stretching (5%, 1Hz) for 2–4 days, and DNA microarray analysis revealed global gene expression changes, including an increase in smooth muscle marker calponin 1 and a decrease in cartilage matrix markers (biglycan, collagen type X a1, collagen type XI a1, and cartilage oligometric matrix protein (COMP)) and MMP-1. Cyclic uniaxial stretching increased the contractile marker calponin but not SMC mature marker MHC, suggesting that additional factors, possibly soluble factors may be required for

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the terminal differentiation of MSCs. Also, the increase in contractile markers and the decrease in cartilage markers by cyclic uniaxial stretching suggest that a more tension-bearing rather than compression-bearing tissue phenotype is promoted. Taken together, these studies show that mechanical forces can induce or enhance the differentiation of stem cells into a specific cell lineage. However, cellular responses by mechanical forces must always be evaluated in the context of other microenvironmental factors such as culture media and ECM. A recent study has demonstrated that cyclic stretching inhibits human embryonic stem cell (hESC) differentiation in cells cultured on plates coated with Matrigel (Saha et al., 2006). Cyclic biaxial stretching (10%) applied to the deformable substrate was found to inhibit hESC cell differentiation and to promote self-renewal, as measured by an increase in Oct4 and SSEA-4 expression. Mechanical stretching significantly repressed differentiation of hESCs cultured in mouse embryonic fibroblast-conditioned medium but had no effect on the differentiation of hESCs cultured in unconditioned medium (Saha et al., 2006). Therefore, it appears that chemical signals act synergistically with mechanical loads to regulate the differentiation of hESCs. The differential effect of different mechanical loading conditions and the cross talk between mechanical loading and chemical factors in stem cell differentiation open up important avenues for further research. In summary, various types of mechanical loading can be effectively applied to direct stem cell differentiation. The upregulation of specific differentiation markers greatly depends on magnitude, duration, and type of mechanical loading. For example, low magnitude, short duration biaxial stretching promotes markers of osteoblast-type gene expression, while high magnitude, long duration promotes markers typical of tendon/ligament type. In addition, uniaxial versus biaxial stretch has differential effects on stem cell differentiation, and the same is true with compressive loading and shear stress, which specifically promote chondrogenic phenotype or endothelial phenotype, respectively. Furthermore, several other mediators such as growth factors and matrix environment (ECM substrates/scaffolds) act in concert with mechanical loading to direct stem cell differentiation. Mechanical regulation of stem cells provides a rational basis for tissue engineering and regeneration.

5. Mechanotransduction Mechanisms Mechanical forces have a myriad of effects on cells, and the whole field of mechanotransduction is devoted to investigating how various forms of mechanical forces are transduced into biochemical cascades and cellular events

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(Alenghat and Ingber, 2002; Davies, 1995; Ingber, 1997; Wang et al., 2001c). Years of intensive research have concluded that the basic mechanotransduction components include integrins, FAs, cytoskeleton, ion channels, cell surface receptors, and various secondary signaling molecules, irrespective of cell type. Many excellent reviews on the mechanotransduction mechanisms are available (e.g., Davies, 1995; Duncan and Turner, 1995; Huang et al., 2004b; Osol, 1995; Silver and Siperko, 2003), and interested readers should consult them for a more in-depth understanding of the topic. One of the major components involved in mechanotransduction is integrins, which act as mechanosensors by themselves or in concert with cytoskeletal proteins (Ingber, 2002; Katsumi et al., 2004; Schwartz et al., 1995). Integrins in adherent cells, such as fibroblasts, are direct mechanosensors by physically connecting the ECM to the cytoskeleton (Stupack, 2007). Binding to ECM proteins, such as collagen and fibronectin, integrins also function as signaling receptors. ECM-attached integrins also physically link FAs to the actin cytoskeleton (Geiger and Bershadsky, 2002). Mechanical forces promote the assembly of FAs (Sawada and Sheetz, 2002; Wang et al., 2001a) and trigger integrin-dependent signaling and activation of MAPKs. FA molecules may sense mechanical forces due to an altering of the relative positions of specific FA components (Geiger and Bershadsky, 2002). Another possible mechanism by which FAs sense mechanical forces involves the conformational changes of specific FA molecules. Interestingly, several FA proteins such as vinculin and fibronectin exist in active and inactive conformations. The transition from inactive to active conformation may occur in response to mechanical forces, which may then trigger the cascade of signaling events (Balaban et al., 2001; Geiger et al., 2001). The actin cytoskeleton, on the contrary, is responsible for the transmission of tension to the nucleus. Tensional force within the actomyosin contractile system is regulated in part by the degree of phosphorylation of the myosin light chain (Kamm and Stull, 2001; Pfitzer, 2001). The activation site of RhoA, a member of a family of GTP-binding proteins, controls the development of FAs and stress fibers in adherent fibroblasts (Chrzanowska-Wodnicka and Burridge, 1996). The activities of RhoA on the actin cytoskeleton are mediated primarily through its downstream effector ROCK, which inactivates myosin phosphatase to induce the stabilization of filamentous actin and formation of stress fibers (Bershadsky et al., 2006). Mechanical stretching causes a remodeling of the microfilament and microtubule networks, and prevention of this cytoskeletal remodeling mitigates stretch-induced increases in gene transfer and expression (Geiger et al., 2006). Fluid shear stress also induces remodeling of the actin cytoskeleton with the formation of stress fibers through an array of signaling molecules including RhoA and Rac GTPase (McCue et al., 2004; Noria et al., 2004; Tzima et al., 2002).

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Besides the changes in the actin cytoskeleton in response to applied mechanical forces, an early cellular response is the influx of Ca2þ through stretch-activated channels (Wu et al., 1999). The Ca2þ influx may then lead to intracellular activation of many molecules, including NF-kB, cAMPresponse element binding protein (CREB), and membrane kinases, which specifically phosphorylate other signaling molecules, such as EGFR, which activates downstream MAPK signaling pathways (Iqbal and Zaidi, 2005; Rosen and Greenberg, 1996; Sadoshima and Izumo, 1997). Stretchinginduced Ca2þ influx upregulates COX-2 expression via activation and translocation of NF-kB into the nucleus (Inoh et al., 2002). However, it is not clear whether ion channels are indispensable components of the mechanosensor itself because integrin-mediated FA mechanosensor can still respond to applied forces in detergent-treated cells (Sawada and Sheetz, 2002). There are similarities and differences between adhesion-dependent and ion channel-based mechanosensors (Geiger and Bershadsky, 2002). In both cases, a mechano-responsive element is linked via a motor protein (e.g., myosin II), to the cytoskeleton at the cell’s interior and to an extracellular anchor, which is usually the ECM. It is speculated that channel-based mechanosensors induce global cellular signaling events, whereas FA-based mechanosensors elicit more localized events. Another mechanotransduction mechanism is thought to originate at the cell membrane and involve G-protein-coupled receptors (GPCRs) (Sarasa-Renedo and Chiquet, 2005; White and Frangos, 2007). Using time-resolved fluorescence microscopy and GPCR conformation-sensitive fluorescence resonance energy transfer (FRET), fluid shear stress on endothelial cells was shown to increase the activity of bradykinin B2 GPCR (Chachisvilis et al., 2006), which is known to be involved in fluid flowdependent responses in endothelial cells (Groves et al., 1995; LeebLundberg et al., 2005). In addition, single-molecule studies have identified certain structural motifs of proteins such as fibronectin that can be mechanically switched between conformations, thereby exposing potential cryptic sites, which may transduce mechanical force into biochemical signals by changing the relative distance of the two binding sites of the receptor or by changing the geometry of the binding site (Smith et al., 2007; Vogel, 2006; Vogel and Sheetz, 2006). A novel mechanotransduction mechanism that has been proposed recently considers a coupling between an external mechanical force and a protein redox (reduction/oxidation reaction) equilibrium by which the triggering of a biochemical signal is controlled (Grandi et al., 2006; Sandal et al., 2006). The disulfide bonds present in many ECM proteins act as redox switches for protein functions. Angiostatin, which contains disulfide bonds and is located in the basement membrane where mechanical forces act, has been shown to be an ideal model system for addressing the issues of the

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possible triggering of biochemical signals by the combination of mechanical forces and redox equilibrium (Grandi et al., 2006). Mechanical forces can partially unfold the structure of angiostatin, which can activate new binding capabilities and trigger new biochemical signals (Grandi et al., 2006). As noted above, many years of research has greatly increased our understanding of cellular mechanotransduction mechanisms—the centerpiece of cell mechanobiology. However, the precise details of how mechanical forces transduce into signaling events and how a cell decides its response from cross talks of many mechanotransduction signals still remain elusive. Furthermore, whether adult and stem cells use the same mechanotransduction mechanisms remains to be worked out. Thus, more research with novel experimental and theoretical methodologies is required.

6. Concluding Remarks Mechanical forces acting on cells within tissues and organs (e.g., tension, compression, hydrostatic pressure, and fluid shear stress) play a vital role in human health and disease. Years of intensive research have also established that mechanical forces stimulate ECM (e.g., collagen) gene and protein expression and induce cellular inflammatory response (e.g., expression of COX-2 and production of PGE2). Mechanical forces can also interact with soluble factors (e.g., TGF-b and IL-1b) in generating anabolic and catabolic cellular responses. However, while in vitro studies highlight the critical role of mechanical forces in cell biology and consequently tissue physiology and pathophysiology, the proper interpretation of experimental data requires consideration of many factors such as cell source, type, morphology, and organization; the manner of applying mechanical forces (e.g., uniaxial vs biaxial stretching); and the type of matrix protein coating, as these factors can all influence cellular response to mechanical forces. Besides these external mechanical forces on cells, cell-generated forces, known as CTFs, play an important role in many biological processes, including wound healing and angiogenesis. Therefore, measurement methods such as CTFM have been developed to quantify CTFs. Current CTFM methods are limited in that they use a 2D substrate. To have much broader application, CTFM methods should be extended to more physiologically accurate 3D matrices. The critical role of mechanical force in the development of functional tissue engineering constructs for repair, replacement, and regeneration of injured and diseased tissues has also been recognized. Much effort has been focused on the effects of mechanical loading on adult stem cells, such as BMSCs, in an attempt to obtain functional tissue engineering constructs.

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Additionally, the application of mechanical forces in the form of tension, compression, and torsion induces controlled differentiation of MSCs into different cell lineages such as ligament and SMCs. A few studies have also examined how mechanical loading regulates ES cells in terms of selfrenewal and differentiation. However, the molecular mechanisms by which mechanical loads activate or suppress signaling pathways that lead to the self-renewal and/or differentiation of ES cells and adult stem cells are not clear. Furthermore, the detailed mechanisms regarding both the interaction of different signaling pathways in modulating expression of mechano-responsive genes and also the cross talk between mechanical and chemical signaling pathways in ES cells and adult stem cells remain to be worked out. Mechanical forces acting on stem cells may trigger cell surface receptors and adhesion sites that activate signaling cascades responsible for the synthesis and secretion of key ECM components (Sebastine and Williams, 2006). Soluble mediators, along with nonprotein metabolic products, such as calcium, may also affect stem cell differentiation. Although the well-described stem cell niches, including neighboring cells and surrounding matrix, are known to regulate the balance of self-renewal and differentiation of adult stem cells (Moore and Lemischka, 2006; Scadden, 2006), the mechanical loading environment around individual stem cells may also be an important ‘‘niche factor’’ that may have an influence on the division, self-renewal, and differentiation (or the lineage commitment) of adult stem cells, especially those in load-bearing tissues such as bone, tendons, and ligaments (Fig. 7.3). Manipulation of the stem cell niche by targeting the mechanical environment surrounding stem cells may

Division, self-renewal

Differentiation

Niche

Mechanical loading Figure 7.3 Schematic of the concept of mechanical loading as a niche factor of stem cells. Stem cell niche generally refers to local microenvironment that maintains stem cell identity and regulates stem cell function. It is suggested that the niche may also include mechanical loading (curved arrows), which, together with other intrinsic factors (e.g., growth factors), regulates stem cell division, self-renewal, and differentiation.

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constitute an effective therapeutic treatment of musculoskeletal tissue diseases, in which mechanical forces are known to play a critical role in tissue pathophysiology. Finally, it should be noted that compared to numerous studies on adult cell mechanobiology, there are far fewer studies on stem cell mechanobiology; therefore, it is an emerging field to be explored fruitfully in future research.

ACKNOWLEDGMENT We gratefully acknowledge the funding support of NIH grant AR049921 ( JHW).

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Index

A Acanthamoeba castellanii, 111, 281 Actin cytoskeleton, 257, 262–265, 267, 276–277, 285, 330 Actin ring, 100 Adenocarcinomas, 40–41, 43 Adenomatous polyposis coli (APC), 20–21 ADP ribosylation factor (ARF)-binding (GGA) protein, 155 Adult cell mechanobiology, 311–316 Agar overlay technique, 258 Aggrecan, 312, 318–320, 324–326 Ago2 protein, 224–225 Alternate adaptors, 155 Alveolar rhabdomyosarcoma (ARMS). See Rhabdomyosarcoma Alzheimer disease (AD), 165, 182, 184 Amoeba proteus, 120 Angiostatin, 331–332 Anterior cruciate ligament (ACL), 305 APC-Wnt pathway, 20–21 Arabidopsis thaliana, 103–104, 120, 130, 134 chloroplast division, in arc mutants, 104 retromer subunits in, 162 ARF protein, 63 Associated RNA-binding proteins (RBPs), 204, 215, 217–219, 227–228, 235–237 Atf4 regulation, by uORFs, 215 Atriplex semibaccata, 102 Atypical squamous cells of undetermined significance (ASCUS), 47, 50 AU-rich elements (AREs), 217 Auxin influx carrier (AUX1), 170 Auxin movement, in plant cell, 167, 170 Avena sativa, 102 5-Azadeoxycytidine, 72 B Bacillus subtilis, 111 Bacillus thuringiensis, 256 Bacterial division machinery, 111–113 Basic fibroblast growth factor (bFGF), 308 Bethesda system, 43 Biaxial stretching, 305 Bicoid, 217 Bone morphogenetic protein-2 (BMP-2), 323

Bone marrow stem cells (BMSCs), 316, 323–324, 326 BRAF, and nevi development, 19–20 Brefeldin A (BFA), 171 40 -Bromo-30 -nitropropiophenone, 25 bsite amyloid precursor protein (APP) cleaving enzyme (BACE), 165, 182–184 C Caenorhabditis elegans, 4, 118, 163 DRP-1 in, 102 lin-14 mRNA, regulation of, 222–223 retromer’s role, in Wnt-producing cells, 163 role of Rab2, 280 Ca2þ influx, stretching induced, 331 Calponin, 326 Calyptogena okutanii, 113 Cancer, 5–6 in fish, 2–3 gene discovery by Zebrafish as tool, 7 hematologic cancer models, 10–13 melanoma, 18–20 pancreatic cancer, 13–14 p53 pathways and, 15–18 and PTEN gene, 21–23 Candida albicans, 38 Cap-dependent translation initiation, 205–206, 210 Cap-independent translation initiation, 208–209 Cardiovascular differentiation, and mechanical stretching, 328 Cartilage oligometric matrix protein (COMP), 328 Catalase, 113 Caveolae-mediated endocytosis, 107–108 Cell membrane system, for communication, 154 Cell migration, CTFs role in, 310 Cell polarity, types of, 167 Cells, and mechanical forces cell-generated mechanical forces, 306–310 external mechanical force application, 304–306 mechanobiological responses adult cells, 311–316 stem cells, 316–322 three-dimensional (3D) experimental models, 315

347

348 Cell traction force microscopy (CTFM), 308–310 Cell traction forces (CTFs), 303, 306–307 cell migration and tissue morphology, role in, 310 force-loaded images, 308 generation and transmission, 307 intracellular protein and, 307 methods for determination, 308 null-force image, 308 vs. asmooth muscle actin, 307–308 Cervical cancer cervical carcinogenesis, steps of, 38–39 cervical lesion classifications, 46–48 dysplasia, 44–46 epigenetic changes in, 72 DNA methylation, 72–73 gene expression pattern, 74–75 telomerase activation, 74 human papillomaviruses (HPV) characteristics of, 51–53 detection and typing, 43–44 E1 gene, 53–54 E2 gene, 54 E4 gene, 54–55 E5 gene, 55 E6 gene, 55–61 E7, role in carcinogenesis, 61–66 genome regulatory region, 53 and infections, 37–38 role of, 43 incidence and mortality rates, 40 metaplasia, 44 pathology of, 41 precancerous lesions conversion dynamics, 50–51 cytomorphological criteria of, 48–50 and diagnosis, 42–43 tumor node metastasis staging system for, 40–41 types and staging, 40–41 vaccines for, 39–40 viral DNA, role of episomal and integrated HPV DNA interaction, 70 HPV DNA persistence significance, 66–67 HPV DNA status and disease progression, 70–71 integrated and episomal DNA, transcriptional regulation of, 69 in women, 37, 40, 42–44 Cervical cytology screening, 42 Cervical intraepithelial neoplasia (CIN), 37, 42, 46–47 Chediak-Higashi Syndrome, 268 Chironomus sp., 98 Chlamydiae, 113 Chlamydia trachomatis, 38 Chlamydomonas reinhardtii, 118

Index

Chlorella vulgaris, 130 Cholera toxin, 166 Chondrocytes, 303–304, 312–314, 323–324, 326 CIN2 þ CIN3 (HSIL), 49–50 CIN1 (LSIL), 49 Clathrin, 108, 110, 154–155 Clathrin adaptor protein (AP) complexes, 154–155 Clathrin-mediated endocytosis, 105, 107–110 Cleavage polyadenylation factor (CPF), 203 Cleavage stimulatory factor (CstF ), 203 C. merolae, 100–102, 135, 138–139 dynamin (MDM1) gene, 104 FtsZ and dynamin rings of plastids, 131–132 genome sequence, 141 isolation of PDF machineries, 138–139 MD rings in, 120 mitochondrial division system in, 121–122, 121–125 mitochondrion in, 118 plastid division machineries in, 128–131 CMR185C (Mda1), 126–127 Coatomer protein complex I (COPI), 155 Collagen expression, and mechanical loading, 311–313 Collagen type II, 312, 318–320, 324–326 Colposcopic biopsy, 42–43 Comitin, 265, 267 Coronin, 264–265, 271 CpG islands, hypermethylation of, 72 Cremart, 131 Crenarchaeota, 113 Criptococcus neoformans, 281 Cup, as translational regulator, 217 Cyanidium caldarium, 100–102 Cyclase-associated protein (CAP), 267 Cyclic hydrostatic pressure (CHP), 324 Cyclooxygenase-2 (COX-2), 313–314, 331 Cytochalasin, 263 Cytochalasin B, 100, 103, 131 Cytoplasmic polyadenylation element binding protein (CPEB), 216–217 Cytoplasmic processing bodies, 219 and mRNAs movement, 220–221 Cytoplasmic vesicle dividing (cVD) machinery evolution, 106, 109 D Diamond-Blackfan anemia, 8 Dictyostelium caveatum, 255 Dictyostelium cells, 258–259 Arp2/3 complex role, 263–264 axenic strains, choice of, 256 cofilin, 264 coronin, 264–265 ENA-VASP, 263 formin, 263–264

349

Index

heterotrimeric G protein, 268–270 Legionella infection in, 285–287 lysosomal hydrolases role, 267 membrane receptors on, 261–262 multicellular aggregates formation, 255 myosins I and VII, 265 Nramp family in, 287–288 pathogenic bacteria, identification of, 256 phagocytosis of bacteria by, 257–258 phosphatidylinositol polyphosphates interconversion pathways, 272 phosphoinositide dynamics, during particle uptake, 270–275 Phospholipase C (PLC), 271–274 PI3K involvement, 273–274 pore-forming peptides, 267 as professional phagocytes, 255 Rab proteins, 278–281 Rac GTPases, 277 RasS and Rap1, 276 tetraspanins, 269–270 vacuolin A and B, 267–268 VSK3 role, 277–278 zipper model, of phagocytosis, 257 Dictyostelium discoideum, 120, 255 Dictyostelium–pathogen interactions, host cell factors affecting, 282–283 Dipeptidyl aminopeptidase A (DPAP A), 160 DNA methylation, 72–73 Dnm1p, in yeast, 102 Drosophila diaphanous protein, mammalian homolog of (mDia1), 307 Drosphophila, 4, 163 retromer participation, in development DmVps35, importance of, 163–164 Dynamin, 101, 109, 137 Drp1, 101–102 Dynamin-like protein 1 (DLP1), 116 Dyskaryosis, 45, 49 Dysplasia, 44–46 E 4E-BP (4E binding proteins), 215, 217 E6BP/ERC-55 protein, 59 E-cadherin recycling, and SNX1, 181 E2F1/DP1/RB cell cycle regulatory pathway, 58 Embryonal rhabdomyosarcoma (ERMS). See Rhabdomyosarcoma Endocytic pathway, 154, 285 Endocytosis, 106–109, 111, 143, 275 Endosome, 107, 155–156, 165–166, 171, 173, 179, 267, 279 Endosymbiotic phagocytosis, 113 Endothelial cells, 303–304, 313–314, 326–327, 331

Endothelial progenitor cells (EPCs), and fluid shear stress, 327 Entamoeba histolytica, 117, 161–162 Epidermal growth factor receptor (EGFR), 179 Equibiaxial stretching, 315, 326–327 Escherichia coli bacterial cytokinesis, 111–112 bacterial nuclear division, 111 DivIB/FtsQ, 112 FtsZ proteins, role of, 112–113 FtsZ ring, 101, 112 Euglena gracilis, 118, 128 Eukaryotic cells nuclei in, 100 and organelle division (OD) machineries, 104–107 origin and evolution of, 101–103 Eukaryotic initiation factors (eIFs), 205 Exocytic pathway, 154 Exon junction complex (EJC), 204 Exonucleolytic pathway, 220 Extracellular matrix (ECM), 304–305, 307–308, 310–312, 315, 329–331 Extracellular signal-regulated kinase (ERK), 179 F Familial adenomatous polyposis (FAP) syndrome, 20–21 Fanconi anemia pathway, 65 Fet3p-Ftr1p iron transporter complex, 161 Fibroblast-populated collagen gels (FPCGs), 308–309 Fibroblasts, 303–305, 309–311, 313–315, 326, 330 Formin-binding protein (FBP), 109 FtsZ protein, 101 in plants, 103 G Galdieria sulphuraria (Cyanidium caldarium M8), 118–119, 122 Gcn2 pathway, 215 Gene regulation posttranslational control, 200–201 transcriptional control, 200–201 Giardia intestinalis, 117 Giardia lamblia, 117 Glaxo Smith Kline bivalent vaccine, 39–40 Glom, 98 GNOM-dependent pathway, 171 G-protein-coupled receptors (GPCR), 157, 269, 331 Green fluorescent protein (GFP), 231 Growth factor receptors, and mechanical forces, 328 Guanine-nucleotide exchange factor (GEF), 171

350

Index H

Haem-regulated inhibitor (HRI), 213 Hartmanella vermiformis, 281 Hedgehog signaling pathway, 9, 14 Hematopoiesis, and cancer, 10–13 Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), 155 Herpes simplex virus type 2 (HSV-2), 38 High-grade squamous intraepithelial lesion (HSIL), 47–48, 60, 68 Histone deacetylase (HDAC), 64–65 Host–pathogen interactions host genes, susceptible to infection, 281–287 Nramp1 as host defence factor, 287–288 Human embryonic stem cell (hESC) differentiation, and cyclic stretching, 329 Human MSCs (hMSCs), and uniaxial cyclic stretching, 316, 323 Human papillomaviruses (HPV), 36, 43 and cervical carcinogenesis, 38, 42 detection and typing, in cervical epithelium, 43–44 E1 gene, 53–54 E2 gene, 54 E4 gene, 54–55 E5 gene, 55 E6 gene apoptosis inhibition, 59 biological effects, 55–56 and epithelial differentiation, 59–60 E6TP1 and, 60 Jak-STAT pathway and, 60 mitogenic activity of, 58–59 and p63 pathway, 60–61 structure of, 56 transcription and DNA replication deregulation, 58 tumor-suppressor p53, interaction with, 57–58 E7 gene and 17 bestradiol receptor, 66 biological properties, 61–62 B-myb protein and, 65 chromosomal instability, induction of, 64–65 cyclin-dependent kinases and, 63–64 DEK expression and, 65 and histone deacetylase (HDAC), 64 serine/threonine phosphatase 2A (PP2A) and, 66 Skip protein and, 65 structure of, 61 tumor growth suppressors, interaction with, 62–63 and tyrosine phosphatase Cdc25, 65 HPV DNA testing, 39 infections, 37, 39

persistence and hormonal contraception use, 38 role in anogenital and nonanogenital cancers, 37 structure and life cycle of, 51–52 types carcinogenic effect, 39 Hydrogenosomes, 113, 117, 143 Hymenophyllum, pleomorphic plastid division in, 133 I Innate immunity signaling proteins, 255 Inositol-requiring enzyme 1 (IRE-1), 234 Insulin-like growth factor (IGF)-binding protein, 3, 65 Integrated papillomavirus forms (IPFs), 68 Integrins, 59, 314, 330 Intercellular adhesion molecule-1 (ICAM-1), 314 Interferon regulatory factor (IRF)-3, 60 Interleukin-1b (IL-1b), 314 Intermittent hydrostatic pressure (IHP), 312, 324–325 Internal ribosomal entry sites (IRES), 208–209 Invasive cervical cancer, 44 J JAGGED1, 60 Jak-STAT pathway, 60 K Klebsiella, 262, 284 Koilocytosis, 48–49 L Latrunculin, 263 LDL receptor (LDLR). See Sortilin-related receptor (SorLA) Legionella, 281, 284–288 Leukemia, 10, 12–13, 18 Li-Fraumeni syndrome, 15 Lilium longiflorum, 104 Listeria, 113, 256 Long control region (LCR), 51, 53 Low-grade squamous intraepithelial lesion (LSIL), 47–48, 50, 66–68 M Macrophages, 259, 277–280, 284–288 Macropinosomes, 109 Madin-Darby canine kidney (MDCK) cells, 165, 184 pIgR–pIgA transcytosis in, 178–179 polarized traffic in, 181–183 Malignant peripheral nerve sheath tumors (MPNSTs), 7

351

Index

Mallomonas, 103, 120 Mallomonas splendens, 101 Mannose-6-phosphate receptors (MPRs), 155 cation-independent MPR (CI-MPR), 156 MAPK/ERK signaling, 179 Maskin, 216–217 Matrixmetalloproteinases (MMPs), 314 Mdm2-p53 pathway, 16 Mechanical forces, on cells, 303, 316 Mechano-responsive cells, 303 Mechanotransduction, 303–304 mechanisms, 329–332 Medial collateral ligament (MCL), 311 Melanoma, 3, 18–20 Merck Gardasi, 39 Mesenchymal stem cells (MSCs), 304 Messenger ribonucleoprotein particles (mRNPs), 203–204 Metaplasia, 44–45 Metazoan histone mRNAs, 202 Methyl guanine methyl transferase (MGMT), 60 Mi2-b protein, 65 Microbodies as autonomous organelles, 115–116 and endoplasmic reticulum, 115 evolutionary origin of, 113–114 Fis1p and DLP1 role, 116 PEX11 family proteins, role of, 116 structure and function, 113 Microbody division, 113–117 Microgrooved substrate, 305, 315 Micropatterning technology, 314 MicroRNA (miRNA), 203, 219, 222–226 Microsporidia, 117 Mitochondrial division machinery, 100, 105 diversity in modes of mitochondrial division, 118–120 division rings (MD rings), 100 origin and evolution of, 120–122 structure, function, and constriction process of, 122–125 contraction, 126–127 pinching-off, 127–128 positioning, 125–126 Mitochondrial division protein 1 (Mda1), 102 Mitochondriokinesis, 99–100, 125 cytochalasin B, 100 Mitogen-activated protein kinase (MAPK), 3, 55, 179, 307 Mitosome and hydrogenosome division, 117 Monocercomonas sp., 117 Monocyte chemotactic protein-1 (MCP-1), 314 Morpholino toxicity, 16 Mycobacterium tuberculosis, 280 Mycoplasma, 113 Myofibroblasts, 307 Myosin heavy chain (MHC) kinases, 265 Myosin light chain kinase (MLCK), 307

N Nannochloris bacillaris, PD rings in, 103, 131 Nannochloropsis oculata, 120 Natural Resistance Associated Membrane Protein (Nramp)1, 266–267 Neisseria gonorrhoeae, 38 Neisseria meningitidis, 281 Neocallimastix, 117 N-glycosylation, 174 Nitella flexili, 100 Nodal expression, and cancer, 23–24 Nonidet P-40 (NP-40), 138 Nonsense-mediated decay (NMD) pathway, 220 Notch signaling, 60, 177 NOTCH transgenic model, 12 Nuclear factor-kappaB (NF-kB) activation, 314 Nuclear pore complex (NPC), 203 O Open reading frames (ORFs), 51, 214 Ophioglossum, pleomorphic plastid division in, 133 Organelle division (OD) machinery, 98 isolation of, 137–140 proteins and genes involved in, 140–142 Organellokinesis, 98, 125 Ostreococcus tauri, 128 P Pals1-associated tight junction (PATJ) protein, 57 Pancreatic exocrine carcinoma, 13–14 Pap smear screening, 42–44 Particle-tracking method, 258 P-bodies. See Cytoplasmic processing bodies PDF machinery, 105 Pelargonium zonale, 103 PDF machinery in, 131 Peptide mass fingerprinting (PMF), 141 Periodontal ligament (PDL), 312 Phagocytic cup, 257 Phagocytosis, 254–257 cellular mechanisms of actin cytoskeleton, 263–265 killing and digestion of bacteria, 266–268 phagocytosis receptors, 260–263 dynamics of, 257–260 regulatory pathways, 268 heterotrimeric G protein, 268–270 phosphoinositides and calcium ions, 270–275 Rab proteins, 278–281 small G proteins of the Ras and Rac families, 276–277 tyrosine kinases, 277–278 Phagosome, 254 and proteomic fingerprinting, 260 spacious phagosomes, 259

352 Phaseolus vulgaris, 102 Phosphatidylinositides, 270–271 Phosphofurin acidic cluster sorting protein-1 (PACS-1), 182 Phosphoinositide 3-kinases (PI3Ks), 156 Phosphoinositides, 263, 268, 270–275 Phospholipase C (PLC), 271–274 Phospholipase D (PLD) activity, and butan-1-ol treatment, 272–273 Physarum polycephalum, 98, 118, 138 MD ring in, 120 mitochondrial division in, 98–100 Physcomitrella patens, 103 pIgR–pIgA transcytosis, 165, 178–179 PI3K/retromer-dependent pathway, 179 p53-independent pathway, 3, 15–18 Pin-formed (PIN) protein family, 170 Pirellula sp., 113 Pisum sativum, 102 PKR-like endoplasmic reticulum kinase (PERK), 213 Plasma membrane vesicle dividing (pVD) machinery evolution, 106, 109 Plasmodium falciparum, 128, 133, 234 Plastid dividing (PD) ring, 100, 102 Plastid division machinery binary division, 133 cyanobacteria and, 130 evolutionary diversity in plastid division, 128–129 origin and evolution of, 130–131 peptidoglycan biosynthesis and, 130 pleomorphic plastid division, 133 primary plastid endosymbiosis, 130 secondary plastid endosymbiosis, 130 structure, function, and constriction process of contraction, 134–136 pinching-off, 136–137 positioning, 131–134 Plastid division, model for, 140 Plastid nuclei, 100 Platelet endothelial cell adhesion molecule-1 (PECAM-1), 314 Polar auxin transport (PAT), 170 Poly(A)-binding proteins (PABPs), 203–204, 206–207, 217, 235 Polyadenylation, 202–203 Poly(A) polymerase (PAP), 203 Poly(A) tail, 202–203, 207, 216–217, 220, 224, 230–231, 234, 237 Poly(glycolic acid) (PGA) scaffolds, 316 Posttranscriptional gene regulation, 227–228 mRNA turnover, 232–234 proteomic approaches, 231–232 RBPs and mRNAs, 235–237 translational profiling, 228–231 Posttranscriptional regulation mechanisms, 201 Potentiation, 230

Index

Prelysosomal nonacidic vesicles, 266 Professional phagocytes, in metazoa, 254 Promyelocytic leukemia protein (PML), 65 Prosporemembrane, 180 Prostaglandin E2 (PGE2), 308, 313–314 Protein coat complexes, and vesicle transport, 154 Protein kinase, activated by double-stranded RNA (PKR), 213 Protein kinase B/Akt pathway, 66 Protein phosphatase 2A (PP2A), 170 Protein Ser/Thr kinase PINOID (PID), 170 aProteobacterium, 105–106, 114, 120 P-Selectin glycoprotein ligand-1 (PSGL-1), and SNX20, 181 Pseudomonas aeruginosa, 256 Pseudopod formation, trigger model of, 257 Pseudo-polysomes, 224 PTEN gene, and AKT signaling, 22 Pteridium, 133 Putative microbody division (MBD) machinery, 117 Pyruvate kinase type M2, 65 R Rab coupling protein (RCP), 279–280 RAS-MAPK pathway, 5 RAS-RAF-MEK-ERK MAPK signaling cascade, 19 RBP Immunoprecipitation followed by chip analysis (RIP-chip), 235–237 Reticulocytes, 209 Retinoblastoma protein (pRb), 62 Retromer, 155 and Alzheimer’s disease, 181–184 assembly and functioning, 155–160 membrane remodeling, 180–181 multiple roles arthropods, 163–164 mammalian cells, 164–166 mouse, 166–167 nematodes, 163 plants, 162 protozoa, 161–162 yeast, 160–161 polarity establishment in yeast, 179–180 polarized traffic in epithelial MDCK cells and neurons, 181–183 polarized transport and, 167–169 PIN proteins polarity, in plant development, 167, 170–173 transcytosis of, polymeric immunoglobulin receptor, 178–179 Wnt proteins secretion, in animal embryo development, 173–178 SNX1 and/or SNX2 subcomplex, 156 and SNX family, 156–160 Vps subunits in, 155 Vps26–Vps35–Vps29 subcomplex, 156–157

353

Index

Rhabdomyosarcoma, 13–14 RhoA/ROCK pathway, 326 Rho-associated kinase (ROCK), 307 Rho-of-Plant (ROP) proteins, 172–173 Ribosomal protein S6 (rpS6) phosphorylation, 210–211 Ricin, retrograde transport of, 166 Rickettsia felis, 120, 143 RNA-induced silencing complexes (RISCs), 222 RUNX1–CBF2T1 fusion, 12 S Saccharomyces cerevisiae, 102 retromer machinery in, 160 translation in, 205 Salmonella minnesota R595 strain, 261 Schizosaccharomyces pombe, 160, 226 Sentinel cells, 255, 287 Serine/threonine phosphatase 2A (PP2A), 66 Sesamum indicum, 102 Shear stress, and vascular endothelial cells, 314 Shiga toxin, and retromer’s retrograde pathway, 166 Short interfering RNAs (siRNAs), 222 SibA, role in phagocytosis, 262–263 Signal transduction pathways, 268, 270–271, 276 Silk fibroin fibers scaffolds, 316 Skip protein, 65 SM aactin (aSMA), 326 Small nuclear RNA (snRNA), 202 aSmooth muscle actin (a-SMA), 307–308 Smooth muscle cells (SMCs), 303–304, 307, 313, 315–316, 321, 326–327 SNX9, role in membrane remodeling, 180 SNXs dimer, and cargo sorting, 156–157 and cargo-recognition subcomplex, 157–158 Sortilin-related receptor (SorLA), 165, 184 AD patients and, 182 Sorting nexin, 156, 168, 180 Spliceosome, 202 Splicing, of pre-mRNA, 202 43S preinitiation complex formation, 205–208 Squamous cell carcinoma, 38, 40–41, 43, 46–47, 50 Stem cells, mechanobiological responses of, 317–322 Sterol regulatory element binding protein (SREBP), 226 Stress granules (SGs), 222 Synechocystis, 111 T Talin, and membrane binding, 261 Targeting-induced local lesions in genomes (TILLING), 10 T-cell acute lymphoblastic leukemia (T-ALL), 10–12

TEL-AML1 fusion, 12–13 TEL-JAK2 fusion, 12 Telomerase structural subunit (hTERT), 58, 68 Telomeres, 58, 74 Tetraspanins, 269–270 Tissue engineering, 304, 316, 329, 332 Tobacco Bright Yellow 2 (BY-2) cells, 126, 133 Toll Interleukin1 Receptor (TIR)-domain proteins, 287 Toxoplasma gondii, 130 TP53 gene, 15 Transcriptional regulation, 200–201 Transforming growth factor-b (TGF-b), 308, 313, 319, 321, 324–325, 332 Transgenic coinjections, 14–15 trans-Golgi network (TGN), 154 Translation miRNA and translational repression, 223–224 preparation for RNA processing and export, 202–204 regulation, 226–227 AU-rich elements (AREs) and, 217–218 by cytoplasmic polyadenylation element (CPE), 216 by eIF4E inhibitory proteins, 215–217 elongation, 204–205 initiation, 204–205 male-specific-lethal (msl-2) mRNA, in Drosophila, 218–219 multistep mechanism of, 218–219 P-bodies and, 220–222, 225–226 reasons for, 209 by small RNAs, 222–225 targets for, 209–211 termination, 205 ternary complex formation, regulation of, 212–213 by uORFs, 214–215 Translational profiling, 228–231 Translation initiation, mechanisms of cap-independent translation initiation, 208–209 mRNA scanning and AUG recognition, 208 preinitiation complex formation, 205–207 recruitment of, preinitiation complex to mRNA, 207 80S ribosome formation, 208 Treponema pallidum, 38 Trichomanes, 133 Trichomonas galinae, 117 Trichomonas vaginalis, 117 Tritrichomonas augusta, 117 Tritrichomonas foetus, 117 Tritrichomonas suis, 117 Trypanosoma brucei, 116 Tumorigenesis, 2, 5–6, 8, 12, 17, 23 Tyrosine phosphatase Cdc25, 65 Tyrosine phosphatase degradation, 57

354

Index U

Ubiquitin–proteasome pathway, 57, 62–63 Uniaxial stretching, 304–305 limitation of, 305 Upstream open reading frames (uORFs), 214–215 Upstream regulatory region (URR). See Open reading frames (ORFs) Ureaplasma mycetes, 113 V Vacuolar protein sorting (Vps) group, 155 Vacuolar sorting receptor (VSR), 162 Vascular cell adhesion molecule-1 (VCAM-1), 314 Vascular cell differentiation, and hemodynamic forces, 326–327 Vascular endothelial growth factor receptor2 (VEGFR-2), 314 VD machinery, 105 Vesicle division machinery endocytosis, origin, and evolution of, 107–109 role of dynamins, 109 structure and function of, 109–110 vesicles formation models, 110 Vesicle division (VD) ring, 100, 110 V-Hþ ATPase vesicles, 266–267 Virus-associated human diseases, 36 Vps10-domain family protein, 165 Vps26–Vps35–Vps29 subcomplex, 156–157 W Wiskott-Aldrich syndrome protein (WASP), 109, 180 Wnt-bcatenin pathway, 5 Wntless (Wls), retromer interaction with, 163, 174–178 Wnt proteins secretion, and retromer role, 173–178 Wnt signaling pathway, 163, 173–174 Wnt sorting receptor, 176 Wortmannin, 171, 279 X X-box-binding protein 1 (XBP-1), 234 Xenotransplantation, 5, 23–25

Xiphophorus melanoma receptor kinase (Xmrk), 3 Xiphophorus melanomas, 3 Y Yeast La protein (Lhp1p), and translational regulation, 237 retromer machinery in Grd19p, 160–161 Kex2p and DPAP A, 160 polarity establishment in, 179–180 Vps35p, 161 Z Zebrafish, 3–4 cancer gene discovery, use in, 6–10 forward genetic screens, 5–7 genomic instability, and mutant line, 8, 9 G2/M DNA damage checkpoint, 9–10 retroviral insertional mutagenesis, use of, 7 ribosomal protein mutants, 8 RNA in situ hybridization, for cell proliferation, 9 separase mutant line, 8–9 cancer models, 10 APC-Wnt cancer pathway, 20–21 hematologic cancer models, 10–13 melanoma, 18–20 pancreatic cancer, 13–14 p53 pathways, 15–18, 19 PTEN in development and cancer, 21–23, 26 rhabdomyosarcoma, 14 transgenic technology, 14–15 chemical molecule screening in, 26–27 embryonic development, 4 human tumors study, 24–25 tumor development, by carcinogen exposure, 2 tumorigenesis, and human disease, 5–6 and xenotransplantation, 23–24 Zebrafish liver tumor differentially expressed gene set (ZLTDEGS), 6

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