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Intermediate Filaments Are of Intermediate Diameter (10 Nm) THE ROLE OF THE CYTOSKELETON IN THE MORPHOGENESIS OF THE AVIAN ERYTHROCYTE by Bettina Winckler B.A., Swarthmore College 1986 Submitted to the Department of Biologyin partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology at the Massachusetts Institute of Technology February 1994 © Massachusetts Institute of Technology, 1994 All rights reserved Signature of Author Department of Biology ,-2/7 _ December 10, 1993 Certified by -- - - Professor Frank Solomon .. ,, / Thesis Advisor Accepted by . -. Professor Frank Solomon Chairman of the Graduate Committee A~ASS;~f;CH'I'~'EtT2!NST1,UIE 1 JlA,', 8 l. Y994 j.' ~': ," it'' 2 -iF;s.1-;~~~~~~~~~~~~~~~4LS,,g, a943 The Role of the Cytoskeleton in the Morphogenesis of the Avian Erythrocyte by Bettina Winckler Submitted to the Department of Biology on December 10, 1993 in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in Biology Abstract The chicken red blood cell serves as a good model system for studying the role of the cytoskeleton in the determination of cell shape because of its simplicity. Its shape is that of a flattened ellipsoid. A bundle of microtubules, the marginal band, encircles the cell at the equator. This microtubule organelle is rigorously specified, qualitatively and quantitatively. The precursors to this cell can be obtained in synchronous populations from the circulation of embryos. They develop in suspension without cell-cell or cell-substrate contacts. This dissertation analyzes the cytoskeleton of developing red cells and its role in morphogenesis. Disruption of the F-actin in embryonic cells in a narrow developmental window leads to microtubule-dependent shape changes giving rise to highly asymmetric cells. We propose that the establishment of a flattenend cell shape and of the marginal band occurs by a microtubule- autonomous mechanism. We establish that two proteins of the ERM protein family (ezrin-radixin-moesin) are components of the marginal band. Radixin is the major ERM protein associated with the cytoskeleton. The expression pattern and localization of the ERM proteins in chicken erythrocytes limit previous models for ERM function and raise possibilities for their role in erythrocyte morphogenesis. We propose that the ERM proteins play functionally conserved roles in quite diverse organelles. Thesis Advisor: Dr. Frank Solomon Title: Professor of Biology 2 Acknowledgments I have been in graduate school for over six years and have not regretted it (yet). Most of the good times I have had are connected to the people I have met. Frank's lab was a great place for me, and I will certainly miss it. It is intellectually stimulating and challenging (lots of great people with whom to have good scientific conversations). It provides a very friendly and supportive atmosphere. Everybody is multi- dimensional, with lots of different interests, not only restricted to science. Besides scientific conversations, the lab has always been a great place to discuss politics, movies, and life in general, as well as for exchanging the worst puns. And last not least, everybody is addicted to chocolate (and/or caf6 latte)-- and if you are not when you start, Frank will make sure you are by the time you leave. Turning me into a chocolaholicis not the only thing, though, Frank did for me during my time in the lab. (If he should claim that it was me who turned him into a chocolate addict who will stop short of nothing, just say one word: "Napoleon",and he will hold his peace forever.) He taught me not only how to find the focal plane in the microscope, but how to devise controls, how to think about my experiments quantitatively as well as qualitatively, how to play devil's advocate with every experimental outcome and every interpretation, how to do a good experiment. Now and in the future, I always think: what would Frank think of this experiment, this line of experimentation, this model? He has been supportive of me in so many ways, but always challenging, always pushing me just a little farther, as long ago as the walk from Toscaninni's and as recently as his comments on the introduction for this dissertation. There are not enough torronis in the world to pay back the energy that Frank has spent on making me a Ph.D. For his mentorship, his enthusiasm, his encouragement and understanding, his guidance, and his M&M habit, I will be grateful for all times. I also want to thank the other members of the lab-- past and present-- for making life so enjoyable. Margaret Magendantz deserves highest praise not only for teaching me how to do most experiments, but also for her encouraging words, her cheerfulness and friendliness, and her good advice on so many issues. Furthermore, she saved me many hours and days by starting egg incubations on the weekends, changing dialysis buffers or turning my 2D gels up early in the morning, and generally keeping an eye on my experiments while I was not there. Without her, the lab would not be the friendly place it is. Of the past members, I want to thank Wilma Wasco and Laurie Connell for teaching me (among other things) which steps are unnecessary in experimental procedures, Jon Dinsmore for many pieces of good advice and Brant Weinstein for asking: "Was ist los?" so many times, leading to good debates about my project. 3 Of the current members, I want to thank my baymate Vida Praitis, who was usually the first to hear about experimental results and ideas, frustrations and triumphs, for her friendship. I want to thank Suzanne Gu6nette, Charo Gonzalez Agosti, Vida Praitis and Jill Hahn for enduring endless conversations about 13H9 and 904 and about less confused, more fun topics. I want to thank Suzanne Guenette, Charo Gonzalez Agosti, and Julie Archer for putting me up for many nights when I came up from New York. I also want to thank Lisa Urry for her advice and encouragement during my first two years, Megan Grether for endless discussions about science and life before and after exercise classes, my classmates Rachel Meyer, Martha Marvin, Mary Herndon, Dawn Faruggio, Megan Grether, Bronwen Brown, and Jan Carminati for many dinners and a great support network, and Julie Archer, Michael Henry, Letty Vega, Adam Grancell, and David Kirkpatrick for many fruitful conversations. For five years, I lived with several roommates who made it great fun to come home at night and I want to thank Martha Marvin, Martha Lees, Laura Stewart, and Sue Oleski Farrell for their friendship and many pints of Ben&Jerry's. I want to thank my friends Chhaya Rao, Bianca Falbo, Martha Marvin, Charo Gonzalez Agosti and Suzanne Gu6nette for standing by me when I was not a lot of fun, cranky and stressed-out. I hope I will be able to do the same for them some time. I would also like to acknowledge gratefully the time and energy spent by my thesis committee members, Arthur Lander, Paul Matsudaira, and Richard Hynes, and the fruitful discussions we had in our meetings. They have contributed in many ways to making this dissertation and this scientist. Nicht zuletzt m6chte ich meinen Eltern, Susanne Winckler-Kalies und Heino Winckler, danken fiur alles, was sie fuir mich getan haben. Sie haben mich immer umgeben mit Liebe und Beistand. Ohne ihr Vertrauen in meine Fahigkeiten und ihre Unterstiitzung-- emotional, finanziell und in jeder anderen Hinsicht-- ware ich heute nicht an dieser Stelle. I dedicate this dissertation to Jeff Olick for everything he has been and done for me, most of which cannot be put into words. 4 TABLE OF CONTENTS Title page 1 Abstract 2 Acknowledgment 3 Table of contents 6 List of figures and tables 8 CHAPTER ONE: The Role of the Cytoskeleton in the establishment and maintenance of cell shape 10 Introduction 11 The major cytoskeletal elements 14 Interactions among different cytoskeletal elements 20 Determinants of cytoskeletal diversity 23 The chicken erythrocyte as a model system 34 The ERM-proteins: ezrin, radixin, and moesin 45 CHAPTER TWO: Cytochalasin D induces asymmetry in embryonic chicken erythroblasts: Microtubules as a driving force for morphological change 52 Introduction 53 Materials and Methods 55 Results 58 Discussion 88 CHAPTER THREE: The 13H9 Antigen in Chicken Erythrocytes: Death of the Subset Hypothesis 93 Introduction 94 Materials and Methods 98 Results 103 Discussion 139 CHAPTER FOUR: Expression and Localization of ERM-Proteins in a Simple Cell, the Chicken Erythrocyte 143 Introduction 144 Materials and Methods 148 Results 154 Discussion 182 5 CHAPTER FIVE:The Morphogenesis of the Chicken Erythrocyte 188 APPENDICES 198 Appendix one: Association of Ezrin Isoforms With the Neuronal Cytoskeleton 198 Introduction 199 Materials and Methods 200 Results 201 Discussion 207 Appendix two: Testing the Subset Hypothesis Introduction 209 Materials and Methods 211 Results 219 Discussion 231 Appendix three: Nocodazole-sensitivity of the 13H9-Antigen in HeLa Cells Introduction 232 Materials and Methods 234 Results 235 Discussion 246 Appendix four: Identifying Near Neighbors of the 904-Antigenin the Marginal Band by Crosslinking Introduction 248 Materials and Methods 250 Results 252 Discussion 258 BIBLIOGRAPHY 259 6 LIST OF FIGURES AND TABLES 1-1: Domain comparison of members of the band 4.1 superfamily 48 2-1: Microtubule intermediates during chicken primitive series erythropoiesis 60 2-2: Induction of asymmetric morphologies in red blood cells from day 2 embryos 63 2-3: Shape change as a function of development 66 2-4: Morphologies of embryonic red blood cells isolated and incubated in the absence
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