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Deciphering the Role of a Microtubule Severing Protein and a Protein Kinase in Cell Cycle and Ciliogenesis in Chlamydomonas Reinhardtii

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Deciphering the Role of a Microtubule Severing Protein and a Protein Kinase in Cell Cycle and Ciliogenesis in Chlamydomonas Reinhardtii DECIPHERING THE ROLE OF A MICROTUBULE SEVERING PROTEIN AND A PROTEIN KINASE IN CELL CYCLE AND CILIOGENESIS IN CHLAMYDOMONAS REINHARDTII by M. Qasim Rasi B.Sc., Simon Fraser University, 2004 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Molecular Biology and Biochemistry ©M. Qasim Rasi 2009 SIMON FRASER UNIVERSITY Summer 2009 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author APPROVAL Name: M. Qasim Rasi Degree: Doctor of Philosophy Title of Thesis: Deciphering the role of a microtubule severing protein and a protein kinase in cell cycle and ciliogenesis in Chlamydomonas reinhardtii Examining Committee: Chair: Dr. C. Krieger Professor of Kinesiology _______________________________________________ Dr. Lynne Quarmby Senior Supervisor; Professor of Molecular Biology and Biochemistry _______________________________________________ Dr. Michel Leroux Supervisor; Associate Professor of Molecular Biology and Biochemistry _______________________________________________ Dr. Nick Harden Supervisor; Associate Professor of Molecular Biology and Biochemistry _______________________________________________ Dr. Christopher Beh Internal Examiner; Associate Professor of Molecular Biology and Biochemistry _______________________________________________ Dr. Stephen M. King External Examiner; Professor of Biochemistry, Associate Director, Graduate Program in Molecular Biology and Biochemistry, University of Connecticut Health Center Date Defended/Approved: June-5-2009 ii Declaration of Partial Copyright Licence The author, whose copyright is declared on the title page of this work, has granted to Simon Fraser University the right to lend this thesis, project or extended essay to users of the Simon Fraser University Library, and to make partial or single copies only for such users or in response to a request from the library of any other university, or other educational institution, on its own behalf or for one of its users. The author has further granted permission to Simon Fraser University to keep or make a digital copy for use in its circulating collection (currently available to the public at the “Institutional Repository” link of the SFU Library website <www.lib.sfu.ca> at: <http://ir.lib.sfu.ca/handle/1892/112>) and, without changing the content, to translate the thesis/project or extended essays, if technically possible, to any medium or format for the purpose of preservation of the digital work. The author has further agreed that permission for multiple copying of this work for scholarly purposes may be granted by either the author or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without the author’s written permission. Permission for public performance, or limited permission for private scholarly use, of any multimedia materials forming part of this work, may have been granted by the author. This information may be found on the separately catalogued multimedia material and in the signed Partial Copyright Licence. While licensing SFU to permit the above uses, the author retains copyright in the thesis, project or extended essays, including the right to change the work for subsequent purposes, including editing and publishing the work in whole or in part, and licensing other parties, as the author may desire. The original Partial Copyright Licence attesting to these terms, and signed by this author, may be found in the original bound copy of this work, retained in the Simon Fraser University Archive. Simon Fraser University Library Burnaby, BC, Canada Last revision: Spring 09 ABSTRACT Many types of eukaryotic cells are ciliated – they project a microtubule-based structure extending from the cell surface called a cilium, which plays roles in cellular motility and sensing the environment. Cilia are anchored at the cell surface by microtubule-based structures called basal bodies or centrioles. In addition to nucleating cilia, centrioles act as spindle organizing centres during cell division. We are beginning to understand that several proteins are involved in simultaneously regulating cilia and centrioles with cell division. My research focused on the function of two proteins in the ciliated alga, Chlamydomonas. The first protein, katanin, severs microtubules and we predicted that it is required during the disassembly of cilia prior to mitosis. I used a genetic approach to repress expression of the gene encoding katanin in Chlamydomonas , and thereby demonstrated that katanin in fact severs microtubules at a distinct location between basal bodies and the cilium. The other gene that I studied encodes a cyclic GMP-dependent kinase type 2 (PKG2). Mutant cells deficient in PKG2 had either no cilia, or cilia of unequal length. I discovered that PKG2 is expressed in the cytoplasm as well as the flagella and may interact with other proteins to regulate ciliary length and structure. Taken together, this research identified novel mechanisms that help explain the coordinated regulation of cilia during the cell cycle, and a novel gene with roles in ciliary assembly was also identified. Keywords: cilia; flagella; centrioles; basal body; Chlamydomonas Subject Terms: cell biology; algae iii DEDICATION To mom, dad, my sisters, my beautiful fiancée Mariam, Uncle Homy and William Lipsit: This would have been impossible without you! “Pader Jan wa Madar jan, tashakor az hama lotf wa mehrabani-ha” iv ACKNOWLEDGEMENTS I thank my mentor, Dr. Lynne Quarmby; My friends Walid Omar Babrakzai, Jeremy Parker; Lab colleagues, Marc Champigny, Laura Hilton, Jaime Kirschner and Bryan Tennant; Past members of the Quarmby Lab, Moe Mahjoub, Brian Bradley, Nick Inglis, Apollos Kim; Members of the Beh Lab and the Leroux Lab; Supervisory committee members, Dr. Michel Leroux and Dr. Nick Harden; MBB department staff members, Christine Beauchamp, Lisa Chow, Ash Lalani, Nancy Suda. v TABLE OF CONTENTS Approval ............................................................................................................................ ii Abstract ............................................................................................................................. iii Dedication ......................................................................................................................... iv Acknowledgements ............................................................................................................v Table of Contents ............................................................................................................. vi List of Figures ................................................................................................................. viii List of Tables .................................................................................................................... ix List of Abbreviations/Acronyms .......................................................................................x Chapter 1 General Introduction .......................................................................................1 1.1 Introduction .....................................................................................................1 1.2 Basic Structure of a Cilium .............................................................................2 1.3 Ciliary Assembly .............................................................................................7 1.4 Ciliary Disassembly .......................................................................................11 1.5 Cross Talk between Cilia and Cell Cycle ......................................................15 1.6 Cilia and Human Disease ..............................................................................17 1.7 Cyclic GMP-dependent Protein Kinase .........................................................22 1.8 Chlamydomonas as a Model System .............................................................24 1.9 Questions .......................................................................................................25 1.10 Figures ...........................................................................................................27 1.11 References .....................................................................................................31 Chapter 2 Katanin knockdown supports a role for microtubule severing in release of basal bodies prior to mitosis in Chlamydomonas .........................................41 2.1 Abstract ..........................................................................................................41 2.2 Introduction ...................................................................................................42 2.3 Materials and Methods ..................................................................................44 2.4 Results ...........................................................................................................50 2.5 Discussion ......................................................................................................58 2.6 Acknowledgements .......................................................................................63 2.7 Figures and Tables .........................................................................................64 2.8 References .....................................................................................................72
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