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Invivo Significance of Tubulin Glutamylation Generated by Ttll6e

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Invivo Significance of Tubulin Glutamylation Generated by Ttll6e INVIVO SIGNIFICANCE OF TUBULIN GLUTAMYLATION GENERATED BY TTLL6E- LIGASES by SWATI SURYAWANSHI (Under the Direction of Jacek Gaertig) ABSTRACT Glutamylation is one of the most conserved post-translational modifications of α- and β- tubulin and involves addition of variable number of glutamate units as a polypeptide side- chain. Glutamylation is present on the surface of microtubules and is in a position to strongly interact with a large number of microtubule effectors such as motors, severing factors, and microtubule end-depolymerizers. Glutamhylation is present in cilia, mitotic spindle, centrioles and basal bodies. To understand the role of glutamylation in cilia, our research group has used the ciliate model system Tetrahymena thermophila. Recently, the catalytic subunits of tubulin glutamylases were identified as conserved enzymes related to tubulin tyrosine ligase, now known as TTLL proteins. Tetrahymena has 50 TTLLs and among these, the TTLL6 group of proteins are my focus of research. Tetrahymena has 6 paralogs of TTLL6 type - TTLL6A, TTLL6B, TTLL6C, TTLL6D, TTLL6E, and TTLL6F. In the previous studies it was shown that Ttll6Ap is a β-tubulin preferring, elongating enzyme that mainly localizes to cilia and overexpression of this protein leads to ciliary paralysis. As all of the 6 TTLL6 type E-ligases are paralogs, we suspected functional redundancy among them. We created Tetrahymena strains lacking these genes to study the significance of glutamylation contributed by them. We show that TTLL6 type E-ligases mainly catalyze the elongation of glutamate side chains on β-tubulin and that glutamylation contributed by Ttll6Ap and Ttll6Fp, is required for normal ciliary motility. Mutants lacking these E-ligases swim and multiply slowly, exhibit lack of ciliary reversal, altered ciliary waveform and lower beat frequency. We also show that combined glutamylation by Ttll6Ap, Ttll6Bp, Ttll6Dp, Ttll6Ep and Ttll6Fp, at low dose are required for maintaining normal ciliary lengths. Strains lacking these proteins swim and multiply even more slowly as compared to the double mutants and their phagocytosis is impaired. Interestingly, these cilia of these mutants have significantly shorter lengths as compared to wild-type cilia. Together, in the light of our results, we show that glutamylation is required for the restraining activity of inner-dynein arms in ciliary motility and that, in dose-dependent manner, tubulin glutamylation regulates processes involved in regulating lengths of cilia. INDEX WORDS: Microtubule, Cilia, Polyglutamylation, E-ligase, Dynein, TTLL6 INVIVO SIGNIFICANCE OF TUBULIN GLUTAMYLATION GENERATED BY TTLL6E- LIGASES by SWATI SURYAWANSHI B.S., University of Pune, India, 2002 M.S., University of Pune, India, 2004 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2010 © 2010 SWATI SURYAWANSHI All Rights Reserved INVIVO SIGNIFICANCE OF TUBULIN GLUTAMYLATION GENERATED BY TTLL6 E- LIGASES by SWATI SURYAWANSHI Major Professor: Jacek Gaertig Committee: Marcus Fechheimer Edward Kipreos John Shields Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2010 DEDICATION I would like to dedicate this dissertation to my parents Prof. Maruti Suryavanshi and Lata Suryavanshi and my brother Rahul, as without their unconditional support, motivation and love, I would not have been where I am today. I am so grateful to my father for inculcating and nurturing my interest and curiosity in biological sciences. It is solely their dream and tremendous hard work that has facilitated me to visage the challenging task of Ph.D with perseverance. I would like to also dedicate this thesis to my husband, Dr. Pushkarraj, for his love and support. iv ACKNOWLEDGEMENTS This research has been benefitted from the insight and directions from a number of people. I would like to take this opportunity to express my immense gratitude towards them. Firstly I would like to thank my mentor Dr. Jacek Gaertig who has been very supportive and tolerant of me during the long and trying years of my doctorate. He has provided timely and instructive comments at each stage of my research that has enabled me to complete my research. Dorota, our post-doctorate, also lovingly called as ‘wife of our lab’ has always been there at every stage of my lab experience, ranging from ordering reagents to trouble shooting. My committee members Dr. Marcus Fechheimer, Dr. Edward Kipreos and Dr. John Shields have helped me a lot in giving my research the right direction at crucial stages. Dr. John Shields’ electron microscopy classes and guidance has assisted my growing fascination for the ultrastructural aspect of biology. I would like to thank Mary Ard for helping me with transmission electron microscopy. My lab members have a great share in turning some long arduous days into fun- filled time by their mere presence. Kathy, Marian, Beth and Genia have been extremely helpful and supportive in helping me with my application deadlines. I am greatful to my husband Dr. Pushkarraj, who has constantly encouraged and motivated me through the last grilling years of my Ph.D. It was his love that helped me complete my research. Finally, I would like to thank all my friends and family members that I have not mentioned here. It is because of their emotional support and motivation that I have come a long way in overcoming difficult situations. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ............................................................................................................ v LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ...................................................................................................................... viii CHAPTER 1 INTRODUCTION ......................................................................................................... 1 2 REVIEW OF LITERATURE........................................................................................ 5 3 TUBULIN GLUTAMYLATION REGULATES CILIARY MOTILITY BYALTERING DYNEIN ARM ACTIVITY ........................................................... 119 4 TUBULIN GLUTAMYLATION IS REQUIRED FOR NORMAL LENGTH OF CILIA ........................................................................................................................ 153 5 CONCLUSIONS ....................................................................................................... 189 vi LIST OF TABLES Page Table 2.1: Outer and inner dynein arm mutants in Chlamydomonas .......................................... 118 Table 3.1: Primers used for construction of targeting fragments ................................................ 151 Table 3.2: Diagnostic primers used for verification of gene disruptions .................................... 152 Table 3.3: Phagocytic activity is restored by reintroduction of GRP-Ttll6Ap transgene into 6AF- KO cells ........................................................................................................................... 152 Table 4.1: Primers Used for Construction of Targeting Fragments ............................................ 185 Table 4.2: Diagnostic primers used for verification of gene disruptions .................................... 188 vii LIST OF FIGURES Page Figure 2.1: Structure of microtubules ......................................................................................... 109 Figure 2.2: Ribbon diagram of tubulin dimers ............................................................................ 110 Figure 2.3: Cartwheel structure of triplet microtubules .............................................................. 111 Figure 2.4: Phylogenetic tree of murine TTLL group of proteins .............................................. 112 Figure 2.5: Longitudinal and cross-section of a cilium ............................................................... 113 Figure 2.6: Organization of dynein heavy chain subdomains ..................................................... 115 Figure 2.7: Diagrammatic representation of the arrangement of outer and inner dynein arms on axonemal doublets ........................................................................................................... 116 Figure 2.8: Models explaining movement of dynein on MTs ..................................................... 117 Figure 3.1: Deletion of Ttll6Ap and Ttll6Fp Leads to a Loss of Tubulin Glutamylation in Cilia ........................................................................................................................ 138 Figure 3.2: Cells Lacking Ttll6Ap and Ttll6Fp Display a Loss of Cilia-Dependent Functions . 140 Figure 3.3: Ttll6Ap and Ttl6Fp Generate Polyglutamylation Primarily on B-Tubule of Outer Microtubules.................................................................................................................... 142 Figure 3.4: Tubulin Glutamylation Regulates the Velocity of Inner Dynein Arm-Driven Microtubule Sliding in Axonemes In Vitro ..................................................................... 144 Figure 3.5: Loss of Ttll6Ap and Ttll6Fp Leads to a Loss of Elongated Glutamyl Side Chains in Axonemes ........................................................................................................................ 146 Figure 3.6: AF-KO Mutants Respond to Membrane-Depolarizing
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