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The Stabilization of Basal Bodies to Resist Cilia Generated Forces

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The Stabilization of Basal Bodies to Resist Cilia Generated Forces THE STABILIZATION OF BASAL BODIES TO RESIST CILIA GENERATED FORCES by BRIAN ANTHONY BAYLESS B.S., University of California, Irvine, 2010 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Cell Biology, Stem Cells, and Development 2016 This thesis for the Doctor of Philosophy degree by Brian Anthony Bayless has been approved for the Cell Biology, Stem Cells, and Development Program By Michael McMurray, Chair Bruce Appel Jennifer DeLuca Jeff Moore Rytis Prekeris Chad Pearson, Advisor Date: August 19, 2016 ii Bayless, Brian Anthony (Ph.D., Cell Biology, Stem Cells and Development) The Stabilization of Basal Bodies to Resist Cilia Generated Force Thesis directed by Assistant Professor Chad G. Pearson ABSTRACT Centrioles and basal bodies (CBBs) are the major microtubule-organizing centers of the cell. Centrioles nucleate the microtubules of the mitotic spindle, and basal bodies anchor and nucleate the ciliary/flagellar axoneme. In both contexts, centrioles and basal bodies experience mechanical force from the pulling of chromosomes during mitosis and the movement of the cilia and flagella, respectively. Failure to stabilize against mechanical force causes CBBs to fragment or disassemble, and may lead to multipolar mitosis and disassembly of cilia. Structurally, CBBs are radially symmetric cylinders comprised of nine sets of modified triplet microtubules arranged around a cartwheel shaped structure at its proximal end. The triplet microtubules that make up CBBs are hyperstable as evidenced by their resistance to various microtubule stressors. The factors that stabilize the CBB from mechanical force are unknown. The ciliate Tetrahymena thermophila serves as a promising model organism to study mechanical forces on basal bodies. Using this system I have shown that basal body stability proteins Bld10 and Fop1 stabilize basal bodies against the forces of ciliary beating. I have also developed a novel method to measure the incorporation dynamics of proteins into the basal body. Additionally, I have shown that Fop1 and post- tubulin glutamylation localize asymmetrically at the basal body coincident with the site of highest predicted cilia generated compression forces. This work highlights my achievements and contributions to the understanding of CBB stabilization against mechanical forces. The form and content of this abstract are approved. I recommend its publication. Approved: Chad G. Pearson iii TABLE OF CONTENTS CHAPTER I: INTRODUCTION ............................................................................................................... 1 Centrioles and Basal Bodies ............................................................................................. 1 Centriole and Basal Body Stabilization ............................................................................11 Motile Cilia and Flagellar Beating .....................................................................................20 Tetrahymena as a Model Organism for Basal Body Research .........................................30 Conclusions and Thesis Aims ..........................................................................................37 II: BLD10/CEP135 STABILIZES BASAL BODIES AGAINST CILIA-GENERATED FORCE.39 Introduction ......................................................................................................................39 Results ............................................................................................................................42 Discussion .......................................................................................................................62 Materials and Methods ....................................................................................................67 III: MOLECULAR ASYMMETRIES STABILIZE BASAL BODIES AGAINST CILIARY BEATING FORCES.............................................................................................................74 Introduction ......................................................................................................................74 Results ............................................................................................................................77 Discussion .......................................................................................................................96 Materials and Methods ....................................................................................................99 IV: CONCLUSIONS AND FUTURE DIRECTIONS ............................................................ 105 APPENDIX A..................................................................................................................... 110 APPENDIX B..................................................................................................................... 113 APPENDIX C .................................................................................................................... 117 REFERENCES .................................................................................................................. 118 iv LIST OF FIGURES 1.1: CBB structure ........................................................................................................... 5 1.2: Axoneme structure ................................................................................................. 22 1.3: Polarized organization of Tetrahymena basal bodies ............................................. 31 1.4: Tetrahymena basal body structure ......................................................................... 32 1.5: Schematic representation of Tetrahymena basal bodies and associated accessory structures ...................................................................................................................... 34 2.1: TtBld10 is a member of a highly conserved family of CBB proteins ........................ 43 2.2: TtBld10 localizes to the basal body outer cartwheel domain ................................... 45 2.3: TtBld10 loss causes decreased cellular growth ...................................................... 48 2.4: TtBld10 knockout causes a loss of basal bodies..................................................... 49 2.5: Maturation and disassembly of K-like-Antigen ........................................................ 50 2.6: TtBld10 is required for basal body assembly .......................................................... 51 2.7: TtBld10 is required for the maintenance of basal bodies ........................................ 53 2.8: Triplet microtubule assembly and stability defects in TtBld10 cells ....................... 55 2.9: TtBld10 protein stably accumulates at basal bodies ............................................... 58 2.10: GFP-TtBld10 temporally matures at basal bodies ................................................. 60 2.11: Cilia generated forces destabilize basal bodies in TtBld10 cells ......................... 61 3.1: Fop1 is a basal body stability protein ...................................................................... 78 3.2: Supplemental to Figure 3.1 .................................................................................... 79 3.3: Fop1 stabilizes basal bodies to resist ciliary beating ............................................... 81 3.4: Fop1 localizes asymmetrically at the basal body .................................................... 83 3.5: Supplemental to Figure 3.4 .................................................................................... 86 3.6: Poc1 promotes normal incorporation of Fop1 into the basal body .......................... 87 3.7: Supplemental to Figure 3.6 .................................................................................... 89 3.8: Basal body microtubule glutamylation increases in basal body stability mutants .... 90 v 3.9: Supplemental to Figure 3.8 .................................................................................... 93 3.10: Basal bodies are stabilized through distinct pathways .......................................... 94 3.11: Supplemental to Figure 3.10 ................................................................................. 95 vi ABBREVIATIONS CBB: Centriole and Basal Body PCM: Pericentriolar Material MAP: Microtubule Associated Protein IEM: Immuno-Electron Microsopy Kl-Ag: K-like-Antigen TEM: Transmission Electron Microscopy FRAP: Fluorescent Recovery After Photobleaching PEO: Poly Ethelene Oxide PTM: Post-Translational Modification SIM: Structured Illumination Microscopy CR: Conserved region vii CHAPTER I INTRODUCTION1 Centrioles and Basal Bodies Centrioles and basal bodies (CBBs) are self-assembling cellular structures that act as microtubule organizing centers. As microtubule organizing centers, CBBs are responsible for organizing the microtubules of mitotic spindles and ciliary axonemes, respectively. CBBs are composed of modified triplet blades of microtubules that extend to make up the length of their barrel shaped structure. The integrity of this structure is essential for centrioles and basal bodies to serve their function as microtubule organizing centers. In order to ensure proper structural integrity of centrioles and basal bodies, their assembly and maturation is highly regulated and organized. Centriole and basal body function CBBs are evolutionarily conserved microtubule organizing centers. During mitosis centrioles are key components of centrosomes,
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