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The Multiple Functions of Efhc1 in Tetrahymena and Xenopus

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The Multiple Functions of Efhc1 in Tetrahymena and Xenopus THE MULTIPLE FUNCTIONS OF EFHC1 IN TETRAHYMENA AND XENOPUS By Ying Zhao B.S., University of Science and Technology of China A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Doctor of Philosophy Department of Molecular, Cellular, and Developmental Biology 2015 i This thesis entitled: The Multiple Functions of EFHC1 in Tetrahymena and Xenopus written by Ying Zhao has been approved for the Department of Molecular, Cellular, and Developmental Biology Mark Winey, Ph.D., Advisor Gia Voeltz, Ph.D., Chair of Committee Date The final copy of this thesis has been examined by the signatories, and we Find that both the content and the form meet acceptable presentation standards Of scholarly work in the above mentioned discipline. ii Zhao, Ying (Ph. D., Molecular, Cellular, and Developmental Biology) The Multiple Functions of EFHC1 in Tetrahymena and Xenopus Dissertation directed by Professor Mark Winey The basal body is the microtubule-organizing center that nucleates the axoneme of the cilium. Both basal bodies and cilia are complex intracellular organelles composed of hundreds of polypeptides found throughout the eukaryotes. Different types of cilia have various and often over-lapping functions, from driving fluid flow and cellular motility, to the establishment of distinct cellular domains and morphologies, to mediating and modulating intracellular signaling involved in tissue patterning. Dysfunction of basal body or ciliary components has been implicated in a variety of human diseases. EFHC1 is conserved from single cell alga to human beings. Members of this protein family contain three tandem DM10 domains and a putative EF-hand Ca2+ binding motif at the C terminal end. In some organisms, the EF-hand motif appears to be missing. Mutations of EFHC1 have been linked to juvenile myoclonic epilepsy (JME). The EFHC1 paralog, EFHC2, is also present in many species and mutant forms are reported to be pathological. Despite their wide spread conservation, the cellular functions of EFHC family proteins and the underlying mechanisms associated with the pathology of mutant forms remains obscure. In this study, both the ciliate Tetrahymena thermophila and the frog Xenopus laveis were used as distinct systems to investigate the functions of EFHC1. Here I have shown that there are two EFHC1- like genes in Tetrahymena. Both of them encode proteins that localize to the basal bodies and the cilia, and their localization is independent on each other. Cells lacking either individual gene do not exhibit obvious growth or basal body defects. However, both of them have structural defects, missing the MIP1a complex inside the axonemal doublet microtubules. Unlike Tetrahymena, only the EFHC1b in X.laveis was confirmed to be expressed. I have demonstrated XlEFHC1 was associated with both motile and immotile cilia. It is involved in ciliated cell formation and cilia genesis. Knockdown of XlEFHC1 by morpholino resulted in disrupted central nervous system patterning and deficient neural crest formation and migration. Reduction of XlEFHC1 leads to increased level of Wnt8a RNA, suggesting XlEFHC1 acts as an antagonist of canonical Wnt signaling. Together, this thesis work uncovered unexpected roles of EFHC1 in maintaining axoneme structure, in ciliogenosis and in regulating Wnt signaling pathway, which might well be relevant to the etiology of JME. iii Acknowledgments First, I would like to thank my advisor Mark for his intellectual and moral support over the past six years. Mark had made himself available to answer my numerous questions. He always had suggestions on the experiments that I could be doing and at the same time gave me a lot of freedom to pursue my own ideas. I have also learned from him that never give up easily. None of this work would have happened without Mark’s support to follow the stories where they took me. Thanks for all the current and previous members of Winey lab for providing such a fun place to learn how to do good science and making my graduate school experience very enjoyable. And I also thank my collaborators, without whom the work would not have been as successful. I would like to express my thanks to my committee members, Gia, Min, Mike and Dan, for providing me all the guidance and suggestions for my research. I would like to thank my family for their love, encouragement and support over the years, especially my mom and dad, who always believe in me; without them, I definitely would not make this far today. Finally, I would like to thank my fiancé, Qian, for his support over the years we have been together. I’m looking forward to share new adventures with you in the future. iv Table of Contents Chapter 1. Introduction ................................................................................................................................ 1 I. Cilia structure and function ......................................................................................................... 1 II. Cilia signaling ............................................................................................................................... 3 III. Basal body structure and function ............................................................................................ 5 IV. Microtubule inner protein ......................................................................................................... 7 V. Ciliopathies .................................................................................................................................. 9 VI. Juvenile myoclonic epilepsy .................................................................................................... 11 VII. EFHC1 protein family .............................................................................................................. 12 VIII. EFHC1 function ....................................................................................................................... 16 Chapter 2. Localization of four conserved basal body component proteins ........................................... 20 I. Introduction ................................................................................................................................ 20 II. Material and method ................................................................................................................ 24 III. Results ...................................................................................................................................... 27 IV. Discussion ................................................................................................................................. 41 Chapter 3. Genetic analysis of BBC73 in Tetrahymena thermophila........................................................ 46 I. Introduction ................................................................................................................................ 46 II. Material and Method ................................................................................................................ 47 III. Results ...................................................................................................................................... 52 IV. Discussion: ................................................................................................................................ 72 Chapter 4. Functional analysis of EFHC1 in Xenopus embryos ................................................................. 79 I. Introduction ................................................................................................................................ 79 II. Material and Methods .............................................................................................................. 81 III. Results: ..................................................................................................................................... 83 IV. Discussion ............................................................................................................................... 105 Chapter 5. Conclusion .............................................................................................................................. 108 References: ............................................................................................................................................... 115 v List of Tables Table 2-1. The four BBC protein candidates are conserved ....................................................................... 28 List of Figures Figure1-1. Diversity of ciliary axonemes ....................................................................................................... 2 Figure1-2. The Structures and function of centrioles/basal body ................................................................ 6 Figure1-3. 3D structures of DMT and MIP1–4 organization in Chlamydomonas axonemes........................ 8 Figure1-4. Genes involved in ciliopathies ................................................................................................... 10 Figure1-5. Phylogenetic analysis and sequence alignment of EFHC family ................................................ 13 Figure2-1. Tetrahymena basal body organization ...................................................................................... 22 Figure2-2. Bbc31 is associated with the accessory structures of basal bodies .......................................... 29 Figure2-3. Bbc31 is
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