Molecular Dissection of Centrosome Biogenesis and its Implication in Human Disease by Yi Liu A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Yi Liu 2020 Molecular Dissection of Centrosome Biogenesis and its Implication in Human Disease Yi Liu Doctorate of Philosophy Department of Molecular Genetics University of Toronto 2020 Abstract Centrosomes function as the primary microtubule-organizing centers in animal cells and play instrumental roles in the control of cell signaling, motility, division and polarity. A typical centrosome consists of a centriole pair that recruits the microtubule-nucleating pericentriolar material, organizes mitotic spindle assembly, and templates the formation of cilia/flagella. In cycling cells, centriole number is tightly controlled and aberrations in this process are associated with a set of human disorders including cancer, microcephaly and ciliopathies. Although factors essential for centriole assembly, such as STIL and PLK4, have been identified, the underlying molecular mechanisms that drive this process are incompletely understood. Herein, I characterize ANK2 and CEP85 as novel proteins that are essential for centriole formation. Firstly, I show that ANK2 localizes to centrosomes and interacts with CEP120. Functionally, ANK2 is required for CEP120 centrosomal localization and centriole duplication. ANK2 also plays a role in regulating microtubule stability and the distribution of centriolar satellites. Using protein proximity mapping and high-resolution structural methods, I identify CEP85 as a novel centriole duplication factor that directly interacts with STIL. Structure-guided mutational analyses indicate that this interaction is critical for centriole loading of STIL, robust PLK4 activation and efficient centriole assembly. In addition, I also find that CEP85 and STIL play important roles in cancer ii cell migration, probably through the ARP2/3 mediated actin organization. Taken together, my PhD research advances the mechanistic understanding of centriole duplication and thus paves the way for new therapeutic approaches to treat human diseases with abnormal centrosomes. iii Acknowledgments I would like to express my sincere gratitude to my supervisor, Dr. Laurence Pelletier for his valuable and constructive suggestions throughout my Ph.D. studies. I have benefited a lot from his great mentorship and endless support on both academic and personal levels to establish my scientific career. Many thanks to my committee members, Dr. Jason Moffat and Dr. Jim Dennis for their guidance, encouragement and insightful comments on my research work to make me become a better scientist. I am also grateful to all of our collaborators – Dr. Mark van Breugel‟s lab, Dr. Etienne Coyaud, Dr. Brian Raught, Dr. Megha Chandrashekhar, and Dr. Jason Moffat. My research would not be conducted so successfully without their help and contributions. Many thanks to all members of the Pelletier lab, past and present and my friends at the LTRI. Special thanks to Dr. Gagan Gupta, Dr. Johnny Tkach, Dr. Yi Luo, Sally Cheung, and Dr. Fikret Gurkan Agircan for their help and suggestions on my research projects. Thanks to Monica for teaching me advanced microscopy skills. Also thanks Dr. Qiazhu Wu, Dr. Bahareh Adhami Mojarad, Dr. Ladan Gheiratmand, Dr. Mikhail Bashkurov, Dr. Christina Yeh, Dr. João Goncalves, Dr. David Comartin for being such amazing colleagues and giving me memorable experience. It‟s my fortune to gratefully acknowledge the support of my family, mom, dad, uncle, aunt, our dog Jacob, cousins, and close friends. Thank you all for your love, encouragement and suggestions during my Ph.D. Above all, I would like to sincerely thank Erika for her generous love and encouragement. In the past twelve years, Erika is my most enthusiastic cheerleader to motivate me to become a well- rounded person and to help me go through the hard time together. She has shared this entire amazing Ph.D. journey with me, and I couldn‟t have done it without you. iv Table of Contents Acknowledgments .......................................................................................................................... iv Table of Contents ............................................................................................................................ v 1 Chapter I: Introduction ............................................................................................................... 1 1.1 The Centrosome Structure .................................................................................................. 1 1.1.1 Centriole Structure .................................................................................................. 1 1.1.2 Pericentriolar Material ............................................................................................ 3 1.1.3 Centriolar Satellites ................................................................................................. 5 1.2 Centriole Biogenesis ........................................................................................................... 6 1.2.1 Establishing a New Centriole .................................................................................. 8 1.2.2 Defining Centriole Length .................................................................................... 14 1.3 Centriole Number Control ................................................................................................ 17 1.3.1 Centriole Disengagement ...................................................................................... 17 1.3.2 Centriole to Centrosome Conversion .................................................................... 18 1.4 Cell-Cycle-Based Mechanisms ......................................................................................... 19 1.4.1 De Novo Centriole Duplication Pathway ............................................................. 20 1.5 Centriole Duplication & Cell Proliferation ....................................................................... 21 1.5.1 Centrosome Loss and Surveillance Pathway ........................................................ 21 1.5.2 Sensing Centrosome Amplification ...................................................................... 22 1.6 The Centrosome in Health ................................................................................................ 23 1.6.1 The Role of Interphase Centrosomes .................................................................... 23 1.6.2 The Role of Mitotic Centrosomes ......................................................................... 24 1.6.3 The Role of Centrosomes in Ciliogenesis ............................................................. 27 1.7 The Centrosome Defects in Disease ................................................................................. 28 1.7.1 Centrosomes and Cancer ....................................................................................... 28 v 1.7.2 Centrosomes and Microcephaly ............................................................................ 32 1.8 Summary and Rationale of this Thesis ............................................................................. 35 2 Chapter II: Direct binding of CEP85 to STIL Ensures Robust PLK4 Activation and Efficient Centriole Assembly ................................................................................................... 37 2.1 Statement of Contributions ............................................................................................... 38 2.2 Summary ........................................................................................................................... 39 2.3 Introduction ....................................................................................................................... 40 2.4 Results ............................................................................................................................... 43 2.4.1 CEP85 is a Regulator of Centriole Duplication .................................................... 43 2.4.2 CEP85 Is Required for STIL Localization and PLK4 Activation ........................ 49 2.4.3 CEP85 and STIL Recruitment during Centriole Duplication ............................... 54 2.4.4 CEP85 Interacts Directly With the N-terminal Domain of STIL ......................... 57 2.4.5 Structural Characterisation of the STIL-CEP85 Interaction Region .................... 60 2.4.6 CEP85-STIL Interaction is required for STIL Localization and Centriole Duplication ............................................................................................................ 63 2.4.7 CEP85 and STIL Binding is Required for PLK4 Activation ................................ 70 2.5 Discussion ......................................................................................................................... 75 2.5.1 The Evolutionary Analysis of the CEP85-STIL Complex ................................... 76 2.5.2 Structural and Functional Characterization of the CEP85-STIL Complex .......... 76 2.5.3 Placing CEP85 and STIL in the Centriole Duplication Pathway ......................... 77 2.6 Materials and Methods ...................................................................................................... 82 3 Chapter III: Direct interaction between CEP85 and STIL mediates PLK4-driven directed migration .................................................................................................................................