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Mechanisms of Centriole Duplication and Spindle Assembly in Human Cells Mechanisms of centriole duplication and spindle assembly in human cells by Qianzhu Wu A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy Molecular Genetics University of Toronto © Copyright by Qianzhu Wu (2019) Mechanisms of centriole duplication and spindle assembly in human cells Qianzhu Wu Doctorate of Philosophy Molecular Genetics University of Toronto 2019 Abstract The centrosome is the major microtubule-organizing center in animal cells. Centrosomes duplicate once and only once per cell cycle thereby ensuring that two centrosomes are present in mitosis, each of them organizing one of the two spindle poles. Numerical and structural centrosomal abnormities lead to a number of devastating human diseases, including ciliopathies, microcephaly and cancer. Despite extensive effort at elucidating the molecular mechanisms underpinning centriole assembly, our understanding of this process remains incomplete and many novel factors remain to be identified. Here, using a high-throughput, semi-automated centriole duplication screen, I identified TRIM36 as a novel regulator of centriole duplication and mitotic spindle assembly. TRIM36 is a microtubule-binding RING-type E3 ligase. In centriole duplication, TRIM36 regulates daughter centriole assembly in a manner that is dependent on the RING domain. Using super-resolution microscopy, I discovered that TRIM36 plays a role in the early steps of centriole formation, where it mediates the recruitment of SAS6. TRIM36 depletion ii also caused severe defects in mitotic spindle assembly and chromosome congression. My results indicated that the dual role of TRIM36 in centriole duplication and spindle formation involves, at least in part, the recruitment of γ-tubulin to centrosomes. Co-depletion of RBM14, a negative regulator of centriole duplication, compensated for the loss of γ-tubulin recruitment and rescued both the centriole duplication and spindle assembly defects. These results provide insights into the critical role of the E3 ligase TRIM36 in centriole duplication, mitotic spindle assembly and cell cycle progression. iii Acknowledgments I would like to begin by thanking my supervisor, Dr. Laurence Pelletier. It was truly a wonderful experience working in your lab, with your enthusiasm and guidance, and with a team of talented scientists. I would not go this far without your support and guidance. Thank you! A big thank you to my committee members, Dr. Brian Raught & Dr. John Brumell. You always ask great and thoughtful questions, providing inspiring and insightful. Your comments have been very much appreciated. Thank you to all of our collaborators - Brian, Étienne, Anne-Claude, Frank, Nero and Dan - This work would not have been possible without your contributions. A big shout out to all members of the Pelletier lab, past and present and my friends at the LTRI. You made every day a fun and memorable experience. You have been amazing colleagues, friends and bench-mate (yes talking about you, Suzy). Thank you Sally & Johnny for going through my paper manuscript, which took a huge portion in this thesis. Thank you Ladan for being there in difficult times. Thank you Dave, Bahareh and João for great suggestions. A special thank you to Yi 1, Yi 2 and Gagan for all their support and help in the past years. You always kept me motivated and set me back on track. I will miss the baking days and the lab lunches. Thank you! Thank you to all my friends, Yiwang & Huayun, Kai & Huijuan, who have been supportive and generous with your time during my Ph.D. The entire experience would be so different without you. To my family, THANK YOU! Mom, Dad & Jianjin – I don’t think I can ever express how grateful I am for your love and support. I am incredibly fortunate to have you in my life. You all make me feel deeply blessed. Table of Contents Abstract ...................................................................................................................................... ii Acknowledgments ..................................................................................................................... iv Table of Contents ....................................................................................................................... v List of Tables ............................................................................................................................ ix List of Figures ............................................................................................................................ x List of Abbreviations ............................................................................................................... xiv Chapter 1. Introduction ............................................................................................................... 1 Introduction ........................................................................................................................... 2 1.1 The Centrosomes ............................................................................................................ 2 1.1.1 Organization of the human centrosome ................................................................ 2 1.1.2 Centrosome biogenesis ........................................................................................ 8 1.1.3 Spindle assembly ................................................................................................17 1.1.4 Centrosome structure/numerical abnormalities and diseases ...............................22 1.2 The Ubiquitination System ............................................................................................26 1.2.1 The covalent conjugation of ubiquitin with other cellular proteins regulates diverse eukaryotic cell functions.........................................................................26 1.2.2 Ubiquitination and de-ubiquitination in centriole duplication and spindle assembly ............................................................................................................30 1.3 The TRIM E3 ligases .....................................................................................................34 1.3.1 Sub-classes of TRIM family ...............................................................................34 1.3.2 TRIM36 .............................................................................................................36 1.4 Rationale of the Thesis ...................................................................................................37 Chapter 2. Identification of novel centriole duplication regulators .............................................39 Identification of novel centriole duplication regulators ..........................................................40 2.1 Statement of Contributions ............................................................................................40 2.2 Summary .......................................................................................................................41 2.3 Introduction ...................................................................................................................41 2.3.1 BioID .................................................................................................................41 2.4 Results ...........................................................................................................................44 2.4.1 Generation of the centriole duplication interactome ............................................44 2.4.2 Functional characterization of centriole duplication interactome and identification of novel centriole duplication regulators .......................................54 2.5 Discussion .....................................................................................................................66 2.6 Material and Methods ....................................................................................................68 2.6.1 Cell lines and Tissue culture ...............................................................................68 2.6.2 Sample preparation for BioID .............................................................................69 2.6.3 BioID followed by mass spectrometry ................................................................69 2.6.4 SAINT analysis ..................................................................................................70 2.6.5 U-2 OS S-phase arrested centriole over-duplication assay ..................................70 2.6.6 Statistical methods .............................................................................................71 Chapter 3. TRIM36 is a novel regulator of centriole duplication and microtubule organization factor ....................................................................................................................................72 TRIM36 is a novel regulator of centriole duplication and a microtubule organization factor ....................................................................................................................................73 3.1 Statement of Contributions ............................................................................................73 3.2 Summary .......................................................................................................................73 3.3 Introduction ...................................................................................................................74 vi 3.3.1 TRIM36 participates
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