Molecular Mechanisms Controlling Centriole Duplication Item Type text; Electronic Dissertation Authors Boese, Cody Citation Boese, Cody. (2020). Molecular Mechanisms Controlling Centriole Duplication (Doctoral dissertation, University of Arizona, Tucson, USA). Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction, presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 05/10/2021 17:34:06 Link to Item http://hdl.handle.net/10150/648667 MOLECULAR MECHANISMS CONTROLLING CENTRIOLE DUPLICATION by Cody Boese ____________________________ Copyright © Cody Boese 2020 A Dissertation Submitted to the Faculty of the GRADUATE INTERDISCIPLINARY PROGRAM IN CANCER BIOLOGY In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2020 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by: Cody Boese titled: Molecular mechanisms controlling centriole assembly and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy. Gregory C Rogers Date: Jul 21, 2020 Gregory C Rogers Date: Jul 21, 2020 Anne E Cress Date: Oct 22, 2020 Keith Maggert Date: Jul 21, 2020 Ghassan Mouneimne Date: Aug 3, 2020 Nathan Ellis Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. Gregory C Rogers Date: Jul 21, 2020 Gregory C Rogers Cellular and Molecular Medicine 2 ACKNOWLEDGEMENTS I would like to thank my P.I., Greg Rogers for mentoring me and teaching me, but mostly for being so supportive of all my ideas. I would like to thank my committee, Nathan Ellis, Keith Maggert, Gus Mouneimne, and Anne Cress for always being willing to meet with me and for helping me think critically about my research. I would like to thank all members of the Rogers lab for their ideas and help with technical aspects of my project, but mostly for being an all-around good group to be around. Lastly, a special thanks to my family and friends, especially my fiancé, Irene Moreno for her never ending, all-around support throughout these years. 3 TABLE OF CONTENTS LIST OF FIGURES………...……………………………………………………………………7 LIST OF TABLES………………………...…………………………………………….……….9 ABSTRACT…………………………………………………………………………….……….10 CHAPTER ONE: CENTROSOMES – AN OVERVIEW 1.1 Introduction…………………………………………………………………………………12 1.2 Centrosome Dysregulation and Cancer………………………….………………………..12 1.3 The Centriole – The Key to Centrosome Duplication……………………………………17 1.4 Molecular Regulation of Centrosome Duplication……………………………………….17 A. Forming the Spot of Centriole Assembly……………………………………………...17 B. From Procentriole Spot to Cartwheel Formation…………………………………….20 C. Centriole Growth………………………………………………………..……………...22 D. Centriole Disengagement and Licensing for Reduplication………..………………...23 1.5 Molecular Regulation of Centrosome Function…………………………………………..25 A. Pericentriolar Material Assembly……………………………………………………..25 B. Centrosome Separation………………………………………………………………...27 1.6 Figures……………………………………………………………………………………….29 1.7 Tables………………………………………………………………………………………..33 4 CHAPTER TWO: ASTERLESS IS A POLO-LIKE KINASE 4 SUBSTRATE THAT BOTH ACTIVATES AND INHIBITS KINASE ACTIVITY DEPENDING ON ITS PHOSPHORYLATION STATE 2.1 Abstract…………………………………………………………………………………...…35 2.2 Introduction…………………………………………………………………………………36 2.3 Results……………………………………………………………………………………….38 2.4 Discussion…………………………………………………………………………………...50 2.5 Materials and Methods……………………………………………………………………..54 2.6 Figures……………………………………………………………………………………….62 2.7 Tables………………………………………………………………………………………..83 CHAPTER THREE: ASTERLESS PHOSPHORYLATION PROMOTES SINGLE SITE OF CENTRIOLE ASSEMBLY 3.1 Abstract………………………………………………………………………….………..…85 3.2 Introduction……………………………………………………………………….……..….85 3.3 Results and Discussion…………………………………………………………………...…88 3.4 Materials and Methods…………………………………………………………………..…98 3.5 Figures……………………………………………………………………………………...103 5 CHAPTER 4: FUTURE DIRECTIONS AND CONCLUSIONS 4.1 Identification of a Cellular Plk4 Inhibitor………...………………………………..……119 4.2 Formation of the Procentriole Spot by Wdb……………………………...………..……121 4.3 Further Molecular Characterizations of the Asl-Plk4 Interaction …………….……...123 4.4 Figures…………………………………………………………………………………...…125 APPENDIX A: PUBLICATIONS …….……………….………………………………….…131 COMPLETE LIST OF REFERENCES…………………………..…………………….…...132 6 LIST OF FIGURES 1.1 The Centrosome: A Mother-Daughter Centriole Pair Surrounded my Pericentriolar Material…………………………………………..………………………………………….29 1.2 Two Centrosomes Facilitate Bipolar Mitotic Spindle Formation……………………….30 1.3 Centriole Duplication Cycle – An Overview……………………………………………...31 1.4 Centrioles Exhibit a Nine-Fold Radial Symmetry………………………………………..32 2.1 Plk4 Phosphorylates the N-terminal Domain of Asl….……………………………..……62 2.2 Asl-A Phosphomutants are Largely α-helical and Self-Oligomerize……………..……..64 2.3 The Phosphorylation State of Asl-A Controls Plk4 Activity……………………………..67 2.4 Nonphosphorylatable Asl-A (13A) Stimulates Plk4 Activity In Vitro……………….….69 2.5 Asl-A Phosphomutants Control Kinase Activity by Modulating Plk4 Inhibition……...71 2.6 Plk4 Phosphorylates Its Kinase domain and Asl-A, Generating a State that Inhibits Kinase Activity…..……………………………………………………………………………...73 S2.1 Full-length Asl Phosphomutants Induce Centriole Amplification……….…………….75 S2.2 The Phosphorylation State of Asl-A Does Not Influence Cellular Aggregate Formation and has Little Effect on Binding to Plk4 PB1-PB2………………….………………………..77 S2.3 Plk4 S228 Phosphomutants are Catalytically Active but do not Bind Asl-A When Lacking PB1-PB2…………………………………….………………………………………....80 3.1 Phosphorylation of T3 and S7 in Asterless-A is Necessary and Sufficient to Block Centriole Duplication………………………..……………………………………………...…103 7 3.2 Asl-A-Plk4 Interaction is Required for Asl-A-2PM to Block Centriole Duplication…106 3.3 Phospho-Asl Forms a Ring of Puncta at the Mother Centriole that may Prohibit Daughter Centriole Assembly.………………………………………………………………..108 3.4 Wdb Interacts with Asl and Promotes Active Site of Centriole Assembly…………….110 S3.1 Development of Plk4 and Asl Phospho-Specific Antibodies…..………………………113 S3.2 Wdb and Wrd Localize to Centrioles and Cause Centriole Amplification When Overexpressed…………………………...…………………………………………………….115 S3.3 Wdb Interacts with Ana2 and Forms a Complex with Plk4 and Asl-A.……………..117 4.1 Screen to Determine Co-Inhibitor of Plk4……………..………………………………...125 4.2 Utilization of a Proximity-Dependent Biotinylation Assay……………………………..126 4.3 Centriolar Localization of Wdb and Ana2………………………………………………128 4.4 Asl-A dimerization is Required for its Interaction with Plk4…………………………..129 8 LIST OF TABLES 1.1 A List of Centrosome Associated Proteins and Their Functions……….….…………….33 2.1 Phosphorylation Sites of Asl-A……………...………………………………………….….83 9 ABSTRACT Centrioles are barrel shaped, non-membrane bound organelles that typically exist in pairs where the younger ‘daughter’ centriole emanates orthogonally off of the proximal end of the older ‘mother’ centriole. The mother-daughter centriole pair and their surrounding proteins constitute the centrosome, which is the primary regulator of chromosome separation and cell division in animal cells. The centrosome controls these processes by acting as the microtubule organizing center (MTOC) of the cell – it nucleates microtubules during mitosis to form the mitotic spindle required to promote chromosome segregation. In order for proper chromosome segregation to occur, each cell needs to contain only two centrosomes entering mitosis – one at each pole to achieve spindle bipolarity. To achieve this, each centrosome must duplicate itself throughout the cell cycle. This duplication event is orchestrated by the centrioles – a single daughter centriole is built off of each mother centriole as the cell cycle progresses. The formation of excess daughter centrioles around a mother is a mechanism of centriole amplification, which is a driver of aneuploidy and tumor formation. Because of this, the process of centriole duplication is tightly controlled on a molecular level. The process of centriole duplication is coordinated in part by two conserved proteins: the Ser/Thr kinase Plk4 and its multifunctional regulator, Asterless. Previous work has shown that an excess of Asl protein levels, Plk4 protein levels and Plk4 catalytic activity can contribute to centriole amplification. Thus, it is crucial to obtain a complete understanding of how Plk4 and Asl coordinate their functions to ensure centrioles duplicate properly. It is known that Asl can regulate Plk4 by targeting it to the centriole as well as control its stability in a cell-cycle dependent manner. However, we still have an incomplete understanding of exactly how these two proteins promote the formation of a single daughter centriole during each cell cycle. Here we 10 identify multiple new regulatory mechanisms that Plk4 and Asl utilize in order to control centriole assembly. First, we identify Asl as a multifunctional phosphorylation-dependent regulator