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Investigating the Role of Cdk11in Animal Cytokinesis

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Investigating the Role of Cdk11in Animal Cytokinesis Investigating the Role of CDK11 in Animal Cytokinesis by Thomas Clifford Panagiotou A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto © Copyright by Thomas Clifford Panagiotou (2020) Investigating the Role of CDK11 in Animal Cytokinesis Thomas Clifford Panagiotou Master of Science Department of Molecular Genetics University of Toronto 2020 Abstract Finely tuned spatio-temporal regulation of cell division is required for genome stability. Cytokinesis constitutes the final stages of cell division, from chromosome segregation to the physical separation of cells, abscission. Abscission is tightly regulated to ensure it occurs after earlier cytokinetic events, like the maturation of the stem body, the regulatory platform for abscission. Active Aurora B kinase enforces the abscission checkpoint, which blocks abscission until chromosomes have been cleared from the cytokinetic machinery. Currently, it is unclear how this checkpoint is overcome. Here, I demonstrate that the cyclin-dependent kinase CDK11 is required for cytokinesis. Both inhibition and depletion of CDK11 block abscission. Furthermore, the mitosis-specific CDK11p58 kinase localizes to the stem body, where its kinase activity rescues the defects of CDK11 depletion and inhibition. These results suggest a model whereby CDK11p58 antagonizes Aurora B kinase to overcome the abscission checkpoint to allow for successful completion of cytokinesis. ii Acknowledgments I am very grateful for the support of my family and friends throughout my studies. I would also like to express my deep gratitude to Wilde Lab members, both past and present, for their advice and collaboration. In particular, I am very grateful to Matthew Renshaw, whose work comprises part of this thesis. I would also like to thank the Canadian Institute of Health Research for funding. I am also very appreciative for the financial support from the Faculty of Medicine’s Leave of Absence Stipendiary Fund. Thank you to my committee members, Drs. Laurence Pelletier and Chris McCulloch, for their incisive comments and helpful direction through my studies. Not least of all, I would like thank my supervisor Dr. Andrew Wilde. Your guidance and mentorship are deeply appreciated. iii Table of Contents Abstract ............................................................................................................................................ ii Acknowledgements .......................................................................................................................... iii Table of Contents ............................................................................................................................. iv List of Figures..................................................................................................................................vii List of Tables...................................................................................................................................viii Abbreviations ................................................................................................................................... ix Preamble ........................................................................................................................................... x Chapter 1. Introduction .................................................................................................................. 1 1.1. The Eukaryotic Cell Cycle ................................................................................................... 1 1.1.1. Overview ....................................................................................................................... 1 1.1.2. Phases and Checkpoints ................................................................................................ 1 1.1.3. Mitosis (M phase) ........................................................................................... ..............2 1.2. Cyclin-dependent Kinases (CDKs) and Cyclins .................................................................. 5 1.2.1. Overview ....................................................................................................................... 5 1.2.2. Discovery ...................................................................................................................... 6 1.2.3. Cyclins .......................................................................................................................... 6 1.2.4. Structural Features of Cyclin-CDK complexes ............................................................ 7 1.2.5. Expression Patterns ....................................................................................................... 8 1.2.6. General Principles of CDK Regulation ......................................................................... 8 1.2.7. Regulation of Cell Cycle Checkpoints ......................................................................... 9 1.2.7.1. Regulation of G1/S ................................................................................................. 9 1.2.7.2. Regulation of S phase .......................................................................................... 10 1.2.7.3. Regulation of G2/M .............................................................................................. 10 1.2.7.4. Regulation of M phase ......................................................................................... 11 1.2.8. Other Functions of CDKs ........................................................................................... 12 1.3. CDK11 ............................................................................................................................... 13 1.3.1. Background ................................................................................................................. 13 1.3.2. Structural Features and Interacting Factors ................................................................ 14 1.3.3. Isoforms and their Associated Functions .................................................................... 15 iv 1.3.3.1. p110 ...................................................................................................................... 15 1.3.3.2. p58 ........................................................................................................................ 16 1.3.3.3. p46 ........................................................................................................................ 17 1.3.4. Implication in Human Disease .................................................................................... 18 1.4. Cytokinesis ......................................................................................................................... 19 1.4.1. Overview .................................................................................................................... 19 1.4.2. Cytokinesis and Cancer ............................................................................................... 20 1.4.3. Anaphase ..................................................................................................................... 21 1.4.3.1. Central Spindle Assembly .................................................................................... 21 1.4.3.2. Contractile Ring Assembly and Furrow Ingression ............................................. 22 1.4.4. Telophase .................................................................................................................... 22 1.4.4.1. Intercellular Bridge Maturation ........................................................................... 22 1.4.4.2. Abscission ............................................................................................................ 23 1.4.4.3. Regulation of Abscission ..................................................................................... 25 1.5. Research Question ............................................................................................................. 27 Chapter 2. CDK11p58 Kinase Activity is Required for Cytokinesis ....................................... 28 2.1. Introduction ........................................................................................................................ 28 2.1.1. Known Roles of CDK11 in Cell Division .................................................................. 28 2.1.2. Objectives and Strategies ............................................................................................ 28 2.2. Results ................................................................................................................................ 29 2.2.1. CDK11p58, but not CDK11p110, Localizes to Stem Body ............................................ 29 2.2.2. Knockdown of CDK11 Induces a Multinucleate Phenotype ...................................... 31 2.2.3. Depletion of CDK11 Stalls Cells Before Abscission ................................................. 36 2.2.4. Expression of CDK11p110 and/or CDK11p58 Rescue Defects of CDK11 Depletion ... 38 2.2.5. Inhibition of CDK11 Kinase Activity by OTS964 Phenocopies Depletion Defects .. 41 2.2.6. Kinase dead mutant of CDK11p58 Cannot Rescue CDK11 Depletion Defects .......... 43 2.3. Discussion .......................................................................................................................... 44 Chapter 3. Summary .................................................................................................................
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