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Control of Cleavage Furrow Formation During Cytokinesis in Human Cells Control of Cleavage Furrow Formation During Cytokinesis in Human Cells Kuan-Chung Su University College London and Cancer Research UK London Research Institute PhD Supervisor: Mark Petronczki PhD A thesis submitted for the degree of Doctor of Philosophy University College London September 2012 Declaration I Kuan-Chung Su confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Cytokinesis is the final stage of the cell cycle. It partitions sister genomes and separates the cytoplasm of nascent daughter cells. Cytokinesis is initiated by the formation of a cleavage furrow whose ingression is powered by an actomyosin network known as the contractile ring. Following furrow ingression, the process of cell separation is completed by a membrane scission reaction. For the accurate inheritance of genetic information, it is crucial that furrow formation is initiated at the cell equator between segregating chromosomes and that this occurs after chromatin has cleared the cleavage plane. In animal cells, the mitotic spindle plays a pivotal role in the formation and placement of the cleavage furrow. The coupling of cytokinesis and chromosome segregation to the mitotic spindle ensures that nuclear and cytoplasmic division are tightly coordinated. The spindle midzone, a structure that is formed at anaphase onset between segregating sister genomes, is thought to play an important instructive role during cleavage furrow formation. How the mitotic spindle controls cytokinetic events at the cell envelope is a key challenge in cell division research. Formation of the cytokinetic furrow in animal cells requires activation of the GTPase RhoA by the conserved guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cell periphery during anaphase is not understood. Using a genetic complementation system, I have been able to replace the endogenous Ect2 protein with a fluorescently-tagged transgene to study its dynamic localization during cytokinesis. Using live-cell time-lapse microscopy, I found that Ect2 concentrates not only at the spindle midzone but also accumulates at equatorial plasma membrane during cytokinesis. The association of Ect2 with the plasma membrane in vivo involves a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids in vitro. I further demonstrated that both guanine nucleotide exchange activity and the membrane targeting domains of Ect2 are essential for RhoA activation, contractile ring formation and cleavage furrow ingression in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2’s spindle midzone anchor complex, and is also temporally coordinated with chromosome segregation through the activation state of Cdk1. My results suggest that targeting of Ect2 to the equatorial membrane may represent a key step in the delivery of the cytokinetic signal to the cortex. 3 Acknowledgements I would like to thank my supervisor Mark Petronczki, for the continuous encouragement and for always having time for discussions about ideas especially, when troubleshooting is required. I am also thankful to my thesis committee, Frank Uhlmann and Julian Downward for their valuable advice and guidance through my time at the London Research Institute. I am very grateful to Tohru Takaki, with whom I was working closely for my project. I also thank him for having taught me almost every single technique I learnt during my time as a PhD student. Thank you also to all Lab members of the Cell Division and Aneuploidy lab, Sergey Lekomtsev, Lola Vazquez-Novelle, Sriramkumar Sundaramoorthy, Laurent L’Epicier-Sansregret, Kristyna Kotynkova and Murielle Serres for providing such a cheerful and inspiring environment and also for being a great sounding board for all my thoughts about the Clare Hall catering. Thanks are due to Nicola Brown and Katharina Willmann for sharing the invaluable experience and ups and downs of being students at Clare Hall. I would like to thank all friends and family who supported me through the years of my studies. I was a recipient of the DOC-fellowship of the Austrian academy of science for which I am very grateful. 4 Table of Contents Abstract ................................................................................................................ 3 Acknowledgements ............................................................................................. 4 Table of Contents ................................................................................................ 5 Table of figures .................................................................................................... 7 Abbreviations ....................................................................................................... 9 Chapter 1. Introduction ................................................................................... 11 1.1 The Cell cycle ..................................................................................................... 11 1.2 Regulation of cell cycle progression ................................................................. 14 1.2.1 Cyclin-Cdks .................................................................................................. 14 1.2.2 Checkpoints .................................................................................................. 17 1.3 Cell division ........................................................................................................ 18 1.3.1 Mitosis .......................................................................................................... 20 1.3.2 Regulation of mitosis .................................................................................... 21 1.3.3 Cytokinesis and cytokinesis failure in animal cells ...................................... 28 1.4 Epithelial cell transforming sequence 2 (Ect2) ................................................ 45 1.4.1 Ect2 is required for cytokinesis .................................................................... 45 1.4.2 Ect2 domain structure ................................................................................... 46 1.4.3 Regulation of Ect2 activity ........................................................................... 49 1.4.4 Ect2 functions beyond cytokinesis ............................................................... 50 1.5 Goal of this research .......................................................................................... 52 Chapter 2. Materials & Methods .................................................................... 53 2.1 Plasmids, DNA Transfection and Generation of Stable Cell Lines ............... 53 2.2 siRNA transfection and Drug Treatment ........................................................ 54 2.3 Preparation of cell lysates ................................................................................. 54 2.4 Immunofluorescence microscopy (IF) ............................................................. 55 2.5 Antibodies and Dyes .......................................................................................... 55 2.6 Live Cell Imaging, FRAP and Image Quantification ..................................... 56 2.7 Recombinant Protein Purification and Protein Assays .................................. 59 Chapter 3. Result 1 - A genetic complementation system to study Ect2 localization in live cells ..................................................................................... 60 3.1 Ect2 is required for cytokinesis ........................................................................ 60 3.2 Construction and testing of the genetic complementation system for Ect2 in human cells .................................................................................................................. 61 3.3 Ect2 localization during cytokinesis ................................................................. 63 3.4 Conclusions: Result 1 - A genetic complementation system to study Ect2 localization in live cells ............................................................................................... 64 Chapter 4. Results 2 - Mechanistic basis of Ect2 association with the cell periphery 72 4.1 Domain requirements for peripheral Ect2 localization .................................. 72 4.2 Ect2CT associates to cell membrane independently of the actin cortex ....... 74 4.3 Recombinant Ect2 protein interacts with polyanionic phosphoinositides in vitro 74 4.4 The PH domain and tail region are not required for GEF activity of Ect2 on RhoA in vitro .......................................................................................................... 75 5 4.5 Conclusions: Results 2 - Mechanistic basis of Ect2 association with the cell periphery ...................................................................................................................... 76 Chapter 5. Results 3 – Role of Ect2 GEF activity and plasma membrane binding domains during cytokinesis ............................................................... 87 5.1 The GEF activity and membrane targeting domains of Ect2 are required for cytokinesis .............................................................................................................. 87 5.2 Importance of Ect2’s GEF activity and membrane targeting domains for cleavage furrow formation and Ect2 protein
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