MOLECULAR MECHANISMS UNDERLYING THE REGULATION OF THE POSITIONING AND FORMATION OF THE CLEAVAGE FURROW IN CYTOKINESIS IN MAMMALIAN CELLS XIAODONG ZHU (M.Sc., University of Science and Technology of China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY AND DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2009 THIS THESIS IS DEDICATED TO MY FAMILY i ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my highly respected supervisor Dr Maki Murata-Hori It is my great honor to work with Dr Maki as her first Ph.D student She offered me the rigorous scientific training from the fundamental techniques to the creation of the great ideas Her encouragement and guidance helped me move through all the up and downs of my research Without her, I would have been lost and this work would not be possible I wish to express my sincerely gratitude to my co-supervisor Associate Professor Dr Sohail Ahmed His deep knowledge of the small GTPases helped my work immensely He always gave me encouragement and confidence to pursue my PhD research in the past years I would sincerely like to thank my committee members, Professor Mohan K Balasubramanian and Dr Snezhana Oliferenko for their constructive criticism, warm encouragement and valuable suggestions I would like to thank Ms Er Poh Nee for technical assistance and help with confocal microscopy I would like to extend my thanks to the confocal facility, sequence facility and all my friends in the TLL for their support I also would like to thank all the past and current members of mammalian cell biology group at the TLL, specially Dr Svetlana Mukhina, Ms Shethva Sankaran, Ms Charlene Foong, Dr Ramirez Hernandez Tzutzuy, Mr Vinayaka Srinivas, Ms Shyan Huey Low, Ms Shazmina Rafee, and Mr Sriramkumar Sundaramoorthy ii They provide a stimulating environment for me to carry out my research Particularly, I would like to thank Ms Charlene Foong and Dr Hiroshi Hosoya and his colleagues (Hiroshima University, Japan) for collaborating with me I am especially indebted to my family, my parents, sisters, brother-in-laws, and my nephew, nieces for their endless love and support This thesis is dedicated to them iii TABLE OF CONTENTS: Introduction…………………………………………………………… 1.1 The cell cycle and cell division…………………………………… … 1.2 Cytokinesis………………………………………………………….… 1.2.1 Cleavage plane determination ………………………………… 1.2.1.1 Roles of microtubules in positioning of the cleavage furrow……………………………………………………… 1.2.1.2 Molecular mechanisms for the determination of the position of the cleavage furrow …………………… 1.2.2 Cleavage furrow formation….………………… 16 1.2.3 Cleavage furrow ingression…………………………………… 19 1.2.4 Abscission………………………………………………….…… 20 Materials and Methods…………………………………………………… 23 2.1 Cell Biology……………………………………………………………23 2.1.1 Cell line…………………………………………………… 23 2.1.2 Reagents…………………………………………………… .23 2.1.2.1 Solution……………………………………………………23 2.1.2.2 Drugs………………………………………………………24 2.1.2.3 Antibodies…………………………………………………26 2.1.2.4 F-actin and DNA markers…………………………………27 2.1.3 Cell culture condition……………………………… ………… 28 2.1.4 Transfection………………………………………… …… 29 2.1.5 Microinjection………………………………………… … 29 2.2 Molecular biology………………………………………………………29 2.2.1 E.coli strain……………………………………………… …… 29 2.2.2 Transformation of E coli……………………………………… 30 2.2.3 Growth and maintenance of E.coli………………… 30 2.2.4 Plasmid construction…………………………… …… .30 2.3 Microscopic imaging………………………………………… … .31 2.3.1 Sample preparation for live-cell imaging………… .31 2.3.2 Sample preparation for immunofluorescence staining……… .31 2.3.3 Image acquisition ………………………….…………… … 32 2.3.4 Fluorescence recovery after photobleaching (FRAP)……… .32 2.4 Image analyses……………………………………………………….… 32 2.4.1 Quantification of fluorescence intensity……………… .32 2.4.2 Kymographic analysis………………………………… .33 2.4.3 Determination of the turnover rate……………………………… 33 Molecular mechanism for the regulation of cleavage furrow positioning………………………………………………………………… 34 3.1 Introduction…………………………………………………………… 34 3.2 Results……………………… ………………………………………….38 3.2.1 Effects of inhibition of aurora B kinase activity on cytokinesis……………………………………………………… 38 3.2.2 Effects of inhibition of aurora B kinase activity on cytokinetic regulators………………………………………………….…… 40 3.2.3 Effects of inhibition of aurora B kinase on the microtubule dynamics………………………………………………………… 42 iv 3.2.4 Effects of inhibition of aurora B kinase on localization of the spindle midzone components………………………………………….…46 3.3 Discussion………………………………………………………… 48 Molecular mechanism of the regulation of cleavage furrow formation…………………………………………………………………….52 4.1 Introduction…………………………………………………………….52 4.2 Results………………………………………………………………….59 4.2.1 Effects of modulation of Cdc42 activity on cytokinesis……………………………………………………….59 4.2.2 Effects of modulation of Cdc42 activity on RhoA localization………………………………………………………61 4.2.3 Effects of modulation of Cdc42 activity on the dynamics and organization of actin cytoskeleton………………………………63 4.2.3.1 Effects of inhibition of Cdc42 activity on actin dynamics and organization…………… 63 4.2.3.2 Effects of overstimulation of Cdc42 activity on actin dynamics and organization………………… 67 4.2.4 Involvement of N-WASP in de novo actin assembly at the equator……………………………………………… .70 4.2.5 Localization of Cdc42 during cytokinesis…………………… 73 4.3 Discussion……………………………………………………… 75 Discussion……………………………………………………………… 84 References………………………………………………………………… 87 v SUMMARY Cytokinesis is the final step of cell division crucial for cell growth and development A clear understanding of the spatial and temporal regulatory mechanisms of cytokinesis is important not only for basic knowledge of the cellular function but also for developing effective countermeasures against various diseases such as cancer and birth defects Animal cell cytokinesis consists of four steps: cleavage plane determination, cleavage furrow formation, ingression and abscission The mitotic spindle is responsible for the determination of the position of the cleavage plane After the division plane is determined, cytokinetic machinery such as actin and myosin II are assembled and form the actomyosin contractile ring Constriction of the contractile ring drives furrow ingression Abscission occurs after a furrow has fully ingressed Numerous studies have focused on the functions of the proteins that localized at the cleavage furrow in order to elucidate the molecular mechanisms that regulate cytokinesis However, there is an increasing body of research suggesting that cytokinesis in animal cells likely involves entire cortex in addition to equatorial cortex Thus, it is important to identify the functions of each protein involved in cytokinesis at a high spatial and temporal resolution In this thesis, I have studied the molecular mechanisms that regulate the determination of the position of the cleavage furrow and the cleavage furrow formation in cytokinesis in mammalian cells using molecular manipulations and microscopy-based techniques vi Correct positioning of the cleavage plane requires proper regulation of the microtubule dynamics in mitotic spindle It has been suggested that stable microtubules at the equator stimulate the formation of the cleavage furrow, while dynamic astral microtubules likely inhibit cortical ingression in the polar region While many studies have focused on the molecular mechanisms that stabilize microtubules at the equator, little is known on how astral microtubules maintain their dynamic status In Chapter III, I have shown that the kinase activity of aurora B, a member of the chromosomal passenger complex, is required not only for stabilization of microtubules at the equator but also for the maintenance of the dynamic status of astral microtubules to ensure that the cleavage furrow forms at the equator Cleavage furrow formation involves flux-dependent transport of pre-existing actin filaments and de novo assembly activities Although functions of Rho family GTPase RhoA in this process have been established, additional mechanisms are likely involved Another Rho family GTPase Cdc42 has been suggested to be involved in the regulation of actin cytoskeleton during cytokinesis However, its detailed functions remain obscure In Chapter IV, I have shown that Cdc42 contributes to actin assembly by stabilizing actin filaments, promoting de novo assembly through N-WASP and negatively cross-talking with RhoA during cytokinesis of mammalian epithelial cells vii LIST OF TABLES: Table 1: A Cell line used in this study……………………………………………23 Table 2A: A list of drugs used in this study: sources, storage and usage……… 24 Table 2B: A list of the drugs used in this study: molecular mechanisms of action…………………………………………………………………………… 25 Table 3A: Primary antibodies used in this study……………………………… 26 Table 3B: Secondary antibodies used in this study…………………………… 27 Table 4: F-actin and DNA markers used in this study………………………… 28 viii LIST OF FIGURES: Figure1: Organization of microtubules during M phase in typical tissue cultured cells…………………………………………………………………… Figure2: Classic experiments demonstrate that astral microtubules position cleavage furrow in embryos whereas midzone microtubules stimulate cytokinesis in tissue cultured cells ………………………………………………… Figure 3: Rho GTPases cycle……………………………………………………14 Figure 4: Time-lapse imaging of dividing HeLa cells treated with Hesperadin 39 Figure 5: Localization of cytokinetic regulators in cells treated with Hesperadin……………………………………………………… .41 Figure 6: Microtubule organization during cytokinesis in cells treated with Hesperadin…………………………………………………………….43 Figure 7: Live-cell analyses of microtubules in cells treated with Hesperadin during cytokinesis…………………………………………………………….45 Figure 8: Localization of MKLP1 and ECT2 during cytokinesis in cells treated with Hesperadin…………………………………………………………….47 Figure 9: Schematic representation of domains of Cdc42………………………58 Figure 10: Modulation of Cdc42 activity affects cytokinesis………………… 60 Figure 11: Modulation of Cdc42 activity affects RhoA localization………… 62 Figure 12: Inhibition of Cdc42 activity affects actin dynamics and organization……………………………………………………………65 Figure 13: Constitutive activation of Cdc42 induces the formation of abnormal actin bundles………………………………………………………….70 Figure 14: N-WASP is involved in de novo actin assembly at the equator…… 73 Figure 15: Localization of Cdc42 during cytokinesis………………………… 75 Figure 16: Proposed model for the regulation of cleavage furrow formation by RhoA and Cdc42………………………………………………………84 ix Kosako, H., Yoshida, T., Matsumura, F., Ishizaki, T., Narumiya, S., and Inagaki, M (2000) Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow Oncogene 19, 6059-6064 Kroschewski, R., Hall, A., and Mellman, I (1999) Cdc42 controls secretory and endocytic 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RhoA and Cdc42 may have similar functions in the regulation of the cleavage furrow formation (see also the introduction section in Chapter 4) 18 1.2.3 Cleavage furrow ingression In the classic ‘intracellular