Regulation of cytokinesis in the fission yeast schizosaccharomyces pombe

REGULATION OF CYTOKINESIS IN THE FISSION YEAST SCHIZOSACCHAROMYCES POMBE MITHILESH MISHRA (M. Sc., Jawaharlal Nehru University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2007 DEDICATED TO MY PARENTS: MEERA MISHRA AND DR. R. P. MISHRA; DR(s). S. BULCHAND AND Maj. H.O. BULCHAND ii ACKNOWLEDGEMENTS This work would not have been possible without the contribution of many people. I would especially like to thank my supervisor Prof. Mohan Balasubramanian for giving me the opportunity to work in his lab. I will always cherish his gentle mentorship, his infectious enthusiasm for science and unflagging support through the years. Many people collaborated with me during the course of this work. I am grateful to Dr. Jim Karagiannis for collaboration on the project described in chapter and especially for the results presented in Figure 9. I would like to thank Maya Sevugan and Pritpal Singh for their help with the GST-pull down assay, Chang Kai Chen and Ventris M. D’souza for the characterization of the swo1-w1 mutant and Huang Yinyi for help with the yeast two-hybrid assay. Many thanks are due to Drs. Dan McCollum and Susan Trautmann for their helpful discussions and sharing of unpublished observations. During the course of this work various people generously made antibodies and yeast strains available to me. I wish to thank Drs. Juan Jimenez, Keith Gull, Paul Russell, Paul Nurse, Koei Okazaki, Matthew O’Connell, Tony Carr, Kathy Gould, Dan McCollum and all the contributors of the Cell Division Laboratory strain and plasmid collection for reagents. iii I would like to thank my thesis committee members Drs. Naweed Naqvi, Uttam Surana and William Chia for their comments and suggestions. I would like to thank all the members past and present of the yeast and fungal laboratories at the Temasek Life Sciences Laboratory and at the Institute of Molecular Agrobiology, specially Suniti Naqvi, Srividya Rajagopalan, Snezhka Oliferanko, Volker Wachtler, Naweed Naqvi, Jim Karagiannis, Andrea Bimbo, Chew Ting Gang, Ge Wanzhong, Huang Yinyi, Loo Tsui Han, Hong Yan, Maya Sevugan, Liu Jianhua, Anup Padmanabhan and Ramanujam Srinivasan for helpful discussions and help with experiments in addition to creating a wonderful environment to work in. I am thankful to IMA/TLL facilities and staff for general support. Thanks are due to Volker Wachtler, Ramanujam Srinivasan and Sarada Bulchand for critical reading of my thesis. I am grateful to the National Science and Technology board; Agency for Science, Technology and Research; Temasek Life Sciences Laboratory and the Singapore Millennium Foundation for financial support. This work would not have been possible without the support of my family and friends. iv TABLE OF CONTENTS: Introduction . 1.1 The cell division cycle and cytokinesis . 1.2 Cytokinesis: early insights 1.2.1 The contractile ring . 1.3 Cytokinesis in animal cells . 10 1.4 Cytokinesis in plant cells 11 1.5 Cytokinesis in budding yeast 12 1.6 Cytokinesis in fission yeast 13 1.6.1 The fission yeast Schizosaccharomyces pombe . 13 1.6.2 The cell cycle and its regulation 14 1.6.3 Division site selection . 15 1.6.4 Actomyosin-ring assembly . 16 1.6.5 Ring contraction, membrane addition and division septum deposition 20 1.6.6 Coordination of cytokinesis with the nuclear cycle . 21 1.6.7 The septation initiation network (SIN) 22 1.6.8 The Cdc14 family phosphatase Clp1p . 25 1.6.9 The cytokinesis checkpoint . 28 Materials and Methods . 32 2.1 Yeast 32 2.1.1 Yeast strains . 32 2.1.3 Drugs used . 36 2.1.4 Yeast genetics and molecular methods 36 2.2 Escherichia coli . 37 2.2.1 Growth and maintenance of bacteria . 38 2.2.2 Transformation of E. coli 38 2.3 Cell biology and microscopy 38 2.3.1 Reagents . 38 2.3.2 Nuclei, F-actin and septum staining 39 2.3.3 Immunofluoresence microscopy . 39 2.3.4 Time lapse microscopy . 41 2.4 Protein and immunological methods . 42 2.4.1 Extraction of protein from S. pombe . 42 2.4.2 Immunoprecipitation 43 2.4.3 Protein electrophoresis, immunoblotting and detection . 43 2.5 Two-hybrid system . 44 2.5 Association of Clp1p-HA and GST-Rad24P . 45 Swo1p facilitates the actomyosin ring formation and myosin II assembly and function . 46 v 3.1 Introduction 46 3.2 Results . 47 3.2.1 Characterization of the cytokinetic phenotype in swo1-w1 cells 47 3.2.2 The swo1-w1 mutation leads to defects in actomyosin ring assembly 50 3.2.3 The swo1-w1 mutant shows a genetic interaction with myo2-E1 and rng3-65 alleles 52 3.2.4 myo2-E1 and rng3-65 strains show a heightened sensitivity to the Hsp90 inhibitor geldanamycin . 55 3.2.5 Swo1p-GFP persists at the actomyosin ring only in the myo2-E1 strain 57 3.2.6 Swo1p physically interacts with the Myo2p-head domain and with Rng3p 60 3.2.7 Myo2-E1p levels are reduced in absence of Swo1p and Rng3p . 63 3.3 Discussion 65 Clp1p ensures completion of cytokinesis in response to minor perturbation of the cell division machinery in S. pombe . 71 4.1 Introduction 71 4.2 Results . 73 4.2.1 Clp1p is essential when the cytokinetic apparatus is mildly perturbed 73 4.2.3 Clp1p-dependent maintenance of the actomyosin ring upon perturbation of cytokinesis . 82 4.2.4 Active SIN signaling greatly bypasses the need for Clp1p function upon perturbation of the cell division machinery 88 4.2.5 Clp1p localizes to the cytoplasm and is important for the maintenance of active SIN cascade when cytokinesis is perturbed 91 4.2.6 Cells lacking the 14-3-3 protein Rad24p are sensitive to perturbation of cell division structures . 94 4.2.7 Rad24p is required for cytoplasmic retention of Clp1p and for prolonged SIN signaling 98 4.2.8 Rad24p physically associates with phosphorylated Clp1p in vitro 101 4.2.9 Ectopic activation of the SIN compensates for the loss of Rad24p during cytokinesis 104 4.2.10 Rad24p localizes to the actomyosin ring and the spindle pole bodies in cells undergoing mitosis and cytokinesis in a SIN-dependent manner. 107 4.3 Discussion 110 References: 122 vi Summary Cytokinesis is the terminal phase of the cell cycle through which the cellular constituents of mother cells are partitioned into two daughter cells resulting in an increase in cell number. The spatio-temporal regulation of cytokinesis is important for successful division, the failure of which may result in cells with altered ploidy and loss of viability. In recent years, the fission yeast Schizosaccharomyces pombe has emerged as an attractive model organism for the study of cytokinesis. S. pombe is a rod-shaped unicellular fungus that grows by elongation at the tips. Like the metazoan S. pombe cells divide by medial fission through an actomyosin based contractile ring producing two daughter cells of equal size. The actomyosin ring consists of about 50 different proteins including actin, actin regulatory proteins, type II myosin heavy and light chains. The mechanism by which various components of the actomyosin ring associate with each other to form a functional complex remains to be determined. The ordered execution of cell cycle events requires surveillance mechanisms, or cell cycle checkpoints, which ensure that the initiation of later events is coupled to the completion of earlier cell cycle events (Hartwell and Winert, 1989). Checkpoints that monitor completion of DNA synthesis, DNA damage and mitotic spindle assembly have been extensively characterized in various eukaryotes. However, the existence of a similar monitoring mechanism for the completion of cytokinesis has remained an open question. Fission yeast mutants defective in actomyosin ring formation and function exhibit a delay in entry into vii the subsequent nuclear division cycle following cytokinetic failure. This G2 delay depends on the Septation Initiation Network (SIN), a signaling network essential for cytokinesis, and on the non-essential Cdc14p family phosphatase Clp1p / Flp1p and has been proposed to signify a “cytokinesis checkpoint”. However the physiological significance of such a checkpoint and the molecular role of Clp1p in the process remain to be determined. In the first half of this study I demonstrate that fission yeast heat-shock protein 90 (Swo1p) is essential for actomyosin ring assembly. I provide evidence that Swo1p together with the UCS domain protein Rng3p modulates the stability and function of type II myosin Myo2p of fission yeast. Temperature sensitive alleles of swo1, show strong genetic interaction with a specific mutant allele of the fission yeast type II myosin head myo2-E1, but not with two other alleles of myo2 or with mutations affecting 14 other genes important for cytokinesis. myo2-E1 and rng3-65 mutants show heightened sensitivity to the Hsp90 inhibitor geldanamycin. I further show that Swo1p and Rng3p physically associate with the head domain of Myo2p, and Myo2-E1p levels are reduced in the absence of Swo1p and Rng3p. My analysis establishes that Swo1p and Rng3p collaborate in vivo to modulate myosin II stability and function. In the second half of this study I demonstrate the physiological significance of the cytokinesis checkpoint in fission yeast. I show that delays in cytokinesis caused by minor perturbations to different components of the cytokinetic machinery, viii which normally causes only mild defects, become lethal in the absence of Clp1p. The cell division apparatus is repaired and reinforced in response to activation of the cytokinesis checkpoint. Ectopic activation of SIN significantly bypasses the requirement of Clp1p for the G2 delay as well as completion of cytokinesis. I further show that Clp1p, normally nucleolar in interphase is maintained in the cytoplasm until completion of cytokinesis and the cytoplasmic retention of Clp1p is essential for checkpoint activation. This cytoplasmic retention of Clp1p is dependent on the SIN and the 14-3-3 protein Rad24p. Rad24p binds to the phosphorylated form of Clp1p. This physical interaction depends on the function of the SIN component Sid2p. I conclude that the Clp1p-dependent cytokinesis checkpoint provides a previously unrecognized cell survival advantage when the cell division apparatus is mildly perturbed. ix LIST OF TABLES AND FIGURES: Table 1: Schizosaccharomyces pombe strains used in this study……………… 25 Figure 1: Characterization of the swo1-w1 mutant strain 49 Figure 2: Swo1p function is essential for assembly of a normal actomyosin ring. 51 Figure 3: The swo1-w1 mutant shows a synthetic lethal interaction with both myo2-E1 and rng3-65 mutant strains. . 54 Figure 4: The mutant strains myo2-E1, rng3-65 and swo1-w1 show a heightened sensitivity to geldanamycin. . 56 Figure 5: Swo1p-GFP is detected at the division site in myo2-E1 cells but not in myo2-S1 or myo2-S2 cells. . 59 Figure 6: Physical interactions between Myo2p and Rng3p with Swo1p. . 62 Figure 7: Swo1p and Rng3p are required for the stability of Myo2-E1p . 64 Figure 8: Clp1p is essential when cell division structures are mildly perturbed and ensures the completion of cytokinesis. . 77 Figure 9: Nuclear cycle arrest is insufficient to allow completion of cytokinesis upon perturbation of the cell division machinery. . 81 Figure 10: Clp1p is important for the maintenance of the actomyosin ring at the medial region of the cell . 84 Figure 11: Dynamics of Clp1p dependent actomyosin ring maintenance upon perturbation of the cytokinetic machinery. . 87 Figure 12: SIN mutants are hypersensitive to mild perturbations of the cell division machinery whereas ectopic activation of the SIN compensates for the loss of Clp1p. . 90 Figure 13: Clp1p localizes to the cytoplasm and is important for the maintenance of an active SIN cascade upon checkpoint activation 93 Figure 14: rad24 Δ cells are sensitive to perturbation of cell division structures. 97 x Motegi, F., M. 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Mol Cell Biol 19, 7410-9. 160 161 [...]... implicated Pom1p in inhibition of the actomyosin-ring assembly in the polar region of cells by restricting the localization of Mid1p to the medial cortex (Celton-Morizur et al 2006; Padte et al 2006) 1.6.4 Actomyosin-ring assembly Genetic screens have identified a number of proteins, which are essential for the formation and maintenance of the actomyosin ring They include the myosin 16 heavy chain Myo2p;... Coordination of cytokinesis with the nuclear cycle 21 Cytokinesis is tightly coupled with the nuclear division cycle in fission yeast (Chang and Nurse 1996; Wu et al 2003) Fission yeast cells assemble the actomyosin ring only after activation of the CDK, Cdc2p-Cdc13p complex Constriction of the actomyosin ring and septum assembly on the other hand depend on the attenuation of CDK activity and on cyclin... 2003) 1.6.7 The septation initiation network (SIN) The SIN is a GTPase-activated protein-kinase cascade, which is assembled at the SPBs (Krapp et al 2004; Morrell et al 2004) In SIN mutants, actomyosin rings are formed but ring constriction fails The actomyosin ring disassembles and continuous rounds of nuclear division occur in the absence of cytokinesis resulting in formation of multinucleated cells... cytoplasmic retention of Clp1p and for prolonged SIN signaling upon perturbation of the cytokinetic machinery 100 Figure 16: Rad24p binds to phosphorylated Clp1p in vitro 103 Figure 17: Ectopic activation of the SIN compensates for the loss of Rad24p during cytokinesis 106 Figure 18: Rad24p localizes to the actomyosin ring and the spindle pole body in cells undergoing mitosis in a SIN-dependent... system in the context of myosin assembly into the actomyosin ring during cytokinesis (Wong et al 2000) rng3 mutants fail to assemble a proper actomyosin ring and are nonviable because of cytokinesis failure rng3 mutants show strong genetic interactions with one of the alleles of myosin, myo2-E1, which has a mutation in the catalytic head domain Rng3p, which is not detected in any specific structure in. .. and cytokinesis, in which the mother cell physically divides to give rise to two independent daughters Cytokinesis is the final event of the cell cycle that physically separates the mother cell into two independent daughter cells While the absence of cytokinesis is sometimes necessary under certain specialized circumstances, for example in the slime mould Physarium or during the early development of. .. alters the overlying cortical surface and then becomes dispensable In the second 4 step the altered surface organizes the division mechanism and in the last step the division mechanism actively constricts the cell (Rappaport 1971) Micromanipulation of fertilized eggs of marine invertebrates was instructive in addressing issues concerning the source of the signal as well as the time and duration of the interaction... indicating that a force larger than the surface tension of the oil droplet in the cytoplasm was generated in the furrow The extent of the force was measured directly in a cleaving sand dollar egg (Rappaport 1967) Two micro-needles one rigid and the other flexible and calibrated, were inserted in a dividing sand dollar egg and the force in the range of 1.5-9 X 10 –3 dyn/cm2 was measured by determining... these proteins remains to be understood Myosin-II is believed to be a molecular motor that generates force for cytokinesis by interacting with actin filaments at the actomyosin ring The fission yeast has two homologs of myosin heavy chain, Myo2p and Myp2p/Myo3p (Bezanilla et al 1997; Kitayama et al 1997; May et al 1997; Motegi et al 1997), both of which localize at the actomyosin ring during cytokinesis. .. actin cytoskeleton to control polarized growth and regulate the assembly of the septin ring Septins are a family of GTP binding proteins, which form filaments and are conserved from yeast through mammals (Trimble 1999) Myo1p (type-II myosin of budding yeast) is recruited to the bud 12 neck early in the cell cycle at the G1/S transition, whereas actin and its regulatory components join the ring late in . 1 1.2 Cytokinesis: early insights 3 1.2.1 The contractile ring 7 1.3 Cytokinesis in animal cells 10 1.4 Cytokinesis in plant cells 11 1.5 Cytokinesis in budding yeast 12 1.6 Cytokinesis in fission. function. In the second half of this study I demonstrate the physiological significance of the cytokinesis checkpoint in fission yeast. I show that delays in cytokinesis caused by minor perturbations. signify a cytokinesis checkpoint”. However the physiological significance of such a checkpoint and the molecular role of Clp1p in the process remain to be determined. In the first half of this