Understanding the septation initiation network (SIN) function during meiotic cytokinesis in fission yeast

UNDERSTANDING THE SEPTATION INITIATION NETWORK (SIN) FUNCTION DURING MEIOTIC CYTOKINESIS IN FISSION YEAST YAN HONGYAN A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABOTORY NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENT I would like to thank my supervisor Prof. Mohan Balasubramanian for providing me the opportunity to pursue a PhD in his laboratory. I am extremely grateful for his excellent guidance, constant encouragement, and continuous support to my projects. I also thank my thesis committee members, Drs. Naweed Naqvi, Liu Jianhua and Gregory Jedd for their valuable suggestions and comments on my thesis projects. I would like to thank Drs Nojima H, Shimoda C, McCollum D, Pollard T and the Yeast Genetic Resource Center (YGRC, Japan) for generously providing several yeast strains and the plasmids used in this study. I am also thankful to Drs Neiman AM and McCollum D for their help and contribution on this work. I would like to express my thanks to all present and past members of the Cell Division Laboratory for their help with experiments, helpful discussions and valuable suggestions. Special thanks to Drs Chew Tinggang, Ge Wanzhong, Huang Yinyi and Loo Tsui Han for their help and guidance when I just joined the laboratory. I am also thankful to Drs. Tang Xie, Loo Tsui Han, Mishra Mithilesh and Srinivasan Ramanujam for their encouragement and useful comments on my projects. I also thank the community of Yeast and Fungal biology for valuable discussion. ii Many thanks to Drs Ng Kianhong, Mishra Mithilesh, Srinivasan Ramanujam, Tang Xie and Ms Dhivya Subramaniam, Mr Anup Padmanabhan for their critical reading of this thesis. I am thankful to TLL facilities and staff for general support. I acknowledge Temasek Life Sciences Laboratory and Singapore Millenium Foundation for their financial support. Finally, I would like to thank my family: my parents, my parents-in-law, my husband and my lovely kids for all the encouragement. iii TABLES OF CONTENTS TITLE PAGE.…………………………………………………………………… i ACKNOWLEDGEMENT ………………………………………………… .ii TABLE OF CONTENTS ……………………………………………………… iv SUMMARY………………………………………………………………….… . vii LIST OF TABLES…………………………………………………………….… x LIST OF FIGURES .………………………………………………………… xi LIST OF ABBREVIATIONS.……………………………………………………xiii LIST OF PUBLICATIONS …………………………………………………… xiv Chapter 1. Introduction…………………………….……………………………. 1.1 Cytokinesis……………………………………………………………… 1.1.1 New membrane/cell wall formation ………………………………… .2 1.1.2 Actomyosin ring assembly in animal cells…………………… . 1.1.3 Spindle/ Phragmph formation in plants………………………… .… …… . 1.1.4 Meiotic cytokinesis during sporulation in yeast………………………. 1.1.4.1 Sporulation in budding yeast………………………………… 1.1.4.2 Actin cytoskeleton during sporulation in budding yeast……….11 1.2 Fission yeast as a model organism…………………………………………… . 13 1.2.1 The fission yeast cell cycle …………………………………………….14 1.2.1.1 Mitotic cell cycle……………………………………………….14 1.2.1.2 Meiotic cell cycle…………………………………………… 15 1.2.1.3 Cell cycle regulation in fission yeast…………………… 15 1.2.2 Actomyosin ring assembly in fission yeast………………………… 17 1.2.3 Sporulation in fission yeast ……………………………………… . 20 1.2.3.1 Meiotic SPBs function as FSM-organizing centres…… … 21 1.2.3.2 FSM assembly during sporulation………………………… … 22 iv 1.2.3.3 Leading edge proteins at the tip of the FSM………………… . 24 1.2.4 Septation initiation network in mitotic cell cycle……… ……………. 26 1.3 Aim and objectives of the thesis……………………………………………… 29 Chapter 2. Materials and Methods…………………………………………….…30 2.1 S. pombe strains, media, reagents and culture condition………………………. 30 2.2 Molecular and Genetic methods of S. pombe cells…………………………… 35 2.2.1 Yeast transformation……………………………………………… .… 35 2.2.2 Spore viability test…………………………………………………… 35 2.2.3 Iodine staining assay……………………………………………… .… 35 2.2.4 rad21 promoter swapping of promoter of endogenous sid4………… . 36 2.2.5 The slk1K191R mutant strain generation…………………………… . 37 2.2.6 Generation of epitope tagged strains………………………………… .37 2.2.7 Plasmid constructs…………………………………………………… .39 2.3 Microscopy…………………………………………………………………… 41 2.3.1 Immunofluorescence staining…………………………………………. 41 2.3.2 Fluorescence microscopy ………………………………………… . 41 2.3.3 Time-lapse microscopy……………………………………………… . 41 2.3.4 Confocal microscopy ……………………………………………….… 42 2.3.5 Electron Microscopy………………………………………………… . 42 2.4 Bioinformatics……………………………………………………………….….43 2.4.1 Sequence alignment……………………………………………… … 43 2.4.2 Domain analysis…………………………………………………….…. 43 Chapter 3. The meiosis-specific sid2-related protein slk1 regulates forespore membrane assembly in fission yeast ……………………………………………. 44 3.1 Introduction…………………………………………………………………… 44 3.2 Results………………………………………………………………………… 46 3.2.1 Slk1p is a protein kinase related to the SIN-component Sid2p……… 46 3.2.2 Slk1p localizes to the SPBs and spore periphery during meiosis………46 3.2.3 Slk1p localization to the SPBs and spore membranes during meiosis depends on the SIN scaffold proteins Cdc11p and Sid4p…………………… 50 v 3.2.4 Localization of SIN components to the SPBs is unaffected in slk1∆, Slk1p localization to the SPBs is independent of Spo3p and Spo15p………. 54 3.2.5 Slk1p is required for proper spore formation………………………… 54 3.2.6 Spore membrane assembly is aberrant in slk1∆ cells…………………. 57 3.2.7 The localization of Spo15p and Bgs2p is unaffected in slk1∆ cells… . 62 3.2.8 Sid2p and Slk1p perform overlapping roles in spore formation………. 64 3.2.9 Overexpression of psy1 rescues sporulation defect of the sid2-250 slk1∆ mutant……………………………………………………………………… 69 3.2.10 Genetic interactions between spo3 and slk1 mutants…………………71 3.3 Discussion …………………………………………………………………… . 76 3.3.1 The protein kinases Slk1p and Sid2p localize to the SPB during meiosis…………………………………………………………………………… . 76 3.3.2 Slk1p and Sid2p are essential for sporulation and regulate FSM assembly…………………………………………………………………………….78 Chapter 4. A Meiotic Actin Ring (MeiAR) essential for sporulation in fission yeast……………………………………………………………………………… . 83 4.1 Introduction…………………………………………………………………… 83 4.2 Results………………………………………………………………………… 85 4.2.1 F-actin assembled into rings that follow the leading edge during FSM assembly in S. pombe……………………………………………………… . 85 4.2.2 Localization of actin nucleators to the leading edge during FSM formation in S. pombe……………………………………………………… 86 4.2.3 Actin is required for proper spore morphology in S. pombe………… .89 4.2.4 SIN is required for MeiAR constriction……………………………… 96 4.3 Discussion…………………………………………………………………… .101 Chapter 5. Conclusion and future directions………………………………… .103 References………………………………………………………… …………….105 vi SUMMARY Cytokinesis in all organisms involves the creation of membranous barriers that demarcate individual daughter cells. In recent years, the fission yeast Schizosaccharomyces pombe has emerged as an attractive model organism for the study of cytokinesis. It uses two distinct mechanisms of cytokinesis depending on the mode of cell cycle regulation. During mitosis, it divides using actomyosin ring constriction of which is coordinated with the formation of new membrane/cell wall during cytokinesis. During meiosis, however, four daughter cells (spores) are generated through a unique form of cytokinesis, called sporulation. The de novo synthesis of a double-layered membrane, termed the forespore membrane (FSM) is initiated during meiosis II, which encapsulates the meiotic nuclei. In fission yeast, a signaling module termed the Septation Initiation Network (SIN) plays an essential role in the assembly of new membranes and cell wall during mitotic cytokinesis. Krapp et al (2006) has recently shown that SIN proteins localize to SPBs during meiosis and the pathway is activated during meiosis II. Some SIN temperature sensitive mutant cells are defective in FSM assembly which results in unencapsulated nuclei (Krapp et al., 2006). However, as in the case of mitotic cells, the precise mechanism linking SIN and FSM assembly during meiotic cytokinesis is not fully understood. How F-actin participates in this process and whether SIN regulates actin cytoskeletal function during meiosis are yet poorly understood. In order to study how SIN regulates the FSM assembly, in Chapter III, I investigated the role of a conserved serine-threonine protein kinase Slk1p. Slk1p belongs to the vii Ndr subfamily of the AGC group of kinases and is analogous to the SIN component Sid2p. Slk1p is expressed specifically during meiosis and localizes to the spindle pole bodies (SPBs) during meiosis I and II in a SIN dependent manner. Slk1p also localizes to the forespore membrane during sporulation. Cells lacking Slk1p display defects associated with sporulation, leading frequently to the formation of asci with smaller and / or fewer spores. The ability of slk1∆ cells to sporulate, albeit inefficiently, is fully abolished when function of Sid2p is compromiised, suggesting that Slk1p and Sid2p play overlapping roles in sporulation. Moreover, increased expression of the syntaxin Psy1p rescues the sporulation defect of sid2-250 slk1∆. Thus, it is likely that Slk1p and Sid2p play a role in FSM assembly by facilitating recruitment of components of the secretory apparatus, such as Psy1p, to allow membrane expansion. These studies thereby suggested that SlN is required to couple the growth of FSM to the meiotic nuclear division. In order to investigate the role of F-actin and how SIN regulates actin cytoskeleton during meiosis, in Chapter IV, I analyzed the dynamics of F-actin and characterized the role of actin nucleators during sporulation in sin mutant cells. F-actin assembles into ring structures per ascus, referred to as the MeiAR (meiotic actin ring). The actin nucleators Arp2p/3p and formin-For3p assemble into ring structures that overlap with the leading edge protein Meu14p, whereas F-actin makes rings that occupy a larger region behind the leading edge. Time-lapse microscopy shows that the MeiAR assembles near the spindle pole bodies and undergoes an expansion in diameter during the early stages of meiosis II, followed by closure in later stages of meiosis II. MeiAR closure completes the process of forespore membrane assembly. viii Loss of MeiAR leads to excessive assembly of forespore membranes with a deformed appearance. The rate of closure of the MeiAR is dictated by the function of the septation initiation network (SIN). These experiments established the fact that the MeiAR ensures proper targeting of the membrane biogenesis machinery to the leading edge, thereby ensuring the formation of spherically shaped spores. 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PLoS One 5, e13323. 131 [...]... et al., 1994) The new membrane addition in fission yeast is also mediated by t-SNARE protein Psy1p (Nakamura et al., 2001; Shimoda, 2004)) In yeast, the membrane addition machinery and the ring constriction machinery are dependent on each other mechanistically In budding yeast, cell cycle-regulated trafficking of the chitin synthase Chs2p controls actomyosin ring stability during cytokinesis (Cabib,... during cell growth in interphase During cytokinesis, only a small fraction of new membrane and new cell wall materials are deposited at the division site In fission yeast, Brefeldin A (BFA), a drug that blocks membrane trafficking of newly synthesized proteins from the Endoplasmic Reticulum (ER) to the Golgi apparatus (Turi et al., 1994), blocks cytokinesis (Brazer et al., 2000) In budding yeast, the. .. to be involved in the formation of cleavage furrow in metazoan cells (Clayton and Johnson, 1998) Hence, these proteins participate in various aspects including cross-linking and/or stabilization of F-actin, in order to anchor the actomyosin ring to the plasma membrane and/or organize F-actin and myosin II into a dynamic contractile ring at the cleavage furrow 1.1.3 Spindle/ Phragmph formation in plants... 1978), the function of some actin regulating proteins including profilin, alpha-actinin, tropomyosin, anillin, filamin, talin, radixin, and cofilin, have also been revealed in cytokinesis (Field and Alberts, 1995; Fujiwara et al., 1978; Gunsalus et al., 1995; Mabuchi et al., 1985; Nunnally et al., 1980; Oegema et al., 2000; Sanger et al., 1994; Sanger et al., 1984) Profilin, an actin-monomer binding protein,... 4.1 F-actin dynamics during meiosis in S pombe 87 Figure 4.2.1 Localization of actin nucleators to the leading edge during FSM formation in S pombe 90 xi Figure 4.2.2 Localization of actin nucleators to the leading edge during FSM formation in S pombe 91 Figure 4.3 Time-lapse images showing the dynamics of actin nucleators during FSM assembly in cells treated with 100µm Lat A 93 Figure 4.4 Actin is required... coordinated with mitosis (Jurgens, 2005a, b) 6 1.1.4 Meiotic cytokinesis during sporulation in yeast In yeast, mitotic cytokinesis is driven by a combination of actomyosin ring-mediated ingression of the plasma membrane and the deposition of septum materials, to create two daughter cells endowed with a complete set of chromosomes and cytoplasmic organelles During meiosis, the equivalent event in yeast. .. furrow ingression Satterwhite et al (1992) reported that the phosphorylation state of the regulatory light chain of myosin (MRLC) changes during the cell cycle, suggesting that the phosphorylation of MRLC, which produces the ingression force, is critical for the progression of cytokinesis (Satterwhite et al., 1992; Trotter and Adelstein, 1979) With the identification of F-actin involved in actomyosin ring... pathways regulating the process of cytokinesis (Balasubramanian et al., 2004; Glotzer, 2001; Jurgens, 2005a, b) The subsequent sections will review cytokinesis in several organisms and discuss the function of a signalling pathway (Septation Initiation Network, SIN) that pertains this study 1.1.1 New membrane/cell wall formation Cytokinesis in all organisms depends on extensive remodelling of the cell membranes... division, and then constricts during anaphase Decades of studies have identified more than 50 associated proteins that are involved in the actomyosin ring assembly (Balasubramanian et al., 2004; Wu et al., 2003) They include actin; the myosin heavy chain Myo2p; essential myosin light chain 17 Cdc4p; myosin assembly factor Rng3p; IQGAP-related protein Rng2p; formin Cdc12p; profilin Cdc3p; tropomyosin Cdc8p;... shortly after anaphase onset when the mitotic spindle begins to elongate An equatorial actomyosin ring is assembled at the division site in late anaphase This dynamic actomyosin contractile ring is essential for the progression of cytokinesis in animal cells F-actin and non-muscle myosin II were identified to be the main components of the actomyosin contractile ring at the cleavage furrow (Forer and Behnke, . UNDERSTANDING THE SEPTATION INITIATION NETWORK (SIN) FUNCTION DURING MEIOTIC CYTOKINESIS IN FISSION YEAST YAN HONGYAN A THESIS SUBMITTED FOR THE DEGREE OF. 1978), the function of some actin regulating proteins including profilin, alpha-actinin, tropomyosin, anillin, filamin, talin, radixin, and cofilin, have also been revealed in cytokinesis (Field. appearance. The rate of closure of the MeiAR is dictated by the function of the septation initiation network (SIN). These experiments established the fact that the MeiAR ensures proper targeting of the