REGULATION OF MITOTIC SPINDLE BIOGENESIS IN BUDDING YEAST CRASTA KAREN CARMELINA (B. Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 Acknowledgements ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation to A/P Uttam Surana, to whom I am much indebted for his guidance, insightful and influential conversations, valuable advice and the freedom to explore my curiosities. My sincere thanks also go out to members of my PhD Supervisory Committee, A/P Mohan Balasubramanian and A/P Yang Xiaohang, for their constructive comments and encouragement. Special Thanks to my extended family, my labmates – Hong Hwa, Chee Seng, Wei Chun, Jonathon, Joan, Zhang Tao, Saurabh, Jenn Hui, San Ling, Wee Kheng and Vaidehi for sharing in the thrill along the path to discovery, help in various ways and for making lab life fun! A big Thank You to all in CMJ lab for being such wonderful neighbours! I am also grateful to Suniti Naqvi, Mithilesh Mishra and all in Mohan’s lab at TLL for their time in teaching me fission yeast techniques. I wish to thank Prof. Mark Winey for providing technical expertise in electron microscopy for various projects. Thanks also to the EM Unit, NUS for my training and Chee Peng for help despite his busy schedule. I am grateful to Drs Mark Winey, David Morgan, Wolfgang Zachariae, John Kilmartin, Matthias Peter, David Pellman, Chris Hardy, Kyung Lee, Jiri Lukas and Michel Bouvier for providing me with valuable reagents and Dr Mark Hall for helpful advice. Special Thanks to Ram, Trich, Jaya, Xianwen, Lee Thean, Kar Lai, Rida, Foong May, Suniti, Vani, Srini, Shal, Nee, Indra for their friendship, the fun times and encouragement. Thanks to all at Opus Dei for their friendship and prayers, and for bringing out the best in me in my daily work. Most importantly, this thesis is dedicated to my loving FAMILY especially my parents for having always encouraged me to aim high, for much-valued support, understanding, advice, sacrifices, prayers and constant cheer that made this journey of scientific discovery possible. Thank You Daddy, Mummy, Sharon and Renita! Karen Crasta, June 2007 i Abbreviations Abbreviations Ab Antibody 1NM-PP1 4-amino-1-tert-butyl-3-(1-naphthylmethyl) pyrazolo [3, 4-d] pyrimidine BSA bovine serum albumin CDK cyclin-dependent kinase cpm counts per minute ˚C degree Celsius D Glucose DAPI 4’, 6-diamidino-2-phenylindole DNA deoxyribonucleic acid DTT dithiothreitol ECL Enhanced chemiluminescence EDTA ethylenediamine tetraacetic acid g gram Gal Galactose GFP Green Fluorescent Protein Glu Glucose h hour HA haemagglutinin HRP Horseradish peroxidase IP immunoprecipitation kb Kilobases kDa KiloDalton M Molar MAP microtubule-associated protein xi Abbreviations Met Methionine mCi Millicurie mg milligram µg microgram minute ml milliliter µl microliter mM millimolar MOPS 3-[N-Morpholino] propane-sulfonic acid MT microtubule nm nanometer OD optical density PAGE polyacrylamide gel electrophoresis PBS Phosphate-buffered saline PCR polymerase chain reaction PEG polyethylene glycol PMSF phenylmethylsulfonylfluoride Raff Raffinose RNA ribonucleic acid SDS sodium dodecyl sulfate SSC saline sodium citrate TE Tris-EDTA buffer ts temperature-sensitive YEP yeast extract-peptone xii Table of Contents TABLE OF CONTENTS Acknowledgements…………………………………………………………………………………i Table of Contents………………………………………………………………………………….ii Summary………………………………………………………………………………………… vi List of Tables…………………………………………………………………………………….viii List of Figures…………………………………………………………………………………… ix Abbreviations…………………………………………………………………………………… .xi Chapter Introduction……………………………………………………………………………1 1.1 Introductory Remarks………………………………………………………………………1 1.2 Overview of Budding Yeast Cell Cycle ………………………………………………… .2 1.2.1 Saccharomyces cerevisiae cell cycle and cyclin-dependent kinase Cdc28……… 1.2.1.1 Inhibitory Phosphorylation on Cdc28-Tyr 19 ……………………………4 1.2.1.2 Structural basis for Cdk activation …………….…………………………5 1.2.1.3 Conditional cdc28 mutants……………………………………………… 1.2.2 1.3. Coordination of cell cycle events and checkpoints……………………………… Protein Degradation in cell cycle control ……………………………………………… .10 1.3.1 Ubiquitin-dependent proteolysis ……………………………………………… .10 1.3.2 SCF……………………………………………………………………………….11 1.3.3 APC………………………………………………………………………………12 1.3.3.1 Selective substrate recognition by APC…………………………………12 1.3.3.2 APC-Cdc20 at metaphase-to-anaphase transition……………………….15 1.3.3.3 APC-Cdh1 at the end of mitosis…………………………………………15 1.4 The bipolar mitotic spindle……………………………………………………………… 17 1.5 The centrosome cycle…………………………………………………………………… 20 1.6 The spindle pole body (SPB) cycle……………………………………………………….25 ii Table of Contents Chapter Materials and Methods………………………………………………………………29 2.1 Materials………………………………………………………………………………… 30 2.2 Methods………………………………………………………………………………… .35 2.2.1 Strains and Culture Conditions………………………………………………… 35 2.2.2 Cell Synchronization Procedures……………………………………………… .36 2.2.3 GAL-HO induction for construction of polyploidy strains………………………36 2.2.4 Yeast Transformation…………………………………………………………….36 2.2.5 Isolation of plasmid DNA from yeast cells………………………………………37 2.2.6 High-copy Suppression Screen………………………………………………… 37 2.2.7 Preparation of Yeast Chromosomal DNA……………………………………… 38 2.2.8 Southern Blot Analysis………………………………………………………… .39 2.2.9 Northern Blot Analysis………………………………………………………… .39 2.2.10 Immunofluorescent Staining…………………………………………………… 40 2.2.11 Visualization of Fluorescent Protein Signals…………………………………….41 2.2.12 Flow Cytometric Analysis……………………………………………………… 42 2.2.13 Transmission Electron Microscopic Analysis……………………………………42 2.2.13.1 Chemical Fixation and Embedding of Yeast Cells…………………… 42 2.2.13.2 Microtome Sectioning, Staining and Viewing under TEM…………… 43 2.2.14 Bioluminescence Resonance Energy Transfer (BRET2) assay………………….44 2.2.15 Preparation of Cell Extracts for Protein Analysis……………………………… 44 2.2.15.1 Cellular lysis using acid-washed glass beads………………………… .44 2.2.15.2 Protein Precipitation using Tri-Chloroacetic Acid (TCA)…………… .45 2.2.16 Western Blot Analysis……………………………………………………………45 2.2.17 Pulse-chase experiments………………………………………………………….46 2.2.18 Immunoprecipitation of HA3 and cmyc3-tagged proteins……………………… 46 iii Table of Contents 2.2.19 Kinase Assays……………………………………………………………………47 2.2.20 Detection of Cdc28 tyrosine phosphorylation……………………………………48 2.2.21 Detection of ubiquitin conjugates in vivo……………………………………… .48 2.2.22 Coomasie Blue Staining………………………………………………………….49 2.2.23 Silver Staining……………………………………………………………………49 2.2.24 Expression and Purification of GST-tagged proteins…………………………….50 Chapter The Regulatory Role of Cdc28 in SPB Separation…………………………………52 3.1 Background……………………………………………………………………………….52 3.2 Results…………………………………………………………………………………….54 3.2.1 cdc28Y19E and cdc28-as1 cells are unable to separate SPBs……………………54 3.2.2 cdc28 mutants defective in SPB Separation Do Not Activate the Spindle Checkpoint………………………………………………………… ………… .56 3.2.3 Genetic Screen to Identify Downstream Targets of Cdc28 in SPB Separation….57 3.2.4 Ectopic Expression of Microtubule-Associated Proteins Induces Spindle Formation………………………………………………………….……… ……59 3.2.5 SPB separation does not require Cdc28-mediated phosphorylation of microtubuleassociated proteins……………………………………………………………… 63 3.2.6 Low Endogenous Levels of Cin8, Kip1 and Ase1 in cdc28Y19E and cdc28-as1 63 3.2.7 Defect in SPB separation is due to proteasomal degradation of microtubule associated proteins……………………………………………………………… 68 3.2.8 APCCdh1, but not APCCdc20, Prevents SPB Separation……………………………72 3.2.9 Cdh1 phosphorylation and Cin8 ubiquitylation in cdc28 mutants defective in spindle assembly…………………………………………………………………76 3.2.10 Cdc28/Clb activity controls Cdh1 subcellular localization and spindle assembly.77 3.2.11 Cdc28-phosphorylation sites in Cdh1 and stability of Cin8 and Clb2………… .79 3.2.12 Microtubule bundling activity, not motor activity, is required for SPB iv Table of Contents Separation……………………………………………………………………… .80 3.2.13 Tyrosine dephosphorylation of Cdc28 temporally precedes spindle assembly during a normal cell cycle……………………………………………………… 83 3.3 Discussion……………………………………………………………………………… .83 Chapter Inactivation of Cdh1 by synergistic action of Cdc28 and Cdc5 is essential for spindle assembly………………………………………………………………………………….94 4.1 Background……………………………………………………………………………….94 4.2 Results…………………………………………………………………………………….94 4.3 4.2.1 Effects of ectopic expression of Cdc5 in cdc28-as1 cells……………………… 94 4.2.2 Phosphorylation of Cdh1 by Cdc5 requires priming by Cdc28………………….99 4.2.3 Cdc5 has a role in bipolar spindle assembly in budding yeast………………….101 4.2.4 Cdc5 degradation in cdc28-as1 cells……………………………………………110 Discussion……………………………………………………………………………….112 Chapter Matters Arising…………………………………………………………………… .119 5.1 Synergistic action of Cdk1 and Plk1 on Mammalian Cdh1 …………………………….119 5.2 The Paradox: Active Cdh1 Is Degraded…………………………………………………121 5.3 Role of Cdc20 in SPB separation……………………………………………………… 124 5.4 Temporal regulation of satellite formation by inactivation of mitotic kinase………… .128 Chapter Conclusion and Perspectives…… .……………………………………………… 133 References……………………………………………………………………………………….138 Appendices v List Of Figures List Of Figures Figure 1. Schematic diagram of the budding yeast cell division cycle………………………3 Figure 2. Cdc28 function is regulated by inhibitory phosphorylation by Swe1 and dephosphorylation by Mih1……………………………………………………….6 Figure 3. Schematic representation of a centrosome and spindle pole body (SPB)……… 21 Figure 4. The centrosome and spindle pole body (SPB) duplication cycles……………….23 Figure 5. cdc28Y19E and cdc28-as1 cells fail to separate SPBs………………………… .55 Figure 6. The G2/M arrest phenotype of cdc28-as1 and cdc28Y19E cells is not due to activation of the spindle checkpoint…………………………………………… .58 Figure 7. Ectopic expression of microtubule-associated proteins induces spindle assembly………………………………………………………………………….61 Figure 8. Cdc28-mediated phosphorylation of Cin8, Kip1 and Ase1 is not required for SPB separation……………………………………………………………………… .64 Figure 9. Low endogenous levels of microtubule-associated proteins in cdc28-as1 and cdc28Y19E cells………………………………………………………………….66 Figure 10. Proteasomal degradation of microtubule-associated proteins in cdc28 mutants…70 Figure 11. APCCdh1-mediated degradation of microtubule-associated proteins prevents spindle assembly…………………………………………………………………74 Figure 12. Phosphorylation status of Cdh1 determines its subcellular localization and Cin8 ubiquitylation…………………………………………………………………….78 Figure 13. Phosphorylation sites in Cdh1 essential for spindle formation………………… 81 Figure 14. Cin8-bundling activity, not its motor activity, is required for SPB separation… 82 Figure 15. Tyrosine dephosphorylation correlates with the timing of SPB separation during normal cell cycle…………………………………………………………………84 Figure 16. Model depicting role of activated Cdc28 (Cdk1) in SPB separation in budding yeast………………………………………………………………………………93 Figure 17. Ectopic expression of Cdc5 causes hyperphosphorylation of Cdh1 and SPB separation……………………………………………………………………… .97 Figure 18. Phosphorylation of Cdh1 by Cdc5 requires priming by Cdc28……………… .102 Figure 19. 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DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior. Mol Cell 16: 687-700. [...]... Cdc28 in SPB separation We show that the ubiquitin ligase APCCdh1 acts as a potent inhibitor of spindle formation by promoting degradation of microtubuleassociated proteins Cin8, Kip1 and Ase1 which are essential for SPB separation Activated Cdc28 kinase causes inactivation of APCCdh1 during S phase, resulting in the accumulation of these SPBseparation promoting proteins Since ectopic expression of Cin8,... sufficient tension across sister kinetochores, the spindle checkpoint is activated to inhibit onset of anaphase The spindle checkpoint is also activated when cells encounter errors in spindle assembly (reviewed in Musacchio and Hardwick, 2002; Tan et al., 2005) Another checkpoint called the spindle positioning checkpoint, delays mitotic exit and cytokinesis when spindles are misaligned with respect... ubiquitin, forming a covalent thioester bond between the terminal glycine of ubiquitin (Gly76) with a cysteine in the active-site of E1 In the second step, an ubiquitin-conjugating enzyme (E2) transiently receives the activated ubiquitin from E1, again on a conserved cysteine residue Finally, a ubiquitin ligase (E3) transfers ubiquitin from E2 to a lysine side-chain on the target protein E3 ubiquitin ligases,... bipolar spindle The BimC family of plus-end kinesin motor proteins (members show strong sequence similarity in an aminoterminal motor domain) like Eg5 in mammalian cells and Cin8 and Kip1 in S cerevisiae, crosslink and slide apart antiparallel microtubules to form bipolar spindles by binding and bundling MTs (Kashina et al., 1997) This plus-end directed force pushing spindle poles apart appears to be... function of the mitotic spindle In a typical metaphase spindle, the two spindle poles are in a “face-to-face” configuration, separated by a set of overlapping microtubules emanating from each spindle pole towards the other (pole-to-pole microtubules) A second set of MTs termed astral MTs radiate from each spindle pole towards the cell cortex A third set, kinetochore MTs, emanate from the spindle pole with... cyclin-dependent kinase Cdc28 The cell cycle of the budding yeast Saccharomyces cerevisiae is currently the best understood of all eukaryotes Cell division in budding yeast is accomplished by the coordinated control of the cell cycle clock consisting of four distinct phases (G1, S, G2, M) with the G2 phase being extremely short (Fig 1) These phases are a temporally organized series of interlocking... threonine-14 and tyrosine-15 (equivalent to Tyr-19 in S cerevisiae) within the ATP-binding domain also have important functions in the regulation of Cdk activity The phosphorylation state of these residues, first described in fission yeast Schizosaccharomyces pombe, is controlled by a balance of opposing kinase and phosphatase activities acting at these sites which influence initiation of mitosis Tyr-15... Weinert, 1989) In budding yeast, four major checkpoint controls have been described: Morphogenetic checkpoint, DNA replication checkpoint, DNA damage checkpoint and Spindle checkpoint While the morphogenetic checkpoint delays cell cycle progression in response to perturbations of cell polarity that prevent bud formation, the DNA replication checkpoint prevents entry into mitosis in response to the inhibition... impossible and would lead to genomic instability and aneuploidy, often associated with cancers (reviewed in Jallepalli and Lengauer, 2001) Faithful chromosome segregation is thus critically dependent upon the formation of a bipolar mitotic spindle Hence, understanding the regulation of mitotic spindle biogenesis, the subject of this dissertation, is crucial for gaining insights into the chromosome segregation... separation even in the absence of Cdc28-Clb activity, we propose that stabilization of these mechanical force-generating proteins is highly likely to be the predominant role of Cdc28-Clb in vi Summary SPB separation Interestingly, our results also indicate that SPB separation is dependent on the microtubule-bundling activity of Cin8 (a plus-end motor protein belonging to the conserved BimC family of spindle . the formation of a bipolar mitotic spindle. Hence, understanding the regulation of mitotic spindle biogenesis, the subject of this dissertation, is crucial for gaining insights into the chromosome. cyclin is bound, whereas in budding Chapter1 Introduction 5 yeast, phosphorylation precedes cyclin binding (Kaldis et al., 1998; Ross et al., 2000). In both cases however, cyclin binding. REGULATION OF MITOTIC SPINDLE BIOGENESIS IN BUDDING YEAST CRASTA KAREN CARMELINA (B. Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR