Regulation of nuclear division during mitotic stagnation

REGULATION OF NUCLEAR DIVISION DURING MITOTIC STAGNATION ZHANG TAO INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 REGULATION OF NUCLEAR DIVISION DURING MITOTIC STAGNATION ZHANG TAO (M.B.B.S., M.Med., GUANGXI MEDICAL UNIVERSITY) 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 earnest thanks to Assoc/Prof. Uttam Surana for his continuous guidance, great support and stimulating discussions which helped me sustain my interest throughout this study. I am grateful to the members of my PhD Supervisory Committee members A/Prof. Cai MingJie, and A/Prof. Wang Yue for their constructive comments, timely suggestions and encouragement. My deepest gratitude to Asist/Prof. Lim Hong Hwa for her continuous help, guidance and advice, without which it would be difficult to complete these projects; to Miss Karen Crasta for useful discussions and sharing of constructs; to members of CMJ and WY laboratory for interesting discussions and generous help; to Saurabh Nirantar, Cheng CheeSeng, Khong JennHui, Wee Kheng, Cher WeiLing, Wong SzeMing and the past members of the Uttam Surana laboratory for their cooperation, friendship and technical assistance; their support and collaboration have made my efforts far more efficient. I would like to thank Dr. Sihoe SanLing for her patience in teaching me the mammalian technique and to Dr. Indrajit Sinha for teaching me 2-dimension gel technique. I am also grateful to Drs. Kim Nasmyth, Frank Uhlmann, Tomo Tanaka, Piatti Simonetta, Yolanda Sanchez, David Morgan, Jim Haber, Wolfgang Zachariae and David Balasundaram for yeast strains, constructs and other reagents which were important for many experiments. Finally, I would like to express my earnest gratitude to my parents and family; without their constant support and encouragement, it would be so much more difficult to complete this thesis. Last but not the least, I would like to extend my gratitude to my wife Ling Ling for endless support and help throughout this study. i Table of Contents TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………… i TABLE OF CONTENTS……………………………………………… ii SUMMARY…………………………………………………………… v LIST OF TABLES………………………………………………… .… viii LIST OF FIGURES ……………………………………………… ix LIST OF SYMBOLS………………………………………………… . xi CHAPTER Introduction 1.1 Cell Cycle of budding yeast Saccharomyces cerevisiae……………. 1.2 Onset of mitosis………………………………………………… .… 1.3 Regulation of chromosome segregation…………………………… 1.3.1 The cohesin complex………………………………… …… 1.3.2 Non- cohesin components……………………………….……. 1.4 The kinetochore……………………………………………… ……. 1.4.1 Kinetochores of budding yeast Saccharomyces cerevisiae… 1.4.2 Kinetochores of other organisms……………………………… 1.5 Chromosome condensation and topoisomerse II……………………. 1.6 Chromosome segregation during mitosis in budding yeast…………. 1.7 Chromosome segregation of vertebrate cells……………………… . 1.8 Chromosome segregation in the meiosis of budding yeast……… . 1.9 Kinetochores and pole-ward forces during mitosis……………….… 1.10 Dynamic kinetochore protein Slk19…………………………….…. 1.10.1 The function of Slk19 in mitosis and meiosis…………… 1.11 Checkpoints and mitotic stagnation…………………………… .… 1.11.1 Spindle assembly checkpoint and spindle position checkpoint……………………………. 1.11.2 DNA replication and DNA damage checkpoint………… . 1.11.3 Targets of DNA damage checkpoint……………………… 1.12 Rationale for revisiting DNA-damage checkpoint……………… 9 11 12 13 19 20 22 25 26 28 30 30 32 32 34 35 37 CHAPTER Materials and methods 2.1 Materials……………………………………………………….……. 2.2 Methods…………………………………………………………… . 2.2.1 Escherichia coli strains and culture conditions…………….…. 2.2.2 Yeast strains and culture conditions………………….……… 39 49 49 49 ii Table of Contents 2.2.3 Cell cycle synchronization……………………………….……. 2.2.3.1 G1 phase synchronization………………………….… 2.2.3.2 Stationary phase synchronization………………… .… 2.2.3.3 G2-M phase synchronization………………………… 2.2.3.4 Early S phase synchronization………………………… 2.2.4 Yeast Manipulations………………………………….……… 2.2.4.1 Yeast transformation………………………………… . 2.2.4.2 Yeast chromosomal DNA extraction……………….…. 2.2.4.3 PCR- based strategy for fluorescent protein and epitope tagging of yeast genes……………………. 2.2.5 Immunofluorescent staining……………………………… …. 2.2.6 Flow cytometric analysis……………………………………… 2.2.7 Microscopy……………………………………………………. 2.2.8 Protein analysis……………………………………………… . 2.2.8.1 Protein extraction…………………………………… 2.2.8.1.1 Protein extraction using TCA……………… 2.2.8.1.2 Protein extraction using acid washed glass beads……………….….… 2.2.8.2 Immunoprecipitation………………………………… 2.2.8.3 in vitro kinase assay………………………………… 2.2.9 Recombinant protein expression and purification…………… 2.2.10 Southern blot analysis……………………………………… . 2.2.11 Chromatin Immunoprecipitation (Chromatin IP)………… . 2.2.12 RNA preparation and Real-time PCR……………………… 2.2.13 Two- dimensional gel electrophoresis (2D gel)…………… . 2.2.14 Pulse- chase assay…………………………………….……… 50 50 50 51 51 52 52 52 53 54 55 56 57 57 57 57 58 59 59 60 61 61 63 64 CHAPTER Kinetochore protein SLK19 and nuclear division 3.1 Introduction…………………………………………………………. 3.2 The absence of Slk19 causes chromatin mass deformation……… 3.3 The absence of Slk19 does not affect amphitelic attachment……… 3.4 Mobile nucleus in Slk19 deficient cells…………………….……… 3.5 Spindle behaviour in Slk19 deficient cells………………………… 3.6 Loss of centromeric elasticity in Slk19 deficient cells……………… 3.7 Physical association between Scc1 and Slk19………………………. 3.8 The absence of Slk19 does not affect the loading of Scc1 at centromeric regions and its cleavage during cell cycle progression…………………………………… .… 3.9 Effect of Scc1 inactivation on localization and cleavage of Slk19.… 3.10 Loss of viability in the absence of Slk19 recovery from Nocodazole arrest……………………………………………. 3.11 Discussion……………………………………………………….…. 65 66 74 78 82 86 91 96 100 104 105 iii Table of Contents CHAPTER Regulation of spindle elongation by DNA damage checkpoint 4.1 Introduction………………………………………………… .…… 4.2 Cohesin cleavage in DNA damaged cells fails to trigger anaphase B…………………………………………….… 4.3 Instability of motor proteins Cin8 and Kip1 in DNA damaged cells 4.4 The roles of cdh1 and polo kinase Cdc5 in DNA damaged cells…… 4.5 Over-expression of ‘active Cdh1’ (not phosphorylatable by Cdc5) inhibits spindle extension in checkpoint-defective cells………….… 4.6 Over-expression of Rad53 suppresses spindle elongation in a ‘non-checkpoint’ context……………………………………… 4.7 Discussion…………………………………………………… …… 110 111 119 124 132 135 138 CHAPTER Perspective 5.1 Maintaining the centromeric elasticity…………………………… 5.2 Cdh1 and DNA damage checkpoint………………………………… 142 144 CHAPTER Conclusion and future work 6.1 Slk19 and mitotic arrest………………………………………….… 6.2 DNA damage checkpoint and the regulation of spindle elongation… 148 149 Bibliography Appendices I Appendices II iv Summary SUMMARY Chromosome duplication and equal partitioning of chromosomes to progeny cells are the central events in cell division. An ordered set of cellular events has to be executed in a highly coordinated fashion to ensure proper alignment and segregation of the duplicated chromosomes (sister chromatids). Kinetochores (a multi-protein complex assembled on the centromeric DNA) and the mitotic spindle (a microtubule-based assembly where microtubules radiate from two centrosomes or spindle pole bodies in yeast) play important roles in the separation of sister chromatids. Prior to anaphase, sister-chromatids are held together by a protein complex known as cohesin which prevents premature segregation. Some time during late S phase, microtubules emanating from the centrosomes establish amphitelic (or bipolar) attachment to sisterkinetochores. During metaphase to anaphase transition, ubiquitin-dependent destruction of securin Pds1 by APC (Anaphase Promoting Complex) liberates the caspase-like protease known as the separase (Esp1 in yeast), which in turn cleaves the cohesin subunit Scc1 leading to the successful separation of duplicated sister chromatids. Once bipolar attachment is established in mid-to-late S phase, sister kinetochores experience a pole-ward pull causing transient separation of centromeric chromatin prior to anaphase termed ‘elastic deformation’ of chromosomes. This force is presumably countered by the cohesin complex which holds sister chromatids together until the onset of anaphase. It is important for cells to resist this pole-ward pull prior to anaphase for the maintenance of genomic stability; this becomes particularly important when cells face relatively long periods of stagnation during mitosis, for example, due to activation of checkpoints. It is not clear whether cohesin v Summary complexes alone are sufficient to resist the pole-ward tug at the centromeric region or whether auxiliary proteins are required to augment or bolster the resistance. It is with this in mind that we started an investigation into the role of the kinetochore protein Slk19. Slk19 has previously been shown to be the only other known target of separase Esp1 other than the cohesions. It localizes to kinetochores and, after cleavage by the separase, translocates to the spindle midzone. We find that during pre-anaphase arrest, the spindle in cells deficient in kinetochore protein Slk19 is excessively dynamic and the nucleus moves prematurely into the mother-daughter junction. As a result, the chromatin mass undergoes a partial division which does not require either APC activity or Scc1 cleavage. Partial division of chromatin mass is accompanied by the loss of centromeric region’s ability to resist pole-ward pull by the spindle. Slk19 was found to physically associate with Scc1 and this association appears necessary for Slk19’s efficient cleavage by separase. Based on our observations, we propose that Slk19 participates in regulating nuclear migration and, in conjunction with the cohesin complex, is involved in the maintenance of centromeric tensile strength required to resist the pole-ward pull. Mitotic stagnation can also be imposed on cells when the DNA-damage-inducible checkpoint is activated. It is now well established that upon DNA damage, the checkpoint activates Mec1 kinase (human ATM/ATR-like kinase) which in turn leads to activation of two other kinases, namely Chk1 and Rad53 (similar to human Chk2), to impose G2/M arrest, thus allowing the cells sufficient time for DNA repair. It is generally believed that the DNA damage checkpoint inhibits segregation of damaged chromosomes by preventing cohesin cleavage via phosphorylation of securion Pds1 vi Summary rendering it resistant to proteolytic destruction by APCCdc20. However, we find that removal of cohesins alone does not lead to spindle extension or complete separation of damaged chromosomes in most cells. We document evidence which show that DNA damage checkpoint also actively prevents mitotic spindle elongation via regulation of the microtubule associated proteins (MAPs). Our data suggest that the checkpoint kinase Rad53 inhibits polo kinase Cdc5 to maintain APC activator Cdh1 in a partially active state which prevents accumulation of Cin8 and Kip1, thus precluding spindle elongation. 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Appendices I Appendices II DNA damage checkpoint maintains Cdh1 in quasi-active state to prevent spindle elongation and Anaphase B Tao Zhang¶, Saurabh Nirantar¶, Indrajit Sinha and Uttam Surana* Institute of molecular and Cell Biology Proteos 61, Biopolis Drive Singapore 138673 ¶ These authors contributed equally *Corresponding author: Uttam Surana Institute of Molecular and Cell Biology Proteos 61, Biopolis Drive Singapore 138673 Tel: (65) 6586 9503 Fax: (65) 6779 1117 Email: mcbucs@imcb.a-star.edu.sg Appendices II Summary DNA damage checkpoint imposes cell cycle arrest to prevent segregation of damaged chromosomes1. In budding yeast, Mec1, Chk1 and Rad53 (homologous to human ATM/ATR, Chk1 and Chk2 kinase, respectively) are among the main effectors of this surveillance pathway2. The DNA damage checkpoint is thought to inhibit segregation of damaged chromosomes by preventing separase-mediated cleavage of cohesins3,4. Here, we describe a new regulatory network that prevents segregation of damaged chromosomes in the absence of sister-chromatid cohesion by inhibiting spindle elongation. This control circuit involves Rad53, polo kinase, anaphase promoting complex (APC) activator Cdh1 and the bimC kinesin family proteins Cin8 and Kip1. We show that inhibition of polo kinase by Rad53 maintains Cdh1 in partially active state which in turn prevents accumulation of Cin8 and Kip1, thus precluding spindle elongation. Hence, DNA damage checkpoint suppresses both cohesin cleavage and spindle elongation to preserve chromosome stability. -------------------------------- Cells respond to chromosomal injuries by triggering DNA damage checkpoint which activates repair processes, induces transcription of genes that facilitate recovery from the damage and arrests cell cycle progression to allow sufficient time for repairs. In mammalian cells5,6 and fission yeast7, inhibition of mitotic kinase Cdc2 (Cdk1) by the checkpoint pathway is thought to be predominantly responsible for arresting progression to M phase thus preventing onset of mitotic events such as chromosome segregation. In budding yeast Saccharomyces cerevisiae, however, expression of activated Cdc28 (Cdk1) does not abolish the checkpoint-induced G2/M arrest suggesting that inactivation of Cdc28 activity is not central to the mechanism that imposes cell cycle arrest8,9. Instead, the checkpoint directly targets the networks responsible for regulating chromosome segregation and mitotic exit 3,10. During normal division cycle, duplicated chromosomes are held together by cohesin complex to prevent premature segregation of sister chromatids. At metaphase to anaphase transition, separase, a caspase-like protease encoded by the ESP1 gene, cleaves cohesin subunit Scc1/Mcd1 and dissolves sister-chromatid cohesion to allow partitioning of chromosomes by the mitotic spindles. However, separase (Esp1) remains inactive until anaphase because of its association with securin (encoded by [...]... budding yeast kinetochore…… Regulation of chromosome segregation… Nuclear division in slk19Δcells…………………………… Partial nuclear division in slk19Δ cells occurs prior to initiation of anaphase…………………………………… … Partial nuclear division does not occur in ndc10 mutant cells…………………………………………….…… Spindles in slk19Δ cells………………………………… … Bipolar attachment in slk19Δ cells………………………… Nuclear dynamics in Slk19... complement of genetic material during cell division Cellular events leading to partitioning of chromosomes have to be coordinated precisely to maintain the integrity of the genome Gross departure from this precision can compromise genome stability and eventually affects cells’ fitness and survival Cell division cycle can be thought of as a series of ordered cellular events, coordinated by two sets of controls:... mediate the initiation of S phase (Bloom and Cross, 2007) Upon completion of DNA synthesis, Cdc28 forms a complex with mitotic cyclins (Clb1, 2, 3, 4) and triggers the onset of M phase (Bloom and Cross, 2007) Of these four mitotic complexes’, Cdc28-Clb2 contributes ~70% of the total mitotic kinase activity (Surana et al., 1991; Fitch et al., 1992; Richardson et al., 1992) The expression of Cln and Clb cyclins... activation of the mitotic kinase Activation of mitotic kinase complex Cdc28/Clb at G2/M transition 8 CHAPTER 1: Introduction leads to the activation of another critical mitotic regulator, the ubiquitin ligase APCCdc20, most likely involving phosphorylation of Cdc20 and/or APC subunits (Stegmeier et al., 2007; Reddy et al., 2007) APCCdc20 plays an important role in the separation of sister chromatids during. .. role in the progression of the division cycle 5 CHAPTER 1: Introduction Figure 1 A schematic diagram of the budding yeast cell division cycle The division cycle of budding yeast, like other eukaryotes, is divided into four phases, G1, S, G2 and M phase Essentially a set of sequential events, it is driven by the key cyclindependent kinase Cdc28 (Cdk1) and three different sets of cyclins, namely, G1 cyclins... This is necessary for the activation of APCCdh1, which is inhibited by mitotic kinase mediated phosphorylation Reduction in mitotic kinase activity by APCCdc20 paves the way for the activation of APCCdh1 by Cdc14 phosphatase, which is released from the nucleolus under the influence of the Mitotic Exit Network (MEN) Activated APCCdh1 mediates further destruction of mitotic cyclins, thus allowing cells’... pombe because of the ease with which they can be genetically manipulated While there are some notable differences (for example the nuclear membrane remains intact in both yeasts during mitosis unlike that in human cells, where nuclear membrane breakdown 1 CHAPTER 1: Introduction is one of the prominent markers of entry into mitosis), the basic operations and control circuits that regulate the division cycle... for understanding the mechanism of cell division cycles of higher eukaryotes Like in other eukaryotes, the budding yeast cell cycle is divided in four phases: G1, the period when the cell prepares itself for entry into a new division cycle; S, the period of DNA synthesis; G2, the preparation period for entry into mitosis; and M, also called mitotic phase, the period during which duplicated sister chromatids... imaging………………………………………… … The inactivation of Scc1………………………………… … DNA damage induced by GAL-HO……………………….… Motor protein levels in DNA-damaged cells …………….… Real- time PCR…………………………………………… Stability of CIN8 and KIP1 in DNA-damaged cells………… Over- expression of Cin8 relieves inhibition of anaphase B by DNA damage checkpoint………………… Involvement of Cdh1 and Cdc5 in the regulation of spindle elongation in DNA-damaged... only transiently localize to the kinetochores during some stages of the cell cycle (Fukagawa, 2004) 1.5 Chromosome condensation and topoisomerse II The primary purposes of mitotic chromosome condensation in eukaryotic cells are to reduce chromosome arm lengths so that truncation during cell division can be avoided and proper separation and segregation of sister chromatids can be facilitated (Belmont, . REGULATION OF NUCLEAR DIVISION DURING MITOTIC STAGNATION ZHANG TAO INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE. 2007 REGULATION OF NUCLEAR DIVISION DURING MITOTIC STAGNATION ZHANG TAO (M.B.B.S., M.Med., GUANGXI MEDICAL UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. diagram of the budding yeast kinetochore…… 18 Figure 4 Regulation of chromosome segregation… 24 Figure 5 Nuclear division in slk19 Δ cells…………………………… 69 Figure 6 (A) Partial nuclear division