ROLES OF TACC-RELATED PROTEIN, Mia1p IN MTOCs AND MICROTUBULE DYNAMICS IN SCHIZOSACCHAROMYCES POMBE ZHENG LILING A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY TEMASEK LIFE SCIENCES LABORATORY NATIONAL UNIVERSITY OF SINGAPORE 2007 i Acknowledgements I would like to express my heartfelt thanks to my supervisor Dr. Snezhka Oliferenko for her excellent guidance, continuous support and encouragement, especially in the past two years after my son was born. Without her understanding and support, this thesis would not have been possible. I thanks to my co-supervisor Prof. Mohan Balasubramanian who provided opportunity to work as ARO in his lab and continually support me to be JRF in Dr Snezhka’s lab. Many thanks to his valuable commemts on my thesis project. I am also thankful to my thesis committee members Dr.Maki Murata Hori, Dr. Wang Yue and Dr. Thirumaran Thanabalu for the time and effort they put in this project and their helpful suggestions on this thesis project. I thank all past and present members of the cell dynamics laboratory and the cell division laboratory, Fungal Patho-biology for their help and discussions. I am thankful to TLL facilities and staff for general support. I thank Dr. Snezhka Oliferenko,Yuenchayo Ling and Aleksandar Vjestica for critical reading of my thesis. ii I would like to thank Dr Phong Tran for his help with the spinning disk confocal microscope. I am thankful to Temasek Holdings, Singapore for their financial support through to my work. Finally, I would like to thank my family for their constant support and encouragement. iii TABLE OF CONTENTS page i Title page Acknowledgements ii Table of contents iv List of Figures xi List of abbreviations xiii Summary xiv Publications xv CHAPTER I: Introduction 1.1 A general introduction of microtubules 1.1.1 Properties of microtubules 1.1.2 Centrosome 1.1.3 Non-centrosomal microtubule 1.1.4 Schizosaccharomyces pombe (S. pombe) as a model for studying microtubule dynamics 1.2 MTOC and microtubule cytoskeleton in S. pombe 1.2.1 Microtubule nucleating sites—MTOCs in S. pombe 5 1.2.1.1 Spindle pole body (SPB) 1.2.1.2 Non-centrosomal MTOCs 1.2.1.2.1 Interphase microtubule organizing centers (iMTOCs) 1.2.1.2.2 Equatorial microtubule organizing centers (eMTOC) 1.2.1.2.3 γ-TuRC satellites 10 iv 1.2.2 Components of MTOCs 10 1.2.2.1 The γ-TuRC 10 1.2.2.2 Mto1p and Mto2p 12 1.2.3 Organizing microtubule bundles 13 1.2.3.1 Minus-end-directed motor protein—Klp2p 14 1.2.3.2 Bundling protein—Ase1p 14 1.2.3.3 Model of maintaining ordered microtubules 15 1.2.4 Arrangement of microtubule cytoskeleton in S. pombe 1.2.4.1 Interphase microtubule cytoskeleton 18 18 1.2.4.1.1 Architecture and dynamics of interphase microtubule cytoskeleton 18 1.2.4.1.2 Functions of interphase microtubule cytoskeleton 19 1.2.4.1.2.1 Maintaining cell morphology 19 1.2.4.1.2.2 Controlling central localization of nucleus 22 1.2.4.2 Mitotic microtubule cytoskeleton 22 1.2.4.2.1 Architecture of mitotic microtubule cytoskeleton 22 1.2.4.2.2 Functions of mitotic microtubule cytoskeleton 23 1.2.5 Self-organization of fission yeast microtubules 1.3 Mia1p as a TACC-related protein 26 27 1.3.1 TACCs in human 27 1.3.2 TACC in Drosophila 28 1.3.3 Current knowledge about Mia1p in S. pombe 30 1.4 Aim and objectives of this thesis 32 v 1.5 Significance of this study 32 Chapter II Materials and Methods 2.1 Strains, reagents and genetic methods 33 2.1.1 Schizosaccharomyces pombe strains 33 2.1.2 Growth, media and conditions 34 2.1.3 Plasmids 34 2.1.4 Enzymes, antibodies and drugs used 35 2.2 Molecular methods and yeast methods 35 2.2.1 Recombinant DNA techniques 35 2.2.2 Transformation of E.coli by electroporation 36 2.2.3 LiAc transformation of S. pombe 36 2.2.4 Mia1p/Microtubule association experiments 37 2.2.5 Extraction of S. pombe genomic DNA 37 2.3 Construction of knock out mutants and epitope tagging of genes 38 2.3.1 Construction of knock out mutants 38 2.3.2 Construction of epitope tagging of genes 39 2.3.3 Generation of cells overexpressing Mia1p 39 2.4 Cell biology and microscopy 39 2.4.1 Immunofluorescence staining 39 2.4.2 Flow chamber experiments 41 2.4.3 Time-lapse fluorescence microscopy 41 2.4.4 Electron microscopy techniques 42 vi 2.4.5 Laser microsurgery 42 Chapter III: Results 3.1 Mia1p in assembly and maintenance of persisent iMTOCs at nuclear envelope 44 3.1.1 Lack of microtubule binding protein Mia1p results in fewer interphase Microtubules 44 3.1.1.1 Mia1p is a microtubule binding protein 44 3.1.1.2 mia1Δ cells exhibit disordered and heterogeneous microtubule bundles 46 3.1.2 mia1Δ cells are defective in attachment of microtubules to the nucleation sites 48 3.1.2.1 Microtubules detached from the nucleation sites in mia1Δ cells 48 3.1.2.2 Microtubules remained attached to the SPB in alp14Δ cells 51 3.1.3 Mia1p is not required for microtubule nucleation 53 3.1.3.1 Microtubules could nucleate on the preexisting microtubules in mia1Δ Cells 53 3.1.3.2 Microtubules could be nucleated from several sites around the nuclear envelope in mia1Δ cells 56 3.1.4 Mia1p is required for maintenance of microtubule bundles 61 3.1.5 Mia1p is required for sustaining proper post-anaphase array dynamics 63 3.1.5.1 PAA dynamics is abnormal in mia1Δ cells 63 3.1.5.2 Mia1p is not required for the assembly of the eMTOC 66 3.1.6 There are no detectable γ-tubulin-rich iMTOC structures in mia1Δ cells 68 vii viii 3.1.7 Microtubules are required for emergence of iMTOC structures upon entry into the new cell cycle 70 3.1.7.1 Why iMTOC structures could be absent from mia1Δ cells 70 3.1.7.2 Experimental confirmation of hypothesis 72 3.1.7.3 Model of establishment of the iMTOCs in fission yeast 75 3.2 The SPBs facilitate the NE division during closed mitosis in fission yeast 76 3.2.1 Cells overexpressing Mia1p arrest in mitosis with fully elongated spindles and nonsegregated chromosomes 76 3.2.1.1 Mia1p-overexpressing cells arrest in mitosis 76 3.2.1.2 Mia1p-overexpressing cells exhibit fully elongated mitotic spindle with non-segregated chromosomes 78 3.2.2 The SPBs are displaced from spindle poles and NE division fails in Mia1p-overexpressing cells 80 3.2.3 Spindles in Mia1p-overexpressing cells are bipolar and require the microtubule-crosslinking protein, Ase1p, for their assembly 85 3.2.4 The SPBs ensure the NE division by preventing abnormal deformation ix of the NE by mitotic spindle 89 3.2.5 Failure of chromosome segregation was not due to defects in kinetochore attachment 91 3.2.6 Genetic ablation of the NE allows chromosome segregation in Mia1poverexpressing cells 3.3 Role of Mia1p in maintenance of microtubule polarity 93 98 3.3.1 Mia1p was required for symmetrical distribution of cortical tip proteins at cell tips 98 3.3.2 Polarity of microtubules was altered in mia1Δ cells 100 Chapter IV Discussion 4.1 Comparison between mia1Δ cells and other γ-TuRC mutants 105 4.2 Role of Mia1p in microtubule attachment but not in microtubule nucleation 106 4.3 Microtubule attachment sites serve as MTOCs to establish microtubule architecture 107 4.4 Establishment of the iMTOCs requires microtubule attachment to the NE 108 4.5 Relationship between Mia1p and Alp14p 109 4.5.1 Mia1p interacts with Alp14p but not Dis1p 109 4.5.2 Different aspect of Mia1p and Alp14p functions in microtubule x attachment to the NE 110 4.5.3 Lack of Mia1p and Alp14p result in failure of iMTOCs establishment 110 4.6 De novo assembly of the MTOCs is not specific to S. pombe cells 111 4.7 Role of Mia1p in modulating mitotic microtubule dynamics 112 4.8 Conclusion and future perspectives 113 4.8.1 Conclusion 113 4.8.2 Future perspectives 114 4.8.2.1 Microtubules are nucleated by the iMTOCs and the SPB: are they different or similar? 114 4.8.2.2 Distribution of the iMTOCs at the NE: random or specific? 115 4.8.2.3 What’re the partners of Mia1p in terms of anchoring microtubule to the NE? 115 References 119 xi LIST OF FIGURES Figures page Figure3.1.1.1: Microtubule-associated protein Mia1p binds microtubules and is a component of S. pombe MTOCs. 45 Figure3.1.1.2: Cells lacking Mia1p exhibit disordered and heterogeneous microtubule bundles. 47 Figure3.1.2.1: Mia1p is required for attachment of microtubules to the SPBs and the NE. 49-50 Figure3.1.2.2: Although SPBs remain stationary in alp14Δ cells, microtubules are attached to the SPBs. 52 Figure3.1.3.1: Microtubule could be nucleated on the preexisting microtubule in mia1Δ cells. 54-55 Figure3.1.3.2: Microtubule could be nucleated form several sites around the NE in mia1Δ cells. 58-60 Figure3.1.4: Mia1p is required for maintenance of microtubule bundles. 62 Figure3.1.5.1: Mia1p-GFP is required for PAA organization. 65 Figure3.1.5.2: Mia1p is not essential for the assembly of the eMTOC. 67 xii Figure3.1.6: Components of the γ-tubulin complex are not found as prominent structures around the NE. 69 Figure3.1.7.1: A model of the iMTOC formation in S. pombe cells 71 Figure3.1.7.2: Microtubules are required for emergence of iMTOC structures upon entery into the new cell cycle. 74 Figure3.2.1.1: Fission yeast cells over-expressing Mia1p arrest in mitosis with fully extended mitotic spindles. 77 Figure3.2.1.2: Cells overexpressing Mia1p exhibit fully elongated mitotic spindle with non-segregated chromosomes 79 Figur3.2.2: Cells overexpressing Mia1p causes the SPBs to be displaced from the mitotic spindle poles and NE division failure. 82-84 Figure3.2.3: Spindle structures in Mia1p-overexpressing cells are bipolar and anti-parallel. 87-88 Figure 3.2.5: The majority of Mia1p-overexpressing cells exhibit Mad2p-GFP on the NE. 92 xiii Sup-Figure 3.2.6: DNA segregation occurs in cells overexpressing Mia1p when the NE is fragmented. 95-97 Figure 3.3.1: Symmetrical cell tip localization of the cell end marker requires Mia1p. 99 Figure 3.3.2: Orientation of microtubules is altered in mia1Δ cells. Sup-Figure 4.5.3: Localization of Mto1p-GFP in alp14Δ cells. 102-104 118 xiv ABBREVIATIONS TACC the transforming acidic coiled-coil related protein MTOC microtubule organizing center iMTOC interphase microtubule organizing center eMTOC equatorial microtubule organizing center PAA post anaphase array NE: nuclear envelope MT microtubule SPB spindle pole body γ-TuRC γ-tubulin ring complex Mia1 microtubule associated protein DAPI 4’6,-diamidino-2-phenylindole MBC methyl benzimidazol-2-yl-carbamate DNA deoxyribonucleic acid GFP green fluorescent protein GTP guanosine triphosphate GDP guanosine diphosphate PCR polymerase chain reaction ts: temperature sensitive EMM Edinburgh minimal medium xv SUMMARY Microtubule arrays of distinct geometries are important for cell function. Microtubule-organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner. It is essential to understand molecular mechanisms underlying MTOC emergence and maintenance. In this thesis, I use a combination of cell imaging, cell manipulation and genetics experiments in the fission yeast Schizosaccharomyces pombe, to show that the Transforming Acidic Coiled Coil protein, Mia1p, functions in sustaining proper MTOC and microtubule dynamics, both in interphase and mitosis. Briefly, I show that Mia1p is required for microtubule attachment to the nucleation sites. Furthermore, when overexpressed, Mia1p organized ectopic spindle pole like structures that interfered with normal mitotic spindle assembly. These studies led us to identify a role for the spindle pole bodies in segregation of the nuclear envelope during closed mitosis. Key words: S. pombe, microtubule, MTOC, iMTOCs, eMTOC, Mia1p, TACC xvi Publications First author publications: Liling Zheng , Cindi Schwartz , Liangmeng Wee , and Snezhana Oliferenko (2006). The Fission Yeast Transforming Acidic Coiled Coil–related Protein Mia1p/Alp7p Is Required for Formation and Maintenance of Persistent Microtubule-organizing Centers at the Nuclear Envelope. Mol Bio Cell 17(5): 2212-2222. Liling Zheng , Cindi Schwartz , Valentin Magidson, Alexey Khodjakov, Snezhana Oliferenko (2007). The Spindle Pole Bodies Facilitate Nuclear Envelope Division during Closed Mitosis in Fission Yeast. PloS Biology 5(7):1530-1542 xvii [...]...attachment to the NE 11 0 4.5.3 Lack of Mia1p and Alp14p result in failure of iMTOCs establishment 11 0 4.6 De novo assembly of the MTOCs is not specific to S pombe cells 11 1 4.7 Role of Mia1p in modulating mitotic microtubule dynamics 11 2 4.8 Conclusion and future perspectives 11 3 4.8 .1 Conclusion 11 3 4.8.2 Future perspectives 11 4 4.8.2 .1 Microtubules are nucleated by the iMTOCs and the SPB: are they... similar? 11 4 4.8.2.2 Distribution of the iMTOCs at the NE: random or specific? 11 5 4.8.2.3 What’re the partners of Mia1p in terms of anchoring microtubule to the NE? 11 5 References 11 9 xi LIST OF FIGURES Figures page Figure3 .1. 1 .1: Microtubule- associated protein Mia1p binds microtubules and is a component of S pombe MTOCs 45 Figure3 .1. 1.2: Cells lacking Mia1p exhibit disordered and heterogeneous microtubule. .. Figure3 .1. 4: Mia1p is required for maintenance of microtubule bundles 62 Figure3 .1. 5 .1: Mia1p- GFP is required for PAA organization 65 Figure3 .1. 5.2: Mia1p is not essential for the assembly of the eMTOC 67 xii Figure3 .1. 6: Components of the γ-tubulin complex are not found as prominent structures around the NE 69 Figure3 .1. 7 .1: A model of the iMTOC formation in S pombe cells 71 Figure3 .1. 7.2: Microtubules... altered in mia1Δ cells Sup-Figure 4.5.3: Localization of Mto1p-GFP in alp14Δ cells 10 2 -10 4 11 8 xiv ABBREVIATIONS TACC the transforming acidic coiled-coil related protein MTOC microtubule organizing center iMTOC interphase microtubule organizing center eMTOC equatorial microtubule organizing center PAA post anaphase array NE: nuclear envelope MT microtubule SPB spindle pole body γ-TuRC γ-tubulin ring complex... Figure3 .1. 2 .1: Mia1p is required for attachment of microtubules to the SPBs and the NE 49-50 Figure3 .1. 2.2: Although SPBs remain stationary in alp14Δ cells, microtubules are attached to the SPBs 52 Figure3 .1. 3 .1: Microtubule could be nucleated on the preexisting microtubule in mia1Δ cells 54-55 Figure3 .1. 3.2: Microtubule could be nucleated form several sites around the NE in mia1Δ cells 58-60 Figure3 .1. 4:... Microtubule- organizing centers (MTOCs) concentrate microtubule nucleation, attachment and bundling factors and thus restrict formation of microtubule arrays in spatial and temporal manner It is essential to understand molecular mechanisms underlying MTOC emergence and maintenance In this thesis, I use a combination of cell imaging, cell manipulation and genetics experiments in the fission yeast Schizosaccharomyces pombe, ... pombe, to show that the Transforming Acidic Coiled Coil protein, Mia1p, functions in sustaining proper MTOC and microtubule dynamics, both in interphase and mitosis Briefly, I show that Mia1p is required for microtubule attachment to the nucleation sites Furthermore, when overexpressed, Mia1p organized ectopic spindle pole like structures that interfered with normal mitotic spindle assembly These studies... the spindle pole bodies in segregation of the nuclear envelope during closed mitosis Key words: S pombe, microtubule, MTOC, iMTOCs, eMTOC, Mia1p, TACC xvi Publications First author publications: Liling Zheng , Cindi Schwartz , Liangmeng Wee , and Snezhana Oliferenko (2006) The Fission Yeast Transforming Acidic Coiled Coil related Protein Mia1p/ Alp7p Is Required for Formation and Maintenance of Persistent... Spindle structures in Mia1p- overexpressing cells are bipolar and anti-parallel 87-88 Figure 3.2.5: The majority of Mia1p- overexpressing cells exhibit Mad2p-GFP on the NE 92 xiii Sup-Figure 3.2.6: DNA segregation occurs in cells overexpressing Mia1p when the NE is fragmented 95-97 Figure 3.3 .1: Symmetrical cell tip localization of the cell end marker requires Mia1p 99 Figure 3.3.2: Orientation of microtubules... emergence of iMTOC structures upon entery into the new cell cycle 74 Figure3.2 .1. 1: Fission yeast cells over-expressing Mia1p arrest in mitosis with fully extended mitotic spindles 77 Figure3.2 .1. 2: Cells overexpressing Mia1p exhibit fully elongated mitotic spindle with non-segregated chromosomes 79 Figur3.2.2: Cells overexpressing Mia1p causes the SPBs to be displaced from the mitotic spindle poles and . 3 .1 Mia1p in assembly and maintenance of persisent iMTOCs at nuclear envelope 44 3 .1. 1 Lack of microtubule binding protein Mia1p results in fewer interphase Microtubules 44 3 .1. 1 .1 Mia1p. Components of MTOCs 10 1. 2.2 .1 The γ-TuRC 10 1. 2.2.2 Mto1p and Mto2p 12 1. 2.3 Organizing microtubule bundles 13 1. 2.3 .1 Minus-end-directed motor protein—Klp2p 14 1. 2.3.2 Bundling protein—Ase1p. NE 11 0 4.5.3 Lack of Mia1p and Alp14p result in failure of iMTOCs establishment 11 0 4.6 De novo assembly of the MTOCs is not specific to S. pombe cells 11 1 4.7 Role of Mia1p in modulating