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The Kinetics of G2 and M Transitions Regulated by B Cyclins

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The Kinetics of G2 and M Transitions Regulated by B Cyclins THE KINETICS OF G2 AND M TRANSITIONS REGULATED BY B CYCLINS by YEHONG HUANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. James W. Jacobberger Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY January 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Yehong Huang, Candidate for the Doctor of Philosophy degree*. Dr. James W Jacobberger, Ph.D. Thesis Advisor and Committee Member Dr. Jonathan Karn, Ph.D. Committee Chair Dr. Mark W Jackson, Ph.D. Committee Member Dr. You-Wei, Ph.D. Committee Member Date of Defense: November 7th, 2013 *We also certify that written approval has been obtained for any proprietary material contained therein. 2 TABLE OF CONTENTS LIST OF TABLES 3 LIST OF FIGURES 9 LIST OF TABLES 12 LIST OF ABBREVIATIONS 13 ACKNOWLEGEMENTS 16 ABSTRACT 18 CHAPTER1: INTRODUCTION & BACKGROUND 20 1.1 INTRODUCTION TO CELL CYCLE 20 1.1.1 Concept of cell cycle 20 1.1.2 Events of cell cycle 21 1.1.3 The duration of cell cycle 22 1.1.4 Role of cell cycle in tumor formation 22 1.2 CONTROL OF CELL CYCLE 23 1.2.1 The cell-cycle control system 23 1.2.2 Cell cycle Checkpoints 24 1.2.3 Key regulators of cell cycle 25 1.2.4 Two alternative models of regulation of cell cycle by Cdk 28 1.3 REGULATION OF MITOSIS 29 1.3.1 History of discovery of protein kinase complex regulating mitosis 29 1.3.2 Components of MPF 30 1.3.2.1 P34 Cdc2 (Cdk1) 30 3 1.3.2.2 Mitotic cyclins 32 1.3.2.3 B- type cyclins 34 1.3.3 Regulation of M phase entry and exit 38 1.3.3.1 Regulation of M phase entry 38 1.3.3.2 Nuclear translocalization of cyclin B1-Cdk1 of mitotic entry 41 1.3.3.3 Regulation of M phase exit 41 1.3.3.4 Mitotic Checkpoints 43 1.4 STAGES OF MITOSIS 44 1.4.1 Morphologic stages of mitosis 44 1.4.2 Re-staging mitosis 47 1.5 FUNCTIONS OF CYCLIN B 50 1.5.1 Function of cyclin B-Cdk1 during mitosis 50 1.5.2 Rate-limiting function of cyclin B 51 1.5.3 Functional redundancy of B cyclins 52 1.6 HYPOTHESIS AND SPECIFIC AIMS OF THESIS WORK 53 CHAPTER 2: STABLE CELL LINES THAT EXPRESS CYCLIN B1- OR B2-EGFP FUSION PROTEINS 56 2.1 ABSTRACT 56 2.2 INTRODUCTION 57 4 2.3 MATERIALS AND METHODS 59 2.3.1 Plasmids and construction of Tet-Off response plasmids 59 2.3.2 Oligonucleotides of siRNA specific to cyclin B1, B2 and A2, and construction of siRNA-resistant constructs 63 2.3.3 Cell culture, transfection and generation of stable cell lines 64 2.3.4 Cell fixation, intracellular staining 64 2.3.5 Fluorescence activated cell sorting and flow cytometry 65 2.3.6 Immunoblotting analysis 66 2.3.7 Microscopy imaging 66 2.4 RESULTS 67 2.4.1 Construction of Tet-Off response plamsids containing human cyclin B1-, B2- or A2 –EGFP gene that is resistant to siRNA and specific for cyclin B1, B2 or A2 67 2.4.2 Establishment stable cell lines 68 2.4.3 Expression of cyclin B1-EGFP and cyclin B2-EGFP is reduced and tightly regulated under the control of doxycycline 69 2.4.4 Effect of fixation on the intensity of B cyclin EGFP fluorescence 70 2.4.5 Induction of cyclin B1- and B2-EGFP is rapid and regulated by doxycycline in a dose dependent manner 71 2.4.6 Cell line resistance of ectopic cyclin B1-EGFP and cyclin B2-EGFP with synonymous base changes to siRNA 72 2.4.7 Ectopic B cyclin -EGFPs activate Cdk1-mediated Bcl-2 phosphorylation 73 5 2.5 DISCUSSION 74 CHAPTER 3: THE KINETICS OF G2 AND M TRANSITIONS REGULATED BY B CYCLINS 77 3.1 ABSTRACT 77 3.2 INTRODUCTION 78 3.3 MATERIALS AND METHODS 81 3.3.1 List of stable cell lines used for experiments 81 3.3.2 Cell culture and transfection 81 3.3.3 Cell fixation, intracellular staining and flow cytometry 82 3.3.4 BrdU labeling, G2 and M phase transit time analysis 83 3.3.5 Time lapse experiments 83 3.3.6 Immunoblotting 84 3.3.7 Software 84 3.4 RESULTS 85 3.4.1 Characterization of stable cell lines in this study 85 3.4.2 Cyclin B2 is rate controlling for G2 and M transitions 85 3.4.3 Over-expression of B cyclins does not affect the rates of G2 and M transition 87 3.4.4 Over-expression of single B cyclins can restore cell cycling when endogenous B cyclins are depleted 88 3.4.5 Expression of single B cyclins can completely rescue the G2 arrest phenotype caused by cyclin B1 and B2 co-depletion 88 6 3.5 DISCUSSION 91 CHAPTER 4: LOCALIZATION OF CYCLIN B1- OR B2-EGFP WHEN LACK OF CYCLIN B2 OR B1 101 4.1 ABSTRACT 102 4.2 INTRODUCTION 105 4.3 MATERIALS AND METHODS 105 4.3.1 Cell culture 105 4.3.2 Plasmids, small interfering RNA (siRNA) constructs and transfection 105 4.3.3 CellLight Golgi-RFP living cell transduction 106 4.3.4 Cell fixation and intracellular staining 107 4.3.5 Time lapse microscopy 108 4.3.6 Confocal microscopy 108 4.3.7 Laser scanning cytometry 108 4.3.8 Immunoblotting analysis 109 4.4 RESULTS 109 4.4.1 Cytoplasmic cyclin B1-EGFP concentrates on the centrosomes during interphase and translocates to the nucleus at beginning of mitosis 109 4.4.2 Cyclin B2-EGFP localizes to Golgi apparatus and centrosomes in interphase and translocalizes to the nucleus at the beginning of mitosis 112 7 4.4.3 Study the localization of cyclin B1 to the Golgi when cyclin B2 knockdown 115 4.5 DISCUSSION 117 CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS 121 5.1. SUMMARY 121 5.2. FUTURE DIRECTIONS 127 5.2.1 To study the effect of single cyclin B1 or B2 on Golgi disassembly 127 5.2.2 To explore the residues that regulate human cyclin B2 nuclear localization 127 5.2.3 To identify distinct sequences in cyclin B2 that bind to the chromosome, centrosomes, and spindle during mitosis 128 5.2.4 To determine when and where cyclin B2-Cdk1 is first activated 129 5.2.5 To study the role of B cyclin –Cdk1 in regulation of chromosome segregation 129 5.2.6 To explore the co-operation and redundancies of cyclin A2 and B cyclins on G2 and M transition 130 8 LIST OF FIGURES Figure 1-1: The phase of cell cycle and the checkpoint of cell cycle 132 Figure 1-2: Two models for the Cdk control of cell cycle 133 Figure 1-3: positive feedback loop that regulates cyclinB-Cdk1 activity 134 Figure 2-1: Schematic of gene regulation in the Tet-Off Systems 135 Figure 2-2: Schematic representation of Tet-response constructs containing EGFP, cyclinB1-EGFP and cyclin B2-EGFP genes 136 Figure 2-3: Transient expression of the tet-response constructs containing cyclin B1-or B2- and EGFP genes in HeLa Tet-Off cells in the presence of siRNA 137 Figure 2-4: Detection of doxycycline regulated cyclinB1- or B2-EGFP expression in stable cells under immunofluorescence microscopy 138 Figure2-5: Induction of cyclin B-EGFP in selected HeLa Tet-Off clones in the absence of Doxycycline 139 Figure2-6: Effect of fixation and permeabilization on intensity of cyclin B-EGFP expression in the stable cell lines 140 Figure 3-1: Expression of cyclin B1- and cyclin B2-EGFP is reduced and tightly regulated by Dox 141 Figure 3-2: Time and concentration dependence of doxycycline action on a Tet-Off stable cell line 142 Figure 3-3: Effect of doxycycline and siRNA on the ectopic cyclin B-EGFP expressed by stable cells 143 Figure 3-4: Bcl2 is phosphorylated as a function of cyclin B1- and 9 B2-EGFP expression 144 Figure 3-5. G2 and M phase transitions after cyclin B2 knockdown 145 Figure 3- 6: Over-expression of B cyclins does not alter G2 and M phase transition times 146 Figure 3-7: B cyclin-EGFP expression rescues the G2 arrest phenotype of B cyclin co-depletion 147 Figure 3-8: B cyclin co-depletion induces G2 arrest 148 Figure 3-9: Single B cyclin-EGFP can rescue the G2 arrest of endogenous cyclin B1 and B2 co-depletion 149 Figure 3-10: Reanalysis of the primary data of the experiments presented in Figure 9 150 Figure 3-11: Single B cyclin EGFP completely rescues the G2 arrest of cyclin co-depletion as a function of dose 151 Figure3-12: Two possible models of cyclin B1 and B2 co-operation to regulate G2 and M transition and progression 152 Figure 4-1: localization of ectopic cyclinB1-EGFP expressed in p015 through the cell cycle 153 Figure 4-2: Titrations to determine optional concentration of doxycycline to obtain ectopic cyclinB1/B2-EGFP level equivalent to endogenous cyclin B1/B2 in p015 and p017 stable cells 154 Figure 4-3: Localization of ectopic cyclin B1-EGFP when expressed in a level equivalent the endogenous cyclin B1 in the absence of endogenous cyclin B1 155 10 Figure 4-4: Localization of transient transfected EGFP protein in HeLa Tet-Off cells during cell cycle 156 Figure 4-5: Localization of ectopic cyclin B2-EGFP to the Golgi determined by confocal microscopy 157 Figure 4-6: Localization of ectopic cyclinB2-EGFP expressed in p017 through the cell cycle 158 Figure 4-7: Localization of wild type of cyclin B2 to the tubulin (centrosomes) and chromosome determined by indirect immunofluorescence 159 Figure 4-8: Localization of wild type cyclin B1 to the Golgi determined by indirect immunofluorescence 160 Figure 4-9: Determination of the intensity of cyclin on the Golgi and in the whole cell by laser scanning cytometry 161 11 LIST OF TABLES Table 3-1.
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