University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2011-05-24 The Importance of the Centrosomal Localization Sequence of Cyclin E for Promoting Centrosome Duplication: A Dissertation Joshua J. Nordberg University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cell Biology Commons, Cells Commons, and the Enzymes and Coenzymes Commons Repository Citation Nordberg JJ. (2011). The Importance of the Centrosomal Localization Sequence of Cyclin E for Promoting Centrosome Duplication: A Dissertation. GSBS Dissertations and Theses. https://doi.org/ 10.13028/et69-4039. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/547 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. THE IMPORTANCE OF THE CENTROSOMAL LOCALIZATION SEQUENCE OF CYCLIN E FOR PROMOTING CENTROSOME DUPLICATION A Dissertation Presented By JOSHUA JOHN NORDBERG Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY MAY 24, 2011 CELL BIOLOGY THE IMPORTANCE OF THE CENTROSOMAL LOCALIZATION SEQUENCE OF CYCLIN E FOR PROMOTING CENTROSOME DUPLICATION A Dissertation Presented By JOSHUA JOHN NORDBERG The signatures of the Dissertation Defense Committee signifies completion and approval as to style and content of the Dissertation _________________________________________ Greenfield Sluder, Ph.D., Thesis Advisor _________________________________________ Kirsten Hagstrom Ph.D., Member of Committee _________________________________________ Edward Hinchcliffe Ph.D., Member of Committee _________________________________________ Stephen Jones Ph.D., Member of Committee _________________________________________ William Theurkauf Ph.D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee _________________________________________ Dannel McCollum Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the school. _________________________________________ Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences Interdisciplinary Graduate Program May 24, 2011 iv I dedicate this work to my loving and supportive wife, Lauren Copp Nordberg, without whom I could not have found the strength and commitment to complete this endeavor. Thank you. v Acknowledgements It could be said that nothing great comes without great sacrifice. Nothing could be truer regarding the completion of this Ph.D. program. However, the experiences I have had and the knowledge I have gained were worth every bit of pain they caused. Some of the pain and most of the knowledge gained can be attributed to my advisor and mentor, Dr. Greenfield (Kip) Sluder. I had the pleasure of working under Kip’s tutelage for 10 years. In that time I learned to never fear a piece of equipment, to always look at the big picture, and above all else, to enjoy working in the lab. Kip has an unmatched sense of biological significance, and an uncanny ability to make the impossible seem worthy of the challenge. There was never an experiment too crazy to try. I would like to extend my thanks also to my advisory committee: Drs. Dannel McCollum, William Theurkauf, Kirsten Hagstrom and Stephen Jones. Thank you for your guidance over the years and your help in shaping my project appropriately to keep me on track. I also thank Dr. Edward (Ted) Hinchcliffe for helpful discussions and comments and for serving as an outside member of my committee. I would also like to take this opportunity to extend my most sincere thanks to the people who work on the third floor of the Biotech Four building. The researchers, the students, and the support staff have been colleagues of unparalleled excellence. James Carey became a great friend as well as a frequent teacher of molecular biology. Beth Luna was a veritable fountain of vi knowledge of all things biochemical. In addition to the community of researchers, Denise Maclachlan, Cathy Warren and Donna Castellanos have made immeasurable contributions to this work through their kindness and support on all manner of day-to-day matters. All the members of the Sluder Lab, past and present, also left their indelible mark on my scientific career. Rick Miller and Ted Hinchcliffe were the first people I met in the lab, and they gave me the foundation for everything I know about microscopy. Anna Krzywicka-Racka and Yumi Uetake have been wonderful mentors as senior post-docs in the lab. Christopher English allowed me to practice my teaching on him, and he seemed to like it. And although Stephen Douthwright and Toshi Hatano were recent additions to the lab, and as such I did not get to know them as well as I would have liked, they were a pleasure to work with as I was completing my research. And last but not least, I would like to formally thank my family. My wife Lauren, our son Elliot, and our daughter Claire all provided me with a wonderful reason to go home from lab every night. My dad has always been an excellent listener when things weren’t going the way I wanted them to. And finally, my mother has been an amazing source of encouragement and support throughout my entire time in graduate school…and life. My deepest and most sincere thanks to you all. vii Abstract This thesis comprises three separate studies that investigate the consequences of supernumary centrosomes, the effect of centrosome loss, and a control mechanism for regulating CDK2/cyclin E activity in centrosome duplication. The centrosome is the major microtubule-organizing center of the cell. When the cell enters mitosis, it is of critical importance that the cell has exactly two centrosomes in order to properly segregate the chromosomes to two daughter cells. Supernumary centrosomes are a problem for the cell in that they increase the incidence of chromosomal instability. Aberrant centrosome numbers are seen in a number of cancers, and there has been a proposed connection between the loss of function of p53 and multiple centrosomes. We investigated the consequences of multiple centrosomes in p53-null mouse embryonic fibroblasts (MEFs) to determine how cells with multiple centrosomes can continue to propagate and become cancer. We found that even in the face of extra centrosomes, p53-null MEFs are able to divide in a bipolar fashion by bundling extra centrosomes into two spindle poles. The centrosome has also been proposed to play a role in cell cycle control. We followed up on a previous study, which had suggested that centrosome loss causes a G1 arrest. We found that cells did not arrest in G1 due to centrosome removal as previously reported, but instead the arrest was viii dependent on additional stressors, namely the incident light used for our long- term live-cell observations. Our study showed that centrosome loss is a detectable stress that, in conjunction with additional stresses, can contribute to cell cycle arrest. It is known that CDK2/cyclin E activity is required to promote centrosome duplication. But with the discovery of a centrosomal localization sequence (CLS) in cyclin E, we wanted to know if centrosome duplication required a specific sub- cellular localization of CDK2 kinase activity. We found that centrosome duplication in Xenopus extract was dependent on CLS-mediated centrosomal localization of cyclin E, in complex with CDK2. Our results point to a mechanism for regulating centrosome duplication in the face of high cytoplasmic CDK2/cyclin E kinase activity. ix Table of Contents Approval Page iii Dedication iv Acknowledgements v Abstract vii Table of Contents ix List of Tables xi List of Figures xii List of Abbreviations xiv Preface xv Chapter I: 1 General Introduction Chapter II: 14 Practical Aspects of Adjusting Digital Cameras Introduction 15 Measuring Gray-Level Information 15 Camera Settings 19 Contrast Stretching 24 Camera Versus Image Display Controls 31 Chapter III: 33 The Good, The Bad, and The Ugly: Practical Consequences of Centrosome Amplification Abstract 34 Introduction 35 Results/Discussion 37 Conclusions 48 Materials and Methods 50 Chapter IV: 52 The Importance of the Centrosomal Localization Sequence of Cyclin E for Promoting Centrosome Duplication in Xenopus Extract Abstract 53 Introduction 54 Results 57 Discussion 74 Future Directions 80 Materials and Methods 86 x Chapter V: 93 General Discussion Appendix A: 104 Centrosome loss is a stress that, when combined with other stresses, causes cell cycle arrest. Abstract 105 Introduction 106 Results 108 Discussion 113 Materials and Methods 115 Appendix B: 117 Detailed Protocol for Observing Centrosome Duplication In a Cell-Free Extract Made From Xenopus laevis Oocytes Making of Xenopus Egg Extract 118 Quantitation of Centrosome Duplication in Xenopus extracts 123 References 125 xi List of Tables CHAPTER IV Tables Table 4.1 Comparison of average number of centrosome 66 doublings in a 6-hour period Table 4.2 Comparison of Average Centrosome Duplication 70 Times xii List of Figures Chapter I Figures Figure 1.1 Structure