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Cytoskeleton-Associated Protein 2 Is Required for the Maintenance of Chromosomal Stability by Tethering Spindle Microtubules to Their Poles

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Cytoskeleton-Associated Protein 2 Is Required for the Maintenance of Chromosomal Stability by Tethering Spindle Microtubules to Their Poles Cytoskeleton-Associated Protein 2 is Required for the Maintenance of Chromosomal Stability by Tethering Spindle Microtubules to their Poles by Chanelle M. Case B.S. in Biology, May 2006, Villanova University A Dissertation submitted to The Faculty of The Columbian College of Arts and Sciences of The George Washington Unviersity in partial fulfillment of the requirements for the degree of Doctor of Philosophy January 31, 2013 Dissertation directed by Thomas Ried Chief, Section of Cancer Genomics, National Cancer Institute, National Institutes of Health Norman Lee Professor of Pharmacology and Physiology The Columbian College of Arts and Sciences of The George Washington University certifies that Chanelle M. Case has passed the Final Examination for the degree of Doctor of Philosophy as of January 31, 2013. This is the final and approved form of the dissertation. Cytoskeleton-Associated Protein 2 is Required for the Maintenance of Chromosomal Stability by Tethering Spindle Microtubules to their Poles Chanelle M. Case Dissertation Research Committee: Thomas Ried, Chief of the Section for Cancer Genomics, National Cancer Insitute, National Institutes of Health, Dissertation Co- Director Norman Lee, Professor of Pharmacology and Physiology, Dissertation Co-Director Dan Sackett, Staff Scientist, Section on Cell Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Committee Member Susan Ceryak, Associate Research Professor of Pharmacology and Physiology, Committee Member ii © Copyright 2013 by Chanelle M. Case All rights reserved iii Dedication I dedicate my dissertation to my parents and grandparents, for their love, prayers and support. It is on their shoulders I stand, and I would not have completed this work without their unwavering support of my passion for science. I also dedicate this project to my best friend and wonderful husband, John Borden, for encouraging me throughout my academic career to be the best scientist I can be. And although my grandfather, John E. Couch, Jr. did not live to see me earn my doctorate, I thank him for always believing me, and I know he is truly proud of his granddaughter. iv Acknowledgements I would like to thank Dr. Janice Knepper and Dr. Mary Desmond for nurturing my love for science, and shepherding me through my first research experience at Villanova University. Equally important to my development were Dr. Carl June and Dr. Jim Riley, and the entire June lab, who gave me the opportunity to continue developing my skills as a Research Associate in their lab at the University of Pennsylvania. I would like to especially thank Dr. Angel Varela-Rohena and Dr. Samik Basu for being great mentors and friends. I owe a special debt of gratitude to Dr. Thomas Ried, and the Ried lab for opening their minds and hearts to me as I pursed my doctoral degree. In particular, I am grateful for the wisdom and mentorship of Dr. Jordi Camps, who helped me brainstorm, develop, edit and perfect my ideas. I would also like to acknowledge the current and former members of the Ried lab, especially Dr. Danny Wangsa, Dr. Dara Wangsa, Dr. Michael Difillippantonio and Dr. Kundan Sengupta, for their assistance and encouragement as I completed my dissertation. Dr. Dan Sackett deserves special acknowledgment for his advice, assistance and patience in answering the many questions I had throughout this project. I would like to thank my co-mentor, Dr. Normal Lee, for his encouragement, patience and guidance. I also would like to acknowledge my dissertation committee members and participants, Dr. Dan Sackett, Dr. Stan Lipkowitz, Dr. Susan Ceryak, Dr. Travis O’Brien, and Dr. Daniela Cimini. Their v insightful questions and comments were invaluable in rightly guiding my finished product. I am deeply appreciative of Dr. Anne Chiaramello for chairing my dissertation, and the other faculty and staff members that were instrumental in making this a remarkable and memorable experience, including Dr. Linda Werling, Marc Witliff, Amanda Page, and the members of the Department of Pharmacology and Physiology. I would also like to thank the OITE staff at the National Institutes of Health for their support assistance throughout this process, especially Dr. Sharon Milgram. vi Abstract of Dissertation Cytoskeleton-Associated Protein 2 is Required for the Maintenance of Chromosomal Stability by Tethering Spindle Microtubules to their Poles Errors in chromosome segregation lead to aneuploidy. Integrity of the microtubule spindle apparatus and intact cell division checkpoints are essential to ensure the fidelity of chromosome distribution into daughter cells. Cytoskeleton- associated protein 2, CKAP2, is a microtubule-associated protein that colocalizes with spindle poles and aids in microtubule stabilization, but the exact function and mechanism of its action are poorly understood. In the present study, RNA interference was utilized to determine the extent to which the expression of CKAP2 plays a role in chromosome segregation in colorectal cancer cells. CKAP2-depleted cells showed a significant increase of multi-polar mitoses and other spindle pole aberrations. Notably, when interrogated for microtubule nucleation capacity, CKAP2-depleted cells showed a very unusual phenotype as early as two minutes after release from mitotic block, consisting of dispersal of newly polymerized microtubule filaments through the entire chromatin region. Nevertheless, spindle poles were formed after one hour of mitotic release, suggesting that centrosome-mediated nucleation remained dominant. Kinetochore-driven microtubule nucleation was not implicated, as there was no colocalization of nascent microtubule filaments with Hec1. Interestingly, there was no effect on the localization of nuclear mitotic apparatus (NuMA) protein in CKAP2-depleted cells. Finally, we showed that suppression of CKAP2 results in a higher incidence of merotelic attachments, anaphase lagging, and vii chromosomal instability. In conclusion, CKAP2 is involved in tethering the microtubule-minus ends to the spindle pole in early mitosis. Delays in this process may alter the mitotic spindle tension, ultimately promoting merotelic kinetochore- microtubule attachments that result in chromosome lagging and increased chromosomal instability. viii Table of Contents Dedication………………………………………………………………………............iv Acknowledgements……………………………………………………………………..v Abstract of Dissertation………………………………………………………………...vi List of Figures…………………………………………………………………………....x List of Tables…………………………………………………………………………….xi List of Abbreviations……………………………………………………………………xii Chapter 1: Introduction…………………………………………………………………1 Section I. Components of the Mitotic Spindle……..……….……………...2 Section II. Importance of Microtubule Dynamics in Chromosome Segregation…………............................................................4 Section III. MAPs Influence Spindle Pole Integrity and Chromosome Missegregation…………………………………………...……..6 Section IV. Microtubule-Associated Protein, CKAP2, May Influence Microtubule Dynamics and Maintenance of the Genome.....7 Section V. Dissertation goals………………………………………………..12 Chapter 2: Materials and Methods Chapter 3: Results Section I. CKAP2 Expression and Localization in Wild-Type DLD1…...24 Section II. Establishment of shCKAP2 Model……………………………..28 Section III. Evaluation of CKAP2 Function……...………………………….38 Section IV. Differential Expression of CKAP2 in Human and Mouse Cancer Cell Lines…………………………………………….. 65 Chapter 4: Discussion Section I. Depletion of CKAP2 Increases Spindle Pole Defects…..……73 Section II. CKAP2 Plays a Role in Tethering the Centrosome to the Spindle Pole……………………………………………………75 Section III. Loss of Spindle Pole Integrity Results in Chromosome Missegregation………………………………………………...78 Section IV. CKAP2 in the context of cancer………………………………..81 ix References……………………………………………………………………………..84 x List of Figures Figure 1……....Diagram of pGIPZ-shCKAP2 plasmid……………………………. 14 Figure 2.……...Schema of Apoptosis Assays……………………………………... 17 Figure 3………CKAP2 Expression is Restricted to Mitosis……………………… 26 Figure 4………CKAP2 Localizes to and is Associated with the Mitotic Spindle. 27 Figure 5.……...CKAP2 is Not Essential for Cell Viability………………………… 30 Figure 6.……...Generation of Stable shCKAP2 Cell Lines .……………………. 31 Figure 7.……...Validation of CKAP2 Knock-down………………………………... 32 Figure 8………Stable CKAP2 Depletion does not Influence Cell Proliferation or Viability…………………………………………….. 33 Figure 9.……...Depletion of CKAP2 Expression does not Affect Cell Cycle Distribution………………………………………………………….. 36 Figure 10.…….CKAP2 Depletion Results in a Decrease in the Length of Mitosis……………………………………………………………….. 37 Figure 11.…….Reduction of CKAP2 Expression Results in an Increase in Multipolar Spindles………………………………………………… 41 Figure 12.…….CKAP2 Depletion Results in a Dispersal of γ-tubuling Away from the Centrosome……………………………………………… 42 Figure 13……..CKAP2 Depletion Results in an Increase in Centrosome Dislocation………………………………………………………….. 43 Figure 14……..Spindle Pole Defects Result in an Increase in Spindle Tension And Chromosome Misalignment in Metaphase………………… 44 Figure 15……..CKAP2 is Required for the Anchoring of Centrosome- Nucleated Microtubules to the Spindle Pole……………………. 47 Figure 16…….. CKAP2 is Required for the Anchoring of Centrosome- Nucleated Microtubules to the Spindle Pole…………………… 48 Figure 17……..Depletion of CKAP2 Causes a Delay in Spindle Formation…... 49 Figure 18……..CKAP2-Depleted Cells are Capable of Bipolar Spindle Formation…………………………………………………………… 50
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