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Nup2 and a Newly Discovered Nuclear Pore Complex Protein, Nupa, Function at Mitotic Chromatin Controlled by the Nima Kinase

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Nup2 and a Newly Discovered Nuclear Pore Complex Protein, Nupa, Function at Mitotic Chromatin Controlled by the Nima Kinase NUP2 AND A NEWLY DISCOVERED NUCLEAR PORE COMPLEX PROTEIN, NUPA, FUNCTION AT MITOTIC CHROMATIN CONTROLLED BY THE NIMA KINASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sarine W. Markossian Graduate Program in Molecular Genetics The Ohio State University 2011 Dissertation Committee: Stephen A. Osmani, Ph.D., Advisor Anita K. Hopper, Ph.D. Iris Meier, Ph.D. Harold A. Fisk, Ph.D. Copyright by Sarine W. Markossian 2011 ABSTRACT In Aspergillus nidulans the mitotic NIMA kinase is required for the transition from G2 into mitosis. How NIMA regulates this process is not well understood. The nucleoporin Nup2 is of interest because it is essential and has a unique and conserved ability to transfer from nuclear pore complexes (NPCs) to chromatin specifically at mitosis. This unique transition suggests that Nup2 might play mitotic roles downstream of NIMA conserved from fungi to humans. We have determined that NIMA promotes Nup2 phosphorylation and relocation from NPCs onto chromatin. Affinity purifications followed by Mass spectroscopy identified a novel nucleoporin, NupA, which copurified with Nup2. NupA transfers from NPCs to mitotic chromatin and is also essential. Deletion analysis and heterokaryon rescue revealed that deletion of either does not cause major defects in nuclear transport but causes mitotic abnormalities that engage the spindle assembly checkpoint (SAC). We have also defined the essential function of NupA which is to tether Nup2 to both the NPC and mitotic chromatin where we propose Nup2 has a role in promoting efficient generation and function of the mitotic apparatus. In addition, Nup2 and NupA have dynamic locations within mitotic chromatin during different phases of mitosis. They are associated throughout chromatin at early prophase but move to locate at the periphery of the chromatin during the transition from anaphase to telophase. This movement at anaphase is likely functionally significant as deletion of either Nup2 or NupA causes defects in mitotic exit into G1. Hence, Nup2 and NupA may have dual roles in mitosis, the first being during early mitosis, defects in which activates the SAC, and the latter in mitotic exit and early G1. Because Nup2 co- purifies with ii importins α and , we propose that both roles might be linked to proper localizations and functions of importins α and during metaphase and mitotic exit. These studies collectively demonstrate that Nup2 and the newly discovered NupA have important functions during mitosis that are controlled by NIMA. iii To my mother, Iskouhi Shamikian, for providing me the opportunity of higher education and teaching me to believe that everything is possible through hard work. iv ACKNOWLEDGEMENTS I am deeply indebted to my advisor Dr. Stephen A. Osmani for his continual guidance and constant support throughout the years of my graduate studies. I thoroughly enjoyed his mentorship and his friendship. I would also like to thank my current and previous committee members, Dr. Anita K. Hopper, Dr. Iris Meier, Dr. Harold A. Fisk, and Dr. Berl R. Oakley for their valuable inputs into my work and all the encouragement during my graduate studies. In addition, I would like to thank all current and former members of the Osmani lab for their friendship, the assistance provided with experiments, the rewarding discussions about my work, and the continuous and critical feedback they have provided throughout the years. I would like to particularly thank Aysha H. Osmani, Subbulakshmi Suresh and Jessica S. El-Hallal because this work would not have been possible without their fruitful collaborations. I would also like to thank all the Molecular Genetics family, particularly my classmates for all the fun times, their friendship, and the extreme encouragement and support they provided throughout this experience. Finally, I would like to thank my parents, my sister, and all the rest of my extended family, including friends that I was lucky enough to encounter throughout the years in Lebanon and in Ohio. It is through their constant and unconditional love, faith, support and encouragement that this achievement was possible. v VITA 2001………………………………………B.S. Biology, American University of Beirut 2003………………………………………M.S. Biology, American University of Beirut 2004 to present …………………………..Graduate Research Associate, Department of Molecular Genetics, The Ohio State University PUBLICATIONS Liu HL, Osmani AH, Ukil L, Son S, Markossian S, Shen KF, Govindaraghavan M, Varadaraj A, Hashmi SB, De Souza CP, Osmani SA. 2010. Single step affinity purification for fungal proteomics. Eukaryot Cell 9:831-3. Kreydiyyeh S.I., Markossian S., Hodeify R.F. 2006. PGE2 exerts dose-dependent opposite effects on net water and chloride absorption from the rat colon. Prostaglandins and Other Lipid Mediators 79:43-52. vi Kreydiyyeh S.I., Markossian S. 2006. Tumor necrosis factor alpha down-regulates the Na+-K+ ATPase and the Na+-K+2Cl- cotransporter in the kidney cortex and medulla. Cytokine 33:138-44. Markossian S., Kreydiyyeh S.I. 2005. TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2. Cytokine 30:319-27. Markossian S.W. 2003. Effect of TNF-α on electrolyte transport across epithelia : possible mechanism of action. M.S. Thesis. Beirut: American University of Beirut. Kreydiyyeh S.I., Usta J., Knio K., Markossian S., Dagher S. 2003. Aniseed oil increases glucose absorption and reduces urine output in the rat. Life Sciences 74:663-73. FIELDS OF STUDY Major Field: Molecular Genetics vii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii ACKNOWLEDGEMENTS ................................................................................................ v VITA .................................................................................................................................. vi PUBLICATIONS ............................................................................................................... vi FIELDS OF STUDY......................................................................................................... vii TABLE OF CONTENTS ................................................................................................. viii LIST OF TABLES ........................................................................................................... xiv LIST OF FIGURES .......................................................................................................... xv LIST OF ABBREVIATIONS ........................................................................................ xviii CHAPTER 1 ....................................................................................................................... 1 INTRODUCTION .............................................................................................................. 1 1.1 Project Goal .................................................................................................................. 1 1.2 Aspergillus nidulans as a model system to study mitosis ............................................. 2 1.2.1 Biology ................................................................................................................... 2 1.2.2 Genome assembly and annotation .......................................................................... 4 1.2.3 Advantages ............................................................................................................. 5 1.3 The Nucleus .................................................................................................................. 6 1.3.1 Overview ................................................................................................................ 6 1.3.2 Differences in nuclear structure between lower and higher eukaryotes ................. 7 1.3.3 The nuclear envelope (NE) .................................................................................... 7 1.3.4 The Nuclear Pore Complex .................................................................................... 9 1.3.4.1 Overview ......................................................................................................... 9 1.3.4.2 Isolation of Nups ........................................................................................... 10 1.3.4.3 NPC biogenesis into an intact NE ................................................................. 10 1.3.5 Nucleoporin 2 (Nup2) .......................................................................................... 11 1.3.5.1 Protein structure ............................................................................................. 11 viii 1.3.5.2 Isolation and characterization ........................................................................ 12 1.3.6 Nucleo-cytoplasmic transport .............................................................................. 14 1.3.6.1 Overview ....................................................................................................... 14 1.3.6.2. Role of Nup2 in transport ............................................................................. 15 1.3.7 Nuclear chromatin ................................................................................................ 16 1.3.7.1 Overview ......................................................................................................
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