ANALYSIS OF THE EFFECTS OF LEPTOMYCIN B ON CELLS EXITING MITOSIS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Gin-Yun Liu, B.S. ******* The Ohio State University 2006 Dissertation Committee: Dale D. Vandré, Ph.D., Advisor Approved by John M. Robinson, Ph.D. Sissy Jhiang, Ph.D. Doug Kniss, Ph.D. ___________________________________ Advisor Graduate Program in Molecular, Cellular, and Developmental Biology ABSTRACT The mitosis to interphase transition in higher eukaryotic cells is characterized by reassembly of the nuclear envelope, decondensation of the chromosomes, and reformation of the nucleolus. Coincident with reassembly of the nuclear envelope, nucleocytoplasmic trafficking is also restored, which allows for reestablishment of the nuclear and cytoplamsic compartments. To determine whether restoration of nuclear export is critical for completion of the interphase transition and formation of the interphase nucleus, we examined the effect of leptomycin B (LMB) when applied to cells during exit from mitosis. LMB is a Streptomyces metabolite that inhibits chromosomal maintenance region 1 (CRM 1) mediated nuclear export. Cells treated with LMB during the mitosis to interphase transition underwent apoptosis within 8 hours, whereas cells treated with LMB 2 hours after release from the mitotic block remained viable. In addition, interphase cells treated with LMB also showed little or no induction of apoptosis. LMB treatment had no apparent effects on nuclear envelope reconstruction, chromosome decondensation, swelling of the nucleus, or establishment of cytoplasmic microtubule arrays. However, microscopic examination of cells prior to apoptosis revealed defective nucleolar morphology. Immunostaining of the nucleolar proteins B23, C23, and fibrillarin showed colocalization to the nucleolus in untreated cells, interphase cells treated with LMB, and cells treated with LMB 2 hours after release from mitosis. ii However, in cells treated with LMB during the mitosis to interphase transition, B23 appeared to be distributed throughout the nucleoplasm, while C23 and fibrillarin were observed to localize small to phase dense structures within the nucleus. These results indicate that LMB inhibits the nuclear export of a critical factor(s) from the newly reformed nucleus that inhibits nucleologenesis, which will be referred to as a nucleologenesis licensing factor. This licensing factor(s) may become trapped within the nucleus during nuclear envelope reassembly or imported into the nucleus during early stages of nuclear reformation. Furthermore, a comparison of other known nucleolar inhibitors showed the nucleolar disruption effects observed in LMB treated cells are specific and unique effects. Therefore, we conclude that reestablishment of CRM1- mediated nuclear export immediately after mitotic release is a critical mechanism required for nucleologenesis and cell survival. iii ACKNOWLEDGEMENTS This is a turning point in my life as I am about to graduate from OSU and obtain my Ph.D. degree. This has been a long journey that at various moments in my life, I did not think I would ever see the end of it. I continued to work hard despite the mistakes and problems that I had encountered with in many of my experiments. The many “bad” days made me want to cry and shout out “why is this happening?” or “why didn’t the PCR work?” However, there were always “good” days that made all the “bad” days seemed like a distant memory. This glimmer of hope had brought joy and happiness to my life, especially when the experiments worked, or the immunofluorescent pictures had very little background. It is amazing of how one small experiment can instantly change a person’s day from “bad” to “good”. The graduate school experience has taught me to be persistent when encountering a problem, and tackle challenges from all angles rather than just from one side. There are many people who have shared the triumph, the happiness, the sadness, the frustration, the anger, and all other emotions that I can not describe in one word throughout my graduate career. First of all, I would like to thank Dr. Vandre for allowing me to join his lab, to teach me, to guide me, and to help me mature into the scientist that I am today. Thank you! I could not have done this without your help, and I could not have asked for a more helpful advisor! iv Next, I would like to thank the present and past post docs in the lab, Hai Yang, Fred, Jeff, and Arun. Thank you so much for your support, your answers to the many questions that I had, your advice, and your detailed explanations on the techniques used. Your suggestions and advice has helped me tremendously, especially I encountered problems in my experiments. To Rodger, Selvi, and Pam, thank you so much for being there for me. Rodger and Pam, thank you for having lunch with me every week. I enjoy the many “silly” conversations that we had together. Selvi, you are a great workout partner. Thanks for being my gym partner so that I can release my stress. To my parents, Viv, Christy, Emily, Lyndsie, Ebony, Brittan, Anita, Jeff, Matt, and Chiang , you guys are the best supporters in the world. You worried about my safety when I have those crazy late night time point experiments at the lab, even though you knew that I would be safe. You cheered me up when I was frustrated about things and tell me that everything will be alright. You celebrated with me whenever I have good days at the lab. Words just cannot describe how much I appreciate each and every one of you. To Mike, my wonderful soon to be husband, you went above and beyond what a boyfriend/fiancé would do. You went with me when I had the late night time point experiments at the lab, even though you were tired. You listened to me complain about the crazy margins for this thesis. You held me when I cried about losing a portion of my unsaved dissertation. Best of all, you bought me a professional massage session to help release my stress. Your kindness, patience, supports, and love has helped me through this long path. Thank you so much for everything you have done for me. v VITA January 13, 1977…………………………………………………. Born in Taipei, Taiwan June, 1996………………………………………………………... Graduate from Hilliard High School June, 2000………………………………………………………... Bachelor of Science in Molecular Genetics from The Ohio State University 2000-present………………………………………………………Graduate Research Associate, The Ohio State University PUBLICATIONS Journal of Cell Science Submitted in July, 2006 Paper title: “Induction of Apoptosis in Cells After Treatment with Leptomycin B During the Mitosis to Interphase Transition is Preceded by a Failure of Nucleologenesis” Experimental Cell Research Submitted in July, 2006 Paper title: “The Effects of LMB on Nucleolar Structures are Distinct from Other Nucleolar Inhibitors” FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology vi TABLE OF CONTENTS Page Abstract…………………………………………………………………………… …..ii Acknowledgements……………………………………………………………….......iv Vita……………………………………………………………………….……….......vi List of Tables……………………………………………………………………... .....ix List of Diagrams………………………………………………………………….........x List of Figures……………………………………………………………...……... .....xi Introduction……………………………………………………………………….........1 Nuclear membrane………………………………………………………………...2 Nuclear Envelope……………………………………………………………..…...5 Nuclear Reassembly…………………………………………………………..…...7 Nuclear Transport……………………………………………………………. …...9 Transport Receptors…………………………………………………………..….11 Mechanisms of Nuclear Transport……………………………………………….16 Leptomycin B………………………………………………………………….....20 The Nucleolus………………………………………………………………... .....22 Types of Nucleolar Organization……………………………………………..….25 Ribosome Biogenesis…………………………………………………………….27 Nucleolar Proteins…………………………………………………………….….32 Fibrillarin…………………………………………………………….. ….33 C23……………………………………………………………………….35 B23……………………………………………………………………….38 Nucleologenesis……………………………………………………………… ….42 vii Chapters: 1. Inhibition of Nuclear Export During M to G1 Transition Induces Apoptosis Introduction…………………………………………………………………………..68 Materials and Methods…………………………………………………………….....72 Results………………………………………………………………………………..75 Discussion………………………………………………………………………….. ..80 Figures and Tables…………………………………………………………………. ..85 2. The Effects of LMB Are Specific for M to G1 Transition, Which Correlates to Nucleologenesis, a Specific Process to the M to G1 Transition Introduction…………………………………………………………………..........…97 Materials and Methods…………………………………………………………… ..102 Results…………………………………………………………………....................105 Discussion…………………………………………………………………………..113 Figures and Tables……………………………………………………….................118 3. The Effect of LMB on Nucleolar Structures are Distinct from Other Nucleolar Inhibitors Introduction…………………………………………………………………............144 Materials and Methods………………………………………………………...........151 Results……………………………………………………………………………....153 Discussion…………………………………………………………………………..162 Figures and Tables……………………………………………………….................168 4. Summary………………………………………………………………………. .254 References……………………………………………………………………......... .270 viii LIST OF TABLES Table Page 1 Examples of transport receptors of the importin β superfamily………………..….51 2 List of acronyms used in the thesis……………………………………………. ….52 ix LIST OF DIAGRAMS Diagram PAGE 1. Diagram of the nuclear pore complex………………………………………………..53 2. The Ran-GTP cycle…………………………………………………………………..54 3. Nuclear import model with importin