OOCYTE MEIOTIC SPINDLE ASSEMBLY IN CAENORHABDITIS ELEGANS by AMY A. CONNOLLY A DISSERTATION Presented to the Department of Chemistry and Biochemistry and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy September 2014 DISSERTATION APPROVAL PAGE Student: Amy A. Connolly Title: Oocyte Meiotic Spindle Assembly in Ccaenorhabditis elegans This dissertation has been accepted and approved in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Department of Chemistry and Biochemistry by: Tom Stevens Chairperson Bruce Bowerman Advisor Karen Guillemin Core Member Ken Prehoda Core Member Chris Doe Institutional Representative and J. Andrew Berglund Dean of the Graduate School Original approval signatures are on file with the University of Oregon Graduate School. Degree awarded September 2014 ii © 2014 Amy A. Connolly iii DISSERTATION ABSTRACT Amy A. Connolly Doctor of Philosophy Department of Chemistry and Biochemistry September 2014 Title: Oocyte Meiotic Spindle Assembly in Caenorhabditis elegans As in many organisms, Caenorhabditis elegans oocytes assemble bipolar meiotic spindles in the absence of centrosomes. While the assembly of the mitotic spindle in C. elegans has been studied in some detail, how the poles assemble in the absence of centrosomes remains poorly understood. In an ongoing screen for temperature-sensitive (TS), embryonic-lethal mutants, we have identified TS mutations in multiple genes required for oocyte meiotic spindle pole assembly. We have so far identified mutations in four genes: or1178ts in mei-1, which encodes the catalytic domain of the microtubule severing complex katanin; or447ts in klp-18, which encodes a kinesin 12 family member; or645ts in aspm-1, which encodes a microtubule scaffolding protein; and or1092ts and or1292ts in klp-7, which encode a kinesin 13/MCAK family member. By using live cell imaging of oocytes from transgenic strains expressing GFP and mCherry fusion to proteins associated with the spindle, we have found and confirmed other findings that klp-18 promotes spindle bipolarity and that MEI-1 promotes pole assembly both by severing microtubules and by recruiting ASPM-1. More recently, we have found that klp- 7 is required for maintaining bipolarity in the meiotic spindle by preventing the number of poles that can form. In klp-7(-) mutants, we observed in addition to extraneous poles an excess accumulation of microtubules during Meiosis I. Futhermore, reducing klp-7 iv function can restore bipolarity in a klp-18(-) monopolar spindle mutant background. We also observed that disruption of the kinetochore factor KNL-1 in klp-7(-) mutants exacerbates the extra spindle pole phenotype. We suggest that in oocyte meiosis, klp-7 is required to limit microtubule accumulation and pole assembly and that it may carry out these functions in a kinetochore-dependent manner. This dissertation includes previously published co-authored material. v CURRICULUM VITAE NAME OF AUTHOR: Amy A. Connolly GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon, Eugene University of Kansas, Lawrence DEGREES AWARDED: Doctor of Philosophy, Chemistry and Biochemistry, 2014, University of Oregon Bachelor of Science, Biochemistry, 2008, University of Kansas AREAS OF SPECIAL INTEREST: Genetics and Molecular Biology PROFESSIONAL EXPERIENCE: Oral Presentation in July 2014 at the C. elegans Development, Cell Biology and Gene Expression Meeting Adjunct Professor, University of Oregon, present Human Genetics, BI 122 class Co-instructor, University of Oregon 2014 Science and Policy, BI 140 class GRANTS, AWARDS, AND HONORS: Donald E. Wimber Fund Award, University of Oregon, 2014 American Heart Association Predoctoral Fellowship, University of Oregon, 2011- 2013 HHMI Science Literacy Fellowship, University of Oregon 2011, 2014 Kansas Idea Network of Biological Experience (K-INBRE), Undergraduate Fellowship, University of Kansas, 2006 Southwestern Bell Scholarship, University of Kansas, 2005-2008 National Society of Collegiate Scholars, University of Kansas, 2005-2008 Dean’s List, University of Kansas, 2004, 2007-2008 Runner up in the National Endowment for Humanities Essay Contest, 2003 vi PUBLICATIONS: Connolly, A.A., V. Osterberg, S. Christensen, M. Price, C. Lu, K. Chicas-Cruz, S. Lockery, P.E. Mains, and B. Bowerman. 2014. Caenorhabditis elegans oocyte meiotic spindle pole assembly requires microtubule severing and the calponin homology domain protein ASPM-1. Mol Biol Cell. 25:1298-1311. O'Rourke SM, Yochem J, Connolly AA, Price MH, Carter L, Lowry JB, Turnbull DW, Kamps-Hughes N, Stiffler N, Miller MR, Johnson EA, Bowerman B. 2011. Rapid mapping and identification of mutations in Caenorhabditis elegans by restriction site- associated DNA mapping and genomic interval pull-down sequencing. Genetics. 189(3):767-78. vii ACKNOWLEDGMENTS I wish to say thank you to my parents Mark and Heide Connolly who on many family vacations encouraged me to stop off at various universities and talk to faculty about their biochemistry program. We ventured out to Oregon on multiple occasions to visit my older sister who helped convince me that Oregon would be the right fit for me! It’s where I fell in love with the scenic outdoors, the opportunities to hike, camp, bike, eat fresh blackberries and drink the best beer in the country. I visited the UO more than once, and it struck me how excited people were here to do science in a collaborative manner. After my positive interactions with faculty and students here, UO became my top pick for graduate schools. Indeed, my graduate experience has lived up to all my expectations. I would also like to express my appreciation to Professor Bruce Bowerman for the valuable mentorship he has given me. Bruce has supported my growth as a scientist by sending me to conferences each year and encouraging me to apply for talks. He taught me how to communicate science by going through my seminar talks with me in great detail; it is during these sessions where I feel I learned to speak like a geneticist! He provided me the opportunity to teach as a full instructor for the Human Genetics course this past Spring, and he was supportive of my decision to participate in the Science Literacy training program. I am also very grateful that he did not simply write my manuscripts for me, but instead extended the effort and time walking me through the process of writing a manuscript until I understood how to write each section. The first manuscript he gave me significant help, but with the second manuscript I was able to write one from to start to finish that Bruce was pretty pleased with—that was a great feeling! I am also thankful for the right amount of support he has given me; he helped viii guide my project and provided intellectual insight, but remained hands-off allowing me room to include my own hypotheses and experiments. Getting to pursue my own ideas in addition to Bruce’s made my science all the more fun and exciting. All and all I’d say we make a great team! I would also like to thank past and present members of my committee: Dr. Hui Zong (past), Dr. Tom Stevens (chair), Dr. Ken Prehoda, Dr. Karen Guillemin, and Dr. Chris Doe. In addition, special thanks are due to Tom and Karen for letting me rotate in their labs my first year here, and to Chris who allowed me to collect most of my data on his spinning disc confocal microscope. Furthermore, I like to extend appreciation to Hui who provided me with wisdom and encouragement in my first few years at UO. Lastly, I like to say thanks to the Institute of Molecular Biology on the whole for having a supportive group of faculty who were always eager to help. The investigation was supported in part by the American Heart Association Predoctoral Fellowship, #12PRE9610016. ix This dissertation is dedicated to my parents, my three siblings, and my grandmother. Thanks for your love and support. x