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Separase Cleaves the Kinetochore Protein Meikin to Direct the Meiosis I/II Transition

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Separase Cleaves the Kinetochore Protein Meikin to Direct the Meiosis I/II Transition Separase cleaves the kinetochore protein Meikin to direct the meiosis I/II transition by Nolan Kenji Kwaisun Maier B.S. Molecular Biology and Biochemistry Middlebury College (2012) SUBMITTED TO THE DEPARTMENT OF BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BIOLOGY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY FEBRUARY 2021 © 2020 Nolan K Maier. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part in any medium now known or hereafter created. Signature of the Author:_________________________________________________________________ Department of Biology 24 November 2020 Certified by:_____________________________________________________________________ Iain Cheeseman Professor of Biology Member, Whitehead Institute Thesis Supervisor Accepted by:_____________________________________________________________________ Amy Keating Professor of Biology and Biological Engineering Co-Director, Biology Graduate Committee Separase cleaves the kinetochore protein Meikin to direct the meiosis I/II transition by Nolan Kenji Kwaisun Maier Submitted to the Department of Biology, MIT On November 24, 2020 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Abstract To generate haploid gametes, meiotic cells must undergo two consecutive rounds of chromosome segregation without an intervening gap phase. Importantly, because homologous chromosomes are segregated in meiosis I, but sister chromatids are segregated in meiosis II, this requires a dramatic rewiring of the cell division machinery between the two divisions. How meiotic cells coordinate this rapid and substantial change to the cell division machinery is a central mystery at the heart of proper fertility and reproduction. Our work reveals a new paradigm that rewires key cell division processes at the meiosis I/II transition through the action of the protease Separase, which we demonstrate acts by cleaving the meiosis-specific kinetochore protein Meikin. Cleavage of Separase substrates such as cohesin results in their potent and complete inactivation. In contrast, we find that Separase cleavage of Meikin acts as a molecular “scalpel,” providing an elegant mechanism to precisely and irreversibly modulate Meikin activity between the two meiotic divisions without inactivating Meikin function. Our results demonstrate that the C-terminal Meikin cleavage product generated by Separase proteolysis retains substantial activity such that it localizes to kinetochores, binds to Plk1 kinase, and promotes downstream activities such as the cleavage of the meiosis-specific cohesin subunit Rec8, similar to full length Meikin. Importantly, we demonstrate that both the failure to cleave Meikin or the complete inactivation of Meikin at the meiosis I/II transition each result in dramatic defects in the proper execution of meiosis II. Our functional analysis in mouse oocytes demonstrates that precise Meikin cleavage is critical to differentially control meiosis I and II. Thus, in contrast to previous models, Meikin is not just a regulator of meiosis I-specific activities, but differentially coordinates chromosome segregation across both meiotic divisions. Our discovery of Meikin as a new substrate for Separase cleavage represents a novel mechanism for the regulatory control of the meiosis I/II transition. Thesis Supervisor: Iain Cheeseman, Title: Professor of Biology 2 Acknowledgements There are many people who I would like to thank for making my time in graduate school such a great and memorable experience. Foremost I would like to thank Iain for being such a great advisor. Thanks for the open door, the willingness to talk, and all of your helpful suggestions. Your curiosity and enthusiasm are infectious, and you always believed in me even when I didn’t believe in myself. You care deeply about me not only as a student but as a friend. I hope to model your mentorship throughout my career. I would like to thank all members of the Cheeseman lab past and present for their suggestions and advice. I benefitted greatly from being surrounded by such generous and smart scientists. Thanks to everyone for always taking the time to offer helpful suggestions and advice. You made the lab not just a great place to work but also a fun environment to spend endless hours with friends. I cherished Fire-Tong, Halloween costume making, and Saturday mimosas. A major reason I chose to do my PhD at MIT was the deep relationships built between the graduate students. I thank my MIT-Bio cohort who have been on this journey with me from our very first days in the PIT to now. Thank you for indulging me in attending Data Club and putting up with my grilling on Fourth of July. I appreciate all of your scientific advice but mostly your comradery and friendship. Thank you to all of the people within MIT Biology and the Whitehead Institute who keep the place running and take care of everything behind the scenes to ensure that the science can happen smoothly and easily. Too often your work goes unseen and unnoticed. This includes the administrative staff who keep the money flowing and the paperwork off my desk, the facilities and janitorial staff who work long nights keeping the place ticking, the Salt Creek Catering crew who feed us as if we are family, the Media Prep, Glasswash, and Core facilities personnel whose expertise and attention to detail make every experiment possible. I would like to thank my thesis committee, Angelika Amon, David Page, and Steve Bell for their expertise and interest not only in my project but in my personal growth as a scientist. I would also like to thank Silke Hauf for participating in my thesis defense. I’d like to thank my roommates and the whole Middlebury-Boston crowd for their friendship and understanding. Thank you for putting up with my constant house projects and my fascination with whatever weird science I read about this week. Over a decade of knowing each other has only deepened our friendships. Thanks to my parents, Steve and Cherie, and my sister, Malia. You taught me the value of hard work and persistence and nurtured my scientific curiosity from my first elementary school Science Fair to today. Your love and support all these years mean so much to me. Most of all I would like to thank my partner Barbara. You have lived the ups and downs of this PhD with me and made every moment of it brighter. I can’t wait to embark with you on whatever new adventure life has in store. 3 Table of Contents Abstract .....................................................................................................................................2 Acknowledgements ..................................................................................................................3 Table of Contents .....................................................................................................................4 Chapter 1: Introduction ..........................................................................................................6 Sexual Reproduction ..................................................................................................................7 Chromosome Segregation and Kinetochore Function ...............................................................9 Overview of Meiosis ..................................................................................................................9 Figure 1 ..............................................................................................................................11 Diversity of Sexual Reproduction at the Organismal and Molecular Level ............................11 MOKIRS and Meiosis I Activities Across Organisms ............................................................14 Figure 2 ..............................................................................................................................15 Figure 3 ..............................................................................................................................18 Driving Cell Cycle Transitions with the Kinases Cdk and Plk1 ..............................................18 Driving Cell Cycle Transitions with Degradation by the E3 Ligase APC/C ...........................19 Driving Cell Cycle Transitions with the Protease Separase ....................................................21 Figure 4 ..............................................................................................................................25 Open Questions Motivating Thesis Work ................................................................................25 References ................................................................................................................................27 Chapter 2: Separase cleaves the kinetochore protein Meikin to direct the meiosis I/II transition ..........................................................................................................34 Summary ..................................................................................................................................35 Introduction ..............................................................................................................................36 Results ......................................................................................................................................37 Meikin is proteolytically cleaved by
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