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A Dissertation Entitled TRIP13 AAA-Atpase Promotes Spindle A Dissertation Entitled TRIP13 AAA-ATPase Promotes Spindle Assembly Checkpoint Activation through Coordinating with MAD1 at Unattached Kinetochores By Christopher E. Arnst Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology __________________________________________ Dr. Song-Tao Liu, Committee Chair __________________________________________ Dr. William Taylor, Committee Member __________________________________________ Dr. Qian Chen, Committee Member __________________________________________ Dr. Donald Ronning, Committee Member __________________________________________ Dr. Ajith Karunarathne, Committee Member __________________________________________ Dr. Cyndee Gruden, Dean College of Graduate Studies University of Toledo August 2019 Copyright 2019, Christopher E. Arnst This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of TRIP13 AAA-ATPase Promotes Spindle Assembly Checkpoint Activation through Coordinating with MAD1 at Unattached Kinetochores By Christopher E. Arnst Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Biology The University of Toledo August 2019 During mitosis, cells must ensure proper separation of the genome into daughter cells. Failure to evenly divide chromosomes into the daughter cells results in aneuploidy and chromosomal instability (CIN). CIN is common in aggressive cancers as it directly promotes genetic heterogeneity and can lead to oncogenic signaling in cells. To maintain genomic integrity during mitosis, cells employ a signal transduction pathway termed the Spindle Assembly Checkpoint (SAC). SAC signaling delays chromosome segregation until all chromosomal kinetochores are properly bound by microtubules from spindle poles. At the molecular level, the SAC is amplified by unattached kinetochores. These unattached kinetochores prevent premature chromosome segregation by driving the production of the Mitotic Checkpoint Complex (MCC). Kinetochore-produced MCC diffuses into the cytoplasm where it binds to and inhibits the Anaphase Promoting Complex/ Cyclosome (APC/C), an E3 ubiquitin ligase that targets securin and cyclin B for degradation by the proteasome. When the spindle is properly assembled and iii chromosomes are bi-oriented, the SAC must be silenced to allow for anaphase onset and cell division. SAC silencing requires disassembling cytoplasmic MCC and preventing further MCC assembly at kinetochores. Our lab was the first to identify a novel SAC silencing role for Thyroid Hormone Receptor Interacting Protein 13 (TRIP13). TRIP13 is an AAA-ATPase that partners with its adaptor p31comet to disassemble cytosolic MCC by extracting and inactivating the key MCC subunit Mitotic Arrest Deficient 2 (MAD2). MCC disassembly allows for APC/C activation and the metaphase-to-anaphase transition. Despite the bona -fide SAC silencing role of TRIP13 and p31comet, both proteins localize to unattached kinetochores during SAC activation in prometaphase. TRIP13 has also been directly implicated in SAC activation in recent publications. These data seem incongruent with the established roles of TRIP13 and p31comet in SAC silencing. The disparity led us to hypothesize that TRIP13 and p31comet play a different role at unattached kinetochores during prometaphase to help activate the SAC. Here we used RNAi and CRISPR based auxin inducible degron (AID) tagging to show that TRIP13 but not p31comet supports SAC activation through promoting kinetochore localization of the MCC subunit MAD2 during prometaphase. We also identified the SAC components that contribute to TRIP13 kinetochore localization. Lastly, we identified novel interactions between TRIP13 and SAC proteins including MAD1 that delineate a kinetochore specific mechanism for TRIP13 dependent SAC activation. iv Table of Contents Abstract .............................................................................................................................. iii Table of Contents .................................................................................................................v List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix List of Abbreviations ........................................................................................................ xii List of Symbols ................................................................................................................ xiv 1. Overview and Significance ......................................................................................1 1.1 Introduction to Cell Division and Cell Cycle Checkpoints ..............................1 1.2 The Spindle Assembly Checkpoint ...................................................................6 1.3 Centromeres and Kinetochores .........................................................................9 1.4 Molecular Components of SAC Activation ....................................................11 1.5 Mechanism of MCC Assembly .......................................................................17 1.6 Additional SAC Components .........................................................................19 1.7 Regulation of SAC by Mitotic Kinases ..........................................................20 1.8 Molecular Components of SAC Silencing ......................................................21 1.9 The Role of TRIP13 and p31comet in SAC Silencing ......................................24 v 1.10 TRIP13 Functions as a SAC Activator ..........................................................32 1.11 Evolutionary Divergence of TRIP13-p31comet and SAC Signaling ...............32 1.12 Central Hypothesis for TRIP13 but not p31comet in SAC Activation .............34 2. Materials and Methods ...........................................................................................36 3. TRIP13 but not p31comet Activates the Spindle Assembly Checkpoint .................42 3.1 Introduction .....................................................................................................42 3.2 Results .............................................................................................................42 4. TRIP13 ATPase Activity and MAD2 Rescues Mitotic Arrest Defect After TRIP13 Depletion ..................................................................................................49 4.1 Introduction .....................................................................................................49 4.2 Results .............................................................................................................50 5. Determining the Requirements for TRIP13 and p31comet Kinetochore Localization during SAC Activation ..........................................................................................55 5.1 Introduction .....................................................................................................55 5.2 Results .............................................................................................................56 6. TRIP13 Coordinates with MAD1 during SAC Activation ....................................65 6.1 Introduction .....................................................................................................65 6.2 Results .............................................................................................................66 7. Discussion ..............................................................................................................73 References ..........................................................................................................................79 Appendix A OTSSP167 Prevents Mitotic Arrest through Inhibiting CPC Kinase Aurora B .........116 B Attribution of Data ......................................................................................................125 vi C Constructs Generated for the Lab ................................................................................126 vii List of Tables C.1 DNA Constructs Generated for the Lab .................................................................126 C.2 Antibodies ..............................................................................................................134 C.3 RNAi Constructs ....................................................................................................135 C.4 Primers Generated for the Lab ...............................................................................136 viii List of Figures 1-1 The Cell Cycle is Divided into Distinct Phases ...........................................................2 1-2 Temporal Cyclin-CDK Expression Drives Cell Cycle Progression ............................3 1-3 Mitotic Signaling Coordinates Genome Division .......................................................4 1-4 Unattached Kinetochores Promote SAC Signaling .....................................................7 1-5 Centromere Regions of Chromosomes Recruit Kinetochores ...................................11 1-6 MAD2 Adopts either Open or Closed Conformation ................................................12 1-7 MAD1-MAD2 Tetramer Converts MAD2 from Open to Closed Conformation
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