A Dissertation Entitled

TRIP13 AAA-ATPase Promotes Spindle Assembly Checkpoint Activation through

Coordinating with MAD1 at Unattached

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 , cells must ensure proper separation of the genome into daughter cells. Failure to evenly divide 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 segregation until all chromosomal kinetochores are properly bound by from spindle poles.

At the molecular level, the SAC is amplified by unattached kinetochores. These unattached kinetochores prevent premature by driving the production of the Mitotic Checkpoint Complex (MCC). -produced MCC diffuses into the where it binds to and inhibits the 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

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chromosomes are bi-oriented, the SAC must be silenced to allow for anaphase onset and 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 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 -to-anaphase transition. Despite the bona -fide SAC silencing role of TRIP13 and p31comet, both 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.

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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 and Cell Cycle Checkpoints ...... 1

1.2 The Spindle Assembly Checkpoint ...... 6

1.3 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

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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 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 ...... 13

1-8 Unattached Kinetochores Produce C-MAD2 Containing MCC...... 14

1-9 TRIP13 and p31comet Disassemble MCC by Converting C-MAD2 to O-MAD2 ...... 25

1-10 TRIP13 AAA-ATPase Forms a Planar Hexamer ...... 28

1-11 TRIP13 Pore Loop Residues Engage the N-Terminus of MAD2 ...... 31

3-1 Auxin Inducible Degradation of TRIP13 and p31 in DLD1 Cells ...... 45

3-2 Mitotic Duration Assay in TRIP13 and p31comet AID Depleted Backgrounds ...... 46

3-3 Mitotic Index Assay in TRIP13 and p31comet AID Depleted Backgrounds ...... 46

3-4 Phospho-Serine 10 Levels after TRIP13-AID and p31comet AID Depletion ...... 46

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3-5 Mitotic Index Assay in TRIP13 and p31comet RNAi Backgrounds ...... 48

4-1 TRIP13 ATPase Activity Rescues Mitotic Arrest Defect from AID-TRIP13

Depletion ...... 52

4-2 Wild Type or Closed Conformation MAD2 Rescues Mitotic Arrest Defect

from TRIP13-AID Depletion ...... 53

5-1 MAD2 is Mislocalized after TRIP13-AID but not p31comet -AID Depletion ...... 58

5-2 MAD2 is Mislocalized after TRIP13 but not p31comet RNAi Depletion ...... 58

5-3 MAD1, CDC20, and BUBR1 Kinetochore Localization after p31comet and TRIP13

AID Depletion ...... 59

5-4 MAD1, CDC20, and BUBR1 Kinetochore Localization after p31comet and TRIP13

RNAi Depletion ...... 61

5-5 TRIP13 Kinetochore Localization is Abrogated by RNAi of Outer Kinetochore

Components ...... 62

5-6 p31comet Kinetochore Localization is Abrogated by RNAi of Outer Kinetochore

Components ...... 63

5-7 Quantification of TRIP13 and p31comet Depletion from 5-5 and 5-6 ...... 64

6-1 TRIP13 binds to MAD1 NTD and CTD but not MIM ...... 69

6-2 p31comet binds to MAD1 NTD and CTD but not MIM ...... 69

6-3 TRIP13 and p31comet Bind to MAD1 CTD but not CTD-4E Mutants ...... 70

6-4 TRIP13 and p31comet Bind to MAD1 716A and 716E Mutants ...... 70

6-5 mCherry-Mis12-MAD1 Does Not Rescue Checkpoint Defect from TRIP13

Depletion ...... 71

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6-6 mCherry-Mis12-TRIP13 Does Not Prolong Mitotic Duration...... 72

7-1 Model for TRIP13 in SAC Activation ...... 78

A-1 OTSSP167 Abrogates Mitotic Arrest ...... 120

A-2 OTSSP167 but not MELK RNAi Inhibits MCC Formation ...... 120

A-3 OTSSP167 Prevents Histone 3 Serine 10 Phosphorylation by Aurora B ...... 121

A-4 OTSSP167 Prevents CPC Localization to Centromeres ...... 122

A-5 OTSSP167 Prevents BUB1 and Haspin Kinase Activity towards Histones ...... 124

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List of Abbreviations

AAA-ATPase………. ATPase Associated With Diverse Cellular Activities APC/C……………… Anaphase Promoting Complex/ Cyclosome ATM………………... Ataxia-Telangiectasia-Mutated ATP………………… Adenosine Triphosphate ATR…………………Ataxia-Telangiectasia Related Protein

BUB…………………Budding Uninhibited by Benzimidazole

CCAN……………….Constitutive Centromere Associated Network CDC………………... Cell Division Cycle CDK…………...... Cyclin Dependent Kinase CENP………...….…..Centromere Protein CHK………....……... Checkpoint Kinase 1 CIN………….……… Chromosomal Instability CLL………….……... Chronic Lymphocytic Leukemia C-MAD2…………… Closed MAD2 CPC………….……... Chromosome Passenger Complex CTD………..………. C-Terminal Domain

D-box………………. Destruction box DNA…………..……. Deoxyribonucleic Acid

E2F………………… Eukaryotic Transcription Factor 2 EMT………………... Epithelial to Mesenchymal Transition FRAP………………. Fluorescence Recovery After Photobleaching FRET………………. Fluorescence Resonance Energy Transfer

G1………….………. Gap Phase 1 G2……………...... Gap Phase 2

I-MAD2……………. Intermediate MAD2 INCENP….………… Inner Centromere Protein

KNL1………….…… Kinetochore Null 1 xii

MAD……………….. Mitotic Arrest Deficient MCC……………...... Mitotic Checkpoint Complexes MELK……………… Maternal Embryonic Leucine Zipper Kinase MPS1………………. Monopolar Spindle One

NTD………………... N-Terminal Domain

O-MAD2…………… Open MAD2

PLK1……………...... Polo-Like Kinase One PP1………………..... Protein Phosphatase One PP2A………..……… Protein Phosphatase Two-A

RB………….………. Retinoblastoma protein RNA……………..…. Ribonucleic Acid RNAi……...………... Ribonucleic Acid Interference RZZ…………….…... Rod-ZW10-Zwilch

SAC………….…….. Spindle Assembly Checkpoint

TPR………….……... Tetratricopeptide Repeats TRIP13……………... Thyroid Hormone Receptor Interacting Protein Thirteen

USP……………..….. Ubiquitin Specific Protease

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List of Symbols

α ...... alpha β ...... beta Δ ...... delta º ...... degree γ ...... gamma µ ...... micro

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Chapter 1

Overview and Significance

1.1 Introduction to aneuploidy, cell division, and cell cycle checkpoints

Nearly 90% of solid tumors contain cellular populations that have lost or gained extra copies of chromosomes [2, 3]. This state of genetic heterogeneity is referred to as aneuploidy [4, 5]. Aneuploid cancer cells do not usually maintain a stable karyotype, but instead undergo constant genomic “shuffling” where copies of chromosomes are gained or lost. The process of genomic reshuffling that leads to aneuploidy is termed chromosomal instability (CIN) [4, 6]. Multiple studies have confirmed that CIN plays a role in neoplastic transformation, tumorigenesis, and is a causal contributor to diverse genetic backgrounds within single solid tumors and in cancer cell populations [2, 4, 7-9].

To prevent CIN and promote growth and proliferation, cells have several evolutionarily honed series of molecular events that allow for accurate genome duplication and division.

These processes that promote genome duplication and cell division are collectively referred to as the cell cycle (Figure 1-1).

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Figure 1-1 The cell cycle is divided into distinct phases. After a successful division the cell enters interphase which is composed of gap phase 1 (G1), S phase, and gap phase 2 (G2). G1 consists of growth signaling and preparation for S phase for DNA replication [10]. After DNA replication the cell enters G2 where it repairs DNA damage from replication and prepares to divide [11]. Cell division takes place during mitosis (M phase). During G1 cells may also enter gap phase 0 (G0). G0 is a non divisive state. Image from [12] is not copyright protected.

The cell cycle is divided into interphase and mitosis where genome duplication and separation are carried out respectively and with high fidelity. Both interphase and mitosis are subdivided into specific phases [10, 11, 13, 14]. Both interphase and mitosis are driven unidirectionally by the activity of cyclins and cyclin dependent kinases

(CDK’s). Temporally isolated expression of different cyclins regulates signal transduction specific to each phase of the cell cycle (Figure1-2).

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Figure 1-2 Temporal cyclin-CDK Activity Drives Cell Cycle Progression. Cyclins are differentially expressed throughout the cell cycle. Cyclins bind to and activate cyclin dependent kinases (CDK). Cyclin-CDK complexes confer specificity towards substrates. Image from [15] is not copyright protected.

The primary driver of G1 progression is CDK4/6-cyclin D complexes. CDK4/6- cyclin D complexes phosphorylate Retinoblastoma protein (RB) and promote its dissociation from the eukaryotic transcription factor Adenovirus Early 2 Promoter Factor

Binding (E2F). Free E2F family members drive the expression of required for the transition from G1 to S phase. As the cell completes G1, E2F activity drives expression of cyclin E and CDK2. Cyclin E-CDK2 complexes drive the cell into S phase and DNA synthesis [16-18]. Increased levels of cyclin A-CDK2 drives the cell out of S phase into

G2. G2 arrest depends on ATM and ATR kinases which activate Checkpoint Kinases 1 and 2 (CHK1 and CHK2). CHK1 and CHK2 phosphorylate and inactivate the phosphatase CDC25 [19-21]. ATM and ATR driven inactivation of CDC25 prevents

CDC25 from removing inhibitory T14 and Y15 phosphorylation of CDK1. CDK1, when

3 bound to cyclin B, is the primary driver of mitotic signaling and allows for the transition from G2 to mitosis. Once the cell has passed the G2 checkpoint it is irreversibly committed to mitosis [22-24].

Similar to interphase, mitosis is divided into phases including , prometaphase, metaphase, anaphase, and telophase. Collectively these stages ensure coordinated separation of the genome and cellular organelles into identical daughter cells

(Figure 1-3).

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Figure 1-3 Mitotic signaling coordinates genome division. After interphase is complete the cell enters mitotic prophase. During mitotic prophase, the nuclear membrane is degraded, centrosomes migrate to form spindle poles, and chromatin condenses into chromatids. After prophase the cell enters prometaphase wherein microtubules polymerize from spindle poles and stochastically capture kinetochore platforms that assemble on chromatids. This period of spindle formation is referred to as prometaphase. Metaphase marks the period of the cell cycle where all chromatids are bi-oriented to the spindle poles and have captured microtubules. Once the spindle has formed, anaphase is initiated and the chromosomes are pulled to spindle poles. The cell enters telophase shortly after anaphase onset, wherein, the membrane forms a contractile ring that separates the two new cells and the chromosomes decondense. Image from [25] is not copyright protected.

During prophase, centrosomes migrate to opposite poles of the cell and nucleate microtubules to begin mitotic spindle construction. At the same time, DNA condenses into compacted chromosomes. The sister chromatids are paired together and maintain cohesion through Cohesin rings that link matching regions of DNA. To prevent premature chromosome separation, Cohesin rings are maintained around centromeres until metaphase [26-28].

During prometaphase the nuclear envelope breaks down and the microtubules in the mitotic spindle engage in polymerization and depolymerization to align chromosomes. There are three types of microtubules that play important roles in chromosome capture and spindle positioning [6, 29]. Astral microtubules extend from centrosomes to the cell membrane, stabilizing the spindle poles; Interpolar microtubules overlap in the spindle midzone and maintain the spindle; and K-fibers are parallel bundles of 20-40 microtubules that form the end-on attachments with kinetochores. The stochastic capturing of kinetochore platforms by the K-fibers in the spindle organizes the

5 chromosomes into a bi-oriented plate at the center of the cell. Formation of this bi- oriented plate defines the metaphase period of mitosis. After metaphase plate formation the cell transitions into anaphase and mitotic exit. During this transition, the cohesive proteins that link sister kinetochores are degraded allowing for chromatid separation [30].

Simultaneously, the molecular machinery that maintains the mitotic state, primarily cyclin B, is degraded. Once the cohesion and cyclin B has been removed, the cell proceeds into anaphase where chromosomes are pulled towards their respective spindle poles.

Once the sister chromatids are separated the cell enters telophase. During telophase chromatin decondenses and the nuclear envelopes reform. Shortly after telophase onset the cell initiates cytokinesis by forming a cleavage furrow in the membrane. This cleavage furrow expands and pinches off the membrane creating two separate genetically identical daughter cells.

1.2 The Spindle Assembly Checkpoint

To avoid aneuploidy and maintain chromosomal stability, it is critical that anaphase does not begin until all chromosomes are attached to the mitotic spindle. To prevent premature mitotic exit, cells employ an evolutionarily conserved signal transduction pathway that monitors kinetochore- attachment and tension status. This signaling pathway is referred to as the Mitotic Checkpoint or the Spindle

Assembly Checkpoint (SAC) [8, 31-35].

The SAC is primarily activated by unattached kinetochores. In addition, there is a clear link between SAC activity and aneuploidy, tumorigenesis, and oncogenic transformation [4, 9, 36, 37]. Interference with SAC genes by RNAi, antibody

6 microinjection, or genetic depletion results in increased rates of chromosome missegregation. Alterations in SAC genes have been identified in multiple human cancers, mostly in expression levels. Additionally, genetic manipulation of SAC genes in mouse models results in embryonic lethal phenotypes, and heterozygotes of some SAC genes show increased rates of tumorigenesis [38-44]. Individual SAC genes will be discussed in the next few sections (Figure 1-4).

Figure 1-4: Unattached kinetochores promote SAC signaling. Unattached kinetochores generate Mitotic Checkpoint Complexes (MCC) that inhibit the anaphase promoting complex/ cyclosome (APC/C). APC/C inhibition prevents the ubiquitination and degradation of cyclin B and securin, thus promoting mitotic arrest. Spindle formation inhibits MCC formation allowing for APC/C activation and degradation of cyclin B and securin. Image used with permission from [33].

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The target of the SAC is the Anaphase Promoting Complex/ Cyclosome (APC/C), an E3 ubiquitin ligase that targets the machinery maintaining mitotic arrest for degradation [45-47]. The APC/C is composed of over 15 different subunits depending on the organism. These subunits are primarily organized into two major subcomplexes [47-

52]. The first subcomplex contains APC2 and APC11 which make up the catalytic core of the APC/C [53-55]. The second subcomplex contains subunits containing tetratricopeptide repeats (TRPs) that promote binding with WD40 domains in coactivator proteins. Two primary coactivator proteins containing WD40 domains are CDC20 and

CDH1. These coactivators are important for proper APC/C activity and depletion of either causes prolonged metaphase arrest [56-58]. These coactivators present substrates for ubiquitination through their ability to bind to Destruction Box (D-box) and Lysine-

Glutamic Acid-Asparagine (KEN box) motifs in substrates. The resulting APC/C - coactivator - substrate complex collaborates with UBCH5 and UBCH10 ubiquitin conjugating enzymes (E2) to polyubiquitinate mitotic signaling substrates [34, 46, 50, 51,

56, 59-65].

Two key substrates targeted by the APC/C are securin and cyclin B at the metaphase to anaphase transition [33] (Figure 1-4). Degradation of securin activates the proteinase Separase, which cleaves the Scc1 subunit of the Cohesin complexes that hold centromeric regions of chromosomes together. Degradation of cyclin B inactivates CDK1 and results in mitotic exit [32, 46-48, 60, 66-71]. During SAC activation and mitotic arrest, the ubiquitin ligase activity of APC/CCDC20 complexes is inhibited by Mitotic

Checkpoint Complexes (MCC) generated from unattached kinetochores (discussed more

8 below in section 1.4). Proper bipolar attachment silences SAC signaling and frees the

APC/C to target D-box motifs in substrates [72-78].

1.3 Centromeres and Kinetochores

The SAC activation that prevents premature cell division occurs primarily at centromeres and kinetochores (Figure 1-5). Centromeres are regions of genomic DNA characterized by repetitive AT rich DNA sequences [79]. While these alphoid repeats can be used to identify centromeres, centromeres can also spontaneously generate in chromosome areas without alphoid repeats. These are called neocentromeres [80]. A second, more reliable determinant of centromeres is the presence of Centromere Protein

A (CENP-A). CENP-A is a histone H3 variant found in complex with the centromeric

DNA of nearly all . CENP-A targeting to chromatin is sufficient for neocentromere formation and the subsequent recruitment of centromere and kinetochore proteins [81-85]. CENP-A containing nucleosomes in centromeric DNA recruit the

Constitutive Centromere-Associated Network (CCAN) which consists of over a dozen other CENP proteins. The CCAN serves as the foundation for kinetochore construction and can transiently recruit over 200 other proteins during mitosis. These proteins have diverse functions but are largely linked to proper mitotic progression and SAC activity

[86-89].

The kinetochore serves as the interface for direct interaction between chromosomes and K-fibers in the spindle. Kinetochores also regulate chromosome movement along the spindle and recruit proteins necessary for SAC activation. In budding , a single kinetochore binds a single microtubule [90]. Vertebrate kinetochores however each can bear twenty to forty microtubule attachments [91, 92].

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Interestingly, a single unattached kinetochore can activate SAC and mitotic exit does not take place until all unattached chromosomes are attached to the spindle [90, 92-96].

Electron microscopy of the kinetochore revealed that it is divided into two main electron dense layers: the inner and outer kinetochore. The inner layer composed of

CCAN and the outer kinetochore serves as the binding site for microtubules and transiently associated mitotic regulatory proteins [91]. To stop SAC signaling, kinetochores must form stable end-on attachments with spindle microtubules. Several outer kinetochore proteins are involved in generating these attachments. The nuclear division cycle 80kda complex (NDC80) is one of the best characterized microtubule binding complexes at kinetochores. It is composed of Ndc80, Nuf2, Spc24, and Spc25.

The Ndc80 and Nuf2 form stable interactions with microtubules while Spc24 and Spc25 anchor the complex to CENP-T or CENP-C through the Mis12 complex [97-101].

The MIS12 complex is composed of MIS12, PMF1, NSL1, and DSN1. This complex links to CENP-C of the CCAN network and plays a secondary role in stabilizing kinetochore attachments and recruiting Monopolar Spindle 1 (MPS1) kinase to outer kinetochores (described more below)[102, 103]. Another component of the outer kinetochore is the Kinetochore Null 1 (KNL1) protein. KNL1 serves as the master scaffold for transient protein interactions for MCC assembly [104-108].

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Figure 1-5 Centromere regions of chromosomes recruit kinetochores. Conserved centromeric regions of DNA recruit CENP-A proteins that serve as foundations for kinetochore assembly. CENP-A scaffolds the CCAN network composed of other CENP proteins. The kinetochore KMN network is assembled on the CCAN network for microtubule capture and SAC signaling. Image used with permission from [109].

1.4 Molecular Components of SAC activation

It is now widely accepted that MCC formation is integral to SAC activity. MCC is primarily produced at unattached kinetochores and then diffuses into the cytoplasm to inhibit the APC/C [34]. (Figure 1-6, 1-7, and 1-8). MCC formation at unattached kinetochores requires two major steps. First, kinetochore associated Mitotic Arrest

Deficient 1 and 2 (MAD1 and MAD2) promotes a conformational change in additional

MAD2 molecules from inactive open (O-MAD2) to active closed (C-MAD2) state

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(Figure 1.6 and 1-7). The second step is the incorporation of C-MAD2 with Budding

Inhibited by Benzimidazole Related 1 (BUBR1), Budding Inhibited by Benzimidazole 3

(BUB3), and Cell Division Cycle 20 (CDC20). This four-protein complex is the final

Mitotic Checkpoint Complex that inhibits APC/C (Figure 1-8).

Figure 1-6: MAD2 adopts either open or closed conformation. MAD2 C-terminal safety belt motif (shown in yellow) wraps around motifs from MAD2 binding partners (shown in brown). The safety belt motif in the closed conformation is adjacent to the αC helix (shown in blue). Image used with permission from [110].

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Figure 1-7: The MAD1-MAD2 tetramer converts MAD2 from open to closed conformation. MAD2 C-terminal safety belt motif binds the MAD2 Interacting Motif of MAD1 (MIM). MAD2 bound to MAD1 recruits a second MAD2 molecule through its dimerization interface. The second MAD2 molecule is converted from SAC inactive open MAD2 (O-MAD2) to SAC active closed MAD2 (C-MAD2). The conversion creates a brief intermediate MAD2 (I-MAD2). The conversion is coordinated by MPS1 kinase activity towards MAD1. MAD1 NTD and CTD both interact with each other and O and C conformations of MAD2. Image used with permission from [111].

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Figure 1-8: Unattached kinetochores produce C-MAD2 containing MCC. Unattached kinetochores recruit a MAD1:C-MAD2 heterotetramer catalyst. This recruitment requires MPS1 kinase. The MAD1-MAD2 tetramer binds and converts more cytoplasmic MAD2 into the closed conformation for incorporation into MCC with CDC20, BUBR1, and BUB3. Once assembled, MCC diffuses into the cytoplasm to inhibit the Anaphase Promoting Complex/Cyclosome. Image used with permission from [111].

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The core MCC components and MAD1 were first identified through genetic screenings in Saccharomyces cerevisiae. Mutations of MAD1, MAD2, MAD3 (the yeast homolog of BUBR1), BUB1 and BUB3 resulted in yeast cells failing to arrest in mitosis in response to spindle damage [112, 113]. Soon after, Monopolar Spindle 1 (MPS1) was identified as a contributor to mitotic arrest in response to disrupted spindle formation

[112-116]. Homologous counterparts for these SAC components have been identified in nearly all eukaryotic taxa [117, 118]

During SAC activation the MAD1-MAD2 complex is localized to unattached kinetochores through direct interaction with BUB1 and the RZZ complex. BUB1 mediates initial kinetochore saturation and the RZZ complex promotes sustained kinetochore localization of MAD1[119-122]. Constitutive targeting of MAD1 to the kinetochore through fusion to MIS12 is sufficient to bypass BUB1-RZZ mediated localization and cause prolonged checkpoint arrest [123]. Cells expressing constitutively targeted MAD1 AA mutant (K541A, L543A) are defective in MAD2 binding, SAC activation, and mitotic arrest [123]. In further support of the fundamental roles of MAD1 and MAD2 in faithful chromosome segregation, both MAD1 and MAD2 are essential for viability in cell lines and embryos and depletion of either results in inability to maintain mitotic arrest in the presence of spindle poisons [39, 41, 124].

To promote the MAD2 conformational change, MAD1 dimerizes and recruits

MAD2 to a MAD2 interacting motif (MIM) in MAD1. The C-terminal “safety belt” motif in MAD2 is important for this MAD1-MIM binding. This 2:2 MAD1-MAD2 hetero-tetramer complex transiently associates with a cytoplasmic pool of MAD2 through a dimerization domain in MAD2. MAD2 dimerization is essential for SAC activation.

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This is supported by the observation that MAD2 mutants deficient in dimerization or

MAD1 binding are defective in supporting SAC activity and cannot maintain mitotic arrest [125-129]. Additionally, in vitro and in vivo FRAP data indicates that all of MAD1 but only about half of MAD2 stably localizes to unattached kinetochores. This indicates that a pool of free MAD2 must exist for mitotic arrest prior to microtubule attachment

[126-128, 130-137]. The conversion from O-MAD2 to C-MAD2 might go through multiple intermediate MAD2 conformations (I-MAD2). The functional implications of I-

MAD2 generation are still the subject of debate. One possible I-MAD2 conformation was presented as MAD2N10 [138]. MAD2N10 elutes like C-MAD2 in chromatography and has core folding that is structurally similar to C-MAD2 [138, 139]. Despite this,

MAD2N10 is checkpoint defective like O-MAD2 [126, 127, 138, 139].

Recent evidence indicates that MAD2 conversion requires coordination between the N-terminal and C-terminal domains of MAD1 (Figure 1-7). The N-terminal domain of MAD1 spans from 1-485 and was initially implicated in kinetochore localization.

MAD1-CTD spans from 585-718 and is required for SAC activation [125, 137, 140,

141]. MAD2 truncations at the N-terminus or C-terminus, or MAD2 mutants at the dimerization domain still exhibit MAD1 NTD and CTD binding [142]. The conversion from O to C conformation also requires direct phosphorylation of MAD1 by the kinetochore kinase Monopolar Spindle 1 (MPS1). MPS1 can bind directly to both MAD1

NTD and CTD and MPS1 phosphorylation of MAD1 regulates interactions between the

NTD and CTD, as well as direct interaction of MAD1 with MAD2 and CDC20 [142-

144].

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C-MAD2 generation is essential for SAC activation. This is because C-MAD2, but not O-MAD2, can be incorporated into MCC with CDC20, BUBR1, and BUB3

(Figure 1.8). The Closed conformation of the MAD2 uses its “safety belt” motif to selectively bind to CDC20’s KIRL motif, while its dimerization domain binds to BUBR1

[65, 132, 145-148]. Like MAD2, the MCC components BUBR1 and CDC20 localize to kinetochores and their abrogation or mislocalization results in chromosome segregation defects. These defects are the result of defective MCC assembly and premature APC/C activity. BUB3 has debatable importance in MCC signaling. BUB3 is essential for mitotic arrest in budding yeast but is dispensable in fission yeast [45, 112, 149, 150].

While MCC assembly has been proven to robustly inhibit APC/C, there is controversy as to whether all four proteins are essential in this inhibition. MAD2-CDC20 complexes alone, as well as BUBR1-CDC20 complexes alone can be isolated from cell lysates [151]. These subcomplexes even display some inhibitory activity towards APC/C both in vivo and in vitro [152]. Despite this, the full MCC complex has greater APC/C silencing activity than subcomplexes. Fully formed MCC can inhibit APC/C over 3000 times more efficiently than MAD2 alone [151, 153-155].

1.5 Mechanism of MCC Assembly

The efficiency of fully formed MCC in APC/C inhibition relative to MCC subcomplexes alone has prompted in depth investigation into the nature of MCC assembly. Faesen et al have used a Fluorescence Resonance Energy Transfer (FRET) based assay to analyze the interactions between purified recombinant MCC components in vitro [143]. FRET analysis of the rate of subcomplex assembly indicates that preincubating MAD2 and CDC20 in the presence of minimal SAC activation machinery

17

(BUB1, MAD1, MPS1, and ATP) rapidly increases MAD2-CDC20 incorporation into

BUBR1. In these conditions, FRET signals for MAD2-BUBR1 interaction reach saturation within minutes, similar to rapid SAC activation in cells. Without the preincubation of MAD2 and CDC20, full MCC assembly and FRET saturation takes hours [143]. Furthermore, BUBR1 and CDC20 have separate pools that stably or dynamically associate with unattached kinetochores. The dynamic pools have similar

FRAP exchange as MAD2 and are implicated in MCC assembly [87, 128, 156]. These data suggest two things. The first is that kinetochores convert MAD2 from the O to C conformation for incorporation into CDC20 and BUBR1 to form fully functional MCC.

The second is that initial subcomplex formation increases full MCC formation.

Cryo-EM studies and crystal structure analyses suggest that the inhibitory activity

MCC has towards APC/CCDC20 is from the direct binding of the CDC20 coactivator bound to APC/C. When MCC binds to the APC/C it disrupts the CDC20-UBCH10 interaction that is needed for APC/C ubiquitination activity. Additionally, the second

KEN box in BUBR1 is recognized by the KEN box receptor in APC/C coactivator

CDC20. It is theorized that this second BUBR1 KEN box motif acts as a decoy substrate that can bind CDC20, but cannot be ubiquitinated [65, 157].

While it is well established that SAC activation at unattached kinetochores leads to rapid formation of MCC, MCC can also form during interphase. The MAD1-MAD2 complex itself is cell cycle independent and localizes to the nuclear envelope during interphase. There is evidence that nuclear envelope-localized MAD1-MAD2 can convert

MAD2 from O to C conformation and that MCC can assemble during interphase. These

18 observations have prompted a “cell timer” theory where interphase MCC acts as a buffer against premature mitotic exit while the kinetochore is assembled [158-161].

1.6 Additional SAC Components

Despite the core requirements for MAD1-MAD2 signaling and MCC formation, there are many other proteins that support SAC activation and mitotic arrest. One example is the RZZ complex [117, 162-166]. RZZ was first identified in and is conserved only in metazoans. RZZ itself is a three-protein complex composed of Rod,

Zw10 and Zwilch. RZZ localizes to kinetochore pools of BUB1. BUB1 RNAi can mislocalize RZZ from kinetochores and a minimal region of BUB1 spanning from 437-

521 can rescue RZZ localization to kinetochores. This binding region is immediately juxtaposed to the BUBR1 binding region on BUB1 [167, 168]. Depletion of the RZZ subunit ZW10 during mitosis prevents prolonged kinetochore localization of MAD1 and

MAD2. Interestingly, ZW10 and RZZ are dispensable for initial recruitment of MAD1 and MAD2 to kinetochores, but essential for maintaining significant pools of MAD1 and

MAD2 at kinetochores during prolonged periods of SAC activation [166, 169, 170].

Additionally, depletion of ZW10 causes chromosome alignment defects through defective recruitment of the microtubule motor protein to kinetochores [171-173].

Another protein essential for SAC activation and maintenance is the kinesin

CENP-E [174, 175]. CENP-E depletion in Xenopus egg extracts supplemented with sperm nuclei and nocodazole mislocalize MPS1, BUB1, BUB3, MAD1, and MAD2

[176]. CENP-E localizes to unattached kinetochores through direct interaction with the pseudo-kinase domain of BUBR1. Once localized, its kinesin activity slides

19 chromosomes along microtubule filaments to move chromosomes to the spindle midzone, resulting in metaphase plate formation [177-183].

1.7 Regulation of SAC by mitotic kinases

While the previously described proteins support proper mitotic signaling, the activity of these SAC activation proteins is modulated by the activity of mitotic kinases.

The primary kinase driving mitotic signaling is the cyclin B-CDK1 complex. Active

CDK1 phosphorylates Serine/Threonine-Proline motifs [S/T]-P-X-[R/K] on multiple mitotic substrates [184, 185]. These phosphorylation events prevent premature mitotic exit by activating SAC through multiple pathways. For example, CDK1 directly phosphorylates MPS1 at Serine 283, which is juxtaposed to MPS1 catalytic domain. This modification directly elevates MPS1 activity [186]. CDK1 phosphorylates Borealin and

Survivin to promote Shugoshin binding. This upregulates CPC localization to the centromere (discussed in Appendix A) [187, 188]. CDK1 also phosphorylates Haspin kinase directly. This promotes Haspin-Plk1 interaction which upregulates Haspin kinase activity and CPC localization [189, 190]. CDK1 also promotes SAC activation by inhibiting SAC silencing phosphatases. CDK1 directly phosphorylates protein phosphatase 1 (PP1) subunits, preventing SAC silencing [191].

Another kinase essential for SAC activation is MPS1 [116, 192]. MPS1 was initially identified as a regulator of spindle pole duplication but has since been implicated in the kinetochore localization and activity of key SAC proteins such as MAD1, MAD2,

BUB1, and CENP-E. MPS1 phosphorylates KNL1 at Methionine-Glutamic Acid-

Leucine-Threonine (MELT) repeats to recruit BUB3-BUB1, and BUB3-BUBR1 complexes [104-107, 193-195]. Additionally, MPS1 phosphorylates MAD1 directly and

20 coordinates MAD1 domain interactions during MAD2 O-C conversion [120, 142-144,

196, 197]. Interestingly, MPS1 engages in autophosphorylation and can phosphorylate other copies of MPS1 [198-201].

A third mitotic kinase that activates the SAC at outer kinetochores is BUB1.

BUB1’s N-terminal domain complexes with BUB3 through GLEBS motif and binds to

MPS1 phosphorylated MELT repeats on KNL1. KNL1 bound BUB1 recruits SAC activation machinery such as MAD1, MAD2, BUBR1, CDC20, and RZZ. Depletion of

BUB1 prevents the kinetochore localization of these proteins and abrogates SAC [176,

202-204]. BUB1 C-terminal kinase domain phosphorylates T120 on Histone H2A. This phosphorylation recruits Shugoshin protein to the inner centromere. Kinase dead BUB1,

T120 H2A mutants, as well as Shugoshin and Borealin depletion all cause comparable chromosome segregation defects. Artificial targeting of CPC to the centromere bypasses the requirement for histone phosphorylation by BUB1 [187, 205].

1.8 Molecular Components of SAC silencing

The molecular components of SAC activation prevent cell division until a fully formed spindle has attached to chromosomes at the metaphase plate. Once the kinetochores are properly attached by spindle microtubules, the SAC must be silenced for the cell to initiate anaphase and finish mitosis. SAC silencing requires the cessation of further MCC formation at kinetochores, and disassembly of MCC and MCC-APC/C complexes.

Multiple molecular mechanisms have been identified for SAC silencing at kinetochores. These mechanisms can be broadly categorized into phosphatase activity that antagonizes mitotic kinases, and dynein mediated removal of outer kinetochore

21 components to spindle poles (see below). There is also evidence that microtubule capture itself abrogates SAC activation. In budding yeast, microtubule capture by NDC80 directly blocks MPS1 kinase from accessing the yeast KNL1 homolog SPC105 [206-

208].

A key phosphatase contributing to SAC silencing at kinetochores is protein phosphatase 2A (PP2A). PP2A is composed of catalytic, scaffolding, and regulatory subunits. During SAC silencing, the B56 subunit promotes localization to kinetochores and confers specificity towards substrates [209]. PP2A-B56 antagonizes MPS1 phosphorylation of MELT repeats on KNL1. This promotes the removal of BUB1 [210].

PP2A also antagonizes its own kinetochore localization through direct dephosphorylation of BUBR1 [209, 210]. PP2A activity towards these substrates is inhibited by Greatwall kinase to prevent premature mitotic exit. [211-213].

In addition to PP2A, Protein Phosphatase 1 (PP1) antagonizes SAC activation at kinetochores. PP1 is recruited to KNL1’s highly conserved RVSF motif. This localization is downregulated by Aurora B and upregulated by PP2A [214-217]. PP1 localization to

RVSF is essential as RVSF mutants are non-viable in embryos and result in metaphase arrest after mature spindle formation in cell culture [105, 217, 218]. Once localized to kinetochores PP1 antagonizes Aurora B and MPS1 signaling. Like PP2A, PP1 dephosphorylates MELT repeats on KNL1. This dephosphorylation mislocalizes BUB1 and BUB1 associated SAC activation machinery. PP1 can also dephosphorylate BUB1 which abrogates MAD1 binding. PP1 also antagonizes Aurora B phosphorylation of

NDC80 and antagonizes Aurora B localization to centromeres by dephosphorylating

22

Threonine 3 on Histone 3. These combined phosphatase events stabilize kinetochore microtubule attachments [87, 219-221].

The third major SAC silencing pathway at kinetochores is the dynein mediated streaming of SAC machinery away from kinetochores [131, 222]. The RZZ component

Zw10 interacts directly with dynactin, Spindly, and phosphorylated dynein light and intermediate chains [164, 170, 223-225]. During SAC silencing, dynein transports the

SAC components MAD1 and MAD2 to the spindle poles and inhibition of dynein results in partial retention of MAD1 and MAD2 at metaphase kinetochores [226, 227]. Despite this, both MAD1 and MAD2 dissociate from kinetochores in dynein deficient backgrounds, probably through the activity of the kinetochore associated phosphatases described above.

Phosphatase and dynein mediated removal of SAC machinery from kinetochores is only one facet of checkpoint silencing. For mitotic exit to proceed, the APC/C must be freed from MCC inhibition in the cytoplasm. Recent evidence has implicated the APC15 mediated ubiquitination of CDC20 in dissociation of APC/C from the MCC. During the transition to mitotic exit CDC20 is ubiquitinated before APC/C activity towards other substrates [228]. This is the result of the APC/C subunit APC15 (MND2 in yeast) [229-

231]. Crystal structure analysis of the APC/C-MCC super complex indicates that binding of APC15 to APC/C-CDC20 promotes a small change in CDC20 orientation that exposes

Lysine residues 485 and 490 for ubiquitination. At the cellular level, depletion of APC15 prolongs mitotic arrest at the metaphase-anaphase boundary. This delay in mitotic progression can be rescued by depleting MCC components. Depletion of APC15 with

23

RNAi in cells directly prevents CDC20 ubiquitination and results in defective cyclin B and securin degradation and defective MCC-APC/C dissociation. Similar defects are seen when CDC20 is replaced by mutants with lysine residues mutated to alanine. [228, 229,

231-233]. Despite the prolonged mitotic duration, cells lacking APC15 still finish mitosis and the prolonged mitotic arrest seen in APC15 deficient background is not as severe as the delay seen after depletion of core APC/C components. These data suggest that APC15 promotes dissociation of MCC from APC/C to promote mitotic exit but the persistence of

MCC allows for reassociation. In keeping with this, recent studies indicated that p31comet and TRIP13 (discussed more thoroughly below) synergistically silence the SAC by dismantling free MCC after it is removed from APC/C [233, 234]. It is also worth noting that the activity of APC15 is directly antagonized by Ubiquitin Specific Protease 44

(USP44). USP44 deubiquitinates CDC20 and allows for reassembly of MCC-APC/C super complexes to maintain mitotic arrest [235-237].

1.9 The Role of TRIP13 and p31comet in SAC Silencing

MCC must be disassembled prior to anaphase onset. As discussed previously, APC15 is able to dissociate MCC from APC/C but is unable to disassemble free MCC. Cellular and biochemical evidence has implicated p31comet and TRIP13 as SAC silencers that disassemble free MCC by extracting MAD2 from MCC and catalytically converting it to the open, checkpoint inactive conformation (Figure 1-9).

24

Figure 1-9: TRIP13 and p31comet disassemble MCC by converting C-MAD2 to O-MAD2. During SAC silencing, TRIP13 utilizes p31comet as an adaptor protein to catalyze MAD2 conformational changes from Closed, active C- MAD2 to Open, inactive O-MAD2. This conformational change requires TRIP13 AAA-ATPase activity and results in MCC disassembly. Image taken with permission from [238].

The first evidence supporting TRIP13 and p31comet in SAC silencing was obtained when p31comet (initially termed “Caught by MAD Two” (CMT2)) was found to interact directly with MAD2 in yeast two hybrid screens [239]. Additionally, p31comet localizes to kinetochores in a MAD2 dependent manner and has similar FRAP exchange rates with the dynamic pool of MAD2 at kinetochores. P31comet RNAi does not mislocalize MAD2 from kinetochores [240-244]. In cancer cells, high expression of p31comet correlates with increased incidents of mitotic slippage, CIN, and lowered susceptibility to antimitotic drugs like nocodazole and taxol [245-247]. Furthermore, induced overexpression of p31comet promotes premature securin and cyclin B degradation and p31comet RNAi prolongs mitosis at the metaphase-anaphase transition, and prolongs securin and cyclin B degradation. This delay results in increased levels of MAD2-CDC20 interaction [241- 25

244]. In vitro binding assays of p31comet and MAD2 indicate that p31comet only binds the

Closed SAC active conformation of MAD2. Additionally, crystal structure analysis of p31comet indicates that p31comet and C-MAD2 share similar folding patterns. p31comet shares a similar dimerization domain to MAD2 and mutation of these residues (Q83A and F199A) abrogates MAD2 binding. p31comet also has a non-functional safety belt motif in contrast to MAD2 [248]. Further biochemical evidence indicates that p31comet competes with O-MAD2 for access to C-MAD2 during checkpoint signaling [126, 127,

241].

Despite the abundance of evidence supporting p31comet in checkpoint silencing, it does not completely answer how free MCC is disassembled. Multiple reports have shown that mitotic exit and MCC disassembly requires ATP hydrolysis [249, 250]. Since that discovery multiple publications have identified Thyroid Hormone Receptor Interacting

Protein 13 (TRIP13) as the enzyme responsible for MCC disassembly by catalyzing

MAD2 C to O conformation. TRIP13 is an ATPase associated with diverse cellular activities (AAA-ATPase). These proteins have highly conserved Walker A and Walker B

ATPase domains and sensor motifs that couple ATP hydrolysis to the assembly and disassembly of protein complexes. This form of ATPase activity allows for non- destructive recycling of protein complexes by causing small reversible structural and conformational changes in proteins. [251-254].

TRIP13 was first implicated in mitotic regulation when it was identified as a kinetochore associating protein that interacts directly with p31comet [88, 255-259]. Similar to p31comet depletion, TRIP13 immunodepletion or RNAi prevented cyclin B degradation and mitotic exit, and prolonged the interaction of MCC components. Furthermore,

26 overexpression of TRIP13, but not the K185T Walker-A ATPase deficient mutant caused premature cyclin B degradation and caused the dissociation of MAD2 from nuclear envelopes during interphase [259]. Prometaphase cell extracts with excess recombinant

TRIP13, p31comet, and ATP promoted MCC disassembly and cyclin B degradation. This accelerated disassembly was not seen with the addition of non-hydrolysable ATP analogues. Additionally, in vitro incubation of TRIP13 and p31comet with MAD2 or recombinant MCC promotes stable TRIP13- p31comet interaction and TRIP13 and p31comet can be detected in CDC20 or BUBR1 immunoprecipitations after incubation with recombinant MCC and non-hydrolysable ATP analogues [233, 260, 261]. These data heavily implicate TRIP13 as a novel mitotic checkpoint silencing protein that promotes mitotic exit by disassembling MCC.

In addition to the cellular and biochemical evidence, detailed structure analysis of

TRIP13, and its C. elegans homolog Pch2 has confirmed its role in MCC disassembly through catalyzing MAD2 conformational conversion [249, 250]. The crystal structure and cryo-EM of TRIP13 revealed a hexamer with the sensor motifs and ATPase domains in the central channel (Figure 1-10). This is similar to traditional AAA-ATPases [238].

When TRIP13 was incubated with p31comet, MAD2 and ATP, the subsequent ATP hydrolysis moved the substrate binding pore loops of TRIP13 axially further into the central channel of the hexamer [139, 238]. This suggests that subsequent ATP hydrolysis reactions could pull MAD2 substrate farther into the enzymatic pocket of the hexamer.

27

Figure 1-10: TRIP13 AAA-ATPase forms a planar hexamer: Cryo-EM analysis of TRIP13 AAA-ATPase after incubation with the non- hydrolysable ATP analog ATP-γ-s. Individual monomers are labeled in different colors and labeled A-F. Image taken with permission from [262].

28

Further crystal structure analysis and enzymatic assays revealed that the enzymatic channel of the TRIP13 hexamer engages the N-terminus of MAD2 (Figure 1-

11) and the MAD2 ΔN10 mutant cannot stimulate TRIP13 ATPase activity. This suggests that MAD2 NTD is the target of TRIP13 ATPase activity. Additionally, the

MAD2 ΔN10 construct elutes like C-MAD2 and can bind MCC and MAD1 in vitro, but is defective in maintaining SAC activity in cells depleted of endogenous MAD2. This suggests that TRIP13 ATPase activity towards MAD2 is critical for MAD2 function in

SAC signaling [139, 262, 263]. Interestingly TRIP13 enzymatic activity triggers a conformation change in the extreme C-terminal safety belt motif. This indicates that

TRIP13 ATPase activity towards MAD2 NTD causes a conformation change in the CTD

[238]. This mechanism of partial unfolding to trigger conformational change across an entire protein is conserved among other AAA-ATPases. The N-terminal residues of

MAD2, and the C-terminal safety belt β-sheets are conserved features in other substrates of TRIP13 and are more commonly referred to as HORMA domain containing proteins

(HOP1, REV7, MAD2) [139, 238, 262-264].

The structural studies on TRIP13 also clarified the role of p31comet in TRIP13 stimulated MAD2 conversion. p31comet binds to TRIP13 through the N-terminus of two

TRIP13 monomers, thus stabilizing the oligomer. This p31comet binding interface for

TRIP13 is on the opposite side of p31comet away from the conserved MAD2 dimerization interface. Mutation of the p31comet motifs for TRIP13 or MAD2 interaction inhibits

TRIP13 ATPase activity towards MAD2 in vitro. The simultaneous binding of MAD2 and TRIP13 to p31comet positions the N-terminus of MAD2 near the central channel of

TRIP13 for enzymatic conversion. During the remodeling, two key changes take place.

29

The first is in the central channel of the TRIP13 hexamer. TRIP13 sensor motifs interact with MAD2 residues 2-6 to pull the MAD2 NTD into the central channel of TRIP13. At the same time, the ATPase activity of TRIP13 shifts the hexamer itself, promoting a conformational change in p31comet. This change pushes against MAD2 while MAD2’s

NTD is being pulled by the central channel of TRIP13. These two combined forces on

MAD2 destabilizes the MAD2 C-terminal safety belt motif resulting in the MAD2 conformational change [139, 238, 262, 263].

30

Figure 1-11: TRIP13 pore loop residues engage the N-terminus of MAD2. The TRIP13 hexamer (left image) binds p31 (purple) and MAD2 (blue). The C-terminal “safety belt” motif of MAD2 (red) and CDC20 binding motif is exposed. The N-terminal region of MAD2 (blue) is positioned in the central channel of the TRIP13 hexamer. The N-terminal residues are positioned next to and interact with residues from TRIP13 poor loops (K220, W221, D272, E269) (right image). Images taken with permission from [262].

31

1.10 TRIP13 functions as a SAC activator

The cellular, biochemical, and structural data firmly established the role of

TRIP13 in SAC silencing. Paradoxically, recent evidence has also implicated TRIP13 as a mitotic checkpoint activator. In C. elegans, depletion of the TRIP13 homolog PCH2 causes mitotic arrest defects and defective kinetochores localization of MAD2. The defect in MAD2 localization was comparable to the defect seen in MAD1 RNAi backgrounds [265]. In human cells, constitutive knockout of TRIP13 with CRISPR resulted in an accumulation of C-MAD2 in cells and a defect in mitotic arrest in the presence of nocodazole. The C-MAD2 accumulation and defect in mitotic arrest were not seen in cells with CRISPR knockout of p31comet [266, 267]. This C-MAD2 accumulation and defect in mitotic arrest was also observed upon prolonged auxin induced degradation of TRIP13. In this background, depletion of TRIP13 also resulted in more p31comet associating with MAD1 and less MAD2 interacting with CDC20, indicating a partial defect in MCC assembly [233, 267, 268]. The checkpoint defect observed upon TRIP13 depletion could be rescued by overexpressing wild type MAD2, which adopts an open checkpoint inactive conformation upon synthesis [233, 265-269].

1.11 Evolutionary divergence of TRIP13-p31comet and SAC signaling

Genomic sequencing indicates that microtubule – kinetochore mediated chromosome segregation is conserved across all eukaryotic taxa. Despite the functional conservation of spindle mediated chromosome segregation, there is significant variation in conservation of SAC genes [117, 118]. Indeed, there are only a select few SAC genes conserved across all eukaryotic taxa. These genes include core genes for centromere formation, core CPC components for correcting improper microtubule kinetochore

32 attachments, core kinetochore components for microtubule capture, and CDC20 with core

APC/C subunits for promoting mitotic exit [117, 118].

Beyond these core requirements there is significant variation in secondary regulatory mechanisms that control chromosome segregation. MCC formation is one of these secondary regulatory mechanisms. MCC components and MAD1 are well conserved in most eukaryotic organisms but there is a significant number of simple eukaryotic taxa that do not contain these proteins. This suggests that MCC formation contributes to protecting the integrity of more complicated genomes in higher eukaryotes

[117, 118]. Given the variation in conservation of MCC and SAC signaling genes it is not surprising that there is significant divergence of TRIP13-p31comet-MAD2/HORMA proteins. TRIP13-p31comet is conserved in most multicellular eukaryotes. Taxa with significant divergence include but are not limited to fungi and eukaryotic parasites. In these taxa it is not uncommon to have TRIP13 homologs but no p31 homologs, suggesting TRIP13 may have p31comet independent function. In contrast, with the exception of some photosynthetic plankton, there are no observable taxa that have p31comet but not TRIP13 homologs, suggesting p31comet function requires TRIP13. In other taxa like fission yeast and eukaryotic obligate animal parasites, there is often no

TRIP13 or p31 homologs at all despite having MAD1-MAD2 signaling or other HORMA domain containing proteins. This divergence suggests that MAD1-MAD2 signaling and

HORMA domain regulation does not necessarily require TRIP13 or ATPase activity for viability [117, 118].

While TRIP13 might not be necessary for viability in lower eukaryotes, there are almost no eukaryotic taxa that have TRIP13 but not MAD2 or other HORMA domain

33 containing proteins. This suggests that TRIP13 functions in MCC signaling and HORMA signaling pathways with or without p31. In keeping with this, other MCC regulatory proteins are poorly conserved across taxa. One good example is APC15. APC15 is conserved in most multicellular eukaryotes but is absent in plants and most unicellular eukaryotic organisms [117, 118]. These observations collectively suggest that TRIP13 coordinates MCC and HORMA signaling in higher eukaryotes and this signaling can be p31 independent.

1.12 Central Hypothesis for the Role for TRIP13 but not p31comet in SAC activation

Both TRIP13 and p31comet are enriched at unattached kinetochores where the mitotic checkpoint is activated, despite their established roles in mitotic checkpoint silencing. While we were analyzing the kinetochore localization of TRIP13 and p31comet, several publications came out supporting the roles of TRIP13 in mitotic checkpoint activation [233, 265-267]. Currently, these results show that TRIP13 depletion leads to accumulation of C-MAD2 and a defective checkpoint. The publications on TRIP13 in

SAC activation suggest that TRIP13 supplies a pool of O-MAD2 substrate at mitotic onset. Such a model for TRIP13 in SAC activation is incongruent with previous data. The first paradox is that all available evidence supports that C-MAD2 is the checkpoint active conformation that can bind MAD1 and form MCC. Therefore, the C-MAD2 accumulation seen after TRIP13 depletion should not result in a checkpoint defect. The second paradox is that newly synthesized MAD2 folds in O conformation [268], therefore it is surprising that even in TRIP13 depleted cells there is not enough O-MAD2 supply. Third, the current model does not explain how TRIP13 but not p31comet supports checkpoint activation. As previously discussed, cytoplasmic p31comet participates in MCC

34 disassembly by partnering with TRIP13 to convert MAD2 to the open conformation during SAC silencing [238, 242, 243, 245, 250, 260, 261]. This creates a disparity where p31comet is both required for and dispensable for MAD2 conversion. Finally, the

“cytoplasmic MAD2 maintenance model” does not explain why TRIP13 and p31comet localize to unattached kinetochores during prometaphase [242, 259]. These combined observations led us to hypothesize that TRIP13 has a separate and p31comet-independent function in activating the SAC at unattached kinetochores. Here we present evidence supporting this hypothesis. We will show that TRIP13 but not p31comet supports SAC activation, and that the SAC activation role of TRIP13 is through coordination with kinetochore associated MAD1 to promote MAD2 localization and O to C conversion for

MCC assembly.

35

Chapter 2

Materials and Methods

Generation of AID-NEON- p31comet and AID-HA-TRIP13 lines:

AID-HA-TRIP13 an AID-NEON- p31comet cell lines were created and provided by Dr. Vasilisa Aksenova from Dr. Marry Dasso’s lab for collaborative research. To create these constructs, endogenous TRIP13 and p31comet loci in DLD1 cells were targeted with CRISPR-Cas9 genome editing. AID-NEON- p31comet and AID-HA-TRIP13 rescue constructs were stably incorporated in place of the endogenous loci.

Cloning and construct generation:

DNA constructs were generated using Gateway recombination cloning.

Constructs were subcloned from pENTR-D-TOPO vectors to destination vectors using L-

R clonase reactions (Invitrogen). To generate mCherry-Mis12-MAD1 and mCherry-

Mis12-TRIP13 fusion constructs, their sequences were amplified by PCR. Primer sequences for amplification are available in Appendix C. Primers were designed with

NotI and EcoRI restriction sites for directional insertion into mCherry-Mis12 vector originally developed in [123]. TRIP13 was additionally modified with site directed

36 mutagenesis to remove an internal EcoR1 restriction site. Ligations were performed with

T4-DNA Ligase with gel-purified vector and insert sequences. Restriction enzymes and

DNA ligase were purchased from New England Biolabs.

Cell Culture – DNA and RNAi Transfections – and AID Depletion:

HeLa and AID-DLD1 lines were incubated with DMEM containing fetal bovine serum (10%) and Hyclone non-essential amino acid solution (1%). Cell cultures were incubated at 37º Celsius and 5% CO2.

For DNA transfections, DNA constructs were incubated in Opti-MEM serum free medium (Gibco) with Polyethylenimine [238] for twenty minutes at room temperature as described in [270]. All DNA transfections were performed 24 hours prior to imaging.

Gene knockdown with RNAi was achieved by siRNA transfections. Cells were transfected with siRNAs twice over a 48-hour period. The transfections were performed at the 0-hour and 24-hour mark in fresh medium. During transfection siRNAs (100nM) and Oligofectamine (Invitrogen) were pre-incubated separately for five minutes in Opti-

MEM serum free media (Gibco). The separate RNAi and Oligofectamine suspensions were then combined and allowed to incubate for twenty minutes before transfection. All siRNA sequences can be found in Appendix C.

Inducible protein degradation in AID cell lines was performed with Indole Acetic

Acid (Auxin) (Sigma Aldrich) treatment at 1mM for indicated time periods in figure legends. Fresh media and Auxin were added daily in all experiments

Live Cell Imaging:

For mitotic index and mitotic duration assays, cells were treated with or without auxin or RNAi for 48 hours before imaging as indicated in figure legends. HEPES

37

(25mM) and a mineral oil overlay were added at the onset of live cell image acquisition.

Time-lapse videos were taken using an Olympus IX81 epifluorescence microscope equipped with 37ºC weather chamber. Images were acquired in five-minute intervals in all live cell imaging experiments. Mitotic duration in all live cell videos is defined from cell rounding until cell flattening or cell death. Statistical analysis was done using

GraphPad Prism software.

Immunofluorescence:

HeLa cells and AID-DLD1 cells were treated with nocodazole (100 ng/µl) and

MG132 (20µM) for four hours then fixed in freshly prepared 3.5% paraformaldehyde at pH 6.6 - 6.9 for seven minutes. Cells were then cleared with KB buffer (10 mM Tris-

HCl, pH 7.5, 150 mM NaCl, 1 mg/ml BSA) containing 0.5% Triton X-100 for five minutes. Cells were then blocked with KB buffer without Triton X-100 for an additional

10 minutes. The coverslips were then incubated with primary antibodies at 37°C for 30 minutes in a wet chamber. Cells were rinsed for five minutes with KB three times and then incubated with corresponding secondary antibodies for 30 minutes at 37°C. All primary and secondary antibodies used for immunofluorescence are listed in Appendix

C-3. Coverslips were then washed with KB again for five minutes three times and mounted on slides with VectaShield mounting medium containing DAPI (Vector

Laboratories). Mounted coverslips were imaged on a Leica TCS SP8 confocal microscope with a 63 × objective (numerical aperture = 1.40). Z-stacks of 1.0μm were collected and maximum projections of all planes are presented unless otherwise stated in figure legends. For intensity measurements, kinetochore signals of the indicated primary antibody and ACA antibody were collected and background signals were subtracted.

38

Background adjusted kinetochore intensities were normalized to their respective ACA intensities and the normalized values were graphed. All images for comparison were acquired and processed in the same manner. Statistical analysis of normalized kinetochore intensities was done using GraphPad Prism software.

Recombinant Protein Purification:

Recombinant proteins were expressed in E. coli BL21 (DE3) Codon Plus RIPL bacteria (Stratagene). His and GST tagged proteins were expressed using Gateway pDEST-17 and pDEST-15 recombination vectors. Expression of recombinant proteins was induced with 0.5 mM IPTG (Goldbio) once bacterial cultures reached OD600 of 0.5 to 0.7. Induced bacteria expressing His-tagged proteins were pelleted and incubated with lysis buffer for thirty minutes on ice (50 mM Tris pH 7.5, 300 mM NaCl, 0.1% TritonX-

100, and 10 mM Imidazole). The bacterial suspension was sonicated on ice in twenty second intervals for a total of two minutes. Forty second breaks were included between each twenty second sonication interval. Sonicated lysates were clarified by centrifugation at 14,000 rpm for twenty minutes in a Beckman Coulter Avanti J25 centrifuge and

JLA16.250 rotor. The supernatant fractions were run through gravity column containing

ProBond nickel beads (Novex). Flow-through was re-run through the column. The resin was then washed three times with the total column volume of wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.1% TritonX-100, and 30 mM Imidazole). 1.5ml fractions were eluted with His elution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.1% TritonX-100, and 150 mM Imidazole).

Induced GST-tagged constructs were pelleted and lysed for thirty minutes on ice

(50 mM Tris pH 7.5, 300 mM NaCl, 0.1% TritonX-100, and 1 mM DTT). The bacterial

39 suspension was sonicated and clarified as described for His tagged protein purification.

The supernatant fractions were run through a gravity column containing Pierce

Glutathione Agarose Beads (Thermo Scientific). Flow-through was re-run through the column. The resin was then washed three times with the total column volume of wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.1% TritonX-100, and 1 mM DTT). 1.5 ml fractions were eluted and collected with GST elution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.1% TritonX-100, and 15 mM GSH).

The purity of final products was assessed using Coomassie staining. Peak fractions were concentrated and subject to buffer exchange into storage buffer (20 mM

Tris pH 7.5, 100mM KCl, 1 mM TCEP, 10% glycerol). Protein concentration was determined by comparing Coomassie intensity to BSA standards in Image J software.

Protein binding assays:

Four microliters of 5× binding buffer (100 mM Tris-HCl, pH 8.0, 750 mM NaCl,

2.5% NP-40, 50 mM MgCl2, 50% glycerol) was mixed with recombinant GST and His- tagged proteins in 20 µl reactions. Protein concentrations in reactions are provided in figure legends. Reactions were incubated at 37ºC for one hour. 10 µl 50% GSH bead slurry (Thermo Scientific) pre-treated with 1% BSA were added to reactions and lightly vortexed at 4ºC for 1 hour. The beads were washed with GSH wash buffer three times (50 mM TRIS pH 8.0, 150 mM NaCl, 1 mM TCEP, 1 mM EDTA). Reactions were then analyzed by Western blotting.

Immunoprecipitations:

Cell lysate (normally 300 µg) was diluted to 300 µl in cell lysis buffer (1× PBS, 10% glycerol, 0.5% NP40) supplemented with Protease Inhibitor Mixture (Calbiochem) and

40 phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, 60 mM β-glycerophosphate).

Antibodies against target proteins or IgG control antibodies were added to the diluted lysate and rotated at 4ºC for an hour. Protein A-agarose beads pretreated with 1% BSA in

1× PBS were added and rotated for another hour. Beads were then washed three times with IP wash buffer (250 mM NaCl, 10% glycerol, 0.5% NP40). Reactions were then analyzed by Western blotting.

41

Chapter 3:

TRIP13 but not p31comet Activates the Spindle Assembly Checkpoint

3.1 Introduction

Our lab was the first to identify TRIP13 AAA-ATPase as a novel SAC silencing protein that works with its adaptor p31comet to disassemble the MCC during the metaphase-to-anaphase transition [259]. Interestingly, TRIP13 and p31comet also localize to unattached kinetochores during prometaphase when the SAC is active [242, 259]. The unexpected localization led us to hypothesize that TRIP13 and p31comet play a distinct role during prometaphase to help activate the SAC. We tested this hypothesis by probing the effects of TRIP13 and p31comet depletion on mitotic arrest and mitotic duration. We found that TRIP13 but not p31comet was required for mitotic arrest.

3.2 Results

To address how TRIP13 and p31comet might activate the mitotic checkpoint, we exploited auxin inducible degradation constructs (AID). Proteins tagged with an AID motifs are recognized by a ubiquitin ligase complex for rapid ubiquitination and

42 degradation [271] (Figure 3-1). Using CRISPR mediated knock-in, Dr. Vasilisa

Aksenova from Dr. Mary Dasso’s lab at NIH established two DLD1-AID cell lines. One cell line had both alleles of p31comet replaced with AID and mNeon Green fluorescent protein tagged p31comet. The second DLD1 line had both alleles of endogenous TRIP13 replaced with HA-AID tagged TRIP13.

We first confirmed that these constructs could rapidly degrade TRIP13 and p31comet. We treated these AID-tagged cell lines with auxin for three hours and probed for

AID-TRIP13 and AID-p31comet by western blot. TRIP13 and p31comet signals were below detectable levels within three hours of auxin treatment compared to untreated cells

(Figure 3-1). We then tested the checkpoint responses of these cells. AID-NEON- p31comet and AID-TRIP13 DLD1 cells were treated with auxin for 48 hours. We then measured mitotic duration in the presence of the spindle poison nocodazole. AID- p31comet cells showed no change in mitotic arrest duration when incubated with nocodazole whereas AID-TRIP13 cells treated with auxin arrested for an average of 35 +

9 minutes compared to 402 + 243 minutes without auxin (Figure 3-2). This indicates that depletion of TRIP13 causes a defect in SAC activation. To further confirm this, we treated AID- p31comet and AID-TRIP13 DLD1 cells with auxin for 48 hours, then added nocodazole and monitored mitotic indices. Only 5% of AID-TRIP13 cells treated with auxin were arrested in mitosis after twenty-four hours in nocodazole. Approximately 75% of AID-TRIP13 cells without auxin treatment were arrested in mitosis after nocodazole treatment. Again, cells depleted of p31comet could arrest in mitosis at similar rates to cells without auxin treatment (Figure 3-3). As a control for mitotic arrest, we immuno-stained cells from Figure 3-3 after imaging with anti-histone H3 phospho-serine-10 antibody.

43

This histone modification is specific to mitosis [272]. TRIP13 but not p31comet depletion, resulted in loss of phospho-serine-10 signals (Figure 3-4). To further confirm that our observations were not artifact from AID modification, we also depleted TRIP13 and p31comet with RNAi in HeLa cells and measured mitotic indices. TRIP13 RNAi caused a comparable mitotic arrest defect to depletion of TRIP13 with auxin. Depletion of p31comet with RNAi did not impact mitotic arrest (Figure 3-5).

44

3-1

Figure 3-1: Auxin Inducible Degradation of TRIP13 and p31 in DLD1 cells. (Left) Auxin signaling diagram was taken with permission from [1]. Auxin inducible degrons (AID) allow for rapid depletion of tagged proteins by TIR1 ubiquitin ligase in the presence of plant hormone auxin. (Right) CRISPR-Cas9 mediated replacement of TRIP13 and p31comet loci with AID-tagged variants allowed for total depletion of TRIP13 and p31comet protein levels within three hours of auxin exposure. Shown are western blots of tagged TRIP13 and p31comet, and other SAC proteins. Actin was used as a loading control.

45

3-2 3-3

3-4

46

Figure 3-2 - 3-4: AID-TRIP13 but not AIDp31comet depletion prevents mitotic arrest. 3-2 Mitotic duration of TRIP13 and p31comet-AID DLD1 cell lines in the presence or absence of 1 mM auxin for 48 hours prior to imaging. Nocodazole (100ng/ul) was then added and cells were imaged as described in materials and methods for mitotic duration. Unpaired t- tests were used for statistical analysis. “NS” = not significant. *** represents P<0.001. Error bars represent SEM. N=20 in all conditions. 3-3 Mitotic index of TRIP13 and p31comet AID DLD1 cell lines in the presence or absence of 1 mM auxin for 48 hours prior to imaging. Nocodazole (100ng/ul) was then added and cells were imaged as described in materials and methods for mitotic indices. Error bars represent SEM from triplicate experiments. N > 175 cells per replicate 3-4: Validation of mitotic arrest of TRIP13 and p31comet-AID DLD cell lines in the presence or absence of 1 mM auxin. After mitotic index imaging, cells from figure 3-3 were stained with antibodies for phosphorylated Serine 10. Images presented are overlays of brightfield and phosphorylated Serine 10 fluorescent signals. Scale = 50 µM

47

3-5

Figure 3-5: TRIP13 but not p31 RNAi abrogates mitotic arrest. Mitotic indices of HeLa cells were calculated after TRIP13, p31comet, or MAD2 RNAi. HeLa cells were synchronized with a double thymidine block (2mM) and simultaneously treated with indicated RNAi constructs for 48 hours. Nocodazole was added eight hours post thymidine release and mitotic index measurements were collected. “WT” indicates cells that were not treated with siRNA. Graph represents results from single replicate with N > 175 cells. Two experimental replicates were performed.

48

Chapter 4

TRIP13 ATPase activity and MAD2 expression rescues mitotic arrest defect after TRIP13 depletion.

4.1 Introduction

After confirming that TRIP13 but not p31comet abrogated mitotic arrest we sought to determine how TRIP13 could abrogate the checkpoint. During checkpoint silencing,

TRIP13 ATPase activity is required to disassemble MCC by extracting and inactivating

MAD2 [238, 259]. However, TRIP13 depletion also causes a build-up of C-MAD2 and a checkpoint arrest defect [259, 261, 273]. The combined data suggested that TRIP13

ATPase activity is required for SAC activation. We also address how a buildup of C-

MAD2 could abrogate the checkpoint since C-MAD2 is the SAC active conformation

[33, 147]. My results indicate that TRIP13 ATPase activity or overexpression of C-

MAD2 can rescue the checkpoint defect caused by TRIP13 depletion.

49

4.2 Results

We first determined if TRIP13 ATPase activity was required for mitotic arrest. To test this, we depleted TRIP13 in TRIP13-AID cells with auxin for 48 hours and tried to rescue the defect with overexpression of GFP-TRIP13 wild type (WT) and the ATPase deficient Walker A and B motif mutants (K185T and E253Q respectively) in the presence of nocodazole. As shown in figure 4-1, auxin treated AID-TRIP13 cells could stay in mitosis for only 49 + 31 minutes in nocodazole compared to 343 + 244 minutes without auxin treatment. When GFP-TRIP13-WT was expressed in the presence of auxin and nocodazole, transfected cells showed a mitotic duration of 190 + 164 minutes. By contrast, cells transfected with GFP-TRIP13 K185T or E253Q (Walker A or B ATPase defective mutants) stayed in mitosis for 62 + 38 and 55 + 29 minutes respectively. These results suggested that TRIP13 ATPase activity is required for checkpoint activation.

GFP-TRIP13-WT expression only partially rescued the mitotic arrest defect seen in auxin treated untransfected cells. This could possibly be due to TRIP13 expression also increasing SAC silencing in the cytoplasm.

Having established that TRIP13 ATPase activity is required to activate the checkpoint we next tested if MAD2, the substrate of TRIP13 ATPase activity, could rescue the mitotic arrest defect seen after TRIP13 depletion. Multiple labs have reported that TRIP13 depletion leads to C-MAD2 accumulation and a SAC defect. These studies have also shown that expression of MAD2-WT rescues the checkpoint even though nascent translated MAD2 is assumed to adopt the checkpoint inactive “open” conformation [266, 267]. These data create a paradox where O-C conversion is essential for SAC activation, but accumulation of the product of this conversion, C-MAD2, is

50 checkpoint defective. To better understand how MAD2 rescues the checkpoint defect from TRIP13 depletion we first depleted TRIP13 with auxin, then overexpressed GFP-

MAD2 WT in the presence of nocodazole. Overexpression of MAD2 WT was able to partially rescue the checkpoint defect from auxin treatment (mitotic duration of 193 +

185 minutes in GFP-MAD2-WT transfected cells compared to 49 + 31 minutes in untransfected cells) (Figure 4-2). We next overexpressed the MAD2-L13A mutant which is locked in the closed conformation [147, 274]. Interestingly, MAD2-L13A was also able to rescue the checkpoint defect from TRIP13 depletion (mitotic duration 296 + 348 minutes). We noticed that Ma et al defined the buildup of C-MAD2 in TRIP13 depleted cells based on elution profiles in anion exchange chromatography [266, 267]. Our results with MAD2-L13A indicate that it is possible for C-MAD2 to activate the checkpoint. The disparity between these data may be due to the C-MAD2 in Ma et al being incompetent compared to the L13A mutant. We also tested the O-MAD2 locked ΔC10 mutant but it was unable to rescue the checkpoint (71 + 48 minutes). This implicates O-C conversion and C-MAD2 generation as an essential step in SAC activation since MAD2-ΔC10 overexpression is checkpoint defective. Our data also suggests that O-C conversion can by bypassed if the concentration of C-MAD2 is high enough in cells. We have made similar observations when MPS1 kinase is inactivated [275]. MPS1 is required for O-C conversion and SAC activation. Inhibition of MPS1 can be bypassed by overexpression of MAD2-L13A but not MAD2 WT.

51

4-1

52

4-2

53

Figure 4-1: TRIP13 ATPase activity rescues mitotic arrest defect from TRIP13 depletion. (Top) TRIP13-AID-DLD1 cells were treated with 1mM Auxin for 48 hours prior to imaging. Cells were transfected with GFP-TRIP13 Wild Type or Walker A or B ATPase mutants 24 hours prior to imaging. Nocodazole (100ng/µl) was then added at the onset of imaging and mitotic durations were recorded based on cell round-up and flattening time. The GFP images confirmed construct transfection and the mitotic duration of displayed cells is shown in red. (Bottom) Quantification of mitotic durations under different treatments shown above. Unpaired t-tests were used to compare indicated populations. “NS” = not significant. *** = P<.0.001, * = P<0.05. Error bars indicate SEM. N>20 transfected cells for all conditions Figure 4-2: Wild type MAD2 and L13A C-MAD2 mimic rescues mitotic checkpoint from TRIP13 depletion. (Top) TRIP13-AID-DLD1 cells were treated with 1mM Auxin for 48 hours prior to imaging. Cells were transfected with GFP-MAD2-WT or L13A or ΔC10 mutants 24 hours prior to imaging. Nocodazole (100ng/ul) was then added at the onset of imaging and mitotic durations were recorded based on cell round-up and flattening time. The GFP images confirmed construct transfection and the mitotic duration of displayed cells is shown in red. (Bottom) Quantification of mitotic durations under different treatments shown above. Unpaired t-tests were used to compare indicated populations. “NS” = not significant. *** = P<0.001. ** = P<0.01. Error bars indicate SEM. N>20 for all conditions.

54

Chapter 5

Requirements for TRIP13 and p31comet Kinetochore Localization during SAC Activation

5.1 Introduction

As described in Chapter 1, MAD2 O-C conversion takes place at unattached kinetochores through the MAD1-MAD2 heterotetrametric catalyst during checkpoint activation (Figure 1-6, 1-7, 1-8). Previous studies indicated that TRIP13 and p31comet are both enriched at unattached kinetochores [242, 259]. In C. elegans depletion of the

TRIP13 homolog PCH2 abrogates MAD2 kinetochore localization and that MAD2 or

MAD1 depletion prevents PCH2 localization [265]. Despite these findings it is still unclear how TRIP13 localizes to kinetochores, and how TRIP13 depletion affects the localization of other kinetochore proteins during SAC activation. Using fixed cell immunofluorescence of nocodazole arrested HeLa and DLD1 cells we confirmed that

TRIP13 but not p31comet depletion abrogates MAD2 kinetochore localization. We also

55 found that TRIP13 is dispensable for the localization of other MCC components. Finally, we found that BUB1 and the MCC components MAD2, BUBR1, and CDC20 are required for TRIP13 and p31comet kinetochore localization.

5.2 Results

We first depleted TRIP13 and p31comet in AID-DLD1 cells with auxin and then arrested them in nocodazole and MG132. Arrested cells were then fixed and stained for immunofluorescent analysis of kinetochore localization of checkpoint components. As reported in C. elegans, auxin mediated depletion of AID-TRIP13 reduced MAD2 accumulation at unattached kinetochores by approximately 90%. In contrast, p31comet depletion did not mislocalize MAD2 (Fig 5-1). We further confirmed this localization dependency by measuring MAD2 kinetochore localization after depleting TRIP13 and p31comet from HeLa cells with RNAi. Similar to our results with the DLD1 AID lines,

TRIP13 but not p31comet depletion abrogated MAD2 kinetochore localization by approximately 90% (Fig 5-2). Additionally, the MAD2 localization defect in TRIP13 but not p31comet depletion mirrors the mitotic arrest defects seen in our live cell imaging experiments (Figure 3-2 and 3-3).

We next tested whether TRIP13 depletion could abrogate the localization of other checkpoint components to kinetochores. We depleted DLD1 cells of TRIP13 and p31comet with auxin, then treated cells with nocodazole and MG132. We then stained for the localization of the MCC components BUBR1 and CDC20, as well as the outer kinetochore SAC catalyst MAD1. We detected no changes in the levels of these proteins after TRIP13 or p31comet depletion. We confirmed the staining profiles in HeLa cells after depleting TRIP13 and p31comet with RNAi. Again, we detected no change in the levels of

56 these proteins at unattached kinetochores (Fig 5-3 and 5-4). This suggests that TRIP13 depletion abrogates MAD2 kinetochore localization but not the localization of other checkpoint proteins.

We also tested how TRIP13 and p31comet localized to unattached kinetochores.

During SAC activation, the outer kinetochore BUB1 serves as a scaffolding protein for the recruitment of MCC components and the MAD1-MAD2 tetrameric catalyst for MCC formation [104]. Since TRIP13 is required for MAD2 localization we tested whether

BUB1 depletion could mislocalize TRIP13 from kinetochores. We depleted HeLa cells of

BUB1 with RNAi and arrested cells in mitosis with nocodazole and MG132. We then probed for TRIP13 and p31comet at unattached kinetochores using quantitative immunofluorescence. BUB1 RNAi greatly reduced kinetochore localization of both

TRIP13 and p31comet. We next tested TRIP13 and p31comet localization after RNAi of other outer kinetochore components. We depleted BUBR1, CDC20, and MAD2 by RNAi and probed for TRIP13 and p31comet localization. TRIP13 and p31comet kinetochore localization was diminished after RNAi of any of the three MCC components (Figure 5-5 and 5-6). Collectively the localization data supports our hypothesis that TRIP13 and p31comet localize to kinetochores through the BUB1 dependent pools of MCC components. More importantly, TRIP13 but not p31comet supports MAD2 kinetochore localization during SAC activation.

57

5-1

5-2

.

Figure 5-1 – 5-2: TRIP13 but not p31comet depletion abrogates MAD2 kinetochore localization. (5-1) p31comet-AID and TRIP13-AID cells were treated with 1mM Auxin for 48 hours, then with Nocodazole and MG132 for four hours. Arrested cells were fixed and stained as described in Materials and Methods. Kinetochore intensities were measured and normalized to corresponding ACA intensities. Maximum projections of 1 µM sections are presented. Scale bars = 10 µm. Error bars indicate SEM. Unpaired t-tests were used for statistical analysis. “NS” = not significant. *** = P<0.001. Graph represents single data set with N>50 kinetochores. Single data sets were repeated in triplicate. (5-2) HeLa cells were treated with TRIP13 or p31comet siRNA (100 nM) for 48 hours then examined for MAD2 kinetochore localization as in 5-1. 58 5-3

v

59

5-3 cont.

Figure 5-3: AID-TRIP13 and AID-p31comet depletion does not mislocalize BUBR1 CDC20 or MAD1 from kinetochores. p31comet-AID and TRIP13-AID cells were treated with 1mM Auxin for 48 hours, then with Nocodazole and MG132 for four hours. Arrested cells were fixed and stained as described in Materials and Methods. Kinetochore intensities were measured and normalized to corresponding ACA intensities. Maximum projections of 1 µm sections are presented. Scale bars = 10 µm. Error bars indicate SEM. Unpaired t-tests were used for statistical analysis. “NS” = not significant. Graph represents single data set with N>50 kinetochores. Single data sets were repeated in triplicate.

60

5-4

Figure 5-4: TRIP13 and p31comet RNAi do not mislocalize BUBR1, CDC20, or MAD1 from kinetochores. HeLa cells were treated 100nM siRNA for 48 hours, then with Nocodazole and MG132 for four hours. Arrested cells were fixed and stained as described in Materials and Methods. Kinetochore intensities were measured and normalized to corresponding ACA intensities. Merged projections of 1 µm sections are presented. Scale bars = 10 µm. Error bars indicate SEM. Unpaired t-tests were used for statistical analysis. “NS” = not significant. Graphs represents single data sets with N>50 kinetochores. Single data sets were repeated in triplicate.

61

5-5

62

5-6

63

5-7

Figure 5-5 - 5-6: TRIP13 (5-5) and p31comet (5-6) require BUB1 and MCC components for kinetochore localization. HeLa cells were treated with indicated siRNA (100 nM) for 48 hours, then with nocodazole and MG132 for four hours. Arrested cells were fixed and stained with corresponding antibodies as described in Materials and Methods. Single 1 µm sections are presented. Scale bars = 10 µm. Figure 5-7: Quantification of data. Kinetochore intensities were measured and normalized to corresponding ACA intensities. Error bars denote SEM. Unpaired t-tests were used to compare individual siRNA populations to control (WT) cell populations. *** = P<0.001. Graph represents single data set with N>50 kinetochores. Single data sets were repeated in triplicate.

64

Chapter 6

TRIP13 Coordinates with MAD1 during SAC Activation

6.1 Introduction

TRIP13 localizes to kinetochores and is required for SAC activation and MAD2 localization to kinetochores. Furthermore, TRIP13 ATPase activity is required for SAC activation. This suggests that TRIP13 does not simply “sit” at kinetochores to promote

MAD2 localization. Instead the data suggest that TRIP13 plays an active role in SAC.

Previous data established the MAD1-MAD2 heterotetrametric catalyst in O-C conversion and SAC activation (reviewed [111]). We hypothesized that TRIP13 activates the SAC by interacting with this MAD1 catalytic complex. To some degree the localization data in the last chapter supported this hypothesis. Previous FRAP studies have shown that

MAD2 exists in two pools at kinetochores. One pool (about 50%) toggles on and off of kinetochores and the other pool (about 50%) stably associates with kinetochores through direct interaction with MAD1 [128, 136]. Despite this, we show in figures 5-1 and 5-2 that TRIP13 depletion reduced MAD2 localization by approximately 90%. In this chapter, I report the results from our investigation into possible coordination of TRIP13

65 with the MAD1:MAD2 catalyst in converting O-MAD2 to C-MAD2. This could promote the assembly of both the MAD1:MAD2 tetramer catalyst and MCC.

6.2 Results

We tested if TRIP13 could directly interact with MAD1. Our recent study on

MAD1 suggests that MAD1 N-terminal and C-terminal domains (NTD and CTD) coordinate to convert MAD2 during SAC activation and that this coordination is driven by MPS1 kinase activity [142]. Using in vitro binding experiments with recombinant proteins we discovered that both TRIP13 and p31comet could stably interact with both

MAD1 NTD and CTD. We incubated endogenous concentrations of 6×His-TRIP13 (100 nM) or endogenous concentrations of 6×His-p31comet (100 nM) with GST tagged MAD1 fragments at three times endogenous concentration (180nM). The increased MAD1 concentration can be justified due to increased MAD1 concentration saturated at unattached kinetochores. We tested GST-MAD1-NTD, GST-MAD1-MIM (MAD2 interacting motif), GST-MAD1-CTD, and GST-MAD1-MC (MIM and CTD). GST alone was used as a negative control. TRIP13 and p31comet were able to bind directly to all

MAD1 fragments tested except the MIM (Figure 6-1 and 6-2).

We next wanted to test if these interactions could be abrogated by MPS1 activity towards MAD1. We have previously identified four residues in the MAD1 CTD (S598,

S610, T716, Y634) that could be phosphorylated by MPS1 in vitro. From these residues we found that Threonine-716 mutants could affect the mitotic checkpoint response when constitutively targeted to kinetochores through fusion to mCherry-Mis12 [142]. We tested whether the interactions between TRIP13, p31comet, and MAD1 could be modulated by these MPS1 phosphorylation sites. We examined the interactions between endogenous

66 concentrations of 6×His-TRIP13 (100 nM) or endogenous concentrations of 6×His- p31comet (100 nM) with GST-MAD1-CTD 4E, GST-MAD1-CTD-716E and GST-MAD1-

CTD-716A at three times MAD1 endogenous concentration (180 nM). TRIP13 and p31comet stably associated with both the 716A and 716E mutants compared to wild type

GST-MAD1-CTD but showed severely reduced binding to GST-CTD-4E in GST pulldowns (Figure 6-3 and 6-4). This suggests that MPS1 phosphorylation can impact the

TRIP13-MAD1 and p31comet-MAD1 interactions in vitro but not through the established

CTD T716 residue. Interestingly, we noticed that the p31comet-MAD1 interactions mirrored the TRIP13-MAD1 interactions even though results from chapter 3 and 4 suggest that TRIP13 but not p31comet impacts mitotic checkpoint strength and MAD2 kinetochore localization. This disparity is discussed more thoroughly in Chapter 7.

We next probed for TRIP13-MAD1 partnership in cells. To do this we utilized an mCherry-Mis12-MAD1 (MMM) that constitutively targets MAD1 to kinetochores.

Expression of the MMM construct is capable of arresting cells in mitosis without the use of spindle poison drugs as it uncouples SAC activation from microtubule attachment

[123]. In agreement with these previous studies the MMM construct caused extended mitotic duration in AID-TRIP13 DLD1 cells without auxin or nocodazole treatment (236

+ 208 minutes vs 50 + 23 minutes in untransfected cells) (Figure 6-5). We then depleted

TRIP13 in DLD1 cells with auxin for 48 hours and tried to rescue the SAC activation defect by overexpressing MMM. The MMM construct could not rescue the SAC activation defect from TRIP13 depletion (66 + 48 minutes).

Finally, we attempted to create an mCherry-Mis12-TRIP13 fusion construct. The fusion construct expressed at the correct molecular weight in Western blot and localized

67 to kinetochores. Despite this, the construct failed to maintain SAC arrest compared to untransfected cells (65 + 30 minutes vs 69 + 38 minutes) (Figure 6-6). This negative phenotype suggested that constitutively anchoring TRIP13 to kinetochores alone is insufficient to trigger mitotic checkpoint activation. The results in this chapter, especially

6-5 and 6-6 suggest that TRIP13 requires MAD1 at unattached kinetochores to present

MAD2 for conformation conversion from Open to Closed conformation for SAC activation.

68

6-1

6-2

Figure 6-1 - 6-2: TRIP13 (6-1) and p31comet (6-2) interact directly with MAD1 N-terminal and C-terminal domains. GST pulldown of recombinant 6×His- p31comet or TRIP13 (100 nM) with GST- MAD1 N-terminal domain (NTD), C-terminal domain (CTD), MAD2 interacting motif (MIM), and MIM-CTD (MC) (180 nM). GST alone was used as a negative control. Red asterisks indicate GST degradation bands. TRIP13 and p31comet signals are observable in Western blot in lanes with GST NTD, CTD, and MC.

69

6-3

6-4

Figure 6-3: MAD1-CTD-4E does not bind to TRIP13 or p31comet. GST pulldowns of recombinant 6×His- p31comet or TRIP13 (100 nM) with MAD1-CTD-WT or MAD1-CTD-4E fragments. GST alone was used as a negative control. TRIP13 and p31 comet signals are observable in Western blot in lanes containing GST-CTD-WT but not GST-CTD-4E. Figuer 6-4: TRIP13 and p31comet bind to MAD1-CTD-716E and MAD1-CTD- 716A mutants. GST pulldowns of recombinant 6×His- p31comet or TRIP13 (100 nM) with MAD1-CTD-WT or MAD1-CTD-716E and GST-MAD1-CTD-716A fragments. GST alone was used as a negative control. TRIP13 and p31 comet signals are observable in Western blot in lanes containing GST-CTD-WT, and both GST-CTD-716E and GST-CTD-716A.

70

6-5

Figure 6-5: mCherry-Mis12-MAD1 (MMM) does not rescue checkpoint defect from TRIP13 depletion. (Top) TRIP13-AID-DLD1 cells were treated with and without 1mM Auxin for 48 hours. Cells were transfected with MMM 24 hours prior to imaging. Mitotic durations were recorded based on cell round-up and flattening time. The mCherry images and MAD1 signals in Western blot confirm construct transfection. The mitotic duration of displayed cells is shown in red on the right. (Bottom) Quantification of mitotic durations under different treatments shown above. Unpaired t-tests were used to compare indicated populations. “NS” = not significant. *** = P<0.001. Error bars indicate SEM. N>16 for all conditions

71

6-6

Figure 6-6: mCherry-Mis12-TRIP13 (MMT) does not prolong mitotic arrest. (Top) HeLa cells with and without MMT transfection prior to imaging. Cells were transfected with MMT 24 hours prior to imaging. Mitotic durations were recorded based on cell round-up and flattening time. The mCherry image and TRIP13 signals in Western blot confirm construct transfection. Kinetochore localization was confirmed with fixed cell immunofluorescence with ACA staining as a control. The mCherry signals for kinetochore localization were changed to green in Leica imaging software for better contrast with ACA. The mitotic duration of displayed cells is shown in red on the right. (Bottom) Quantification of mitotic durations under different treatments shown above. Unpaired T-tests were used to compare indicated populations. “NS” = not significant. Error bars indicate SEM. N > 50.

72

Chapter 7

Discussion

Following our lab’s initial discovery that TRIP13 AAA-ATPase participates in

SAC silencing, it has been firmly established how TRIP13 coordinates with p31comet to convert C-MAD2 to O-MAD2 to disassemble the MCC. More recent publications revealed a surprising role for TRIP13 in SAC activation (Results in section 3 and [266,

267]). The current model for TRIP13 participating in SAC activation is that TRIP13 maintains a pool of cytoplasmic O-MAD2 for substrate for SAC activation. When

TRIP13 is depleted, cells only have C-MAD2 and are defective in SAC activation when treated with nocodazole [266, 267]. This “cytoplasmic MAD2 maintenance” model raises several questions that must be resolved.

The first is that this model does not explain why p31comet is dispensable for SAC activation. Biochemical and structural studies all show that TRIP13 utilizes p31comet as an adaptor protein to engage and convert C-MAD2 to O-MAD2 during SAC silencing [238,

259]. It does not make sense that p31comet is both required for, and completely dispensable for cytoplasmic O-MAD2 generation. The second potential problem with the

73 current model is that it seems counterintuitive that prolonged TRIP13 depletion results a checkpoint defect while accumulating C-MAD2 [266, 267]. All available evidence on

SAC signal supports C-MAD2 as the checkpoint active conformation found in MCC

[125, 134, 137, 147]. Furthermore, we found that expression of the L13A-MAD2 mutant

(a conformationally locked C-MAD2 mutant) rescues the checkpoint defect from TRIP13 depletion (Figure 4-2).

The most likely interpretation is that the endogenous MAD2 in TRIP13 deficient cells is not a functionally competent C-MAD2 as suggested in previous studies. The

MAD2 in previous studies may be in an intermediate MAD2 conformation that is not competent for SAC activity. Studies on one presumed intermediate conformation of

MAD2 (I-MAD2) suggested that it is checkpoint inactive but displays similar elution profiles to C-MAD2 [139]. The ΔN10 mutant of MAD2 (supposedly in the intermediate conformation) is refractory to TRIP13 ATPase activity and defective in SAC activation in cells [139]. Despite this, MAD2-ΔN10 still binds MCC components and MAD1 fragments in vitro [262, 263]. Taken together, the data indicate that TRIP13 depletion creates an accumulation of checkpoint defective I-MAD2 and that TRIP13 can bind O-

MAD2 or I-MAD2 and catalytically convert it to bona fide C-MAD2 during SAC activation.

A third key problem with the current model is that it does not accommodate the presence of TRIP13 and p31comet at unattached kinetochores during SAC activation, or explain the severe delocalization of MAD2 from kinetochores upon TRIP13 depletion.

These data suggest a model where TRIP13 acts on kinetochore specific pools of MAD2 during SAC activation. In addition to the localization data, we discovered that TRIP13 74 interacts directly with MAD1 and that targeting MAD1 to kinetochores does not rescue the checkpoint defect from TRIP13 depletion (Chapter 6). Taken together, the data suggests that the function of TRIP13 in SAC activation is through coordinating with

MAD1 to promote C-MAD2 conversion at unattached kinetochores. In this new model,

MAD1 presents MAD2 for TRIP13 to engage the N-terminus of MAD2. TRIP13 ATPase activity then converts the checkpoint defective open and intermediate MAD2 to the closed checkpoint active MAD2 conformation.

This new model wherein TRIP13 converts MAD1:C-MAD2 bound MAD2 from

O to C at unattached kinetochores explains many of our new findings. For example,

TRIP13-MAD1-MAD2 coordination explains how the mCherry-Mis12-MAD1 construct is defective in maintaining mitotic arrest in TRIP13 deficient backgrounds. MAD1 at kinetochores cannot trigger the MAD2 O-C conversion necessary for SAC activation without the ATPase activity of TRIP13. This is further supported by our observation that expression of only wild type TRIP13 and not the ATPase deficient mutants can rescue the checkpoint from TRIP13 depletion (Figure 6-5, 4-1). This new model also explains previous observations that TRIP13 depletion results in C-MAD2 build up but L13A-

MAD2 expression rescues the checkpoint from TRIP13 depletion. Without TRIP13 there is a buildup of checkpoint defective I-MAD2 and expression of C-MAD2 locked L13A bypasses the requirement for TRIP13 dependent O-C conversion.

The biochemical and immunofluorescent evidence presented in this publication also fits this model of TRIP13 acting on MAD1-MAD2 complexes at kinetochores. We have reported that TRIP13 binds to both the NTD and CTD of MAD1 (Figure 6-1). Other studies in C. elegans have reported that MAD1 depletion also mislocalizes the TRIP13

75 homolog PCH2 from kinetochores [265]. We also show that BUB1 dependent pools of

MCC are required for TRIP13 localization to unattached kinetochores. Recent studies have identified the precise binding regions on BUB1 for both MCC components and

MAD1. The binding motifs for BUBR1 and MAD1 on BUB1 are closely positioned to each other and are extremely well conserved in eukaryotes [104, 106, 194]. This spatial arrangement would allow for TRIP13 to localize to MCC components at kinetochores while simultaneously engaging MAD1-MAD2 complexes.

Lastly, a TRIP13-MAD1 partnership during SAC activation would also explain the dispensability for p31comet in SAC activation as well as its paradoxical localization to unattached kinetochores. Crystal structures of TRIP13- p31comet -MAD2 indicate that the addition of p31comet and MAD2 in vitro promotes the stability of the TRIP13 oligomer.

Furthermore, crystal structure data of TRIP13- p31comet -MAD2 indicates that ATP hydrolysis promotes disassembly the TRIP13- p31comet -MAD2 complex [139, 238, 262,

263]. We speculate that the novel TRIP13 interactions with MAD1 and kinetochore associated MCC subunits can stabilize the TRIP13 hexamer at kinetochores without requiring p31comet. This would explain why p31comet is dispensable for SAC activation at kinetochores but not SAC silencing in the cytoplasm. It has been shown that prolonged depletion of TRIP13 increases the levels of p31comet -MAD1 subcomplexes in cells during mitosis [266, 267]. It is very enticing to think that TRIP13 ATPase activity towards

MAD2 at unattached kinetochores promotes the disassembly of MAD2 from p31comet.

This would, in theory, either free MAD2 from p31comet for incorporation into the MAD1 catalyst, or free MAD2 from p31comet for incorporation into MCC. The first route would generate excess free C-MAD2 to bind MAD1-MIM to generate the MAD1-MAD2

76 tetramer at kinetochores. The second route would promote excess C-MAD2 for MCC assembly. It is especially important and enticing to note that these two events are not mutually exclusive.

More work is still needed to further consolidate our model. We have yet to solidify the exact spatial arrangement TRIP13 with respect to other proteins at outer kinetochores. We also do not have sufficient evidence that TRIP13 depletion seriously abrogates MCC assembly. This biochemical evidence would provide a direct link between our live cell data (Chapter 3 and 4) and the kinetochore localization and MAD1 interaction data (Chapter 5 and 6). Further studies will be directed towards evaluating

MCC integrity in TRIP13 deficient backgrounds. Additionally, research should be directed towards dissecting the nature of the TRIP13-BUBR1 interactions (not shown), the MAD1-TRIP13 interactions and how MPS1 phosphorylation contributes to those interactions. Special consideration should be given to illuminating kinetochore dynamics of MCC subunits and MAD1-MAD2 complexes after TRIP13 depletion.

77

TRIP13

p31

Figure 7-1: Model for TRIP13 in SAC Activation. The novel interactions between TRIP13 and MAD1 stabilize TRIP13 at outer kinetochores during SAC activation. Kinetochore localized TRIP13 ATPase activity prevents I-MAD2 and checkpoint defective MAD2 containing subcomplexes from accruing. This results in free MAD2 can then participate in O-C conversion with the MAD1-MAD2 tetramer.

78

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115

Appendix A

OTSSP167 Prevents Mitotic Arrest through Inhibiting CPC Kinase Aurora B

OTSSP167 is a kinase inhibiting pharmaceutical compound currently in phase II clinical trials for its ability to prevent cancer cell growth [276]. This inhibitory effect was attributed to its potent ability to inhibit Maternal Embryonic Leucine Zipper Kinase, an

AMP activated kinase [277-279]. Interestingly, our lab found that MELK co-transcribes with core centromere and kinetochore components and its protein level and kinase activity peak during prometaphase [88, 280, 281]. Additionally high MELK expression correlates with CIN [282]. These data led us to hypothesize that MELK was a novel mitotic regulator and we sought to use OTSSP167 to delineate a role for MELK in mitosis. Our results indicate that OTSSP167 totally abrogates mitotic arrest. Furthermore, we show that the anti-mitotic effects of OTSSP167 are MELK independent from off target inhibition of Aurora B kinase. We also show that this off target inhibition of

116

Aurora B results in loss of localization and activity of the chromosome passenger complex (CPC) (described below).

To probe the effects of OTSSP167 and MELK on the mitotic checkpoint we first tested the mitotic arrest proficiency of cells treated with OTSSP167. We arrested HeLa cells in mitosis with nocodazole (100 nM) and then added OTSSP167 (100 nM). In agreement with previous literature, all OTSSP167 treated cells exited mitosis within one hour in the presence of nocodazole. All cells treated with DMSO control remained in mitosis for hours (Figure A.1). We next tested if OTSSP167 was able to abrogate MCC assembly. We arrested HeLa cells in mitosis with nocodazole and MG132 (20 µM)for two hours, then added OTSSP (100 nM) or DMSO control for two hours. Mitotic cells were collected by shake of and lysates were subject to BUBR1 Immunoprecipitation.

OTSSP167 treatment prevented the BUBR1-MAD2 interaction, indicating defective

MCC assembly (Figure A.2). To confirm that the defect in MCC assembly was specific to MELK, we transfected HeLa cells with MELK shRNA and selected for transfected cells with puromycin. Transfected cells were synchronized with nocodazole (100 nM) and MG132 (20 µM) for two hours and then OTSSP167 (100 nM) or DMSO for two hours. Mitotic cells were collected by shake off and lysates were again subject to BUBR1

IP. Interestingly, MELK RNAi did not affect MCC assembly as BUBR1-MAD2 interaction was detectable at a similar level to DMSO treated controls (Figure A.2). This suggested that the inhibitory activity of OTSSP167 was due to off target effects on mitotic kinases.

We next wanted to determine how OTSSP167 abrogated mitotic arrest. To determine how OTSSP167 affected the checkpoint we probed cells for inhibition of

117 canonical mitotic kinases. Fixed cell immunofluorescence was used to probe in-vivo levels of canonical mitotic phosphorylation events. A primary phosphorylation event chosen for analysis was Serine 10 on Histone 3. This residue is phosphorylated by Aurora

B kinase during mitosis [272]. HeLa cells were arrested in mitosis with the spindle poison Taxol and MG132 for two hours then OTSSP167 or DMSO was added for two hours. Arrested cells were fixed as described in Chapter 2 and stained with anti-phospho serine 10 on histone H2A. Interestingly, OTSSP167 totally inhibited Serine 10 phosphorylation. This suggests that Aurora B kinase activity is inhibited by OTSSP167

(Figure A.3).

We next wanted to confirm that OTSSP167 inhibited Aurora B kinase activity.

During mitosis Aurora B contributes to the centromere localization of Chromosome

Passenger Complexes (CPC) composed of Aurora B, Borealin, INCENP, Survivin, and

Shugoshin [283-286]. CPC localization and subsequent Aurora B kinase activity during mitosis prevents aberrant kinetochore-microtubule attachments to ensure that only proper bi-polar attachments form with K-fibers in the spindle [284, 287-291]. As the mature spindle forms however, proper bipolar attachments generate tension on chromatids. This tension pulls Aurora B substrates away from the inner centromere, thus inhibiting their phosphorylation and allowing for mitotic exit.

To confirm that OTSSP167 inhibited Aurora B we arrested HeLa cells in mitosis with the spindle poison Taxol and MG132 for two hours. We then added OTSSP167 or

DMSO for two hours and fixed and stained cells for the CPC components Aurora B,

Borealin, and Shugoshin, as well as the outer kinetochore component BUB1. OTSSP167, but not DMSO treatment totally mislocalized Aurora B, Borealin, Shugoshin, and BUB1

118

(Figure A.4). We next wanted to confirm that OTSSP167 inhibited Aurora B by probing for phosphorylation events that require Aurora B. Two major phosphorylation events chosen for analysis were Histone 3 Threonine 3, and Histone H2A Threonine 120. These are phosphorylated by Haspin and Bub1 kinases respectively and both phosphorylation events are upregulated by Aurora B. These two histone modifications promote CPC localization [187, 290, 292, 293]. To confirm that OTSSP167 inhibited these phosphorylation events we arrested HeLa cells in mitosis with the spindle poison Taxol and MG132 for two hours. We then added OTSSP167 or DMSO for two hours and fixed and stained cells with antibodies against the phosphorylated histones. In agreement with our previous data, OTSSP167 treatment completely inhibited both histone modifications compared to DMSO (figure A.6). These combined data confirm our hypothesis that the mitotic defects seen with OTSSP167 are from Aurora B inhibition of CPC localization and activity and not MELK inhibition.

119

A-1

A-2

Figure A-1 – A-2: OTSSP167 but not MELK RNAi abrogates mitotic arrest. (5-1) Nocodazole arrested MCF7 cells were further treated with DMSO or OTSSP167 (100 nM) and monitored for mitotic exit (cell flattening). OTSSP167 but not DMSO treatment was able to completely abrogate mitotic arrest. (5-2) Thymidine synchronized cells were released into nocodazole for 12 hours and then treated with OTSSP167 (100 nM) or DMSO for an additional 2 hours. Lysates were collected and subject to BubR1 immunoprecipitation. For MELK shRNA, transfected cells were selected for with puromycin while being synchronized with thymidine. Cells were then treated in nocodazole for 12 hours. Lysates were collected and subject to BubR1 IP. OTSSP167 but not MELK RNAi was able to prevent BUBR1- MAD2 interaction indicating defective MCC assembly.

120

A-3

Figure A-3: OTSSP167 prevents histone 3 Serine 10 phosphorylation by Aurora B. Immunofluorescence of HeLa cells treated with Taxol and MG132 after thymidine synchronization. Cells were then treated with either OT or DMSO. Cells were probed with phospho-specific antibody for Histone 3 Serine 10 and with CENP-A and DAPI staining for mitotic controls. Single 1 µM sections are presented. Scale bar = 10 um. N>15 cells

121

A-4

122

A-4 cont.

Figure A-4: OTSSP167 inhibits CPC localization to centromeres. Immunofluorescence of HeLa cells treated with Taxol and MG132 after thymidine synchronization. Cells were then treated with either OT or DMSO. Treated cells were probed with antibodies against Aurora B, Borealin, Shusoshin, and BUB1 with CENP-A and DAPI staining for mitotic controls. Single 1 µM sections are presented. Scale bar = 10 um. Error bars represent SEM from N>50 kinetochore pairs from 5 cells in single replicate experiment. Experiments were repeated in triplicate.

123

A-5

C

Figure A-5: OTSSP167 prevents BUB1 and Haspin kinase activity towards histones. Immunofluorescence of HeLa cells treated with Taxol and MG132 after thymidine synchronization. Cells were then treated with either OT or DMSO. Treated cells were probed with antibodies against phosphorylated Threonine 3 on histone 3 and Threonine 120 on Histone H2A with CENP-A and DAPI staining for mitotic controls. Single 1 µM sections are presented. Scale bar = 10 um. Error bars represent SEM from N>50 kinetochore pairs from 5 cells in single replicate experiment. Experiments were repeated in triplicate.

C 124

Appendix B

Attribution of Data

1. TRIP13 and p31comet interaction with MAD1 NTD and CTD were contributed by

Dr. Ejaz Ahmad from Dr. Song-Tao Liu’s lab.

2. The Cloning of mCherry-Mis-TRIP13 was prepared in collaboration with Dr.

Yibo Luo.

3. CRISPR modified DLD1-AID cell lines were constructed and generously

provided by Dr. Vasilisa Askenova in Dr. Mary Dasso’s Lab.

4. p31comet antibody for immunofluorescent analysis was generously provided by Dr.

Jacob Nilsson’s lab.

5. Mitotic arrest assays and MCC IP’s in Appendix A (Figure A-2-1 and A-2-2)

were performed by Dr. Wenbin Ji from Dr. Song-Tao Liu’s lab

6. All other experiments and data presented in this manuscript were performed and

collected by the primary author Christopher Arnst.

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Appendix C

Constructs generated for the lab

Tab1e C1: DNA Constructs Generated for the Lab BACTERIAL STOCKS Location Location 2 Location 3 Strain Arnst Protein P31 WT DEST 17 - CODON+ Stocks B3 -80 Codon+ P31 WT DEST 17 - DH5 Arnst Protein DH5 ALPHA Stocks D1 -80 Alpha Arnst Protein P31 WT DEST 15 CODON+ Stocks A4 -80 Codon+ Arnst Protein P31 WT DEST 15 CODON+ Stocks A5 -80 Codon+ P31 WT (V2) DEST 15 Arnst Protein CODON+ Stocks B5 -80 Codon+ Arnst Protein P31 WT DEST 17 CODON+ Stocks G5 -80 Codon+ TRIP13 WALKER B DEST17 Arnst Protein ROSSETTA Stocks J4 -80 Rossetta CDC20 111-128 DEST15 Arnst Protein CODON+ Stocks B6 -80 Codon+ CDC20 DELTA 129-136 Arnst Protein DEST 15 CONDON+ Stocks B7 -80 Codon+ BUBR1 1-486 DEST-STREP Arnst Protein CODON + Stocks E6 -80 Codon+ BUBR1 1-486 DEST15 Arnst Protein CODON + Stocks F6 -80 Codon+

126

Tab1e C1: DNA Constructs Generated for the Lab Continued BACTERIAL Location Location 2 Location 3 Strain STOCKS BUBR1 1-486 DEST15 CODON + Arnst Protein Stocks F7 -80 Codon+ BUBR1 1-486 DEST15 CODON + Arnst Protein Stocks G6 -80 Codon+ BUBR1 1-486 DEST15 CODON + Arnst Protein Stocks G7 -80 Codon+ CDC20 DELTA 129- 136 DEST 15 CONDON+ Arnst Protein Stocks C8 -80 Codon+ CDC20 111-138 DEST15 CODON+ Arnst Protein Stocks C9 -80 Codon+ MAD1 NTD DEST15 CODON+ Arnst Protein Stocks B8 -80 Codon+ BUBR1 1-486 DEST15 CODON + Arnst Protein Stocks B9 -80 Codon+ MAD1 CTD 4E DEST15 CODON+ Arnst Protein Stocks B10 -80 Codon+ CDC20 WT DEST17 CODON+ Arnst Protein Stocks C10 -80 Codon+ TRIP13 WT DEST 17 ROSETTA Arnst Protein Stocks E9 -80 Rossetta TRIP13 WT DEST 17 ROSETTA Arnst Protein Stocks E10 -80 Rossetta TRIP13 WT DEST 17 ROSETTA Arnst Protein Stocks F9 -80 Rossetta TRIP13 WT DEST 17 ROSETTA Arnst Protein Stocks F10 -80 Rossetta TRIP13 WT DEST 17 ROSETTA Arnst Protein Stocks F8 -80 Rossetta TRIP13 WALKER A DEST 17 ROSETTA Arnst Protein Stocks H8 -80 Rossetta TRIP13 WALKER A DEST 17 ROSETTA Arnst Protein Stocks H9 -80 Rossetta TRIP13 WALKER A DEST 17 ROSETTA Arnst Protein Stocks H10 -80 Rossetta

127

Tab1e C1: DNA Constructs Generated for the Lab Continued

BACTERIAL STOCKS Location Location 2 Location 3 Strain Pentr BubR1 N750 Arnst Box1 H10 -80 TOP10 GFP Mad1 CTD WT Arnst Box1 I10 -80 TOP10 mCherry BubR1 WT-3 Arnst Box1 H8 -80 TOP10 mCherry BubR1 WT-2 Arnst Box1 I8 -80 TOP10 mCherry BubR1 WT-1 Arnst Box1 J8 -80 TOP10 mCherry BubR1 487-700 -1 Arnst Box1 H7 -80 TOP10 mCherry BubR1 487-700-2 Arnst Box1 I7 -80 TOP10 mCherry BubR1 487-700-3 Arnst Box1 J7 -80 TOP10 CRY Mad2 Arnst Box1 H3 -80 TOP10 CRY Mad2 Arnst Box1 I3 -80 TOP10 CRY Mad2 Arnst Box1 J3 -80 TOP10 CRY Mad2 Arnst Box1 H4 -80 TOP10 CRY Mad2 Arnst Box1 I4 -80 TOP10 CRY Mad2 Arnst Box1 J4 -80 TOP10 LOV CDC20 Arnst Box1 H2 -80 TOP10 LOV CDC20 Arnst Box1 I2 -80 TOP10 LOV MAD2 Arnst Box1 I1 -80 TOP10 LOV MAD2 Arnst Box1 J1 -80 TOP10 ECE-GFP MAD2 L13A Arnst Box1 F8 -80 TOP10 ECE-GFP MAD2 L13A Arnst Box1 G7 -80 TOP10 Pentr-D topo MAD1 WT Arnst Box1 F7 -80 TOP10 Pentr-D topo MAD1 WT Arnst Box1 G8 -80 TOP10 MELK shrna Arnst Box1 A1 -80 TOP10 MELK shrna Arnst Box1 A2 -80 TOP10 MELK shrna Arnst Box1 A3 -80 TOP10 Cibn-Cax optogenetics Arnst Box1 A6 -80 TOP10 Destination vector venus Arnst Box1 A7 -80 TOP10 Destination vector venus Arnst Box1 B7 -80 TOP10 destination vector venus Arnst Box1 C7 -80 TOP10 psuper kiaa shrna Arnst Box1 A8 -80 TOP10 psuper kiaa shrna Arnst Box1 B8 -80 TOP10 psuper vector Arnst Box1 C8 -80 TOP10 psi kiaa shrna Arnst Box1 A9 -80 TOP10

128

Tab1e C1: DNA Constructs Generated for the Lab Continued BACTERIAL STOCKS Location Location 2 Location 3 Strain psuper kiaa shrna Arnst Box1 B10 -80 TOP10 GST Codon + (control) Arnst Box1 B10 -80 Codon+ mCherry-Mis12-Mad1 S598E Arnst Box1 D1 -80 TOP10 mCherry -Mis12-Mad1 WT Arnst Box1 D2 -80 TOP10 mCherry -Mis12-Mad1 Y634E Arnst Box1 D3 -80 TOP10 mCherry-Mis12-Mad1 S432E Arnst Box1 D4 -80 TOP10 Pentr2b Mad1 Y649E Arnst Box1 E1 -80 TOP10 Pentr2b Mad1 Y649E Arnst Box1 E2 -80 TOP10 mCherry-Mis12-Mad1 y649E Arnst Box1 E3 -80 TOP10 pentr2b Mad1 wt Arnst Box1 E4 -80 TOP10 mCherry-Mis12-Mad1 S598E Arnst Box1 E5 -80 TOP10 pentr2b mad1 y649E Arnst Box1 F1 -80 TOP10 Pentr2b Mad1 Y649E Arnst Box1 F2 -80 TOP10 mCherry-Mis12-Mad1 490E Arnst Box1 F3 -80 TOP10 mCherry-Mis12-Mad1 y649E Arnst Box1 F4 -80 TOP10 mCherry-Mis12-Mad1 y649E Arnst Box1 F5 -80 TOP10 GFP-TRIP13 WT Arnst Box2 A1 -80 TOP10 GFP-TRIP13 wt Arnst Box2 A2 -80 TOP10 GFP-TRIP13 wt Arnst Box2 A3 -80 TOP10 GFP-TRIP13 S18A Arnst Box2 A4 -80 TOP10 GFP-TRIP13 S18A Arnst Box2 A5 -80 TOP10 GFP-TRIP13 S20A Arnst Box2 A7 -80 TOP10 GFP-TRIP13 S20A Arnst Box2 A9 -80 TOP10 GFP-TRIP13 S41A Arnst Box2 A10 -80 TOP10 GFP-TRIP13 S41A Arnst Box2 B10 -80 TOP10 GFP-TRIP13 S74A Arnst Box2 B1 -80 TOP10 GFP-TRIP13 S74A Arnst Box2 B2 -80 TOP10 GFP-TRIP13 S147A Arnst Box2 B3 -80 TOP10 GFP-TRIP13 S147A Arnst Box2 B4 -80 TOP10 GFP-TRIP13 S155A Arnst Box2 B6 -80 TOP10 GFP-TRIP13 S155A Arnst Box2 B8 -80 TOP10 GFP-TRIP13 S155A Arnst Box2 B9 -80 TOP10 GFP-TRIP13 S74A Arnst Box2 C1 -80 TOP10 GFP-TRIP13 T183A Arnst Box2 C2 -80 TOP10

129

Tab1e C1: DNA Constructs Generated for the Lab Continued

BACTERIAL STOCKS Location Location 2 Location 3 Strain PENTR-D-TRIP13 E253Q Arnst Box2 G2 -80 TOP10 PENTR-D-TRIP13 E253Q Arnst Box2 I1 -80 TOP10 PENTR-D-TRIP13 S20A Arnst Box2 H2 -80 TOP10 PENTR-D-TRIP13 S384A Arnst Box2 G3 -80 TOP10 PENTR-D-TRIP13 S155A Arnst Box2 G4 -80 TOP10 PENTR-D-TRIP13 S155A Arnst Box2 G6 -80 TOP10 PENTR-D-TRIP13 S147A Arnst Box2 G7 -80 TOP10 PENTR-D-TRIP13 S370A Arnst Box2 G8 -80 TOP10 PSUPER TRIP13 SHRNA Arnst Box2 G9 -80 TOP10 PENTR-D-TRIP13 WT Arnst Box2 H1 -80 TOP10 PENTR-D-TRIP13 S20A Arnst Box2 H3 -80 TOP10 GFP-TRIP13 e253q Arnst Box2 H4 -80 TOP10 PENTR-D-TRIP13 S41A Arnst Box2 H5 -80 TOP10 PENTR-D-TRIP13 S147A Arnst Box2 H6 -80 TOP10 PENTR-D-TRIP13 S147A Arnst Box2 H7 -80 TOP10 PENTR-D-TRIP13 S367A Arnst Box2 H8 -80 TOP10 PSUPER TRIP13 SHRNA Arnst Box2 H9 -80 TOP10 PBABE Arnst Box2 H10 -80 TOP10 PENTR-D-TRIP13 E253Q Arnst Box2 I1 -80 TOP10 GFP-TRIP13 e253q Arnst Box2 I2 -80 TOP10 PENTR-D-TRIP13 E253Q Arnst Box2 I3 -80 TOP10 GFP-TRIP13 e253q Arnst Box2 I4 -80 TOP10 PENTR-D-TRIP13 S74A Arnst Box2 I6 -80 TOP10 PENTR-D-TRIP13 S18A Arnst Box2 I7 -80 TOP10 PENTR-D-TRIP13 S367A Arnst Box2 I8 -80 TOP10 TRIP13-SHRNA + PURO-R Arnst Box2 I9 -80 TOP10 PBABE Arnst Box2 I10 -80 TOP10 GFP-TRIP13 Walker A Arnst Box3 A5 -80 TOP10 GFP-TRIP13 Walker B - ECOR1-R Arnst Box3 A6 -80 TOP10 GFP-TRIP13 WT -ECOR1-R Arnst Box3 A7 -80 TOP10 GFP-TRIP13 WT -ECOR1-R Arnst Box3 A9 -80 TOP10 BBs1 vector Puro- R Arnst Box3 B1 -80 TOP10 GFP-TRIP13 Walker A - ECOR1-R Arnst Box3 B5 -80 TOP10 GFP-TRIP13 Walker A - ECOR1-R Arnst Box3 B6 -80 TOP10 GFP-TRIP13 WT -ECOR1-R Arnst Box3 B7 -80 TOP10

130

Tab1e C1: DNA Constructs Generated for the Lab Continued BACTERIAL STOCKS Location Location 2 Location 3 Strain GFP-TRIP13 T183A Arnst Box2 C4 -80 TOP10 GFP-TRIP13 S367A Arnst Box2 C5 -80 TOP10 GFP-TRIP13 S367A Arnst Box2 C7 -80 TOP10 GFP-TRIP13 S384A Arnst Box2 C8 -80 TOP10 GFP-TRIP13 S384A Arnst Box2 C10 -80 TOP10 GFP-TRIP13 S377A Arnst Box2 D1 -80 TOP10 GFP-TRIP13 S377A Arnst Box2 D2 -80 TOP10 GFP-TRIP13 S370A Arnst Box2 D5 -80 TOP10 GFP-TRIP13 S370A Arnst Box2 D6 -80 TOP10 GFP-TRIP13 Y56F Arnst Box2 D7 -80 TOP10 GFP-TRIP13 Y56F Arnst Box2 D9 -80 TOP10 GFP-TRIP13 Y206F Arnst Box2 D10 -80 TOP10 GFP-TRIP13 S302A Arnst Box2 E1 -80 TOP10 GFP-TRIP13 S302A Arnst Box2 E2 -80 TOP10 PENTR-D-TRIP13 Y56F Arnst Box2 E3 -80 TOP10 GFP-TRIP13 S216A Arnst Box2 E4 -80 TOP10 GFP-TRIP13 S216A Arnst Box2 E6 -80 TOP10 GFP-TRIP13 Y206F Arnst Box2 E7 -80 TOP10 GFP-TRIP13 Y206F Arnst Box2 E8 -80 TOP10 GFP-TRIP13-CTD Arnst Box2 E9 -80 TOP10 GFP-TRIP13-NTD Arnst Box2 E10 -80 TOP10 PENTR-D-TRIP13 S20A Arnst Box2 F1 -80 TOP10 PENTR-D-TRIP13 S20A Arnst Box2 F2 -80 TOP10 PENTR-D-TRIP13 Y56F Arnst Box2 F3 -80 TOP10 PENTR-D-TRIP13 S384A Arnst Box2 F4 -80 TOP10 PENTR-D-TRIP13 S41A Arnst Box2 F5 -80 TOP10 PENTR-D-TRIP13 S155A Arnst Box2 F6 -80 TOP10 PENTR-D-TRIP13 S147A Arnst Box2 F7 -80 TOP10 PENTR-D-TRIP13 S370A Arnst Box2 F8 -80 TOP10 PENTR-D-TRIP13 WT Arnst Box2 F9 -80 TOP10 TRIP13-SHRNA Arnst Box2 F10 -80 TOP10 PENTR-D-TRIP13 WT Arnst Box2 G1 -80 TOP10 PENTR-D-TRIP13 WT Arnst Box2 H1 -80 TOP10

131

Tab1e C1: DNA Constructs Generated for the Lab Continued

BACTERIAL STOCKS Location Location 2 Location 3 Strain GFP-TRIP13 WT -ECOR1 R Arnst Box3 B8 -80 TOP10 GFP-TRIP13 WT -ECOR1 R Arnst Box3 C6 -80 TOP10 PMD-plenti vector Arnst Box3 D1 -80 TOP10 Pspax-plenti vector Arnst Box3 D2 -80 TOP10 Plenti BSMB1 vector Arnst Box3 E1 -80 TOP10 Plenti BSMB1 vector Arnst Box3 E2 -80 TOP10 BBs1 Trip13 sg-RNA 3-2 Arnst Box3 E4 -80 TOP10 BBs1 Trip13 sg-RNA 1-5 Arnst Box3 E5 -80 TOP10 BBs1 Trip13 sg-RNA 3-1 Arnst Box3 F4 -80 TOP10 BBs1 Trip13 sg-RNA 1-4 Arnst Box3 F5 -80 TOP10 BBs1 Trip13 sg-RNA 3-5 Arnst Box3 G4 -80 TOP10 BBs1 Trip13 sg-RNA 3-4 Arnst Box3 H4 -80 TOP10 BBs1 Trip13 sg-RNA 3-3 Arnst Box3 I4 -80 TOP10 BBs1 Trip13 sg-RNA 1-3 Arnst Box3 G5 -80 TOP10 BBs1 Trip13 sg-RNA 1-2 Arnst Box3 H5 -80 TOP10 BBs1 Trip13 sg-RNA 1-1 Arnst Box3 I5 -80 TOP10 Bsmb1 TRIP13 sg-RNA 1-1 Arnst Box3 G1 -80 TOP10 Bsmb1 TRIP13 sg-RNA 1-2 Arnst Box3 G2 -80 TOP10 Bsmb1 TRIP13 sg-RNA 1-3 Arnst Box3 G3 -80 TOP10 Bsmb1 TRIP13 sg-RNA 2-1 Arnst Box3 H1 -80 TOP10 Bsmb1 TRIP13 sg-RNA 2-2 Arnst Box3 H2 -80 TOP10 Bsmb1 TRIP13 sg-RNA 2-3 Arnst Box3 H3 -80 TOP10 Bsmb1 TRIP13 sg-RNA 3-1 Arnst Box3 I1 -80 TOP10 Bsmb1 TRIP13 sg-RNA 3-2 Arnst Box3 I2 -80 TOP10 Bsmb1 TRIP13 sg-RNA 3-3 Arnst Box3 I3 -80 TOP10 mCherry-Mis12-TRIP13 WT Arnst Box3 F8 -80 TOP10 mCherry-Mis12-TRIP13 WT Arnst Box3 H8 -80 TOP10 mCherry-Mis12 TRIP13 W B Arnst Box3 E9 -80 TOP10 mCherry-Mis12 TRIP13 W B Arnst Box3 H9 -80 TOP10 mCherry-Mis12-TRIP13 W A Arnst Box3 H7 -80 TOP10 mCherry-Mis12-TRIP13 W A Arnst Box3 I7 -80 TOP10 mCherry-Mis12-MAD1 WT Simran-Jenna box A1 -80 TOP10 mCherry-Mis12-MAD1 WT Simran-Jenna box B1 -80 TOP10

132

Tab1e C1: DNA Constructs Generated for the Lab Continued

BACTERIAL STOCKS Location Location 2 Location 3 Strain mCherry-Mis12-MAD1 634E Simran-Jenna box A3 -80 TOP10 mCherry-Mis12-MAD1 649E Simran-Jenna box A4 -80 TOP10 mCherry-Mis12-MAD1 490E Simran-Jenna box A5 -80 TOP10 mCherry-Mis12-MAD1 598E Simran-Jenna box A6 -80 TOP10 mCherry-Mis12-MAD1 598E Simran-Jenna box A7 -80 TOP10 mCherry-Mis12-MAD1 432E Simran-Jenna box A8 -80 TOP10 mCherry-Mis12-MAD1 16E Simran-Jenna box B2 -80 TOP10 mCherry-Mis12-MAD1 16E Simran-Jenna box B3 -80 TOP10 mCherry-Mis12-MAD1 16E Simran-Jenna box B4 -80 TOP10 Pentr2b - MAD1- 649E Simran-Jenna box B5 -80 TOP10 Pentr2b - MAD1- 77E Simran-Jenna box C5 -80 TOP10 Pentr2b - MAD1- 77E Simran-Jenna box D5 -80 TOP10 Pentr2b - MAD1- 77E Simran-Jenna box E5 -80 TOP10 Pentr2b - MAD1- 77E Simran-Jenna box F5 -80 TOP10 Pentr2b - MAD1- 77E Simran-Jenna box G5 -80 TOP10 Pentr2b - MAD1- 77E Simran-Jenna box H5 -80 TOP10 Pentr2b - MAD1- 13E Simran-Jenna box D1 -80 TOP10 Pentr2b - MAD1- 92E Simran-Jenna box E1 -80 TOP10 Pentr2b - MAD1- 92E Simran-Jenna box F1 -80 TOP10 Pentr2b - MAD1- 92E Simran-Jenna box G1 -80 TOP10 Pentr2b - MAD1- 16E Simran-Jenna box D2 -80 TOP10 Pentr2b - MAD1- 16E Simran-Jenna box F2 -80 TOP10 Pentr2b - MAD1- 90E Simran-Jenna box I4 -80 TOP10 Pentr2b - MAD1- 90E Simran-Jenna box I5 -80 TOP10 Pentr2b - MAD1- 90E Simran-Jenna box I7 -80 TOP10 Pentr2b - MAD1- 29E Simran-Jenna box I8 -80 TOP10 Pentr2b - MAD1- 29E Simran-Jenna box H4 -80 TOP10 Pentr2b - MAD1- 29E Simran-Jenna box G8 -80 TOP10 Pentr2b - MAD1- 29E Simran-Jenna box F8 -80 TOP10 Pentr2b - MAD1- 29E Simran-Jenna box E8 -80 TOP10 Pentr2b - MAD1- 29E Simran-Jenna box D8 -80 TOP10 Pentr2b - MAD1- 377E Simran-Jenna box I1 -80 TOP10 Pentr2b - MAD1- 377E Simran-Jenna box I2 -80 TOP10

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Tab1e C2: Antibodies

antibodies Loction Location 2 Location 3 Mad2: Bethyl A300-301A, 4 degree common Rabbit 1:100 antibody box B2 4 Mad1: Protein Tech, Rabbit common Immunosoft 1:500 antibody box D4 -20 TRIP13: Homemade (Wang et common antibody box al. 2014) Rabbit 1:50 #2 I6 -20 BubR1: Immunosoft China, common Immunosoft Rabbit 1:100 antibody box E3 -20 Bub1: Immunosoft China, common Immunosoft Rabbit 1:500 antibody box C2 -20 CDC20: Immunosoft China, common Immunosoft Rabbit 1:100 antibody box -20 p31comet: Jacob Nilsson’s lab: Mouse 1:50 STOCK DEPLETED NA NA ACA: Antibodies Incorporated common Immunosoft 9101-02, Human 1:100 antibody box D4 -20 Phospho-Serine-10: Invitrogen, Mouse 1:1000 Arnst Antibodies -20 Secondary antibodies: 4 degree secondary Alexaflour (1:1000) Rabbit-488 antibody box A1 4 Secondary antibodies: 4 degree secondary Alexaflour (1:1000) Mouse-488 antibody box A2 4 Secondary antibodies: 4 degree secondary Alexaflour (1:1000) Rabbit-555 antibody box A3 4 Secondary antibodies: 4 degree secondary Alexaflour (1:1000) Mouse-555 antibody box A4 4 Secondary antibodies: Alexaflour (1:1000v Human- 4 degree secondary 647 antibody box A5 4 Secondary antibodies: 4 degree secondary Alexaflour (1:1000) Mouse-647 antibody box A6 4 Cenp A mouse 1:500 Arnst Antibodies C2 -20 H3T3 rabbit: Invitrogen 1:500 Arnst Antibodies E2 -20 H2A T120 rabbit: Invitrogen 1:500 Arnst Antibodies F2 -20 sgo1 rabbit: Invitrogen 1:750 Arnst Antibodies G3 -20 borealin rabbit: Invitrogen 1:500 Arnst Antibodies H2 -20 aurora B rabbit: Dr. William Taylor’s lab 1:750 Arnst Antibodies J2 -20

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Tab1e C3: RNAi Constructs

RNAi STOCKS Location Location 2 Sequence (5'-3') BubR1 SIRNA ARNST RNAI F2 gucucacagauugcugccuuu TRIP13 SIRNA-1 ARNST RNAI C1 cauuauaccaacugagaaa TRIP13 SIRNA-2 ARNST RNAI C2 acaagaacgucaacagcaauu CDC20 SIRNA-1 ARNST RNAI D1 cggaagaccugccguuacauu P31 SIRNA 1 ARNST RNAI E1 gguaugagaaguccgaaga P31 SIRNA 2 ARNST RNAI E2 ggacacuaguaccgcgagu BUB1 SIRNA ARNST RNAI F1 MAD2 SIRNA ARNST RNAI G1 uacggacucaccuugcuuguu

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Tab1e C4: Primers Generated for the Lab PRIMERS Location Location 2 Sequence Primer - TRIP13 Arnst S384A Fwd Primers 1 J1 ggaagagcgagggcctcgccggccgggtctgag Primer - TRIP13 Arnst S384A Rev Primers 1 J2 ctcagacccggccggcgaggccctcgctcttcc Primer - TRIP13 Arnst S377A FWD Primers 1 I1 cttttgaatgacattgccaggaagagcgagggcc Primer - TRIP13 Arnst S377A Rev Primers 1 I2 ggccctcgctcttcctggcaatgtcattcaaaag Primer - TRIP13 Arnst S370A Fwd Primers 1 H1 acaacgtgtcaaaattggccccttcttttgaatgacatttc Primer - TRIP13 Arnst S370A Rev Primers 1 H2 gaaatgtcattcaaaagaaggggccaattttgacacgttgt Primer - TRIP13 Arnst S367A Fwd Primers 1 G1 gcttcattgaaaacaacgtggccaaattgagccttctttt Primer - TRIP13 Arnst S367A Rev Primers 1 G2 aaaagaaggctcaatttggccacgttgttttcaatgaagc Primer - TRIP13 Arnst T183A Fwd Primers 1 F1 ccacggtcctcctggcgccggaaaaacatccctg Primer - TRIP13 Arnst T183A Rev Primers 1 F2 cagggatgtttttccggcgccaggaggaccgtgg Primer - TRIP13 Arnst S155A Fwd Primers 1 E1 cctcgattatgtgatggccactttactgttttca Primer - TRIP13 Arnst S155A Rev Primers 1 E2 tgaaaacagtaaagtggccatcacataatcgagg Primer - TRIP13 Arnst S74A Fwd Primers 1 D1 ccagaaatgtgcagtctgtggccattattgacacagaatt Primer - TRIP13 Arnst S74A Rev Primers 1 D2 aattctgtgtcaataatggccacagactgcacatttctgg Primer - TRIP13 Arnst Y56A Fwd Primers 1 C1 atattgtgtttggtgatgccacatggactgagtttg Primer - TRIP13 Arnst Y56A Rev Primers 1 C2 caaactcagtccatgtggcatcaccaaacacaatat Primer - TRIP13 Arnst S41A Fwd Primers 1 B1 gacataaacctggccgttagaaagc Primer - TRIP13 Arnst S41A Rev Primers 1 B2 gctttctaacggccaggtttatgtc Primer - TRIP13 Arnst S20A Fwd Primers 1 A1 gtggccgagtcgccagccgtccacgtggaggtgc Primer - TRIP13 Arnst S20A Rev Primers 1 A2 gcacctccacgtggacggctggcgactcggccac

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Tab1e C4: Primers Generated for the Lab Continued PRIMERS Location Location 2 Sequence Primer - TRIP13 Arnst T302A FWD Primers 1 J3 ctgaccacttctaacatcgccgagaagatcgacgtgg Primer - TRIP13 Arnst S302A Rev Primers 1 J4 ccacgtcgatcttctcggcgatgttagaagtggtcag Primer - TRIP13 Arnst S216A Fwd Primers 1 I3 tgaaataaacagccacgccctcttttctaagtg Primer - TRIP13 Arnst S216A Rev Primers 1 I4 cacttagaaaagagggcgtggctgtttatttca Primer - TRIP13 Arnst Y206A Fwd Primers 1 H3 caagcaggtaccgatttggccaattaattg Primer - TRIP13 Arnst Y206A Rev Primers 1 H4 caattaattggccaaatcggtacctgcttg Primer - TRIP13 Arnst Y56F Fwd Primers 1 G3 cataatattgtgtttggtgattttacatggactgagtttg Primer - TRIP13 Arnst caaactcagtccatgtaaaatcaccaaacacaatattat Y56F Rev Primers 1 G4 g Primer - TRIP13 Arnst E253Q Fwd Primers 1 F3 cgtgctgatgatcaggtggagagtctcacagccg Primer - TRIP13 Arnst E253Q Rev Primers 1 F4 cggctgtgagactctccacctgatcatcagcacg Primer - TRIP13 Arnst S18A Fwd Primers 1 E3 ccgccttcgtcagcaactactacaatccacaaa Primer - Mad1 Arnst ggaaaacaccatggttttagagaccctgagatctttgaa S12E Fwd Primers 1 J5 caac Primer - Mad1 Arnst gttgttcaaagatctcagggtctctaaaaccatggtgtttt S12E Rev Primers 1 J6 cc Primer - Mad1 Arnst ggaaaacaccatggttttatccgagctgagatctttgaa T13E Fwd Primers 1 I5 caac Primer - Mad1 Arnst gttgttcaaagatctcagctcggataaaaccatggtgttt T13E Rev Primers 1 I6 tcc Primer - Mad1 Arnst S16E Fwd Primers 1 H5 ccatggttttatccaccctgagagagttgaacaacttc Primer - Mad1 Arnst S16E Rev Primers 1 H6 gaagttgttcaactctctcagggtggataaaaccatgg Primer - Mad1 Arnst ccacccgatctttgaacaacttcatcgagcagcgtgtgg S22E Fwd Primers 1 G5 aggg

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Tab1e C4: Primers Generated for the Lab Continued PRIMERS Location Location 2 Sequence Primer - Mad1 Arnst S22E Rev Primers 1 G6 ccctccacacgctgctcgatgaagttgttcaaagatcgggtgg Primer - Mad1 Arnst S29E Fwd Primers 1 F5 ggagggaggcgagggactggatattctacc Primer - Mad1 Arnst S29E Rev Primers 1 F6 ggtagaatatccagtccctcgcctccctcc Primer - Mad1 Arnst S77E Fwd Primers 1 E5 gcagatggagctggagcacaagagggc Primer - Mad1 Arnst S77E Rev Primers 1 E6 gccctcttgtgctccagctccatctgc Primer - Mad1 Arnst S90E Fwd Primers 1 D5 ggagagagcagccgagaccagtgccagg Primer - Mad1 Arnst S90E Rev Primers 1 D6 cctggcactggtctcggctgctctctcc Primer - Mad1 Arnst S92E Fwd Primers 1 C5 ggagagagcagccagcacgaggccaggaactacg Primer - Mad1 Arnst S92E Rev Primers 1 C6 cgtagttcctggcctcgtgctggctgctctctcc Primer- Mad1 Arnst S185E Fwd Primers 1 J7 ggtgaagcgcctggaggaggagaagcagg Primer- Mad1 Arnst S214E Rev Primers 1 I7 cctgcttctcctcctccaggcgcttcacc Primer- Mad1 Arnst S377E Fwd Primers 1 H7 ccggcaggtcgagggccagctgttgg Primer- Mad1 Arnst S377E Rev Primers 1 H8 ccaacagctggccctcgacctgccgg Primer- Mad1 Arnst Y427E Rev Primers 1 G7 ccccggccgaggagtcacccagctgacgc Primer- Mad1 Arnst T423E Fwd Primers 1 F7 cgacagcgagctggagccggccgagtactcaccc Primer- Mad1 Arnst T432E Fwd Primers 1 E7 cgagtactcaccccagctggagcggcgcatgcggg Primer- Mad1 Arnst T485E Fwd Primers 1 D7 ggaagctctgttcggcagactcggactgagactt Primer- Mad1 Arnst T649E Fwd Primers 1 C7 ccacggagaaccaggagcggctgacctcgctgtacgc Primer- Mad1 Arnst T699E Fwd Primers 1 B7 aaggcagggatctcgtcctggc Primer- Mad1 Arnst T699E Rev Primers 1 B8 gccaggacgagatccctgcctt

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Tab1e C4: Primers Generated for the Lab Continued

Location PRIMERS Location 2 Sequence TRIP13 T155E Arnst FWD Primers 2 C6 cctcgattatgtgatggagactttactgttttca TRIP13 T155E Arnst REV Primers 2 C7 tgaaaacagtaaagtctccatcacataatcgagg TRIP13 S147E Arnst FWD Primers 2 D6 cgatgtggaagtcaaagagcatctcctcgattatg TRIP13 S147E Arnst REV Primers 2 D7 cataatcgaggagatgctctttgacttccacatcg TRIP13 S74E Arnst cagaaatgtgcagtctgtggagattattgacacagaatt FWD Primers 2 E6 a Arnst TRIP13 S74E REV Primers 2 E7 taattctgtgtcaataatctccacagactgcacatttctg TRIP13 Y56D Arnst FWD Primers 2 F6 atattgtgtttggtgatgacacatggactgagtttg TRIP13 Y56D Arnst REV Primers 2 F7 caaactcagtccatgtgtcatcaccaaacacaatat TRIP13 S41E Arnst FWD Primers 2 G6 gacataaacctggaggttagaaagc Arnst TRIP13 S41E REV Primers 2 G7 gctttctaacctccaggtttatgtc TRIP13 T20E Arnst FWD Primers 2 H6 gcacctccacgtggacctctggcgactcggccac Arnst TRIP13 T20E REV Primers 2 H7 gtggccgagtcgccagaggtccacgtggaggtgc Arnst TRIP13 S18e fwd Primers 2 I6 ccctgtgtggccgaggagccaacggtccacgtgg Arnst TRIP13 s18E rev Primers 2 I7 ccacgtggaccgttggctcctcggccacacaggg Benazera TRIP13 Arnst sgRNA FWD Bbs1 Primers 1 J9 caccggtcgccaacggtccacgtgg Benazera TRIP13 Arnst sgRNA Rev Bbs1 Primers 1 J10 ccagcggttgccaggtgcacccaaa Cheeseman 1 T13 Arnst sgRNA FWD Bbs1 Primers 1 H9 caccgtgagtagctttctaacactc Cheeseman 1 T13 Arnst sgRNA Rev Bbs1 Primers 1 H10 cactcatcgaaagattgtgagcaaa

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Tab1e C4: Primers Generated for the Lab Continued

PRIMERS Location Location 2 Sequence TRIP13 Arnst K227A REV Primers 2 B1 cggaaagtggcgcgctggtaaccaag TRIP13 Arnst K231E REV Primers 2 B2 cttctgaaacatctcggttaccagcttgcc TRIP13 Arnst K227 FWD Primers 2 C1 cttggttaccagcgcgccactttccg TRIP13 Arnst K231E Fwd Primers 2 C2 ggcaagctggtaaccgagatgtttcagaag TRIP13 Arnst S384E FWD Primers 2 D1 ggaagagcgagggcctcgagggccgggtcctgag TRIP13 Arnst S384E REV Primers 2 D2 ctcaggacccggccctcgaggccctcgctcttcc TRIP13 Arnst S377E FWD Primers 2 E1 cttttgaatgacattgagaggaagagcgagggcc TRIP13 Arnst S377E Rev Primers 2 E2 ggccctcgctcttcctctcaatgtcattcaaaag TRIP13 Arnst S370E FWD Primers 2 F1 acaacgtgtcaaaattggagcttcttttgaatgacatttc TRIP13 Arnst S370E REV Primers 2 F2 gaaatgtcattcaaaagaagctccaattttgacacgttgt TRIP13 Arnst S367E FWD Primers 2 G1 cttcattgaaaacaacgtggagaaattgagccttcttttg TRIP13 Arnst S367E REV Primers 2 G2 caaaagaaggctcaatttctccacgttgttttcaatgaag TRIP13 Arnst T302E FWD Primers 2 H1 ctgaccacttctaacatcgaggagaagatcgacgtgg TRIP13 Arnst T302E REV Primers 2 H2 ccacgtcgatcttctcctcgatgttagaagtggtcag TRIP13 Arnst S216E FWD Primers 2 I1 tgaaataaacagccacgagctcttttctaagtgg TRIP13 Arnst S216E REV Primers 2 I2 ccacttagaaaagagctcgtggctgtttatttca TRIP13 Arnst Y206D FWD Primers 2 A6 caagcaggtaccgagacggccaattaattg TRIP13 Arnst Y206D REV Primers 2 A7 caattaattggccgtctcggtacctgcttg TRIP13 Arnst T183E FWD Primers 2 B6 ccacggtcctcctggcgagggaaaaacatccctg

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Tab1e C4: Primers Generated for the Lab Continued PRIMERS Location Location 2 Sequence MAD2 RNAI RESISTANCE TRIP13 cgagtgcagaaatacggactcacattacttgtaactact FWD DNA 2 A3 g MAD2 RNAI TRIP13 RESISTANCE DNA 2 REV B3 cagtagttacaagtaatgtgagtccgtatttctgcactcg P31 V1 RNAI TRIP13 RESISTANCE DNA 2 FWD B4 ggagtggtatgagaaatcagaagaaactcacgcc P31 V1 RNAI TRIP13 RESISTANCE DNA 2 REV B5 ggcgtgagtttcttctgatttctcataccactcc TRIP13 ECOR1 TRIP13 RESISTANCE DNA 2 REV A6 cccaaagcccatgaaactcagctgcaggtagaaccc TRIP13 ECOR1 TRIP13 RESISTANCE DNA 2 FWD B6 gggttctacctgcagctgagtttcatgggctttggg CDC20 RNAI TRIP13 RESISTANCE DNA 2 FWD a2 ggctccagccggaagacatgtcgttacattccttccc CDC20 RNAI TRIP13 RESISTANCE DNA 2 REV b2 gggaaggaatgtaacgacatgtcttccggctggagcc TRIP13 LOV-CDC20 DNA 2 E9 cggggtacgaacatcagaaagcctgggc LOV and CRY TRIP13 MAD2 DNA 2 D9 cggggtaccatggcgctgcagctctcc LOV and CRY TRIP13 MAD2 R DNA 2 D10 ggagagctgcagcgccatggtaccccg MCHERRY TRIP13 ANTISENSE DNA 2 E9 ctttgtacaagaaagctgggtctcagatgtaagc MCHERRY TRIP13 FWD DNA 2 F9 ggaggcggtagtatggacgaggccgtgg

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