The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores. The mechanism by which the SAC works is by inhibiting the activity of the Anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase that targets several anaphase inhibitors for proteasome-mediated degradation, including securin and cyclin B. When the SAC is satisfied, the APC/C becomes active and this allows progression through the cell cycle. Work from the last decade identified the mitotic checkpoint complex (MCC) as the main transducer of the SAC. The MCC is composed of BubR1, Bub3, Mad2 and Cdc20 and it is a very potent inhibitor of the APC/C. When the SAC is active, the MCC binds the APC/C and it inhibits its activity. Once the SAC is satisfied, the MCC becomes disassembled, which allows APC/C activation and mitotic progression. However, the mechanisms that dictate MCC assembly and how it inhibits the APC/C remain to be understood. Here, I used a combination of cell biology and in vitro biochemistry to investigate the mechanism by which the MCC component BubR1 participates in the SAC. My data shows that through its interaction with Bub3, BubR1 localises to kinetochores and this event greatly facilitates its assembly onto the MCC and its SAC function. On the other hand, MCC formation and APC/C binding were only dependent on BubR1's N-terminus, therefore questioning the existence of a second Cdc20 binding site. Within this region, TPR domains and an N-terminal motif known as the KEN box (KEN1) mediates these interactions. By contrast, BubR1's second KEN box (KEN2) does not participate in MCC assembly or APC/C binding. However, both in cells and in vitro, the KEN2 box is required for APC/C inhibition. Indeed, I show that this second KEN box promotes SAC function by blocking the interaction of the APC/C with its substrates. Thus, both KEN boxes in BubR1 participate differentially in the SAC, the first to promote MCC assembly and the second one to block substrate recruitment to the APC/C.In addition, I investigated the mechanisms that mediate MCC inactivation, following SAC silencing. I observed that p31comet and APC/C activity cooperate to promote MCC turnover. The implication of these observations in our understanding of the SAC is discussed.

Indiana University-Purdue University Indianapolis (IUPUI) / The Fanconi anemia (FA) pathway consists of 21 genes that maintain genomic stability and prevent cancer. Biallelic mutations within this network cause Fanconi anemia, an inherited bone marrow failure and cancer predisposition syndrome. Heterozygous inborn mutations in FA genes increase risk of breast/ovarian cancers, and somatic mutations occur in malignancies in non-Fanconi patients. Understanding the tumor suppressive functions of FA signaling is important for the study of Fanconi anemia, inherited cancers, and sporadic cancers. The FA network functions as a genome guardian throughout the cell cycle. In addition to the well-established roles of FA proteins in interphase DNA replication/repair, the FA pathway controls mitosis by regulating the spindle assembly checkpoint (SAC) to ensure proper chromosome segregation. The SAC consists of several tumor suppressors, including Mad2, and SAC impairment predisposes to aneuploidy and cancer. However, the in vivo contribution of SAC dysfunction to malignant transformation of FA-deficient cells remains unknown. Furthermore, the mechanisms by which FA proteins regulate the SAC are unclear. To test whether SAC dysfunction drives genomic instability and tumorigenesis in FA, we generated a novel FA-SAC model by intercrossing Fancc-/- and Mad2+/- mice. The intercrossed mice displayed heightened aneuploidy secondary to exacerbated SAC dysfunction. Importantly, these mice were prone to developing hematologic malignancies, particularly leukemia, faithfully recapitulating the clinical phenotype of Fanconi anemia. Upon establishing SAC dysfunction as a driver of tumorigenesis in FA, we next explored the mechanism by which FANCC regulates the SAC. We demonstrated that the mitotic kinase CDK1 phosphorylates FANCC to regulate subcellular localization and SAC function of FANCC during mitosis. Our study highlights the essential role of compromised chromosome segregation in the development of leukemia due to impaired FA signaling. This work furthers our knowledge of FANCC signaling at the SAC, and has implications for future use of mitotic-centered therapies for FA-associated tumors. / 2 years

The development and maintenance of multicellular organisms depends fundamentally on cell division, a series of events largely mediated by the mitotic spindle. Errors in spindle formation and/or function are often associated with severe consequences, most notably cancer. In order to elucidate the cause of such errors and the potential for therapeutic intervention, it is imperative to attain a clear understanding of how cell division normally operates. In this regard, this dissertation focuses on the activity of two microtubule-based motor proteins, Eg5 and dynein, prior to and immediately following nuclear envelope breakdown during mitosis. I show that prophase microtubules are remarkably more dynamic than their metaphase counterparts, moving both toward and away from centrosomes across a wide distribution of rates. Inhibition of Eg5, dynein and Kif2a revealed that a subset of this motion is consistent with microtubule flux, a well-established phenomenon temporally limited to metaphase and anaphase spindles by the preceding literature. My data indicates that flux is operational throughout all of mitosis, possibly functioning at early stages to collect centrosomal components. Immediately following prophase, cells begin assembling bipolar spindles. While the establishment of spindle bipolarity fails in the physical or functional absence of Eg5, I show that co-inhibition of dynein restores a cell’s ability to organize microtubules into a bipolar structure. Despite inhibition of both Eg5 and dynein, these spindles are morphologically and functionally equivalent to controls. Together, these data suggest that Eg5 and dynein share an antagonistic relationship and that a balance of forces, rather than a definitive set of players, is important for spindle assembly and function. To determine how Eg5- and dynein-mediated forces functionally coordinate to bring about antagonism during spindle assembly, I utilize a nocodazole washout assay. I show, via in vivo imaging and in silico modeling, that spindle collapse in the absence of functional Eg5 requires dynein activity and an initial intercentrosomal distance of less than 5.5μm. These data are consistent with a model in which dynein antagonizes Eg5 by crosslinking and sliding antiparallel microtubules, a novel role for dynein within the framework of spindle assembly.

Centrioles are conserved microtubule-based structures that are required for the formation of two important cellular organelles, centrosomes and cilia. Centrosomes form the poles of the mitotic spindle and consist of a pair of centrioles surrounded by a matrix of pericentriolar material (PCM) that has the ability to nucleate and organise microtubules. Centrosome defects are implicated into a variety of human diseases including cancer, microcephaly, and ciliopathies. Therefore it is of great interest to understand the mechanisms that lead to centrosome formation and the consequences that centrosome defects have in cells. I have analysed the roles of several centrosomal proteins in centrosome assembly in Drosophila. My results indicate that Sak/PLK4 is only required for the initial step of centriole duplication, but has no further role in recruitment of PCM. I show that two proteins important for PCM recruitment, Asterless (Asl) and Spd-2, are preferentially phosphorylated when they are integrated into the centrosome and I identified these phosphorylation sites using a phosphoproteomic screen. A phosphorylation site in Asl is specifically phosphorylated in mitosis, and the phosphorylation state of Spd-2 regulates its maintenance at the centrosome, suggesting that phosphorylation of PCM proteins is an important mechanism to ensure PCM assembly specifically at the centrosome and in mitosis. I have performed a global transcriptional analysis of flies lacking centrosomes or having extra centrosomes to investigate the effects of centrosomal defects on a cellular level. Surprisingly, my results indicate that centrosome defects per se do not dramatically alter cellular physiology. Finally, I demonstrate that in the absence of centrioles acentrosomal microtubule-organising centres (aMTOCs) are formed in an Asl- and Cnn-dependent fashion, and I show that these aMTOCs can contribute to spindle focusing in acentrosomal cells.

The centrosome is the major microtubule organising centre in vertebrate cells. CDK5RAP2 is a human protein that localises to the centrosome. At the start of this thesis work, the function of CDK5RAP2 was uncharacterised. Significantly, cdk5rap2 is one of several centrosomal genes that are mutated in the developmental disorder Primary Microcephaly, where affected individuals have smaller brains than expected for the age- and sex-adjusted mean. Orthologues of CDK5RAP2 in the fruit fly (Centrosomin/Cnn) and in fission yeast (Mod20p) have been well characterised and are known to have important roles in maintaining centrosome structure and in regulating microtubule nucleation. CDK5RAP2 shares two evolutionarily conserved domains with Cnn, known as CNN motif 1 and 2. Using the chicken B-cell line, DT40, I have used gene-targeting methods to disrupt both of these domains in CDK5RAP2. This revealed a function for CDK5RAP2 in attaching centrosomes to mitotic spindle poles. Centrosome attachment to spindle poles is mediated by a binding partner of CDK5RAP2, AKAP450. AKAP450 also localises to centrosomes and provides anchorage sites for spindle poles in the centrosome. Disruption of the CNN1 and CNN2 domains of CDK5RAP2 causes mislocalisation of AKAP450 from the centrosome and detachment of centrosomes from spindle poles. My studies in DT40 and in human cell lines revealed that CDK5RAP2 and AKAP450 also cooperate during interphase to maintain the two centrioles in the centrosome as a pair. In addition to a structural role in the centrosome, I also find that CNN motif 1 of CDK5RAP2 plays a role in the cellular response to DNA damage. In the absence of CNN motif 1, cells no longer efficiently arrest the cell cycle in response to damage. Centrosome-mediated mitotic spindle alignment and the DNA damage response have both been implicated in microcephaly. Therefore, defects in these functions of CDK5RAP2 may explain how mutations in cdk5rap2 may lead to microcephaly.

During mitosis, accurate chromosome segregation is essential: gain or loss of genetic information can be detrimental to cell viability, or promote tumourigenesis. The mitotic checkpoint (also known as the spindle assembly checkpoint or SAC) ensures accurate chromosome segregation by delaying cell cycle progression until accuracy can be guaranteed. Mps1 is a protein kinase that is crucial for mitotic checkpoint signalling and also for proper chromosome alignment at metaphase. However, the precise role of Mps1’s catalytic activity is still unclear. Here, I present AZ3146, a novel small molecule inhibitor of Mps1. AZ3146 inhibits recombinant Mps1 in vitro with an IC50 of ~35 nM, and has low activity against a panel of 50 kinases, suggesting a reasonable degree of selectivity. As predicted for an Mps1 inhibitor, AZ1346 treatment led to spindle checkpoint malfunction in cells, accelerated mitotic timing, and perturbed the kinetochore localisation of the checkpoint effector Mad2. AZ3146 has a negative effect on cell viability, suggesting it leads to detrimental missegregations. Thus, the cellular effects of AZ3146 are consistent with Mps1 inhibition, and I was able to use the compound confidently as a tool to further probe the role of Mps1 activity in cells.Strikingly, levels of Mps1 increased at unattached kinetochores following inhibition of its kinase activity, suggesting Mps1’s kinetochore localisation is regulated by its own activity. A kinase-dead GFP-Mps1 fusion protein only accumulated at kinetochores in the absence of endogenous, active Mps1, implicating intra-molecular interactions in regulation of Mps1’s kinetochore localisation. I confirm a role for Mps1 in the mechanism of chromosome alignment, but in contrast to previous reports I did not detect a decrease in Aurora B activity following Mps1 inhibition. On the contrary, both Mps1’s phosphorylation status and its kinetochore localisation were affected by treatment with the Aurora B inhibitor ZM447439, placing Mps1 downstream of Aurora B. As an alternative explanation for the alignment defect in cells with reduced Mps1 activity, I found that levels of the plus-end directed kinesin Cenp-E were markedly decreased at unaligned kinetochores. I propose a model in which catalytically active Mps1’s auto-release from kinetochores simultaneously promotes both mitotic checkpoint signalling and chromosome alignment by facilitating Mad2 dimerisation and Cenp-E binding at unattached kinetochores.

A cell must build a bipolar mitotic spindle in order to faithfully segregate replicated DNA. To do so, multiple microtubule nucleation pathways are utilized to generate the robust spindle apparatus. TPX2, a microtubule binding protein, holds crucial roles in both the Ran-dependent and Augmin-dependent pathways where microtubules are nucleated near the chromosomes and from pre-existing microtubules. However, the exact role TPX2 plays in branching microtubules is less understood. Here, we explored the effect of truncating the essential TPX2 C-terminal 37 amino acids on Augmin localization and branching microtubule activity. First, we depleted LLC-Pk1 cells of the Augmin subunit HAUS6 and show that microtubule nucleation around the chromosomes following a nocodazole washout is strongly reduced leading to exaggerated kinetochore microtubule growth. Next, we depleted endogenous TPX2 in LLC-Pk1 cells harboring full length or truncated TPX2 bacterial artificial chromosome (BAC) DNA. Results show that TPX2 710 LAP cells have reduced Augmin localization on the spindle fibers, which correlates with reduced microtubule regrowth in the chromosomal region. In TPX2 710 LAP cells, regrowth was like Augmin depleted cells. Therefore, we provide evidence that the far C-terminus of TPX2 is required for branching microtubule nucleation and that kinetochore microtubule growth is Augmin-independent. In addition, we investigated cell cycle regulation of TPX2 by mutating the S738 phosphosite in the C-terminal motor interacting region. We utilized BAC recombineering to create phospho-mimetic and phospho-null mutants. In combination with plasmid DNA knockdown/rescue, overexpression and spindle assembly assays, we show that the phosphorylation of the C-terminal domain contributes to early mitotic events. LLC-Pk1 cells showed a significant increase in aberrant spindle morphology and reduced spindle stability in the presence of 738A and absence of endogenous TPX2. While rescue with the alanine mutant caused in an increase in multipolar spindles, overexpression resulted in a strong dominant negative monopolar phenotype. Therefore, S738 appears to contribute to mitotic force regulation during mitosis. In conclusion, the far C-terminus of TPX2 and its regulation play a role in the formation of a proper mitotic spindle.

Indiana University-Purdue University Indianapolis (IUPUI) / Fanconi Anemia (FA), a chromosomal instability syndrome, is characterized by bone marrow failure, genetic malformations, and predisposition to malignancies like acute myeloid leukemia (AML) and solid tumors. FA is caused by germline bi-allelic mutations in one of 21 known FA pathway genes and somatic mutations in FA genes are also found in a variety of sporadic cancers. Recently, numerous reports have discovered that the protective function of the FA pathway extends beyond its canonical role in regulation of DNA repair in interphase. In particular, the FA pathway has been shown to function in essential mitotic processes including spindle assembly checkpoint (SAC), cytokinesis, and centrosome maintenance. Understanding of the mechanistic origins of genomic instability leading to carcinogenesis and bone marrow failure has important scientific and clinical implications. To this end, using a micronucleus assay, we showed that both interphase DNA damage and mitotic errors contribute to genomic instability in FA ex vivo and in vivo. Functional studies of primary FA patient cells coupled with super-resolution microscopy revealed that FANCA is important for centrosome dependent spindle assembly supporting the protective role of FA pathway in mitotic processes. Furthermore, we dissected the interactions between the FA pathway and cellular kinase networks by employing a synthetic lethality sh-RNA screen targeting all human kinases. We mapped kinases that were synthetically lethal upon loss of FANCA, particularly those involved in highly conserved signal transduction pathways governing proliferation and cell cycle homeostasis. We mechanistically show that loss of FANCA, the most abundant FA subtype, results in in premature degradation of the mitotic kinase BUBR1 and faster mitotic exit. We further demonstrate that FANCA is important for PCAF-dependent acetylation of BUBR1 to prevent its premature degradation. Our results deepen our understanding of the molecular functions of the FA pathway in mitosis and uncover a mechanistic connection between FANCA and SAC phosphosignaling networks. These findings support the notion that further weakening the SAC through targeting kinases like BUBR1 in FA-deficient cancers may prove to be a rational therapeutic strategy.

Chromosome alignment is highly conserved in all eukaryotic cell divisions. Microtubule (MT) -based forces generated by the mitotic spindle are integral for proper chromosome alignment and equal chromosome segregation. The kinetochore is a multi-subunit protein complex that assembles on centromeric regions of chromosomes. Kinetochores tether chromosomes to MTs (K fibers) that emanate from opposite poles, in a process called biorientation. This linkage translates K fiber dynamics into chromosome movements during alignment and segregation. Stable, high-affinity kinetochore attachments promote spindle assembly checkpoint (SAC) silencing, which is active when unattached kinetochores are present. During chromosome alignment, 1) K fiber plus-end dynamics decrease, confining chromosome movements near the spindle equator, and 2) electrostatic interactions between kinetochore proteins and MTs increase. Chromosome segregation occurs as soon as all chromosomes are stably attached to microtubules and the SAC has been silenced. SAC silencing and chromosome alignment are temporally coordinated during normal divisions, implying that the mechanisms regulating K fiber dynamics and kinetochore affinity must be linked. Interestingly, HeLa cells depleted of a kinesin-8 motor Kif18A, known for its role in promoting chromosome alignment, display a SAC-dependent mitotic delay due to kinetochore-MT attachment defects. This is puzzling, as Kif18A's function in chromosome alignment is to suppress MT growth by stably associating with MT plus-ends. Whether Kif18A is required for attachment in all cells and how it promotes kinetochore microtubule linkages are not understood. The work presented in this dissertation supports a model in which Kif18A functions as a molecular link that coordinates chromosome alignment and anaphase onset. We find that Kif18A is required to stabilize kinetochore-MT attachments during mammalian germline development, as germline precursor cells in Kif18A mutant mice are unable to divide during embryogenesis due to an active SAC. However, while all cell types require functional Kif18A for chromosome alignment, mouse primary somatic cells can still divide with normal timing. This finding indicates a cell-type specific dependence on Kif18A for stabilizing kinetochore-MT attachments, and provides evidence that this function might be separate from Kif18A's known role in chromosome alignment. Consistent with this idea, we find that an evolutionarily conserved binding motif for protein phosphatase 1 (PP1) is required for Kif18A's novel role in regulating kinetochore microtubule attachments. Kif18A-PP1 interaction is required for Kif18A-mediated dephosphorylation of the kinetochore protein Hec1, which enhances attachment. However, Kif18A's interaction with PP1 is dispensable for chromosome alignment. Thus, point mutations that disrupt PP1 binding separate Kif18A's role in stabilizing kinetochore attachments from its function in promoting chromosome alignment. Additionally, through structure function studies of the motor domain, we identified a long surface loop (Loop2) that is required for Kif18A's unique MT plus-end binding activity, which is essential for its function in confining chromosome movements. Taken together, we find that Kif18A is molecularly tuned to provide temporal control of chromosome alignment and anaphase entry.

The spindle assembly checkpoint (SAC) is an evolutionarily conserved mechanism that delays the initiation of anaphase by inhibiting the Anaphase Promoting Complex (APC) until all kinetochores have achieved bipolar attachment on the mitotic spindle. Mad1-3, Bub1, and Bub3, components of the SAC, are conserved from yeast to humans. These proteins localize to unattached kinetochores, though it is unknown with which kinetochore proteins they interact and how these interactions transduce information about microtubule attachement. Here, purification of the checkpoint proteins from Saccharomyces cerevisiae suggests that Mad1 interacts with the outer kinetochore protein Ndc80 in a SAC, cell cycle, and DNA dependent manner. Ndc80 is thought to mediate attachment of kinetochores to microtubules so the interaction between Mad1 and Ndc80 suggests a mechanism by which cells sense kinetochore-microtubule attachment. The SAC is of special importance in some types of cancer where genetic damage and aneuploidy is correlated with mutated SAC genes. A better understanding of the SAC mechanism will aid in the development of targetted cancer therpeutics.