Thesis (Ph. D.)--University of Virginia, 1997. / Spine title: Control of anaphase onset checkpoint. Includes bibliographical references (172-192). Also available online through Digital Dissertations.

In cells undergoing mitosis with unreplicated genomes (MUG), anaphase is successfully initiated despite the abundance of kinetochores that are attached to microtubules emanating from both spindle poles (merotely). In cultured cells, merotely is associated with lagging at the metaphase plate. Treatment with microtubule-perturbing drugs alters the frequency of lagging, but the effect of these drugs on MUG cells is unclear. In this study, low doses of a microtubule-stabilizing drug, taxol, or a microtubule-destabilizing drug, nocodazole, dramatically increased the frequency of lagging kinetochores in the midbody of MUG daughter cell pairs. Likewise, increasing the kinetochore number increased the frequency of lagging kinetochores. In this thesis, these data are used to propose a model of mitosis in which the bipolar attachments of MUG cells are reduced to monopolar attachments that are stabilized by their perpendicular orientation with respect to the kinetochore, allowing for spindle assembly checkpoint satisfaction without centromeric tension.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available online through Digital Dissertations.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 34-38).

The accurate segregation of DNA during cell division is essential for the viability of future cellular generations. Genetic material is packaged in the form of chromosomes during cell division, and chromosomes are segregated equally into two daughter cells. Chromosome mis-distribution leads to genetic disorders (e.g. Down's syndrome), aneuploidy and cancer. The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor (the Mitotic Checkpoint Complex-MCC). The MCC delays anaphase, thus giving cells time to fix attachment errors. Although the major checkpoint proteins were identified several years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the checkpoint signal. Yeast genetics and proteomics have revealed that kinetochores are highly complex molecular machines with almost 50 kinetochore components and ~10 components of the spindle checkpoint machinery. Such complexity makes the separation of error correction, kinetochore bi-orientation and microtubule attachment functions very challenging. To circumvent this complexity, a synthetic version of the spindle checkpoint (SynCheck), based on tetO array was engineered at an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes that combined targeting, initially of KNL1Spc7 with Mps1Mph1 and later of Bub1 (but not Mad1) with Mps1Mph1 fragments, was able to activate the spindle checkpoint and generate a robust arrest. The system is based on, soluble complexes, which were formed between KNL1Spc7 or Bub1 with Mps1Mph1. The synthetic checkpoint or 'Syncheck' is independent of localisation of the checkpoint components to the kinetochores, to spindle pole bodies (SPBs) and to nuclear pores. By using the synthetic tethering system a Mad1-Bub1 complex was identified for the first time in S.pombe. Bub1- Mad1 complex formation is crucial for checkpoint activation. Bub1-Mad1 gets phosphorylated itself and is thought to act as an assembly platform for MCC production and thereby generation of the "wait anaphase" signal. The ectopic tetO array is an important tool, not only for generating MCC formation and activating the spindle checkpoint, but also for providing a nice system for analysing in vivo protein-protein interactions. The ectopic array is capable of not only recruiting checkpoint components, but also recruiting them in a physiological manner (similar to the unattached kinetochores). For this reason it was decided to adopt this system to examine the role of the conserved Bub1TPR domain in the recruitment of other spindle checkpoint proteins. This work represents two novel functions for the S. pombe Bub1TPR domain. For the first time in S. pombe, both in vivo tethering and in vitro experiments with purified, recombinant proteins showed that the Bub1 has the ability to homodimerise and to form a complex with Mad3BubR1 through its TPR domain. These results revealed that complex formation of Bub1 with Mad3BubR1 is important for checkpoint signalling and that the highly conserved TPR domains in BubR1Mad3 and Bub1 have key roles to play in their interactions.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: References located at the end of each chapter.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 59-68).

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 138-139

The MAP kinase pathway is well known for its key roles in regulating cell proliferation and cell cycle progression. MAP kinases have also been implicated in mitotic functions, however these functions are less-well understood. Recent studies from our laboratory used Xenopus egg extracts to identify B-Raf as an essential activator of the MAPK cascade during mitosis. Therefore, the first objective of my dissertation research was to determine if B-Raf has functional significance during mitosis in human somatic cells. Using RNA interference against B-Raf and various immunofluorescence techniques, I show that B-Raf: (1) localizes to and is phosphorylated at a key mitotic structure, (2) is critical for proper mitotic spindle assembly and chromatin congression, (3) is important for the engagement of microtubules with kinetochores during mitosis, and (4) is necessary for activation of the spindle assembly checkpoint. It has been demonstrated that B-Raf is a prominent oncogene, constitutively activated in the vast majority of melanomas and other cancers. I hypothesized that oncogenic B-Raf expression perturbs mitosis and causes aneuploidy. First, we show that oncogenic B-Raf expression correlates with mitotic abnormalities in human melanoma cells and that spindle defects are induced when oncogenic B-Raf is ectopically expressed. Further, using FISH and karyotype analysis, I demonstrate that oncogenic B-Raf drives aneuploidy and chromosome instability in primary, immortalized, and tumor cells. In summary, my dissertation studies elucidate novel roles for B-Raf in mammalian mitosis. In addition, my studies show for the first time that oncogenic B-Raf disrupts mitosis causing chromosomal instability. I propose that oncogenic B-Raf-induced chromosome instability contributes to tumorigenesis.

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.