Abstract Precise regulation of dynamic microtubules (MTs) is essential for mitotic spindle assembly, chromosome segregation, cell motility, signaling, cell cycle regulation and subcellular patterning. We have established that the kinesin-13 family member MCAK (Mitotic Centromere-associated kinesin), a protein that I discovered 14 years ago, is the most potent regulator of global microtubule dynamics so far measured. We have intensively studied its mechanism of action and its role in cellular processes. We will use these data as a foundation to understand the function of other proteins that modulate microtubule dynamics. Specifically we will investigate a functionally uncharacterized, phylogenetically ubiquitous kinesin that is closely related to the animal-specific MCAK subfamily, Kif24. We will also focus on kinesin-8 family member kif18A, which possesses both motor activity and MT depolymerizing activity and is indispensable for chromosome congression. Each of these kinesins contributes essential activities required for spindle assembly and chromosome segregation during cell division. They represent vital targets for anti-cancer theurapeutics. Specific aims of the proposed studies are as follows: Specific Aim 1: Investigate the degree of conservation between the MT depolymerization mechanisms of kinesin-13/MCAK and kinesin-8/Kif18A subfamilies. We will use TIRF microscopy, mutational analysis, bulk MT depolymerization assays and cryo-EM to determine the contribution of processivity, and inter-head coordination in the ATPase cycle for these motors. In contrast to kinesin-13 motors, we hypothesize that the motile/depolymerizing kinesin- 8s suppress MT dynamics in live microtubules. This contradicts the conjectures drawn from the previously published depolymerization activity of kinesin-8s for stabilized MTs. We will investigate how much of the MCAK MT depolymerization cycle is conserved in the kinesin-8 family to produce this unusual activity. Specific Aim 2: Define the role of kinesin-8 and kinesin-13 family members in controlling chromosome movement and MT spindle fiber dynamics. We hypothesize that MCAK increases K-fiber MT turnover and "loosens" kinetochores from the kinetochore MTs. In contrast, we hypothesize that Kif18A suppresses chromosome movement and decreases kinetochore fiber turnover. We will use RNAi, chimeric contructs and live imaging to investigate the interplay between these two depolymerizing motors that appear to have antagonistic effects on chromosome congression and motility. Specific Aim 3: Investigate the functional interrelationship between the expression of different regulators of MT dynamics. We hypothesize that alterations in the levels and balance of MT +tip proteins (of which MCAK is one) will affect the dynamics of the actin/myosin cytoskeleton resulting in cytokinesis defects and also in modifications to cell motility. We will use high resolution filming, protein depletions and proteomic methods to investigate the mechanism by which this occurs. We further hypothesize that altering the global levels of single regulators of MT dynamics may affect the levels and distributions of other MT regulators via transcriptional and translational feedback. We will use protein and mRNA quantification to score the response of known MT regulators to changes in the potent MT depolymerizer MCAK. PUBLIC HEALTH RELEVANCE The proposed studies are designed to investigate the mechanism of action, regulation and cellular function of kinesins that regulate the dynamic properties of microtubules (MTs). Dynamic MTs are so important for cell division that drugs that alter their dynamic properties even slightly, will usually lead to cell cycle arrest and rapid cell death. Many highly successful anti-cancer drugs, such as paclitaxel and vinblastine that target MTs, exploit this property. In addition, such drugs have been useful in decreasing the motility and invasive potential of cancer cells. Mitotic Centromere-associated Kinesin (MCAK) is the most potent regulator of microtubule (MT) dynamics thus identified. We are interested in exploring its activity and cellular function and also the cellular response to changes in MCAK levels. MCAK has been detected at abnormally high levels in a wide variety of tumor types and is correlated with tumors with an unfavorable prognosis and poor response to anti-cancer drugs. Anti-cancer drugs that target MTs can lead to preferential selection of highly invasive cancers by selecting for cells that modify MT dynamics. Thus, it is extremely important to identify and map the interplay of regulators of MT dynamics during tumor progression and to seek therapies that specifically target these regulators once they are identified. A thorough understanding of their mechanism of action, regulation and cellular function is required to realize this goal.
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NIH ExPorter