Organization of the Mammalian Mitotic Spindle

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DESCRIPTION (provided by applicant): Accurate chromosome segregation is essential for the propagation of species and the viability of cells, and is driven by a complex microtubule-based structure called the spindle. Spindle organization and chromosome movement are driven by the concerted actions of microtubule-associated proteins (motor and non-motor) and the inherent dynamic properties of microtubules. Despite extensive knowledge of the proteins involved in spindle morphogenesis and chromosome movement, very little is known about how the accuracy of chromosome segregation is ensured during mitosis in mammalian cells. The purpose of the experiments proposed here is to combine biochemical methods and live cell imaging to identify the proteins and determine the mechanisms underlying the high fidelity of chromosome segregation during mitosis in human cells. Because chromosomes are linked to spindle microtubules through the kinetochore, a focus will be on defining the molecules and mechanisms that govern the dynamic attachment of spindle microtubules to kinetochores. The specific aims of this research are to: 1) combine live cell imaging with quantitative chromosome segregation assays to define the mechanisms regulating kinetochore- microtubule attachment necessary for accurate chromosome segregation; 2) use live cell imaging to examine how the spatial and temporal sequence of spindle assembly contributes to the accuracy of chromosome segregation; 3) use biochemical methods to determine how the kinetochore-associated microtubule depolymerizing activity of the kinesin-13 protein Kif2b is regulated during mitosis; and 4) use live cell assays to determine the fate of human cells that mis-segregate chromosomes. PUBLIC HEALTH RELEVANCE: Chromosome mis-segregation causes aneuploidy that causes birth defects and is commonly associated with advanced stage cancer. The goal of the experiments proposed here is to combine biochemical methods and live cell imaging to identify the proteins and determine the mechanisms underlying the high fidelity of chromosome segregation during mitosis in human cells. Data generated from this work will provide insight into mechanisms of aneuploidy in tumor cells and may reveal strategies for therapy of chromosomally unstable aneuploid tumors.