The overall objective of this project is to determine the molecular and structural mechanisms which regulate microtubule (Mt) assembly and move chromosomes. Because chromosome movement is tightly coupled to the dynamics of Mt assembly, our approach continues to be focused on establishing the pathways and regulation of microtubule assembly. A major strength of our research effort is, the development and application of new techniques in quantitative optical microscopy, fluorescence analog cytochemistry, caged compounds, laser optical traps, video-enhanced contrast and digital image processing and analysis to measure the dynamics of Mt-associated processes in living cells and reconstituted preparations in vitro. The major specific aims are: (1) to determine the mechanism of dynamic instability of plus and minus ends of Mts (a) using video assays of Mt dynamics in reconstituted preparations in vitro and in extracts of cells obtained in interphase and mitotic phases of the cell cycle, and (b) by identifying cytoplasmic factors in sea urchin eggs and clam oocytes which enhance elongation velocity, regulate the transition frequencies of dynamic instability, and nucleate growth to establish the in vitro conditions which can support life-like spindle assembly and chromosome movement; (2) to clarify the functioning of kinetochores during chromosome movements (a) by doing detailed kinetic studies of kinetochore movements in vivo, (b) using photoactivation of caged fluorescent tubulin to mark regions of kinetochore microtubules and high resolution video microscopy to track fluorescent tubulin and kinetochore motility, (c) analyzing Mt attachment to kinetochores and dynamic instability at the attachment site, and (d) using micromanipulation and laser optical traps to test how changes in tension forces at the kinetochore may modulate dynamic instability of kinetochore Mts; and (3) to identify and characterize molecular motors which participate in meiotic and mitotic processes (a) by continuing our video assays in collaboration with Sharyn Endow of domain functions of Drosopholia segregation motors like the claret and when expressed in bacteria, and (b) using our video assays to test for in vitro motility of centromeric regions of yeast artificial chromosomes generated by Kerry Bloom.
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