Microtubules are important components of the cytoskeleton of eucaryotic cells, and participate in diverse processes such as the development and maintenance of cell shape and in various kinds of intracellular movements (e.g., intraaxonal transport and mitotic chromosome movement during meiosis and mitosis). Microtubule populations differ in cells, from being completely stable such as those found in cilia and flagella, to being extremely dynamic, such as those found in mitotic and meiotic spindles. Microtubules have been found to exhibit a variety of polymerization behaviors in vitro that may reflect the heterogeneous behaviors that they exhibit in cells. Cells may use the various polymerization capabilities of microtubules to accomplish different functions. It seems reasonable to believe that microtubule functions such as those related to the organization and growth of microtubules in cells, and those associated with certain kinds of microtubule-linked motility such as mitotic chromosome movement, are mechanistically determined and regulated through the assembly and disassembly reactions at microtubule ends. Further, it is reasonable to think that diversity in microtubule behavior and function may be related to participation of distinct tubulins and microtubule-associated proteins in different microtubule populations. Thus, the main strategy of this proposal is to investigate the dynamics of tubulin addition and loss at microtubule ends in vitro. A combination of procedures will be employed that can distinguish tubulin addition and loss dynamics at individual microtubule ends, together with analysis by electron microscopy of microtubule length dynamics. Microtubule preparations composed of distinct tubulins and microtubule-associated proteins from brain and sea urchin eggs and sperm will be examined. The goal is to understand the mechanisms responsible for tubulin addition and loss at microtubule ends, and to identify, characterize, and understand the functions of molecules that interact with the surfaces and ends of microtubules and regulate assembly and disassembly dynamics in cells.
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