The long term objective of this proposal is to understand the molecular basis of microtubule organizing center function, which is essential to generating a polar microtubule cytoskeleton to carry out the important tasks of chromosome segregation, cell motility, organelle transport, and nuclear migration. We have only just begun to answer some of the central questions in this field: How does the organizing center nucleate the growth of microtubules? How is nucleation temporally and spatially regulated? Which molecules are important for which functions in the organizing center? Cancer cells often have defects in microtubule organizing center size and number, possibly leading to genetic instability, demonstrating the importance of these questions. To further our understanding of the microtubule organizing center (called the centrosome in animal cells) I will focus primarily on the structure and function of gamma-tubulin and the large macromolecular assembly of which it is part, gamma-tubulin complex (also called the gamma-tubulin ring complex, or gamma-TuRC). The gamma-tubulin complex is required for microtubule nucleation, and has been proposed to act as a direct template for the polymerization of microtubules. The gamma-tubulin complex is made up of gamma-tubulin plus five different polypeptides. This remarkable complex must interact both with microtubules and the centrosome, placing it at the center of microtubule organization. We have found that all five of the gamma-tubulin complex proteins, or GCPs, belong to a new protein superfamily. The importance of gamma-tubulin in microtubule organization has led us to search for new tubulins in the mammalian genome and we have discovered two, d(delta)-tubulin and e(epsilon)-tubulin. Epsilon tubulin has not been described before in any organism, and its properties suggest a role in centrosome function as well. We will attack four specific aims in the next project period: 1) Determine the subunit composition and structure of the gamma-tubulin complex. 2) Define the interactions among the gamma-tubulin complex proteins, and their role in the gamma-tubulin complex. 3) Characterize the interaction of the gamma-tubulin complex with the microtubule and the centrosome. 4) Characterize the role of epsilon-tubulin in centrosome function.
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