The long term objectives are to understand the molecular mechanisms of microtubule organizing center function, including microtubule nucleation, microtubule organization, and cell cycle regulated duplication. These objectives are significant in that the microtubule organizing center is essential to cytoplasmic organization, morphological differentiation and mitotic spindle assembly and function. The general strategy is to focus on one important molecular, gamma-tubulin, characterize its role, and use it to identify other important molecules. The microtubule organizing center is a complex organelle, and gamma-tubulin is one of the only highly conserved, functionally important components known. Two experimental systems with distinct nd non-overlapping advantages for study of the organizing center will be used; the egg of the frog Xenopus laevis, and the fission yeast Schizosaccharomyces pombe.
Four specific aims are proposed to address important questions of organizing center function and the role of gamma-tubulin in those functions: 1) Purify gamma-tubulin and characterize its activities on microtubules. gamma-Tubulin will be purified from frog eggs, a rich source. The effects of gamma-tubulin on microtubule nucleation, and the capping of microtubule ends will be examined,a nd athe molecular details of gamma-tubulin microtubule binding determined. gamma-Tubulin exists as a large complex in the cytoplasm: the composition of this complex will be determined. 2) Determine the requirements for assembly of the microtubule organizing center. An in vitro assay that reconstitutes the organizing center formation reaction that takes place during fertilization in Xenopus will be used to determine what components of the egg and sperm are required for the reaction. gamma-Tubulin is one of the components required for the reactions. The relationship of this assembly process to the cell cycle- regulation change in microtubule nucleation capacity will be examined. 3) Study in vivo role of gamma-tubulin in microtubule organization. Conditional-lethal mutations will be isolated int he S. pombe gamma-tubulin gene, and the phenotypes of these mutations will be analyzed in detail. These phenotypes will reveal the in vivo role of gamma-tubulin in a simple microtubule system. 4) Identify proteins that interact with gamma-tubulin and assess their function. The conditional-lethal gamma-tubulin mutations will be used in genetic screens to identify new components of the organizing center, and these new components will be studied genetically to determine their function and their relationship to gamma-tubulin.
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