Molecular Biology of Microtubule Interacting Proteins

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The importance of gamma-tubulin for the assembly of the microtubule cytoskeleton from microtubule organizing centers is now well established. Data obtained in the current grant period and data obtained in other labs suggest, however, that gamma- tubulin also plays an essential role in the functioning of the assembled mitotic apparatus and in the organization of cytoplasmic microtubules. The role of gamma-tubulin in these processes is poorly understood and, given the universal importance of these processes, worthy of study. In the present grant period our lab has created 41 gamma-tubulin mutations in the filamentous fungus Aspergillus nidulans by alanine-scanning site-directed mutagenesis. The mutations confer a variety of phenotypes. Some of the alleles are conditionally lethal such that gamma-tubulin functions improperly under some conditions. Under these conditions most of these mutations do not block the assembly of mitotic spindles but inhibit the functioning of assembled spindles. At least two alleles inhibit the movement of chromosomes to the the poles in mitosis and the growth of three alleles is restored by antimicrotubule agents. The proposed studies will employ these mutations as tools to explore the role of gamma-tubulin in the functioning of assembled microtubules. The aims of the proposal are 1) to determine the mechanism of benomyl/nocodazole suppression of conditionally lethal gamma- tubulin mutations, 2) to determine if microtubule dynamics are altered by gamma-tubulin mutations, 3) to investigate the role of gamma-tubulin in checkpoint regulation, 4) to determine the effects of gamma-tubulin mutations on mitotic progression, and 5) to identify genes that interact with gamma-tubulin.