The central problems addressed in the proposed research are how the centrosome is assembled, how it duplicates once per cell cycle, and what happens in cells in which centrosome number is aberrant. The centrosome nucleates microtubules and organizes those microtubules to create a useful array. The centrosome is the major microtubule organizing center in animal cells, and is present in a single copy at the beginning of the cell cycle. The centrosome duplicates in S phase, and the two resulting centrosomes help to organize the two poles of the mitotic spindle. Work from my lab and others over the last ten years has identified molecules involved in microtubule nucleation, the central regulators of centrosome duplication, important structural proteins involved in duplication, and control mechanisms that control centrosome number and link the centrosome and the cell cycle. Interest in the centrosome has grown recently because a correlation has been established between centrosome abnormalities and the development of cancer. Cancer cells often have extra centrosomes, which is likely to contribute to the genomic instability and rapid evolution characteristic of this disease. The proposed experiments make use of the strengths that we have developed in reagents and assays for studying centrosome structure, function and duplication. I address four specific aims in this proposal: 1) Characterize the block to centrosome reduplication. We will test models for the mechanism of the block, determine whether cancer cells have defects in the block, and test whether the block can be overcome by manipulation of potential regulators. 2) Investigate the relationship between centrosome number, ploidv, and genome instability. We will use cell fusion methods to create cells of defined centrosome number and ploidy, examine the progression and outcome of mitosis in these cells, and compare normal and cancer cells in their response to centrosome and ploidy abnormalities. 3) Define the molecular interactions of the gamma-tubulin ring complex with microtubules and with the centrosome. We will use purified components to determine the molecular interactions between the gamma-tubulin ring complex, the microtubule, and the centrosome. 4) Investigate the role of delta-tubulin and epsilon-tubulin in centrosome function and duplication. We will characterize the interactions between delta-tubulin, epsilon-tubulin and the other known tubulins, and define the function of delta-tubulin in vitro using assays in frog egg extracts, and in vivo in human cells.
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