(Supported by Council for Tobacco Research Inc. #3157 R2 to R. Kuriyama) The centrosome, which is composed of a pair of centrioles and a surrounding amorphous cloud of pericentriolar material, plays an essential role in microtubule organization. We have recently identified a novel centrosomal protein, termed Cep135, which appears to be important for spindle formation and function during mitosis. To understand the role of Cep135 in the structural organization of the centrosome we are attempting to localize this protein in the pericentriolar material by immunoelectron microscopy. We then plan to assess the temporal and spatial relation of Cep135 with other substructures in the centrosome, including gamma-tubulin rings, basal feet and satellites, employing both high- and intermediate-voltage electron microscope tomography, which has proven to be effective in defining several domains included in ill-defined pericentriolar material. Preliminary observation has pr ovided e vidence that over-expression of Cep135 subdomains results in striking modifications of the centrosomal structure and stability in transfected cells. To evaluate the role of Cep135 subdomains in the structural integrity of the centrosome, we will create a series of deletion constructs to express various regions of Cep135 in CHO cells. The detailed nature of morphological as well as functional alternations of the centrosome will be examined both in vivo and in vitro. Dr. Kuriyama's lab prepared EM blocks together with thin-section micrographs of the cells of interest. The cell locations were marked on the blocks, as well. Serial 0.25 and 0.5(m thick sections were cut and examined on the HVEM. The thinner sections proved to be better for locating the centrioles. Centrioles were photographed from two groups of cells and the negatives were sent to Dr. Kuriyama for selection of centrioles for tomography.
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