The centrosome, which is composed of a pair of centrioles and a surrounding amorphous cloud of pericentriolar material, plays an essential role for 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 will first analyze the detailed localization of the protein in the pericentriolar material by immuno-electron microscopy. Temporal and spatial relation of Cep135 with other substructures in the centrosome, including gamma-tubulin rings, basal feet and satellites, will next be assessed by 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 provided evidence that over expression of Cep135 sub-domains result ed in striking modifications of the centrosomal structure and stability in transfected cells. To evaluate the role of Cep135 sub-domains 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. Eight block were sent for preliminary studies. Sections were cut and stained (0.25 and 0.5 micron) and viewed on the HVEM. The cells are numerous and the location of centrioles is time consuming. The images of the centrioles in 0.25 micron sections appear very """"""""muddy"""""""". The fine structure is not clear. The grids were also looked at on the Zeiss 910 and the recorded images are still lacking in clear detail. We discussed the possibility of sample preparation causing the """"""""muddy"""""""" appearance of the centrioles, and our complete procedure for processing and flat embeddment of cells grown on coverslips was sent to Dr. Kuriyama. We were not able to see any """"""""whorl-like"""""""" structures associated with the centrioles in this preliminary examination.
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