MOLECULAR ANALYSIS OF CEP135 IN THE CENTROSOME

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Microtubules are involved in fundamental cellular processes, such as mitosis/meiosis, cell motility, intracellular transport and morphogenesis. Any perturbation in their functions causes a number of serious human diseases including birth defects and cancers. The key structure to the mechanism of the microtubule function is the MTOC (microtubule-organizing center), or centrosome which is composed of a pair of centrioles and the surrounding amorphous pericentriolar material. The centrosome controls the temporal and spatial distribution of microtubules, and recent evidence has indicated that cancer cells include centrosomes abnormal in structure and function. The purpose of our research is to understand how the centrosome regulates microtubule nucleation, orientation and anchorage at the molecular level. We have recently identified a novel centrosomal component (Cep135) present in a wide range of organisms. It is a structural protein involved in microtubule organization and spindle assembly during mitosis. Experiments are proposed to extend our studies on Cep135 in order to evaluate the working hypothesis that Cep135 is a matrix component of the centrosome important for maintenance of overall integrity of the pericentriolar material. Functional analysis will be done using both stable CHO cell lines expressing different forms of Cep135 and the Xenopus cell free extracts. The role of Cep135 in the process of bipolar spindle formation will be assessed in p53-deficient cells which contain multiple copies of centrosomes. Overexpression of Cep135 results in the formation of extraordinary filamentous structures in the centrosome; the nature of morphological defects caused by the change in the level of Cep135 will be determined using high resolution immunofluorescence microscopy and electron tomography. Finally, we will investigate the pattern of Cep135 interaction with other molecules in the centrosome, and a large Cep135-containing fraction identified in frog extracts will be further characterized.