Assembly and Function of Mammalian Centrosomes

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DESCRIPTION (provided by applicant): The centrosome is a major microtubule-organizing center responsible for initiation of microtubule assembly in animal cells. Microtubules are important for such processes as mitosis/meiosis, cell motility, and morphogenesis, thus centrosome dysfunction causes serious human diseases, including cancers, metastasis, and birth defects. The overall aim of this application is to understand how assembly and function of the centrosome are controlled at the molecular level. The research focuses on three centrosome proteins: Aurora-A, CNN, and Cep135. A mitosis-specific kinase, Aurora-A is involved in centrosome maturation during cell division. It binds to CNN (centrosomin), which has unique properties to induce microtubule-nucleating sites through interacting with the gamma-tubulin complex. CNN also binds to a highly coiled-coiled protein, Cep135 important for maintaining the structural integrity of the centrosome. The first specific aim is to determine how the CNN activity to form microtubule-nucleating sites is controlled by Aurora-A. Effects of the change in CNN interaction with Aurora-A and its phosphorylation by the Aurora-A kinase will be examined. Additional CNN- and Aurora-A-interacting molecules will be searched using a combined two-hybrid screens and functional RNAi assays. The second objective is to dissect molecules involved in organization of microtubule-nucleating sites. Microtubule formation onto cytoplasmic assemblies induced by CNN overexpression will be characterized in vivo and in vitro. Proteins required for microtubule nucleation will be identified by in vitro tests of microtubule polymerization onto isolated CNN assemblies from which different molecules are stripped off and added back. Attempts will be made to polymerize microtubules onto microbeads coated with CNN and other potential molecules. The third objective is to examine assembly of mammalian centrosomes by supplementing Cep135-depleted cells with exogenous Cep135 and CNN. The process of disintegration and reformation of the functional centrosome will be monitored by fluorescence and electron microscopy. The Aurora-A gene is a tumor-susceptibility gene and its overexpression causes cancer formation. As centrosome abnormalities are implicated in the origin of chromosome instability and cancer pathogenesis, the study will be useful in establishing strategies for prevention, diagnosis, and treatment of human cancers.