MOLECULAR ANALYSIS OF A NOVEL MAP/MTOC COMPONENT

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The purpose of this proposal is to study the function of a novel protein interacting with microtubules and microtubule-organizing centers (MTOCs). Microtubules are involved in vital cell processes including cell division and morphogenesis, and elucidation of the mechanism of microtubule functions and its regulation is crucial to an understanding of biological growth, differentiation and its pathologies. The key elements for regulation of microtubule functions are MAPs (Microtubule-associated proteins) and MTOCs which modulate properties of microtubules and direct organization of higher ordered structures which are essential for performance of microtubule functions. This proposal will focus on a molecule that has recently been identified by a monoclonal anti-phosphoprotein antibody, AX3, raised against MTOCs from the cellular slime mole, Dictyostelium discoideum. The AX3 antigen is a novel MAP associated with MTOCs in a wide range of organisms from fungi to human. The genes coding for the protein in different biological systems (Dictyostelium and HeLa cells) will be cloned and their nucleotide sequences will be compared. Phosphorylation properties of the antigen will be examined during the process of cell cycle and cell differentiation, and the effect of phosphorylation on its association with microtubules will be analyzed in The proteins' capacity to interact with other MTOC components will be examined by constructing affinity columns with fusion proteins produced using recombinant DNA technology. The gene corresponding to the AX3 antigen in Dictyostelium will be disrupted using the gene targeting method found to be successful in this organism, and the phenotype of transformed cells and changes in the microtubule organization and mitotic spindle formation will be analyzed by microscopy. The protein's role in HeLa cells will be evaluated by employing in vitro inhibition of microtubule assembly onto isolated centrosomes, and by microinjection of antibodies into living cells at different cell cycle stages. Intracellular concentration of the protein will be modulated either by introduction of antisense RNA, or microinjection of fusion proteins, or transfection of cells with DNAs expressing the AX3 antigen. Such studies on MAPs and their role in MTOC-directed regulation of microtubule organization enable us to analyze the molecular basis of the microtubule-dependent cellular processes, which will be important for understanding the mechanism of uncontrolled cell division and differentiation.