MICROFILAMENT ORGANIZATION DURING MITOSIS

Rutgers The St Univ Of Nj New Brunswick, New Brunswick NJ

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Abstract

The proposed research is aimed at improving our understanding of the molecular mechanisms that regulate the microfilament reorganization seen during mitosis and oncogenic transformation. The disassembly of stress fibers and focal adhesions associated with mitosis and oncogenic transformation are dramatic results of microfilament reorganization. These lead to changes in cell shape (e.g., rounding), inhibition of cell migration, and reduced cell-cell and cell-substrate adhesions. The proposed research will investigate the biological functions of three proteins that play a crucial role in these reorganizational events. These proteins, which undergo changes in their phosphorylation states in synchrony with the cell cycle, are the myosin-targeting subunit of myosin phosphatase (MYPT), citron kinase, and focal adhesion kinase (FAK). To explore the physiological roles of these proteins (particularly regarding their phosphorylation), mutants with altered phosphorylation sites will be designed so as to constitutively activate or block the proteins' biochemical activities. These mutants, and/or antibodies that block the proteins' functions, will be microinjected into cultured cells to determine how the proteins contribute to the regulation of stress fibers and focal adhesions, and whether such mutants alter cellular activities such as cell spreading, migration and proliferation. Interactions of the proteins and mutants with other proteins involved in the function of the actin cytoskeleton will also be studied, and various kinases will be compared as to their kinetic properties and intracellular localization. Because alterations in microfilament structures and functions are closely coupled with cell proliferation in both normal cells and tumor cells, these studies will increase the understanding not only of how cells divide, but also of why cancer cells lose normal control of cell division, and will thus help to develop new cancer therapies. In the longer term, such understanding may also contribute to the understanding of certain processes controlling embryonic development at the cellular level, and thus may be relevant to understanding the etiology of certain birth defects.