MOLECULAR REGULATION OF INTERMEDIATE FILAMENT STRUCTURE

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Intermediate filaments are major structural features of the cytoskeleton of mammalian cells. Although the filaments are dynamic structures and are reorganized during cell division, little is known about the molecular mechanisms which regulate filament structure and organization. The hypothesis of the proposed experiments is that protein phosphorylation plays an important role in the regulation of intermediate filament organization, and that the increased level of filament phosphorylation observed in dividing cells is a direct reflection of this regulation. The proposed experiments will use the intermediate filament protein vimentin as a model for phosphorylation regulated filament organization. We will determine the specific vimentin serine sites that are phosphorylated in non-mitotic and mitotic cultured mouse cells as well as following in vitro phosphorylation with purified protein kinases. We will develop an in vitro model system to study the effect of vimentin phosphorylation on specific interactions with membrane and nuclear envelope proteins. We will evaluate the effect of in vitro phosphorylation on the homotypic associations between vimentin molecules that results in filament disassembly. Finally, we will clone vimentin cDNAs and produce cDNAs which are specifically altered at mitosis specific serine phosphorylation sites by oligonucleotide directed mutagenesis. These recombinant cDNAs will be inserted into an expression vector and used to transfect cells which do not express vimentin. Lines of stablely transfected cells will be isolated and characterized for vimentin filament protein content and phosphorylation. Cell lines with an appropriate phenotype will then be used in cell cycle studies to determine if alteration of specific phosphorylation sites alters filament organization or the ability of cells to remodel intermediate filaments during cell division. The long term goal of these studies is to better understand the role of protein phosphorylation in the control of cytoplasmic organization and the relation of this process to cellular proliferation.