The long-term objectives of this work are to understand the molecular and structural organization of the nuclear lamina and to determine how this is important for nuclear function. The lamina consists of a polymer of intermediate-type filament proteins called lamins, which directly bind to chromatin, and a number of more minor lamina-associated polypeptides. Work in this project period will be focused on analysis in mammalian cells of two lamin-binding integral membrane protein of the inner nuclear membrane, LAP1 and lAP2. These are good candidates for proteins involved in attachment and assembly of lamins at the inner nuclear membrane. The project also will involve detailed functional analysis of the interaction of lamins with chromatin.
A first aim i s directed at dissecting the molecular interactions of LAP1 and LAP2. This ill involve characterization of the region of LAP2 that interacts with chromosomes, the chromosome component that binds to LAP2, and the regions of lamins and LAP1 that associate.
A second aim will examine the roles of lAP1 and LAP2 in nuclear envelope and chromosome organization by functional studies in cultured cells. This will involve expression of mitotic phosphorylation site mutants of lAP2, microinjection of antibodies to LAP1 and lAP2, downregulation of LAP1 and lAP2 expression by antisense approaches, and expression of putative dominant negative mutants of lAPs.
A third aim will examine possible roles of LAP1 and lAP2 in modulating nuclear lamina structure by in vitro assembly studies with purified lamins and lAPs.
A fourth aim will involve in vivo functional analysis of the lamin-chromosome interaction by injection of antibodies into cultured cells and long-term expression of lamin mutants deficient in chromatin binding that may affect genome stability. Together this set of approaches is expected to significantly advance current understanding of the nuclear lamina, and contribute to knowledge of how aberrations in nuclear organization can promote human disease states such as cancer.
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