The nuclear lamina is a meshwork of intermediate-type filaments lining the inner nuclear membrane that is implicated in regulating nuclear envelope architecture and anchoring chromosomes to the nuclear periphery. The long-term goals of this project are to understand the molecular organization of the lamina and its role in structure and functions of the eukaryotic nucleus. This project period will focus on molecular interactions of the lamina with the inner nuclear membrane and chromosomes. The studies will involve the four major components of the lamina in mammalian somatic cells (lamins A, B1, B2, and C) and three integral membrane proteins associated with the lamina. Regions of nuclear lamins A, B1, B2, and C involved in association with mitotic chromosomes will be identified with in vitro binding assays using bacterially expressed lamins that have been modified by deletion and point mutagenesis. Correspondingly, the chromosomal binding site(s) for lamins will be identified using chemical crosslinking approaches and binding assays with fractionated chromosomal components. The role of the lamina-chromosome interaction in nuclear reassembly and interphase chromosome organization will be evaluated by disrupting this interaction in vivo and in vitro with site-specific antibodies, lamin fragments and lamins altered in their chromosome-binding ability. Furthermore, regulation of the lamin-chromatin interaction during mitosis will be evaluated with in vitro biochemical approaches. The molecular associations of integral membrane proteins of the nuclear envelope with specific nuclear lamins will be broadly characterized using a combination of binding and crosslinking studies. Finally the regions of interaction between a specific integral membrane protein and lamins A and C will be mapped in detail and the functions of this association will be examined using a cell-free nuclear assembly system and in vivo studies involving expression of truncated forms of the integral protein. Together this work should provide important new insight on major karyoskeletal functions of the lamina.
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