Biochemical and functional analysis of nuclear complexes

描述（由申请人提供）：

DESCRIPTION (provided by applicant): Organization at the nuclear envelope (NE) is important for nuclear stability and gene expression, and there are many diseases caused by loss, mutation or mislocalization of NE proteins. Loss of emerin, a nuclear membrane protein, specifically causes Emery-Dreifuss muscular dystrophy (EDMD). Emerin forms a variety of oligomeric protein complexes purified from nuclei including a 1.5 MDa complex with actin, spectrin and lamins. The goal of this study is to reconstitute all or parts of this 1.5 MDa emerin-based structural complex in vitro using purified recombinant protein, with characterized emerin mutants as controls. I will determine the relative stoichiometry of reconstituted subcomplex(es), binding affinities and binding sites. In independent experiments I will study the functions of nuclear spectrin and other components in vivo. Each protein will be depleted (by siRNA) in cultured cells. Nuclear architecture and structure will be studied by fluorescence microscopy. Nuclear stability and mechanics will be determined in isolated nuclei by micropipette aspiration. This study will provide fundamental information about the roles of emerin, actin and spectrin in nuclear structure, and will quantify the structural contributions of nuclear proteins to disease.