Biochemical and functional analysis of nuclear complexes

描述（由申请人提供）： 核被膜（NE）上的组织对于核的稳定性和基因表达是重要的，并且存在许多由NE蛋白的缺失、突变或错误定位引起的疾病。Emerin（一种核膜蛋白）的缺失专门导致埃默里-德赖富斯肌营养不良症（EDMD）。Emerin形成多种从细胞核纯化的寡聚蛋白复合物，包括与肌动蛋白、血影蛋白和核纤层蛋白的1.5MDa复合物。本研究的目的是使用纯化的重组蛋白在体外重建全部或部分1.5 MDa的基于Emerin的结构复合物，以特征性Emerin突变体作为对照。我将确定重组子复合物的相对化学计量、结合亲和力和结合位点。在独立的实验中，我将研究核血影蛋白和其他成分在体内的功能。每种蛋白质将在培养的细胞中耗尽（通过siRNA）。核结构和结构将通过荧光显微镜进行研究。核稳定性和力学将通过微管抽吸在分离的细胞核中确定。这项研究将提供有关Emerin，肌动蛋白和血影蛋白在核结构中的作用的基本信息，并将量化核蛋白对疾病的结构贡献。

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.