Spectrin-Actin Junctions of the Erythrocyte Skeleton

红细胞骨架的血影蛋白-肌动蛋白连接

• 批准号: 7086317
• 负责人: SAMUEL E LUX
• 金额: $ 41.26万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7086317
• 关键词: actins; blood tests; electron microscopy; erythrocyte membrane; human tissue; immunoelectron microscopy; intercellular connection; laboratory mouse; membrane proteins; photolysis; protein binding; protein protein interaction; protein purification; spectrin

DESCRIPTION (provided by applicant): The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments cross-linked by a2B2 spectrin tetramers with the assistance of protein 4.1 R. Actin and 4.1 R bind to the actin binding domain (BSpABD) at the tail of the spectrin B chain. The adjacent end of a-spectrin, called the EF hand domain (aSpEF), has been considered inert; however, the sph(1J) mouse, which has very unstable RBC membranes, makes a mutant a-spectrin that lacks the last 13 amino acids (aSpEFAC13), showing that the domain has an important function in spectrin-actin binding. 1) We will determine what binds to aSpEF, using pull-down assays or photoactivatable cross-linking, and extracts containing all RBC membrane proteins. Initial studies show that aSpEF but not aSpEFAC13 binds to protein 4.2 in a Ca (2+)-dependent manner. We will characterize this and other aSpEF-protein interactions and investigate whether post-translational modifications or other factors affect the binding. 2) We will ascertain whether binding site fragments that interfere with aSpEF interactions decrease membrane stability measured in the ektacytometer. 3) We will prepare a series of mini-spectrins consisting of the BSpABD domain and variants of the aSpEF domain attached by the last 4 spectrin repeats in each chain. The binding of these mini-spectrins to F actin and the roles of 4.1 R, 4.2 and other aSpEF binding proteins will be assessed. 4) Finally, we will identify proteins adjacent to 4.1 R or 4.2 on the membrane, or adjacent to aSpEF in actin junctional complexes using photoactivatable crosslinkers, and we will visualize the structure of the normal unstretched membrane skeleton and the skeletal defects in 4.2(-) and sph (1J) mice using high resolution EM and immunoEM. These studies will provide new information about the molecular junctions that form the core of the membrane skeleton and will illuminate the functions of the previously little studied EF hand domain. Since plasma membranes of all cells and membranes of many organelles have spectrin skeletons, these studies will be of broad interest in cell biology.

描述(由申请人提供)：红细胞膜骨架主要由短的F-肌动蛋白细丝组成，由A2B2血影蛋白四聚体在4.1R肌动蛋白和4.1R结合到血影蛋白B链尾部的肌动蛋白结合域(BSpABD)的辅助下交联。A-血影蛋白的相邻末端被称为EF手区(ASpEF)，一直被认为是惰性的；然而，具有非常不稳定的红细胞膜的sph(1J)小鼠产生了一个突变的缺乏最后13个氨基酸的a-血影蛋白(ASpEFAC13)，表明该结构域在血影蛋白-肌动蛋白结合中具有重要功能。1)我们将使用下拉试验或光活化交联法，以及包含所有红细胞膜蛋白的提取物，确定哪些物质与a SpEF结合。初步研究表明，aSpEF而不是aSpEFAC13以钙离子依赖的方式与蛋白质4.2结合。我们将描述这种相互作用和其他aSpEF与蛋白质的相互作用，并调查翻译后修饰或其他因素是否影响结合。2)我们将确定干扰SpEF相互作用的结合位点片段是否会降低在红细胞测定仪中测量的膜稳定性。3)我们将制备一系列由BSpABD结构域和aSpEF结构域的变体组成的迷你血影蛋白，每个链上的最后4个血影蛋白重复序列连接在一起。这些微型光谱蛋白与F肌动蛋白的结合以及4.1R、4.2和其他aSpEF结合蛋白的作用将被评估。4)最后，我们将利用光活化交联剂在肌动蛋白连接复合体中识别膜上4.1R或4.2附近或aSpEF附近的蛋白质，并将使用高分辨率EM和免疫EM显示4.2(-)和Sph(1J)小鼠正常未拉伸的膜骨架和骨骼缺陷的结构。这些研究将提供关于形成膜骨架核心的分子连接的新信息，并将阐明以前很少研究的EF手区的功能。由于所有细胞的质膜和许多细胞器的膜都有幽灵蛋白骨架，这些研究将在细胞生物学中引起广泛的兴趣。