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（RBC）膜骨架主要由短的F-肌动蛋白丝组成，在蛋白4.1 R的帮助下，这些肌动蛋白丝通过α 2B 2血影蛋白四聚体交联。肌动蛋白和4.1 R结合血影蛋白B链尾部的肌动蛋白结合结构域（BSpABD）。α-血影蛋白的相邻末端称为EF手结构域（aSpEF），被认为是惰性的;然而，具有非常不稳定的RBC膜的sph（1 J）小鼠产生了缺少最后13个氨基酸的突变体α-血影蛋白（aSpEFAC 13），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)我们将使用下拉分析或光活化交联以及含有所有RBC膜蛋白的提取物来确定与aSpEF结合的物质。初步研究表明，aSpEF而不是aSpEFAC 13以Ca（2+）依赖性方式结合蛋白4.2。我们将表征这种和其他aSpEF-蛋白质相互作用，并研究翻译后修饰或其他因素是否影响结合。2)我们将确定干扰aSpEF相互作用的结合位点片段是否会降低Ektacytometer中测量的膜稳定性。3)我们将制备一系列微血影蛋白，其由BSpABD结构域和aSpEF结构域的变体组成，所述变体由每条链中的最后4个血影蛋白重复序列连接。将评估这些微血影蛋白与F肌动蛋白的结合以及4.1R、4.2和其它aSpEF结合蛋白的作用。4)最后，我们将使用光活化交联剂鉴定膜上邻近4.1 R或4.2的蛋白质，或肌动蛋白连接复合物中邻近aSpEF的蛋白质，并且我们将使用高分辨率EM和免疫EM可视化4.2（-）和sph（1 J）小鼠中正常未拉伸膜骨架和骨骼缺陷的结构。这些研究将提供有关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手结构域的功能。由于所有细胞的质膜和许多细胞器的膜具有血影蛋白骨架，这些研究将在细胞生物学中具有广泛的意义。