RED CELL MEMBRANE SKELETON: ULTRASTRUCTURE AND FUNCTION

红细胞膜骨架：超微结构和功能

• 批准号: 3344935
• 负责人: BETTY W SHEN
• 金额: $ 10.64万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-12-01 至 1987-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3344935
• 关键词: cell age; chromatography; cytoplasm; electron microscopy; electrophoresis; erythrocyte membrane; evolution; membrane activity; sickle cell anemia

We propose a detailed analysis of the fine structure and deformability of the network of spectrin, actin, and band 4.1 which lines the cytoplasmic surface of the human red blood cell membrane. The goal of this work is a better understanding of normal red cell membrane durability, deformability and shape and its alterations in sickle cell disease. We have recently shown by electron microscopy of negatively-stained specimens that isolated red cell skeletons are constructed of actin filaments approximtely 50 nm in length linked by multiple spectrin tetramers at sites of association with oligomers of band 4.1. We propose to extend these initial studies as follows: (1) Examine skeletons prepared in isotonic saline plus 10 mM Mg++ so as to conserve their native structure and tropomyosin content against dissociations. (2) Generate and examine well-reserved unit fragments of the skeleton which have lost none of their spectrin, actin or band 4.1. (3) Test for the existence of cables of two or more spectrin molecules running between adjacent sites on neighboring actin filaments. (4) Generate, isolate and characterize native spectrin-oligomeric band 4.1 ensembles free of actin. (5) Measure the length of native (undegraded) actin protofilaments from red cells of varied age, phylogenetic origin and disease state. (6) Demonstrate definitively that the slow-growing ends of red cell actin protofilaments are free to elongate (i.e., bind G-actin). (7) Add G-actin onto actin filaments within red cells and determine whether spectrin migrates so as to bind to the new extensions. (8) Develop a quantitative assay for network deformability based on laser diffraction analysis of the shape of networks subjected to controlled shear stress. (9) Examine whether mechanical deformations or treatments which reduce cellular deformability alter the molecular ultrastructure of the skeletons. (10) Examine the network of reversibly and irreversibly sickle cells according to the approaches outlined above.

我们提出了对精细结构和变形能力的详细分析 血影蛋白、肌动蛋白和排列在细胞质中的带 4.1 组成的网络 人体红细胞膜表面。 这项工作的目标是 更好地了解正常红细胞膜的耐久性、变形性 镰状细胞病中的形状及其改变。 我们最近通过电子显微镜显示了负染色 分离的红细胞骨架由肌动蛋白构成的标本 长约 50 nm 的细丝由多个血影蛋白连接 与条带 4.1 寡聚物结合位点的四聚体。 我们建议 将这些初步研究扩展如下：（1）检查准备好的骨架 在等渗盐水中加 10 mM Mg++ 以保存其天然结构 和抗解离的原肌球蛋白含量。 (2)生成并检查 保存完好的骨骼单位碎片，没有丢失任何内容 血影蛋白、肌动蛋白或带 4.1。 (3) 测试两根电缆是否存在 或更多血影蛋白分子在相邻位点之间运行 肌动蛋白丝。 (4) 生成、分离和表征本机 血影蛋白寡聚带 4.1 整体不含肌动蛋白。 (5) 测量 来自不同红细胞的天然（未降解）肌动蛋白原丝的长度 年龄、系统发育起源和疾病状态。 (6)明确论证 红细胞肌动蛋白原丝缓慢生长的末端可以自由地 伸长（即结合 G-肌动蛋白）。 (7) 将G-actin添加到肌动蛋白丝上 红细胞并确定血影蛋白是否迁移以与新细胞结合 扩展。 (8) 开发网络变形性的定量分析方法 基于网络形状的激光衍射分析 受控剪切应力。 (9) 检查是否有机械变形或 减少细胞变形性的治疗会改变分子 骨骼的超微结构。 (10) 检查可逆网络 和根据上述方法不可逆镰状细胞。