RECONSTRUCTION OF RBC CYTOSKELETAL ELEMENTS IN VITRO

红细胞细胞骨架元件的体外重建

• 批准号: 3233557
• 负责人: ANDREAS S ROSENBERG
• 金额: $ 9.5万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3233557
• 关键词: blood proteins; cytoskeleton; elasticity; erythrocyte membrane; erythrocytes; gel electrophoresis; human tissue; mathematical model; membrane lipids; membrane reconstitution /synthesis; nonblood rheology; phosphorylation; solutions; viscosity

We intend to investigate the role of cytoskeletal proteins of human red blood cell (RBC) in formation of structural elements capable of lending elastic properties to the intact RBC membrane. Our approach is to reconstitute in vitro possible elements of the cytoskeleton using highly purified cytoskeletal proteins. We will use, as a first step, the elastic modulus measured by an oscillating rheometer as a measure of elasticity in vitro. We have shown that spectrin and actin assemble spontaneously to form gels possessing respectable elastic modulus. We will investigate the role of other proteins such as band 4.1 as well as the role of phosphorylation and specific ion binding to spectrin, actin and other cytoskeletal components. We will determine the temperature dependence of the elastic modulus of our multiprotein gels. As the next step we propose to develop a 2-D model for reconstitution utilizing monolayers of phospholipids and a Wilhelmy balance. We hope to insert purified band 3 into such monolayer and attach reconstituted elements of spectrin-actin-band 4.1 to the solution side of the monolayer. Although the absolute elasticity and viscosity of such a monolayer is difficult to measure due to the presence of solvent, the apparent elastic modulus determined by oscillating Pt ring provides a good way to follow the effects of changing protein composition and phosphorylation. Our goal is to determine quantitatively the elasticity of such reconstructed protein complexes and determine by comparison to appropriate data for whole red cells whether the structures are likely to represent the cytoskeletal architecture in vivo.

我们打算研究人类红色细胞骨架蛋白的作用。 血细胞(RBC)在形成能够放贷的结构元素 完整的红细胞膜的弹性性质。我们的方法是 利用高效细胞因子在体外重建细胞骨架的可能元件 纯化的细胞骨架蛋白。作为第一步，我们将使用弹性 用振荡式流变仪测量的弹性系数 体外培养。我们已经证明了幽灵蛋白和肌动蛋白自发地组装成 形成的凝胶具有可观的弹性模数。我们将调查 其他蛋白质的作用，如Band 4.1以及 与血影蛋白、肌动蛋白等的磷酸化和特异性离子结合 细胞骨架组件。我们将确定温度的依赖关系 我们的多蛋白凝胶的弹性模数。 作为下一步，我们建议开发一个用于重建的二维模型 利用磷脂的单层和Wilhelmy平衡。我们希望 将纯化的条带3插入到这种单层中并连接重组的 光影蛋白-肌动蛋白带4.1的元件连接到单层的溶液侧。 尽管这种单层的绝对弹性和粘度是 由于溶剂的存在难以测量，表观弹性 由振荡铂环确定的模数提供了一种很好的方法来跟踪 改变蛋白质组成和磷酸化的影响。我们的目标是 定量测定这种重组蛋白的弹性 并通过与整个红色的适当数据进行比较来确定 细胞结构是否可能代表细胞骨架 活生生的建筑。