CYTOSKELETON-MEMBRANE INTERACTIONS

细胞骨架-膜相互作用

• 批准号: 2176822
• 负责人: ELIZABETH J. LUNA
• 金额: $ 43.91万
• 依托单位: WORCESTER FOUNDATION FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176822
• 关键词: Dictyostelium; actins; animal tissue; antisense nucleic acid; binding proteins; biological signal transduction; cell adhesion; cell cell interaction; cell motility; chemotaxis; cytoskeleton; diacylglycerols; enzyme linked immunosorbent assay; high performance liquid chromatography; immunoprecipitation; laboratory rabbit; membrane activity; membrane potentials; membrane proteins; molecular cloning; phosphorylation; polymerase chain reaction; polymerization; protein structure function; protoplasm motility; western blottings; Dictyostelium; actins; animal tissue; antisense nucleic acid; binding proteins; biological signal transduction; cell adhesion; cell cell interaction; cell motility; chemotaxis; cytoskeleton; diacylglycerols; enzyme linked immunosorbent assay; high performance liquid chromatography; immunoprecipitation; laboratory rabbit; membrane activity; membrane potentials; membrane proteins; molecular cloning; phosphorylation; polymerase chain reaction; polymerization; protein structure function; protoplasm motility; western blottings

Dynamic changes in cell motility and in the actin-based membrane skeleton occur during leukocyte migration, neuronal outgrowth, wound healing, and the transformation of cells into invasive cancers. Although these changes are thought to involve rearrangements of the proteins at the actin-membrane interface, the relevant interacting proteins are largely uncharacterized. The proposed research will continue the characterization of Dictyostelium ponticulin, the only integral membrane protein known that binds actin and nucleates actin filament assembly. A number of techniques, including those of molecular genetics, biochemistry, cell biology, and lipid chemistry, will be used to determine the primary structure of ponticulin and to ascertain how and when it functions in vivo. The biochemical mechanisms by which ponticulin activity is regulated also will be elucidated. The specific aims of the proposed research are: (1) to clone and sequence the cDNA and genomic DNA for Dictyostelium ponticulin; (2) to prepare domain- specific and conformation-specific antibodies against Dictyostelium ponticulin and structurally-related proteins; (3) to examine the role of Dictyostelium ponticulin in living cells by generating loss-of-function mutants using homologous recombination, anti-sense RNA, and/or overexpression of defective or truncated ponticulin; (4) to screen existing motility mutants for defects in ponticulin; (5) to examine mutant cell lines for alterations in stimulus-mediated actin polymerization, pseudopod extension, cell translocation, chemotaxis, phagocytosis, and cell-cell cohesion; (6) to explore the mechanisms regulating ponticulin activity by looking for Dictyostelium proteins associated with ponticulin and by examining the regulatory roles of diacylglycerols, phosphorylation, oligomerization, disulfide reduction, and transmembrane pH and/or membrane potential; and (7) to characterize the actin-binding and nucleating activities of a 16-kD integral membrane protein found in bovine granulocyte membranes that may be bovine ponticulin. Eventually, we hope to use the tissue distribution of this protein as a guide to the generality of ponticulin function in higher organisms. The long-term goals of this project are to understand the molecular basis and control of the actin- membrane interactions involved in motile processes and to apply this knowledge to the understanding of pathological conditions such as developmental abnormalities, cancer cell invasion, and HIV-induced dementia.

细胞运动和基于肌动蛋白的膜骨骼的动态变化 发生在白细胞迁移，神经元出现，伤口愈合和 细胞转化为侵入性癌症。 尽管这些变化 被认为涉及肌动蛋白膜上蛋白质的重排 界面，相关的相互作用蛋白在很大程度上没有表征。 拟议的研究将继续表征dictyostelium Ponticulin，是唯一结合肌动蛋白和 成核肌动蛋白丝组件。 许多技术，包括这些技术 分子遗传学，生物化学，细胞生物学和脂质化学， 将用于确定ponticulin和to的主要结构 确定它如何以及何时在体内发挥作用。 生化机制 还将阐明哪些ponticulin活性。 这 拟议的研究的具体目的是：（1）克隆并序列 poctyostelium ponticulin的cDNA和基因组DNA； （2）准备域 - 针对DICTYOSTELIUM的特异性和构象特异性抗体 ponticulin和与结构相关的蛋白质； （3）检查 通过产生功能丧失，活细胞中的dictyostelium ponticulin 使用同源重组，抗敏感RNA和/或的突变体 有缺陷或截短的庞蒂蛋白的过表达； （4）筛选现有 运动性突变体用于ponticulin中的缺陷； （5）检查突变细胞 改变刺激介导的肌动蛋白聚合，假脚架的线 延伸，细胞易位，趋化性，吞噬作用和细胞细胞 凝聚; （6）探索通过 寻找与ponticulin相关的dictyostelium蛋白 检查二酰基甘油的调节作用，磷酸化， 寡聚，减少二硫化物和跨膜pH和/或膜 潜在的; （7）表征肌动蛋白结合和成核 在牛粒细胞中发现的16 kD整体膜蛋白的活性 可能是牛ponticulin的膜。 最终，我们希望使用 该蛋白的组织分布作为通用性的指南 ponticulin在较高生物体中的功能。 这个长期目标 项目将了解分子基础和控制肌动蛋白 运动过程中涉及的膜相互作用并应用此 了解理解病理状况的知识 发育异常，癌细胞侵袭和HIV诱导的 失智。