CYTOSKELETON-MEMBRANE INTERACTIONS

细胞运动和肌动蛋白膜骨架的动态变化 发生在白细胞迁移、神经元生长、伤口愈合和 细胞转化为侵袭性癌症。 虽然这些变化 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质在很大程度上是未知的。 拟议的研究将继续表征网骨藻 脑桥蛋白是唯一已知的结合肌动蛋白的膜蛋白， 使肌动蛋白丝装配成核。 一些技术，包括 分子遗传学、生物化学、细胞生物学和脂质化学， 将用于确定脑桥蛋白的一级结构， 确定它如何以及何时在体内发挥作用。 生物化学机制， 还将阐明哪种脑桥蛋白活性受到调节。 的 本研究的具体目标是：（1）克隆和测序 （2）制备桥脑网骨藻蛋白的结构域- 抗网骨藻特异性和构象特异性抗体 脑桥蛋白和结构相关蛋白;（3）研究 活细胞中的网状骨桥蛋白通过产生功能丧失 使用同源重组、反义RNA和/或 过度表达缺陷或截短的脑桥蛋白;（4）筛选现有的 桥脑蛋白缺陷的运动突变体;（5）检测突变细胞 刺激介导的肌动蛋白聚合的改变，伪足 延伸、细胞移位、趋化性、吞噬作用和细胞-细胞 （6）探讨脑桥蛋白活性的调控机制。 寻找与脑桥蛋白相关的网骨桥蛋白， 研究二酰基甘油，磷酸化， 寡聚化、二硫键还原和跨膜pH和/或膜 潜在的;和（7）表征肌动蛋白结合和成核 牛粒细胞中16 kD膜整合蛋白的活性 可能是牛脑桥蛋白的膜。 最终，我们希望利用 这种蛋白质的组织分布作为指导的一般性， 脑桥蛋白在高等生物中的作用 长期目标是 项目是了解肌动蛋白的分子基础和控制- 膜的相互作用参与能动的过程，并应用这一点， 知识，以了解病理条件， 发育异常、癌细胞侵袭和HIV诱导的 痴呆

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.