CYTOSKELETON-MEMBRANE INTERACTIONS

细胞运动性和基于肌动蛋白的膜骨架的动态变化 发生在白细胞迁移、神经元生长、伤口愈合和 细胞转化为浸润性癌症。尽管这些变化 被认为涉及肌动蛋白膜上蛋白质的重排。 在界面上，相关的相互作用蛋白在很大程度上是未知的。 拟议的研究将继续Dictyostelius的特征 肌动蛋白是目前已知的唯一结合肌动蛋白和肌动蛋白的膜蛋白。 使肌动蛋白细丝组装成核。许多技术，包括那些 分子遗传学、生物化学、细胞生物学和脂类化学， 将被用来确定黄连蛋白的一级结构和 确定它在体内的作用方式和时间。生物化学机制通过 还将阐明哪些脑桥蛋白活性受到调控。这个 拟议研究的具体目标是：(1)克隆和测序 (2)制备结构域 抗网囊藻的特异性和构象特异性抗体 脑桥蛋白和结构相关蛋白；(3)研究其作用 在活细胞中通过产生功能丧失而产生的网柄扁茎线虫蛋白 使用同源重组、反义RNA和/或 过表达缺陷或截短的脑桥蛋白；(4)筛选现有的 针对脑桥蛋白缺陷的运动性突变；(5)检测突变细胞 刺激介导的肌动蛋白聚合的改变线，伪足 延伸、细胞移位、趋化、吞噬和细胞间 凝聚力；(6)探讨脑桥蛋白活性的调节机制 寻找与Pitticlin相关的Dictyostelials蛋白，并通过 研究二酰基甘油、磷酸化、 齐聚、二硫化物还原和跨膜pH和/或膜 潜力；和(7)表征肌动蛋白的结合和成核 牛粒细胞中发现的16kD完整膜蛋白的活性 可能是牛脑桥蛋白的膜。最终，我们希望使用 这种蛋白质的组织分布作为指导一般情况的指南 脑桥蛋白在高等生物中的作用。这样做的长期目标是 项目是了解肌动蛋白的分子基础和控制- 涉及运动过程的膜相互作用及其应用 对病理情况的了解，如 发育异常、癌细胞侵袭和艾滋病毒诱导 痴呆症。

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.