Phosphoinositide Regulation of Focal Adhesion Structure

磷酸肌醇对粘着斑结构的调节

• 批准号: 7118241
• 负责人: Juliet A GREENWOOD
• 金额: $ 21.07万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118241
• 关键词: X ray crystallography; alpha actinin; cell adhesion; cell migration; fibroblasts; focal adhesion kinase; green fluorescent proteins; high performance liquid chromatography; immunoprecipitation; mass spectrometry; neoplastic cell; phosphatidylinositols; platelet derived growth factor; protein binding; protein localization; protein protein interaction; protein structure function; site directed mutagenesis

DESCRIPTION (provided by applicant):Cell adhesion and migration is important for development, tissue morphogenesis, wound healing, tumor invasion and metastasis. Focal adhesions are essential loci for regulation of cell motility. The long-term goal of this project is to understand the signaling mechanisms regulating cellular adhesive strength and migration. Activation of PI 3-kinase and production of Ptdlns (3,4,5)-P3 induce the restructuring of focal adhesions in PDGF treated fibroblasts. Using this model system, a-actinin was identified as a key target for Ptdlns (3,4,5)-P3 involved in the restructuring of focal adhesions. a-Actinin also bound Ptdlns-P and Ptdlns-Ps, and PDGF induced temporally distinct interactions with these phosphoinositides potentially regulating localization and function. a-Actinin modulates cell adhesion and motility by bundling and linking actin filaments to integrins. The overall goal of this proposal is to test the hypothesis that differential regulation of alpha-actinin function by phosphoinositide binding modulates cellular adhesive strength and motility. To accomplish this goal, two contrasting cellular systems will be used. Experiments will be carried out using slow migrating fibroblasts, which contain large focal adhesions and stress fibers and have high basal levels of Ptdlns (4,5)-Ps, and rapidly migrating U-87MG glioblastoma cells, which contain few focal adhesions and stress fibers and have high levels of Ptdlns (3,4,5)-P3. The following specific aims will be carried out: 1) Identify and quantify phosphoinositide binding to alpha-actinin in PDGF treated fibroblasts. 2) Determine the localization of specific phosphoinositides in PDGF treated fibroblasts. 3) Design and generate site-directed mutants and use these to characterize the interaction of phosphoinositides with a-actinin. 4) Elucidate phosphoinositide regulation of ?-actinin bundling and interaction with other proteins. 5) Determine the influence of phosphoinositide binding to a-actinin on cell adhesion and motility. 6) Examine the structural consequences of phosphoinositide binding to a-actinin. Results from these studies will determine for the first time how phosphoinositides regulate alpha-actinin within the cell and the influence on adhesion and motility. Understanding these mechanisms could lead to the development of therapeutic agents targeted at mimicking the interaction of specific phosphoinositides with a-actinin to control desirable or undesirable cell migration in human disease.

描述（由申请人提供）：细胞粘附和迁移对于发育，组织形态发生，伤口愈合，肿瘤侵袭和转移至关重要。局灶性粘连是调节细胞运动的必要基因座。该项目的长期目标是了解调节细胞粘合强度和迁移的信号传导机制。 PI 3-激酶的激活和PTDLN的产生（3,4,5）-3诱导PDGF处理的成纤维细胞中局灶性粘连的重组。使用此模型系统，将A-肌动蛋白确定为参与焦点粘连重组的PTDLN（3,4,5）-3。 A-肌动蛋白还结合了PTDLNS-P和PTDLNS-PS，PDGF诱导了与这些磷酸肌醇的时间不同的相互作用，从而可能调节定位和功能。 A-肌动蛋白通过捆绑并将肌动蛋白丝与整联蛋白联系起来调节细胞粘附和运动。该提案的总体目标是检验以下假设：磷酸肌醇结合对α-肌动蛋白功能的差异调节调节细胞粘合强度和运动性。为了实现这一目标，将使用两个对比的蜂窝系统。实验将使用缓慢迁移的成纤维细胞进行，其中包含较大的局灶性粘附和应力纤维，并具有较高的PTDLN（4,5）-PS，并且迅速迁移的U-87MG胶质母细胞瘤细胞迁移，含有焦点纤维和压力纤维的含量很少，并且具有PTDLNS的高水平（3,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4,4。将执行以下特定目的：1）识别和量化PDGF处理的成纤维细胞中与α-肌动蛋白结合的磷酸肌醇。 2）确定在PDGF处理的成纤维细胞中特定磷酸肌醇的定位。 3）设计和生成位置定向的突变体，并使用它们来表征磷酸肌醇与A-肌动蛋白的相互作用。 4）阐明 - 肌动蛋白捆绑和与其他蛋白质相互作用的磷酸肌醇调节。 5）确定磷酸肌醇与a-肌动蛋白对细胞粘附和运动性的影响。 6）检查磷酸肌醇与A-肌动蛋白的结构后果。这些研究的结果将首次确定磷酸肌醇如何调节细胞内α-肌动蛋白以及对粘附和运动的影响。了解这些机制可能会导致用于模仿特定磷酸肌醇与A-肌动蛋白相互作用的治疗剂的发展，以控制人类疾病中的理想或不良细胞迁移。