Phosphoinositide Regulation of Focal Adhesion Structure

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、肿瘤侵袭和转移非常重要。粘着斑是调节细胞运动的重要位点。该项目的长期目标是了解调节细胞粘附强度和迁移的信号机制。PI 3-激酶的激活和Ptdlns（3，4，5）-P3的产生诱导PDGF处理的成纤维细胞中的粘着斑的重构。使用该模型系统，α-辅肌动蛋白被鉴定为参与局部粘连重建的Ptdlns（3，4，5）-P3的关键靶标。α-辅肌动蛋白还结合Ptdlns-P和Ptdlns-Ps，并且PDGF诱导与这些磷酸肌醇的时间上不同的相互作用，从而潜在地调节定位和功能。α-肌动蛋白通过将肌动蛋白丝与整联蛋白成束和连接来调节细胞粘附和运动性。这个建议的总体目标是测试的假设，差异调节α-辅肌动蛋白功能的磷酸肌醇结合调节细胞的粘附强度和运动。为了实现这一目标，将使用两种对比的蜂窝系统。将使用慢迁移成纤维细胞和快速迁移U-87 MG成胶质细胞瘤细胞进行实验，所述慢迁移成纤维细胞含有大的粘着斑和应力纤维并且具有高的Ptdlns（4，5）-Ps基础水平，所述快速迁移U-87 MG成胶质细胞瘤细胞含有很少的粘着斑和应力纤维并且具有高水平的Ptdlns（3，4，5）-P3。将进行以下具体目标：1）鉴定和定量PDGF处理的成纤维细胞中与α-辅肌动蛋白结合的磷酸肌醇。2)确定PDGF处理的成纤维细胞中特异性磷酸肌醇的定位。3)设计并产生定点突变体，并利用这些突变体来表征磷酸肌醇与α-辅肌动蛋白的相互作用。4)阐明磷酸肌醇调节？-辅肌动蛋白捆绑和与其他蛋白质的相互作用。5)测定磷脂酰肌醇与α-辅肌动蛋白结合对细胞粘附和运动的影响。6)检查磷脂酰肌醇与α-辅肌动蛋白结合的结构后果。这些研究的结果将首次确定磷酸肌醇如何调节细胞内的α-辅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治疗药物的发展，靶向模拟特定的磷酸肌醇与α-辅肌动蛋白的相互作用，以控制人类疾病中的期望或不期望的细胞迁移。

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.