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.
