Project Summary/Abstract This proposal seeks to elucidate the mechanism of PI(3,4)P2's role in migration. Phosphatidylinositols serve as critical mediators of cellular signaling. PI(3,4)P2 has emerged as a critical regulator of cell migration. It has recently been proposed that a mutually inhibitory feedback loop between Ras and PI(3,4)P2 regulates the dynamic extension and retraction of cellular protrusions. This proposal will elucidate the mechanism by which Ras regulates PI(3,4)P2 and establish PI(3,4)P2 as a bona fide regulator of Ras-dependent leading-edge dynamics. We will also provide insight into the regulation of PI(3,4)P2 in migrating cells by investigating the hypothesis that class PI- 3kinase PIKF regulates the biosynthesis of PI(3,4)P2 in Dictyostelium. Our work will also demonstrate that PIKF's role in migration is attributable to its ability to synthesize PI(3,4)P2. This work will provide significant insight into the feedback loops that regulate Ras-dependent activity. The molecular tools generated by this work will be instrumental in the pursuit of our long-term goals of elucidating the structure of the signaling networks that control migration. Phosphoinositide signaling plays an integral role in cellular signaling and the pathophysiology of numerous diseases. Therefore, this work is immediately relevant to our understanding of disease and the mission of the National Institutes of Health. Our proposed studies will provide undergraduate students with the opportunity to engage in rigorous and impactful science and will bolster the already robust research environment at Amherst College.