Chemotaxis, or directed cell movement toward a small molecule ligand, plays a key role in many cellular and physiological responses, including metastasis of cancer cells, movement of neutrophils and macrophage in immunity, migration of embryonic cells during development, and aggregation of Dictyostelium during development. In each of these varied cell types and processes, the responding cells are able to amplify a shallow extracellular chemoattractant gradient into a very steep intracellular gradient and thus translate the directional signal into directional cell movement. Fulfillment of this requirement occurs through an integrated circuit of signaling pathways that are pathways are highly conserved between Dictyostelium and man. Recent findings establish that the phosphatidylinositol 3-kinase (PI3K) mediated pathway is pivotal to this circuit. This proposal focuses on the further analysis of the PI3K dependent signal pathway using Dictyostelium cells, which are amenable to biochemical, genetic, and cell biological approaches. Our goal is to identify and examine potential upstream regulators, including those required for localizing PI3K to the leading edge of chemotaxing ceils. We will examine the function of two already identified PI3K effectors, PhdA and PhdB, and identify the proteins with which they interact to better understand how these PI3K effectors contribute to the regulation of chemotaxis. The application also focuses on the role of Ras in controlling the spatial-temporal regulation of PI3K activity. Additional Ras effectors that control the ability of cells to establish polarized intracellular gradients, which are essential for directional cell movement, will also be examined. Finally, we propose to discover and examine the function of additional PI3K regulators through application of mutant screens, two-hybrid screens, and biochemical approaches. The work proposed in this application should provide new and important insights into mechanisms that control this highly evolutionarily conserved cell biological process, and thus provide the needed background to elucidate the cellular basis underlying a variety of human diseases, including those affecting innate immunity and metastasis of cancer cells.
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