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 macrophages in immune system reactions, 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 highly conserved through evolution between Dictyostelium and man. Recent findings establish that the Ras and the related GTPase Rap1 are important for directional sensing, pseudopod formation, cell polarization, and cell attachment. Both Ras and Rap1 are preferentially activated at the leading edge of chemotaxing Dictyostelium cells and abrogation of their function impairs this process. This proposal focuses on the further examination of the roles of Ras and Rap1 of using Dictyostelium cells, which are amenable to biochemical, genetic, and cell biological approaches. We propose to identify the mechanisms by which the activations of Ras and Rap1 are regulated and spatially restricted to the leading edge of chemotaxing cells. This will be achieved through the analysis of a RasGEF complex and by examining defects in chemotaxis resulting from disruptions of specific GTPase activating proteins (GAPs) for Ras and for Rap1. Through the examination of the spatial and temporal regulation of activated Ras and Rap1 strains in which normal Ras and Rap1 activity is altered, we will elucidate the mechanisms by which cells orient themselves in a chemoattractant gradient. We propose to determine how Ras and Rap1 help mediate chemotaxis through the identification of downstream effectors. The function of these will be examined through the analysis of their null mutations with the aid of real-type fluorescent reporters and biochemical assays that reveal different components of directional sensing, pseudopod formation, and cell polarization. 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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