UC San Diego

In response to a chemoattractant signal, amoeboid cells such as neutrophils, macrophages, and Dictyostelium cells are able to polarize and move towards the chemoattractant source. During Dictyostelium chemotaxis, it is known that cells amplify and differentially localize specific signaling responses at the future anterior and posterior of the cell leading to outwardly directed F-actin polymerization and myosin II-mediated contractility at the front and back, respectively. However, how cells initially detect and orient themselves in chemoattractant gradients remains largely unknown. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium and inhibition of Ras signaling results in directional migration defects. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2. To investigate the role of Ras in directional sensing, I studied the effect of its misregulation using cells with disrupted RasGAP activity. I identified an orthologue of mammalian NF1, DdNF1, as a major regulator of Ras activity in Dictyostelium. nfaA- cells fail to spatially and temporally regulate Ras activity, which leads to misregulated downstream PI3K activity and F-actin polymerization. As a result, severe cytokinesis and chemotaxis defects in nfaA- cells are observed. Through both genetic and biochemical approaches, I identified RasG as the major target of DdNF1 GAP activity in chemotaxis. Using unpolarized, latrunculin-treated cells, I showed that tight regulation of Ras is required for directional sensing. It is speculated that the uniformly distributed DdNF1 functions as a global inactivator of Ras activity, coupled to putative local activators (e.g. RasGEFs), unidentified global inhibitors as well as positive feedback signaling, leads to persistence and amplification of the Ras signal at the front and promotes leading edge formation. Consequently, cells migrate up the gradients. Together, it is suggested that Ras is part of the cell's compass, and that the RasGAP-mediated regulation of Ras activity affects directional sensing. Further, growing nfaA- cells exhibit elevated Ras activity and display enhanced random cellular movement, consistent with the model that a G protein-independent Ras/PI3K/F-actin circuit regulates basic cell motility