Ras activation and symmetry breaking during Dictyostelium chemotaxis.

Central to chemotaxis is the molecular mechanism by which a shallow spatial gradient of chemoattractant induces symmetry breaking of activated signaling molecules. Previously, we have used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signaling module providing activation of Ras and F-actin at the leading edge. Here, we show that Ras activation after application of a pipette releasing the chemoattractant cAMP has three phases, each depending on specific guanine-nucleotide-exchange factors (GEFs). Initially a transient activation of Ras occurs at the entire cell boundary, which is proportional to the local cAMP concentrations and therefore slightly stronger at the front than in the rear of the cell. This transient Ras activation is present in gα2 (gpbB)-null cells but not in gβ (gpbA)-null cells, suggesting that Gβγ mediates the initial activation of Ras. The second phase is symmetry breaking: Ras is activated only at the side of the cell closest to the pipette. Symmetry breaking absolutely requires Gα2 and Gβγ, but not the cytoskeleton or four cAMP-induced signaling pathways, those dependent on phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3], cGMP, TorC2 and PLA2. As cells move in the gradient, the crescent of activated Ras in the front half of the cell becomes confined to a small area at the utmost front of the cell. Confinement of Ras activation leads to cell polarization, and depends on cGMP formation, myosin and F-actin. The experiments show that activation, symmetry breaking and confinement of Ras during Dictyostelium chemotaxis uses different G-protein subunits and a multitude of Ras GEFs and GTPase-activating proteins (GAPs).