The aimless RasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium

Background: Ras proteins are small GTP-binding proteins that play an essential role in a wide range of processes, particularly in mammalian growth control. They act as molecular switches, being inactive when GDP is bound, and active when associated with GTP. Activation is accomplished by guanine nucleotide exchange factors (RasGEFs); when RasGEFs interact with Ras proteins, GDP is allowed to escape, and is replaced by GTP. Dictyostelium responds to chemoattractants through typical seven transmembrane domain receptors and heterotrimeric G proteins. There are at least five different Dictyostelium Ras genes, whose functions are not yet known. . Results: We have isolated the aimless gene, which encodes the Dictyostelium homologue of RasGEFs, during a screen for insertional mutants that fail to aggregate. We found that aimless null mutants grew at a normal rate, but were severely impaired in both chemotaxis and activation of adenylyl cyclase, both of which are critical for the early stages of development. Although coupling between receptors and their G proteins is unaffected, and several cyclic AMP (cAMP)-mediated responses appear normal, activation of adenylyl cyclase by receptors and GTPγS (a non-hydrolyzable GTP analogue) is reduced by up to 95 %. The motility of mutant cells appears normal, suggesting a true defect in gradient sensing. Conclusion: The discovery of the aimless gene adds an interesting new member to the family of RasGEFs. Our data suggest an unforeseen role for a RasGEF, and therefore presumably a complete Ras pathway, in the processing of chemotactic signals through G-protein-coupled receptors

Two Phases of Actin Polymerization Display Different Dependencies on PI(3,4,5)P(3) Accumulation and Have Unique Roles during Chemotaxis

The directional movement of cells in chemoattractant gradients requires sophisticated control of the actin cytoskeleton. Uniform exposure of Dictyostelium discoideum amoebae as well as mammalian leukocytes to chemoattractant triggers two phases of actin polymerization. In the initial rapid phase, motility stops and the cell rounds up. During the second slow phase, pseudopodia are extended from local regions of the cell perimeter. These responses are highly correlated with temporal and spatial accumulations of PI(3,4,5)P(3)/PI(3,4)P(2) reflected by the translocation of specific PH domains to the membrane. The slower phase of PI accumulation and actin polymerization is more prominent in less differentiated, unpolarized cells, is selectively increased by disruption of PTEN, and is relatively more sensitive to perturbations of PI3K. Optimal levels of the second responses allow the cell to respond rapidly to switches in gradient direction by extending lateral pseudopods. Consequently, PI3K inhibitors impair chemotaxis in wild-type cells but partially restore polarity and chemotactic response in pten(-) cells. Surprisingly, the fast phase of PI(3,4,5)P(3) accumulation and actin polymerization, which is relatively resistant to PI3K inhibition, can support inefficient but reasonably accurate chemotaxis