An attenuating role of a WASP-related protein, WASP-B, in the regulation of F-actin polymerization and pseudopod formation via the regulation of RacC during Dictyostelium chemotaxis.

The WASP family of proteins has emerged as important regulators that connect multiple signaling pathways to regulate the actin cytoskeleton. Dictyostelium cells express WASP, as well as a WASP related protein, WASP-B, endoded by wasB gene. WASP-B contains many of the domains present in WASP. Analysis of wild type, wasB null cells revealed that WASP-B is required for proper control of F-actin polymerization in response to a cAMP gradient. Due to the lack of tight control on actin polymerization, wasB null cells exhibited higher level of F-actin polymerization. wasB(-) cells extend more de novo pseudopods laterally and their average life span is longer than those of wild type cells, causing more turns and inefficient chemotaxis. YFP-WASP-B appears to be uniformly distributed in the cytosol and shows no translocation to cortical membrane upon cAMP stimulation. Active RacC pull-down assay reveals that the level of active RacC in wasB(-) cells is significantly higher than wild type cells. Moreover, the distribution of active RacC is not localized in wasB(-) cells. We conclude that chemotaxis defects of wasB(-) cells are likely to result from the aberrant regulation of RacC activation and localization.

Functional roles of VASP phosphorylation in the regulation of chemotaxis and osmotic stress response.

Vasodilator-stimulated phosphoprotein (VASP) plays crucial roles in controlling F-actin-driven processes and growing evidence indicates that VASP function is modulated by phosphorylation at multiple sites. However, the complexity of mammalian system prevents the clear understanding of the role of VASP phosphorylation. In this study, we took advantage of Dictyostelium which possesses only one member of the Ena/VASP family to investigate the functional roles of VASP phosphorylation. Our results demonstrated that hyperosmotic stress and cAMP stimulation cause VASP phosphorylation. VASP phosphorylation plays a negative role for the early steps of filopodia/microspikes formation. VASP phosphorylation appears to modulate VASP localization at the membrane cortex and its interactions with WASP and WIPa. Analysis of chemotaxis of cells expressing VASP mutants showed that VASP phosphorylation is required for the establishment of cell polarity under a cAMP gradient.