Microglia play crucial roles in increased inflammation in the CNS upon brain injuries and diseases. Extracellular ADP has been reported to induce microglia chemotaxis and membrane ruffle formation through P2Y12 receptor. In this study, we examined the role of ERK1/2 activation in ADP-induced microglia chemotaxis. ADP stimulation increases the phosphorylation of ERK1/2 and paxillin phosphorylation at Tyr31 and Ser83. Inhibition of ERK1/2 significantly inhibited paxillin phosphorylation at Ser83 and the retraction of membrane ruffles, causing inefficient chemotaxis. Close examination of dynamics of focal adhesion formation with GFP-paxillin revealed that the disassembly of focal adhesions in U0126-treated cells was significantly impaired. Depletion of β-Arr2 with shRNA markedly reduced the phosphorylation of ERK1/2 and Pax/Ser83, indicating that β-Arr2 is required for ERK1/2 activation upon ADP stimulation. A large fraction of phosphorylated ERK1/2 and β-Arr2 were translocated and co-localized at focal contacts in the newly forming lamellipodia. Examination of kinetics and rate constant of paxillin formation and disassembly revealed that the phosphorylation of paxillin at Tyr31 by c-Src appears to be involved in adhesion formation upon ADP stimulation while Ser83 required for adhesion disassembly.

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.

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.