We are investigating the following questions: 1. how a GPCR chemosensing network regulates the polarized reorganization of the actin cytoskeleton required for protrusion of the cell's front and retraction of its back during chemotaxis. 2. What are the molecular mechanisms underlying phagosome maturation process during phagocytosis? 1. In recent years, the Elmo (Engulfment and Motility) protein family has been implicated in actin cytoskeleton reorganization during both phagocytosis and chemotaxis. ELMO was first discovered as an essential component involved in engulfment of dead cells in C. elegans. DOCK180 and ELMO form a complex that functions as a bipartite GEF to optimally activate Rac and promote actin cytoskeleton rearrangement. DOCK-ELMO complexes also receive signals from GPCRs, integrins and tyrosine kinases to activate Rac, leading to cell migration. However, how GPCRs and other receptors activate DOCK-ELMO has not been determined in any system. As both DOCK and ELMO are evolutionarily conserved, we have been using D. discoideum, as a model system to study the functions of ELMO (A-F) proteins in GPCR-mediated signaling network leading to chemotaxis. We have identified six Elmo homologs in D. discoideum, and have reported that ElmoA functions to maintain cell polarization by preventing excessive actin polymerization around the cell periphery during phagocytosis and chemotaxis. We have been investigating the roles of ELMO-E in chemotaxis. We performed gene disruption, replacement, and over-expression experiments. We found that cells lacking ELMO-E display chemotaxis defect. Visualization of YFP tagged ELMO-E by confocal microscopy revealed ELMO-E translocates to regions of newly forming actin filaments, colocalizing at the leading edge of dominant pseudopod in chemotaxing cells. ELMO-E transiently translocates to membrane under cAMP stimulation by the facility of actin polymerization. This translocation is PI3K independent. More interestingly, we found that ELMO-E interacts with G protein beta subunit and may associate with Rac family proteins as well. Taken together, we propose that ELMO-E protein may be the missing connection between the GPCR/G-protein, the chemoattractant sensing machinery, and the actin cytoskeleton, the machinery of cell movement. 2. Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered from phagosomes to lysosomes for degradation, but the molecular mechanism regulating phagosome maturation remains unclear. Using D. discoideum as a model system, we plan to reveal important components involved in phagosome maturation. We have identified 3 novel vesicle-associated receptor tyrosine kinases, VSK1-3, in D. discoideum. Our previous study suggests that localized VSK3 tyrosine kinase signaling on the surface of endosome/lysosomes represents a new control mechanism for phagosome maturation. We are identifying targets of VSK 2 and 3. This study will provide a foundation for understanding the molecular mechanism of VSK signaling that regulate phagosome maturation.