The long term goal of this grant is to advance understanding of chemotaxis, the ability of cells tosense chemical gradients and move directionally. Studies oi Dictyostelium discoideum, with itsaccessible biochemistry, cell biology, and genetics, have contributed enormously to the currentunderstanding of chemotaxis. Briefly, in Dictyostelium and human neutrophils, chemotaxis ismediated by GPCRs linked to specific heterotrimeric G-proteins, which trigger downstreamsignaling events to occur selectively on the side of the cell facing the higher concentration ofchemoattractant. For example, the accumulation of phosphatidylinositol 3,4, 5-tris phosphate(PIP3) is sharply localized to the leading edge of the cell. Recent studies have shown that PIP3 actsin parallel with other signaling events in a network that biases the motility machinery of the cell. Inthe Progress Report, we describe our studies of tsunami which plays a key role in cell polarity. Wedefine me central roles of pianissimo (piaA), a subunit of Tor Complex 2 (TorC2), and aphospholipase A2, plaA. We report single molecule imaging studies of receptors and G-proteinsrevealing the earliest steps in gradient sensing. We describe the results of a large forward geneticscreen that uncovered over twenty novel chemotaxis genes. We also report on a genomic librarycomplementation approach to identify chemically-induced mutants. The latter two projects,involving considerable risk and long term commitment, were made possible by the MERIT award.The plans for the second half of the grant are presented. We will continue to unravel the network ofsignaling pathways that mediate chemotaxis, exploiting the ability to combine gene disruptions inDictyostelium, along with careful biochemical and cell biological experiments. We will ask whetherkey elements of the pathways are conserved in neutrophils. To define the roles of the newchemotaxis genes we have identified, we will assess a series of well-characterized physiologicalresponses associated with chemotaxis such as PIP3 production, actin polymerization, andphosphorylation of PKB substrates and carry out and analyze time-lapse images of the cellsundergoing directed migration. We will pursue the single molecule imaging studies to achieve andrefine direct imaging individual G-protein activation events, hopefully creating as powerful anexperimental tool as single channel recording of ion channel openings.
Chemotaxis mediates many physiological processes including embryogenesis, immune response,wound healing, and stem cell homing and plays a key role in the pathology of inflammatorydiseases and cancer metastasis. A detailed understanding of chemotaxis, which involves linksbetween signal transduction and cell motility networks, would provide an incredibly powerful toolfor designing rational interventions for improving health and defeating disease.
Showing the most recent 10 out of 86 publications
