NOTE: THE SPECIAL CHARACTERS IN THIS ABSTRACT CANNOT BE DISPLAYED CORRECTLY ON THIS PAGE. PLEASE REFER TO ABSTRACT IN THE ATTACHED FILE FOR AN ACCURATE DISPLAY. In this study, we report the characterization of Dock/Elmo proteins, a novel family of RacGEF's in Dictyostelium discoideum and describe their role in phagocytosis, macropinocytosis and CAMP mediated chemotactic cell movement. Aggregation competent Dictyostelium cells can detect and move up CAMP gradients. cAMP stimulation causes a transient localized increase in phosphatidyl inositol (3,4,5) tris phosphate (PIP3) concentration in the plasma membrane at the cell's leading edge. PIP3 binding domain containing RacGEF's are thought to mediate the cAMP signal through the activation of Racs to localised actin polymerization, which results in further extension of the leading edge in the direction of the CAMP gradient. However inhibition of PIP3 formation, through inhibition of P13K activity,results only in a partial defect of chemotaxis, implying that there may exist other signalling pathways that mediate the CAMP signal to the cytoskeleton. Members of the Dock family form a novel class of Rac activators and deletion of dock in Drosophila and C. elegans results in chemotaxis and phagocytosis defects. We have identified the 8 dock genes and 4 elmo genes in the Dictyostelium genome, which we named dockA-H and elmo1-4. To investigate the role of Dock/Elmo, we generated null mutant strains (dockA""ll, dockBn"ºi, dockA/Bnuu, dockCii°, dockD'dockGuil, dockI(wfl, elmolni° and elmo1/dockB' ) by gene disruption using homologous recombination.In our study, knockout dock genes in Dictyostelium results in numerous defects. dockA"""cells show a slow chemotactic response to CAMP and reduced rate in macropinocytosis. dockA""ll slugs show poor phototaxis. dock8""`l cells grow slowly in the axenic medium and are defective in chemotaxis, macropinocytosis and phagocytosis. dockA/8 double mutant cells show stronger defects in chemotaxis than either mutant by itself. In the dockN8""ll double mutant chemotaxis is almost completely inhibited in presence of the P13K inhibitor LY294002. doCkAnu#' dockBn1 dockA/8""0 cells all show reduced actin polymerization upon CAMP stimulation. dockC"'l' cells also show poor chemotaxis to CAMP. However there is no obviously defect in chemotaxis, macropinocytosis and phagocytosis for dockD""ll, dockGnr°, and dockI-P cells. Although Dictyostelium Docks do not contain the conventional DH domain for Rac activation, we show that the DockerB domain (Docker domain of DockB protein) interacts with RaclA in the nucleotide free state in vitro.The DockerA domain (Docker domain of DockA protein) preferentially interacts with RacH and RacL. Expression of RaclA in dockBIul background can effectively rescue the defect of phagocytosis. These findings show that Dictyostelium Docks most likely act as RacGEF's involved in a P13K independent chemotaxis signalling pathway and also play important roles in the regulation of endocytosis in Dictyostelium.In this study we also investigated the role of Elmol in the regulation of cell behavior. The elmo1""m cells grow slowly in the axenic medium and are defective in chemotaxis, macropinocytosis and phagocytosis. The phenotype of elmolia0 cells is extremely similar to that of nuii cells. Cells of the elmo1/dockBniD double mutant did not show a much more severe phenotype than either parent stain (elmol""" or dockBnu") by itself. These data strongly support the idea that in Dictyostelium Elmol and DockB are involved in same signalling pathways.Our data thus strongly suggest that Dock/Elmo complexes function as RacGEF's during chemotaxis and endocytosis in Dictyostelium.
|Date of Award||2006|
|Supervisor||Kees Weijer (Supervisor)|