dictyNews Electronic Edition Volume 28, number 13 May 18, 2007 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= A CHS/Beige homologue is involved in biogenesis of Dictyostelium secretory lysosomes Steve J. Charette and Pierre Cosson Universite de Geneve, Centre Medical Universitaire, Departement de Physiologie Cellulaire et Metabolisme, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland. Journal of Cell Science, in press The Chediak-Higashi syndrome (CHS) is characterized at the cellular level by a defect in the ability of cells to secrete lysosomes. However the precise step affected in the secretion process is unclear to date. This question was addressed by characterizing Dictyostelium discoideum cells where LVSB, the homologue of the CHS gene, was mutated. As observed in mammalian cells, secretion of lysosome-derived compartments was affected in lvsB mutant cells. This defect was mirrored by a decrease in the number of fusion-competent post-lysosomal compartments, which in Dictyostelium can be clearly distinguished from lysosomes. In addition, the transfer of endocytosed particles from lysosomes to post-lysosomes was strongly diminished in lvsB mutant cells compared to wild-type cells. These results suggest that LvsB is primarily involved in transport from lysosomes to post-lysosomes, and thus plays a critical role in the maturation of lysosomes into fusion-competent post-lysosomal compartments. Submitted by: Steve Charette [steve.charette@medecine.unige.ch] -------------------------------------------------------------------------------- GrlJ, a Dictyostelium GABAB-like receptor with roles in post-aggregation development Yogikala Prabhu 1,4, Rolf Mueller 1, Christophe Anjard 3 and Angelika A.Noegel 1,2* 1Institute of Biochemistry I, Centre for Biochemistry, Medical Faculty, University of Cologne, Joseph-Stelzmann-Str. 52, D-50931 Kšln, Germany 2Centre for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Str. 52, 50931 Kšln, Germany 3Center for Molecular Genetics, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0368, USA 4Present address: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA BMC Developmental Biology, in press Background: The G-protein-coupled receptor (GPCR) family represents the largest and most important group of targets for chemotherapeutics. They are extremely versatile receptors that transduce signals as diverse as biogenic amines, purins, odorants, ions and pheromones from the extracellular compartment to the interior via biochemical processes involving GTP-binding proteins. Until recently, the cyclic AMP receptors (cARs) were the only known G protein coupled receptors in Dictyostelium discoideum. The completed genome sequence revealed the presence of several families of GPCRs in Dictyostelium, among them members of the family 3 of GPCRs, the GABAB/ glutamate like receptor family, which in higher eukaryotes is involved in neuronal signaling. Results: D. discoideum has seventeen Family 3 members of GPCRs, denoted GrlA through GrlR. Their transcripts are detected throughout development with increased levels during early and late development. We have examined here GrlJ. GFP-tagged GrlJ localises to the plasmamembrane and to internal membranes. Inactivation of the grlJ gene leads to precocious development, and the mutant completes development ~6 hours earlier. Alterations were also noted at the slug stage and in spore formation. grlJ- slugs were longer and broke apart several times on their way to culmination forming smaller but proportionate fruiting bodies. Spores from grlJ- fruiting bodies were malformed and less viable, although the spore differentiation factors were synthesized and sensed normally. Expression of a GFP-tagged full length GrlJ rescued the phenotype. Conclusion: Our data suggest that GrlJ acts at several stages of Dictyostelium development and that it is a negative regulator in Dictyostelium development. Submitted by: Angelika A.Noegel [noegel@uni-koeln.de] -------------------------------------------------------------------------------- Possible roles of the endocytic cycle in cell motility David Traynor and Robert R. Kay J. Cell Sci., in press Starving, highly motile Dictyostelium cells maintain an active endocytic cycle, taking up their surface about every 11 minutes. Cell motility depends on a functional NSF protein Ð also essential for endocytosis and membrane trafficking generally Ð and we therefore investigated possible ways in which the endocytic cycle might be required for cell movement. First, NSF, and presumably membrane trafficking, are not required for the initial polarization of the leading edge in a cyclic-AMP gradient. Second, we can detect no evidence for membrane flow from the leading edge, as photobleached or photoactivated marks in the plasma membrane move forward roughly in step with the leading edge, rather than backwards from it. Third, we find that the surface area of a cell Ð measured from confocal reconstructions Ð constantly fluctuates during movement as the cell projects pseudopodia and otherwise changes shape; increases of 20-30% can often occur over a few minutes. These fluctuations cannot be explained by reciprocal changes in filopodial surface area and they substantially exceed the 2 Ð3% by which membranes can stretch. We propose that the endocytic cycle has a key function in motility by allowing adjustment of cell surface area to match changes in shape; and that without this function, movement is severely impaired. Submitted by: Rob Kay [rrk@mrc-lmb.cam.ac.uk] -------------------------------------------------------------------------------- Locally controlled inhibitory mechanisms are involved in eukaryotic GPCR-mediated chemosensing Xuehua Xu1, Martin Meier-Schellersheim2, Jianshe Yan1 and Tian Jin1* J. Biol. Chem., in press GPCR signaling mediates a balance of excitatory and inhibitory activities that regulate Dictyostelium chemosensing to cAMP. The molecular nature and kinetics of these inhibitors are unknown. We report that transient cAMP stimulations induce PIP3 responses without a refractory period, suggesting that GPCR-mediated inhibition accumulates and decays slowly. Moreover, exposure to cAMP gradients leads to asymmetric distribution of the inhibitory components. The gradients induce a stable accumulation of the PIP3 reporter PHCrac-GFP in the front of cells near the cAMP source. Rapid withdrawal of the gradient led to the re-association of G-protein subunits, and the return of the PIP3 phosphatase PTEN and PHCrac-GFP to their pre-stimulus distribution. Reapplication of cAMP stimulation produces a clear PHCrac-GFP translocation to the back but not to the front, indicating that a stronger inhibition is maintained in the front of a polarized cell. Our study demonstrates a novel spatiotemporal feature of currently unknown inhibitory mechanisms acting locally on the PI3K activation pathway. Submitted by: Tian Jin [TJIN@niaid.nih.gov] ============================================================== [End dictyNews, volume 28, number 13]