Dicty News Electronic Edition Volume 11, number 3 August 8, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== SCAR, a WASP-related protein, isolated as a suppressor of receptor defects in late Dictyostelium development James E. Bear , John F. Rawls, and Charles L. Saxe, III Department of Cell Biology, Emory University School of Medicine J. Cell Biology, in press. Abstract G protein-coupled receptors trigger the reorganization of the actin cytoskeleton in many cell types, but the steps in this signal transduction cascade are poorly understood. During Dictyostelium development, extracellular cAMP functions as a chemoattractant and morphogenetic signal which is transduced via a family of G protein-coupled receptors, the cAR's. In a strain where the cAR2 receptor gene is disrupted by homologous recombination, the developmental program arrests prior to tip formation. In a genetic screen for suppressors of this phenotype, a gene encoding a protein related to the Wiskott-Aldrich Syndrome protein (WASP) was discovered. Loss of this protein, which we call SCAR (for Suppressor of cAR), restores tip formation and most of later development to cAR2- strains, and causes a multiple-tip phenotype in a cAR2+ strain, as well as leading to the production of extremely small cells in suspension culture. SCAR- cells have reduced levels of F-actin staining during vegetative growth, and abnormal cell morphology and actin distribution during chemotaxis. Uncharacterized homologs of SCAR have also been identified in humans, mouse, C. elegans, and Drosophila. These data suggest SCAR may be a conserved negative regulator of G protein-coupled signaling and that it plays an important role in regulating the actin cytoskeleton. ------------------------------------------------------------------------- Selection of Gb Subunits with Point Mutations that Fail to Activate Specific Signaling Pathways in vivo: Dissecting Cellular Responses Mediated by a Heterotrimeric G protein in Dictyostelium discoideum Tian Jin1, Mario Amzel2, Peter N Devreotes1*, Lijun Wu3 1Department of Biological Chemistry, and 2 Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe Street, MD21205 and 3LeukoSite, Inc., 215 First Street, Cambridge, MA 02142 Mol. Biol. Cell, in press. Abstract In Dictyostelium discoideum, a unique Gb subunit is required for a G protein coupled receptor system that mediates a variety of cellular responses. Binding of cAMP to cAR1, the receptor linked to the G protein G2 triggers a cascade of responses, including activation of adenylyl cyclase, gene induction, actin polymerization and chemotaxis. Null mutations of the cAR1, Ga2 or Gb genes completely impair all these responses. To dissect specificity in Gbg signaling to downstream effectors in living cells, we screened a randomly mutagenized library of Gb genes and isolated Gb alleles that lacked the capacity to activate some effectors but retained ability to regulate others. These mutant Gb subunits were able to link cAR1 to G2, support gene expression, mediate cAMP-induced actin polymerization, and some were able to mediate to chemotaxis toward cAMP. None were able to activate adenylyl cyclase and some did not support chemotaxis. Thus, we separated in vivo functions of the Gbg by making point mutations on Gb. Using the structure of the heterotrimeric G protein displayed in the computer program (CHAIN), we examined the positions and the molecular interactions of the amino acids substituted in each of the mutant Gbs, and analyzed the possible effects of each replacement. We identified several residues that are crucial for activation of the adenylyl cyclase (ACA). These residues formed an area that overlaps but is not identical with regions where bovine Gtbg interacts with its regulators, Ga and phosducin. ------------------------------------------------------------------------- Production and activity of spore differentiation factors (SDFs) in Dictyostelium Christophe Anjard1, Wen Tsan Chang2, Julian Gross2 and Wolfgang Nellen1* 1 Universität Kassel, Abt. Genetik, Heinrich-Plett-Str. 40, D-34 132 Kassel, Germany. 2 University of Oxford, South Park Road, Oxford OX1 3 QU, England Development, in press. Summary SDF-1 and SDF-2 are peptides that promote terminal spore differentiation under submerged conditions. The present study shows that they accumulate differentially and are released during the development of wild-type cells and can promote spore formation in cells disaggregated from wild-type culminants. SDF-1 accumulates during the slug stage and is released in a single burst at the onset of culmination while SDF-2 accumulates during early culmination and is released in a single burst from mid-culminants. The effects of SDF-1 and SDF-2 on stalk cell formation in cell monolayers were investigated. SDF-1 by itself induces stalk cell formation in some strains and also synergizes with the stalk-cell-inducing factor, DIF-1. cAMP has an inhibitory effect on stalk cell formation when either DIF-1 or SDF-1 are present on their own but is almost not inhibitory when both are present. SDF-2 alone does not induce stalk cell formation and appears to inhibit the response to DIF-1. At the same time it increases the extent of vacuolization of the stalk cells that are produced. We propose that the release of SDF-1 and then of SDF-2 may mark irreversible steps in the developmental programme associated respectively with culmination and spore maturation. ------------------------------------------------------------------------- [End Dicty News, volume 11, number 3]