dictyNews Electronic Edition Volume 27, number 5 August 18, 2006 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 ============= Selective membrane exclusion in phagocytic and macropinocytic cups Valentina Mercanti1#, Steve J. Charette1#, Nelly Bennett2, Jean-Jeacques Ryckewaert3, Franois Letourneur4 and Pierre Cosson1 1. Universite de Geneve, Centre Medical Universitaire, Departement de Physiologie Cellulaire et Metabolisme, 1 rue Michel Servet, CH-1211 Geneve 4, Switzerland. 2. Laboratoire de Biochimie et Biophysique des Systemes Integres, Departement de Reponse et Dynamique Cellulaires, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. 3. Laboratoire de Chemie des Proteines, ERM 201 INSERM/CEA/UJF, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. 4. Institut de Biologie et Chimie des Proteines (IBCP UMR 5086); CNRS; Univ. Lyon1; IFR128 BioSciences Lyon-Gerland; 7, passage du Vercors, 69367 Lyon cedex 07, France #These authors contributed equally to this work. Journal of Cell Science, in press Specialized eukaryotic cells can ingest large particles and sequester them within membrane-delimited phagosomes. Many studies have described the delivery of lysosomal proteins to the phagosome, but little is known about membrane sorting during the early stages of phagosome formation. Here we used Dictyostelium discoideum amoebae to analyze the membrane composition of newly formed phagosomes. The membrane delimiting the closing phagocytic cup was essentially derived from the plasma membrane, but a subgroup of proteins was specifically excluded. Interestingly the same phenomenon was observed during the formation of macropinosomes, suggesting that the same sorting mechanisms are at play during phagocytosis and macropinocytosis. Analysis of mutant strains revealed that clathrin-associated adaptor complexes AP-1, -2 and -3 were not necessary for this selective exclusion, and accordingly ultrastructural analysis revealed no evidence for vesicular transport around phagocytic cups. Our results suggest the existence of a new, as yet uncharacterized, sorting mechanism in phagocytic and macropinocytic cups. Submitted by: Steve Charette [steve.charette@medecine.unige.ch] ----------------------------------------------------------------------------- The function of the Dictyostelium Atg1 kinase during autophagy and development Turgay Tekinay, Mary Y. Wu1, Grant P. Otto, O. Roger Anderson and Richard H. Kessin Eukaryotic Cell, in press When starved, the amoebae of Dictyostelium discoideum initiate a developmental process that results in the formation of fruiting bodies, in which stalks support balls of spores. The nutrients and energy necessary for development are provided by autophagy. Atg1 is a protein kinase that regulates the induction of autophagy in budding yeast. In addition to a conserved kinase domain, Dictyostelium Atg1 has a C-terminal region that has significant homology to the Caenorhabditis elegans and mammalian Atg1 homologues, but not to the budding yeast Atg1. We investigated the function of the kinase and conserved C-terminal domains of DdAtg1 and showed that these domains are essential for autophagy and development. Kinase-negative DdAtg1 acts in a dominant-negative fashion, resulting in a mutant phenotype when expressed in the wild-type cells. GFP-tagged kinase-negative DdAtg1 co-localizes with RFP tagged DdAtg8, a marker of preautophagosomal structures and autophagosomes. The conserved C-terminal region is essential for localization of kinase-negative DdAtg1 to autophagosomes labeled with RFP-tagged Dictyostelium Atg8. The dominant-negative effect of the kinase-defective mutant also depends on the C-terminal domain. In cells expressing dominant-negative DdAtg1, autophagosomes are formed and accumulate but seem not to be functional. By using a temperature-sensitive DdAtg1, we showed that DdAtg1 is required throughout development; development halts when the cells are shifted to the restrictive temperature, but resumes when cells are returned to the permissive temperature. Submitted by: Richard Kessin [rhk2@columbia.edu] ----------------------------------------------------------------------------- RacG regulates morphology, phagocytosis and chemotaxis Baggavalli P. Somesh(1), Georgia Vlahou(1), Miho Iijima(3), Robert H. Insall(2), Peter Devreotes(3) and Francisco Rivero(1) 1. Center for Biochemistry and Center for Molecular Medicine Cologne, Medical Faculty, University of Cologne. 2. School of Biosciences, The University of Birmingham. 3. Department of Cell Biology, Johns Hopkins University School of Medicine. Eukaryotic Cell, in press. RacG is an unusual member of the complex family of Rho GTPses in Dictyostelium. We have generated a knockout strain (KO) as well as strains that overexpress the wild-type (WT), constitutively active (V12) or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 µM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in RacG-V12 and RacG-N17 mutants, in part due to interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system RacG induced actin polymerization upon GTPgammaS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or more Dictyostelium Rho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility and phagocytosis. Submitted by: Francisco Rivero [francisco.rivero@uni-koeln.de] ============================================================================== [End dictyNews, volume 27, number 5]