Dicty News Electronic Edition Volume 17, number 13 December 1, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ========================= Published Book Review ========================= The Whole Organism, and nothing but the Organism Robert H. Insall School of Biosciences, Birmingham University, Edgbaston, Birmingham B15 2TT, United Kingdom Cell, Vol 107, 279-281, November 2001 A review of Rich Kessin's Dicty Monograph ============= Abstracts ============= A cell number-counting factor regulates the cytoskeleton and cell motility in Dictyostelium Lei Tang2, Tong Gao1, Catherine McCollum2, Wonhee Jang2, Michael G. Vicker3, Robin R. Ammann1 and Richard H. Gomer1,2 1Howard Hughes Medical Institute and 2Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 3Department of Biology/Chemistry University of Bremen 28359 Bremen, Germany PNAS in press Abstract Little is known about how a morphogenetic rearrangement of a tissue is effected by the individual cells. Starving Dictyostelium discoideum cells aggregate to form dendritic streams which then break up into groups of ~2 x 104 cells. Cell number is sensed at this developmental stage using counting factor (CF), a secreted complex of polypeptides. A high extracellular concentration of CF indicates that there is a large number of cells, which then causes the aggregation stream to break up. Computer simulations indicated that stream breakup could be caused by CF decreasing cell-cell adhesion and/or increasing cell motility, and we observed that CF does indeed decrease cell-cell adhesion. We find here that CF increases cell motility. In Dictyostelium, motility is mediated by actin and myosin. CF increases the amounts of polymerized actin and the ABP-120 actin- crosslinking protein. Partially inhibiting motility using drugs which interfere with actin polymerization reduces stream dissipation, resulting in fewer stream breaks and thus larger groups. CF also potentiates the phosphorylation and redistribution of myosin, while repressing its basal level of assembly. The computer simulations indicated that a narrower distribution of group sizes results when a secreted factor modulates both adhesion and motility. CF thus appears to induce the morphogenesis of streams into evenly-sized groups by increasing actin polymerization, ABP-120 levels, and myosin phosphorylation, and decreasing adhesion and myosin polymerization. ----------------------------------------------------------------------------- RNAi in Dictyostelium: the role of RdRPs and dsRNase Henrik Martens, Jindrich Novotny, Jrgen Oberstrass, Theodore L. Steck1, Pamela Postlethwait1 and Wolfgang Nellen* Abt. Genetik, Universitt Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany, 1 Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637-1432 Mol. Biol. Cell, in press Abstract We show that in Dictyostelium discoideum an endogenous gene as well as a transgene can be silenced by introduction of a gene construct which is transcribed into a hairpin RNA (RNAi). Gene silencing was accompanied by the appearance of sequence specific RNA ~23mers and seemed to have a limited capacity. The three Dictyostelium homologs of the RNA directed RNA polymerase gene (RrpA, RrpB and DosA) all contain an N-terminal helicase domain homologous to the one in the dicer nuclease, suggesting exon shuffling between RdRP and the dicer homolog. Only the knock-out of rrpA resulted in a loss of the RNAi effect and simultaneously in a loss of detectable ~23mers. However, ~23mers were still generated by the Dictyostelium dsRNase in vitro with extracts from rrpA-, rrpB- and DosA- cells. Both RrpA and a target gene were required for production of detectable amounts of ~23mers suggesting that target sequences are involved in ~23mer amplification. ----------------------------------------------------------------------------- Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum Kissia Ravanel1, Benoit de Chassey2, Sophie Cornillon1, Mohammed Benghezal1, Laurence Zulianello1, Leigh Gebbie1, Franois Letourneur2, Pierre Cosson1* 1 Universit de Genve, Centre Mdical Universitaire, Dpartement de Morphologie, 1 rue Michel Servet, CH-1211 Genve 4, Switzerland. 2 Institut de Biologie et de Chimie des Protines, UMR 5086 CNRS, Universit Lyon I, 7 passage du Vercors, 69367 Lyon Cedex 07, France. European J. Cell Biol., in Press ABSTRACT To study sorting in the endocytic pathway of a phagocytic and macropinocytic cell, monoclonal antibodies to membrane proteins of Dictyostelium discoideum were generated. Whereas the p25 protein was localized to the cell surface, p80 was mostly present in intracellular endocytic compartments as observed by immunofluorescence as well as immunoelectron microscopy analysis. The p80 gene was identified and encodes a membrane protein presumably involved in copper transport. Expression of chimeric proteins revealed that the cytoplasmic domain of p80 was sufficient to cause constitutive endocytosis and localization of the protein to endocytic compartments. Dileucine and tyrosine-based endocytic signals described previously in mammalian systems were also capable of targeting chimera to endocytic compartments. In phagocytosing cells no membrane sorting was observed during formation of the phagosome. Both p25 and p80 were incorporated non-selectively in nascent phagosomes, and then retrieved shortly after phagosome closure. Our results emphasize the fact that very active membrane traffic takes place in phagocytic and macropinocytic cells. This is coupled with precise membrane sorting to maintain the specific composition of endocytic compartments ----------------------------------------------------------------------------- Two members of the beige/CHS (BEACH) family are involved at different stages in the organization of the endocytic pathway in Dictyostelium. Sophie Cornillon1, Annick Dubois2, Franz Brckert3, Yaya Lefkir2, Anna Marchetti1, Mohammed Benghezal1, Arturo De Lozanne4, Franois Letourneur2, and Pierre Cosson1, 5 1 Universit de Genve, Centre Mdical Universitaire, Dpartement de morphologie, 1 Rue Michel Servet, CH1211 Genve 4, Switzerland; 2 Institut de Biologie et de Chimie des Protines, UMR5086-CNRS, Universit Lyon I, 7 Passage du Vercors, 69367 Lyon cedex 07, France 3 Laboratoire de Biochimie et Biophysique des Systmes Intgrs, UMR314 CNRS, CEA, 38054 Grenoble, France 4 Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, 241 Patterson Bldg., Mail code C0930, University of Texas, Austin, TX 78712, USA J. Cell Sci., in press SUMMARY Proteins of the Chediak-Higashi/Beige (BEACH) family have been implicated in the function of lysosomes as well as in signal transduction but their molecular role is still poorly understood. In Dictyostelium at least six members of the family can be identified. Here cells mutated in two of these, LVSA and LVSB, were analyzed. Interestingly both mutants exhibited defects in the organization of the endocytic pathway, albeit at distinct stages. In lvsB mutant cells the regulated secretion of lysosomal enzymes was enhanced, a phenotype reminiscent of the Chediak-Higashi syndrome. LvsA mutant cells exhibited alterations in the organization and function of the early endocytic and phagocytic pathway. The LvsA protein may participate in the signaling pathway which links adhesion of a particle to the subsequent formation of a phagocytic cup. Further genetic analysis will be necessary to determine if other members of the BEACH family of proteins are also involved in controlling the organization of the endocytic pathway. ---------------------------------------------------------------------------- QUANTITATIVE ANALYSIS OF THE BEHAVIOR OF DICTYOSTELIUM DISCOIDEUM AMOEBAE: STRINGENCY OF PTERIDINE RECEPTION. Jared L. Rifkin, Biology Department, Queens College of CUNY, Flushing, NY 11367-1597 Cell Motility and the Cytoskeleton, in press Abstract A convenient, sensitive, quantitative assay for the measurement of chemotaxis of populations of D. discoideum vegetative amoebae is presented. A strategy for determining the boundary of the bulk of a population of migrating amoebae was devised and is described. This assay employs a dynamic gradient and is independent of deaminase activity. Measurements of chemoattractant capabilities of various pteridines, folates, and mixtures of folate fragments are reported. 2-Amino 4-quinazolinone, a pterin analog without the pyrazine ring nitrogens, is chemotactic. Lumazine, deaminated pterin, inhibits chemotaxis towards pterin but not towards folic acid. Deaminofolic acid is a chemoattractant as are mixtures of lumazine plus aminobenzoylglutamic acid or deaminopteroic acid plus various amino acids. Separately, the components of these mixtures exhibit no ability to stimulate chemotaxis. These mixtures are of fragments that together comprise most of the folate structure. Our results are in accord with separate receptors for pterin vs. folic acid and with a high stringency for pterin reception but a relative tolerance for folate reception. The possibility of using such mixtures to investigate the requirements of various parts of the folate structure for competent signalling is discussed. ----------------------------------------------------------------------------- [End Dicty News, volume 17, number 13]