Dicty News Electronic Edition Volume 12, number 2 January 16, 1999 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 ============== Cytokinesis mediated through the recruitment of cortexillins into the cleavage furrow Igor Weber, Günther Gerisch, Christina Heizer, John Murphy, Kim Badelt, Alexander Stock, Jean-Marc Schwartz and Jan Faix Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany. EMBO Journal, in press Abstract: The fact that substrate-anchored Dictyostelium cells undergo cytokinesis in the absence of myosin II underscores the importance of other proteins in enabling the cleavage furrow to constrict. Cortexillins, a pair of actin- bundling proteins, are required for normal cleavage. They are targeted to the incipient furrow in wild-type and, more prominently, in myosin II-null cells. No other F-actin bundling or crosslinking protein tested is co-localized. GFP-fusions show that the N-terminal actin-binding domain of cortexillin I is dispensable and the C-terminal region is sufficient for translocation to the furrow and the rescue of cytokinesis. Cortexillins are supposed to have a targeting signal for coupling to a myosin II-independent system that directs transport of membrane proteins to the cleavage furrow. ---------------------------------------------------------------------------- RacF1, a novel member of the Rho protein family in Dictyostelium discoideum, associates transiently with cell contact areas, macropinosomes and phagosomes. Francisco Rivero1,2; Richard Albrecht2, Heidrun Dislich1, Enrico Bracco2,3; Laura Graciotti2, Salvatore Bozzaro3 and Angelika A. Noegel1,2. 1Institut für Biochemie I, Medizinische Fakultät, Universität zu Köln, Joseph-Stelzmann-Str. 52, 50931 Köln, FRG 2Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, FRG 3Dipartimento di Scienze Cliniche e Biologiche, Ospedale S. Luigi Gonzaga, Regione Gonzole 10, 10043 Orbassano-Torino, Italy Molecular Biology of the Cell, in press. Abstract: Using a PCR approach we have isolated racF1, a novel member of the Rho family in Dictyostelium. The racF1 gene encodes a protein of 193 amino acids and is constitutively expressed throughout the Dictyostelium life cycle. Highest identity (94%) was found to a RacF2 isoform, to Dictyostelium Rac1A, Rac1B and Rac1C (70%), and to Rac proteins of animal species (64-69%). To investigate the role of RacF1 in cytoskeleton-dependent processes, we have fused it at its amino-terminus with green fluorescent protein (GFP) and studied the dynamics of subcellular redistribution using a confocal laser scanning microscope and a double view microscope system. GFP-RacF1 was homogeneously distributed in the cytosol and accumulated at the plasma membrane, especially at regions of transient intercellular contacts. GFP-RacF1 also localized transiently to macropinosomes and phagocytic cups, and was gradually released within less than one minute after formation of the endocytic vesicle or the phagosome, respectively. Upon stimulation with cAMP, no enrichment of GFP-RacF1 was observed in leading fronts, from which it was found to be initially excluded. Cell lines were obtained using homologous recombination that expressed a truncated racF1 gene lacking sequences encoding the carboxyl-terminal region responsible for membrane targeting. These cells displayed normal phagocytosis, endocytosis and exocytosis rates. Our results suggest that RacF1 associates with dynamic structures that are formed during pinocytosis and phagocytosis. Although RacF1 appears not to be essential, it might act in concert and/or share functions with other members of the Rho family in the regulation of a subset of cytoskeletal rearrangements that are required for these processes. ---------------------------------------------------------------------------- Glycogen Synthase Kinase-3 (GSK-3) is regulated during Dictyostelium development via the serpentine receptor cAR3 Simon E. Plyte, Emma O’Donovan, James R. Woodgett, and Adrian J. Harwood Development 126, 325-333. Abstract Glycogen Synthase Kinase-3 (GSK-3) is required during metazoan development to mediate the effects of the extracellular signal wingless/Wnt-1 and hence is necessary for correct cell type specification. GSK-3 also regulates cell fate during Dictyostelium development, but in this case it appears to mediate the effects of extracellular cAMP. By direct measurement of GSK-3 kinase activity during Dictyostelium development, we find that there is a rise in activity at the initiation of multicellular development which can be induced by cAMP. The timing of the rise correlates with the requirement for the Dictyostelium homologue of GSK-3, gskA, to specify cell fate. We show that loss of the cAMP receptor cAR3 almost completely abolishes the rise in kinase activity and causes a mis-specification of cell fate that is equivalent to that seen in a gskA- mutant. The phenotype of a cAR3- mutant however is less severe than loss of gskA and ultimately gives rise to an apparently wild type fruiting body. These results indicate that in Dictyostelium extracellular cAMP acts via cAR3 to cause a rise in GSKA kinase activity which regulates cell type patterning during the initial stages of multicellularity. ---------------------------------------------------------------------------- The Small Mr Ras-like GTPase Rap1 Interacts with the Phospholipase C Pathway to Regulate Phagocytosis in Dictyostelium discoideum David J. Seastone1, Linyi Zhang1, Greg Buczynski1, Patrick Rebstein2, Gerald Weeks2, George Spiegelman2, and James Cardelli1 3 * 1 Department of Microbiology and Immunology and 3 Feist-Weiller Cancer Center Louisiana State University Medical Center Shreveport, LA 71130 2 Department of Microbiology University of British Columbia Vancouver, B.C. Canada V6T 1Z3 Molecular Biology of the Cell (in press) ABSTRACT The function of the small Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium, and the oxidative burst in mammalian neutrophils. To test if Rap1 regulates phagocytosis, we biochemically analyzed cell-lines that conditionally overexpressed wild-type (Rap1 WT(+)), constitutively active (Rap1 G12T(+)), and dominant negative (Rap1 S17N(+)) forms of D. discoideum Rap1 at levels less than 6 fold higher levels endogenous Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT (+) and Rap1 G12T (+) cells, and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase or PI 3-kinase to wild-type cells did not affect phagocytosis rates. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor) and BAPTA-AM (an intracellular Ca2+ chelator) to control cultures reduced phagocytosis rates by 90%, 50% and 65%, respectively, suggesting both arms of the PLC pathway played a role in this process. Other PKC-specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol (DAG)-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative DAG-binding protein acted upstream in a signalling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells as compared to control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 2]