Dicty News Electronic Edition Volume 14, number 4 February 12, 2000 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" ====================== Position Available ====================== GRADUATE STUDENT TEACHING ASSISTANTSHIP Teaching assistantship available August 2000 for Ph.D. student to study molecular basis for signal transduction and gene expression in Dictyostelium discoideum in the laboratory of Dr. Maureen Brandon. Assistantship includes stipend plus tuition and fees. Responsibilities include teaching undergraduate laboratories, so excellent English speaking skills are essential. Idaho State University is located in southeastern Idaho with easy access to Nordic and alpine ski areas, hiking, camping and mountain biking trails, and Yellowstone and The Grand Teton National Parks. Application materials and instructions are available online at www.isu.edu/departments/bios/. Contact Dr. Maureen Brandon at branmaur@isu.edu prior to submission for further information. ============= Abstracts ============= Cloning and characterization of beta-COP from Dictyostelium discoideum Eur. J. Cell Biol. Martina R. Mohrs2+, Klaus-Peter Janssen1+, Thomas Kreis3#, Angelika A. Noegel2* and Michael Schleicher1 1 Institut für Zellbiologie, Ludwig-Maximilians-Universität, Schillerstrasse 42, 80336 München, FRG, 2 Institut für Biochemie I, Medizinische Einrichtungen der Universität zu Köln, Joseph-Stelzmann-Str. 52, 50931 Köln, FRG, 3 Department of Cell Biology, CH-1211 Geneva, Switzerland Abstract We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum by polymerase chain reaction using degenerate primers derived from rat beta-COP. The complete cDNA clone has a size of 2.8 kb and codes for a protein with a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between the highly conserved N- and C-terminal domains. Dictyostelium beta-COP is encoded by a single gene, which is transcribed at moderate levels into two RNAs that are present throughout development. To localize the protein, full length beta-COP was fused to GFP and expressed in Dictyostelium cells. The fusion protein was detected on vesicles distributed all over the cells and was strongly enriched in the perinuclear region. Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this compartment was identified as the Golgi apparatus. beta-COP distribution in Dictyostelium was not brefeldinA sensitive being most likely due to the presence of a brefeldinA resistance gene. However, upon DMSO treatment we observed a reversible disassembly of the Golgi apparatus. In mammalian cells DMSO treatment had a similar effect on beta-COP distribution. ---------------------------------------------------------------------------- Characterization and heterologous expression of cDNAs encoding two novel closely related Ca2+-binding proteins in Dictyostelium discoideum Magdalena Dorywalska, Barrie Coukell and Akil Dharamsi Department of Biology, York University, 4700 Keele St., Toronto, Ontario, M3J 1P3, CANADA Biochimica et Biophysica Acta (in press) Abstract During a yeast two-hybrid screen of a Dictyostelium cDNA library using the Ca2+-binding protein CBP1 as bait, we isolated a full-length cDNA encoding a novel Ca2+-binding protein (termed CBP4a). The protein is composed of 162 amino acids and contains four consensus EF-hands. PCR amplification of Dictyostelium genomic DNA using primers specific for the cDNA sequence resulted in the isolation of a gene encoding a different Ca2+-binding protein of 162 amino acids (designated CBP4b) with 90 % amino acid sequence identity to CBP4a. Southern blot analysis confirmed the presence of two closely related genes in the Dictyostelium genome. CBP4a and CBP4b mRNAs are expressed at the same stages of development as CBP1 mRNA. In addition, both novel proteins bind 45Ca2+ and interact with CBP1 in vitro in a Ca2+-dependent manner. ---------------------------------------------------------------------------- The tail domain of myosin M catalyses nucleotide exchange on Rac1 GTPases and can induce actin-driven surface protrusions. Heidrun A. Geissler, Ronald Ullmann, and Thierry Soldati Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany Correspondence and requests should be addressed to Thierry Soldati Tel: +49-6221-486407, Fax: +49-6221-486325 E-mail: soldati@mzf.mpimf-heidelberg.mpg.de Traffic, in press (May 2000 issue) Abstract Members of the myosin superfamily play crucial roles in cellular processes including management of the cortical cytoskeleton, organelle transport and signal transduction. GTPases of the Rho family act as key control elements in the reorganization of the actin cytoskeleton in response to growth factors, and other functions such as membrane trafficking, transcriptional regulation, growth control and development. Here, we describe a novel unconventional myosin from Dictyostelium discoideum, MyoM. Primary sequence analysis revealed that it has the appearance of a natural chimera between a myosin motor domain and a guanine nucleotide exchange factor (GEF) domain for Rho GTPases. The functionality of both domains was established. Binding of the motor domain to F-actin was ATP-dependent and potentially regulated by phosphorylation. The GEF domain displayed selective activity on Rac1-related GTPases. Overexpression rather than absence of MyoM affected the cell morphology and viability. Particularly in response to hypoosmotic stress, cells overexpressing the MyoM tail domain extended massive actin-driven protrusions. The GEF was enriched at the tip of growing protuberances, likely through its pleckstrin homology domain. MyoM is the first unconventional myosin containing an active Rac-GEF domain, suggesting a role at the interface of Rac-mediated signal transduction and remodeling of the actin cytoskeleton. ---------------------------------------------------------------------------- Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella Sonja Haegele,1 Rolf Koehler,1 Hilde Merkert,1 Michael Schleicher,2 Joerg Hacker, 1 Michael Steinert 1* 1 Institut fuer Molekulare Infektionsbiologie, Universitaet Wuerzburg, Roentgenring 11, D-97070 Wuerzburg, Germany. 2 Institut für Zellbiologie, Ludwig-Maximilians-Universitaet, Schillerstrasse 42, D-80336 Muenchen, Gemany. Accepted: Cellular Microbiology Summary The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single-cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared to their growth in the natural host Acanthamoeba castellanii. Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy it was observed that replicating, intracellular, GFP-tagged legionellae did rarely co-localize with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum. This indicates that the bacteria inhibit fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co-culture studies with profilin-minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared to infections of wild type amoebae. Because the amoebae are amenable to genetic manipulation due to their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system to study intracellular pathogenesis of microbial pathogens. ---------------------------------------------------------------------------- Role of cAMP-dependent protein kinase during growth and early development of Dictyostelium discoideum Gerd Primpke1, Vasiliki Iassonidou1, Wolfgang Nellen2, Birgit Wetterauer1* 1Zoologisches Institut der Universität München, Luisenstraße 14, 80333 München, Germany 2University of Kassel, Abt. Genetik, Heinrich-Plett-Str. 40, 34132 Kassel, Germany * to whom correspondence should be addressed Develop. Biol., in press. cAMP dependent protein kinase (PKA) is an essential regulator of gene expression and cell differentiation during multicellular development of Dictyostelium discoideum. Here we show that PKA activity also regulates gene expression during the growth phase and at the transition from growth to development. Overexpression of PKA leads to overexpression of the discoidinIg promoter, while expression of the discoidinIg promoter is reduced when PKA activity is reduced, either by expression of a dominant negative mutant of the regulatory subunit, or by disruption of the gene for the catalytic subunit (PKA-C). The discoidin phenotype of PKA-C null cells is cell autonomous. In particular, normal secretion of discoidin inducing factors was demonstrated. In addition, PKA-C null cells are able to respond to media conditioned by PSF and CMF. We conclude that PKA is a major activator of discoidin expression. However, it is not required for production or transduction of the inducing extracellular signals. Therefore PKA dependent and PKA independent pathways regulate the expression of the discoidin genes. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 4]