CSM News Electronic Edition Volume 4, number 10 March 18, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmsbio.nwu.edu/dicty.html" =========== Abstracts =========== Comparative Analysis of Chemotaxis in Dictyostelium using a Radial Bioassay Method: Protein tyrosine kinase activity is required for chemotaxis to folate but not to cAMP Darren D. Browning1, Tony The, and Danton H. O'Day2 Department of Zoology, Erindale College, University of Toronto, Mississauga, Ontario, Canada L5L 1C6. 1Current address: Department of Immunology, IMM2, The Scripps Research Institute, La Jolla, CA 92032, 2Author to whom all correspondence should be addressed Phone: (905) 828-3897; fax: (905) 828-3792 Cellular Signaling, in press. Abstract The role of signal transduction during chemotaxis of Dictyostelium discoideum cells to cAMP and folic acid was investigated using a radial bioassay technique. The effects of signalling agonists were assessed by measuring the diameters of visible rings formed by the outward migration of amoebae up radial gradients of chemoattractant. This rapid and simple bioassay method yields chemotactic rates equivalent to more complex assay systems. In support of previous studies, chemotaxis toward both cAMP and folic acid was inhibited in a dose dependent manner by LaCl3, EDTA, chlorotetracycline and AlFl3 supporting the importance of calcium ions and G protein-mediated signalling in both chemotactic events. The work was extended by examining the effects of the protein tyrosine kinase inhibitor genistein. This agent inhibited chemotaxis to folate in a dose dependent manner but had no observable effect on chemotaxis toward cAMP. The notion that phosphorylation of proteins on tyrosine residues is critical for chemotaxis to folic acid was supported by western blotting experiments with monoclonal anti-phosphotyrosine antibodies which detected two candidate proteins of Mr 52 kDa and 38 kDa in the membranes of folate responsive amoebae. These two bands disappeared with sta rvation which leads to the loss of responsiveness to folic acid and the acquisition of responsiveness to cAMP. Time-lapse videomicrography also revealed some unique differences in chemotactic response. Starved cells responded to cAMP as individuals but feeding cells chemoattracted to folic acid on a populational basis. The ability to compare two different types of chemotaxis using a simple, rapid and accurate bioassay system should enhance future studies of chemotaxis in wild type and mutant strains of D. discoideum. -------------------------------------------------------------------- Mitochondrial DNA Replication but no Nuclear DNA Replication During Development of Dictyostelium Gad Shaulsky and William F. Loomis* Center for Molecular Genetics, Department of Biology, University of California, San Diego, La Jolla CA 92093 * To whom reprint requests should be addressed Proc. Natl. Acad. Sci. USA, in press Abstract Dictyostelium discoideum cells initiate development when nutrients are depleted. DNA synthesis decreases rapidly thereafter but resumes during late aggregation, only in prespore cells. This observation has been previously interpreted as indicating progression of prespore cells through the cell cycle during development. We show that developmental DNA replication occurs only in mitochondria and not in nuclei. We also show that the prestalk morphogen DIF-1 can inhibit mitochondrial respiration. A model is proposed for cell type divergence, based on competition to become prespores, that involves mitochondrial replication in prespore cells and reduction of mitochondrial activity in prestalk cells. ------------------------------------------------------------------------- An MDR Transporter / Serine Protease Gene is Required for Prestalk Specialization in Dictyostelium Gad Shaulsky, Adam Kuspa* and William F. Loomis** Center for Molecular Genetics, Department of Biology, University of California, San Diego, La Jolla CA 92093, *Present address: Department of Biochemistry, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. **Corresponding author Genes & Development, in press Abstract The prestalk specific gene, tagB, was disrupted by REMI mutagenesis. Mutant aggregates exhibit a cell autonomous defect in specialization of PST-A cells, a prestalk sub-population that eventually forms the tip. Cooperative (non-cell-autonomous) defects were found in sporulation and in specialization of prestalk cells that eventually form the upper cup (PST-O). The pattern of ecmA::lacZ expression in mutant tagB- cells defines a primary prestalk population, PST-I, from which other prestalk cells differentiate. After PST-A cells differentiate, they induce remaining PST-I cells to become PST-O cells. Subsequently, prestalk cells induce encapsulation of prespore cells during culmination. tagB is homologous to serine protease and to MDR transporter genes, implying a mechanism of action that includes proteolysis and export of peptide signals. Intercellular communication via TagB may mediate integration of cellular differentiation with morphogenesis. ------------------------------------------------------------------------- Overexpression of wild-type and mutant NDP-Kinase in Dictyostelium discoideum Olivier Sellam, Michel Veron and Martin Hildebrandt Mol. Microbiol., in press. Abstract Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxi-)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed a genomic clone encoding the enzyme NDP kinase in Dictyostelium discoideum. The increased level of RNA and enzyme activity defines a 5' noncoding genomic region of 0.9 kb as a complete promoter. Overexpression of wild type NDP kinase has no effect on development. The same is true for an inactive mutant H122C, that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase which is known to be affected in its stability in vitro, only leads to a small increase in total NDP kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild type and mutant monomers. ---------------------------------------------------------------------- Agonist-Induced Loss of Ligand Binding is Correlated with Phosphorylation of cAR1, a G Protein-Coupled Chemoattractant Receptor from Dictyostelium Michael J. Caterina, Peter N. Devreotes, Jane Borleis, and Dale Hereld. Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 J. Biol. Chem., in press. The parallel agonist-induced phosphorylation, alteration in electrophoretic mobility, and loss of ligand binding of cAR1 depend upon a cluster of five C- terminal-domain serine residues (Caterina, M.J., Hereld, D., and Devreotes, P.N., J. Biol. Chem., in press). Analysis of mutants lacking combinations of these serines revealed that either S303 or S304 is required; mutants lacking both serines are defective in all of these responses. Interestingly, several mutants, including those substituted at only S299, S302 or S303 or at non-serine positions within the third cytoplasmic loop, displayed an unstable mobility shift--the alteration was rapidly reversed upon cAMP removal. These mutants also exhibited subnormal extents of loss of ligand binding, which is assessed after removal of the ligand. For the wild-type receptor, we found that the stability of phosphorylation depends upon the concentration and duration of agonist pretreatment. This suggests that, following phosphorylation of S303 or S304, cAR1 undergoes a further transition (EC50=140 nM, t1/2=4 min) to a relatively phosphatase- resistant state. We used this insight to show that, under all conditions tested, the extent of loss of binding is correlated with the fraction of cAR1 in the altered mobility form. We discuss possible relationships between cAR1 phosphorylation and loss of ligand binding. ------------------------------------------------------------------------- Purification and Characterization of a Dictyostelium protein kinase required for actin-activation of the Mg2+ATPase activity of Dictyostelium myosin ID. Sheu-Fen Lee and Graham P. Cote Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6 J. Biol. Chem., in press ABSTRACT We have isolated a protein from Dictyostelium with a molecular mass of 110 kDa as judged by SDS gel electrophoresis that can stimulate the actin- activated MgATPase activity of Dictyostelium myosin ID (myoD). In the presence of MgATP the 110 kDa protein incorporated phosphate into itself and into the heavy chain, but not the light chain, of myoD. Phosphorylation to 0.5 mol Pi/mol increased the myoD actin-activated MgATPase rate from 0.2 to 3 umol/min/mg. Renaturation following SDS gel electrophoresis demonstrated that the 110 kDa protein contained intrinsic protein kinase and autophosphorylation activity. Autophosphorylation to 1 mol of Pi/mol enhanced the rate at which the 110 kDa protein kinase phosphorylated myoD by 40-fold. The rate of autophosphorylation was strongly dependent on the 110 kDa protein kinase concentration, indicating an intermolecular reaction. Synthetic peptides of 9 to 11 residues corresponding to the heavy chain phosphorylation site of Acanthamoeba myosin IC and the homologous sites in Dictyostelium myosin IB (myoB) and myoD were poor substrates for the 110 kDa protein kinase. The 110 kDa protein kinase was unable to phosphorylate the myoB isozyme suggesting that it may be specific for myoD. ---------------------------------------------------------------------- [End CSM-News, volume 4, number 10]