CSM News Electronic Edition Volume 5, number 12 October 28, 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" ============================ Postdoc Position Available ============================ Postdoctoral position available for studies on the role of the cell cycle in cell type determination in Dictyostelium. Experience with gene manipulation techniques and protein and immunological methodologies would be an asset. The position is available immediately but the exact starting date is negotiable. Send curriculum vitae with names of references to Gerry Weeks, Department of Microbiology and Immunology, University of British Columbia, Vancouver, B.C., V6T 1Z3; fax 604-822-6041 =========== Abstracts =========== Almuth Behrisch*, Christian Dietrich*, Angelika A. Noegel", Michael Schleicher& and Erich Sackmann* *Technische Universitaet Muenchen, "MPI f. Biochemie, Martinsried, &Ludwig-Maximilians-Universitaet Muenchen THE ACTIN-BINDING PROTEIN HISACTOPHILIN BINDS IN VITRO TO PARTIALLY CHARGED MEMBRANES AND MEDIATES ACTIN COUPLING TO MEMBRANES Biochemistry, in press Summary The interaction of the actin-binding protein hisactophilin from Dictyostelium discoideum amoebae to partially charged lipid membranes composed of mixtures of L-alpha-dimyristoylphosphatidylcholine (DMPC) with L-alpha-dimyristoylphosphatidylglycerol (DMPG) and L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is studied by film balance experiments, microfluorescence and lateral diffusion measurements at low ionic strengths (~20mM). Excess surface concentrations and adhesion energies of the protein are evaluated by the application of Gibbs law of surface excess as a function of charged lipid content. Protein expressed in E. coli lacking a myristic acid chain (EC-HIS) and natural protein with a fatty acid (DIC-HIS) isolated from Dictoystelium cells are compared. For mixtures of DMPG and DMPC protein binding leads to an increase in lateral pressure of the monolayser (at constant area) and causes strong lipid immobilization pointing to partial penetration of the protein into the lipid layer. The natural protein causes a much stronger immobilization than EC-HIS. For a given bulk concentration the adsorbed protein-to-lipid molar ratio increases with the molar fraction x(PG) of charged lipid but saturates at about 50 mole% of DMPG. Natural hisactophilin (DIC-HIS) binding to PIP2-containing monolayers is purely electrostatic at low bulk concentration c(b) and protein penetration dominates only at c(b) >68nM. Fluorescence experiments demonstrate that the natural protein (DIC-HIS) can mediate binding of monomeric actin or very small oligomers to membranes, showing that the adsorbed protein remains functional. In contrast, the recombinant hisactophilin (EC-HIS) can mediate only the membrane coupling of larger actin structures. ----------------------------------------------------------------------- tagB Expression and Prestalk Divergence in Dictyostelium are Independent of DIF-1 Gad Shaulsky and William F. Loomis Center for Molecular Genetics, Department of Biology, University of California, San Diego, La Jolla CA 92093 Developmental Biology, in press Summary Differentiation in Dictyostelium leads to divergence of prespore and prestalk cells. Cell type specific genes can be used as diagnostic and predictive tools for studying cell type divergence. Here we describe the cell type specificity of the recently discovered gene tagB. The 5' untranslated region of tagB was used to express E. coli =DF-galactosidase in wild type cells and in tagB- mutants. The results showed that tagB is a prestalk specific gene which is expressed early in development. tagB expression can be used to mark the differentiation of PST-I cells, the initial prestalk population, PST-A cells that will eventually form the stalk, and PST-O cells that will eventually form the upper and the lower cups surrounding the sorus. Anterior-like cells and basal disk cells were not efficiently labeled with tagB::lacZ. The expression of tagB was independent of the prestalk morphogen DIF-1 and so was the expression of another prestalk specific gene, carB. We conclude that the initiation of prestalk development is not dependent on DIF-1 and suggest that the morphogen participates in maintaining a differentiated prestalk stage rather than inducing it. ----------------------------------------------------------------------- Dictyostelium myosin II is regulated during chemotaxis by a novel protein kinase Abu Elneel, K., Karchi, M., and Ravid, S. Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem 91120, Israel J. Biol. Chem., in press Summary The myosin II heavy chain (MHC) specific protein kinase C (MHC-PKC) isolated from Dictyostelium discoideum has been implicated in the regulation of myosin II assembly in response to the chemoattractant, cAMP (Ravid, S., and Spudich, J. A. ( 1989) J. Biol. Chem. 264, 15144-15150). Here we report that elimination of MHC-PKC results in the abolishment of MHC phosphorylation in response to cAMP. Cells devoid of MHC-PK C exhibit substantial myosin II overassembly, as well as aberrant cell polarization, chemotaxis and morphological differentiation. Cells overexpressing the MHC-PKC contains highly phosphorylated MHC and exhibit impaired myosin II localization and no apparent cell polarization and chemotaxis. The results presented here provide direct evidence that MHC-PKC phosphorylates MHC in response to cAMP and plays an important role in the regulation of myosin II localization during chemotaxis. ------------------------------------------------------------------------- CALMIDAZOLIUM LEADS TO AN INCREASE IN THE CYTOSOLIC CA2+ CONCENTRATION IN DICTYOSTELIUM DISCOIDEUM BY INDUCTION OF CA2+-RELEASE FROM INTRACELLULAR STORES AND INFLUX OF EXTRACELLULAR CA2+ Christina Schlatterer and Ralph Schaloske Fakultaet fuer Biologie, Universitaet Konstanz, D-78434 Konstanz Biochem J, in press Summary The Ca2+-stores of Dictyostelium discoideum amoebae take part in the control of the [Ca2+]i-homeostasis and the cAMP-induced [Ca2+]i- signaling cascade. In order to characterize regulatory mechanisms of these stores we incubated cells with the calmodulin antagonist calmidazolium. Measurement of permeabilized and intact cells in suspension with a Ca2+- sensitive electrode revealed that calmidazolium induced Ca2+-release from intracellular stores, influx of Ca2+ across the plasma membrane and subsequent efflux. In single, fura-2 loaded cells calmidazolium evoked rapid and global transient elevations of [Ca2+]i. Other calmodulin antagonists (trifluoperazine, chlorpromazine, fendiline and W7) also induced transient elevations of [Ca2+]i, which were, however, slower and observed in fewer cells. The calmidazolium-induced influx of extracellular Ca2+ was inhibited by preincubation with BHQ and NBD-Cl, both known to interact with pumps of the IP3-sensitive store, and by the V-type H+-ATPase inhibitor bafilomycin A1 which affects the acidosomal Ca2+ store. Incubation with pump inhibitors did not induce changes in [Ca2+]i per se. We conclude that the effects of calmidazolium are, at least in part, mediated by its calmodulin-antagonizing properties, that it acts by inducing Ca2+-release from filled storage compartments and that its target of action is both the IP3-sensitive store and the acidosome; emptying of these stores leads to influx of extracellular Ca2+. --------------------------------------------------------------------- Antagonistic effects of signal transduction by intracellular and extracellular cAMP on gene regulation in Dictyostelium. Ingrid Endl, Angelika Konzok, and Wolfgang Nellen* MPI f. Biochemie, D-82152 Martinsried, and Univ. of Kassel, Dept. Genetics, Heinrich-Plett-Str. 40, D-34109 Kassel, Germany Summary In Dictyostelium, cAMP plays a role as an intracellular second messenger and in addition, as an extracellular first messenger. Both functions are thought to be tightly linked because adenylyl cyclase is coupled via G-proteins to the cell surface cAMP receptor cAR 1. Using the discoidin I gene family as a molecular marker for the first stages of development, we show here that induction of transcription requires the G-protein subunit a2 and thus a yet unidentified surface receptor, CRAC (cytosolic regulator of adenylyl cyclase) and PKA. Induction can be conferred by an increase in intracellular cAMP. In contrast, transcriptional down-regulation occurs by stimulation of cAR 1 with extracellular cAMP (Blusch et al, 1995) and a subsequent, G-protein independent Ca2+ influx. In a Ga2 gene disruption mutant, discoidin I expression can be efficiently modulated by analogs simulating intracellular cAMP (discoidin induction) and extracellular cAMP (discoidin down-regulation). We thus demonstrate possible antagonistic functions of intra- and extracellular cAMP. ---------------------------------------------------------------------- [End CSM News, volume 5, number 12]