Dicty News Electronic Edition Volume 9, number 7 27 September 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== Changes in Actin Filament Organization during Pseudopod Formation Eunkyung Lee, Eric A. Shelden* and David A. KnechtÝ Department of Molecular and Cell Biology University of Connecticut, Storrs, CT 06269 Experimental Cell Research, in press ABSTRACT Fluorescent phalloidin has been introduced into Dicytostelium amoebae in order to visualize dynamic changes in the localization of F-actin during pseudopod extension. Phalloidin was initially localized to the peripheral cortex of the cell. Newly formed pseudopods were not fluorescent indicating that phalloidin was tightly bound to existing F-actin filaments and could not rapidly relocalize to newly formed filaments. As pseudopod extension proceeded, the fluorescent signal disappeared from the region directly underlying the expansion zone leaving a gap in the actin cortex. Similar results were obtained in both wild-type cells and those lacking myosin II heavy chain. The disappearance of the fluorescent signal from the cortical region underlying the new pseudopod is presumed to be due to breakdown of the actin cortex and dispersion of the remnants. These results suggest that new pseudopods are not built upon the existing actin cortex but rather the cortex is locally solated as part of the construction of the new actin network. ------------------------------------------------------------------------- A Novel Cytosolic Regulator, Pianissimo, Is Required for Chemoattractant Receptor and G Protein-mediated Activation of the Twelve Transmembrane Domain Adenylyl Cyclase in Dictyostelium Mei-Yu Chen, Yu Long, and Peter N. Devreotes Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, Maryland 21205-2185 Genes and Development, in press Abstract Genetic analysis was applied to identify novel genes involved in G-protein linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, PiaA, involved in cAMP signaling in D. discoideum. PiaA encodes a 130 kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the twelve transmembrane domain adenylyl cyclase. In piaA- null mutants, neither chemoattractant stimulation of intact cells nor GTPgS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPgS stimulation of adenylyl cyclase activity in piaA- lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme since lysates of crac-piaA- double mutants require both proteins for reconstitution. Homologs of PiaA are found in S. cerevisiae and S. pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G-protein linked pathways are likely to exist in higher eukaryotes. -------------------------------------------------------------------------- Phosphorylation of Chemoattractant Receptors is Not Essential for Chemotaxis or Termination of G-Protein-Mediated Responses Ji Yun Kim#, Ron Soede%, Romi Valkema&, Jane A. Borleis#, Peter J.M. VanHaastert&, Peter Devreotes#, and Dale Hereld, #,@ #Departmentment of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; %Institute of Molecular Plant Sciences, University of Leiden, Netherlands; &Department of Biochemistry, University of Groningen, Netherlands; @Present address: Department of Microbiology and Molecular Genetics, The University of Texas Health Science Center, P.O. Box 20708, Houston, Texas 77225. J. Biol. Chem., in press Abstract In several G-protein-coupled signaling systems, ligand-induced receptor phosphorylation by specific kinases is suggested to lead to desensitization via mechanisms including receptor/G-protein uncoupling, receptor internalization, and receptor down-regulation. We report here that elimination of phosphorylation of a chemoattractant receptor of Dictyostelium, either by site-directed substitution of the serines or by truncation of the C-terminal cytoplasmic domain, completely prevented agonist-induced loss-of -ligand binding but did not impair the adaptation of several receptor-mediated responses including the activation of adenylyl and guanylyl cyclases and actin polymerization. In addition, the phosphorylation-deficient receptors were capable of mediating chemotaxis, aggregation, and differentiation. We propose that, for chemoattractant receptors, agonist-induced phosphorylation regulates surface binding activity but other phosphorylation-independent mechanisms mediate response adaptation. ------------------------------------------------------------------------- Photosensory and thermosensory responses in Dictyostelium slugs are specifically impaired by absence of the F-actin cross-linking gelation factor (ABP-120). P.R. Fisher, A.A. Noegel, M. Fechheimer, F. Rivero, J. Prassler and G. Gerisch. Current Biology, In Press. Chemotactic aggregation of starving amoebae of Dictyostelium discoideum leads to formation of a motile, multicellular organism - the slug - whose anterior tip controls its phototactic and thermotactic behaviour. To determine whether proteins that regulate the in vitro assembly of actin are involved in these responses, we tested phototaxis and thermotaxis in mutant slugs in which the gene encoding one of five actin-binding proteins had been disrupted. Of the proteins tested - severin, a-actinin, fimbrin, the 34 kD actin-bundling protein and the F-actin cross-linking gelation factor (ABP-120) - only ABP-120 proved necessary for normal phototaxis and thermotaxis in the multicellular slugs. The related human protein ABP-280 is required for protein phosphorylation cascades initiated by lysophosphatidic acid and tumor necrosis factor a. The repeating segments constituting the rod domains of ABP-120 and ABP-280 may be crucial for the function of both proteins in specific signal transduction pathways by mediating interactions with regulatory proteins. ------------------------------------------------------------------------- Origin and Evolution of the Slime Molds (Mycetozoa) Sandra L. Baldauf and W. Ford Doolittle Canadian Institute for Advanced Research, and Department of Biochemistry,Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7 ABSTRACT The Mycetozoa include the cellular (dictyostelid), acellular (myxogastrid) and protostelid slime molds. However, available molecular data are in disagreement on both the monophyly and phylogenetic position of the group. We have sequenced the elongation factor-1a genes from one member of each division of the Mycetozoa, including Dictyostelium discoideum for which cDNA sequences were previously available. Phylogenetic analyses of these sequences strongly support a monophyletic Mycetozoa, of which the myxogastrid and dictyostelid are most closely related to each other. All phylogenetic methods used also place this coherent Mycetozoan assemblage as emerging among the multicellular eukaryotes, tentatively supported as more closely related to animals+fungi than are green plants. With our data there are now three proteins that consistently support a monophyletic Mycetozoa and at least four which place these taxa within the crown of the eukaryote tree. We suggest that ribosomal RNA data should be more closely examined with regard to these questions, and emphasize the importance of developing multiple sequence data sets. ------------------------------------------------------------------------- Intracellular Ca2+ signals in Dictyostelium chemotaxis are mediated exclusively by Ca2+ influx. T. Nebl and P.R. Fisher School of Microbiology, La Trobe University, Bundoora VIC 3083, AUSTRALIA. J. Cell Sci., In press. SUMMARY We measured folate- and cAMP-induced changes in cytoplasmic free calcium concentration ([Ca2+]i) using recombinant aequorin reconstituted in living Dictyostelium cells with coelenterazine-h. The resulting semi-synthetic protein displayed increased sensitivity to Ca2+ allowing accurate measurement of chemoattractant-induced transients at low resting levels. Both folate-and cAMP-induced Ca2+ responses were developmentally regulated, exhibited remarkably similar kinetics and were dependent on the relative rather than the absolute magnitude of increases in attractant concentration. They began after a short delay of 5-10s, leading to a maximum increase in cytosolic calcium concentration after ~25 s and a return to basal level within ~60s after stimulation. Responses elicited by the two chemoattractants were dose-dependent and saturated between 4-20 uM. They depended on the presence of free extracellular calcium ions and were inhibited in a concentration- dependent manner between 10-4 and 10-5 M. In accordance with 45Ca2+- uptake measurements by Milne and Coukell (J. Cell Biol. 112, 103-110; 1991), both responses were also completely inhibited by 15 uM Ruthenium Red, 15 uM carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and 500 uM gadolinium ions. Under conditions that prohibited influx of Ca2+ from the extracellular medium there were no detectable changes in [Ca2+]i that could be realted to a separate release of the ion from intracellular stores. Together these results show that the Ca2+ signals involved in chemotaxis correlate temporally with actin depolymerization (not polymerization) and are mediated by Ca2+ influx, not IP3-mediated intracellular release. ------------------------------------------------------------------------- Functional activity and developmental regulation of DdRBP1, a RNA binding protein in Dictyostelium discoideum Peter Oberosler and Wolfgang Nellen* Dept. Genetics, Universität Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel and Max-Planck-Institut f. Biochemie, D-82152 Martinsried, Germany Biol. Chemistry, in press Summary In an attempt to find potential components of natural antisense mechanisms in Dictyostelium, we investigated RNA binding protein (RBD) genes of the RNP-CS* family. RBD proteins can enhance hybridization of complementary RNAs and may thus mediate the interaction of sense and antisense RNA. Using the conserved RNP1 and RNP2 motifs as primers, we cloned 4 PCR fragments containing ORFs and additional homologies to known members of the RNP-CS family. We cloned a full length cDNA for one (DdRBP1) which showed similarities to hnRNP A1. Recombinant protein synthesized in E. coli displayed binding to single stranded RNA and a weak annealing activity for partially complementary RNAs in vitro. Deletion of the RNP1 motif reduced RNA binding considerably but not completely. DdRBP1 is thus one of the few members of the RNP-CS family for which binding and annealing activity have been experimentally demonstrated. Polyclonal antisera directed against recombinant DdRBP1 detected a protein of approx. 40 kDa. In whole cell extracts, this protein was present in equal amounts throughout the developmental cycle of Dictyostelium while differential accumulation was observed in nuclei during early and late development. ------------------------------------------------------------------------- [End CSM News, volume 9, number 7]