CSM News Electronic Edition Volume 8, number 5 February 15, 1997 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Molecular cloning of a cDNA encoding the nucleosome core histone H3 from Dictyostelium discoideum by genetic screening in yeast Monika Bukenberger(1), Jens Horn(2), Theodor Dingermann(2), Robert P. Dottin(1) and Thomas Winckler(2) (1) Hunter College, Dept. of Biological Sciences, 695 Park Avenue, New York, NY 10021 U.S.A. and (2) Institut fur Pharmazeutische Biologie, Universitat Frankfurt. (Biozentrum), Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany Biochim. Biophys. Acta, in press Summary The one-hybrid method for genetic screening in yeast was used to search a Dictyostelium discoideum cDNA library for DNA-binding proteins that interact with the C-module of the Dictyostelium repetitive element. The C-module was formerly shown to contain two high affinity, sequence-specific binding sites for a nuclear protein factor of unknown function (CMBF). The bait DNA sequence was bound in vivo by a cDNA-encoded protein whose derived amino acid sequence showed high homology to nucleosome core histone H3, but not to partially available CMBF sequences. The D. discoideum histone H3 homolog is encoded by a single gene and shows significant sequence variation at the amino terminus of the protein, including a triple-serine insertion not found in any other histone H3. --------------------------------------------------------------------- Microfilament dynamics in cell motility and chemotaxis, monitored by a GFP-actin fusion Monika Westphal, Andreas Jungbluth, Manfred Heidecker, Bettina Muehlbauer, Christina Heizer, Jean-Marc Schwartz, Gerard Marriott, and Guenther Gerisch* Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany Current Biol., in press Abstract Background: The microfilament system in the cortex of highly motile cells, such as neutrophils and cells of the eukaryotic microorganism Dictyostelium discoideum, is subject to rapid re-organization, either spontaneously or in response to external signals. In particular, actin polymerization induced by chemoattractant leads to local accumulation of F-actin and protrusion of a leading edge in the direction of a gradient. To study the dynamics of actin in these processes, the actin was tagged at its N-terminus with green fluorescent protein (GFP) and observed with fluorescence microscopy in living cells of Dictyostelium discoideum. Results: Purified GFP-actin was capable of co-polymerizing with actin. In the transfected cells of D. discoideum studied, GFP-actin made up 10 to 20 percent of the total actin. Fluorescence observations of single living cells showed that the fusion protein was enriched in cell projections including filopodia and leading edges, and reflected the dynamics of the microfilament system in cells that were freely moving, being chemotactically stimulated or aggregated. Superimposition of a phalloidin label to the GFP-actin fluorescence in confocal sections of fixed cells revealed a good correlation, however there were distinct sites more prominent in GFP-actin. Conclusion: GFP-actin containing microfilaments are still capable of generating force with myosin in vitro and of interacting with myosin in vivo. In highly motile cells, the local accumulation and disassembly of actin parallels the protrusion and retraction of pseudopods on a time scale of seconds. Re-organization of the actin system is steered by chemotactic signal inputs. Double labeling with GFP-actin and other probes provides a cue to the various states of actin in a motile cell. A major portion of the actin assemblies visualized by GFP-actin are networks or bundles of filamentous actin. Other clusters of GFP-actin might represent stores of monomeric actin complexed with sequestering proteins. --------------------------------------------------------------------- Localization of a DNA Topoisomerase II to Mitochondria in Dictyostelium discoideum: Deletion Mutant Analysis and Mitochondrial Targeting Signal Presequence Kayoko Komori*, Fumiaki Maruo, Takahiro Morio, Hideko Urushihara, Yoshimasa Tanaka<1> Institute of Biological Sciences and <1> Center for TARA, University of Tsukuba, Tsukuba, Ibaraki, 305 Japan * Present address: Department of Molecular Biology, Biomolecular Engineering Research Institute (BERI), Furuedai 6-2-3, Suita, Osaka, 565 Japan J. Plant Res. in press. Abstract DNA topoisomerase II of Dictyostelium discoideum (TopA), the gene (topA) encoding which we cloned, was shown to have an additional N-terminal region which contains a putative mitochondrial targeting signal presequence. We constructed overexpression mutants which expressed the wild-type or the N-terminally deleted enzyme, and examined its localization by immunofluorescence microscopy and proteinase K digestion experiment. These experiments revealed that the enzyme is located in the mitochondria by virtue of the additional N-terminal region. Furthermore, in the cell extract depleted the enzyme by immunoprecipitation, nuclear DNA topoisomerase II activity was not decreased. These results confirmed that TopA is located in the mitochondria, even though its amino acid sequence is highly similar to those of nuclear type topoisomerase II of other organisms. Thus, this report is the first to establish the location of the mitochondrial targeting signal presequence in DNA topoisomerase II and in proteins of D. discoideum directly by analyzing a deletion mutant. --------------------------------------------------------------------- [Ed. Note--The following appeared in the last issue, but was apparently truncated in some of the messages. It is reprinted here in its entirety] Trehalase of Dictyostelium discoideum: Inhibition by Amino-containing Analogs of Trehalose and Affinity Purification L.A. Temesvari (1,2) and D.A. Cotter (1) (1) Department of Biological Sciences, University of Windsor, Windsor, Ontario, Canada N9B 3P4. (2 Present Address) Department of Microbiology and Immunology and Center for Excellence in Cancer Research, Louisiana State University Medical Center, Shreveport, LA 71130. Biochimie, in press. Summary The inhibitory effects of three nitrogen-containing analogs of trehalose, Validamycin A, MDL 25,637 and castanospermine, on Dictyostelium discoideum trehalase were examined. Prior to this study, the effects of glycohydrolase inhibitors on D. discoideum trehalase have not been reported. Validamycin A, MDL 25,637 and castanospermine were found to be potent, reversible, competitive inhibitors of D. discoideum vegetative trehalase in vitro with IC50 values of 1 X 10-9 M, 2 X 10-8 M and 1.25 X 10-4 M, respectively. Validamycin A and MDL 25,637 also exhibited time dependent inhibition of D. discoideum trehalase, whereby the potencies of these two inhibitors were observed to increase when pre-incubated with the enzyme for up to 60 minutes. The competitive natures of Validamycin A and MDL 25,637 were also altered during pre-incubation with enzyme such that the compounds behaved as mixed inhibitors under these conditions. Taken together these results suggest that the inhibitory action of Validamycin A and MDL 25,637 on trehalase is of a slow-binding nature. A trehalase-specific affinity resin was synthesized by covalently coupling Validamycin A to Sepharaose 6B. This resin was used to purify D. discoideum trehalase to near homogeneity in a two-step procedure. SDS-PAGE of affinity purified trehalase, and silver staining or in situ staining for trehalase activity, revealed a major protein species of 42 kDa, exhibiting trehalase activity, and 2 minor protein species of approximately 45 kDa and 49 kDa. Since Validamycin A demonstrates strict binding specificity for trehalase, Validamycin A-Sepharose has potential and novel applications in rapid, large scale, purification of trehalases from a variety of species origins. --------------------------------------------------------------------- Coronin and vacuolin identify consecutive stages of a late, actin-coated endocytic compartment in Dictyostelium Robert Rauchenberger, Ulrike Hacker, John Murphy, Jens Niewhner and Markus Maniak Abt. Zellbiologie, Max-Planck-Institut fur Biochemie, D-82152 Martinsried Current Biology, in press Cells of the unicellular eukaryote Dictyostelium discoideum take up all their nutrients by endocytosis. Both particle- and fluid-containing vacuoles are transiently surrounded by a cytoskeletal coat [1,2] . When the coat has dissociated, acidification and digestion of the vesicle contents occurs, followed by exocytosis of indigestible remnants after 60 to 90 minutes. For mathematical modelling of endocytic transit at least nine compartments are needed [3] , suggesting that markers associate only for a few minutes with a specific endocytic compartment. Among the proteins that have been identified as components of endocytic vesicles are actin, subunits of the V-H+ATPase and small GTP-binding proteins of the Rab family [4-7]. Using a monoclonal antibody produced against Dictyostelium endocytic vesicles, we have isolated a cDNA corresponding to a novel protein that we have named vacuolin. In order to determine the precise step along the endocytic pathway that involves vacuolin, we generated a fusion of the green fluorescent protein (GFP) with vacuolin. GFP-vacuolin decorated vesicles were identified as a post-lysosomal compartment, that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-lysosomal compartment was identified by the binding of a tagged cytoskeletal protein, coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-lysosomal pathway and the integrity of this coat wss required for exocytosis. --------------------------------------------------------------------- Genetics of phototaxis in a model eukaryote, Dictyostelium discoideum. P.R. Fisher. School of Microbiology, La Trobe University, Bundoora, VIC 3083, Australia. Bioessays, in press. Summary The life cycle of Dictyostelium discoideum offers a unique opportunity to study signal transduction in eukaryotic cells at both the unicellular and the multicellular levels of organization. Adding to the already extensive knowledge of the unicellular stages, classical and molecular genetics have begun to unravel transduction of signals controlling morphogenesis and behaviour (phototaxis and thermotaxis) in the multicellular "slug" stage of the life cycle. Distributed over all seven genetic linkage groups are probably about 20 but possibly as many as 55 genes of importance for slug behaviour. The encoded proteins appear from pharmacological studies and mutant phenotypes to govern transduction pathways involving the intracellular second messengers cAMP, cGMP, IP3 and Ca2+. Pathways from the photo- and thermoreceptors converge first with each other and thence, at the level of the second messengers, with those from extracellular tip activation (cAMP) and inhibition (Slug Turning Factor and/or ammonia and/or adenosine) signals that control slug movement and morphogenesis. --------------------------------------------------------------------- [End CSM-News, volume 8, number 5]