Dicty News Electronic Edition Volume 14, number 2 January 22, 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" ============= Abstracts ============= A Xestospongin C-sensitive Ca2+ store is required for cAMP-induced Ca2+-influx and cAMP-oscillations in Dictyostelium Ralph Schaloske, Christina Schlatterer and Dieter Malchow Faculty of Biology, University of Konstanz, Postfach 5560, 78457 Konstanz, Germany J. Biol. Chem., in press Abstract Xestospongin C (XeC) is known to bind to the IP3-sensitive store in mammalian cells and to inhibit IP3- and thapsigargin-induced Ca2+-release. In this study we show that this is also true for Dictyostelium. In addition, XeC inhibited Ca2+-uptake into purified vesicle fractions and induced Ca2+-release. This suggests that in the case of Dictyostelium XeC opens rather than plugs the IP3-receptor-channel as was proposed for mammalian cells (Gafni et al., Neuron (1997) 19, 723-733). In order to elucidate the function of the XeC-sensitive Ca2+-store in Dictyostelium during differentiation, we applied XeC to the cells and found that it caused a time-dependent increase of basal [Ca2+]i and inhibited cAMP-induced Ca2+-influx in single cells as well as in cell suspensions. Moreover, XeC blocked light scattering spikes and pulsatile cAMP-signalling. ---------------------------------------------------------------------------- Disruption of myosin function by a single charge change. Steffen Nock, Wenchuan Liang, Hans M. Warrick & James A. Spudich* Department of Biochemistry, Beckman Center B405, Stanford University Medical Center, Stanford CA 94305-5307, USA FEBS Letters, 12-23-99 issue ABSTRACT The dynamic assembly / disassembly of nonmuscle myosin II filaments is critical for the regulation of enzymatic activities and localization. Phosphorylation of three threonines, 1823, 1833 and 2029 in the tail of Dictyostelium discoideum myosin II has been implicated in control of myosin filament assembly. By systematically replacing the three threonines to aspartates, mimicking a phosphorylated residue, we found that position 1823 is the most critical one for the regulation of myosin filament formation and in vivo function. Surprisingly a single charge change is able to perturb filament formation and in vivo function of myosin II. ---------------------------------------------------------------------------- The mitochondrial DNA of Dictyostelium discoideum: Complete sequence, gene content and genome organization. Shinji Ogawa1+, Ryuji Yoshino1, Kiyohiko Angata1, Masao Iwamoto1, Min Pi1, Kenji Kuroe1, Kuniko Matsuo1, Takahiro Morio1, Hideko Urushihara1, Kaichiro Yanagisawa1 and Yoshimasa Tanaka1,2,* 1Institute of Biological Sciences and 2Center for TARA#, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Mol. Gen. Genet. in press. Abstract We present an overview of the gene content and genome organization of the mitochondrial genome of Dictyostelium discoideum. The mitochondria genome consists of 55,564 base-pairs with an A + T content of 72.6%. The identified genes are two ribosomal RNAs (rnl and rns), 18 tRNAs, ten subunits of the NADH dehydrogenase complex (nad1, 2, 3, 4, 4L, 5, 6, 7, 9 and 11), apocytochrome b (cytb), three subunits of the cytochrome oxidase (cox1/2 and 3), four subunits of the ATP synthase complex (atp1, 6, 8 and 9), 15 ribosomal proteins, and five open reading frames excluding intronic ORFs. Notable features of D. discoideum mtDNA include: (1) All genes are encoded on the same strand of the DNA and a universal genetic code is used. (2) The cox1 gene has no termination codon and is fused with downstream cox2 . The 13 ribosomal protein and four ORF genes form a cluster 15.4 kb long with several overlapping regions. (3) The number of tRNAs encoded in the genome is not sufficient to support the synthesis of mitochondrial protein. (4) In total, five group I introns reside in rnl and cox1/2, and three of which in cox1/2 contain four free-standing ORFs. We compare the genome to other sequenced mitochondrial genomes, particularly that of Acanthamoeba castellanii. ---------------------------------------------------------------------------- The molecular genetics of chemotaxis: Sensing and responding to chemoattractant gradients Richard A. Firtel and Chang Y. Chung BioEssays, in press Summary Chemotaxis plays a central role in various biological processes, including movement of neutrophils and macrophage during wound healing and aggregation of Dictyostelium cells. During the past few years, new understanding of the mechanisms controlling chemotaxis has been obtained through the molecular genetic and biochemical studies of Dictyostelium and other experimental systems. This review outlines our present understanding of the signaling pathways that allow a cell to sense and respond to a chemoattractant gradient. In response to chemoattractants, cells either become polarized in the direction of the chemoattractant source, resulting in the formation of a leading edge, or reorient their polarity in the direction of the chemoattractant gradient and move with a stronger persistence. Models are presented to explain this directional response. They include a localized activation of pathways at the leading edge and an "inhibition" of these pathways along the lateral edges of the cell. One of the primary pathways that may be responsible for such localized responses is the activation of phosphatidyl inositol-3 kinase (PI3K). Evidence suggests that a localized formation of binding sites for PH (pleckstrin homology) domain-containing proteins produced by PI3K leads to the formation of "activation domains" at the leading edge, producing a localized response. ---------------------------------------------------------------------------- The cellulose synthase gene of Dictyostelium Richard L. Blanton, Danny Fuller, Negin Iranfar, Mark J. Grimson, and William F. Loomis Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, U.S.A. Department of Biology, University of California San Diego, La Jolla, CA 92093, U.S.A. Proc. Natl. Acad. Sci , in press ABSTRACT Cellulose is a major component of the extracellular matrices formed during development of the social amoeba, Dictyostelium discoideum. We isolated insertional mutants that failed to accumulate cellulose and had no cellulose synthase activity at any stage of development. Development proceeded normally in the null mutants up to the beginning of stalk formation, at which point the culminating structures collapsed onto themselves, then proceeded to attempt culmination again. No spores or stalk cells were ever made in the mutants, with all cells eventually lysing. The predicted product of the disrupted gene (dcsA) showed significant similarity to the catalytic subunit of cellulose synthases found in bacteria and putative homologs in plants. Enzyme activity and normal development were recovered in strains transformed with a construct expressing the intact dcsA gene. Growing amoebae carrying the construct accumulated the protein product of dcsA, but did not make cellulose until they had developed for at least 10 hours. These studies show directly that the product of dcsA is necessary, but not sufficient, for synthesis of cellulose. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 2]