CSM News Electronic Edition Volume 7, number 1 July 6, 1996 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 =========== CHOICE OF PARTNERS: Sexual cell interactions in Dictyostelium discoideum Hideko Urushihara Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305 Japan Cell Struct. Funct., in press. ABSTRACT Recognition of mating partners are of central importance in the sexual processes. In consideration that the most important function of sexuality is to shuffle genetic materials to generate wider variation of characters, mating among different genetic backgrounds is preferable. Wild isolates of cellular slime mold Dictyostelium discoideum are predominantly heterothallic, but homothallic ones also exist. In addition, there are bisexual strains which are compatible with either mating type of heterothallic strains but are self-incompatible. How cells of these organisms choose proper mating partners may include the essential mechanisms for sexual cell recognition in general. This minireview addresses studies on sexual cell interactions of D. discoideum with special attention to cell recognition and evolution of the mating system. --------------------------------------------------------------------- The Dictyostelium Dual-Specificity Kinase splA is Essential for Spore Differentiation Glen H. Nuckolls1,3, Nir Osherov2, William F. Loomis2, and James A. Spudich1 1 Depts. of Biochemistry and Developmental Biology, Stanford Univ. School of Med., Stanford, CA 94305, 2 Center for Molecular Genetics, Dept. of Biology, UCSD, LaJolla, CA 92093, 3 Craniofacial Development Section, NIH/NIAMS, Bethesda, MD 20892-2745 Development, in press Summary We have studied the structure and function of the Dictyostelium kinase splA. A truncated form of the splA protein exhibited primarily tyrosine kinase activity in vitro; however it also autophosphorylated on serine and threonine residues. The kinase domain of splA exhibits approximately 38% identity to the CTR1 kinase of Arabidopsis, which is a member of the Raf family. Outside its kinase domain, splA shares homology with the byr2 kinase of S. pombe. By aligning the sequences of splA, byr2, and STE11, a homologue of byr2 in S. cerevisiae, we have identified a conserved motif that is also found in members of the Eph family of growth factor receptor tyrosine kinases. SplA is expressed throughout development with a peak during the mound stage of morphogenesis. Strains in which the splA gene had been disrupted completed fruiting body formation; however spore cells spontaneously lysed before completing their differentiation. Northern analysis revealed the expression of the prespore marker cotB and the prestalk markers ecmA and ecmB in the mutant strain during development. The spore differentiation marker spiA was detected in the mutant spores both by Northern and immunoblotting, but these cells failed to assemble spore coats. Immunoblot analysis of the developmental pattern of tyrosine phosphorylation revealed a protein that was phosphorylated in mutants but was not phosphorylated in the wild-type cells. SplA is a novel dual specificity kinase that regulates the differentiation of spore cells. ---------------------------------------------------------------------------- A SLOW SUSTAINED INCREASE IN CYTOSOLIC Ca2+ LEVELS MEDIATES STALK GENE INDUCTION BY DIFFERENTIATION INDUCING FACTOR IN DICTYOSTELIUM Pauline Schaap1, Thomas Nebl2 and Paul R. Fisher2 1. Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. 2. School of Microbiology, La Trobe University, Bundoora, WIC 3083, Australia. EMBO J., in press ABSTRACT During Dictyostelium stalk cell differentiation, cells vacuolate, synthesize a cellulose cell wall and die. This process of programmed cell death is accompanied by expression of the prestalk gene ecmB and induced by the differentiation inducing factor DIF. Using cell lines expressing the recombinant Ca2+- sensitive photoprotein apoaequorin, we found that 100 nM DIF increases cytosolic Ca2+ ([Ca2+]i) levels from about 50 to 150 nM over a period of 8 h. The Ca2+-ATPase inhibitor 2,5-di(tert- butyl)-1,4-hydroquinone (BHQ) induced a similar increase in [Ca2+]i levels and induced expression of the prestalk gene ecmB to the same level as DIF. The [Ca2+]i increases induced by DIF and BHQ showed similar kinetics and preceded ecmB gene expression by about 1-2 hours. The Ca2+ chelator 1,2-bis(o-aminophenoxy)- ethane-N,N,N'N'-tetraacetic acid (BAPTA) efficiently inhibited the BHQ induced [Ca2+]i increase and blocked DIF-induced expression of the ecmB gene. These data indicate that the effects of DIF on stalk gene expression are mediated by a sustained increase in [Ca2+]i. Sustained [Ca2+]i elevation mediates many forms of programmed cell death in vertebrates. The Dictyostelium system may be the earliest example of how this mechanism developed during early eukaryote evolution. ------------------------------------------------------------------- EXTRACELLULAR cAMP DEPLETION TRIGGERS STALK GENE EXPRESSION IN DICTYOSTELIUM; DISPARITIES IN DOSE DEPENDENCY AND DEVELOPMENTAL TIMING INDICATE THAT PRESPORE INDUCTION AND STALK REPRESSION BY cAMP ARE MEDIATED BY SEPARATE SIGNALING PATHWAYS. Ron D.M. Soede1, Neil A. Hopper2, Jeffrey G. Williams2 and Pauline Schaap1*. 1Cell Biology Section, Institute for Molecular Plant Sciences,University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, TheNetherlands. 2Medical Research Council Laboratory for Molecular Cell Biology and Department of Biology, University College London, GowerStreet, London WC1E6B, United Kingdom. Dev. Biol., in press. ABSTRACT During Dictyostelium development, amoebae differentiate into spores and stalk cells. Earlier studies showed that extracellular cAMP is essential for induction of prespore differentiation and that cAMP represses stalk gene expressionin vitro. We show that the repressive pathway is operative in vivo, because activation of the stalk-specific promoter region of the ecmB gene is strongly enhanced by overexpression of a phosphodiesterase that depletes extracellular cAMP. To test whether a single cAMP transduction pathway controls the choice between prespore or stalk cell differentiation, we compared the timing and dose-dependency of the effects of cAMP on both responses. Cells acquire competence for cAMP repression of ecmB promoter activity 4 hr later than for prespore gene induction. Half maximal prespore induction requires 30 uM of the stable cAMP analog Sp-cAMPS, while ecmB induction is halfmaximally repressed by 200 nM of Sp-cAMPS, which is equivalent to about 3 to 13 nM cAMP. At concentrations exceeding 10 uM, Sp-cAMPS stimulates ecmB expression from the intact promoter,but not from the stalk specific subregion. These data suggest that distinct signaling pathways operating at different developmental stages control induction of prespore genes on one hand and repression of stalk genes on the other. Both stalk gene repression and prespore gene induction by Sp-cAMPS are antagonized by millimolar adenosine concentrations. However, an adenosine analog that is resistant to extracellular metabolism is active at 10 uM. Since adenosine inhibits cAMP binding to cAMP receptors, it may facilitate stalk gene expression by reducing the perceived cAMP concentration. -------------------------------------------------------------------- BINDING OF HISACTOPHILIN I AND II TO LIPID MEMBRANES IS CONTROLLED BY A pH DEPENDENT MYRISTOYL-HISTIDINE SWITCH Frank Hanakam*, Guenther Gerisch*, Sandra Lotz@, Thomas Alt@, and Anna Seelig * MPI-Biochemie, Abt. Zellbiologie, Am Klopferspitz 18a, D-82152 Martinsried, Germany. @ University of Basel, Klingelbergerstr. 70, CH-Basel, Switzerland. Biochemistry, in press. Abstract The interaction of the two N-terminally myristoylated isoforms of Dictyostelium hisactophilin with lipid model membranes was investigated by means of the monolayer expansion method and high sensitivity titration calorimetry. The two isoforms, hisactophilin I and hisactophilin II, were found to insert with their N-terminal myristoyl residue into an electrically neutral POPC monolayer corresponding in its lateral packing density to that of a lipid bilayer. The partition coefficient for this insertion process was Kp=3D(1.1+/-0.2) 104 /M. The area requirement of the protein in the lipid membrane was estimated as 44+/-6 A2 which corresponds to the crosssectional area of the myristoyl moiety with an additional small contribution from amino acid side chains. The interaction of hisactophilin I (hisactophilin II) with negatively charged membrane surfaces is modulated in a pH dependent manner by charged amino acid residues clustered around the myristoyl moiety. The electrostatic binding site consists of 3 lysine (1 arginine, 2 lysine), 7 (9) hisidine, and 4 (4) glutamic acid residues and has an isoelectric point of 6.9 (7.1). For small unilamellar POPC/POPG (75/25 mol/mol) vesicles an apparent binding constant, Kapp =3D (8+/- 1)/M was measured at pH 6.0 by means of high sensitivity calorimetry. Electrostatic interactions hence increase the binding constant by about two orders of magnitude compared to hydrophobic binding alone. With increasing pH the electrostatic attraction decreases and turns into an electrostatic repulsion at pH > 7.0 +/- 0.1. The area occupied by the cluster of charged residues constituting the membrane binding region was 280 +/- 20 A2 as derived from monolayer measurments in close agreement with molecular modelling data derived from the NMR structure of hisactophilin I (Habazettl et al., 1992). -------------------------------------------------------------------- Molecular Cloning and Characterization of DdCAD-1, a Ca2+-dependent Cell-Cell Adhesion Molecule, in Dictyostelium discoideum* Estella F.S.Wong, Simuran K. Brar, Hiromi Sesaki, Chunzhong Yang and Chi-Hung Siu Banting and Best Department of Medical Research and Department of Biochemistry University of Toronto, Toronto, Ontario M5G 1L6, Canada J. Biol. Chem., in press. SUMMARY Dictyostelium discoideum expresses EDTA-sensitive cell-cell adhesion sites soon after the initiation of development, and a Ca2+-binding protein of Mr 24,000 (designated DdCAD-1) has been implicated in this type of adhesiveness. We have previously purified DdCAD-1 to homogeneity and characterized its cell binding activity (Brar and Siu (1993) J. Biol. Chem. 268, 24902-24909). In this report, we describe the cloning of DdCAD-1 cDNAs. DNA sequencing revealed a single open reading frame coding for a polypeptide containing 213 amino acids. The identity of the cDNA was confirmed by amino acid sequences of two cyanogen bromide peptides. The deduced amino acid sequence of DdCAD-1 exhibits a relatively high degree of sequence similarity with members of the cadherin family and proteins of Myxococcus xanthus. Unlike the other cadherins, the carboxy-terminal region of DdCAD-1 contains a Ca2+-binding motif. Although analyses of the sequence suggest that the polypeptide lacks a signal peptide sequence and a transmembrane domain, immuno-fluorescence microscopy demonstrates the association of DdCAD-1 with the ecto-surface of the plasma membrane. To investigate the structure/function relationships of DdCAD-1, GST-fusion proteins containing different DdCAD-1 fragments were expressed and assayed for their 45Ca2+ and cell binding activities. These studies revealed that the cell binding activity is dependent on the amino-terminal segment and not the carboxy-terminal Ca2+-binding domain and showed additional Ca2+-binding site(s) within the amino-terminal segment. --------------------------------------------------------------------- Novel Redistribution of the Ca2+-dependent Cell Adhesion Molecule DdCAD-1 during Development of Dictyostelium discoideum Hiromi Sesaki* and Chi-Hung Siu*@ *Banting and Best Department of Medical Research and Department of Biochemistry University of Toronto, Toronto, Ontario M5G 1L6, Canada Develop. Biol., in press. SUMMARY Dictyostelium discoideum cells express DdCAD-1, a Ca2+-dependent cell-cell adhesion molecule, soon after the initiation of development. DdCAD-1 is a soluble protein which shares a significant degree of sequence similarity with E-cadherin. Laser scanning confocal microscopy of the subcellular localization of DdCAD-1 has revealed a nonrandom pattern of DdCAD-1 distribution. DdCAD-1 is present mostly as diffusely stained material in the cytoplasm during the initial stage of development. However, a drastic redistribution takes place before the onset of cell aggregation, when DdCAD-1 becomes localized predominantly at the cell periphery and an enrichment of DdCAD-1 occurs on membrane ruffles. A high concentration of DdCAD-1 also becomes associated with lamellipodia and filopodia, which often appear to participate in cell contact formation. Although DdCAD-1 is present in high concentrations in contact regions during early development, it disappears rapidly from these areas during cell aggregation. This redistribution is accompanied by an accumulation of the Ca2+-independent cell adhesion molecule gp80 in contact regions. During chemotactic migration, DdCAD-1 is present primarily on cells at the tip and on the outer margin of cell streams. In contrast, gp80 is concentrated in contact regions among cells within well developed streams. This dynamic redistribution suggests a unique role for DdCAD-1 in the recruitment of cells into streams and in the formation of initial contacts, but it may not be required to maintain stable contacts in the presence of gp80. ----------------------------------------------------------------------- [End CSM News, volume 7, number 1]