Dicty News Electronic Edition Volume 16, number 12 June 23, 2001 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============== Abstracts ============== Inhibition of multicellular development switches cell death of Dictyostelium discoideum towards mammalian-like unicellular apoptosis. Irène Tatischeff1) a, Patrice Xavier Petit b, Alain Grodet c, Jean-Pierre Tissier d, Isabelle Duband-Goulet e, Jean-Claude Ameisen f a: Laboratoire de Physicochimie Biomoléculaire et Cellulaire, CNRS ESA 7033, Université Pierre et Marie Curie, Paris/France b: Institut Cochin de Génétique Moléculaire, INSERM U129 - CHU Cochin Port-Royal, Paris/France c: Unité 82 INSERM, Faculté de Médecine Xavier Bichat, Paris/France d: INRA LGPTA, Villeneuve d'Ascq/France e: Institut Jacques Monod, Laboratoire de Biologie Structurale du Noyau Cellulaire, Paris/France f: INSERM EPI 9922, CHU Bichat, Université Paris VII, Paris/France Eur. J. Cell Biol. (2001) 80, (6), 428-441 Abstract The multicellular development of the single celled eukaryote Dictyostelium discoideum is induced by starvation and consists of initial aggregation of the isolated amoebae, followed by their differentiation into viable spores and dead stalk cells. These stalk cells retain their structural integrity inside a stalk tube that support the spores in the fruiting body. Terminal differentiation into stalk cells has been shown to share several features with programmed cell death (Cornillon et al (1994), J. Cell Sci. 107, 2691- 2704). Here we report that, in the absence of aggregation and differentiation, D. discoideum can undergo another form of programmed cell death that closely ressembles apoptosis of most mammalian cells, involves mitochondrial transmembrane potential loss, phosphatidylserine surface exposure, and engulfment of dying cells by neighboring D. discoideum cells. This death has been studied by various techniques (light microscopy and scanning- or transmission-electron microscopy, flow cytometry, DNA electrophoresis), in two different conditions inhibiting D. discoideum multicellular development. The first one, corresponding to an induced unicellular cell death, was obtained by starving the cells in a "conditioned" cell-free buffer, prepared by previous starvation of another D. discoideum cell population in potassium phosphate buffer (pH 6.8). The second one, corresponding to death of D. discoideum after axenic growth in suspension, was obtained by keeping stationary cells in their culture medium. In both cases of these unicellular-specific cell deaths, microscopy revealed morphological features known as hallmarks of apoptosis for higher eukaryotic cells and apoptosis was further corroborated by flow cytometry. The occurrence in D. discoideum of programmed cell death with two different phenotypes, depending on its multicellular or unicellular status, is further discussed. ----------------------------------------------------------------------------- A non-Golgi a1,2-Fucosyltransferase that Modifies Skp1 in the Cytoplasm of Dictyostelium Hanke van der Wel@, Howard R. Morris§, Maria Panico§, Thanai Paxton§, Simon J. North§, Anne Dell§, J. Michael Thomsoný and Christopher M. West@ @ Dept. of Anatomy & Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235 USA, §Dept. of Biochemistry, Imperial College, London SW7 2AY United Kingdom J. Biol. Chem., in press Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose a1,2- linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDP-Fuc:Skp1 a1,2-fucosyltransferase. Tryptically-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker-PCR method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1-fucosylation, based on biochemical analysis of FT85-mutant cells and E. coli expressing FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known a1,2- fucosyltransferases, and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting family 2 glycosyltransferase domains, and its expression in FT85-mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote family 2 glycosyltransferases are membrane-bound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1- fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus. ----------------------------------------------------------------------------- Expression Patterns of Cell-type Specific Genes in Dictyostelium Negin Iranfar, Danny Fuller, Roman Sasik@, Terence Hwa@, Michael Laub* and William F. Loomis# Division of Biology, and @ Department of Physics, U.C.S.D. La Jolla, CA 92093 * Department of Cell and Developmental Biology, Stanford Medical School, Stanford, CA Molecular Biology of the Cell, in press Abstract Cell-type specific genes were recognized by interrogating microarrays carrying Dictyostelium gene fragments with probes prepared from fractions enriched in prestalk and prespore cells. Cell-type specific accumulation of mRNA from 17 newly identified genes was confirmed by Northern analyses. DNA microarrays carrying 690 targets were used to determine expression profiles during development. The profiles were fit to a biologically based kinetic equation to extract the times of transctription onset and cessation. While the majority of the genes that were cell type enriched at the slug stage were first expressed as the prespore and prestalk cells sorted out in aggregates, some were found to be expressed earlier before the cells had even aggregated. These early genes may have been initially expressed in all cells and then preferentially turned over in one or the other cell type. Alternatively, cell type divergence may start soon after the initiation of development. ----------------------------------------------------------------------------- The protein kinase YakA regulates G-protein linked signaling responses during growth and development of Dictyostelium Saskia van Es, Karin E. Weening, and Peter N. Devreotes Department of Cell Biology and Anatomy, Johns Hopkins University, School of Medicine, Baltimore, MD 21205 J. Biol. Chem. In press. Abstract A genetic screen for Dictyostelium mutants that phenotypically resemble cells lacking the G-protein b-subunit yielded the protein kinase YakA. Like gb null cells, yakA null cells fail to enter development and display slow growth on bacterial lawns. We created a temperature sensitive YakA mutant and showed that YakA activity is required not only at the onset but also during development. The yakA null cells have strong defects in folic acid induced responses, such as actin polymerization and cGMP accumulation, indicating that they play a role in G-protein mediated signaling responses. We propose that YakA acts downstream of G-proteins, because cAMP receptors still couple to G-proteins in the yakA mutant. In addition, the previously observed growth arrest induced by overexpression of YakA also occurs in gb mutants. We localized YakA-GFP to the cytosol suggesting that YakA may be a functional homologue of its mammalian counterparts Dyrk2 and Dyrk3, a subclass of Dual-specificity Yak Related Kinases (Dyrk) with unknown function. ----------------------------------------------------------------------------- Sphingosine-1-phosphate lyase has a central role in the development of Dictyostelium discoideum Guochun Li, Christopher Foote, Stephen Alexander and Hannah Alexander Division of Biological Sciences, University of Missouri, Columbia, MO 65211-7400 Development, in press. SUMMARY Sphingosine-1-phosphate, a product of sphingomyelin degradation, is an important element of signal transduction pathways regulating cell proliferation and cell death. In this work we demonstrate additional roles for sphingosine-1-phosphate in growth and multicellular development. The specific disruption in Dictyostelium discoideum of the sphingosine-1-phosphate lyase gene, which encodes the enzyme catalyzing sphingosine-1-phosphate degradation, results in a mutant strain with aberrant morphogenesis as well as an increase in viability during stationary phase. The absence of sphingosine-1-phosphate lyase affects multiple stages throughout development including the cytoskeletal architecture of aggregating cells, the ability to form migrating slugs, and the control of cell-type specific gene expression and terminal spore differentiation. This pleiotropic effect due to the loss of sphingosine-1-phosphate lyase establishes sphingolipids as pivotal regulatory molecules in a wide range of processes in multicellular development. ======================== Technical innovation ======================== [Editor's note: Although not yet in press, many of you may be interested in the following abstract of what promises to be a valuable technical advance] Making ts mutants in essential genes in Dictyostelium Mark S Bretscher and Chris Thompson, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England. We decided to try to make a ts mutant in an essential gene in Dictyostelium discoideum Ax2, and selected the single gene encoding NSF (the NEM-sensitive factor which appears to be required in all membrane fusion processes) that exists in Dicty (Weidenhaupt et al, 1998) as a target. Our strategy was basically to adapt the current procedure used for gene knockouts, hoping to replace the endogenous gene with one putatively encoding a temperature sensitive allele. There are several temperature-sensitive mutants in NSF in Drosophila and two of these, comatose 4 and 6, occur in highly conserved regions. We therefore constructed a Dicty NSF vector which contained, in the linear mutagenic portion: about 300bp upstream of the gene, the gene itself (including a 100 bp intron, ~2200bp in all) containing the comatose 4 site (marked with a BssHII site, at position1270 from the 5' end of the vector) or 6 site (marked with an AflII site, at position1640), followed by 200bp trailer, a blasticidin casette (1350bp) and a further 1300bp of 3' genomic sequence. These constructs were electroporated into Ax2 in the same manner as for gene knockouts. The cells were then parcelled out into wells and clones selected for drug resistance. These were recloned and grown up andthe DNA from 18 of each mutant subjected to Southern blotting. This showed that in each case, 17/18 clones had arisen by homologous recombination. In the case of comatose 4, none of the clones contained the desired mutation; conversely, in the case of comatose 6, all of them contained the desired site. Furthermore, in clones in which an in-frame termination codon had been placed just after the comatose 6 site, all 12 clones examined had recombined homologously, but none now carried the comatose 6 mutation. This shows that the gene is essential and suggests that when it contains the comatose 4 site, the encoded NSF is non-functional. The comatose 6 clones had, unfortunately, no new phenotype. We therefore made a mutagenic library in which the last 1000bp of coding sequence was made by pcr mutagenesis. Ax2 cells were transformed as before and positive wells recloned. About 1500 such clones were generated and each was screened for growth at 22°C and 26.5°C. From this, 11 ts mutants were recovered; they all arose by homologous recombination. Their properties are under study. Reference Weidenhaupt,M., Bruckert,F. and Satre, M. (1998) Gene 207, 53-60. We have been greatly assisted in this by David Traynor and, especially, Rob Kay. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 12]