CSM News Electronic Edition Volume 6, number 9 April 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 =========== Dictyopterin appears at amoebae emergence during spore germination in Dictyostelium Roger Klein 1, Irene Tatischeff 1* and David Cotter 2 (1) Laboratoire de Physicochimie Biomoleculaire et Cellulaire, CNRS URA 2056, Universite P. et M. Curie, 4, Place Jussieu, Case 138, 75252 Paris Cedex 05, France (2) Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B CA3P4, Canada Comptes Rendus de l'Academie des Sciences, in press Summary A pteridine pathway has been previously discovered, which functions during growth and aggregation of Dictyostelium discoideum cells . A natural isomer of L-biopterin was isolated from vegetative cells, identified and named dictyopterin. Its reduced form, tetrahydrodictyopterin, was the main intracellular form in Dictyostelium discoideum cells. Dictyolumazine, resulting from the enzymatic deamination of dictyopterin was identified as a major pteridine present in extracellular media of both vegetative cells and starved cells. Comparison of intracellular and extracellular pteridines during growth and aggregation leads one to assume that tetrahydrodictyopterin might be associated with growth. The present work was designed to complete the above observations by studying the levels of intracellular tetrahydrodictyopterin and extracellular dictyolumazine during spore germination. Dictyostelium discoideum, strain SG2 (ATCC 44841) was used because this variant autoactivates after about 2-3 days of aging post-culmination versus at least 14=CAdays for the wild-type. Two different methods of activation of SG2 spores were used: autoactivation or heat-shock preactivation. Dictyolumazine was found associated with a spore matrix, corresponding to the washing of 2-day-old dormant spores. The concentration of dictyolumazine in the matrix was about 0.4 nanomoles per 1x107 spores. During germination, no extracellular pteridine, neither dictyopterin, nor dictyolumazine could be detected, whatever the mode of activation. The limit of detection of dictyopterin was estimated as 1 picomole per 1x107 spores. Intracellular dictyopterin could neither be detected in dormant spores nor in unswollen or swollen activated spores. Synthesis of tetrahydrodictyopterin starts only with emergence of nascent amoebae during spore germination. Then, tetrahydrodictyopterin levels vary as a function of the percent emerged amoebae in spore suspension. These data are consistent with a requirement for tetrahydrodictyopterin during vegetative growth. Thus, this spore germination study underscores the fact that the reduced form of dictyopterin, already known to be associated with vegetative growth, is biosynthesized in nascent amobae. The absence of any extracellular dictyolumazine during spore germination and the biosynthesis of tetrahydrodictyopterin before initiation of a new cycle of growth emphasize the idea that tetrahydrodictyopterin itself might be one component of the programmed series of effectors needed for mitosis. Some biochemical functions, which could imply tetrahydrodictyopterin, are the matter of discussion. ------------------------------------------------------------- Isolation And Characterization Of A Novel Cytokinesis-Deficient Mutant In Dictyostelium discoideum. Kalpa Vithalani, J. Daniel Shoffner, and Arturo De Lozanne J. Cell. Biochem. in press Cytokinesis is a dramatic event in the life of any cell during which numerous mechanisms must coordinate the legitimate and complete mechanical separation into two daughter cells. We have used Dictyostelium discoideum as a model system to study this highly orchestrated event through genetic analysis. Transformants were generated using a method of insertional mutagenesis known as restriction enzyme mediated integration (REMI) and subsequently screened for defects in cytokinesis. Mutants isolated in a similar screen suffered a disruption in the myosin II heavy chain gene, a protein known to be essential for cytokinesisand in a novel gene encoding a rho-like protein termed racE (Larochelle et al., 1995) . In the screen reported here we isolated a third type of mutant, called 10BH2, which also had a complete defect in cytokinesis. 10BH2 mutant cells are able to propagate on tissue culture plates by fragmenting into smaller cells by a process known as traction-mediated cytofission. However, when grown in suspension culture, 10BH2 cells fail to divide and become large and multinucleate. Phenotypic characterization of the mutant cells showed that other cytoskeletal functions are preserved. The distribution of myosin and actin is identical to wild type cells. The cells can chemotax, phagocytose, cap crosslinked receptors and contract normally. However, the 10BH2 mutants are unable to complete the Dictyostelium developmental program beyond the finger stage. The mutant cells contain functional genes for myosin II heavy and light chains and the racE gene. Thus, based on these findings, we conclude that 10BH2 represents a novel cytokinesis-deficient mutant. ---------------------------------------------------------------------- A NOVEL MEMBER OF THE RHO FAMILY OF SMALL GTP-BINDING PROTEINS IS SPECIFICALLY REQUIRED FOR CYTOKINESIS Denis A. Larochelle, Kalpa Vithalani and Arturo De Lozanne J. Cell Biol. in press Several members of the rho/rac family of small GTP-binding proteins are known to regulate the distribution of the actin cytoskeleton in various subcellular processes. We describe here a novel rac protein, racE, which is specifically required for cytokinesis, an actomyosin-mediated process. The racE gene was isolated in a molecular-genetic screen devised to isolate genes required for cytokinesis in Dictyostelium. Phenotypic characterization of racE mutants revealed that racE is not essential for any other cell motility event, including phagocytosis, chemotaxis, capping, or development. Our data provide the first genetic evidence for the essential requirement of a rho-like protein specifically in cytokinesis and suggest a role for these proteins in coordinating cytokinesis with the mitotic events of the cell cycle. --------------------------------------------------------------------- A Two-Component Histidine Kinase Gene that Functions in Dictyostelium Development Nancy Wang, Gad Shaulsky, Ricardo Escalante and William F. Loomis Center for Molecular Genetics, Department of Biology University of California San Diego, La Jolla CA 92093 EMBO J. (in press) Summary A mutant which fails to complete development was isolated from a population of cells that had been subjected to insertional mutagenesis using Restriction Enzyme Mediated Integration (REMI). The disrupted gene, dhkA, encodes the conserved motifs of a histidine kinase as well as the response regulator domain. It is likely that the histidine in DhkA is autophosphorylated and the phosphate passed to one or more response regulators. Such two-component systems function in a variety of bacterial signal transduction pathways and have recently been characterized in yeast and Arabidopsis. In Dictyostelium we find that DhkA functions both in the regulation of prestalk gene expression and in the control of terminal differentiation of prespore cells. ---------------------------------------------------------------------- ============ Correction ============ The following abstract, which appeared in the last issue of the newsletter (v6, #8), somehow became garbled in transmission. The following is corrected version. Myosin-based Cortical Tension in Dictyostelium Resolved into Heavy and Light Chain-Regulated Components. Thomas T. Egelhoff, Teresa V. Naismith, and Frank V. Brozovich Department of Physiology and Biophysics Case Western Reserve School of Medicine Cleveland, OH 44106-4970 J. Muscle Research and Cell Motility, in press Summary Cortical tension in most nonmuscle cells is due largely to force production by conventional myosin (myosin II) assembled into the cytoskeleton. Cytoskeletal contraction in smooth muscle and nonmuscle cells is influenced by the degree of myosin filament assembly, and by activation of myosin motor function via regulatory light chain (RLC) phosphorylation. Recombinant Dictyostelium discoideum cell lines have been generated bearing altered myosin heavy chains, resulting in either constitutive motor function or constitutive assembly into the cytoskeleton. Analysis of these cells allowed stiffening responses to agonists, measured on single cells, to be resolved into an RLC-mediated component reflecting activation of motor function, and a myosin heavy chain (MHC) phosphorylation-regulated component reflecting assembly of filaments into the cytoskeleton. These two components can account for all of the cortical stiffening response seen during tested in vivo contractile events. ---------------------------------------------------------------------- [End CSM-News, volume 6, number 9]