Dicty News Electronic Edition Volume 19, number 4 August 10, 2002 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 ============= MECHANISTICS OF AMOEBOID LOCOMOTION: SIGNAL TO FORCES Yoshio Fukui Cell Biology International, accepted for publication, 08/08/2002 Dictyostelium serves as an ideal model system for studying the molecular and structural properties of the actomyosin and microtubule systems. This organism also has been the vehicle on which the gene-targeting technique was pioneered. Dictyostelium also represents a small number of organisms whose chemotactic ligand-receptor mechanism has been well characterized. This article reviews recent advances in studies of the actin-based cytoskeletal system in Dictyostelium, focusing on the mechanistic aspects of the amoeboid motion. Special emphasis is placed on the recently identified cell-substrate-anchoring structures eupodia , and the measurement of single-cell migration forces. The recent advances in signal transduction cascade is also discussed with relevance to the mechanistics in amoeboid locomotion. ----------------------------------------------------------------------------- Identification of a novel type of cGMP phosphodiesterase that is defective in the chemotactic stmF mutants Marcel E. Meima, Ricardo M. Biondi and Pauline Schaap. School of Life Sciences, University of Dundee, MSI/WTB complex, Dow Street, Dundee DD1 5EH, UK. Mol. Biol. Cell, in press. ABSTRACT StmF mutants are chemotactic mutants that are defective in a cGMP phosphodiesterase (PDE) activity. We identified a novel gene, PdeD, which harbours two cyclic nucleotide binding domains and a metallo-$-lactamase homology domain. Similar to stmF mutants, pdeD null mutants displayed extensively streaming aggregates, prolonged elevation of cGMP levels after chemotactic stimulation and reduced cGMP-PDE activity. PdeD transcripts were lacking in stmF mutant NP377, indicating that this mutant carries a PdeD lesion. Expression of a PdeD-YFP fusion protein in pdeD null cells restored the normal cGMP response and showed that PdeD resides in the cytosol. When purified by immunoprecipitation, the PdeD-YFP fusion protein displayed cGMP-PDE activity, which was retained in a truncated construct that contained only the metallo-$-lactamase domain. ----------------------------------------------------------------------------- A secreted cell-number counting factor represses intracellular glucose levels to regulate group size in Dictyostelium Wonhee Jang, Binh Chiem, and Richard H. Gomer Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005-1892 Journal of Biological Chemistry, in press Summary Developing Dictyostelium cells form evenly sized groups of ~2 x 104 cells. A secreted 450 kDa protein complex called counting factor (CF) regulates group size by repressing cell-cell adhesion and myosin polymerization, and increasing cAMP-stimulated cAMP production, actin polymerization, and cell motility. We find that CF regulates group size in part by repressing internal glucose levels. Transformants lacking bioactive CF and wild-type cells with extracellular CF depleted by antibodies have high glucose levels, while transformants oversecreting CF have low glucose levels. A component of CF, countin, affects group size in a manner similar to CF, and a 1-minute exposure of cells to countin decreases glucose levels. Adding 1 mM exogenous glucose negates the effect of high levels of extracellular CF on group size and mimics the effect of depleting CF on glucose levels, cell- cell adhesion, cAMP pulse size, actin polymerization, myosin assembly, and motility. These results suggest that glucose is a downstream component in part of the CF signaling pathway, and may be relevant to the observed role of the insulin pathway in tissue size regulation in higher eukaryotes. ----------------------------------------------------------------------------- Identification and Characterization of Two Unusual cGMP-stimulated Phoshodiesterases in Dictyostelium Leonard Bosgraaf, Henk Russcher, Helena Snippe, Sonya Bader, Joyce Wind, and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen,Nijenborgh 4, 9747 AG Groningen, the Netherlands Molecular Biology of the Cell, in press Recently we recognized two genes, gbpA and gbpB, encoding putative cGMP- binding proteins with a Zn2+-hydrolase domain and two cyclic nucleotide binding (cNB) domains. The Zn2+-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously, and shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and appears to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP about 9-fold faster than cGMP, and is activated by cAMP and cGMP with a Ka of about 0.7 and 2.3 m M, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels, and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn2+-hydrolase domain, while the cNB domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP-activation. ----------------------------------------------------------------------------- TEMPERATURE-SENSITIVE INHIBITION OF DEVELOPMENT IN DICTYOSTELIUM DUE TO A POINT MUTATION IN THE piaA GENE Barbara Pergolizzi, Barbara Peracino, James Silverman#, Adriano Ceccarelli, Angelika Noegel*, Peter Devreotes# and Salvatore Bozzaro ABSTRACT The Dictyostelium mutant HSB1 is temperature-sensitive for development, undergoing aggregation and fruiting body formation at temperatures below 18C but not above. In vivo G protein-linked adenylylcyclase activation is defective in HSB1 and the enzyme is not stimulated in vitro by GTPgS; stimulation is restored upon addition of wild-type cytosol. Transfection with the gene encoding the cytosolic regulator PIA rescued the mutant. We excluded the possibility that HSB1 cells fail to express PIA and show that the HSB1 piaA gene harbours a point mutation, resulting in the aminoacid exchange G917D. Both wild-type and HSB1 cells were also transfected with the HSB1 piaA gene. The piaAHSB1 gene product displayed a partial inhibitory effect on wild-type cell development. We hypothesize that PIA couples the heterotrimeric G protein to adenylyl cyclase via two binding sites, one of which is altered in a temperature-sensitive way by the HSB1 mutation. When overexpressed in the wild-type background, PIAHSB1 competes with wild-type PIA via the non-mutated binding site, resulting in dominant-negative inhibition of development. Expression of GFP-fused PIA shows that PIA is homogeneously distributed in the cytoplasm of chemoctically moving cells. ----------------------------------------------------------------------------- [End Dicty News, volume 19, number 4]