dictyNews Electronic Edition Volume 30, number 2 January 11, 2008 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= A first glimpse at the transcriptome of Physarum polycephalum 1*Gernot Gloeckner, 2Georg Golderer, 2Gabriele Werner-Felmayer, 3Sonja Meyer, 3Wolfgang Marwan 1Leibniz Institute for Age Research - Fritz Lipmann Institute, Beutenbergstr. 11, D-07745 Jena, Germany 2Sektion für Biologische Chemie, Biozentrum der Medizinischen Universitaet Innsbruck, Fritz-Pregl-Str 3, A-6020 Innsbruck, Austria 3Magdeburg Centre for Systems Biology, Otto von Guericke University and Max-Planck-Institut fuer Dynamik komplexer technischer Systeme, Sandtorstr. 1, D-39106 Magdeburg, Germany *corresponding author BMC Genomics, in press Background Physarum polycephalum, an acellular plasmodial species belongs to the amoebozoa, a major branch in eukaryote evolution. Its complex life cycle and rich cell biology is reflected in more than 2500 publications on various aspects of its biochemistry, developmental biology, cytoskeleton, and cell motility. It now can be genetically manipulated, opening up the possibility of targeted functional analysis in this organism. Methods Here we describe a large fraction of the transcribed genes by sequencing a cDNA library from the plasmodial stage of the developmental cycle. Results In addition to the genes for the basic metabolism we found an unexpected large number of genes involved in sophisticated signaling networks and identified potential receptors for environmental signals such as light. In accordance with the various developmental options of the plasmodial cell we found that many P. polycephalum genes are alternatively spliced. Using 30 donor and 30 acceptor sites we determined the splicing signatures of this species. Comparisons to various other organisms including Dictyostelium, the closest relative, revealed that roughly half of the transcribed genes have no detectable counterpart, thus potentially defining species specific adaptations. On the other hand, we found highly conserved proteins, which are maintained in the metazoan lineage, but absent in D. discoideum or plants. These genes arose possibly in the last common ancestor of Amoebozoa and Metazoa but were lost in D. discoideum. Conclusion This work provides an analysis of up to half of the protein coding genes of Physarum polycephalum. The definition of splice motifs together with the description of alternatively spliced genes will provide a valuable resource for the ongoing genome project. Submitted by: Gernot Gloeckner [gernot@fli-leibniz.de] -------------------------------------------------------------------------------- Rap1 activation in response to cAMP occurs downstream of Ras activation during Dictyostelium aggregation Parvin Bolourani, George B. Spiegelman and Gerald Weeks Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada Running head: Role of Ras in chemotaxis and cAMP relay Correspondence to: Gerald Weeks, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada. Tel: 604-822-6649 ; Fax: 604-822-6041 ; E-mail: gerwee@interchange.ubc.ca J. Biol. Chem., in press We have used a doubly disrupted rasC-/rasG- strain of Dictyostelium discoideum, that ectopically expresses the carA gene, to explore the relationship between the activation of RasC and RasG, the two proteins that are necessary for optimum cAMP signaling, and the activation of Rap1, a Ras subfamily protein, that is also activated by cAMP. The ectopic expression of carA restored early developmental gene expression to the rasC-/rasG- strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through both the cAMP chemotactic pathway and the adenylyl cyclase activation pathway in the rasC-/rasG-/[act15]:carA strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The position of Rap1 in the signal transduction cascade was clarified by the finding that Rap1 activation was totally abolished in rasC-/rasG-/[act15]:carA and rasG- cells, but only slightly reduced in rasC- cells. Rap1 activation, therefore, occurs downstream of the Ras proteins and predominantly, if not exclusively, downstream of RasG. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC-/rasG-/[act15]:carA strain identifies RasG/RasC as the presumptive monomeric GTPases required for this activation. Submitted by: Gerald Weeks [gerwee@interchange.ubc.ca] -------------------------------------------------------------------------------- Dictyostelium MEGAPs: F-BAR domain proteins that regulate motility and membrane tubulation in contractile vacuoles Robert JW Heath1* and Robert H Insall1,2 1 School of Biosciences, University of Birmingham, Edgbaston, Birmingham, England, B15 2TT, UK 2The Beatson Institute for Cancer Research, Glasgow, Scotland, G61 1BD *Corresponding author PCH family proteins are fundamentally important proteins, linking membrane curvature events with cytoskeletal reorganisation. One important group, the MEGAPs (also called srGAPs and WRPs) contain RhoGAP domains in addition to the F-BAR domain. We have disrupted MEGAP1 and MEGAP2 in Dictyostelium both singly and in combination. We find a strong cytoskeletal phenotype in MEGAP1- cells and a subtle phototaxis defect in MEGAP2- slugs. MEGAP1-/2- cells have an overabundance of filopodia and serious effects in motility and slug function. The most dramatic changes, however, are on contractile vacuoles. MEGAP1-/2- cells empty their contractile vacuoles less efficiently than normal and consequently have three times the usual number. GFP tagged MEGAP1 localizes to tubules of the contractile vacuole network, when vacuoles start to empty they recruit cytosolic GFP-MEGAP1. Mutants in the Saccharomyces homologues RGD1 and RGD2 also show abnormal vacuoles, implying a role conserved through evolution. Thus MEGAP is an important regulator for the contractile vacuole network, and we propose that tubulation of the contractile vacuole by MEGAP1 represents a novel mechanism for driving vacuole emptying. Submitted by: Robert Heath [rjh451@bham.ac.uk] -------------------------------------------------------------------------------- Facultative cheater mutants reveal the genetic complexity of cooperation in social amoebae Lorenzo A. Santorelli, Christopher R. L. Thompson2, Elizabeth Villegas, Jessica Svetz, Christopher Dinh, Anup Parikh, Richard Sucgang, Adam Kuspa, Joan E. Strassmann, David C. Queller and Gad Shaulsky Nature, in press Cooperation is central to many major transitions in evolution, including the emergence of eukaryotic cells, multicellularity and eusociality1. Cooperation can be destroyed by the spread of cheater mutants that do not cooperate but gain the benefits of cooperation from others. However, cooperation can be preserved if cheaters are facultative, cheating others but cooperating among themselves. Several cheater mutants have been studied before, but no study has attempted a genome-scale investigation of the genetic opportunities for cheating. Here we describe such a screen in a social amoeba and show that cheating is multifaceted by revealing cheater mutations in well over 100 genes of diverse types. Many of these mutants cheat facultatively, producing more than their fair share of spores in chimeras, but cooperating normally when clonal. These findings indicate that phenotypically stable cooperative systems may nevertheless harbor genetic conflicts. The opportunities for evolutionary moves and countermoves in such conflicts may select for the involvement of multiple pathways and numerous genes. Submitted by: Gad Shaulsky [gadi@bcm.tmc.edu] -------------------------------------------------------------------------------- Abi Mutants in Dictyostelium Reveal Specific Roles for the SCAR/WAVE Complex in Cytokinesis Alice Y. Pollitt & Robert H. Insall School of Biosciences, University of Birmingham, and Beatson Institute for Cancer Research, Glasgow Current Biology, in press Actin polymerisation drives multiple cell processes involving movement and shape change. SCAR/WAVE proteins connect signalling to actin polymerization through the activation of the Arp2/3 complex. SCAR/WAVE is normally found in a complex with four other proteins, PIR121, Nap1, Abi2 and HSPC300 (supplementary Figure 1A) [1-3]. However, there is no consensus as to whether the complex functions as an unchanging unit or if it alters its composition in response to stimulation as originally proposed by Eden et al. [1]. It is also unclear wither complex members exclusively regulate SCAR/WAVEs, or if they have additional targets [4-6]. Here we analyze the roles of the unique Dictyostelium Abi. We find that abiA null mutants show less severe defects in motility than scar null cells, indicating – unexpectedly - that SCAR retains partial activity in the absence of Abi. Furthermore, abiA null mutants have a serious defect in cytokinesis, which is not seen in other SCAR complex mutants, and is only seen when SCAR itself is present. Detailed examination reveals that normal cytokinesis requires SCAR activity, apparently regulated through multiple pathways. Submitted by: Robert Insall [r.insall@beatson.gla.ac.uk] -------------------------------------------------------------------------------- Four key signaling pathways mediating chemotaxis in Dictyostelium. Douwe M. Veltman, Ineke Keizer-Gunnik and Peter J.M. Van Haastert J. Cell Biology, in press Chemotaxis is the ability of cells to move in the direction of an external gradient of signaling molecules. Cells are guided by actin-filled protrusions in the front, while myosin filaments retract the rear of the cell. Previous work demonstrated that chemotaxis of unpolarized amoeboid Dictyostelium cells is mediated by two parallel pathways, PI3K and PLA2. Here we show that polarized cells exhibit very good chemotaxis with inhibited PI3K and PLA2 activity. Using genetic screens we demonstrate that this activity is mediated by a soluble guanylyl cyclase (sGC), providing two signals: the protein localizes to the leading edge where it interacts with actin filament, while the cGMP product induces myosin filaments in the rear of the cell. We conclude that chemotaxis is mediated by multiple signaling pathways regulating protrusions at the front and rear of the cell. Cells that express only rear activity are polarized but do not exhibit chemotaxis, while cells with only front signaling are unpolarized but do chemotax. Submitted by: Peter J.M. Van Haastert [p.j.m.van.haastert@rug.nl] -------------------------------------------------------------------------------- Altered composition and secretion of lysosome-derived compartments in Dictyostelium AP-3 mutant cells. Steve J. Charette(1)* and Pierre Cosson(2) (1)Centre de recherche en cancerologie de l'Universite Laval, L'Hotel-Dieu de Quebec, Centre hospitalier universitaire de Quebec, 9, rue McMahon, Quebec, Canada G1R 2J6 (2)Universite de Geneve, Centre Medical Universitaire, Departement de Physiologie cellulaire et metabolisme, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland. *Corresponding author TRAFFIC, in press Genetic alteration of the AP-3 complex is responsible for the type 2 Hermansky-Pudlak syndrome (HPS2), a lysosomal storage disease similar to the Chediak-Higashi syndrome (CHS). AP-3 presumably participates in the biogenesis of late endosomal compartments, and may also be critical for the regulated secretion of lysosomes by specialized cells. Here, Dictyostelium discoideum cells defective for the µ3 subunit of the AP-3 complex were used and their phenotype analyzed. In µ3 mutant cells, endosomal maturation and lysosome secretion were markedly slower than in wild-type cells. This phenotype is similar to that reported previously in lvsB mutant cells, where the ortholog of the LYST gene, involved in CHS, is mutated. Detailed analysis revealed however significant differences between these two isogenic mutant cells: in lvsB mutant cells the primary defect is an inefficient biogenesis of otherwise normal secretory lysosomes, while in µ3 mutant cells the biogenesis, and also the composition and the fusion properties of secretory lysosomes are affected. These results suggest that in D. discoideum, AP-3 controls both the efficiency and the specificity of post-lysosome maturation, which represent two critical elements in the control of lysosome secretion. Submitted by: Steve Charette [steve.charette@crhdq.ulaval.ca] ============================================================== [End dictyNews, volume 30, number 2]