Dicty News Electronic Edition Volume 19, number 2 July 27, 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. ======================= Positions Available ======================= A Postdoc position funded by the Deutsche Forschungsgemeinschaft (Epigenitcs Program) is available in the Genetics Group at Kassel University. The aims of the project are 1. to understand the mechanisms of RNA interference and 2. the mechanisms and function of DNA methylation. In both cases, Dictyostelium provides an excellent model system which is both "lean" (in terms of the small genome) and "general" (in that apparently all or most components found in higher eukaryotes also exist in Dictyostelium). Some more details are outlined on our web page (http://www.uni-kassel.de/fb19/genetics/projects/projects.html). Within the DFG Schwerpunkt Programm, we closely cooperate with other groups working on epigenetics in plants, flys, and mammals. Applicants should have obtained their Ph.D in a relevant field of molecular biology or biochemistry. Applications containing CV, a brief description of research experience, research interests and the names and e-mail address of two potential referees may be sent by e-mail to Nellen@hrz.uni-kassel.de A position for a Ph.D. student (funded by an OBF stipend) is available in the Genetics Group at Kassel University in the new laboratory of Christian Hammann. The project is aimed at understanding the role of different RNA directed RNA polymerases (RdRP) in posttranscriptional gene silencing. Biochemical, biophysical and molecular biology methods will be applied. The project will be carried out in close cooperation with the Epigenetics Program in the Genetics group and with the Center for Interdisciplinary Nanostructure Science and Technology (CINSaT). Applicants should have a diploma or equivalent qualification in biology, biochemistry or biophysics. Applications containing CV, a brief description of research experience, research interests and the names and e-mail address of two potential referees may be sent by e-mail to c.hammann@uni-kassel.de ============= Abstracts ============= A Rho GDP-dissociation inhibitor is involved in cytokinesis of Dictyostelium Keita Imai,a Toshirou Kijima,b,c Yoichi Noda,a Kazuo Sutoh,b Koji Yoda,a and Hiroyuki Adachi a,* a Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan and b Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 Japan *Corresponding author. Fax: +81-3-5841-8139. E-mail address: adachih@mail.ecc.u-tokyo.ac.jp (H. Adachi). c On leave from Department of Applied Chemistry, Kogakuin University, Shinjuku-ku, Tokyo 163-8677, Japan. Biochem. Biophys. Res. Commun. in press Abstract Homology searches toward the EST databases of Dictyostelium discoideum identified two putative Rho GDP-dissociation inhibitors (RhoGDIs), RhoGDI1 and RhoGDI2. In this study, the roles of RhoGDI1 in cytokinesis were examined. The RhoGDI1-null Dictyostelium strains produced by homologous recombination were viable but generated multinucleate giant cells in suspension culture, suggesting that RhoGDI1 is involved in cytokinesis. The expression of green fluorescent protein (GFP)-tagged RhoGDI1 complemented the defects of the RhoGDI1-null cells, and the GFP-RhoGDI1 is predominantly present in cytoplasm of the cell like yeast RhoGDI. Of fifteen Rho family GTPases in Dictyostelium currently known, Dictyostelium versions of Rac1 proteins (Rac1A, Rac1B and Rac1C) and RacE that are reportedly involved in Dictyostelium cytokinesis, showed two-hybrid interactions with RhoGDI1 as well as human and yeast Cdc42. These results suggest that RhoGDI1 is involved in cytokinesis of Dicytostelium through the regulation of Rho family GTPases Rac1s and/or RacE. ---------------------------------------------------------------------------- Defects in cytokinesis, actin reorganization and the contractile vacuole in cells deficient in RhoGDI Francisco Rivero(1), Daria Illenberger(2), Baggavalli P. Somesh(1), Heidrun Dislich(1), Nicola Adam(2) and Ann-Kathrin Meyer(1) (1)Institut fr Biochemie I, Medizinische Fakultt, University of Cologne. Joseph-Stelzmann-Strasse 52, D-50931 Kln, Germany (2)Department of Pharmacology and Toxicology, University of Ulm, Albert- Einstein-Allee 11, D-89081 Ulm, Germany EMBO J., in press Rho GDP-dissociation inhibitors (RhoGDIs) modulate the cycling of Rho GTPases between active GTP-bound and inactive GDP-bound states. We identified two RhoGDI homologues in Dictyostelium. GDI1 shares 51-58% similarity to RhoGDIs from diverse species. GDI2 is more divergent (40-44% similarity) and lacks the N-terminal regulatory arm characteristic for RhoGDI proteins. Both are cytosolic proteins and do not relocalize upon reorganization of the actin cytoskeleton. Using a two-hybrid approach we identified Rac1a/b/c, RacB, RacC and RacE as interacting partners for GDI1. Cells lacking GDI1 are multinucleate, grow slowly and display a moderate pinocytosis defect, but rates of phagocytosis are unaffected. Mutant cells present prominent actin- rich protrusions, and large vacuoles that are continuous with the contractile vacuole system. The actin polymerization response upon stimulation with cAMP was reduced but the motile behavior toward the chemoattractant was unaffected. Our results indicate that GDI1 plays a central role in the regulation of signal transduction cascades mediated by Rho GTPases. ---------------------------------------------------------------------------- Endosome fusion and microtubule-based dynamics in the early endocytic pathway of Dictyostelium Margaret Clarke1*, Jana Khler2, John Heuser3, and Gnther Gerisch2 1Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma; 2Max-Planck-Institut fr Biochemie, D82152 Martinsried, Germany; 3Washington University School of Medicine, St. Louis, Missouri. *Author for correspondence. E-mail: clarkem@omrf.ouhsc.edu Traffic, in press. Abstract Dictyostelium amoebae, like mammalian macrophages, take up fluid by macropinocytosis. The present study used fluorescent fluid phase markers and GFP-labeled microtubules to visualize the uptake, dynamics, and fusion of early endosomes in Dictyostelium. Consecutive labeling with two fluorescent fluid phase markers demonstrated that within the first few minutes after uptake, new macropinosomes underwent fusion with pre-existing endosomes. The fusing endosomes, which represent the mixing compartment, displayed extreme shape changes and rapid transport about the cell in association with microtubules. The great plasticity of endosomes at this stage of maturation was also evident by electron microscopy. The constant undulatory motion of microtubules was implemental in establishing contact with endosomes. Treatment of cells with agents that selectively disrupted either actin filaments or microtubules confirmed that endosome dynamics were microtubule-based. Further maturation of endosomes led to loss of pleiomorphy in favor of a spherical shape, inability to fuse with new macropinosomes, and diminished motility. ----------------------------------------------------------------------------- Evidence for a role for the Dictyostelium monomeric GTPase, Rap1, in cell viability and the response to osmotic stress Rujun Kang, Helmut Kae, Hermia Ip, George B. Spiegelman and Gerald Weeks Department of Microbiology and Immunology, University of British Columbia, 300 - 6174 University Blvd., Vancouver, BC V6T 1Z3, Canada J. Cell Science, in press. Summary The Dictyostelium genome contains a single gene, rapA, encoding a Rap1 monomeric G protein. Since attempts at generating rapA null Dictyostelium cells had been unsuccessful, expression of antisense RNA from the rapA gene under control of the folate repressible discoidin promoter was used to reduce cellular levels of the Rap1 protein. As Rap1 levels gradually decreased following antisense rapA RNA induction, growth rate and cell viability also decreased, a result consistent with the idea that rapA is an essential gene. The Rap1 depleted cells exhibited reduced viability in response to osmotic shock. The accumulation of cGMP in response to 0.4M sorbitol was reduced after rapA antisense RNA induction and was enhanced in cells expressing the constitutively activated Rap1(G12V) protein, suggesting a possible role for Rap1 in the generation of cGMP. Dictyostelium Rap1 was shown to complex to the Ras binding domain of RalGDS only when it was in a GTP-bound state. This assay was used to demonstrate that activation of Rap1 in response to 0.4M sorbitol occurred with initial kinetics similar to those observed for the accumulation of cGMP. Furthermore, the addition of 2mM EDTA to osmotically shocked cells, a treatment that enhances cGMP accumulation, also enhanced Rap1 activation. These results suggest a direct role for Rap1 in the activation of guanylyl cyclase during the response to hyperosmotic conditions. Rap1 was also activated in response to low temperature, but was not activated in response to low osmolarity or high temperature. ----------------------------------------------------------------------------- OSBPa, a predicted oxysterol binding protein of Dictyostelium, is required for regulated entry into culmination Masashi Fukuzawa and Jeffrey G. Williams* School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, DUNDEE, DD1 5EH, UK FEBS Letters, in press Abstract The oxysterol binding (OSBP) proteins are believed to control cholesterol homeostasis but their precise mechanism of action is not well understood. The Dictyostelium osbA gene encodes a predicted OSBP, OSBPa, which lacks the PH domain that in most other OSBPs directs targetting to the Golgi. OSBPa instead localises selectively to the cell periphery and also, in some cells, to the peri-nuclear region. OSBPa null strains form normal fruiting bodies but are defective in the regulation of the transition from slug migration to culmination. Thus a plasma membrane-enriched OSBP family member is essential for correct regulation of the slug-fruiting body switch. ----------------------------------------------------------------------------- [End Dicty News, volume 19, number 2]