Dicty News Electronic Edition Volume 22, number 4 February 13, 2004 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Chimeric analysis of the small GTPase RacE in cytokinesis signaling in Dictyostelium discoideum Madhavi Agarwal, Nicholas A Guerin, Denis A Larochelle Department of Biology, Clark University, Worcester, MA 01610, USA. Experimental Cell Research, in press RacE is a small GTPase required for cytokinesis in Dictyostelium discoideum. To investigate RacEās potential binding and signaling interfaces that allow its function in cytokinesis, 10 different chimeras were created between RacE and the closely related small GTPase, RacC. RacE/RacC chimeras, containing various combinations of four RacE regions, EI-IV: E-I (aa 1-67), E-II (aa 68-124), E-III (aa 125-184), and E-IV (aa 185-224), were tested in their ability to rescue the multinucleated, cytokinesis-defective phenotype of RacE null cells grown in suspension. Regions E-II and E-IV were essential but not sufficient for the rescue of RacE null cells. These two regions, in combination with either region E-I or E-III, resulted in rescue. Results presented here suggest that region E-II contains a crucial, yet incomplete, binding site. Regions E-I or E-III separately provide additional, necessary elements for RacEās function. The extended E tail of RacE (E-IV) may act as a Īsensorā of the bound nucleotide state of RacE and facilitate GDP to GTP exchange (possibly through interactions with a GEF molecule), thereby resulting in activation of RacE. This study provides new evidence for small GTPases engaging several distinct protein interfaces to mediate signaling in various cellular processes. Submitted by: Madhavi Agarwal [magarwal@clarku.edu] ----------------------------------------------------------------------------- Global change in Escherichia coli gene expression in initial stage of symbiosis with Dictyostelium cells Shin-Ichi Matsuyama1*, Chikara Furusawa2*, Masahiko Todoriki3, Itaru Urabe3 and Tetsuya Yomo1,,3,4,5 * Both authors equally contributed to this work. 1 Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 2-1 Yamada-oka, Suita City, Osaka 565-0871 Japan 2 Center for Developmental Biology, The Institute of Physical and Chemical Research (RIKEN), 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, JAPAN 3 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita City, Osaka 565-0871 Japan 4 Intelligent Cooperation and Control Project, PRESTO, JST, 2-1 Yamada-oka, Suita City, Osaka 565-0871 Japan 5 Department of Pure and Applied Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo, 153-8902 Japan Biosystems, in press Genome-wide gene expression profiling was performed to investigate the early formation of symbiosis using an artificial symbiosis of Escherichia coli and Dictyostelium discoideum. We have previously reported that when these two species were allowed to grow on minimal agar plates, they achieved a stable state of coexistence, in which the emerging E. coli colonies housing Dictyostelium cells were of a mucoidal nature that was not observed originally. We used this microbiological system as a model to study the initial stages of the development of the symbiotic relationship. The E. coli gene expression profiles of symbiotic cells and non-symbiotic cells captured using GeneChip technology were compared. It was clearly shown that the gene expression profile was substantially altered in E. coli cells undergoing symbiotic transition. The genes responsible for central energy metabolism as well as those responsible for translation apparatus were down-regulated in symbiotic E. coli. The transcriptional patterns of genes coding for the E. coli cell surface structure were drastically altered, and this alteration may determine the mucoidal nature and unique structure of coexisting colonies. General stress inducible genes were expressed at low levels in symbiotic E. coli. These observed changes in the transcription profile indicate that the central metabolism of symbiotic E. coli is attenuated as a whole, and the cells are probably under less stress because of the benefits brought by coexistence with the symbiotic counterpart Dictyostelium. Submitted by: Shin-Ichi Matsuyama [sxm67@bio.eng.osaka-u.ac.jp] ----------------------------------------------------------------------------- Structure-Function Analysis of the Beach Protein LvsA Wei-I Wu, Juhi Yajnik, Michael Siano, and Arturo De Lozanne Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712. Traffic, in press ABSTRACT Most eukaryotes have several members of the BEACH family of proteins but the molecular function of these large proteins remains unknown. The Dictyostelium BEACH protein LvsA is essential for cytokinesis and contractile vacuole activity. The functional contribution of different portions of LvsA was tested here by deletion analysis. The C-terminal WD domain was important for protein stability and C-terminal deletions resulted in loss of LvsA function. In contrast, N-terminal deletions yielded abundant protein expression that could be assayed for function. Despite very low sequence conservation of the N-terminal portion of LvsA, this region is important for its function in vivo. Deletion of 689 N-terminal amino acids produced a protein that was functional in cytokinesis but partially functional in osmoregulation. Further deletions resulted in the complete loss of LvsA function. Using in vitro fractionation assays we found that LvsA sedimented with membranes but this association does not require the N-terminal portion of LvsA. Interestingly, the association of LvsA with the contractile vacuole was perturbed by the loss of drainin, a protein important for vacuole function. In drainin-null cells LvsA bound irreversibly to engorged contractile vacuoles that fail to expel water. These experiments help delineate the biochemical and physiological requirements for function of one important BEACH protein, LvsA. Submitted by: Arturo De Lozanne [a.delozanne@mail.utexas.edu] ----------------------------------------------------------------------------- RasC Plays a Role in the Transduction of Temporal Gradient Information in the cAMP Wave of Dictyostelium Deborah Wessels1, Rebecca Brincks1, Spencer Kuhl1, Vesna Stepanovic1, Karla J. Daniels1, Gerald Weeks2, Chinten J. Lim2, George Spiegelman2, Danny Fuller3, Negin Iranfar3, William F. Loomis3 and David R. Soll1 W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, The University of Iowa, Iowa City, IA 522421; Department of Microbiology and Immunology, The University of British Columbia, Vancouver, British Columbia2; Department of Biology, University of California, San Diego, La Jolla, California 920393 Eukaryotic Cell, in press To define the role RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC -, was analyzed in buffer and in response to the individual spatial, temporal and concentration components of a natural cAMP wave, using computer-assisted 2D and 3D motion analysis systems. These quantitative studies reveal that rasC - cells translocate at the same velocity and chemotax up spatial gradients of cAMP with the same efficiency as control cells. However, rasC - cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of attractant. rasC - cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and back of a wave. These defects result in the inability of rasC - cells to chemotax in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in higher levels of coritcal F-actin in both buffer and the front of the wave, and the failure of myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. The behavioral defect in the front of the wave is similar to that of the myosin I double mutant myoA -/myoF -, while the behavioral and cytoskeletal defects in the back of the wave are similar to those of the myosin II regulatory light chain phosphorylation mutant S13A. Expression array data support the premise that the behavioral defects exhibited by the rasC - mutant are the immediate result of the absence of RasC function. Submitted by: Deborah Wessels [deborah-wessels@uiowa.edu] ----------------------------------------------------------------------------- Identification of genes dependent on the MADS-boxtranscription factor SrfA in Dictyostelium development R. Escalante, N. Iranfar, L. Sastre, and W.F. Loomis Eukaryotic Cell, in press Analysis of microarrays containing 6345 Dictyostelium discoideum genes has identified twenty one whose expression is dependent on the MADS-box transcription factor SrfA. In wild type cells, all these genes are induced late in development. At least four of them are necessary for proper spore differentiation, stability and/or germination. Submitted by: Leandro Sastre [lsastre@iib.uam.es] =============================================================================== [End Dicty News, volume 22, number 4]