Dicty News Electronic Edition Volume 20, number 6 March *, 2003 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 ============= RacB regulates cytoskeletal function in Dictyostelium Eunkyung Lee1+, David J. Seastone2, Ed Harris2 James A. Cardelli2 and David A. Knecht1* 1Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269 2Department of Microbiology and Immunology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130 *to whom correspondence should be addressed phone-860-486-2200 fax- 860-486-4331 Email knecht@uconn.edu Eukaryotic Cell, in press ABSTRACT Thus far 14 homologues of mammalian Rac proteins have been identified in Dictyostelium. It is unclear whether each of these genes has a unique function or to what extent they play redundant roles in actin cytoskeletal organization. In order to investigate the specific function of RacB, we have conditionally expressed wild type (WT-RacB), dominant negative (N17-RacB) and constitutively activated (V12-RacB) versions of the protein. Upon induction, cells expressing V12-RacB stopped growing, detached from the surface and formed numerous spherical surface protrusions, while cells overexpressing WT-RacB became flattened on the surface. In contrast, cells overexpressing N17-RacB did not show any significant morphological abnormalities. The surface protrusions seen in V12-RacB cells appear to be actin driven protrusions because they were enriched in F-actin and were inhibitable by cytochalasin A treatment. The protrusions in V12-RacB cells did not require myosin II activity, which distinguishes them from blebs formed by wild type cells under stress. Finally, we examined the functional consequences of expression of wild type and mutant RacB. Phagocytosis, endocytosis and fluid phase efflux rates were reduced in all cell-lines expressing RacB proteins but the greatest decrease was observed for cells expressing V12-RacB. From these results, we conclude that like other members of the Rho family, RacB induces polymerization of actin, but the consequences of activation appears to be different from other Dictyostelium Rac proteins so far investigated, resulting in different morphological and functional changes in cells. ---------------------------------------------------------------------------- A STAT regulated, stress-induced signalling pathway in Dictyostelium (Dictyostelium STAT and the stress response) Tsuyoshi Araki*, Masatsune Tsujioka*, Tomoaki Abe, Masashi Fukuzawa, Marcel Meima, Pauline Schaap, Takahiro Morio+, Hideko Urushihara+, Mariko Katoh+, Mineko Maeda^, Yoshimasi Tanaka+, Ikuo Takeuchi@ and Jeffrey G. Williams School of Life Sciences, University of Dundee, Wellcome Trust Biocentre Dow Street, DUNDEE, DD1 5EH, UK + Institute of Biological Sciences, University of Tsukuba,Tsukuba, Ibaraki 305-8572, JAPAN ^Department of Biology, Osaka University, Machikaneyama 1-16, Toyonaka, Osaka 560-0043 JAPAN @Novartis Foundation (Japan) for the Promotion of Science, Roppongi, Minato-ku, Tokyo 106-0032, JAPAN *These authors contributed equally to this work Author for correspondence (e-mail address j.g.williams@dundee.ac.uk) J. Cell Science, in press. SUMMARY The Dictyostelium stalk cell inducer DIF directs tyrosine phosphorylation and nuclear accumulation of the STAT (Signal Transducer and Activator of Transcription) protein Dd-STATc. We show that hyper-osmotic stress, heat shock and oxidative stress also activate Dd-STATc. Hyper-osmotic stress is known to elevate intracellular cGMP and cAMP levels and the membrane permeant analogue 8-bromo-cGMP rapidly activates Dd-STATc, while 8-bromo- cAMP is a much less effective inducer. Surprisingly, however, Dd-STATc remains stress activatable in null mutants for components of the known cGMP-mediated and cAMP-mediated stress-response pathways and in a double mutant affecting both pathways. Also, Dd-STATc null cells are not abnormally sensitive to hyper-osmotic stress. Micro-array analysis identified two genes, gapA and rtoA, that are induced by hyper-osmotic stress. Osmotic stress induction of gapA and rtoA is entirely dependent upon Dd-STATc. Neither gene is inducible by DIF but both are rapidly inducible with 8-bromo-cGMP. Again, 8-bromo-cAMP is a much less potent inducer than 8-bromo-cGMP. These data show that Dd-STATc functions as a transcriptional activator in a stress response pathway and the pharmacological evidence, at least, is consistent with cGMP acting as second messenger. ---------------------------------------------------------------------------- Synergistic control of cellular adhesion by TM9 proteins Mohammed Benghezal, Sophie Cornillon, Leigh Gebbie, Laeticia Alibaud, Franz Brckert, Franois Letourneur and Pierre Cosson Molecular Biology of the Cell (in press) ABSTRACT The transmembrane 9 (TM9) family of proteins contains numerous members in eukaryotes. Although their function remains essentially unknown in higher eukaryotes, the Dictyostelium discoideum Phg1a TM9 protein was recently reported to be essential for cellular adhesion and phagocytosis. Here the function of Phg1a and of a new divergent member of the TM9 family called Phg1b was further investigated in Dictyostelium discoideum. The phenotypes of PHG1a, PHG1b and PHG1a/PHG1b double knockout cells revealed that Phg1a and Phg1b proteins play a synergistic but not redundant role in cellular adhesion, phagocytosis, growth and development. Complementation analysis supports a synergistic regulatory function rather than a receptor role for Phg1a and Phg1b proteins. Altogether these results suggest that Phg1 proteins act as regulators of cellular adhesion, possibly by controlling the intracellular transport in the endocytic pathway and the composition of the cell surface. submitted by Benghezal@athelas.com ---------------------------------------------------------------------------- Dictyostelium discoideum transformation by oscillating electric field electroporation Laeticia Alibaud, Pierre Cosson, and Mohammed Benghezal Biotechniques (in press) ABSTRACT Dictyostelium discoideum has been used as a genetically tractable model organism to study many biological phenomena. High efficiency transformation is a prerequisite for successful genetic screens such as mutant complementation, identification of suppressor genes or insertional mutagenesis. Although exponential decay electroporation is the standard transformation technique for Dictyostelium discoideum, its efficiency is relatively low and its reproducibility weak. In this study we optimized the oscillating electroporation technique for Dictyostelium discoideum transformation and compared it to the exponential decay electroporation. A twenty-fold increase of the efficiency was reproducibly achieved. This alternative electroporation technique should facilitate future genetic approaches in Dictyostelium discoideum. submitted by Benghezal@athelas.com ---------------------------------------------------------------------------- [End Dicty News, volume 20, number 6]