dictyNews Electronic Edition Volume 28, number 5 March 2, 2007 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 ========= Diverse Cytopathologies in Mitochondrial Disease Are Caused by AMPK Signalling Paul B. Bokko1, Lisa Francione1, Esther Bandala-Sanchez1, Afsar U. Ahmed1, Sarah J. Annesley1, Xiuli Huang2, Taruna Khurana2, Alan R. Kimmel2 and Paul R. Fisher1* 1Department of Microbiology, La Trobe University, Victoria 3086, Australia. 2National Institutes of Health, Bethesda, Maryland MD20892, USA. Molecular Biology of the Cell, in press The complex cytopathology of mitochondrial diseases is usually attributed to insufficient ATP. AMP-activated protein kinase (AMPK) is a highly sensitive cellular energy sensor that is stimulated by ATP-depleting stresses. By antisense-inhibiting chaperonin 60 expression we produced mitochondrially diseased strains with gene dose-dependent defects in phototaxis, growth and multicellular morphogenesis. Mitochondrial disease was phenocopied in a gene dose-dependent manner by overexpressing a constitutively active AMPK alpha subunit (AMPKalphaT). The aberrant phenotypes in mitochondrially diseased strains were suppressed completely by antisense-inhibiting AMPKalpha expression. Phagocytosis and macropinocytosis, although energy-consuming, were unaffected by mitochondrial disease and AMPKalpha expression levels. Consistent with AMPKÕs role in energy homeostasis, mitochondrial "mass" and ATP levels were reduced by AMPKalpha antisense inhibition and increased by AMPKalphaT overexpression, but near normal in mitochondrially diseased cells. We also found that AICAR, a pharmacological AMPK activator in mammalian cells, mimics mitochondrial disease in impairing Dictyostelium phototaxis and that AMPKalpha antisense-inhibited cells were resistant to this effect. The results show that diverse cytopathologies in Dictyostelium mitochondrial disease are caused by chronic AMPK signalling not by insufficient ATP. Submitted by Paul R. Fisher [P.Fisher@latrobe.edu.au] -------------------------------------------------------------------------------- Role of PI3 kinases in Chemotaxis in Dictyostelium Kosuke Takeda, Atsuo T. Sasaki, Hyunjung Ha, Hyun-A Seung, and Richard A. Firtel J. Biol. Chem., in press Experiments in several cell types revealed that local accumulation of phosphatidylinositol 3,4,5-triphosphate [PI(3,4,5)P3] mediates the ability of cells to migrate during gradient sensing. We took a systematic approach to characterize the functions of the six putative Class I PI3Ks (PI3K1-6) in Dictyostelium by creating a series of gene knockouts. These studies revealed that PI3K1-PI3K3 are the major PI3Ks for chemoattractant-mediated PI(3,4,5)P3 production. We studied chemotaxis of the pi3k1/2/3 triple knockout strain (pi3k1/2/3 null cells) to cAMP under two distinct experimental conditions, an exponential gradient emitted from a micropipette and a shallow, linear gradient in a Dunn chamber, using four cAMP concentrations ranging over a factor of 10,000. Under all conditions tested, pi3k1/2/3 null cells moved slower and had less polarity than wild-type cells. pi3k1/2/3 null cells moved towards a chemoattractant emitted by a micropipette, although persistence was lower than that of wild-type or pi3k1/2 null cells. In shallow linear gradients, pi3k1/2 null cells had greater directionality defects, especially at lower chemoattractant concentrations. Our studies suggest that although PI3K is not essential for directional movement under some chemoattractant conditions, it is a key component of the directional sensing pathway and plays a critical role in linear chemoattractant gradients, especially at low chemoattractant concentrations. The relative importance of PI3K in chemotaxis is also dependant on the developmental stage of the cells. Our data suggest that the output of other signaling pathways suffices to mediate directional sensing when cells perceive a strong signal, but PI3K signaling is crucial for detecting weaker signals. Submitted by Rick Firtel [rafirtel@ucsd.edu] -------------------------------------------------------------------------------- cAMP signaling in Dictyostelium: G-proteins activate separate Ras pathways using specific RasGEFs Helmut Kae, Arjan Kortholt, Holger Rehmann, Robert H. Insall, Peter J.M. Van Haastert, George B. Spiegelman, and Gerald Weeks EMBO Reports, In Press In general, mammalian RasGEFs display little substrate specificity, even though they are often thought to regulate specific pathways. Here we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium development, RasC and RasG are activated in response to cAMP, with each regulating different downstream functions; RasG regulating chemotaxis, and RasC responsible for adenylyl cyclase activation. RasC activation was abolished in a gefA- mutant, while RasG activation was normal in this strain, indicating that RasGEFA activates RasC, but not RasG. Conversely, RasC activation was normal in a gefR- mutant, whereas RasG activation was greatly reduced, indicating that RasGEFR activates RasG. These results were confirmed by the finding that RasGEFA and RasGEFR specifically released GDP from RasC and RasG, respectively, in vitro. This RasGEF target specificity provides a mechanism for one upstream signal to regulate two downstream processes using independent pathways. Submitted by: Helmut Kae [hkae@interchange.ubc.ca] -------------------------------------------------------------------------------- Mitochondrial carrier family. Repertoire and peculiarities of the cellular slime mould Dictyostelium discoideum Michel Satre1, Sara Mattei2, Laurence Aubry1, Pascale Gaudet3, Ludovic Pelosi1, Gerard Brandolin1, Gerard Klein1 1Laboratoire de Biochimie et Biophysique des Systemes Integres, iRTSV/BBSI, UMR 5092, CNRS-CEA-UJF, CEA-Grenoble, Grenoble, France 2Department of Cellular and Molecular Medicine, Howard Hughes Medical Institute, UCSD, School of Medicine, 9500 Gilman Drive, CMM-W318, La Jolla, CA 92093-0668, USA 3dictyBase, Center for Genetic Medicine, Northwestern University, Chicago, IL, 60611, USA. * To whom correspondence should be sent at the Laboratoire de Biochimie et Biophysique des Systemes Integres, iRTSV/BBSI, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France tel : +33 438 784 661 fax : +33 438 784 499 e-mail : gklein@cea.fr Biochimie, in press Proteins of the mitochondrial carrier family (MCF) mediate the transport of a large range ofcompounds, including metabolites and cofactors. They are localized mainly in the innermitochondrial membrane, except for a few members found in the membranes of peroxisomes. Similarity searches among Dictyostelium discoideum protein sequences identified a total of 31MCF members. All these are membrane proteins that possess three characteristic repeats of adomain of approximately 100 residues. Among them, three proteins have supplementarystructural domains consisting of Ca2+-binding motifs made up of 2 or 4 EF-hand unitslocalized on the N-terminal end, facing the mitochondrial intermembrane space. The nature oftransported substrates is proposed on the basis of sequence comparison with orthologscharacterized biochemically in other organisms, of phylogenetic analysis, and of theconservation of discriminating amino acid residues belonging to the substrate binding sites. Carriers have been grouped in subclasses based on their specificity for the transport ofnucleotides, amino acids or keto acids. Furthermore, we have identified an iron carrier of themitoferrin type, an inorganic phosphate carrier, and three carriers with similarity to uncouplerproteins. This study provides a focus for mitochondrial carrier analysis in Dictyostelium discoideum. Submitted by: Gerard Klein [ gerard.klein@cea.fr] ============================================================ [End dictyNews, volume 28, number 5]