Dicty News Electronic Edition Volume 24, number 17 June 24, 2005 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 ============= Release of Ca2+ from the endoplasmic reticulum contributes to Ca2+ signalling in Dictyostelium. Zofia Wilczynska1, Kathrin Happle2, Annette MŸller-Taubenberger3, Christina Schlatterer2, Dieter Malchow2 and Paul R. Fisher1,* 1 Department of Microbiology, La Trobe University, Victoria 3086, Australia. 2 UniversitŠt Konstanz, 78464 Konstanz, Federal Republic of Germany. 3 Max-Planck-Institut fŸr Biochemie, 82152 Martinsried bei MŸnchen, Federal Republic of Germany. Eukaryotic Cell, in press Ca2+ responses to two chemoattractants (folate and cAMP) were assayed in Dictyostelium mutants deficient in one or both of two abundant Ca2+-binding proteins of the endoplasmic reticulum (ER) - calreticulin and calnexin. Mutants deficient in either or both proteins exhibited enhanced cytosolic Ca2+ responses to both attractants. Not only were the mutant responses greater in amplitude, but they also exhibited earlier onsets, faster rise rates, earlier peaks and faster fall rates. Correlations amongst these kinetic parameters and the response amplitudes suggested that key events in the Ca2+ response are autoregulated by the magnitude of the response itself ie. by cytosolic Ca2+ levels. This autoregulation was sufficient to explain the altered kinetics of the mutant responses - larger responses are faster in both mutant and wild type cells in response to both folate (vegetative cells) and cAMP (differentiated cells). Searches of the predicted Dictyostelium proteome revealed 3 putative Ca2+ pumps and 4 putative Ca2+ channels. All but one contained sequence motifs for Ca2+- or calmodulin-binding sites, consistent with Ca2+ signals being autoregulatory. Although cytosolic Ca2+ responses in the calnexin and calreticulin mutants are enhanced, the influx of Ca2+ from the extracellular medium into the mutant cells was smaller. Compared to wild type cells, Ca2+ release from the ER in the mutants thus contributes more to the total cytosolic Ca2+ response while influx from the extracellular medium contributes less. These results provide the first molecular genetic evidence that release of Ca2+ from the ER contributes to cytosolic Ca2+ responses in Dictyostelium. Submitted by: Paul Fisher [fisher@lumi.latrobe.edu.au] ----------------------------------------------------------------------------- Engineered gene over-expression as a method of drug target identification Christopher J. Sugden +, Janine R. Roper + and Jeffrey G. Williams* School of Life Sciences University of Dundee Wellcome Trust Biocentre Dow Street DUNDEE, DD1 5EH, UK +contributed equally to this work *Corresponding Author Professor Jeffrey Williams, School of Biological Sciences, University of Dundee MSI/WTB Complex, Dow Street DUNDEE, DD1 5EH Fax (44) 01382 345386 Email j.g.williams@dundee.ac.uk Biochem. and Biophys. Res. Comm., in press The proposed target of aminobisphosphonate (aBP) bone resorption inhibitors, both in mammalian osteoclasts and in Dictyostelium, is the enzyme farnesyl diphosphate synthase (FDP synthase). The genetic evidence, obtained with Dictyostelium, derives from variant strains that over-express FDP synthase and that are relatively resistant to aBPs. We show that forced FDP synthase over-expression also leads to aBP resistance; by placing FDP synthase under control of a semi-constitutive promoter, transforming it into Dictyostelium cells and selecting with the aBP alendronate. This combination of drug and dominant selectable marker provides a novel selection system for transformation. We further show that, when a population of Dictyostelium cells expressing an entire growth stage cDNA library is placed under alendronate selection, FDP synthase is the only cDNA insert that confers drug resistance. This confirms FDP synthase as the primary target of aBPs and suggests a general method of drug target identification based upon engineered gene over-expression. Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk] ----------------------------------------------------------------------------- Loss of SMEK, a novel, conserved protein, suppresses mek1 null cell polarity, chemotaxis, and gene expression defects Michelle C. Mendoza, Fei Du, Negin Iranfar, Nan Tang, Hui Ma, William F. Loomis, and Richard A. Firtel Molecular and Cellular Biology, in press. MEK/ERK MAP kinase signaling is imperative for proper chemotaxis. Dictyostelium mek1- (MEK1 null) and erk1- cells exhibit severe defects in cell polarization and directional movement, but the molecules responsible for the mek1- and erk1- chemotaxis defects are unknown. Here, we describe a novel, evolutionarily conserved gene, smkA (suppressor of mek1-), whose loss partially suppresses the mek1- chemotaxis phenotypes. SMEK also has MEK1-independent functions: SMEK,but not MEK1, is required for proper cytokinesis during vegetative growth, timely exit from the mound stage during development, and myosin II assembly. SMEK localizes to the cell cortex through an EVH1 domain at its N-terminus during vegetative growth. At the onset of development, SMEK translocates to the nucleus via an NLS (nuclear localization signal) at its C-terminus. The importance of SMEKâs nuclear localization is demonstrated by our findings that a mutant lacking the EVH1 domain complements SMEK deficiency, whereas a mutant lacking the NLS does not. Microarray analysis reveals that some genes are precociously expressed in mek1- and erk1- cells. The mis-expression of some of these genes is suppressed in the smkA deletion. These data suggest that loss of MEK1/ERK1 signaling compromises gene expression and chemotaxis in a SMEK-dependent manner. Submitted by: Rick Firtel [rafirtel@ucsd.edu] ----------------------------------------------------------------------------- The effect of the disruption of a gene encoding a PI4 kinase on the developmental defect exhibited by Dictyostelium rasC- cells Meenal Khosla, George B. Spiegelman and Gerald Weeks Department of Microbiology and Immunology University of British Columbia Vancouver, BC, V6T 1Z3 Canada Developmental Biology, in press The disruption of the gene encoding the Dictyostelium Ras sub-family protein, RasC results in a strain that fails to aggregate with defects in both cAMP signal relay and chemotaxis. Restriction Enzyme Mediated Integration disruption of a second gene in the rasC- strain resulted in cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene, designated pikD1, encodes a member of the phosphatidyl-inositol-4-kinase °ñ subfamily. Although the rasC-/pikD1 cells were capable of progressing through early development, when starved on a plastic surface under submerged conditions, they did not form aggregation streams or exhibit pulsatile motion. The rasC-/pikD1 cells were extremely efficient in their ability to chemotax to cAMP in a spatial gradient, although the reduced phosphorylation of PKB in response to cAMP observed in rasC- cells, was unchanged. In addition, the activation of adenylyl cyclase, which was greatly reduced in the rasC- cells, was only minimally increased in the rasC-/pikD1 strain. Thus, although the rasC-/ pikD- cells were capable of associating to form multicellular structures, normal cell signaling was clearly not restored. The disruption of the pikD gene in a wild type background resulted in a strain that was delayed in aggregation and formed large aggregation streams, when starved on a plastic surface under submerged conditions. This strain also exhibited a slight defect in terminal development. In conclusion, disruption of the pikD gene in a rasC- strain resulted in cells that were capable of forming multicellular structures, but which did so in the absence of normal signaling and aggregation stream formation. Submitted by: Gerald Weeks [gerwee@interchange.ubc.ca] ============================================================================== [End Dicty News, volume 24, number 17]