Dicty News Electronic Edition Volume 23, number 6 August 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 ============= Identification of new modes of Dd-STATa regulation of gene expression in Dictyostelium by in situ hybridisation NAO SHIMDA1, MINEKO MAEDA2, HIDEKO URUSHIHARA3 and TAKEFUMI KAWATA1, 4 1Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan TEL & FAX: +81-47-472-5156, E-mail: tkawata@bio.sci.toho-u.ac.jp 2 Department of Biology, Graduate School of Science, Osaka University , Toyonaka, Osaka 560-0043, Japan 3 Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan 4 Corresponding author Int. J. Dev. Biol., in press. STATs (signal transducers and activators of transcription) are transcription factors, which lie at the end of cytokine and growth signal transduction pathways. Dictyostelium Dd-STATa is a functional homologue of metazoan STATs. It is activated by cAMP and, at the slug stage, it translocates into the nuclei of the tip cells : a sub-set of the anterior, prestalk A (pstA) cells. Here we searched for novel Dd-STATa regulated genes by in situ hybridisation. A set of 54 cDNA clones, whose gene expressions patterns are known to be prestalk-specific (Maeda et al., 2003), were chosen as probes and we compared their expression patterns in parental and Dd-STATa-null strains. We identified 13 genes that are candidates for direct induction by Dd-STATa. In the parental strain, most of these genes are expressed in the cone shaped mass of pstAB cells that is located within the prestalk region region. These cDNAs show little or no expression in the Dd-STATa-null strain. This contrasts markedly with the paradigmatic, ecmB gene which is expressed in pstAB cells in parental cells but which is expressed throughout the prestalk zone in the Dd-STATa null strain. We also identified several genes that are normally expressed in the pstA cells, or throughout the prestalk region, but whose expression is markedly down-regulated in the null mutant. Again, this contrasts with markers derived from the paradigmatic, ecmA gene which are expressed normally in the Dd-STATa-null strain. The identification of these novel genes provides valuable tools to investigate the role of Dd-STATa. Submitted by: Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp] ----------------------------------------------------------------------------- Developmental control of cAMP-induced Ca2+-influx by cGMP: Influx is delayed and reduced in a cGMP-phosphodiesterase D deficient mutant of Dictyostelium discoideum Daniel F. Lusche and D. Malchow Cell Calcium, in press It was previously shown that cGMP enhances cAMP-induced Ca2+-influx in Dictyostelium discoideum. This finding is based on experiments done with strains defective in cGMP-hydrolysis, the streamer F cells. In this work, we show that these chemically mutagenized cells display different properties in their cAMP-induced light-scattering response and cAMP-induced Ca2+-influx compared with a cGMPphosphodiesterase knock-out strain, pdeD KO, generated by homologous recombination. PdeD KO cells possess a reduced Ca2+-influx that is developmentally regulated. This finding contradicts the result of streamer F cells, where cAMP-induced Ca2+-influx is prolonged and elevated. Both mutants, however, showed a three to four-fold delayed response to cAMP at 3ö4 h of starvation. Thus, the consequence of an elevated cGMP concentration is a delay and an inhibition of Ca2+-influx and not an enhancement. Results obtained with streamer F cells should therefore be interpreted with caution because the mutation(s) responsible for the divergent phenotype to pdeD KO cells has not been identified. We show by the use of membrane-permeant cGMP-analogues in wild type (wt) cells, permeabilized cells and measurements on isolated vesicles that the cause for the reduced Ca2+-influx seems to be due to developmentally regulated Ca2+-channel inhibition by cGMP. Submitted by: Daniel Lusche [Daniel.Lusche@uni-konstanz.de] ----------------------------------------------------------------------------- Multichannel apparatus for parallel monitoring of light scattering in Dictyostelium discoideum cell suspensions Daniel F. Lusche*, Hanni Rštzer*, Rudolf Merz, Hubert Fink, Rupert Mutzel+, Christina Schlatterer Faculty for Biology, University of Konstanz, 78457 Konstanz, FRG * these authors contributed equally to the work + Institute of Biology-Microbiology, Free University of Berlin, Kšnigin-Luise-Strasse 12-16, 14195 Berlin, FRG BioTechniques, in press Suspensions of Dictyostelium discoideum amoebae display free-running light scattering oscillations at the onset of development. We describe a device to monitor these oscillations in several samples in parallel. The apparatus consists of a thermostatted cuvette holder where up to eight cuvettes containing cell suspension are inserted. Cells are aerated and kept in suspension via an airlift. Infrared light emitted from a five-diode array passes through the suspension and is detected by an array of five light detecting diodes. The resulting signal is digitized and recorded with a sampling rate of two measuring points/sec. The parallel analysis approach allows determination of the effects of adding of agents or of variations in the external conditions in the same batch of amoebae at the same developmental time point. This represents an advantage over the conventional single cuvette approach as oscillation characteristics themselves are developmentally regulated. Moreover, as the new experimental setup enables simultaneous analyses of up to eight samples the behaviour of wild type and several mutant strains can be compared under identical experimental conditions. Submitted by: Christina Schlatterer [Christina.Schlatterer@uni-konstanz.de] ----------------------------------------------------------------------------- Translocation of the Dictyostelium TRAP1 homologue to mitochondria induces a novel prestarvation response Tsuyoshi Morita*, Aiko Amagai and Yasuo Maeda Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan * Present address: Department of Neuroscience (D13), Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita City, Osaka 565-0871, Japan J. Cell Science, in press Dd-TRAP1 is a Dictyostelium homologue of tumor necrosis factor receptor associated protein 1 (TRAP-1). Dd-TRAP1 is located in the cortex of cells growing at a low density, but was found to be translocated to mitochondria with the help of a novel prestarvation factor that was accumulated in growth medium along with increased cell densities. The knockdown mutant of Dd-TRAP1 (TRAP1-RNAi cells) exhibited a significant defect in prestarvation response. Although TRAP1-RNAi cells showed normal expressions of classical prestarvation genes (discoidin I and car1), the expressions of differentiation-associated genes (dia1 and dia3) induced by prestarvation response were markedly repressed. On the other hand, transformants overexpressing Dd-TRAP1 showed a precocious prestarvation response and also augmented expressions of dia1 and dia3 in a cell density-dependent manner. Importantly, introduction of Dd-TRAP1 antibody into D. discoideum Ax-2 cells by electroporation inhibited the translocation of Dd-TRAP1 from the cortex to mitochondria and greatly inhibited the initiation of differentiation. Taken together, these results indicate that Dd-TRAP1 is translocated to mitochondria by sensing the cell density in growth medium and enhances the early developmental program through a novel prestarvation response. Submitted by: Y. Maeda [ymaeda@mail.tains.tohoku.ac.jp] ----------------------------------------------------------------------------- Cell shape regulation and co-translocation of actin and adenosyl homocysteinase in response to intermediate hypertonicity Yohko Yamada1.*Ű and Masazumi Sameshima1,2 1 The Tokyo Metropolitan Institute of Medical Science, Electron Microscopy Center 2 Department of Biofunctional Science, Faculty of Agriculture and Life Science, Hirosaki University, FEMS Microbiol Lett, in press Hypertonic stimulation induced association of S-adenosyl-L-homocysteine hydrolase (SAHH) with the F-actin-rich cell cortex in Dictyostelium. At intermediate, but not higher, levels of hypertonicity, SAHH further translocated from the cortex to the cytosol in company with a fraction of actin and cofilin. At the same time the cells rounded up. Acidification of the cells stimulated both the cell rounding and the translocation of actin and SAHH, whereas alkalinization retarded these responses, suggesting that cellular pH is involved in their control. On the other hand, mutant analysis suggested that neither cGMP signaling nor conventional myosin is required. Submitted by: Yoko Yamada [yyamada@chaos.bio.sci.osaka-u.ac.jp] ============================================================================== [End Dicty News, volume 23, number 6]