Dicty News Electronic Edition Volume 11, number 5 September 12, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== Cytoskeletal association of an esterase in Dictyostelium discoideum Catherine P. Chia1,*, Lothar Bomblies2, Kristy K. Taylor 1School of Biological Sciences, University of Nebraska-Lincoln Lincoln, NE 68588-0118 2Max-Planck-Institüt für Biochemie, D-8033 Martinsried, Federal Republic of Germany Experimental Cell Research, in press Abstract A 70 kDa glycoprotein, gp70, was found enriched in the detergent- insoluble cytoskeletal fraction of axenically-grown Dictyostelium discoideum cells. Its N-terminal amino acid sequence identified it as ‘crystal protein’ (Bomblies, et al., 1990 J. Cell Biol. 110:669). This finding was corroborated when antibody to crystal protein cross- reacted with gp70 and its deglycosylated form. The postulated esterase activity of gp70/crystal protein was verified through comparative enzyme assays of extracts derived from cells that either overexpressed or lacked gp70. Gp70 co-sedimented with cytoskeletons on sucrose gradients, suggesting an interaction with the cytoskeleton. Co-isolation of gp70 with detergent-extracted cells, observed by immunofluorescence microscopy, also implied a gp70-cytoskeletal association. These data supported the idea that the localization or secretion of gp70, or both, was cytoskeletally-mediated. Although axenically-grown cells contained high levels of gp70, the same cell lines had reduced levels of gp70 when grown in bacterial suspension or in nutrient media containing bacteria. Bacterially-grown cells, compared to axenically-grown cells, had lower fluid-phase uptake rates even when nutrient media was present, indicating that phagocytosis was a preferred mode of feeding. Thus, bacteria inhibited gp70 expression, which suggested a role for prestarvation factor, in regulating its synthesis. ------------------------------------------------------------------------- Identification of Darlin, a Dictyostelium Protein with Armadillo-like Repeats that Binds to Small GTPases and is Important for the Proper Aggregation of Developing Cells. Kalpa K. Vithalani, Carole A. Parent, Eric M. Thorn, Marcus Penn, Denis A. Larochelle, Peter N. Devreotes and Arturo De Lozanne Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 Mol. Biol. Cell, in press Abstract: We purified from Dictyostelium lysates an 88 kDa protein that bound to a subset of small GTPases including racE, racC, cdc42Hs, and TC4ran, but did not bind to R-ras or rabB. Cloning of the gene encoding this 88 kDa protein revealed that it contained multiple armadillo-like repeats most closely related to the mammalian GTP exchange factor smgGDS. We named this protein darlin (Dictyostelium armadillo-like protein). Disruption of the gene encoding darlin demonstrated that this protein is not essential for cytokinesis, pinocytosis, phagocytosis or development. However, the ability of darlin null cells to aggregate in response to starvation is severely affected. When starved under liquid medium, the mutant cells were unable to form aggregation centers and streams, possibly due to a defect in cAMP relay signaling. This defect was not due to an inability of the darlin mutants to activate adenylate cyclase in response to G protein stimulation. These results suggest that the darlin protein is involved in a signaling pathway that may modulate the chemotactic response during early development. ------------------------------------------------------------------------- Dictyostelium TRFA Homologous to Yeast Ssn6 is Required for Normal Growth an d Early Development Junichi Saito, Takahide Kon, Akira Nagasaki, Hiroyuki Adachi, and Kazuo Sutoh Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153, Japan J. Biol. Chem., in press Abstract The TPR (tetratricopeptide repeat) family became widespread during evolution, being found from bacteria to mammalians. By means of restriction enzyme-mediated integration (REMI), we have identified a Dictyostelium gene (trfA) highly homologous to a Saccharomyces cerevisiae gene encoding a TPR protein, Ssn6 (Cyc8), which functions as a global transcriptional repressor for diverse genes. The deduced amino acid sequence of the Dictyostelium gene product, TRFA, contains ten consecutive TPR units as well as Gln repeats, Asn repeats, and a region rich in Glu, Lys, Ser and Thr. The sequences of some of the ten TPR units in TRFA are more than 70% identical to the corresponding units in Ssn6. The trfA- cells produced smooth plaques on a bacterial lawn, and also failed to aggregate normally when starved on a plain agar plate. Individual cells showed weaker chemotactic responces to cAMP, although adenylyl cyclases of the trfA- cells were activated by cAMP as in the wild-type cells. In a rich medium, they grew more slowly and stopped growing at a lower density than the wild-type cells. Furthermore, they divided into cells of various sizes and tended to be much smaller than the wild-type cells. These pleiotropic defects of the trfA- cells suggest the possibility that Dictyostelium TRFA may regulate the transcription of diverse genes required for normal growth and early development. ------------------------------------------------------------------------- Underexpression of a Novel Gene, Dia2, Impairs the Transition of Dictyostelium Cells from Growth to Differentiation Soo-Choen Chae*, Yuji Inazu, Aiko Amagai, and Yasuo Maeda1 Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-8578, Japan Biochem. Biophys. Res. Commun., in press Abstract In Dictyostelium discoideum Ax-2 cells, a specific point (PS-point) in the cell cycle from which they initiate differentiation in response to starvation has been specified. Using synchronized Ax-2 cells and the differential display method, a novel gene (differentiation-associated gene 2; dia2) was isolated as one of genes expressed specifically during the shift of Ax-2 cells from growth to differentiation. The dia2 gene codes a lysine- and leucine-rich protein with a predicted molecular mass of 16.9 kDa. Northern blot analys is has shown that the dia2 mRNA, of 0.7kb, accumulates in differentiating cells starved just before the PS-point, while that there is no detectable expression in vegetatively growing cells. Antisense- mediated gene inactivation of dia2 greatly inhibited the progress of differentiation, presumably through the reduced expression of cAMP receptor 1 (car1). Thus, the DIA2 expression was suggested to have an essential role in the initiation of differentiation , closely relating to the cAMP signaling system. ------------------------------------------------------------------------- A Molecular Network that Produces Spontaneous Oscillations in Excitable Cells of Dictyostelium Michael T. Laub and William F. Loomis Center for Molecular Genetics, Department of Biology, University of California, San Diego, La Jolla, CA 92093 Mol. Biol. Cell, in press Abstract A network of interacting proteins has been found that can account for the spontaneous oscillations in adenylyl cyclase activity that are observed in homogenous populations of Dictyostelium cells 4 hours after the initiation of development. Previous biochemical assays have shown that when extracellular cAMP binds to the surface receptor, CAR1, the adenylyl cyclase, ACA, and the MAP kinase, ERK2, are transiently activated. A rise in the internal concentration of cAMP activates protein kinase A, PKA, such that it inhibits ERK2 and leads to a loss-of-ligand binding by CAR1. ERK2 phosphorylates the cAMP phosphodiesterase, REG A, that reduces the internal concentration of cAMP. A secreted phosphodiesterase, PDE, reduces external cAMP concentrations between pulses. Numerical solutions to a series of nonlinear differential equations describing these activities faithfully account for the observed periodic changes in cAMP. The activity of each of the components is necessary for the network to generate oscillatory behavior, however, the model is robust in that 25 fold changes in the kinetic constants linking the activities have only minor effects on the predicted frequency. Moreover, constant high levels of external cAMP lead to attenuation while a brief pulse of cAMP can advance or delay the phase such that interacting cells become entrained. ------------------------------------------------------------------------- [End Dicty News, volume 11, number 5]