Dicty News Electronic Edition Volume 9, number 14 November 22, 1997 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 =========== Mutual Relation between the Cell-cycle Progression and Prespore Differentiation in Dictyostelium Development Tsuyoshi Araki and Yasuo Maeda* Biological Institute, Graduate School of Science, Tohoku University, Aoba, Sendai 980-77, Japan Zool. Sci., in press Abstract: In Dictyostelium discoideum Ax-2, the cell-cycle progression from the early aggregate to mound stage has been proposed to have some connection with prespore differentiation. Hereupon, we examined the role of cell-cycle progression during the development on cell differentiation, using two kinds of cell-cycle inhibitors. Nocodazole, an inhibitor of microtubule formation, was found to inhibit greatly cell division around the mound stage as well as during the vegetative growth phase, when applied to exponentially growing Ax-2 cells. Essentially the same inhibition was attained by treatment of starved Ax-2 cells with calyculin A, an inhibitor of serine/threonine protein phosphatases. It noteworthy that the nocodazole- or calyculin A-treated cells exhibit abnormal morphogenesis to form a stick-like multicellular structure on non-nutrient agar, and also that prespore differentiation as exemplified by the prespore-specific Dp87 gene expression and prespore specific vacuole (PSV) formation was greatly suppressed. In contrast, the differentiation of prestalk (pstA) cells was scarcely affected by the drug treatments. Taken together these results seem to indicate that the cell-cycle progression around the mound stage is important for prespore differentiation. ---------------------------------------------------------------------- Cells at the centre of Dictyostelium aggregates become spores Hao-Jen Huang, David Tanagawa, Gerald Weeks and Catherine Pears Dev. Biol in press Abstract: The cellular slime mould Dictyostelium discoideum undergoes a developmental life cycle on starvation to generate a fruiting body consisting of a mass of spores supported on a stalk of dead, vacuolated cells. The choice between alternative cell fates is influenced by a variety of factors including cell cycle position at the onset of starvation. We present evidence to suggest that the cell cycle position influences cell fate by determining the position of cells in the early aggregate. The existence of a strain which cannot initiate development on its own but which can respond to signals generated by non-mutant cells has allowed us to investigate the eventual cell fate of the initiating cells which are, by definition, at the centre of the early aggregate. Cells which have a propensity to become prespore cells show an increased efficiency in initiating development of this strain. Labelling the initiating cells by the expression of green fluorescent protein reveals that these cells become spores. The higher levels of expression of genes characteristic of early development in cells with a prespore tendency is consistent with the earlier expression of the components of relay in prespore cells. ------------------------------------------------------------------------- Coupling between catalysis and oligomeric structure in NDP kinase. Sebastien MESNILDREY, Fabrice AGOU, Anna KARLSSON, Dominique DEVILLE-BONNE and Michel VERON. Unite de Regulation Enzymatique des Activites Cellulaires, Institut Pasteur, CNRS UMR 321, 25 rue du Docteur Roux, 75724 Paris cedex 15, France J. Biol. Chem., in press Absract: A dimeric Dictyostelium NDP kinase has been stabilized by the double mutation P100S-N150stop which targets residues involved in the trimer interface (Karlsson et al., (1996) J. Biol. Chem., 271, 19928-19934). The reassociation of this dimeric form of into a hexamer similar to the wild type enzyme is induced by the presence of a nucleotide substrate. Equilibrium sedimentation and gel filtration experiments, as well as enzymatic activity measurements, show that reactivation of the enzyme closely parallels its reassociation. A phosphorylatable intermediate with low activity participates in the association pathway while the dimeric form is shown totally devoid of enzymatic activity. Our results support the hypothesis that different oligomeric species of NDP kinase are involved in different cellular processes where the enzymatic activity is not required. ------------------------------------------------------------------------- The in vitro DNA-binding properties of NDP kinase are related to its oligomeric state. S�bastien MESNILDREY, Fabrice AGOU and Michel VERON. Unite de Regulation Enzymatique des Activites Cellulaires, Institut Pasteur, CNRS UMR 321, 25 rue du Docteur Roux, 75724 Paris cedex 15, France FEBS Letters, in press. Abstract: Genetic and biochemical evidences suggest that the enzymatic activity of NDP kinase is necessary but not sufficient for its biological function. While the human NDPK B binds specifically single-strand poly-pyrimidines sequences, the hexameric enzyme from Dictyostelium does not. We demonstrated by Electrophoretic Mobility Shift Assay and Filter Binding Assay that a dimeric mutant from Dictyostelium binds to an oligodesoxynucleotide while the wild type does not. These data suggest that the differences in the DNA-binding properties of several eucaryotic NDP kinases might be correlated to the differences in the stability of their hexameric structure. ------------------------------------------------------------------------- The biosynthesis of DIF, a chlorinated signal molecule regulating Dictyostelium development Robert R. Kay MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK J. Biol. Chem., in press. Summary DIF-1 is a chlorinated alkyl phenone released by developing Dictyostelium amoebae, which induces them to differentiate into stalk cells. A biosynthetic pathway for DIF-1 is proposed from labeling, inhibitor and enzymological experiments. Cells incorporate 36C1- into DIF-1 during development, showing that the chlorine atoms originate from chloride ions; peak incorporation is at the first finger stage. DIF-1 synthesis can be blocked by cerulenin, a polyketide synthase inhibitor, suggesting that it is made from a polyketide. This is most likely the C12 polyketide THPH. Feeding experiments confirm that living cells can convert THPH to DIF-1. Conversion requires both chlorination and methylation of THPH and enzymatic activities able to do this exist in cell lysates. The chlorinating activity, assayed using 36C1-, is stimulated by H2O2 and requires both soluble and particulate components. It is specific for THPH and does not use this compound after O-methylation. The methyltransferase is soluble, uses AdoMet as a co-substrate, has a Km for dichloro-THPH of about 1.1 micro-molar and strongly prefers this substrate to close analogues. Both chlorinating and methyltransferase activities increase in development, in parallel with DIF-1 production and both are greatly reduced in a mutant strain that makes little DIF-1. It is proposed that DIF-1 is made by the initial assembly of a C12 polyketide skeleton, which is then chlorinated and methylated. ------------------------------------------------------------------------- Cloning and transcriptional regulation of the gene encoding the vacuolar/H+ ATPase B subunit of Dictyostelium discoideum Enrico Bracco1, Barbara Peracino1, Angelika A. Noegel2$, Salvatore Bozzaro1* FEBS Lett., in press Summary: The main function of vacuolar H+-ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments. We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H+-ATPase (vatB) and analyzed its transcriptional regulation. The deduced protein comprises 493 aminoacids with a calculated molecular mass of 54,874 Da. The predicted protein sequence is highly homologous to previously determined V/H+ ATPase B subunit sequences. The protein is encoded by a single gene in the Dictyostelium genome. The gene is maximally expressed during growth and it decreases during the first hours of development. Gene expression is rapidly enhanced by phagocytosis not however by fluid-phase endocytosis. Acidic and alkaline conditions affect differently vatB gene expression. ------------------------------------------------------------------------- cGMP potentiates receptor-stimulated Ca2+ influx in Dictyostelium discoideum Hidekazu Kuwayama and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Biochimica et Biophysica Acta, in press. Abstract Binding of extracellular cAMP to surface receptors induces at least two responses in Dictyostelium discoideum, the G-protein-dependent activation of guanylyl cyclase, and the opening of a plasma membrane Ca2+ channel. Some experiments suggest that intracellular cGMP opens the Ca2+ channel, while others demonstrate that the channel can open in the absence of functional G-proteins (and thus in the absence of cGMP formation). We have analyzed 45Ca2+ uptake in three mutants with altered cGMP formation. Mutant stmF shows a prolonged cGMP response due to deletion of an intracellular phosphodiesterase. Uptake of receptor-stimulated 45Ca2+ is enhanced about 2-fold in this mutant if compared to wild-type cells, suggesting that cGMP regulates the opening of the channel. Mutant KI-7 has very low levels of surface cAMP receptors, but nevertheless an enhanced receptor-stimulated cGMP response due to a defect in the turn-off of guanylyl cyclase. This mutant shows poor receptor-stimulated 45Ca2+ uptake, suggesting that cGMP alone is not sufficient to open the Ca2+ channel. Finally, mutant KI-8 has no cGMP due to the absence of nearly all guanylyl cyclase activity. The mutant shows significant but reduced 45Ca2+ uptake (19 % of wild-type; 60 % if corrected for the reduced level of surface cAMP receptors), suggesting that the channel can open in the absence of cGMP. Taken together, the results demonstrate that receptor-stimulated Ca2+ influx is not directly induced by cGMP formation; it can occur in the absence of cGMP, but is potentiated 2-4 fold by cGMP. ------------------------------------------------------------------------- [End Dicty News, volume 9, number 14]