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Cross-induction of cell types in Dictyostelium: evidence that DIF-1 is made by prespore cells

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK

View larger version (15K): [in a new window]   Fig. 1. Outline of the DIF-1 biosynthetic pathway. The C12 skeleton of DIF-1 is proposed to be assembled by a polyketide synthase and then modified, first by chlorination and then by methylation, to give DIF-1 (Kay, 1998). The methylation step is carried out by the methyltransferase encoded by the dmtA gene (Thompson and Kay, 2000).

View larger version (155K): [in a new window]   Fig. 2. Distribution of dmtA mRNA in developing structures of strain Ax2. This mRNA encodes the methyltransferase catalysing the last step in DIF-1 biosynthesis and is clearly prespore specific from the time when a specific signal can be first detected. (A) Mound just before tip formation; (B) tipped mound; (C,D) slugs (a graded distribution of dmtA mRNA is apparent in the prespore zone); (E) Mexican hat; (F) early culminant.

View larger version (114K): [in a new window]   Fig. 3. Expression pattern of a green fluorescent protein reporter driven by the dmtA promoter. (A,B) Fruiting body squash, in phase contrast (A) and fluorescence (B), the upper cup is arrowed (it derives from AL cells); (C) mature spores.

View larger version (18K): [in a new window]   Fig. 4. Regulation of DIF-1 synthetic capacity by cAMP and DIF-1. Cells of strain Ax2 were starved in shaken suspension at 2x107/ml in KK2 buffer and after the first hour, pulsed with 50 nM cAMP every 6 minutes for a further 6 hours. These cells were subdivided, 4 mM cAMP and 100 nM DIF-1 added as indicated, and samples taken for northern analysis and enzyme assays. A second experiment gave a similar result.

View larger version (29K): [in a new window]   Fig. 5. Synthesis of DIF-1 made from the labelled polyketide, [3H]THPH, by prestalk and prespore cells. The products made from [3H]THPH by disaggregated slug cells (top two panels) or the prestalk and prespore fractions purified from them, were analysed by HPLC. THPH is successively converted to Cl-THPH, dM-DIF-1, DIF-1 and then to DIF-3, all of which are resolved (the unlabelled peaks preceding THPH are either impurities or produced in the work up). It can be seen that the prestalk fraction is capable of chlorinating THPH to Cl-THPH and dM-DIF-1, but methylation to give DIF-1/3 is barely detectable. By contrast, prespore cells and the starting mixture of cells (Disagg. cells) can perform all of these reactions. Cells were incubated at 107/ml in 1 ml NS/MES containing 1.9 nM [3H]THPH and 40 µM ancymidol (to inhibit breakdown of DIF-3). After 5 or 10 minutes at 22°C, labelled compounds were extracted, concentrated and resolved by HPLC, together with unlabelled standards. Each sample was analysed in duplicate.

View larger version (26K): [in a new window]   Fig. 6. Rates of chlorination and methylation of [3H]THPH by living cells. (A) Rate of chlorination of THPH by disaggregated slug cells or the prestalk and prespore cells purified from them. (B) Rate of methylation of [3H]THPH by disaggregated slug cells or the prestalk and prespore cells purified from them. (C) Effect of 100 nM DIF-1 or 100 nM DIF-3 on chlorination and methylation by disaggregated slug cells and the prestalk and prespore cells separated from them. Cells were incubated at 107/ml in 1 ml NS/MES containing 1.9 nM [3H]THPH and 40 µM ancymidol (to inhibit breakdown of DIF-3). After 10 minutes at 22°C, labelled compounds were extracted, concentrated and resolved by HPLC, together with unlabelled standards (see Materials and Methods for the method of calculating the chlorination and methylation rates). The prespore fraction was 96% and the prestalk fraction 88% pure.

View larger version (58K): [in a new window]   Fig. 7. The regulation of DIF-1 production by DIF in living cells. (A) TLC showing the inhibition of DIF-1 synthesis by DIF-1. Accumulation of the DIF-1 metabolites, DIF-3 and DM3, is inhibited in parallel with DIF-1. (B) Quantitation of the results shown in A; (C) effect of compounds related to DIF-1 on DIF-1 synthesis. All these compounds are produced during development and therefore are potential regulators of DIF-1 synthesis: DIF-2 is a homologue of DIF-1 with one fewer carbon atom in the alkyl side chain, DIF-3 is produced from DIF-1 by a single dechlorination; DM3 is produced from DIF-3 by an oxidation of the side chain. Cells of strain V12M2 were incubated for 12 hour in submerged culture with cAMP and 36Cl–. DIF-1 or related compounds were added at the start of the experiment, as indicated. Labelled compounds were extracted from cells and media, resolved by TLC and quantitated using a Phosphorimager. Data are typical of three experiments.

View larger version (11K): [in a new window]   Fig. 8. Speed of inhibition of DIF-1 synthesis by DIF-1 in living cells. Cells of strain V12M2 were incubated in submerged culture with cAMP and 36Cl–. At 12 hours, fresh medium (still containing 36Cl–) was substituted, with 100 nM DIF-1 added as indicated. Labelled compounds were extracted from cells and media, resolved by TLC and quantitated using a Phosphorimager. Data are typical of three experiments.

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