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First published online 2 July 2003
doi: 10.1242/jcs.00649

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Superoxide signalling required for multicellular development of Dictyostelium

Gareth Bloomfield* and Catherine Pears{ddagger}

Biochemistry Department, Oxford University, South Parks Road, Oxford OX1 3QU, UK



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  		Fig. 1. Generation of superoxide during early development. (A) Ax2 cells were starved after plating onto plastic dishes under buffer at a density of 1.4x106 cells/cm2, and superoxide generation was measured using the XTT assay. The increase in optical density results from the accumulation of the brightly coloured product of the reaction of XTT and superoxide. XTT reduction in the absence of cells was subtracted from each time point. Data are the means ± s.d. of five experiments. (B) Ax2 cells were starved in shaking suspension at a density of 2x107 cells/ml in the presence ({blacksquare}) and absence ({circ}) of 100 units/ml Cu/Zn SOD. Data are the means ± s.d. of three experiments. (C) Ax2 cells were developed exactly as for A but with the addition of various concentrations of sodium diethyldithiocarbamate (DEDTC). The absorbance was measured after 7 hours. The data are expressed relative to the control lacking DEDTC, and are the means ± s.e.m. of three experiments.

 


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  		Fig. 2. Contribution of mitochondria to superoxide generation. (A) Ax2 cells were starved in shaking suspension for 6 hours at a density of 2x107 cells/ml, then resuspended at 108 cells/ml, and 0.5 mM XTT and the indicated concentration (µM) of rotenone added. After 10 minutes the absorbance at 470 nm was measured. The data are the means ± s.e.m. of three experiments. (B) Exponentially growing Ax2 cells were washed and resuspended in phosphate buffer at a density of 2x106 cells/ml. Duplicate samples were treated with rotenone or DMSO carrier for 10 minutes and then for a further 10 minutes with 10 µg/ml rhodamine 123. Cells were washed briefly three times in phosphate buffer and the degree of rhodamine sequestration determined as the relative fluorescence measured using a spectrofluorimeter. One experiment typical of three is shown. Fluorescence microscopy confirmed that rhodamine 123 accumulated in punctate, filamentous structures as expected for mitochondria in cells not treated with rotenone (Matsuyama and Maeda, 1995Go), but no such staining was detectable in cells treated with 2 µM rotenone (data not shown).

 


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  		Fig. 3. Stimulation of superoxide production by conditioned medium. Ax2 cells were starved in shaking suspension, and after 6 hours were resuspended at 108 cells/ml in fresh buffer, in conditioned medium (CM), or heat-treated conditioned medium (CM h/t), all containing 0.5 mM XTT. After 10 minutes the absorbance at 470 nm was measured. XTT reduction in the presence of the relevant medium (buffer or CM) and the absence of cells was subtracted. The data are the means ± s.e.m. of three experiments.

 


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  		Fig. 4. The effect of superoxide scavangers on aggregation and XTT reduction. (A) Ax2 cells were allowed to develop after plating onto plastic dishes at a density of 9.3x105 cells/cm2 in buffer alone, or buffer plus 500 units/ml SOD, 1.5 mM MTT, 0.5 mM NBT, 10 mM tiron, or 1 mM XTT. The cells were photographed after 12 hours. The bar represents 0.5 mm. (B) Ax2 cells were developed in tissue culture dishes at a density of 1.4x106 cells/cm2 in the presence of the stated concentration of MTT, NBT or tiron, along with 0.5 mM XTT, for 24 hours, when the buffer was collected and the absorbance at 470 nm measured. The data are expressed as percentage reductions in absorbance relative to a control from cells developed in the presence of 0.5 mM XTT alone, and are the means ± s.e.m. of three experiments. Asterisks indicate concentrations of scavengers that were sufficient to inhibit aggregation at this cell density. (C) Exponentially growing Ax2 cells were diluted 1:12 in phosphate buffer and superoxide scavenger added (XTT 1 mM, MTT 1.5 mM, NBT 0.2 mM, tiron 20 mM). These concentrations of scavenger all inhibited aggregation. The cultures were incubated for 18 hours at 22°C, 180 rpm before serial dilution in phosphate buffer and 300 cells of each were plated over 2x 15-cm SM plates, in association with Klebsiella aerogenes. Cell survival was determined by counting the number of clonal Dictyostelium colonies appearing on the bacterial lawn. Data are the means ± s.e.m. of triplicate plates within one experiment. One experiment representative of 3 independent experiments is shown. (D) Exponentially growing Ax2 cells were harvested and plated to aggregate under buffer as described in legend to Fig. 4A, but at three different cell densities, as indicated below. 1.5 mM MTT was sufficient to inhibit aggregation at the two lower cell densities, but not at the highest cell density. Similar density dependence was observed for the inhibition of aggregation in the presence of XTT, NBT and tiron (data not shown).

 


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  		Fig. 5. Verification of cells overexpressing SodA. (A) Exponentially growing parental Ax2 (A) and SOD-OE (B) cells (grown in 20 µg/ml G418) were lysed in SDS sample buffer and resolved by 12% SDS-PAGE, along with cell lysate from human Jurkat T cells (C) as a positive control, before being transferred to a PVDF membrane. The membrane was probed with polyclonal antisera raised against SOD (FL-154). A band of around 20 kDa, the expected size for SOD, was detected in both Ax2 and Jurkat cells. Increased immunoreactivity was seen for a doublet in the SOD-OE cells. The blot was then stripped and reprobed with antibody specific for the human c-myc epitope which had been inserted at the N terminus of the exogenous SodA protein, using the 9E10 antibody. This reacted with a single band, only in the SOD-OE cells, co-migrating with the upper of the two bands from the anti-SOD blot. It is likely that the lower band of the doublet seen with the anti-SOD antibody (co-migrating with the band seen in Ax2 cells) is SOD protein in which the N-terminal myc tag has been cleaved. The blot was then reprobed using the C-11 anti-actin antibody as a loading control. (B) 24-hour XTT assays were performed on the following cells aggregating on plastic as described in Fig. 1. GAL indicates Ax2 cells expressing ß-galactosidase, after selection at 100 µg/ml G418; 20, 50, 100 indicate Ax2 cells overexpressing SodA, (SOD-OE) after selection at 20, 50 or 100 µg/ml G418, respectively. Data are the means ± s.e.m. of three experiments.

 


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  		Fig. 6. Phenotype of cells overexpressing SodA. (A) Exponentially growing cells (Ax2, control cells expressing ß-galactosidase and SOD-OE cells, the latter two grown in 50 µg/ml G418) were diluted to a density of 3x105/ml in fresh HL5 medium in triplicate. The cultures were incubated at 22°C and 180 rpm and aliquots were taken in duplicate every day to determine cell density. One experiment typical of three independent experiments is shown. (B) Control cells (expressing ß-gal from the actin 15 promoter) grown in 100 µg/ml G418 and SOD-OE cells grown in 20 and 50 µg/ml G418 were harvested, washed in phosphate buffer and plated on phosphate-buffered agar at the cell densities indicated. The plates were incubated at 22°C in the dark and photographs were taken after 12 hours. No aggregates subsequently formed on the two plates of SOD-OE 50 cells at the two lowest cell densities and no aggregates were seen at any of these cell densities for SOD-OE cells grown in 100 µg/ml G418 (data not shown).

 


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  		Fig. 7. Regulation of gene expression during early development of cells overexpressing superoxide dismutase. SOD-OE and Ax2 cells were separately developed in shaking suspension at 2x107 cells/ml and aliquots taken for RNA extraction at the indicated times after starvation. The RNA was blotted onto a nylon membrane, and probed with 32P-labelled fragments of the indicated genes. carA, cAMP receptor cAR1; pdsA, extracellular phosphodiesterase; dscA, discoidin 1; acaA, aggregation stage adenylyl cyclase A; pkaC, the catalytic subunit of cAMP dependent protein kinase; IG7, loading control.

 


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  		Fig. 8. Rescue of aggregation defect of SOD-OE cells by conditioned medium. SOD-OE cells were developed in tissue culture dishes at a density of 5.7x105 cells/cm2 under (A) buffer, (B) conditioned medium from AX2 cells, (C) heat-treated conditioned medium, (D) conditioned medium from SOD-OE cells.

 

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© The Company of Biologists Ltd 2003