Extracellular Polyphosphate Inhibits Proliferation in an Autocrine Negative Feedback Loop in Dictyostelium discoideum

Abstract

Polyphosphate is a polymer of phosphate residues linked by high energy phosphoanhydride bonds. Despite being highly conserved throughout nature, its function is poorly understood. Here we show that Dictyostelium cells accumulate extracellular polyphosphate, and this acts to inhibit proliferation at high cell densities. In shaking culture, extracellular polyphosphate concentrations increase as cell density increases, and if the concentration of polyphosphate observed at the stationary phase is added to cells at mid-log, proliferation is halted. Adding an exopolyphosphatase to cell cultures or stationary phase conditioned medium decreases polyphosphate levels and abrogates the anti-proliferative effect. The cells show saturable binding of polyphosphate, suggesting the presence of a cell surface polyphosphate receptor. Extracellular polyphosphate accumulation is potentiated by decreased nutrient levels, potentially as a means to anticipate starvation. Loss of the Dictyostelium polyphosphate kinase DdPpk1 causes intracellular polyphosphate levels to become undetectable and negatively affects fitness, cytokinesis, and germination. However, cells lacking DdPpk1 accumulate ∼50% normal levels of extracellular polyphosphate, suggesting an additional means of synthesis. We found that cells lacking inositol hexakisphosphate kinase, which is responsible for the synthesis of the inositol pyrophosphates IP7 and IP8, reach abnormally high cell densities and show decreased extracellular polyphosphate levels. Two different enzymes thus appear to mediate the synthesis of Dictyostelium extracellular polyphosphate, which is used as a signal in an autocrine negative feedback loop to regulate cell proliferation.

Keywords: Dictyostelium; cell biology; cell growth; cell proliferation; inositol hexakisphosphate kinase; polyphosphate; polyphosphate kinase; stress.

FIGURE 1.

Wild type cells accumulate increasing amounts of extracellular polyphosphate as cell density increases. A and B, cells were cultured, and the polyphosphate (polyP) concentration in the conditioned medium was measured. C, polyP from stationary phase conditioned medium was resolved on a 25% polyacrylamide gel and stained with toluidine blue (representative image of three gels). D and F, cells were cultured with 150 μm polyP (D) or the indicated concentration of polyP (F) and density (D), and the percentage of inhibition after 24 h (F) was determined. E, wild type cells were cultured in the presence or absence of 150 μm polyP for 18 h, washed twice in HL5, resuspended in fresh HL5, and counted daily. G, wild type cells were cultured in the presence or absence of triphosphate, pyrophosphate, or sodium phosphate and counted daily. All values are means ± S.E., n ≥ 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (one-way analysis of variance).

FIGURE 2.

Effect of nutrient levels on extracellular polyphosphate accumulation. A and B, SIH was generated at concentrations of 1×, 2×, and 0.67×; wild type cells were cultured and counted daily (A); and CM samples were harvested to determine polyP content (B). C, cells were cultured in the indicated conditions, and the polyP concentration in the conditioned medium was measured. D, wild type cells were starved for 5 h in PBM, and conditioned medium was harvested and concentrated using a 2-kDa size exclusion filter. Wild type cells were then cultured in the presence or absence of 50% of the resulting concentrated, starved CM and counted daily. E, polyP from starved CM was resolved using a 25% polyacrylamide gel, stained with toluidine blue, and destained with 20% methanol. All values are means ± S.E., n ≥ 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (one-way analysis of variance).

FIGURE 3.

The proliferation inhibiting activity is reduced by exopolyphosphatase. A, wild type cells were treated with ScPPX1 or buffer (0) daily, and cell density was determined. B and C, stationary phase conditioned medium was treated with 0.15 μg/ml ScPPX1 for 3 h. The samples were then assayed for polyP content (B) and the ability to inhibit cell proliferation (C). D, starved CM was treated with ScPPX1 for 3 h, and polyP content was determined using DAPI. E and F, stationary phase CM was treated with phytase or DDP1, polyP fluorescence was determined using DAPI (E), and inhibitory activity was determined by culturing cells with 50% CM (treated or untreated) with daily counts (F). The values are means ± S.E., n ≥ 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the no-PPX1 control (unpaired two-tailed t tests).

FIGURE 4.

Polyphosphate binds wild type cells. A, mid-log wild type cells were incubated with the indicated amounts of biotinylated polyP and a streptavidin-conjugated fluorophore. The cells were washed, and fluorescence was measured using a flow cytometer. B, cells were incubated with 200 μm medium chain biotinylated polyP for the indicated times, and binding was measured as in A. C, D, and E, competition assays were performed by incubating wild type cells with 200 μm medium chain biotinylated polyP, streptavidin-conjugated fluorophore, and either stationary phase conditioned medium (C), unlabeled polyP (D), or sodium sulfate (E) binding was measured as in A. Concentrations of unlabeled polyP (D) are shown as whole polyphosphate molecules as opposed to phosphate monomers. The values are means ± S.E., n ≥ 4.

FIGURE 5.

Inositol hexakisphosphate kinase regulates extracellular polyphosphate accumulation. A and B, the indicated strains were cultured in SIH, and extracellular polyP accumulation was measured. C and D, conditioned medium from cells at stationary (WT) or maximum density (i6kA⁻, ppk1⁻) was assayed for polyP. E, polyP from conditioned medium was resolved on a 25% polyacrylamide gel and stained with toluidine blue. 14P indicates 14-mer polyP size standard. F, conditioned medium from cells at the indicated densities was assayed for their ability to inhibit the proliferation of wild type cells. G, proliferation of the indicated strains or of i6kA⁻ cells with 30 μm polyP added to the culture at day 1, 20 μm added at days 2 and 3, and 10 μm added at days 4 and 5. H, wild type or cells lacking DdPpk1 were cultured in SIH in the presence or absence of polyP. Cell density was measured daily. I, the indicated strains were cultured in HL5, and intracellular polyP accumulation was measured. The values are means ± S.E., n ≥ 4. *, p < 0.05, compared with the Ax2 wild type control (unpaired two-tailed t tests in most panels; unpaired one-tailed t test in F).

FIGURE 6.

Mutant proliferation phenotypes correlate with extracellular polyphosphate accumulation. A, wild type cells or transformants were cultured in SIH and counted daily, and the maximal density was recorded. B, the extracellular polyP concentration was measured at the maximal density and normalized to the cell density. The values are means ± S.E., n ≥ 4. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with the wild type control (unpaired two-tailed t tests).