An Autocrine Negative Feedback Loop Inhibits Dictyostelium discoideum Proliferation through Pathways Including IP3/Ca2+

ABSTRACT Little is known about how eukaryotic cells can sense their number or spatial density and stop proliferating when the local density reaches a set value. We previously found that Dictyostelium discoideum accumulates extracellular polyphosphate to inhibit its proliferation, and this requires the G protein-coupled receptor GrlD and the small GTPase RasC. Here, we show that cells lacking the G protein component Gβ, the Ras guanine nucleotide exchange factor GefA, phosphatase and tensin homolog (PTEN), phospholipase C (PLC), inositol 1,4,5-trisphosphate (IP3) receptor-like protein A (IplA), polyphosphate kinase 1 (Ppk1), or the TOR complex 2 component PiaA have significantly reduced sensitivity to polyphosphate-induced proliferation inhibition. Polyphosphate upregulates IP3, and this requires GrlD, GefA, PTEN, PLC, and PiaA. Polyphosphate also upregulates cytosolic Ca2+, and this requires GrlD, Gβ, GefA, RasC, PLC, IplA, Ppk1, and PiaA. Together, these data suggest that polyphosphate uses signal transduction pathways including IP3/Ca2+ to inhibit the proliferation of D. discoideum.

D. discoideum grows on soil surfaces and eventually overgrows its food supply and starves. D. discoideum accumulates extracellular polyphosphate as cells grow and proliferate (9). At cell densities corresponding to mid-log phase, the extracellular polyphosphate causes some cells to store rather than digest phagocytosed bacteria, possibly in anticipation of possible starvation (22). At very high cell densities, when the cells are about to starve, the accumulated extracellular polyphosphate reaches ;150 mM. This concentration of polyphosphate contributes to the inhibition of cytokinesis (and, thus, cell proliferation) (9), possibly to prevent the formation of small cells. Therefore, just before starvation, the percentage of large cells with relatively large reserves of stored nutrients is increased (9).
Polyphosphate regulates the proliferation of D. discoideum by different signaling pathways depending on nutrient levels (23). In rich media, the loss of the G proteincoupled receptor GrlD, a metabotropic glutamate receptor-like receptor, partially reduced the sensitivity of cells to polyphosphate, and the loss of the small GTPase RasC did not reduce the sensitivity of cells to polyphosphate (23). However, under lownutrient conditions, the loss of GrlD or RasC blocked the sensitivity of cells to polyphosphate (23).
The above-mentioned results suggest that polyphosphate uses a signal transduction pathway to inhibit D. discoideum proliferation under low-nutrient conditions. To elucidate additional signaling components in the polyphosphate proliferation inhibition pathway, we screened 52 available signal transduction pathway mutants for insensitivity to polyphosphate-induced proliferation inhibition under low-nutrient conditions. In combination with biochemical assays, we found evidence for a pathway involving inositol 1,4,5-trisphosphate (IP3) and cytosolic calcium that may mediate autocrine proliferation inhibition in Dictyostelium.

RESULTS
In addition to a G protein-coupled receptor and a Ras protein, a Ras GEF potentiates polyphosphate inhibition of cell proliferation. We previously observed that polyphosphate inhibits the proliferation of wild-type D. discoideum cells and that the loss of GrlD, RasC, or polyphosphate kinase 1 (Ppk1) reduces the ability of polyphosphate to inhibit proliferation (9,23), suggesting the existence of a polyphosphate signal transduction pathway. To identify additional components of the polyphosphate proliferation inhibition pathway, 52 available mutants were screened for sensitivity to polyphosphate-induced proliferation inhibition under the low-nutrient condition of 25% HL5. The data were graphed in 9 groups: commonly used parental/wild-type cells (Ax2 to HPS400) and previously reported polyphosphate signal transduction pathway components (Fig. 1A), G protein subunits (Fig. 1B), AprA pathway components (Fig. 1C), selected cAMP pathway components (Fig. 1D), phospholipase C (PLC)/IP3 pathway components (Fig. 1E), mitogen-activated protein kinase (MAPK) pathway/polyphosphate synthesis pathway components (Fig. 1F), D. discoideum developmentrelated proteins (Fig. 1G), TOR complex components/protein kinases (Fig. 1H), and mechanotransduction components (Fig. 1I). The initial cell density was 1.5 Â 10 6 cells/ ml, and cells were counted 24 h later. The data were plotted as 100 Â (density with polyphosphate 2 1.5 Â 10 6 cells/ml)/(density with no added polyphosphate 2 1.5 Â 10 6 cells/ml). This value would then be 100 if the polyphosphate had no effect on cell proliferation and 0 if the polyphosphate completely inhibited cell proliferation. Compared to no added polyphosphate, 125 mM and 150 mM polyphosphate reduced the increase in the cell density of Ax2 wild-type cells to ;30% and ;18%, respectively (Fig. 1A). At 24 h, the density of the Ax2 cells with no polyphosphate was 3.9 Â 10 6 6 0.1 Â 10 6 cells/ml (mean 6 standard error of the mean [SEM]) (n = 7) (see Table S2 in the supplemental material), so the 18% cell density increase at 24 h represents a change in the doubling time from the control value of 17.7 6 0.7 h to 81.3 6 16.7 h. Polyphosphate also reduced the proliferation of all the other commonly used parental/wild-type strains (Fig. 1A). The proliferation of these strains in the absence of added polyphosphate, and all of the mutant strains described below, is shown in Table S2.
As previously reported, compared to Ax2 cells, cells lacking the putative polyphosphate receptor GrlD (23) or the Ras protein RasC (24) showed abolished sensitivity (no significant difference compared to no added polyphosphate by a t test) to 125 and 150 mM polyphosphate (Fig. 1A). Cells lacking GefA, a Ras guanine nucleotide exchange factor (GEF) for RasC but not RasB, RasD, or Rap1 (25), also showed reduced sensitivity to polyphosphate (Fig. 1A). The density of cells lacking RasG after 24 h was 80% 6 18% (mean 6 SEM) (n = 3) of the initial cell density, suggesting that cells lacking rasG (rasG 2 cells) do not grow in 25% HL5.
The Gb subunit potentiates polyphosphate inhibition of cell proliferation. Cells lacking the heterotrimeric G protein subunit Gb (26) showed reduced sensitivity to polyphosphate inhibition of cell proliferation (Fig. 1B). Cells lacking Ga2, -3, -4, -5, -7, -8, or -9 did not have significantly abnormal sensitivity to polyphosphate. Cells lacking Ga1 (27) showed increased sensitivity to polyphosphate at 150 mM compared to their FIG 1 Some signal transduction pathway components are needed for polyphosphate (polyP) inhibition of proliferation in 25% HL5. The indicated cell lines were tested for proliferation with 0, 125, or 150 mM polyphosphate for 24 h. The increase in cell density over 24 h was normalized to the value with no added polyphosphate for the indicated strain. For each strain, the left bar is with 125 mM, and the right bar is with 150 mM polyphosphate. All values are means 6 SEM (n $ 3 independent experiments). * indicates a P value of ,0.05 compared to the parental wild-type cells with the same concentration of polyphosphate (by 2-way ANOVA, with multiple comparisons with Dunnett's test within the panel). 0 indicates not significantly different from 100, and thus, the associated concentration of polyphosphate does not significantly inhibit proliferation in that mutant (by a two-tailed one-sample t test).
Autocrine Proliferation Inhibition ® parental strain HPS400. Whereas cells lacking the putative receptor GrlD appeared to be completely insensitive to polyphosphate, none of the Ga mutants showed complete insensitivity. Comparing the values for grlD 2 cells in Fig. 1A to those for the G protein mutants in Fig. 1B, although at 125 mM, the difference for gb 2 was not significant, at 150 mM, the differences for gb 2 were significant, with a P value of ,0.01 (by t tests). These results suggest that there is an additional pathway downstream of GrlD that does not involve the single characterized Gb in Dictyostelium (26) and that GrlD may activate multiple Ga subunits or untested Ga subunits.
The MAPK/Erk pathway components Erk1 and MekA potentiate polyphosphate inhibition of cell proliferation. Compared to their parental KAx3 or JH10 cells, cells lacking the extracellular signal-regulated kinase Erk1 (52) or the Erk1 kinase MekA (53) showed reduced sensitivity to 125 mM or 150 mM polyphosphate inhibition of cell proliferation (Fig. 1F). Deleting the suppressor of MekA, SmkA (53), did not significantly alter sensitivity to polyphosphate (Fig. 1F). These results suggest that the MekA-Erk1 pathway is involved in polyphosphate proliferation inhibition.
The polyphosphate synthesis pathway components I6kA and Ppk1 potentiate polyphosphate inhibition of cell proliferation. The inositol phosphate kinase I6kA does not appear to affect intracellular polyphosphate levels at cell densities below Tang et al. ® ;1 Â 10 7 cells/ml but plays a role in upregulating intracellular polyphosphate at cell densities of $2 Â 10 7 cells/ml (9). The polyphosphate kinase Ppk1 is essential for intracellular polyphosphate production at all cell stages (12). Compared to their parental Ax2 cells, cells lacking I6kA showed reduced sensitivity to 125 mM polyphosphate. Cells lacking Ppk1 showed abolished sensitivity to 125 mM polyphosphate and strongly reduced sensitivity to 150 mM polyphosphate (Fig. 1F). The correlation between intracellular polyphosphate synthesis and sensitivity to extracellular polyphosphate suggests that intracellular polyphosphate plays a role in polyphosphate inhibition of cell proliferation.
The development-related Gdt proteins potentiate polyphosphate inhibition of cell proliferation. Members of the growth-differentiation transition family of proteins (Gdts) are Dictyostelium-specific tyrosine kinase-like proteins, classified by their sequence similarity and their participation in development (54). Gdt1 and Gdt2 are negative regulators of the Dictyostelium growth-differentiation transition process (54,55), but there is no report about the function of Gdt4 yet. Compared to their parental Ax4 cells, cells lacking growth-differentiation transition family member 2, or both Gdt1 and -2, showed reduced sensitivity to both 125 mM and 150 mM polyphosphate (Fig. 1G). Cells lacking Gdt4 had reduced sensitivity to 125 mM polyphosphate. Cells lacking the protein contact site A CsaA (56) or the ammonium transporter AmtA (57) did not show significantly altered sensitivity to polyphosphate. These results suggest that Gdt2 and Gdt4 may play a role in cell proliferation.
The cell aggregate size regulator SmlA attenuates polyphosphate inhibition of cell proliferation. The small-aggregate formation protein SmlA regulates the size of cell aggregates and fruiting bodies during development by inhibiting the extracellular accumulation of the group size-regulating factor counting factor (58,59). Compared to their parental strain DH1, for unknown reasons, cells lacking SmlA showed increased sensitivity to 125 mM polyphosphate and appeared to be hypersensitive to 150 mM polyphosphate (after 24 h, this polyphosphate concentration caused the cell density to decrease from 1.5 Â 10 6 cells/ml to 1.2 Â 10 6 6 0.2 Â 10 6 cells/ml [mean 6 SEM] [n = 4]) (Fig. 1G).
The TORC2 component PiaA and the protein kinase PKA-C potentiate polyphosphate inhibition of cell proliferation. Dictyostelium Tor complex 2 (TORC2), composed of Tor, PiaA, Lst8, and Rip3, regulates adenylyl cyclase ACA (60,61) and protein kinase B/Akt activation (60,62) and is essential for cell aggregation (60,63). Cells lacking the TORC2 component PiaA (Rictor) but not Lst8 showed abolished sensitivity to both 125 mM and 150 mM polyphosphate, suggesting that PiaA is an essential component of the polyphosphate proliferation inhibition pathway (Fig. 1H). Compared to their parental JH10 cells, cells lacking the cAMP-dependent protein kinase catalytic subunit PKA-C (64) showed reduced sensitivity to polyphosphate inhibition of cell proliferation, suggesting that cAMP might be a messenger in the polyphosphate proliferation inhibition pathway (Fig. 1H). Compared to wild-type cells, cells lacking Lst8 or protein kinase C (PKCA) did not show significantly abnormal sensitivities to polyphosphate (Fig. 1H), indicating that some components of the PKCA pathway are dispensable for polyphosphate to inhibit proliferation.
Four mechanotransduction components do not significantly affect polyphosphate inhibition of cell proliferation. Testing a variety of other signal transduction pathway components, we observed that cells lacking the mechanotransduction components SibA (an integrin beta-like protein) (65), TPC2 (two-pore calcium channel protein 2) (65), TrpP (the transient receptor potential cation channel protein) (65), or Mcln (an ortholog of mucolipin) (65) did not show significantly altered sensitivities to polyphosphate compared to their parental DH1 cells (Fig. 1I). These results suggest that many components of the mechanotransduction pathway are dispensable for polyphosphate to inhibit proliferation.
To further test the effects of the genes encoding Gb, GefA, PTEN, PLC, IplA, Ppk1, and PiaA on the cells' sensitivity to polyphosphate, mutant and available complemented strains were tested for sensitivity to polyphosphate with a more extensive dose-response curve in 25% HL5 (Fig. S1) (these assays were previously done for GrlD and RasC [17]). Compared to their respective parental wild-type cells, cells lacking Gb, GefA, PTEN, PLC, IplA, Ppk1, or PiaA showed reduced sensitivity to physiological levels of polyphosphate (150 mM or lower) (Fig. S1). The 50% inhibitory concentrations (IC 50 s) of polyphosphate proliferation inhibition of these knockout mutant strains were higher than that of parental wild-type cells in 25% HL5 (Table 1). Expressing PTEN in pten 2 cells and PLC in plC 2 cells rescued or partially rescued the decreased sensitivity to polyphosphate (Table 1 and Fig. S1C and D).
To determine if these proteins are also involved in the polyphosphate signal transduction pathway under nutrient-rich conditions, the corresponding knockout strains were tested for sensitivity to polyphosphate with dose-response curves in 100% HL5 (Fig. S2). In 100% HL5, compared to parental wild-type cells, cells lacking Gb, GefA, PTEN, PLC, IplA, Ppk1, or PiaA also showed reduced sensitivity to polyphosphate (Fig. S2). In 100% HL5, the proliferation inhibition curve fits for gb 2 and pten 2 cells could not be generated, and the curve fits for gefA 2 , plC 2 , iplA 2 , ppk1 2 , and piaA 2 cells were ambiguous. The IC 50 s of polyphosphate proliferation inhibition of these knockout mutant strains were higher than that of parental wild-type cells (Table 1). Expressing PTEN in pten 2 cells and PLC in plC 2 cells appeared to partially rescue or rescue the decreased sensitivity to polyphosphate (Table 1 and Fig. S2C and D). Together, these results support the idea that Gb, GefA, PTEN, PLC, IplA, Ppk1, and PiaA affect the polyphosphate proliferation inhibition signal transduction pathway under both low-and high-nutrient conditions.
Gb, PTEN, PLC, IplA, Ppk1, and PiaA affect cell proliferation. To assess the effect of the disruption of these genes on general cell proliferation, we assayed proliferation curves of the above-described strains in 100% HL5 in a shaking culture (Fig. 2), except for gb 2 cells, which were assayed previously (66). The doubling times at a low cell a IC 50 s were calculated from the data in Fig. S1 and S2 in the supplemental material, using Prism with nonlinear regression (sigmoidal dose-response, variable slope, and the top constrained to 100). All values are means 6 SEM (n $ 3 independent experiments). *, P , 0.05; **, P , 0.01; ***, P , 0.001 (compared to the parental wildtype strain Ax2 or DH1 [by a two-tailed t test or one-way ANOVA followed by Tukey's test among DH1, plC 2 , and plC 2 /plC cells or among Ax2, pten 2 , and pten 2 /pten-GFP cells]). @, P , 0.001 (compared to plC 2 or pten 2 cells [by one-way ANOVA with Tukey's test among DH1, plC, and plC 2 /plC cells or among Ax2, pten 2 , and pten 2 /pten-GFP cells]). density (;0.5 Â 10 6 to 6 Â 10 6 cells/ml) and a high cell density (6 Â 10 6 cells/ml to the maximal cell density or plateau) were calculated. At low cell densities, where the extracellular polyphosphate concentration is expected to be low, cells lacking PTEN or Ppk1 had a longer doubling time than Ax2 cells (Table 2), and cells lacking Gb or PLC had a shorter doubling time than the parental wild-type DH1 cells (66). Expressing PTEN in pten 2 cells rescued the long-doubling-time phenotype, and expressing PLC in plC 2 cells further shortened the doubling time (Table 2), possibly because too little or too much PLC potentiates cell proliferation. At high cell densities, where the extracellular polyphosphate concentration is expected to be high, cells lacking IplA, Ppk1, or PiaA had shorter doubling times than Ax2 cells (66), and cells lacking PLC had a longer doubling time than DH1 cells ( Table 2). Expressing PLC in plC 2 cells caused a shorter doubling time than in DH1 cells (Table 2). These data suggest that PTEN and Ppk1 promote cell proliferation at low cell densities; PLC promotes cell proliferation, and IplA,  Table 2.
Autocrine Proliferation Inhibition ® Ppk1, and PiaA slow cell proliferation at high cell densities. The maximal cell density is abnormally high in cells lacking Gb, GefA, IplA, Ppk1, or PiaA (66) ( Fig. 2A, D, E, and F and Table 2) and is abnormally low in cells lacking PTEN or PLC ( Fig. 2B and C and Table 2). Expressing PTEN in pten 2 cells and PLC in plC 2 cells rescued or reversed the phenotype ( Fig. 2B and C and Table 2). These data suggest that these genes affect the proliferation of D. discoideum cells.
Polyphosphate upregulates inositol 1,4,5-trisphosphate. PLC catalyzes the hydrolysis of PIP2 to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (50,67). PLC and the putative IP3 receptor IplA potentiate polyphosphate inhibition of cell proliferation, suggesting that IP3 might mediate polyphosphate proliferation inhibition. To examine this, we measured the effect of polyphosphate on IP3 levels with an IP3 enzyme-linked immunosorbent assay (ELISA) kit. IP3 levels in Ax2 cells were increased with 125 mM polyphosphate at 4 and 8 h and were increased with 150 mM polyphosphate at 1, 2, 4, 8, and 24 h (Fig. 3A and B). At 4 h, 150 mM polyphosphate increased IP3 in gb 2 , rasC 2 , iplA 2 , and ppk1 2 cells (Fig. 3C). The upregulation of IP3 for gb 2 cells is slight but statistically significant. Polyphosphate did not significantly affect IP3 levels in grlD 2 , gefA 2 , pten 2 , plC 2 , and piaA 2 cells, and expressing PTEN in pten 2 cells and PLC in plC 2 cells partially rescued the response (Fig. 3C), possibly because the complementation, with the expression of the cDNA from an actin promoter, causes abnormally high or low levels of the complementing mRNA. Compared to Ax2 cells, the baseline IP3 levels of grlD 2 , plC 2 /plC, and ppk1 2 cells were significantly higher, and the baseline IP3 level of piaA 2 was significantly lower (Fig. 3C). These results indicate that polyphosphate upregulates IP3 in D. discoideum; that this upregulation requires GrlD, GefA, PTEN, PLC, and PiaA; and that Gb, RasC, IplA, or Ppk1 is dispensable for polyphosphate-induced upregulation of IP3.
Polyphosphate upregulates cytosolic free Ca 2+ . IP3 activates IP3 receptors on the endoplasmic reticulum, leading to Ca 21 release from the endoplasmic reticulum lumen to the cytosol in many organisms (50). In D. discoideum, the putative IP3 receptor IplA is localized mostly in cytoplasmic organelles and at very low levels at the plasma membrane and is involved in Ca 21 entry into the cytosol in response to chemoattractants (48,68). As a partial test of the hypothesis that the GrlD-PLC-IP3-IplA-Ca 21 pathway is required for the inhibition of proliferation by polyphosphate, we examined the effect of polyphosphate on cytosolic Ca 21 . 1,2-Bis(2-aminophenoxy)ethane-N,N,N9, N9-tetraacetic acid (BAPTA-1) dextran, which shows increased fluorescence in the presence of Ca 21 (69), was loaded into Dictyostelium cells by electroporation. This technique loads BAPTA-dextran into the cytosol (69, 70). The BAPTA-1 dextran-loaded cells a For the data in Fig. 2, doubling times were calculated for low cell densities (0.5 Â 10 6 to 6 Â 10 6 cells/ml) and high cell densities (6 Â 10 6 cells/ml to the maximal density reached). Values are means 6 SEM (n $ 3 independent experiments). *, P , 0.05; **, P , 0.01; ***, P , 0.001 (compared to their parental strains [by a t test or one-way ANOVA with Dunnett's test among DH1, plC 2 , and plC 2 /plC cells or among Ax2, pten 2 , and pten 2 / pten-GFP cells]). @, P , 0.001 (compared to plC 2 or pten 2 cells [by one-way ANOVA with Dunnett's test among DH1, plC 2 , and plC 2 /plC cells or among Ax2, pten 2 , and pten 2 /pten-GFP cells]).
Tang et al.
® were then incubated with or without polyphosphate, and Ca 21 levels were analyzed based on the total fluorescence per cell (representing the total Ca 21 amount) ( Fig. 4A and C) and the mean fluorescence per square micrometer of cells ( Fig. 4B and D) to exclude the impact of cell size/surface area. By both measurements, polyphosphate increased cytosolic free Ca 21 in Ax2 cells ( Fig. 4 and Fig. S3). The polyphosphateinduced Ca 21 increase happened in 1 h and was maintained for at least 8 h ( Fig. 4A and B). These data suggest that polyphosphate upregulates the resting Ca 21 level of cells.
To test if GrlD, Gb, GefA, RasC, PTEN, PLC, IplA, Ppk1, and PiaA affect the polyphosphate-induced Ca 21 increase, we measured the Ca 21 levels of the related mutant cells with or without polyphosphate for 4 h. Polyphosphate did not significantly affect cytosolic free Ca 21 in cells lacking GrlD, GefA, RasC, IplA, Ppk1, or PiaA ( Fig. 4C and D); increased Ca 21 in cells lacking PTEN; and reduced Ca 21 in cells lacking Gb or PLC ( Fig. 4C and D). Expressing PLC in plC 2 cells rescued the response to polyphosphate (Fig. 4C and D). Overall, these data suggest that polyphosphate upregulates cytosolic free Ca 21 of D. discoideum, and this requires GrlD, Gb, GefA, RasC, PLC, IplA, Ppk1, and PiaA.
Polyphosphate inhibits cytokinesis. Polyphosphate inhibits the proliferation of cells by inhibiting cytokinesis, causing an increased number of multinucleated cells (9). To determine if the signal transduction components identified above are needed for the effect of polyphosphate on cytokinesis, we measured the number of nuclei per cell in the presence or absence of polyphosphate. For wild-type cells (Ax2, Ax3, KAx3, Ax4, DH1, and JH10), polyphosphate increased the number of nuclei per cell (Table 3). This effect was not observed in cells lacking GrlD, Gb, RasC, PTEN, PLC, IplA, Ppk1, and PiaA (Table 3). Expressing PTEN in pten 2 cells and PLC in plC 2 cells rescued or partially rescued the sensitivity to polyphosphate (Table 3). These data suggest that most of the Autocrine Proliferation Inhibition ® potential signaling components identified above are needed for polyphosphate inhibition of cytokinesis.
Polyphosphate does not upregulate total Ras activity. Ras is activated when it binds to GTP and inactivated when it binds to GDP (71). As RasC is required for the polyphosphate effect on proliferation, we hypothesized that polyphosphate might affect RasC activation. Due to the lack of a RasC-specific detection method, we tested the effect of polyphosphate on the total Ras activity of Ax2 cells. There are 11 Ras proteins in Dictyostelium (72). We did not observe any significant difference in active-Ras levels between cells cultured with 0 and those cultured with 150 mM polyphosphate for 1, 4, and 24 h (Fig. S4). This suggests that the RasC activity needed for the polyphosphate proliferation inhibition pathway might be only a small fraction of the total Ras activity.

DISCUSSION
We screened 52 signal transduction pathway mutants for sensitivity to polyphosphate-induced proliferation inhibition. We found that in addition to the previously reported GrlD receptor and RasC (17), Gb, GefA, PakD, PlaA, PTEN, PLC, IplA, Dd5p4, Erk1, MekA, I6kA, Ppk1, Gdt1, Gdt2, Gdt4, PiaA, and PKA-C potentiate polyphosphate inhibition of cell proliferation, suggesting that a complex signal transduction pathway mediates this example of an autocrine proliferation inhibition mechanism (Fig. 5). Compared to their respective parental cells, gb 2 , gefA 2 , rasC 2 , pten 2 , plC 2 , iplA 2 , ppk1 2 , and piaA 2 cells showed strongly reduced sensitivity to polyphosphate proliferation inhibition but not as abolished as that of grlD 2 cells. This suggests that there might be branched pathways downstream of the receptor GrlD. We observed that the lack of any tested Ga subunit did not abolish the cells' sensitivity to polyphosphate inhibition of cell proliferation (Fig. 1B). This is possibly because multiple Ga subunits are  Table S2 in the supplemental material) do not appear to be part of the polyphosphate signal transduction pathway, indicating that, as expected, many other factors besides polyphosphate affect proliferation.  The polyphosphate signal transduction pathway appears to use components that regulate proliferation in other systems. Ras-, PLC-, and IP3-induced Ca 21 release promotes proliferation, and PTEN and PKA inhibit proliferation in mammalian systems (73)(74)(75)(76)(77)(78)(79). Inhibition of Ras-, PLC-, or IP3-induced Ca 21 release inhibits cell proliferation in various cell types (75,(80)(81)(82). The overexpression of PTEN inhibits cell proliferation in many cancer cell lines (76,77,83), and the activation of PKA inhibits vascular smooth cell proliferation induced by injury (78,79).
Consistent with the observation that polyphosphate induces Erk phosphorylation (17), we found that cells lacking Erk1 showed reduced sensitivity to polyphosphate. Polyphosphate-induced Erk phosphorylation requires RasC (17). Combined with the data in this report, this suggests that RasC-Erk1 is part of a pathway involved in polyphosphate proliferation inhibition.
Polyphosphate is a prestarvation factor that primes Dictyostelium cells for development (17). Polyphosphate induces the expression of the early-onset development protein CsaA (17). Cells lacking the polyphosphate receptor GrlD showed an impaired response to the starvation-induced expression of the aggregation markers CsaA, Car1 (cyclic AMP receptor 1), and AcaA (adenylyl cyclase A) and could not perform normal development (23). Many signal transduction pathway components affecting the Dictyostelium growth-development transition also affect polyphosphate inhibition of proliferation (Fig. 1G). As starvation causes both the cessation of proliferation and the initiation of development, many components involved in the initiation of development might also affect the proliferation inhibition response. Inducing these developmentrelated components could be part of the mechanism whereby high concentrations of extracellular polyphosphate allow cells to anticipate starvation. How bacteria, by either consuming the polyphosphate secreted by Dictyostelium cells or secreting their own polyphosphate, interfere with Dictyostelium polyphosphate signaling is unclear. An intriguing observation is that Dictyostelium cells can proliferate on lawns  of Pseudomonas aeruginosa bacteria that lack the bacterial polyphosphate kinase PPK1 but not on lawns of wild-type P. aeruginosa cells (84). One possibility for this result is that the polyphosphate from wild-type P. aeruginosa cells causes Dictyostelium cells to stop proliferating.
Many components of the AprA and cAMP signal transduction pathways (some components, such as the cAMP receptor cAR1 [85], were not examined) did not affect polyphosphate inhibition of cell proliferation. For those components in these two pathways that potentiated polyphosphate-induced proliferation inhibition, the effect on polyphosphate inhibition was relatively mild. PiaA and Lst8 are both Tor complex 2 components (60), but piaA 2 cells showed some impairment of polyphosphate signaling, while lst8 2 cells showed no significant inhibition, suggesting that PiaA and Lst8 have independent functions.
We tested the effect of the mutants that attenuate polyphosphate-mediated inhibition of proliferation in a shaking culture. pten 2 and ppk1 2 cells proliferated abnormally slowly and gb 2 (66) and plC 2 cells proliferated abnormally quickly at low cell densities, and iplA 2 , ppk1 2 , and piaA 2 cells proliferated abnormally quickly at high cell densities. The maximal cell densities of gb 2 , gefA 2 , iplA 2 , ppk1 2 , and piaA 2 cells were abnormally high, and those of pten 2 and plC 2 cells were abnormally low. Compared to wildtype cells, we expected that mutant cells with reduced sensitivity to polyphosphate would proliferate faster and reach higher maximal cell densities. However, pten 2 cells proliferated slower, and pten 2 and plC 2 cells had a lower maximal cell density. The accumulated extracellular polyphosphate levels of these four mutants might be abnormally high, or the genes knocked out could be required for regulating proliferation through other pathways. Compared to DH1 or plC 2 cells, plC 2 /plC cells (overexpressing PLC in plC 2 cells) proliferate faster and reach a higher maximal density (Table 2), suggesting that the overexpression of PLC does more than just restore the function of the lost gene. As the concentration of accumulated extracellular polyphosphate is low when the cell density is low (9), the faster-proliferation phenotype at this stage supports the hypothesis that plC regulates proliferation through pathways other than polyphosphate. The lack, or overexpression, of PLC caused a faster-proliferation phenotype, indicating that the PLC effect on cell proliferation is dependent on PLC levels.
Polyphosphate proliferation inhibition is potentiated by proteins in the PLC/IP3 pathway. We found that polyphosphate upregulates cellular IP3 levels; that this requires GrlD, GefA, PTEN, PLC, and PiaA; and that Gb, RasC, IplA, and Ppk1 are not required for polyphosphate to upregulate IP3. Together, these results suggest that polyphosphate activates a signal transduction pathway that upregulates IP3 levels. IP3 activates IP3 receptors on the endoplasmic reticulum, leading to Ca 21 release from the endoplasmic reticulum lumen to the cytosol in many organisms (50). We found that polyphosphate upregulates cytosolic Ca 21 levels and that this requires GrlD, Gb, GefA, RasC, PLC, IplA, Ppk1, and PiaA. Polyphosphate thus appears to upregulate the resting cytosolic Ca 21 of Dictyostelium cells, similar to the effects of other signals on resting cytosolic Ca 21 in other systems (86,87).
Polyphosphate upregulated both IP3 levels and cytosolic Ca 21 levels of Ax2 cells but did not significantly alter either IP3 levels or cytosolic Ca 21 levels of grlD 2 , gefA 2 , and piaA 2 cells. These results suggest that GrlD, GefA, and PiaA function upstream of elevating IP3 in the polyphosphate pathway (Fig. 5). As expected, polyphosphate upregulated IP3 levels and did not alter cytosolic Ca 21 levels in cells lacking the inositol 1,4,5-trisphosphate receptor-like protein IplA. In cells lacking RasC or Ppk1, polyphosphate upregulated IP3 but did not affect cytosolic Ca 21 . In cells lacking Gb, polyphosphate upregulated IP3 but downregulated cytosolic Ca 21 . A possible explanation is that Gb, RasC, and Ppk1 are required for IP3 to activate the IplA receptor to release Ca 21 to the cytosol and that GrlD might use components in addition to G proteins to transduce extracellular signals. Unexpectedly, the IP3 levels of cells lacking PTEN or PLC were not altered by polyphosphate, but the cytosolic Ca 21 of cells lacking PTEN or PLC was upregulated or downregulated, respectively. These results suggest that polyphosphate can regulate cytosolic Ca 21 levels through a pathway not involving IP3.
Ppk1 mediates intracellular polyphosphate production, and the intracellular polyphosphate of ppk1 2 cells is undetectable (12). How intracellular (as opposed to extracellular) polyphosphate or Ppk1 affects extracellular polyphosphate-induced proliferation inhibition is unclear. As polyphosphate can bind to free divalent cations such as Ca 21 and Mg 21 (10), one hypothesis is that intracellular polyphosphate might bind to the extracellular polyphosphate-induced elevated cytosolic free Ca 21 , and the intracellular polyphosphate-Ca 21 complex could then function as a second messenger. If this is the case, compared to Ax2 cells, cells lacking Ppk1 should show a higher increase of the fluorescence signal with the BAPTA-1 dextran method after stimulating cells with polyphosphate, as polyphosphatebound Ca 21 could not be detected by BAPTA-1. However, cells lacking Ppk1 lost the polyphosphate-induced cytosolic free Ca 21 increase (Fig. 4) while still showing a polyphosphateinduced IP3 increase (Fig. 3). This result disproves the hypothesis of a polyphosphate-Ca 21 elevation and a Ca 21 -bound polyphosphate pathway. This indicates that Ppk1/intracellular polyphosphate functions downstream of IP3 and upstream of Ca 21 elevation.
Besides proliferation inhibition, polyphosphate inhibits proteasome activity, promotes aggregation, and regulates actin polymerization in D. discoideum cells (23). In both 25% and 100% HL5, polyphosphate reduces proteasome activity, and this requires GrlD and RasC (23). However, in 25% HL5 but not 100% HL5, MG132-induced inhibition of proteasome activity inhibits proliferation (23). In human colon cancer HCT116 cells, the proteasome inhibitor MG132 increases intracellular Ca 21 levels (88), and in mouse embryonic fibroblasts, chelating calcium by BAPTA-acetoxymethyl ester (AM) decreases proteasome activity, while increasing intracellular Ca 21 with 2 mM extracellular Ca 21 and ionomycin treatment increases proteasome activity (89). In Dictyostelium, whether there is cross talk in the polyphosphate signal transduction pathway between proteasome activity and IP3/Ca 21 levels is unclear.
In this report, we identified 7 signaling components in the polyphosphate pathway and showed that polyphosphate appears to inhibit Dictyostelium proliferation through pathways including the IP3/Ca 21 pathway. An intriguing possibility is that similar mechanisms may be used in other eukaryotes for autocrine proliferation inhibition and group and tissue size regulation.
Proliferation inhibition and counts of nuclei. Polyphosphate was prepared by dissolving 0.474 g of ;46-mer (average length) S0169 sodium polyphosphate (Spectrum, New Brunswick, NJ) in 10 ml of PBM (20 mM KH 2 PO 4 , 0.01 mM CaCl 2 , 1 mM MgCl 2 [pH 6.1]) (23) to make a 10 mM stock; the final pH was 6.1, and the pH was thus not adjusted. Mid-log-phase cells (1 Â 10 6 to 4 Â 10 6 cells/ml) cultured in HL5 were collected by centrifugation at 1,000 Â g for 3 min, washed once by resuspension of the cells in PBM and centrifugation at 1,000 Â g for 3 min, and then resuspended in fresh HL5 to 6 Â 10 6 cells/ml. Cell cultures were started by mixing 100 ml of these cells with 300 ml of PBM or HL5 containing the indicated concentrations (adjusted for the dilution with cells) of polyphosphate in the well of a type 353047 24-well plate (Corning, Corning, NY) and incubated in a humid box for 24 h at 21°C. For work with cells in 25% HL5, HL5 was diluted by mixing 1 volume of HL5 with 3 volumes of PBM. Cells were counted at 24 h, and the cell density normalized to the density with no added polyphosphate was calculated. The doubling time and maximal density of each strain were calculated as described previously (23), and the numbers of nuclei per cell were counted as described previously (28). Curve fits and IC 50 calculations were done using Prism (GraphPad, San Diego, CA) with nonlinear regression (sigmoidal dose-response, variable slope, and top constrained to 100).
Extraction and measurement of inositol (1,4,5)-trisphosphate. Cells were grown to mid-log phase and counted, and ;2 Â 10 7 cells were collected by centrifugation, washed with PBM as described above, and then resuspended and incubated in 10 ml 25% HL5 (diluted with PBM) with 0 or 150mM polyphosphate in a shaking culture at 175 rpm. After 1, 2, 4, 8, or 24 h, cells were collected by centrifugation at 1,000 Â g for 3 min and resuspended in 110ml of the supernatant from the centrifugation step in 1.7-ml Eppendorf tubes. From the resuspended cells, 10ml was taken out for cell counts, and the remaining cells were mixed with 100ml 3.5% perchloric acid and incubated on ice for 15 min as described previously (111). Half-saturated KHCO 3 (50ml) was then added to the 200-ml mix to neutralize the lysates, and CO 2 was allowed to escape. The material was then clarified by centrifugation at 14,000 Â g for 5 min at 4°C. The supernatant (200ml) of each tube was transferred to new prechilled 1.7-ml tubes and stored at 0°C. The IP3 levels in the clarified lysates were measured with a type 2515875 IP3 ELISA kit (MyBioSource, San Diego, CA) less than 1 week after extraction. The baseline IP3 levels that we measured ( Fig. 3A and C) are far lower than the levels previously reported using an isotope dilution kit that has been discontinued by the manufacturer (picograms versus micrograms per 10 7 cells) (112,113). Both kits detect IP3 levels based on a competition binding strategy, but the isotope kit used an IP3 binding protein prepared from bovine adrenal cortex, and the ELISA kit uses an anti-IP3 antibody. We hypothesize that the difference between the measured IP3 levels could be caused by the specificity of the anti-IP3 antibody being much higher than that of the bovine IP3 binding protein.
Measurement of cytosolic free Ca 2+ . Mid-log-phase cells (3 Â 10 6 ) were collected by centrifugation at 1,000 Â g for 3 min, washed with ice-cold Sorensen's buffer (14.7 mM KH 2 PO 4 , 2 mM Na 2 HPO 4 [pH 6.1]) twice (each time collecting cells by centrifugation and resuspension), and then resuspended in 95ml icecold Sorensen's buffer. As described previously (70), 90ml of washed cells was then mixed with 10ml 25 mg/ml BAPTA-1 dextran at a 10,000 molecular weight (MW) (Invitrogen, Eugene, OR), loaded into an EC2L 2-mm electroporation cuvette (Midsci, Valley Park, MO), and pulsed once with 850 V at 10mF and 200 X in a GenePulser XCell electroporator (Bio-Rad, Hercules, CA). The cells were then collected by centrifugation, resuspended in 1 ml HL5, and incubated for 30 min at 21°C in a shaking culture at 175 rpm. The cells were then diluted and incubated at 1 Â 10 6 cells/ml with 150 mM polyphosphate or an equal volume of PBM in 25% HL5 for 0.5, 1.5, 3.5, or 7.5 h. The cells were then diluted to 0.3 Â 10 6 cells/ml with 150mM polyphosphate or an equal volume of PBM in 25% HL5, and 300 ml of diluted cells was allowed to adhere in the well of a type 94.6190.802 8-well tissue culture chamber (Sarstedt, Nümbrecht, Germany) for 30 min. Cells were imaged with a 40Â objective on a Ti2 Eclipse inverted epifluorescence microscope (Nikon, Melville, NY). The fluorescence intensity was analyzed by using ImageJ.
Measurement of active Ras. Cells were grown to mid-log phase (1 Â 10 6 to 4 Â 10 6 cells/ml) and counted, and 1 Â 10 6 cells were collected by centrifugation, washed with PBM as described above, and then resuspended and incubated in 1 ml 25% HL5 (diluted with PBM) with 0 or 150 mM polyphosphate. After 1, 4, or 24 h, cells were lysed, and the active Ras levels in the lysates were measured with a Ras activation assay kit (Cytoskeleton, Denver, CO). All the procedures were performed according to the manufacturer's manual except that the cell lysate with 30 mg protein was mixed with 30 mg Raf-RBD protein beads for active Ras pulldown.
Statistics. Statistical analyses were done using Prism (GraphPad). Significance was defined as a P value of ,0.05.

SUPPLEMENTAL MATERIAL
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