Dictyostelium discoideum is a unicellular eukaryote that eats bacteria, and eventually overgrows the bacteria. D. discoideum cells accumulate extracellular polyphosphate (polyP), and the polyP concentration increases as the local cell density increases. At high cell densities, the correspondingly high extracellular polyP concentrations allow cells to sense that they are about to overgrow their food supply and starve, causing the D. discoideum cells to inhibit their proliferation. In this report, we show that high extracellular polyP inhibits exocytosis of undigested or partially digested nutrients. PolyP decreases plasma membrane recycling and apparent cell membrane fluidity, and this requires the G protein-coupled polyP receptor GrlD, the polyphosphate kinase Ppk1, and the inositol hexakisphosphate kinase I6kA. PolyP alters protein contents in detergent-insoluble crude cytoskeletons, but does not significantly affect random cell motility, cell speed, or F-actin levels. Together, these data suggest that D. discoideum cells use polyP as a signal to sense their local cell density and reduce cell membrane fluidity and membrane recycling, perhaps as a mechanism to retain ingested food when the cells are about to starve.

For protein content determination, the 1% Triton X-100 insoluble fraction (pellet) was resuspended in 1X SDS sample buffer containing protease and phosphatase inhibitors, and heated at 95ºC for 5 minutes. Samples were loaded onto 4-20% polyacrylamide gel, electrophoresed until the bromophenol blue in the sample buffer had migrated 5 mm from the bottom of the wells, and the portion of the gel from the bottom of the well to the dye front was excised with a clean razor blade, diced into 2 mm2 cubes, and transferred to Eppendorf tubes pre-rinsed with ethanol. Sample preparation for LC-MS was performed using the in-gel protein digestion protocol at the Department of Chemistry mass spectrometry core facility at Texas A&M University (https://mass-spec.chem.tamu.edu/proteomics/proteomics-protocols.php). Mass spectrometry proteomics was performed on a Thermo Scientific Orbitrap Fusion tribrid mass spectrometer equipped with a Dionex UltiMate 3000 reverse-phase nano-UHPLC system.

The LC-MS results were using Proteome Discoverer 2.4 - please find attached an Excel summary. The identified proteins were run against the Dictyostelium discoideum AX4 reference database rather than the AX2, as the AX2 genome sequence doesn't seem to be available on UniProt. Columns AA to AE describe the abundance ratio of the identified proteins in all 3 independent experiments; Columns AF to AK describe the adjusted p-value; Columns AL to AQ describe the abundance variability; Columns AR to AY describe the coefficient of variation; the remaining columns describe which of the four samples a given protein was detected in.

Biological processes, molecular functions, and cellular locations were determined for identified proteins using the Gene ontology resource (http://geneontology.org/), and PANTHER analysis and enrichment analysis were performed using Fisher’s Exact test and Bonferroni correction for multiple testing. Membrane raft proteins were identified in Triton X-100 insoluble membrane fraction by searching the Uniprot database and PubMed.

All the proteins or lipids analyzed from three independent experiments are listed and can be found in the provided Microsoft Excel. Please refer to the README file.