SRA

Dictyostelium discoideum behavior depends on nutrients1. When sufficient food is present these amoebae exist in a unicellular state, but upon starvation they aggregate into a multicellular organism2,3. This unique biology makes D. discoideum an ideal model for investigating how fundamental metabolic pathways command cell differentiation and function. We show here that reactive oxygen species (ROS), generated as a consequence of nutrient limitation, lead to the sequestration of the amino acid cysteine in the antioxidant glutathione, limiting the use of its sulfur atom for processes such as protein translation and FeS cluster-containing enzyme activity that contribute to mitochondrial metabolism and cellular proliferation. Such regulated sulfur sequestration maintains D. discoideum in a non-proliferating state that paves the way for multicellular development. This new mechanism of ROS signaling highlights oxygen and sulfur as simple, early evolutionary signaling molecules dictating cell fate, with implications for responses to nutrient fluctuations in higher eukaryotes. Overall design: Dictyostelium discoideum (D. discoideum) strain Ax4 was purchased from the Dictybase stock center. Vegetatively growing cells were axenically maintained in shaking culture in HL5 nutrient medium (14.3 g/L bacto peptone, 7.15 g/L yeast extract, 18 g/L maltose monohydrate, 0.641 g/L Na2HPO4, 0.49 g/L KH2PO4, supplemented with biotin, cyanocobalamin, folic acid, lipoic acid, riboflavin and thiamine-HCl). Starvation and consequent aggregation were induced by washing D. discoideum four times in development buffer (DB; 5 mM Na2HPO4, 5 mM KH2PO4, 1 mM CaCl2, 2 mM MgCl2 in autoclaved H2O) and plating at a density of 2 x 106 cells/ml in DB on tissue culture-treated plates, without shaking. As a control, vegetatively growing cells were plated at a density of 2 x 106 cells/ml in HL5 on tissue culture-treated plates, without shaking. Cells were starved for different lengths of time with or without treatment with reduced glutathione (1 – 20 mM) with 3 biological replicates per treatment