Figure 1

Dictyostelium cells in cAMP-induced clumps were primed for vacuolization and cellulose encasing upon second signaling. (a) Induction of cell death. DH1 cells starved for 8 h in SB saline in the presence of cAMP were further incubated for the indicated times, either in SB alone, or in SB containing the inducers DIF-1, c-di-GMP, or DIF-1 and c-di-GMP. SB and cAMP led to enveloped clumps, and addition of inducers led to vacuolization. Note the integrity of c-d-GMP-treated enveloped clumps within which vacuolization occurred. This pattern was found in each of close to 200 independent experiments including groups with or without c-di-GMP. From another point of view, each LabTek chamber seeded with 3 × 10⁵ cells showed about 100 clumps, each containing an average of 30 cells, thus only a minority of cells were recruited into clumps. (b) Vacuolization in but not outside clumps. Cells were starved in the presence of cAMP for 8 h, further incubated for 16 h with c-di-GMP, and examined by confocal microscopy. Isolated cells close to the glass substrate staid non-vacuolated, whereas cells in the enveloped clump vacuolized and acquired pericellular encasings. Extracted from a Z stack, three slices 10 μm apart. (c) Periclump envelopes included cellulose. DH1 (upper) or mutant cellulose-less DH1.DcsA- cells (lower) were starved in the presence of cAMP for 8 h, then further incubated for 40 h in SB and labeled with calcofluor. The DH1.DcsA- clump was negative for calcofluor fluorescence in spite of deliberate overexposure, confirming that the parental DH1 periclump envelope included cellulose. In this and further experiments, at least five clumps were examined per group. (d) Calcofluor staining of periclump envelopes and pericellular encasings. Cells were starved in the presence of cAMP for 8 h, further incubated for 16 h in SB in the absence (upper) or in the presence of c-di-GMP (lower), stained with calcofluor, and examined by confocal microscopy. Slices were extracted from confocal videos. In the absence of c-di-GMP, cells clumped without vacuolization and showed a calcofluor-positive periclump envelope. In the presence of c-di-GMP, cells clumped and vacuolized and showed calcofluor-positive periclump envelopes and pericellular encasings. Scale bars, 10 μm