A Ca(2+)-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry

When freshly starved amoebae of Dictyostelium discoideum are loaded with the Ca(2+)-specific dye indo-1/ AM and analyzed in a fluorescence-activated cell sorter, they exhibit a quasi-bimodal distribution of fluorescence. This permits a separation of the population into two classes: H, or "high Ca(2+)-indo-1 fluorescence," and L, or "low Ca(2+)-indo-1 fluorescence." Simultaneous monitoring of Ca(2+)-indo-1 and Ca(2+)-chlortetracycline fluorescence shows that by and large the same cells tend to have high (or low) levels of both cytoplasmic and sequestered Ca2+. Next we label H cells with tetramethylrhodamine isothiocyanate (TRITC) and mix them in a 1:4 ratio with L cells. In the slugs that result, TRITC fluorescence is confined mainly to the anterior prestalk region. This implies that amoebae with relatively high Ca2+ at the vegetative stage tend to develop into prestalk cells and those with low Ca2+ into prespores. Polysphondylium violaceum, a cellular slime mold that does not possess prestalk and prespore cells, also does not display a Ca(2+)-dependent heterogeneity at the vegetative stage or in slugs. Finally, confirming earlier findings with the fluorophore fura-2 (Azhar et al., Curr. Sci. 68, 337-342 (1995)), a prestalk-prespore difference in cellular Ca2+ is present in the cells of the slug in vivo. These findings are discussed in light of the possible roles of Ca2+ for cell differentiation in D. discoideum.