A Ca²⁺-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry

Abstract

When freshly starved amoebae of Dictyostelium discoideumare loaded with the Ca²⁺-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 Ca²⁺-indo-1 fluorescence," and L, or "low Ca²⁺-indo-1 fluorescence." Simultaneous monitoring of Ca²⁺-indo-1 and Ca²⁺-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 Ca²⁺-dependent heterogeneity at the vegetative stage or in slugs. Finally, confirming earlier findings with the fluorophore fura-2 (Azharet al., Curr. Sci.68, 337-342 (1995)), a prestalk-prespore difference in cellular Ca²⁺is present in the cells of the slugin vivo.These findings are discussed in light of the possible roles of Ca²⁺for cell differentiation in D. discoideum.