Cell Responsiveness to cAMP during the Aggregation Phase of Dictyostelium discoideum: Comparison between the Inhibitory Action of Progesterone and the Stimulatory Action of EDTA and Ionophore A23187

Abstract

Amoebae of the cellular slime mould Dictyostelium discoideum undertake a developmental program under conditions of food deprivation. After a lag period of a few hours, called the interphase, differentiated amoebae express the capacity to migrate toward central collection points where they form multicellular aggregates able to generate fruiting bodies. Aggregation-competent amoebae communicate positional information via cAMP pulses. Oscillating levels of cAMP play a dual role in the differentiation of the cells, since they are also responsible for the induction of several cellular properties characteristic of aggregation competence: chemotactic responsiveness, cell cohesiveness and high levels of cAMP binding sites on the plasma membrane. In previous studies, conditions were defined under which the differentiation of amoebae into aggregation-competent cells was either inhibited or stimulated. 4 × 10 −5 M progesterone was shown to prevent aggregation, while 10 −3 M EDTA plus 5 × 10 −3 M MgCl 2 , or 7 × 10 −7 M ionophore A23187 appeared to accelerate this process by shortening the interphase period. In the present work, experiments were carried out to investigate the molecular level at which these compounds exert their action. Progesterone is shown to inhibit aggregation by rendering amoebae unresponsive to the chemotactic signal, cAMP pulses. Even after prolonged periods of starvation, steroid-treated cells do not develop aggregation competence, but retain the properties characteristic of growth phase cells: they fail to respond to cAMP in the chemotactic tests, but remain sensitive to folic acid; they fail to develop EDTA-resistant contacts and cAMP binding sites on their plasma membrane. Steroid-treated amoebae starved in the presence of exogenously generated cAMP pulses cannot be induced to express any of the characteristics of differentiated cells. The stimulation of cell aggregation obtained by starving cells in the presence of EDTA, MgCl 2 or ionophore A23187, is accompanied by an early development of the properties characteristic of aggregation-competent amoebae: increase of the chemotactic sensitivity to cAMP, loss of the sensitivity to folic acid and increase in the number of cAMP binding sites. Although externally applied cAMP pulses do not act cooperatively with these compounds in stimulating cell differentiation, a synergistic effect is observed with a differentiation stimulating factor (DSF), which is known to make amoebae precociously sensitive to cAMP pulses. Incubation of two aggregateless mutants with EDTA, MgCl 2 , or ionophore, phenotypically suppresses their morphogenetic block. The differentiation of these strains has previously been shown to be fully induced by imposed cAMP pulses. The data are interpreted on the basis of a stimulation of adenyl cyclase by EDTA and ionophore A23187, and further emphasize the importance of this enzyme in the differentiation of Dictyostelium cells.