Ammonia differentially suppresses the cAMP chemotaxis of anterior-like cells and prestalk cells in Dictyostelium discoideum.

A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum in drop assays. Chemotaxis suppression is mediated by the unprotonated (NH3) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells.

Cell prints on the surface of the slug of Dictyostelium discoideum: a Nessler-positive matrix substance.

A substance or mixture of substances that stains positively with Nessler's reagent is localized above the surface layer of cells of the migrating slug of Dictyostelium discoideum, possibly incorporated into the slime sheath and coating its surface as well. The substance is manifested as outlines ("cell prints") conforming to the profiles of the surface cells and as small globules over the external face of these surface cells. The entire circumference of the slug is covered by the Nessler-positive cell print substance, which is present from the slug tip to the rear of the slug; presumably all cells at the surface of the slug produce the substance. Cell prints are also present on the surface of the culminating slug as well as on the stalk sheath. No cell prints are seen on the slime sheath or in the slime trail, the collapsed sheath behind the slug. Either the Nessler-positive substance is not incorporated into the sheath or it is uniformly smeared into the sheath as the slug migrates through it. Analysis of size and orientation of cell prints reveals characteristic regional differences that correlate with the prestalk/prespore boundary but not with the location of "zones of adhesion." These patterns of cell prints are compared with some current models of slug cell patterns and movements. Nessler's reagent, a sensitive detector of free ammonia, is shown to react with the amino acids asparagine and glutamine as well as with peptides containing these amino acids. Nessler's reactivity to N-acetylglucosamine suggests that polysaccharides containing this amino sugar also react with Nessler's reagent. Several matrix proteins and polysaccharides are enriched in asparagine, and sheath polysaccharides contain N-acetylglucosamine. It is likely that Nessler's reagent is reacting with such matrix proteins and polysaccharides when cell prints are visualized. The possibility is raised of an interaction between such molecules and the ammonia concentration and flux at the slug surface.

Regulation of the anterior-like cell state by ammonia in Dictyostelium discoideum.

Ammonia appears to be an important regulatory signal for several aspects of the Dictyostelium life cycle. The postulated role of ammonia in the determination of the prespore pathway in cells of the slug stage has led us to examine the effect of ammonia on the prestalk/prespore ratio of migrating slugs. In the presence of 10(-3) M ammonium chloride, the volume of the prestalk region decreases by 40.8%. The kinetics of the process make it unlikely that this is due to a shift in the differentiation pathway. A test of the hypothesis that the decrease in volume of the prestalk region is due to the conversion of prestalk cells to anterior-like cells shows that the percent of anterior-like cells in the posterior region increases by the amount predicted by the hypothesis. This suggests that ammonia may be the molecular signal, produced by the tip, that prevents anterior-like cells from chemotactically migrating to the tip and thereby becoming anterior cells. The effect of enzymatic removal of ammonia from vitally stained migrating slugs is the appearance of a series of dark stripes beginning at the posterior end and progressing forward. We interpret this as a result of progressive removal of anterior-like cells from tip dominance and essentially as the formation of new potential tips. Indeed, in a few cases one or even two of the stripes separate from the posterior of the cell mass and form small fruiting bodies. We consider the phenomenon of stripe formation further evidence that the tip acts on anterior-like cells through ammonia.

Ammonia can stimulate or inhibit aggregate density in Dictyostelium mucoroides: a quantitative test of the hypothesis that ammonia is the aggregation-suppressing gas.

Ammonia, at moderate concentrations, stimulates aggregate density of Dictyostelium mucoroides. The range of stimulatory concentrations includes ammonia concentrations established by populations of amebae. At higher concentrations, ammonia inhibits aggregate density. A quantitative test of the hypothesis that ammonia is the aggregation-suppressing gas has been carried out. The concentration of ammonia established over defined populations of amebae is one or two orders of magnitude lower than the concentration of ammonia required to exert the same degree of inhibitory effect as the populations of amebae exert. An additional difference between ammonia and the aggregation-suppressing gas is the fact that increasing concentrations of the aggregation-suppressing gas cause progressively larger aggregation streams, while increasing concentrations of ammonia have no such effect. The stimulatory effect of ammonia at concentrations established by ameba populations indicates that ammonia must be included in the variables affecting the aggregation process and that this ammonia effect must be taken into account in any quantitative modelling of the aggregation process.

Induction of stalk and spore cell differentiation by cyclic AMP in slugs of Dictyostelium discoideum.

Multicellular masses of the cellular slime mold Dictyostelium discoideum, under conditions which ordinarily suppress cell differentiation, develop clusters of stalk cells and spore cells when implanted with Sephadex particles that had been soaked in 5 X 10(-3)M cyclic adenosine monophosphate (AMP). A possible relation exists between oxygen gradients, cyclic AMP gradients, and the pattern of morphogenesis and cell differentiation during fruiting.