Separation of Dictyostelium discoideum cells into density classes throughout their development and their relationship to the later cell types

This paper describes a fast, non-destructive method for the separation of large quantities of Dictyostelium discoideum cells into density classes at all stages of development. The cells were separated by low-speed centrifugation on preformed, linear Percoll density gradients. On these gradients, cells at all developmental stages showed a unimodal variation in density and this variation in density rapidly increased during the first hours of development. The density was affected by the amount of salt present in the gradient medium, which suggests that it is regulated by a permeability property of the cells. Slug cells showed a unimodal variation in density and did not form bands corresponding to the cell types. However, were able to isolate density fractions which showed a good enrichment of prespore and prestalk cells: 95% and 90%, respectively. Preaggregation cells separated on density gradients yielded fractions which contained different amounts of three developmentally regulated enzymes. Hence, cells at this stage are already heterogeneous in their enzymatic content. Sorting experiments showed a strong correlation between density and developmental fate; the least dense (light) cells preferentially became prestalk cells, and the dense (heavy) cells became prespore cells. This was found for cells at all developmental stages; even vegetative-stage cells showed considerable heterogeneity with regard to density, which was related to their developmental fate. The light cells become prestalk cells, and the heavy cells become prespore cells. Vegetative cells from the various density fractions differed in their DNA content and temporal onset of mitotic activity when resuspended in medium. Therefore, we suggest that the separation of vegetative cells on density gradients results in a separation of cells into cell-cycle phases. Hence, there appear to be cell-cycle-linked differences among vegetative cells, which bias their differentiation towards either the spore or stalk pathway.