A density sensing mechanism in the eukaryote Dictyostelium discoideum
Yuen, Ita Shien
Doctor of Philosophy
I am interested in understanding a mechanism by which multicellular organisms sense number of cells of the same type. The model selected is the conditioned medium factor secreted by Dictyostelium discoideum. In submerged monolayer culture, Dictyostelium cells can differentiate into prespore and prestalk cells at high cell densities in response to cAMP but this process does not occur at low cell densities. However, cells at low densities will differentiate in medium taken from developing cells starved at a high density. The putative factor in the medium was designated CMF for Conditioned Medium Factor (Mehdy and Firtel 1985). In this report, we show that the CMF activity can be separated into high and low molecular weight fractions. The large conditioned medium factor can be purified to a single 80 kD protein with N- and O-linked glycosylation and has CMF activity at a concentration of $\sim$4 pM (0.3 ng/ml). This 80 kD CMF can undergo size reduction to a $\sim$100-fold more active set of smaller peptides with molecular weight less than 10 kD. Glycosylation is required for the activity of the low molecular weight CMF. Starvation triggers the release of CMF from a precursor pool already present in vegetative cells, and diffusion calculations indicate that the CMF level in the vicinity of a single isolate will not accumulate to the threshold concentration $\sim$0.3 ng/ml. CMF antisense transformants do not aggregate, whereas normal development is restored by the addition of purified 80 kD CMF. These results suggest that CMF is a secreted factor that functions in vivo as an indicator of cell density in starved cells. The developing cells simultaneously secrete CMF and monitor its extracellular level. When a majority of the cells in a given area have starved as indicated by the high level of CMF, aggregation is triggered to ensure the onset of development is synchronized. When present below a threshold concentration, the expression of genes required for early development is blocked or not induced. This factor plays an essential role in the regulatory pathway necessary for cells to obtain the developmental competence to induce prestalk and prespore gene expression in response to cAMP.