Purification and the mode of actions of differentiation inducing factors in a cellular slime mold

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Development of the cellular slime mold Dectyostelium discoideum greagly depends on the formation of multicellular complexes. Using this organism, we have undertaken to clarify the nature of the substance(s) which is secreted by cells and induces differentiation. The results are summarised as follows:(1) Cells starved for 0 h (t_0) or 5 h (t_5) in the shake culture at a high cell density do not undergo prespore differentiation when incubated as an isolated, single cell. However, 10 h (t_<10>) starved cells under the same conditions do so in the presence of cAMP and BSA. This indicated that the cellular interactions between t5 and t10 is essential to provide cells an ability to differentiate under isolated conditions.(2) Isolated t_5-cells can undergo prespore differentiation in the conditioned medium obtained from the t_<11>-cell culture, indicating the presence of a substance(s) which renders the t_5-cells competent for differentiation. This substance(s) is heat stable, pronase resistant and water soluble. Amicon fractionation demonstrates the presence of more than two components having different molecular masses. The substances exert thier effects only on the cells starved for more than 5 h.(3) The ability of cells to differentiate under isolated conditions decreases after 11 h of starvation. However, this decrease is restored by addition of another factor(s) obtained from the cells or medium of the late stages of development. This latter factor(s) is heat resistant, soluble in acidic organic solvents and separable into two distinct components by HPLC, both of which have a molecular mass of about 300. These two appear to work at different stages.From these results, it is concluded that the development of D. discoideum proceeds through the action of multiple substances which are secreted by cells at different stages.