Fig. 3. Development of tagA mutants. Cells were allowed to develop on filters as pure populations for 14 hours (A), for 16-18 hours to form slugs (B), or 24 for hours to form fruiting bodies (C). Wild-type (Ax4[ecmA/GFP]) or mutant (tagA^-[ecmA/GFP]) cells expressing green fluorescent protein (GFP) under the control of the ecmA promoter were used to visualize prestalk cells in slugs (B) and spore heads (C) during development. The arrow indicates the lower cup of tagA mutants that appear to contain an excess number of cells. (D) Developing cells were scraped from filters, dissociated into single cells and observed by bright-field and fluorescence microscopy to determine percentage of ecmA/GFP-positive cells. Similar results were obtained at 14 and 18 hours of development whether an entire filter of cells was harvested for counting (D), or 10 individual developing structures were picked from filters, disrupted and counted. (E) Wild-type (Ax4) or tagA^- cells were washed, plated at low density (1x10⁴ cells/cm²) in 24-well plates and incubated with 5 mM cAMP in stalk buffer for 24 hours. Cells were then washed free of cAMP and incubated with DIF or DIF + 5 µM cerulenin for another 24 hours and examined by fluorescence microscopy for the expression of prestalk-specific expression of GFP (ecmA/GFP). Three independent determinations were carried out for each condition and results are given as the mean±s.e.m.

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