Figure 3

rRNA synthesis in non-clumped cells and its inhibition in cells in cAMP-induced clumps. (a) rRNA synthesis in starved cells in the absence of cAMP. In all, 24 h-starved DH1 cells were incubated with EU then fixed, permeabilized, treated with the Alexafluor 488 Click-It reagent, counterstained with DAPI and examined by confocal microscopy. DAPI (pseudocolor blue) and Alexafluor 488 (pseudocolor yellow) pictures were merged. Nucleoli containing newly synthesized rRNA appeared as white bodies at the edge of blue nuclei. Two slices 1.5 μm apart were extracted from the confocal Z stack of Supplementary Video 1, either at lower (close to substrate) or higher levels. White dots in the upper slice, that is, nucleoli containing neo-synthesized rRNA, corresponded to indentations in the DAPI-labeled nuclei in the lower slice. This pattern was found in 17 separate experiments. (b) In a separate experiment, DH1 (left) and DH1.DmtA- cells (right) were starved in the presence of cAMP for 8 h, then without cAMP for a total of 24 h. They were then processed as above. Isolated cells outside clumps, lying on the substrate thus best visible in the lower level slices, showed nucleoli labeled through RNA synthesis. Cells in clumps, best visible in the upper level slices, showed no such labeled nucleoli. Upper and lower slices were 10 and 6 μm apart for DH1 cells and DH1.DmtA- cells, respectively. For DH1 cells, slices were extracted from Supplementary Video 2. This pattern was found for each of a total of about 50 examined clumps in 14 (for DH1) and 3 (for DH1.DmtA-) separate experiments. In this and further experiments, nucleolus negativity of a clump usually means that each confocally visible cell in a clump showed no stained nucleolus. Scale bars, 10 μm