Figure 2.
Intercellular Communication Ceases upon Ionophore Treatment in a Reversible Manner
(A) FRAP analysis of cells that were stained with fluorescent calcein. In the control experiment (control cells were treated with DMSO to exclude effects of the solvent), all cells showed full recovery of fluorescence after bleaching. After treatment with the ionophore CCCP (50 μM in DMSO), the bleached cells showed four different types of FRAP responses: “full recovery,” “slow increase” (delayed recovery to <50% of original fluorescence), “only two cells” (exchange of calcein only with one neighboring cell), and “no communication” (no recovery). For each FRAP response, representative images are shown at three time points (5 s before bleaching, ∼0.5 s after bleaching, 30–60 s after bleaching). Arrowheads point to the bleached cells. Anabaena was apparently able to control communication upon challenging the proton motif force, because the majority of CCCP-treated cells showed “no communication.” Bar, 5 μm.
(B) Fluorescence recovery curves corresponding to the four FRAP responses that were observed in (A) (color scheme identical to A). Time point t = 0 shows the analyzed cell directly after bleaching.
(C) Cell-cell communication after increasing the concentration of CCCP (color scheme identical to A). The effect of CCCP on cell-cell communication was concentration-dependent for CCCP concentrations between 0.5–50 μM. In the control experiment, cells were treated with 0.002% DMSO. Numbers within the bars indicate the number of analyzed cells (n) from different filaments and represent cumulated results from at least two independent cultures (except for 0.1 μM CCCP).
(D) Recovery of cell-cell communication after incubation in fresh medium lacking CCCP and in the presence of chloramphenicol (color scheme identical to A). Regaining cell-cell communication was independent of de novo protein synthesis, “+” and “−” indicate the presence and absence of CCCP, washing in fresh medium, and chloramphenicol. Numbers in bars indicate number of analyzed cells (n). Shown are cumulated results from at least two independent cultures.