Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance
Fig 2
Chemical inhibition of formins impairs EPEC motility and colonization.
(A) NIH3T3 cells stably expressing mCherry-actin (red) were infected with EPEC (left) or KC12+EspFU (right) for 3.5–4.0 h, treated with the indicated inhibitors, and imaged live for 20–30 min. Bacteria with pedestals were tracked over time (top panels, scale bar, 2 µm), and white lines highlight the paths taken by representative bacteria during imaging. These experiments were used to determine actin-based motility rates (lower panels). Each point represents a single bacterium associated with a pedestal, and lines show the mean speed +/- SEM (n = 25–40 pedestals, 3–4 cells per condition). *** p < 0.001 (ANOVA, Tukey post-hoc tests). (B) Polarized Caco-2 monolayers were treated with the indicated inhibitors for 15 min prior to and during infection with EPEC or KC12+EspFU for 6 h. Monolayers were then fixed and treated with antibodies to detect LPS (red), phalloidin to visualize F-actin (green), and DAPI to label DNA (blue). Scale circles, 500, 100 µm2. (C-D) Experiments shown in B were quantified. Each bar represents the mean macrocolony area +/- SEM (n = 70–124 EPEC colonies, 750–1177 KC12+EspFU colonies). Only macrocolonies larger than 100 µm2 were included in the analysis. * p < 0.05, ** p < 0.01, *** p<0.001 (ANOVA, Dunnett’s multiple comparison test). Significance asterisks in A, C, and D are in reference to the DMSO treated conditions.