Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance
Fig 7
mDia1 and the Arp2/3 complex localize to distinct subregions within EPEC pedestals.
(A) HeLa cells were infected with EPEC for 4 h, fixed, and treated with antibodies to detect mDia1 (green) and Arp3 (red), as well as phalloidin to visualize F-actin (magenta), and DAPI to label DNA. Scale bars, 2 µm. (B) Pixel intensity plots were generated from 3 actin pedestals selected from A (white lines i, ii, and iii). Each point represents the relative fluorescence intensity of Arp3 (red) or mDia1 (green) along the 2–3 µm line. All lines were normalized so that a distance of 0 represents the brightest fluorescence of DNA, with the pedestal positioned to the right of 0, and black points represent the maxima. (C) Line scan analyses of EPEC pedestals stained as in A and analyzed as in B were used to calculate the distance from the maximum DNA signal (positioned at 0) to the distance of the maximum signal for F-actin, Arp3, and mDia1 staining. Each point represents the distance within an individual pedestal (n = 18 pedestals), and black lines indicate the mean distance +/- SEM. ** p<0.01 (ANOVA, Tukey post-hoc test). (D) ArpC2 Flox and KO cells were treated with control or mouse mDia1 siRNAs, infected with EPEC, fixed, and stained with antibodies to detect HA-Tir (red) and mDia1 (green), as well as phalloidin to visualize F-actin (magenta), and DAPI to label DNA (blue). The arrow indicates mDia1 enrichment near an accumulation of F-actin adjacent to Tir. Scale bar, 2 µm. (E) HeLa cells were pre-infected for 4 h with EPECΔeae, then washed and challenged with E. coli expressing intimin for 10 min. Cells were fixed and stained with antibodies to detect mDia1 (green) and Arp3 (red), phalloidin to visualize F-actin (magenta), and DAPI to label bacterial DNA. The arrow points to a bacterium lacking a pedestal, and the arrowhead indicates a pedestal. Scale bar, 2 µm.