Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance
Fig 4
Depletion of mDia1 inhibits actin pedestal assembly by EPEC but not by KC12+EspFU.
(A) HeLa cells were treated with control siRNAs or independent siRNAs targeting mDia1 and infected with EPEC or KC12+EspFU for 4 h. Cells were fixed and treated with antibodies to detect HA-Tir (red) and mDia1 (green), and with phalloidin to visualize F-actin (magenta). Scale bar, 10 µm. (B) The whole cell fluorescence intensity for mDia1 from experiments shown in A was measured. Each point represents the average pixel intensity of a single cell, and black lines show the mean intensity +/- SD for 28–32 cells. *** p<0.001, ns = not significant (ANOVA, Tukey post-hoc tests). (C) Lysates from uninfected cells treated in parallel to those in A were analyzed by immunoblotting with antibodies to detect mDia1, actin, and GAPDH. mDia1 band intensity was calculated relative to actin and GAPDH and normalized to 100 in control extracts. Data represent the mean +/- SEM quantified from 9 blots encompassing 7 experiments. (D) The % of adherent bacteria (determined by HA-Tir staining) associated with pedestals was quantified from experiments performed in A. Each point represents a single infected cell (n = 15–20 cells) harboring 10–50 bacteria, and lines display the mean % +/- SD. *** p<0.001, ns = not significant (ANOVA, Tukey post-hoc tests). (E-F) The whole-cell intensity of mDia1 staining (for control and mDia1 depleted cells) was plotted against the % of EPEC (E) or KC12+EspFU (F) forming pedestals on that cell. Each point represents a single cell (n = 45–46 cells). Data were subjected to linear regression analysis, and linear trend lines, including R2 values, are displayed on the plot with p-values describing whether the slopes are significantly non-zero. (G) Cells were treated with control siRNAs or individual siRNAs targeting ArpC4 or mDia1. After fixation, cells were treated with antibodies to detect HA-Tir (red), with phalloidin to visualize F-actin (green), and with DAPI to label DNA (blue). Scale bar, 2 µm. (H) Pixel intensity plots were generated from cells treated as in G. Lines were drawn through the pedestal-forming region (indicated by Tir staining and displayed as white lines in panel G) and F-actin intensity along the 3 µm line was plotted. All plots were normalized so that a distance of 0 represents the brightest fluorescence of HA-Tir, with the bacteria positioned to the left of 0. Points represent the normalized mean fluorescence of F-actin +/- SEM (n = 21 pedestal-forming regions per condition from 4–5 cells).