Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance
Fig 5
mDia1 is recruited to EPEC pedestals in a Tir phosphotyrosine-mediated manner.
(A) HeLa cells (upper panels), or HeLa cells transiently expressing GFP-mDia1 (lower panels) were infected with EPEC for 4 h, fixed, and treated with antibodies to detect HA-Tir, antibodies to stain mDia1 (top panels only), and with phalloidin to visualize F-actin. Arrowheads highlight mDia1-positive pedestals. Scale bars, 10 µm. (B) HeLa cells were infected, fixed, and stained as in A. Lines were drawn through pedestals to measure pixel intensity profiles for HA-Tir, F-actin, and mDia1. The brightest pixel from HA-Tir staining was set to a distance of 0 (indicated by the black triangle) for each pedestal to normalize the F-actin and mDia1 curves (n = 15 pedestals, 4 cells). Each point represents the mean normalized pixel intensity (+/- 95% CI), and black points indicate the maxima. (C) The % of Tir-positive EPEC or KC12+EspFU associated with an enrichment in mDia1 staining was calculated from control siRNA experiments described in Fig 4A. Each point represents a single infected cell, and the mean % +/- SEM is indicated in black (n = 16–20 cells). *p<0.05 (unpaired t test). (D) HeLa cells were infected with the indicated strains of EPEC or KC12 for 3.5 h, fixed, and treated with antibodies to detect HA-Tir (red) and mDia1 (green), with phalloidin to visualize F-actin (magenta), and with DAPI to label DNA. Scale bar, 5 µm. (E) 3 adherent bacteria from each panel in D were selected for line scan analysis as in A-B. Each point displays the normalized pixel intensity of F-actin (magenta) or mDia1 (green) along the 2–3 µm line in the corresponding panel. (F) HeLa cells treated with siRNAs against mDia1 were infected with EPEC expressing EspFU-myc, then fixed and stained with antibodies to detect mDia1 (green) and EspFU-myc (red), and with phalloidin to visualize F-actin (magenta). The “+” indicates a cell with detectable levels of mDia1, and the “-” indicates a cell with substantially less mDia1 because of successful knockdown. Scale bar, 25 µm; inset, 5 µm.