Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance
Fig 6
The Arp2/3 complex is essential for all forms of actin pedestal assembly.
(A) ArpC2-floxed mouse fibroblasts were treated with DMSO (ArpC2 Flox) or tamoxifen (ArpC2 KO) for 6 days to delete arpC2 and deplete the Arp2/3 complex. Following 2–6 days of culturing in normal growth media, cells were infected with EPEC or KC12+EspFU for 4 h, fixed, and stained with antibodies to detect HA-Tir (magenta), phalloidin to visualize F-actin (green), and DAPI to label DNA (blue). The arrow denotes a weak actin punctum. The bottom row shows an F-actin basket. Scale bar, 5 µm. (B) ArpC2 Flox and KO cell populations cultured as in A were treated with control siRNAs or independent siRNAs targeting mouse mDia1. Cell lysates were analyzed by immunoblotting with antibodies to detect mDia1, ArpC2, tubulin, and actin. Mean mDia1 and ArpC2 band intensities +/- SEM were normalized to tubulin and actin and quantified from 2 experiments. ND, not detectable. (C) ArpC2 KO cells were treated with control or mDia1 siRNAs, infected with EPEC, fixed, and stained with antibodies to detect HA-Tir (red), phalloidin to visualize F-actin (green), and DAPI to label DNA (blue). Scale bar, 2 µm. (D) Pixel intensity plots were generated from cells treated as in C. Lines were drawn through the pedestal-forming region (indicated by Tir staining and displayed as white lines in panel C) and F-actin intensity along the line was plotted. All lines 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 = 9 pedestal-forming regions per condition from 3 cells).