Enteropathogenic E. coli relies on collaboration between the formin mDia1 and the Arp2/3 complex for actin pedestal biogenesis and maintenance
Fig 8
mDia1 is important for Src-family kinase activation and phosphorylation of EPEC Tir Y474.
(A) HeLa cells were treated with control or mDia1 siRNAs and infected with EPEC for 4 h. Cells were fixed and stained with antibodies to detect HA-Tir (red) and phosphotyrosine residues (pY, green). Scale bar, 10 µm. (B) The fluorescence intensities of HA-Tir and pY from cells treated as in A were quantified in the pedestal forming regions by outlining the area encompassing HA-Tir and measuring the average pixel intensity within that area. Intensities were normalized to a bacteria-free area of the cell, and then to the mean for control siRNA treated cells, which was set to 1. Each point represents the pY/HA-Tir intensity ratio for an individual bacterium, and black lines show the mean intensity +/- SEM for 131–165 Tir foci from 10 cells per condition. *** p<0.001, ns = not significant (ANOVA, Tukey post-hoc tests). (C) Cells treated with an mDia1 siRNA were fixed and stained with antibodies to detect mDia1 (green) and pY (red), as well as phalloidin to visualize F-actin (magenta), and DAPI to label DNA (blue). The “+” indicates a cell with detectable levels of mDia1, and the “-” indicates a cell with substantially less mDia1 because of successful knockdown. The arrow denotes a bacterium with no pY staining or actin assembly in an mDia1-depleted cell, and the arrowhead highlights a bacterium with pY staining and an actin pedestal in an adjacent mDia1-expressing cell. Scale bar, 25 µm. (D) Cells were treated as in A, but stained with antibodies to phosphotyrosine 416 in active Src-family kinases (pSrc, green). Scale bar, 25 µm. (E) The normalized HA-Tir intensity and pSrc intensity at pedestal forming regions from cells treated as in D were quantified and normalized as in B. Each point represents the normalized pSrc/HA-Tir intensity ratio for an individual bacterium, and black lines show the mean intensity +/- SEM for 70–94 Tir foci from 8 cells per condition. *** p<0.001, ** p<0.01, ns = not significant (ANOVA, Tukey post-hoc tests). (F) HeLa cells were treated with control siRNAs or independent siRNAs targeting mDia1 and either left uninfected (“-”) or infected with EPEC expressing wild type Tir (“WT”) or the Tir Y474F mutant (“F”). Cell lysates were analyzed by immunoblotting with antibodies to mDia1, phosphotyrosine (pTir), HA-Tir, Src phosphotyrosine-416 (pSrc), total Src, actin, and GAPDH. (G) mDia1 band intensities from immunoblots described in F were quantified relative to actin and GAPDH and normalized to the control, which was set to 100. Each point represents the intensity calculated for a single mDia1 band, and the mean +/- SEM is indicated in black. *** p<0.001 (ANOVA, Tukey post-hoc tests). (H) A Tir phosphorylation index was determined from immunoblot band intensities described in F by calculating the pTir to Tir ratio for each condition and normalizing to the control, which was set to 1.0. Each point represents the Tir phosphorylation index within a single sample, and the mean +/- SEM is indicated in black. *** p<0.001 (ANOVA, Tukey post-hoc tests). (I) The mDia1 densitometry points from G were plotted against the Tir phosphorylation indices from H. Data were subjected to linear regression analysis, and the linear trend line and R2 value are displayed on the plot, with the p-value indicating that the slope is significantly non-zero. (J) A Src activation index was determined by calculating the pSrc to Src ratio for each condition and normalizing to the control, which was set to 1.0. Each point represents the Src activation index within a single sample, and the mean +/- SEM is indicated in black. *** p<0.001 (ANOVA, Tukey post-hoc tests).