Figure 4

NHERF1 is required for cpERM-dependent dynamic reorganization of the cell cortex. (a) The role of ERM phosphorylation in LPA-induced remodeling of the cell cortex. OVCAR-3 cells were transfected with VSVG-tagged Ezrin constructs, including wild-type Ezrin and the phosphorylation-defective mutant (Ezrin-T567A). The transfected cells were treated with 1 μM LPA for 10 min. The fixed cells were immunostained with α-VSVG epitope and α-cpERM antibody, and then observed by confocal microscopy with the focus adjusted to either the top or the bottom side of the cells. CpERM proteins co-localized with VSVG-tagged Ezrin wild-type (arrows), but not with its dominant-negative mutant Ezrin-T567A (arrowheads). All images are from a single experiment, which was representative of three independent experiments. Scale bars, 20 μm. (b–d) Effect of NHERF1 depletion on cpERM-dependent reorganization of the cell cortex. OVCAR-3 cells were transfected with siRNA duplexes specific for NHERF1 or −2. The transfected cells were re-plated onto collagen-coated glass coverslips and serum-deprived for 24 h prior to 1 μM LPA treatment for 10 min. Generation and subcellular localization of cpERM protein was visualized by immunostaining with anti-cpERM antibody (b). Images were acquired by confocal microscopy with the focus adjusted to either the top or the bottom side of the cells. CpERM localization is indicated at surface protrusions (arrows) and at the cell margin (arrowheads). All images are from a single experiment that was representative of three independent experiments. Scale bars, 20 μm. Specific gene silencing of NHERF1 and NHERF2 with transfection with isoform-specific siRNAs (c). Protein levels of NHERF1 and NHERF2 were determined using specific antibodies. Representative western blot images for each group and NHERF isoform are shown. Proportion of LPA-stimulated cells with heavy protrusions (d). Images in each slide were captured in 5–7 randomly selected high-power fields (HPF, × 20 objective) with constant settings. Number of total cells and cells with heavy protrusions defined as those with typical protrusive structures covering the cell edge of the bottom region by more than 1/4. Representative images of the cells with or without typical heavy protrusions are as shown in c. The results from each slide show the percentage of the cells with heavy protrusions. Each bar represents the mean±s.e.m. from three independent experiments. All data represent mean±s.e.m. Pair-wise post hoc test, ^#P<0.001, *P<0.05, NS, non-significant.