FIGURE 2.

NHERF2 anchors PMCA2w/b to the actin cytoskeleton beneath the apical membrane of polarized MDCK cells. A and B, MDCK cells co-expressing GFP-PMCA2w/b (green) and NHERF2 were formaldehyde- fixed, permeabilized with Triton X-100, and stained with anti-NHERF2 antibody (red) and either TRITC-labeled phalloidin (blue in A panels) or anti-ezrin antibody (blue in B panels). In panels A5–A8 and B5–B8, cells were treated with 2.5 μm cytochalasin D for 10 min and in panels A9–A12 and B9–B12 with 1 μm latrunculin B for 30 min before the addition of formaldehyde. The apical sections of typical cells expressing the appropriate constructs are shown. Panels A4, A8, A12, B4, B8, and B12 show merged images, where white indicates overlap of the green, red, and blue staining. C, Pearson's co-localization coefficients of the GFP-PMCA2w/b (2wb), NHERF2 (NH), ezrin, and phalloidin (ph) signals in MDCK cells without (−Cyto) or with (+Cyto) cytochalasin D treatment were determined as described under “Experimental Procedures.” Values represent the mean ± S.D. of calculations from 15 to 20 cells of three independent experiments. D, schematic showing anchorage of PMCA2w/b to the apical actin cytoskeleton by NHERF2 via ezrin. PMCA2w/b binds via its C-terminal ETSL sequence to the PDZ protein NHERF2, which interacts through its ERM domain with ezrin.