OL-pc alters the distribution of junctional F-actin and N-cadherin. (A) Double immunostaining for N-cadherin and HA tag. N-cadherin signals are linearly arranged along the cell borders in control and OL-pcΔNBS transfectants. N-cadherin is concentrated along the apical-most region of the obliquely formed cell–cell contacts, which was identified by differential focusing. In OL-pc transfectants, however, N-cadherin assumes a streaklike distribution over the OL-pc–positive cell–cell contact areas (arrows). (B) Double immunostaining for N-cadherin and F-actin. In control cells or OL-pcΔNBS transfectants, N-cadherin colocalizes with nonfibrous F-actin signals (arrowheads), whereas in OL-pc transfectants, N-cadherin is associated with the terminals of radial actin fibers (arrows). (C) Ca2+-switch experiment. Cells were preincubated with 1 mM EGTA to inactivate cadherins and were incubated in normal culture medium for 15 min. Double immunostaining for N-cadherin and F-actin shows that their distribution patterns unique to each transfectants are already observable at this time point (arrows). (D) Cells were treated with DMSO (control) or 0.4 μg/ml cytochalasin D for 30 min at 37°C and were stained for N-cadherin and F-actin. The unique distributions of N-cadherin in OL-pc transfectants were abolished by cytochalasin D (arrowheads). (E) Control and OL-pc-HA transfectants were treated with control, Nap1, or WAVE1 siRNA and double stained for N-cadherin (red) and F-actin (green; shown in each of the merged images). Arrows point to OL-pc transfectant-specific distribution of N-cadherin, and arrowheads indicate the restored distribution of N-cadherin. All images were taken with a confocal microscope. Bars, 10 μm.
