Figure 4. Accurate location of CRB2 in the cell membrane of RPE cells.

(A–L) Confocal microscopy Z-stack images of a triple immunolabeling for CRB2 (red), p120 catenin (green) and nuclei (blue) in RPE flatmounts obtained from the basal to apical cell sides with a step size of 300 nm. In the lateral membrane, p120 catenin is expressed more basally (B,E,H) and CRB2 is located apically (G,J). None of these proteins is located at the basal side of the cell membrane (arrowheads in A–C). Towards the apical side, p120 catenin appears (arrowhead in E,F), but not CRB2 (arrowhead in D). Both proteins colocalize in the cell membrane across a short range (arrowheads in G–I). CRB2, but not p120 catenin, is localized at the apical-most side of the cell membrane analyzed (arrowheads in J–L). In (C,F,I,L) an RPE cell scheme represents the relative position where each image was acquired. The black line in C represents the portion of the RPE where none of the proteins studied are located. The green line in F is where only p120 catenin is located. The yellow line in (I) is where both proteins (p120 catenin, green and CRB2, red) colocalize. The red line in (L) represents the area of the RPE cell where CRB2, but not p120 catenin, is present. (M) maximal projection and orthogonal view of the Z-stacks shows the location of the two proteins (arrowheads) in the apical side of the cells. (N) a three-dimensional reconstruction of the Z-stack shows that CRB2 is located apically to p120 catenin. (O) intracellular staining was removed to facilitate the examination of the membrane labeling. Arrows in the upper right margin in (N,O) indicate the orientation from basal (base of the arrow) to apical side of the cells (arrowhead). A movie of this 3D reconstruction is available on “Video file”. Scale bars: 20 μm.