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Fig. 6. Distribution of occludin, but not claudin-1 and claudin-2, is altered by tMLCK expression. (A) Like ZO-1, arc-like occludin profiles are also remodeled to include irregular undulations 4 hours after induction of tMLCK expression. Although redistributed, occludin remains colocalized with ZO-1 (see merged images). Representative images are shown. (B) Rare claudin-1 undulations can be found before and 4 hours after induction of tMLCK expression. However, overall, tMLCK expression did not cause significant changes in claudin-1 localization. This is confirmed by quantitative analysis (Fig. 7). Representative images are shown. (C) Like claudin-1, focal claudin-2 undulations can be found before and 4 hours after induction of tMLCK expression. Also like claudin-1, the overall distribution of claudin-2 was not changed after induction of tMLCK expression. This is confirmed by quantitative analysis (Fig. 7). Representative images are shown. (D) Triple-label image of claudin-1 (green), ZO-1 (red) and F-actin (blue) in cells expressing tMLCK. Although undulations of the ZO-1 profile can be easily seen (arrows), no such undulations are apparent in the claudin-1 image. F-actin shows some minimal undulations, but to a far lesser degree than ZO-1. The absence of claudin-1 at sites of undulations can also be appreciated in the merged image. (E) High-magnification analysis of undulations in monolayers expressing tMLCK shows that, at these sites, F-actin (green) staining intensity is reduced (arrows). Claudin continues directly across the site of F-actin undulation, without deviating from the arc of the junction. However, claudin-1 labeling is decreased at this site (also seen in panel D). Bars, 10 µm (A-C); 5 µm (D); 2.5 µm (E).
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