Figure 5.

ZO-1/ZO-2 regulates epithelial polarity. (A–D) Immunofluorescence analyses of polarity markers. (A₁–F₁) In MDCK II cells, ezrin (A₁), gp135 (B₁), and Forssman antigen (C₁) were selectively localized to the apical membrane, while Na-K ATPase α1 subunit (D₁) and Scribble (E₁) were restricted to the basolateral membrane. γ-Tubulin staining (F₁) shows that centrosomes are aligned in the apical cytoplasm. (A₂–F₂) In ZO-1/ZO-2 dKO cells, epithelial polarity was disorganized, and ezrin (A₂), Forssman antigen (C₂), Na-K ATPase α1 subunit (D₂), and Scribble (E₂) were detected on both the apical and basolateral membranes, while gp135 (B₂) or γ-tubulin (F₂) localization was not severely perturbed. (A₃–F₃) Expression of ZO-1–GFP rescued the epithelial polarity phenotypes of ZO-1/ZO-2 dKO cells. (A₄–F₄) No epithelial polarity defects were observed in claudin quinKO cells. (G–J) Quantitation of polarization index of ezrin (G), gp135 (H), Na-K ATPase α1 subunit (I), and Scribble (J). Graphs represent mean ± SD (n = 3–9). (K) Forssman antigen localization in apical (K₁–K₄) and lateral (K′₁–K′₄) confocal sections. (L) Quantitation of polarization index of Forssman antigen. Graphs represent mean ± SD (n = 6–15). All cells were cultured on Transwell filters for 5–7 d. *, P < 0.05; **, P < 0.005; ***, P < 0.0005, compared by t test. Scale bars: 10 µm.