Figure 10.
CAR acts in conjunction with claudins and JAM-A to regulate the nanometer-scale ordering of ZO-1 and apical junction integrity. (A) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A KO cells. The N-terminus was localized at the membrane-proximal region, while the C-terminus was located on the cytoplasmic side. Dotted lines indicate the plasma membrane. The line scan shows that the N- and C-termini were separated. (B) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A/occludin KO cells. The N-terminus was localized at the membrane-proximal region while the C-terminus was located on the cytoplasmic side. Dotted lines indicate the plasma membrane. The line scan showed that the N- and C-termini were separated. (C) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A/CAR KO cells. The N- and C-termini of ZO-1 were randomly oriented at the membrane-proximal region. Dotted lines indicate the plasma membrane. The line scan shows that the localization of the N- and C-termini was random. It should be noted that a thick line was used as the region of interest for the line scans shown in A–C, resulting in the averaging of multiple puncta. (D) Quantification of the order parameter S. The angle of the HA/Flag pairs was measured, and the deviation angle θ for individual vectors from the average vector was determined. The order parameter S was defined as S = (3cos2θ − 1)/2 and showed that the nanometer-scale ordering of ZO-1 molecules at cell junctions was disorganized in claudin/JAM-A/CAR KO cells (n = 5–6 junctions). (E–L) ZO-1 staining in MDCK II (E), CAR KO (F), JAM-A KO (G), claudin quintuple KO (H), claudin/JAM-A KO (I), claudin/CAR KO (J), claudin/JAM-A/occludin KO (K), and claudin/JAM-A/CAR KO (L) cells. Junction breakage was not observed in MDCK II (E), CAR KO (F), JAM-A KO (G), or claudin KO (H) cells. Claudin/JAM-A KO (I) and claudin/JAM-A/occludin KO (K) cells showed junction breakage with occasional large gaps (asterisks), while claudin/CAR KO cells (J) showed discontinuity in cell junctions (arrow). Claudin/JAM-A/CAR KO cells (L) showed an exaggerated junction breakage phenotype, and numerous large gaps were observed (asterisks). (M) Quantification of the junction breakage phenotype. Data represent the numbers of endpoints of ZO-1 staining per unit area and are presented as mean ± SD (n = 9). ***P < 0.0005, compared by one-way ANOVA with Tukey post hoc test. Scale bars: (A–C) 200 nm; (E–L) 10 μm.

CAR acts in conjunction with claudins and JAM-A to regulate the nanometer-scale ordering of ZO-1 and apical junction integrity. (A) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A KO cells. The N-terminus was localized at the membrane-proximal region, while the C-terminus was located on the cytoplasmic side. Dotted lines indicate the plasma membrane. The line scan shows that the N- and C-termini were separated. (B) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A/occludin KO cells. The N-terminus was localized at the membrane-proximal region while the C-terminus was located on the cytoplasmic side. Dotted lines indicate the plasma membrane. The line scan showed that the N- and C-termini were separated. (C) STED observation of double staining for the N-terminal HA tag (green) and C-terminal Flag tag (magenta) of HA-ZO-1-Flag expressed in claudin/JAM-A/CAR KO cells. The N- and C-termini of ZO-1 were randomly oriented at the membrane-proximal region. Dotted lines indicate the plasma membrane. The line scan shows that the localization of the N- and C-termini was random. It should be noted that a thick line was used as the region of interest for the line scans shown in A–C, resulting in the averaging of multiple puncta. (D) Quantification of the order parameter S. The angle of the HA/Flag pairs was measured, and the deviation angle θ for individual vectors from the average vector was determined. The order parameter S was defined as S = (3cos2θ − 1)/2 and showed that the nanometer-scale ordering of ZO-1 molecules at cell junctions was disorganized in claudin/JAM-A/CAR KO cells (n = 5–6 junctions). (E–L) ZO-1 staining in MDCK II (E), CAR KO (F), JAM-A KO (G), claudin quintuple KO (H), claudin/JAM-A KO (I), claudin/CAR KO (J), claudin/JAM-A/occludin KO (K), and claudin/JAM-A/CAR KO (L) cells. Junction breakage was not observed in MDCK II (E), CAR KO (F), JAM-A KO (G), or claudin KO (H) cells. Claudin/JAM-A KO (I) and claudin/JAM-A/occludin KO (K) cells showed junction breakage with occasional large gaps (asterisks), while claudin/CAR KO cells (J) showed discontinuity in cell junctions (arrow). Claudin/JAM-A/CAR KO cells (L) showed an exaggerated junction breakage phenotype, and numerous large gaps were observed (asterisks). (M) Quantification of the junction breakage phenotype. Data represent the numbers of endpoints of ZO-1 staining per unit area and are presented as mean ± SD (n = 9). ***P < 0.0005, compared by one-way ANOVA with Tukey post hoc test. Scale bars: (A–C) 200 nm; (E–L) 10 μm.

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