Figure 7.
Actin polymerization is required for the apical junction integrity in claudin/JAM-A KO cells. (A) ZO-1 staining of MDCK II cells treated with DMSO. ZO-1 formed a continuous chicken-wire pattern. (A′) ZO-1 staining of claudin/JAM-A KO cells treated with DMSO. Junction breakage was occasionally observed (asterisks). (B) ZO-1 staining of MDCK II cells treated with ROCK inhibitor Y-27632 (10 μM, 3 h). ZO-1 staining was continuous. (B′) ZO-1 staining of claudin/JAM-A KO cells treated with ROCK inhibitor Y-27632 (10 μM, 3 h). The junction breakage phenotype was exaggerated (asterisks). (C) ZO-1 staining of MDCK II cells treated with myosin II inhibitor blebbistatin (100 μM, 3 h). ZO-1 staining was continuous. (C′) ZO-1 staining of claudin/JAM-A KO cells treated with myosin II inhibitor blebbistatin (100 μM, 3 h). The junction breakage phenotype (asterisk) was comparable to that in DMSO-treated claudin/JAM-A KO cells. (D) ZO-1 staining of MDCK II cells treated with LIMK inhibitor BMS-5 (10 μM, 3 h). ZO-1 staining was reduced but remained continuous. (D′) ZO-1 staining of claudin/JAM-A KO cells treated with ROCK inhibitor LIMK inhibitor BMS-5 (10 μM, 3 h). Extensive fragmentation of cell junctions was observed (asterisks). (E) ZO-1 staining of MDCK II cells treated with actin polymerization inhibitor latrunculin A (0.3 μM, 1 h). ZO-1 staining remained continuous. (E′) ZO-1 staining of claudin/JAM-A KO cells treated with actin polymerization inhibitor latrunculin A (0.3 μM, 1 h). Extensive fragmentation of cell junctions was observed (asterisks). (F) 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 < 0.005, ***P < 0.0005, compared by one-way ANOVA followed by Bonferroni’s post hoc test. (G) Snapshots from a two-color time-lapse movie of claudin/JAM-A KO cells expressing ZO-1-GFP (green) and LifeAct-mCherry (magenta) showing a junction breakage event following treatment with Y-27632. Junction breakage is accompanied by loosening of the actin bundles (arrows). (H) Snapshots from a two-color time-lapse movie of claudin/JAM-A KO cells expressing ZO-1-GFP (green) and LifeAct-mCherry (magenta) showing a junction breakage event following treatment with BMS-5. Actin bundles dissolved simultaneously to the junction breakage (arrowheads). Scale bars: 10 μm.

Actin polymerization is required for the apical junction integrity in claudin/JAM-A KO cells. (A) ZO-1 staining of MDCK II cells treated with DMSO. ZO-1 formed a continuous chicken-wire pattern. (A′) ZO-1 staining of claudin/JAM-A KO cells treated with DMSO. Junction breakage was occasionally observed (asterisks). (B) ZO-1 staining of MDCK II cells treated with ROCK inhibitor Y-27632 (10 μM, 3 h). ZO-1 staining was continuous. (B′) ZO-1 staining of claudin/JAM-A KO cells treated with ROCK inhibitor Y-27632 (10 μM, 3 h). The junction breakage phenotype was exaggerated (asterisks). (C) ZO-1 staining of MDCK II cells treated with myosin II inhibitor blebbistatin (100 μM, 3 h). ZO-1 staining was continuous. (C′) ZO-1 staining of claudin/JAM-A KO cells treated with myosin II inhibitor blebbistatin (100 μM, 3 h). The junction breakage phenotype (asterisk) was comparable to that in DMSO-treated claudin/JAM-A KO cells. (D) ZO-1 staining of MDCK II cells treated with LIMK inhibitor BMS-5 (10 μM, 3 h). ZO-1 staining was reduced but remained continuous. (D′) ZO-1 staining of claudin/JAM-A KO cells treated with ROCK inhibitor LIMK inhibitor BMS-5 (10 μM, 3 h). Extensive fragmentation of cell junctions was observed (asterisks). (E) ZO-1 staining of MDCK II cells treated with actin polymerization inhibitor latrunculin A (0.3 μM, 1 h). ZO-1 staining remained continuous. (E′) ZO-1 staining of claudin/JAM-A KO cells treated with actin polymerization inhibitor latrunculin A (0.3 μM, 1 h). Extensive fragmentation of cell junctions was observed (asterisks). (F) 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 < 0.005, ***P < 0.0005, compared by one-way ANOVA followed by Bonferroni’s post hoc test. (G) Snapshots from a two-color time-lapse movie of claudin/JAM-A KO cells expressing ZO-1-GFP (green) and LifeAct-mCherry (magenta) showing a junction breakage event following treatment with Y-27632. Junction breakage is accompanied by loosening of the actin bundles (arrows). (H) Snapshots from a two-color time-lapse movie of claudin/JAM-A KO cells expressing ZO-1-GFP (green) and LifeAct-mCherry (magenta) showing a junction breakage event following treatment with BMS-5. Actin bundles dissolved simultaneously to the junction breakage (arrowheads). Scale bars: 10 μm.

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