Figure 4. Changes in AJs upon p120 and... | Rockefeller University Press

Figure 4.

$Changes in AJs upon p120 and pkp4 knockout. (a) Western blot of A431-EcGFP cells (EcGFP) and their pkp4-KO and p120-KO derivatives probed for EcGFP (Ec), p120, and pkp4. β-Tubulin staining (tbl) serves as a loading control. Molecular weight markers (in kDa) are on the right. The EcGFP band intensities in the KO cells (relatively to the parental EcGFP cells) are quantified at the bottom. (b) Projections of all x–y optical slices of the same cells stained for EcGFP (GFP, green) and PLEKHA5 (PLEKHA, red). Low-magnification panels show merged images only. Bar, 20 μm. Zoomed regions (dashed boxes) are shown below in both channels. (ap and bl indicate apical and basal ends of the lateral membranes). Bar, 12 μm. Arrows indicate a few remaining PLEKHA5-positive AJs in pkp4-KO cells. (c) Quantification of heterochromatic EcGFP/PLEKHA5-positive pixels (lateral AJs) or EcGFP/vinculin-positive pixels (apical/basal AJs), expressed as fractions of total EcGFP-positive pixels (n = 6 images from three experiments). Means ± SD are shown. (d and e) Time-lapse imaging of EcGFP-pkp4-KO (d) and EcGFP-p120-KO (e) cells at 30-s intervals. Only the first (0 min) and last (60 min) frames are shown (see Fig. 3). Bar, 20 μm. Corresponding zoomed regions (white dashed boxes) are shown below (see Videos 2 and 3). Apical (ap) and basal (bl) edges are indicated. Additional zoomed areas of these contacts (blue dashed boxes) taken 2 min apart (bottom row) illustrate lateral AJ instability in pkp4-KO cells and stability in p120-KO cells. The overlay (right) shows the first frame in green and the second in red; arrows mark AJs that disassembled. Bar, 4 μm. Tracked AJ trajectories are shown as in Fig. 3 b. (f) Schematic representation of net displacements of major AJs according to the trajectories in d and e. The AJs are depicted as in Fig. 3 c. (g) FRAP analysis of lateral AJs in cell lines indicated as in a (n = 15; mean ± SD). (h) Left: Western blot of total cell lysates from control cultures (−) and from parallel cultures extracted for 5 min with 1% Triton X-100 (+), probed for EcGFP, p120, and pkp4 (cells are indicated as in a) Right: Quantification of EcGFP intensities in the extracted cells relative to the corresponding non-extracted controls (five independent experiments). Statistical significance for all graphs was calculated using two-tailed Student’s t tests: ns, nonsignificant; *P < 0.05; **P < 0.01; ****P < 0.0001. The means ± SD are indicated by bars. Source data are available for this figure: SourceData F4.$

Changes in AJs upon p120 and pkp4 knockout. (a) Western blot of A431-EcGFP cells (EcGFP) and their pkp4-KO and p120-KO derivatives probed for EcGFP (Ec), p120, and pkp4. β-Tubulin staining (tbl) serves as a loading control. Molecular weight markers (in kDa) are on the right. The EcGFP band intensities in the KO cells (relatively to the parental EcGFP cells) are quantified at the bottom. (b) Projections of all x–y optical slices of the same cells stained for EcGFP (GFP, green) and PLEKHA5 (PLEKHA, red). Low-magnification panels show merged images only. Bar, 20 μm. Zoomed regions (dashed boxes) are shown below in both channels. (ap and bl indicate apical and basal ends of the lateral membranes). Bar, 12 μm. Arrows indicate a few remaining PLEKHA5-positive AJs in pkp4-KO cells. (c) Quantification of heterochromatic EcGFP/PLEKHA5-positive pixels (lateral AJs) or EcGFP/vinculin-positive pixels (apical/basal AJs), expressed as fractions of total EcGFP-positive pixels (n = 6 images from three experiments). Means ± SD are shown. (d and e) Time-lapse imaging of EcGFP-pkp4-KO (d) and EcGFP-p120-KO (e) cells at 30-s intervals. Only the first (0 min) and last (60 min) frames are shown (see Fig. 3). Bar, 20 μm. Corresponding zoomed regions (white dashed boxes) are shown below (see Videos 2 and 3). Apical (ap) and basal (bl) edges are indicated. Additional zoomed areas of these contacts (blue dashed boxes) taken 2 min apart (bottom row) illustrate lateral AJ instability in pkp4-KO cells and stability in p120-KO cells. The overlay (right) shows the first frame in green and the second in red; arrows mark AJs that disassembled. Bar, 4 μm. Tracked AJ trajectories are shown as in Fig. 3 b. (f) Schematic representation of net displacements of major AJs according to the trajectories in d and e. The AJs are depicted as in Fig. 3 c. (g) FRAP analysis of lateral AJs in cell lines indicated as in a (n = 15; mean ± SD). (h) Left: Western blot of total cell lysates from control cultures (−) and from parallel cultures extracted for 5 min with 1% Triton X-100 (+), probed for EcGFP, p120, and pkp4 (cells are indicated as in a) Right: Quantification of EcGFP intensities in the extracted cells relative to the corresponding non-extracted controls (five independent experiments). Statistical significance for all graphs was calculated using two-tailed Student’s t tests: ns, nonsignificant; *P < 0.05; **P < 0.01; ****P < 0.0001. The means ± SD are indicated by bars. Source data are available for this figure: SourceData F4.