Figure 4.
Figure 4. AC point mutant (R2834H) in C-tail alters DP border localization. (A) Colocalization of WT and R2834H DP-GFP SCC9 stable cell lines with keratin, assessed by immunofluorescence. Bars, 10 µm. (A′) Pearson’s correlation coefficients were calculated for DP and keratin where R2834H exhibits increased colocalization ∼2.5-fold compared with WT. *, P < 0.001. Error bars indicate SEM. (B) R2834H DP weakens intercellular adhesion compared with WT DP, assessed by dispase assay. Confluent cell monolayers were lifted with dispase enzyme and subjected to mechanical stress. (B′) Quantification of the number of total particles in each well between each condition was compared (t test; *, P < 0.001 and P < 0.005, respectively) from three independent experiments in which each condition was tested in triplicate. Error bars indicate SEM. (C) FRAP for WT (32% recovery after 11 min) and R2834H (29% recovery after 11 min) DP-GFP in stable lines. The bleach point is indicated as time 0. Images were taken at regular intervals (0.5–10 s) to monitor recovery. Representative series of FRAP regions depicted here. Bars, 1 µm. (C′) FRAP quantification. The means of the relative fluorescence intensities are plotted as functions of time for WT and R2834H DP-GFP from 0–11 min (n = 5 FRAP regions imaged of each condition from three independent experiments). (D and D′) WT and R2834H DP cells, fixed at 0, 0.5, and 2.5 h after being incubated in high calcium media, assessed by immunofluorescence. Bars, 10 µm. *, P < 0.001. Error bars indicate SEM. (E) Monolayers of SCC9 cells stably expressing WT, R2834H, or S2849G DP-GFP were subjected to scratch wounding and imaged. Shown are still frames from Videos 4–6. White arrows mark the forming borders. Red arrow in the R2834H DP image denotes filamentous alignment of DP, further highlighted in Video 5. Bars, 10 µm. (E′) Quantification of fluorescence intensities of DP-GFP expressing cell borders imaged under the same conditions. Fluorescent intensities over time were calculated for representative borders from Videos 4–6 and correspond to borders shown in E. Results are representative of data obtained from >30 videos for each condition. (F and F′) Phosphorylation (red arrow) of R2834H and WT DP S-tag (DP CT), assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (G) WT, S2849G, and R2834H DP S-tag (DP CT) were compared by analyzing band shifts (red arrow denotes 36 kD), assessed by phos-tag gel immunoblotting.

AC point mutant (R2834H) in C-tail alters DP border localization. (A) Colocalization of WT and R2834H DP-GFP SCC9 stable cell lines with keratin, assessed by immunofluorescence. Bars, 10 µm. (A′) Pearson’s correlation coefficients were calculated for DP and keratin where R2834H exhibits increased colocalization ∼2.5-fold compared with WT. *, P < 0.001. Error bars indicate SEM. (B) R2834H DP weakens intercellular adhesion compared with WT DP, assessed by dispase assay. Confluent cell monolayers were lifted with dispase enzyme and subjected to mechanical stress. (B′) Quantification of the number of total particles in each well between each condition was compared (t test; *, P < 0.001 and P < 0.005, respectively) from three independent experiments in which each condition was tested in triplicate. Error bars indicate SEM. (C) FRAP for WT (32% recovery after 11 min) and R2834H (29% recovery after 11 min) DP-GFP in stable lines. The bleach point is indicated as time 0. Images were taken at regular intervals (0.5–10 s) to monitor recovery. Representative series of FRAP regions depicted here. Bars, 1 µm. (C′) FRAP quantification. The means of the relative fluorescence intensities are plotted as functions of time for WT and R2834H DP-GFP from 0–11 min (n = 5 FRAP regions imaged of each condition from three independent experiments). (D and D′) WT and R2834H DP cells, fixed at 0, 0.5, and 2.5 h after being incubated in high calcium media, assessed by immunofluorescence. Bars, 10 µm. *, P < 0.001. Error bars indicate SEM. (E) Monolayers of SCC9 cells stably expressing WT, R2834H, or S2849G DP-GFP were subjected to scratch wounding and imaged. Shown are still frames from Videos 4–6. White arrows mark the forming borders. Red arrow in the R2834H DP image denotes filamentous alignment of DP, further highlighted in Video 5. Bars, 10 µm. (E′) Quantification of fluorescence intensities of DP-GFP expressing cell borders imaged under the same conditions. Fluorescent intensities over time were calculated for representative borders from Videos 4–6 and correspond to borders shown in E. Results are representative of data obtained from >30 videos for each condition. (F and F′) Phosphorylation (red arrow) of R2834H and WT DP S-tag (DP CT), assessed by immunoblotting. Densitometry quantification of three independent experiments. *, P < 0.001. Error bars indicate SEM. (G) WT, S2849G, and R2834H DP S-tag (DP CT) were compared by analyzing band shifts (red arrow denotes 36 kD), assessed by phos-tag gel immunoblotting.

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