Figure 1.
Selective expansion of apical membrane of epithelial cells induced by hypo-osmotic stress. (A) Wild-type MDCK cells were treated with hypo-osmotic buffer (150 mOsm/liter) for indicated times. Cells were stained with anti-GP135/podocalyxin mAb (green) and anti-E-cadherin mAb (red). Scale bar, 10 µm. (B) Quantification of the changes of apical plasma membrane area stained with anti-GP135/podocalyxin mAb (red line) and lateral plasma membrane area stained with anti-E-cadherin mAb (blue line). The ratio of surface area under hypo-osmotic stress (SHypo) to surface area under iso-osmotic stress (SIso) from N = 3 independent experiments were plotted. Surface rendering of the plasma membrane area (S) by each antibody staining was performed using Imaris 9.6 software (Bitplane, Inc.). (C) Quantification of E-cadherin fluorescence intensities in (A) under iso-osmotic and hypo-osmotic (2 min) conditions. N = 3 independent experiments; error bar, SD; ns, not significant by Student’s t test. (D) Quantification of podocalyxin fluorescence in A under iso-osmotic and hypo-osmotic (2 min) conditions. N = 3 independent experiments; error bar, SD; **, P = 0.0016 by Student’s t test. (E) EpH4 cells expressing GFP-tagged podocalyxin or YFP-tagged E-cadherin were treated with hypo-osmotic buffer (150 mOsm/liter) for 6 mins. Scale bar, 7 µm. (F) Quantification of the change of the surface area positive for GFP-tagged podocalyxin (red line) or YFP-tagged E-cadherin (blue line). The ratio of surface area at indicated time (S) to surface area at time point = 0 (S0) were plotted from N = 6 independent experiments for GFP-tagged podocalyxin and N = 7 independent experiments for YFP-tagged E-cadherin. Surface rendering of the plasma membrane area (S) by each antibody staining was performed using Imaris 9.6 software (Bitplane, Inc.). (G) Time course change in the ratio of fluorescence intensity (F) under hypo-osmotic stress to fluorescence intensity at time point = 0 (F0) of GFP-tagged podocalyxin (red line) or YFP-tagged E-cadherin (blue line) from data obtained with (Fig. 1 E). (H) Time-lapse imaging of EpH4 cells stained with purified GFP-Lysenin. Hypo-osmotic buffer (150 mOsm) containing the same concentration of purified GFP-Lysenin as in iso-osmotic medium was added at time 0:00. Scale bar, 5 µm. (I) Time course change in the fluorescence intensities of GFP-Lysenin normalized to the value at t = 0 min. Cells cultured in iso-osmotic medium were exposed to 150 mOsm medium at t = 1 min. Frames were taken every 10 s. Means with standard deviations from N = 3 independent experiments are shown.

Selective expansion of apical membrane of epithelial cells induced by hypo-osmotic stress. (A) Wild-type MDCK cells were treated with hypo-osmotic buffer (150 mOsm/liter) for indicated times. Cells were stained with anti-GP135/podocalyxin mAb (green) and anti-E-cadherin mAb (red). Scale bar, 10 µm. (B) Quantification of the changes of apical plasma membrane area stained with anti-GP135/podocalyxin mAb (red line) and lateral plasma membrane area stained with anti-E-cadherin mAb (blue line). The ratio of surface area under hypo-osmotic stress (SHypo) to surface area under iso-osmotic stress (SIso) from N = 3 independent experiments were plotted. Surface rendering of the plasma membrane area (S) by each antibody staining was performed using Imaris 9.6 software (Bitplane, Inc.). (C) Quantification of E-cadherin fluorescence intensities in (A) under iso-osmotic and hypo-osmotic (2 min) conditions. N = 3 independent experiments; error bar, SD; ns, not significant by Student’s t test. (D) Quantification of podocalyxin fluorescence in A under iso-osmotic and hypo-osmotic (2 min) conditions. N = 3 independent experiments; error bar, SD; **, P = 0.0016 by Student’s t test. (E) EpH4 cells expressing GFP-tagged podocalyxin or YFP-tagged E-cadherin were treated with hypo-osmotic buffer (150 mOsm/liter) for 6 mins. Scale bar, 7 µm. (F) Quantification of the change of the surface area positive for GFP-tagged podocalyxin (red line) or YFP-tagged E-cadherin (blue line). The ratio of surface area at indicated time (S) to surface area at time point = 0 (S0) were plotted from N = 6 independent experiments for GFP-tagged podocalyxin and N = 7 independent experiments for YFP-tagged E-cadherin. Surface rendering of the plasma membrane area (S) by each antibody staining was performed using Imaris 9.6 software (Bitplane, Inc.). (G) Time course change in the ratio of fluorescence intensity (F) under hypo-osmotic stress to fluorescence intensity at time point = 0 (F0) of GFP-tagged podocalyxin (red line) or YFP-tagged E-cadherin (blue line) from data obtained with (Fig. 1 E). (H) Time-lapse imaging of EpH4 cells stained with purified GFP-Lysenin. Hypo-osmotic buffer (150 mOsm) containing the same concentration of purified GFP-Lysenin as in iso-osmotic medium was added at time 0:00. Scale bar, 5 µm. (I) Time course change in the fluorescence intensities of GFP-Lysenin normalized to the value at t = 0 min. Cells cultured in iso-osmotic medium were exposed to 150 mOsm medium at t = 1 min. Frames were taken every 10 s. Means with standard deviations from N = 3 independent experiments are shown.

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