Figure 6.

Effect of stretch on endocytosis. Umbrella cells were surface biotinylated and incubated for 0, 5, 15, 30, 60, or 120 min without stretch (A) or with stretch (B). MESNA-protected biotinylated proteins were isolated and Western blots were probed with streptavidin-HRP. Representative blot of control tissue (A) shows no visible endocytosis by the lack of signal in MESNA-treated lanes. Stretched tissue (B) exhibits large MESNA-protected signal, indicating endocytosis. (C) Quantification of endocytosis in control and stretched tissue. Shown is mean ± SEM (n = 5). Values that are significantly different (p < 0.05) from matched control values are marked with an asterisk. (D) WGA-FITC was internalized for 15 min in control unstretched (a) or stretched (b) tissue. Surface-bound WGA-FITC was removed by incubation at 4°C with N-acetyl glucosamine, and the samples were fixed, stained with 4,6-diamidino-2-phenylindole, and then examined byepifluorescence microscopy. A projection of digitally deconvoluted sections is shown. (Dc) Bladder cells were stretched for 2 h, WGA-FITC was added to the chamber and the samples stretched for an additional 15 min. Surface WGA-FITC was removed, the samples were fixed, and then examined in a confocal microscope. A projection of multiple XY sections is shown. (Dd) WGA-FITC was added to an excised but otherwise intact bladder for 15 min. Surface WGA-FITC was removed, the samples were fixed, and then examined in a confocal microscope. A projection of multiple XY sections is shown. (E) Apical surface proteins were biotinylated and the tissue was stretched for 0–300 min. The cells were lysed and biotinylated proteins detected by probing Western blots with streptavidin-HRP. Treatment with 40 μM leupeptin (Leup) prevented the degradation, demonstrating a lysosomal-mediated pathway, whereas treatment with lactacystin (Lact) had no effect. (F) Quantification of apical membrane protein degradation upon stretch. Values are relative to unstretched tissue biotinylated at t = 0. Shown is mean ± SEM (n = 4). Values that are significantly different (p < 0.5) from unstretched tissue at t = 0 are marked with an asterisk.