H2O2 Slows GFP-Occludin Dynamic Mobility into MDCK Cell Tight Junctions

Tight junctions (TJ) located at the apicolateral border of adjacent epithelial cells form the primary barrier to the paracellular movement of water and solutes. Oxidative stress of renal epithelia causes increased permeability of the TJ. Previous studies have demonstrated that multiple TJ proteins move dynamically into and out of the TJ structure. We hypothesized that H2O­2 treatment alters the dynamic mobility of the TJ protein, occludin, leading to increased TJ permeability. In this study, we examined the effect of H2O2 treatment on the mobility of occludin protein into and out of the TJ region by Fluorescence Recovery After Photobleaching. GFP-occludin expressed in MDCK cells was localized within the TJ region and along the lateral and apical membranes. A portion of the TJ region was photobleached and the rate of recovery of fluorescence into the photobleached region was monitored. Following photobleaching, GFP-occludin diffused progressively into the TJ region. Kinetic parameters for GFP-occludin (t1/...