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H 2 O 2 Slows GFP‐Occludin Dynamic Mobility into MDCK Cell Tight Junctions
Author(s) -
Amsler Kurt,
Janosevic Danielle,
Axis Josephine
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.673.5
Subject(s) - occludin , tight junction , photobleaching , fluorescence recovery after photobleaching , paracellular transport , chemistry , biophysics , green fluorescent protein , permeability (electromagnetism) , claudin , fluorescence , biochemistry , membrane , biology , optics , physics , gene
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 H 2 O 2 treatment alters the dynamic mobility of the TJ protein, occludin, leading to increased TJ permeability. In this study, we examined the effect of H 2 O 2 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 (t 1/2 , immobile fraction) were estimated by curve fitting. The halftime for recovery from photobleaching (t 1/2 ) in control cells was 51 + 11 seconds (mean + standard deviation, n=10). Treatment of MDCK cells with H 2 O 2 produced a concentration‐dependent increase in t 1/2 for fluorescence recovery (55 mM H 2 O 2 – t 1/2 = 78 + 11 seconds (n=16, p<0.001 to 0 mM H 2 O 2 ); 110 mM H 2 O 2 – 119 + 18 seconds (n=9, p<0.001 to both 0 mM and 55 mM H 2 O 2 )). The immobile fraction was not affected by H 2 O 2 treatment (0 mM – 46 + 8%, 55 mM – 37 + 14%, 110 mM – 44 + 11%, p>0.1). These results indicate that oxidative stress slows the dynamic mobility of GFP‐occludin into the TJ region. (supported by NIH grant 1R15DK091749)