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The Role of Tight Junction Membrane Dynamics in the Serine Protease‐Induced Increase in Epithelial Barrier Function
Author(s) -
Ronaghan Natalie Jane,
Colarusso Pina,
Shen Le,
Turner Jerrold R.,
MacNaughton Wallace K.
Publication year - 2016
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.30.1_supplement.1250.1
Subject(s) - occludin , tight junction , fluorescence recovery after photobleaching , proteases , microbiology and biotechnology , barrier function , trypsin , chemistry , serine , serine protease , confocal microscopy , cell junction , biophysics , protease , cell , biology , biochemistry , membrane , phosphorylation , enzyme
One of the main contributors to intestinal epithelial barrier function is the composition of the tight junction (TJ) between epithelial cells. In IBD, inflammatory mediators induce disruption of the TJ and may contribute to pathogenesis. We have found that the apical addition of the serine proteases trypsin and matriptase to colonic epithelial cell lines induces a rapid and sustained increase in barrier function. However, the mechanism is incompletely understood. Tight junction proteins are continuously trafficked in and out of the junction to maintain and modulate barrier properties. We aimed to determine if serine proteases alter tight junction dynamics in the membrane using fluorescence recovery after photobleaching (FRAP). Previous studies using siRNA suggest that occludin is an important TJ protein in the serine protease‐mediated increase in TER. EGFP‐occludin was stably expressed in SCBN intestinal epithelial cells, plated on transwells and mounted on a Live Cell Instruments flow chamber, placed on a closed Olympus FV1000 confocal microscope at 37°C, and the chamber perfused continuously on the apical side with heated Krebs that was equilibrated with 5% CO 2 . Triplicate baseline FRAP runs were conducted along the junction between two epithelial cells strongly expressing EGFP‐occludin. Cells were then perfused apically with trypsin for 15 minutes, and then follow‐up triplicate FRAP experiments were conducted. The mobile fraction, half time of recovery, and direction of recovery were then determined. FRAP recovery curve analysis shows SCBN cells treated with trypsin exhibit significantly increased EGFP‐occludin in the photobleached area after 90 seconds of recovery compared to untreated cells. The mobile fraction is increased but not significantly different (before treatment 58.08 ± 6.99% and after trypsin treatment 73.35 ± 4.59% (SEM) p=0.1164 n=6). Half time of recovery is also not changed (before trypsin 100.8 ± 8.6s and after trypsin 82.42 ± 10.4s p=0.279). From observation and kymograph analysis, there was also no change in direction of occludin recovery, which was from the lateral membrane, after serine protease treatment. Our results suggest that serine proteases may induce an increased trafficking of occludin into the membrane, or change how occludin binds to other tight junction proteins. Future experiments will investigate phosphorylation and binding of occludin with other TJ proteins and the change in membrane dynamics of other TJ proteins such as ZO‐1, tricellulin, and claudin‐1. Support or Funding Information CIHR, NSERC