The Role of Tight Junction Membrane Dynamics in the Serine Protease‐Induced Increase in Epithelial Barrier Function
Notice bibliographique
Résumé
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
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Comment cette classification a été obtenuedéplier
Prédiction distillée sur la base complète
Imitation des enseignantsNi prévalence calibrée, ni vérité terrain. Validation humaine à venir. Apprise à partir de 10 348 étiquettes directes de Codex et de 10 348 étiquettes directes de Gemma. Le mode candidate est l'union des têtes enseignantes seuillées; le consensus est leur intersection. Ces sorties portent le statut machine_predicted_unvalidated et ne sont ni des étiquettes humaines ni des étiquettes directes de modèles de pointe.
Scores Codex et Gemma par catégorie
| Catégorie | Codex | Gemma |
|---|---|---|
| Métarecherche | 0,001 | 0,001 |
| Méta-épidémiologie (sens strict) | 0,000 | 0,000 |
| Méta-épidémiologie (sens large) | 0,000 | 0,000 |
| Bibliométrie | 0,000 | 0,000 |
| Études des sciences et des technologies | 0,001 | 0,000 |
| Communication savante | 0,000 | 0,000 |
| Science ouverte | 0,000 | 0,000 |
| Intégrité de la recherche | 0,000 | 0,000 |
| Charge utile insuffisante (le modèle a refusé de juger) | 0,000 | 0,000 |
Scores machine (provisoires)
Les deux têtes enseignantes du modèle étudiant, lues sur ce travail. Un score ordonne la base pour la relecture; il n'affirme jamais une catégorie, et le statut de validation accompagne chaque rangée tel quel.
Scores de référence d'un modèle non mature (critères de maturité non atteints, 7 itérations). Un score ordonne; il n'affirme jamais une catégorie.
score_only:v0-immature-baseline · tel quel depuis la passe de notation : score_only signifie que le nombre peut ordonner les travaux, et qu'aucune étiquette de catégorie n'en découleClassification
machine, non validéePrédiction automatique; un appel candidat d’une seule tête enseignante, pas un consensus.
Le détail, modèle par modèle et score par score, se trouve en fin de page sous « Comment cette classification a été obtenue ».