Claudins (cldns) represent the largest family of transmembrane tight junction (TJ) proteins, determining organ‐specific epithelial barrier properties. Because methods for the analysis of multiple cldn interaction are limited, we have established the heterologous Xenopus laevis oocyte expression system for TJ protein assembly and interaction analysis. Oocytes were injected with cRNA encoding human cldn‐1, ‐2, or ‐3 or with a combination of these and were incubated in pairs for interaction analysis. Immunoblotting and immunohistochemistry were performed, and membrane contact areas were analyzed morphometrically and by freeze fracture electron microscopy. Cldns were specifically detected in membranes of expressing oocytes, and coincubation of oocytes resulted in adhesive contact areas that increased with incubation time. Adjacent membrane areas revealed specific cldn signals, including “kissing‐point”–like structures representing homophilic trans‐interactions of cldns. Contact areas of oocytes expressing a combination markedly exceeded those expressing single cldns, indicating effects on adhesion. Ultra‐structural analysis revealed a self‐assembly of TJ strands and a cldn‐specific strand morphology.—Vitzthum, C., Stein, L., Brunner, N., Knittel, R., Fallier‐Becker, P., Amasheh, S. Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins. FASEB J. 33, 5312–5319 (2019). http://www.fasebj.org

Epithelial barriers constitute a fundamental requirement in every organism, as they allow the separation of different environments and set boundaries against noxious and other adverse effectors. In many inflammatory and degenerative diseases, epithelial barrier function is impaired because of a disturbance of the paracellular seal. Recently, the Xenopus laevis oocyte has been established as a heterologous expression model for the analysis of transmembrane tight junction protein interactions and is currently considered to be a suitable screening model for barrier effectors. A prerequisite for this application is a physiological anchoring of claudins to the cytoskeleton via the major scaffolding protein tjp1 (tight junction protein 1, ZO-1). We have analyzed the oocyte model with regard to the interaction of heterologously expressed claudins and tjp1. Our experiments have revealed endogenous tjp1 expression in protein and mRNA analyses of unfertilized Xenopus laevis oocytes expressing human claudin 1 (CLDN1) to claudin 5 (CLDN5). The amphibian cell model can therefore be used for the analysis of claudin interactions. Graphical Abstract

Claudin-5 determines the sealing properties of blood-brain barrier tight junctions and its function is impaired in neurodegenerative and neuroinflammatory disorders. Focusing on the contribution of claudin-5 to the trans-interaction within the tight junction seal, we used Xenopus laevis oocytes as an expression system. Cells were clustered and challenged in a novel approach for the analysis of claudin interaction. We evaluated the strengthening effect of claudin-5 to cell-cell-connection in comparison to claudin-3. Application of a hydrostatic pressure impulse on clustered control oocyte pairs revealed a reduction of contact areas. In contrast, combinations with both oocytes expressing claudins maintained an enhanced connection between the cells (cldn5-cldn5, cldn3-cldn3). Strength of interaction was increased by both claudin-3 and claudin-5. This novel approach allowed an analysis of single claudins contributing to tight junction integrity, characterizing homophilic and hetrophilic trans-interaction of claudins. To test a new screening approach for barrier effectors, exemplarily, this 2-cell model of oocytes was used to analyze the effect of the absorption enhancer sodium caprate on the oocyte pairs.

The proinflammatory cytokine tumor necrosis factor (TNF) has been described as one of the main mediators of intestinal inflammatory diseases, affecting the composition of tight junction (TJ) proteins and leading to a disruption of the epithelial barrier. An intact intestinal barrier is mandatory, because the follicle-associated epithelium of Peyer’s patches represents the first defense line of the intestinal immune system and ensures a controlled uptake of antigens from the gut lumen. In the current study, we have analyzed the detailed effects of TNF on the follicle-associated epithelium of porcine Peyer’s patches by applying the Ussing chamber technique. Epithelial tissue specimens of Peyer’s patches and the surrounding villus epithelium were mounted into conventional Ussing chambers and incubated with TNF for 10 h. The transepithelial resistance, representing epithelial barrier function of the tissue, was recorded. A reduction of transepithelial resistance was detected after 8 h in Peyer’s patch tissue specimens, whereas the villus epithelium was not significantly affected by TNF. Subsequent molecular analysis of TJ protein expression revealed a marked decrease of claudin-1 and -4, and an increase of claudin-2. In neighboring villus epithelium, no significant changes in the expression of TJ proteins could be shown. A strong increase of TNF receptor-2 (TNFR-2) could also be detected in Peyer’s patches, in agreement with the major role of this receptor in Peyer’s patches. Our findings were in accordance with changes detected by confocal laser scanning immunofluorescence microscopy. The regulation of TNF effects via myosin light chain kinase (MLCK) was analyzed in blocking experiments. Our detailed analysis is the first to show that TNF affects the barrier function of the follicle-associated epithelium of porcine Peyer’s patches but has no effects on the villus epithelium. These findings reveal not only the basic differences of epithelial barrier function between the two structures, but also the significance of Peyer’s patches as a primary mucosal immune defense.

The epithelial barrier is crucial for proper gastrointestinal function, preventing the unwanted passage of solutes and therefore representing a prerequisite for vectorial transport. Claudin-4 and claudin-18.2, two critical tight junction proteins of the gastric epithelium, seal neighboring cells in a physically and mechanically challenging environment. As the Xenopus laevis oocyte allows the functional and molecular analyses of claudin interaction, we have addressed the hypothesis that this interaction is not only dependent on mechanical force but also on pH. We expressed human claudin-4 and claudin-18 in Xenopus oocytes, and analyzed them in a two-cell model approach. Cells were clustered in pairs to form contact areas expressing CLDN18 + CLDN18, CLDN4/18 + CLDN4/18, and compared to controls, respectively. Contact areas in cells incubated in medium at pH 5.5 and 7.4 were quantified by employing transmitted light microscopy. After 24 h at pH 5.5, clustering of CLDN18 + CLDN18 and CLDN4/18 + CLDN4/18-expressing oocytes revealed a contact area reduced by 45% and 32%, compared with controls, respectively. A further approach, high-pressure impulse assay, revealed a stronger tight junction interaction at pH 5.5 in oocyte pairs expressing CLDN18 + CLDN18 or CLDN4/18 + CLDN4/18 indicating a protective role of claudin-18 for tight junction integrity during pH challenge. Thus, our current analysis of gastric tight junction proteins further establishes oocytes as an expression and two-cell screening model for tight junction integrity analysis of organ- and tissue-specific claudins by the characterization of homo- and heterophilic trans-interaction dependent on barrier effectors.