Functional Analysis of Gastric Tight Junction Proteins in Xenopus laevis Oocytes

Abstract

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.

Cannabidiol Strengthening of Gastric Tight Junction Complexes Analyzed in an Improved Xenopus Oocyte Assay

“…We therefore achieved for the first time a successful expression of the three gastric claudins after three days of injection. Recently, the two gastric claudins -4 and -18.2 have already been reported to be successfully co-expressed in the oocyte membrane [ 13 ].…”

“…With this study, we have expanded our established stomach model [ 13 ]. The heterologous expression of human gastric-specific claudins in the Xenopus laevis oocyte allows the direct examination of secondary plant compounds as well as other extracellular factors to the gastric mucosa and provides new possibilities for trans -interaction and TJ integrity analyses of barrier-modulating mechanisms.…”

“…Performing interaction studies by pairing oocytes with heterologously expressed gap junction proteins such as connexins or tight junction (TJ) proteins such as claudins extended the possibilities of using Xenopus oocytes, providing important insight into the physiological function and composition of protein complexes [ 7 , 8 , 9 , 10 , 11 , 12 ]. Moreover, the oocyte is capable of tolerating different physical and chemical environments for analyses, e.g., in acidic pH ranges [ 13 ], and it endogenously expresses the scaffolding protein ZO-1 regarded as a prerequisite for proper membrane insertion and functionality [ 14 ].…”

Selective Role of TNFα and IL10 in Regulation of Barrier Properties of the Colon in DMH-Induced Tumor and Healthy Rats

“…TJ protein levels were analyzed in the colon segments, as described in detail earlier [ 70 ], and stain-free immunoblotting was performed, as described previously [ 71 , 72 , 73 ].…”

The Xenopus Oocyte: A Tool for Membrane Biology

“…By generating substitution point mutations, they demonstrated the functional importance of Pro residues [11]. Finally, Stein et al used the oocyte to express tight junction proteins in a two-cell model [12]. Their data show that the activity of claudin proteins is largely pH-dependent.…”