Brunner N et al. (2023), Cellular Distribution Pattern of tjp1 (ZO-1) in...

XB-ART-59176

J Membr Biol 2023 Feb 01;2561:51-61. doi: 10.1007/s00232-022-00251-z.

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Cellular Distribution Pattern of tjp1 (ZO-1) in Xenopus laevis Oocytes Heterologously Expressing Claudins.

Brunner N , Stein L , Amasheh S .

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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.

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Species referenced: Xenopus laevis

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	Fig. 1. Immunoblot analysis of tight junction (TJ) proteins in X. laevis oocytes Cell membrane lysates applied to 10% stain-free acrylamide gel and transferred onto PVDF membranes. All claudin-injected oocytes membranes revealed claudin-specific signals at the predicted protein mass in accordance with the injected cRNAs (20–27 kDa). RNAse-free water-injected oocytes were treated identically and showed no signal for endogenous expression of claudins. However, specific signals for both tjp1 isoforms α+ (195 kDa) and α− (187 kDa) in claudin-expressing oocytes and water-injected controls confirmed endogenous tjp1 protein expression
	Fig. 2. Immunohistochemical staining of TJ proteins in X. laevis oocytes All claudin-injected oocytes revealed claudin-specific signals at their cell membranes in accordance with the injected CLDN cRNAs (green). RNAse-free water-injected oocytes were treated identically and showed no signal for the endogenous expression of claudins (* representative image of water-injected oocyte screened for endogenous CLDN3 expression). Additionally, immunofluorescent staining in claudin- and water-injected oocytes revealed specific tjp1 signals (red) in oocytes, whereas in no primary antibody controls, no specific signals were detected by confocal microscopy. Tjp1 signals were concentrated in the submembranous space and appeared as a belt-like structure. In CLDN2- and CLDN3-expressing oocytes, claudin and tjp1 signals were selectively colocalized at the plasma membrane and resulted in a yellow signal (arrows). Scale bars: 20 mm
	Fig. 3. Qualitative PCR of tjp1 and housekeeping genes odc1, gapdh, and h4c4 PCR products were loaded onto a 2% agarose gel in TBE buffer. Pooled samples of claudin-injected oocytes showed gene products in accordance with the predicted amplicon size (Table 2) of the housekeeping genes and the gene of interest
	Fig. 4. Quantitative real-time PCR of tjp1 in claudin-expressing X. laevis oocytes PCR delta Ct values in control oocytes were indistinctive from claudin-expressing oocytes at all three tested concentrations of 0.5, 1, and 2 ng cRNA/ oocyte, ANOVA: p ≥ 0.9

References [+] :

Agarwal, Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase-2 activity. 2005, Pubmed