Xenopus laevis oocyte cell model for barrier research of tight junction proteins

Abstract

Tight junctions (TJs) ring the lateral membrane of polarized epithelial cells and thus establish a border at the apical plasma membrane allowing the epithelium to perform its main function, namely to serve as a selectively permeable barrier. TJs are mainly formed by small (20- 27kDa) transmembrane claudins that span the cellular membrane four times, with the N-terminus and C-terminus being located in the cytoplasm of the cells. The barrier properties of the epithelium are mainly attributed to the claudin protein family (Markov et al. 2015). Various claudin family members can associate into functional groups of sealing or barrier-forming and pore- or channel-forming claudins, but this classification is in part fluent, as claudins can fulfill a range of functions (Günzel and Yu 2013).

The work in this thesis focused on three aspects of a new barrier model:

1. Establishment and validation of a new model for the analysis of claudins by heterologous expression in Xenopus laevis oocytes The functional characterization of claudins and claudin-claudin interactions is of fundamental importance for understanding the physiological properties of barrier function and relevant mechanisms in health and disease. The intention of the research detailed here was to establish a barrier research model as an alternative for transgenic mouse models or primary cell lines. The heterologous expression of claudins was therefore examined in X. laevis oocytes. Claudin-1, claudin-2, and claudin-3 were used for the establishment of the model system. When co-expressed in a single oocyte, the claudins colocalized, with indications of functional heterophilic cis-interactions. Not only were the injected claudins physiologically targeted into the oocyte plasma membrane and formed heterophilic and homophilic trans-interactions, but membrane freeze fracture images also revealed claudin-specific strand assembly patterns for the examined claudins in the oocyte membranes. Moreover, Xenopus oocytes were found to provide many advantages for the implementation of the 3Rs in barriology research.

2. Application of the expression model for the analysis of blood-brain barrier-specific claudin-5 interaction Following the verification of the general suitability of the germ cells for the analysis of claudin cis- and trans-interactions, the expression model was applied to the analysis of blood-brain barrier-specific claudin-5 interactions. In addition to the establishment of a paired oocyte assay in which oocyte contact areas were evaluated, the connection forces of the oocyte junctions were measured by a hydrostatic pressure impulse (HPI) assay. This novel approach allowed for an evaluation of the fraction that the single claudins contribute to the junction of the oocytes and revealed the strength of trans-interaction.

3. Examination of elementary interactions of heterologously expressed claudins with the oocyte scaffolding protein tjp1 In the third set of experiments, interactions were examined between tjp1 and heterologously expressed claudins in Xenopus oocytes. The functional interplay between the scaffolding proteins of the cell and the heterologously expressed claudins is mandatory for further applications of the model system. Immunoblots and immunohistochemical staining confirmed and visualized this functional interplay between the two binding partners, as X. laevis oocytes showed a specific signal for tjp1 in the sub-membranous space without influence of claudin cRNA injection on endogenous tjp1 mRNA expression.