Blood-Brain Barrier Protein Claudin-5 Expressed in Paired Xenopus laevis Oocytes Mediates Cell-Cell Interaction

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.

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

“…Using our established protocols, oocytes were paired for the analysis of claudin trans -interactions (Brunner et al 2020 ). Briefly, vitelline membranes were removed, and claudin-expressing oocytes were clustered to induce adhering contact areas.…”

“…The establishment of an alternative amphibian model system for barrier research has recently been described by our group (Vitzthum et al 2019 ), which has shown that oocytes of the African claw frog Xenopus laevis can be employed for the analysis of claudin–claudin interactions. Recently, we have been able to expand this heterologous expression system to the blood–brain barrier protein CLDN5 and to extend the analytical approach by using hydrostatic pressure impulses for the further characterization of claudin trans -interactions (Brunner et al 2020 ). Our current study focuses on fundamental aspects involved in the application of Xenopus oocytes for barrier research in the context of the cytoskeleton of the oocyte.…”

Functional Analysis of Gastric Tight Junction Proteins in Xenopus laevis Oocytes

“…Oocytes were prepared for pairing and treated with pH 5.5 ORi as described before. To test the strength of the contact area, a hydrostatic pressure impulse Assay (HPI) established by Brunner et al [ 14 ] was carried out with the oocyte combinations: CLDN18 + CLDN18 and ctrl + ctrl, such as CLDN4/18 + CLDN4/18, CLDN4/18 + ctrl, ctrl + ctrl.…”

“…Common analyses have included measurement of the uptake of radiolabeled substrates, and electrophysiological experiments such as voltage clamp and patch clamp approaches [ 11 , 12 ]. In recent years, these studies have been extended regarding analyses of gap junction and TJ proteins in paired oocyte approaches [ 9 , 13 , 14 ], which have enhanced the possibilities of single cell interaction and tissue analyses. However, the paired cell approaches do not encompass the effects of tissue factors, and thus questions remain regarding the role of endogenous effectors, including further membrane and membrane-associated proteins.…”

Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4+

“…Xenopus laevis frogs were anaesthetized in a bath solution containing 0.2% MS222 (ethyl 3-aminobenzoate methanesulfonate, Sigma-Aldrich, Taufkirchen, Germany) for 5-10 min at 20 °C. After sufficient anaesthesia was reached, ovarian lobes were obtained by partial ovariectomy [10,78]. X. oocytes were injected with 50 nl RNAsefree water containing 15 to 30 ng of HA-Strep-hTRPV3 cRNA (WPI Nanoliter 2010, World Precision Instruments, Sarasota, FL, USA) to overexpress hTRPV3.…”