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Front Physiol
2020 Jan 01;11:857. doi: 10.3389/fphys.2020.00857.
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Blood-Brain Barrier Protein Claudin-5 Expressed in Paired Xenopus laevis Oocytes Mediates Cell-Cell Interaction.
Brunner N, Stein L, Cornelius V, Knittel R, Fallier-Becker P, Amasheh S.
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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.
Figure 1. Experimental setup of hydrostatic pressure impulse assay (A) schematic top view of the well: central positioning of the oocyte pair was checked before application of the hydrostatic pressure. (B) Schematic side view of the well: 250 μl ORi was added using a single channel electronic pipette. The dispensing speed was uniformely set to maximum speed. The angle (45°) and distance of application was uniformely applied. Ambient pressure, viscosity of ORi and diameter of pipette tip opening were kept under constant conditions.
Figure 2. Detection of heterologously expressed claudins in Xenopus laevis oocytes. (A) Immunoblot analysis of tight junction protein claudin-5 in X. laevis oocytes of three animals (d1–d3). Cell membrane lysates were subjected to SDS-PAGE followed by immunoblot onto PVDF membranes. The membranes were incubated with primary antibodies and secondary peroxidase-conjugated antibodies (n = 3). (B) Immunofluorescent staining revealed specific claudin-5 signals (green) in oocyte membranes of all cRNA-injected oocytes, whereas in water-injected controls, no claudin-specific signals were detected in confocal microscopy. Representative images of oocytes derived from three animals. Scale bars: 50 μm. (C) Immunofluorescent staining of claudin-5 and claudin-3 expressing oocytes revealed specific claudin-3 signals (green) und claudin-5 signals (red) in oocyte membranes of cRNA-injected oocytes, whereas in water-injected controls, no claudin-specific signals were detected. Colocalization of expressed claudin proteins within the oocyte plasma membrane is revealed by double immunofluorescent staining (yellow). Scale bar: 10 μm.
Figure 3. Freeze fracture electron microscopy. (A) Freeze fracture electron microscopy reveals tight junction protein cldn-5 as a meshwork of angular discontinous fibrils in rows in Xenopus laevis oocytes. (B) Freeze fracture electron microscopy reveals tight junction protein cldn-3 as a meshwork of rounded fibrils in X. laevis oocytes. (C) Freeze fracture electron microscopy of claudin-3 and claudin-5 coexpressing oocytes reveal fibrils that both bear properties of claudin-3 and claudin-5. (D) Water injected control oocytes have a smooth surface. Representative images of oocytes derived from three animals. Scale bar: 250 nm.
Figure 4. Median contact areas of clustered oocyte combinations cldn5–cldn5, cldn3–cldn3, cldn3,5–cldn3,5 and control–control 24 and 48 h after clustering in % of initial contact areas shortly after clustering (n = 8–38).
Figure 5. Contact areas of claudin-5, claudin-3, and coexpressing claudin-3 and claudin-5 oocytes in hydrostatic pressure impulse (HPI) challenge after stabilization period and 30 min after HPI (n = 16–70, *p < 0.05, **p < 0.01, Kruskal-Wallis followed by a Dunn–Bonferroni correction).
FIGURE S1 | Contact areas of claudin-5 expressing oocyte pairs in μm2 during incubation with sodium caprate in different concentrations (n = 6–8, respectively) and water-injected oocytes as controls (n = 5–7, respectively).
FIGURE 1. Experimental setup of hydrostatic pressure impulse assay (A) schematic top view of the well: central positioning of the oocyte pair was checked before application of the hydrostatic pressure. (B) Schematic side view of the well: 250 μl ORi was added using a single channel electronic pipette. The dispensing speed was uniformely set to maximum speed. The angle (45°) and distance of application was uniformely applied. Ambient pressure, viscosity of ORi and diameter of pipette tip opening were kept under constant conditions.
FIGURE 2. Detection of heterologously expressed claudins in Xenopus laevis oocytes. (A) Immunoblot analysis of tight junction protein claudin-5 in X. laevis oocytes of three animals (d1–d3). Cell membrane lysates were subjected to SDS-PAGE followed by immunoblot onto PVDF membranes. The membranes were incubated with primary antibodies and secondary peroxidase-conjugated antibodies (n = 3). (B) Immunofluorescent staining revealed specific claudin-5 signals (green) in oocyte membranes of all cRNA-injected oocytes, whereas in water-injected controls, no claudin-specific signals were detected in confocal microscopy. Representative images of oocytes derived from three animals. Scale bars: 50 μm. (C) Immunofluorescent staining of claudin-5 and claudin-3 expressing oocytes revealed specific claudin-3 signals (green) und claudin-5 signals (red) in oocyte membranes of cRNA-injected oocytes, whereas in water-injected controls, no claudin-specific signals were detected. Colocalization of expressed claudin proteins within the oocyte plasma membrane is revealed by double immunofluorescent staining (yellow). Scale bar: 10 μm.
FIGURE 3. Freeze fracture electron microscopy. (A) Freeze fracture electron microscopy reveals tight junction protein cldn-5 as a meshwork of angular discontinous fibrils in rows in Xenopus laevis oocytes. (B) Freeze fracture electron microscopy reveals tight junction protein cldn-3 as a meshwork of rounded fibrils in X. laevis oocytes. (C) Freeze fracture electron microscopy of claudin-3 and claudin-5 coexpressing oocytes reveal fibrils that both bear properties of claudin-3 and claudin-5. (D) Water injected control oocytes have a smooth surface. Representative images of oocytes derived from three animals. Scale bar: 250 nm.
FIGURE 4. Median contact areas of clustered oocyte combinations cldn5–cldn5, cldn3–cldn3, cldn3,5–cldn3,5 and control–control 24 and 48 h after clustering in % of initial contact areas shortly after clustering (n = 8–38).
FIGURE 5. Contact areas of claudin-5, claudin-3, and coexpressing claudin-3 and claudin-5 oocytes in hydrostatic pressure impulse (HPI) challenge after stabilization period and 30 min after HPI (n = 16–70, *p < 0.05, **p < 0.01, Kruskal-Wallis followed by a Dunn–Bonferroni correction).
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