Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Front Physiol
2020 Jan 01;11:857. doi: 10.3389/fphys.2020.00857.
Show Gene links
Show Anatomy links
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
.
???displayArticle.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.
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).
Amasheh,
Inflamed pouch mucosa possesses altered tight junctions indicating recurrence of inflammatory bowel disease.
2009,
Pubmed
Amasheh,
Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells.
2005,
Pubmed
Comper,
Expression pattern of claudins 5 and 7 distinguishes solid-pseudopapillary from pancreatoblastoma, acinar cell and endocrine tumors of the pancreas.
2009,
Pubmed
Del Vecchio,
Sodium caprate transiently opens claudin-5-containing barriers at tight junctions of epithelial and endothelial cells.
2012,
Pubmed
Dittmann,
Laurate permeabilizes the paracellular pathway for small molecules in the intestinal epithelial cell model HT-29/B6 via opening the tight junctions by reversible relocation of claudin-5. [Corrected].
2014,
Pubmed
Engelhardt,
Involvement of the choroid plexus in central nervous system inflammation.
2001,
Pubmed
Florian,
Claudins in the tight junctions of stria vascularis marginal cells.
2003,
Pubmed
Furuse,
Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.
2002,
Pubmed
Garcia-Hernandez,
Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation.
2017,
Pubmed
Greene,
Claudin-5: gatekeeper of neurological function.
2019,
Pubmed
Günzel,
Claudin-16 affects transcellular Cl- secretion in MDCK cells.
2009,
Pubmed
Hashimoto,
Claudin-5-Binders Enhance Permeation of Solutes across the Blood-Brain Barrier in a Mammalian Model.
2017,
Pubmed
Koda,
Novel expression of claudin-5 in glomerular podocytes.
2011,
Pubmed
Konrad,
Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement.
2006,
Pubmed
Krug,
Sodium caprate as an enhancer of macromolecule permeation across tricellular tight junctions of intestinal cells.
2013,
Pubmed
Leduc-Nadeau,
Elaboration of a novel technique for purification of plasma membranes from Xenopus laevis oocytes.
2007,
Pubmed
,
Xenbase
Lindmark,
Absorption enhancement through intracellular regulation of tight junction permeability by medium chain fatty acids in Caco-2 cells.
1998,
Pubmed
Maher,
Safety and efficacy of sodium caprate in promoting oral drug absorption: from in vitro to the clinic.
2009,
Pubmed
Milatz,
Claudin-3 acts as a sealing component of the tight junction for ions of either charge and uncharged solutes.
2010,
Pubmed
Mineta,
Predicted expansion of the claudin multigene family.
2011,
Pubmed
Nenni,
Xenbase: Facilitating the Use of Xenopus to Model Human Disease.
2019,
Pubmed
,
Xenbase
Nitta,
Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice.
2003,
Pubmed
Ohtsuki,
Exogenous expression of claudin-5 induces barrier properties in cultured rat brain capillary endothelial cells.
2007,
Pubmed
Piontek,
Elucidating the principles of the molecular organization of heteropolymeric tight junction strands.
2011,
Pubmed
Piontek,
Formation of tight junction: determinants of homophilic interaction between classic claudins.
2008,
Pubmed
Radloff,
Caprate Modulates Intestinal Barrier Function in Porcine Peyer's Patch Follicle-Associated Epithelium.
2019,
Pubmed
Reinhold,
Barrier function in the peripheral and central nervous system-a review.
2017,
Pubmed
Resnick,
Claudin expression in gastric adenocarcinomas: a tissue microarray study with prognostic correlation.
2005,
Pubmed
Rossa,
Claudin-3 and claudin-5 protein folding and assembly into the tight junction are controlled by non-conserved residues in the transmembrane 3 (TM3) and extracellular loop 2 (ECL2) segments.
2014,
Pubmed
Session,
Genome evolution in the allotetraploid frog Xenopus laevis.
2016,
Pubmed
,
Xenbase
Soini,
Claudins in lung diseases.
2011,
Pubmed
Suzuki,
Crystal structure of a claudin provides insight into the architecture of tight junctions.
2014,
Pubmed
Tebbe,
Tight junction proteins: a novel class of integral membrane proteins. Expression in human epidermis and in HaCaT keratinocytes.
2002,
Pubmed
Tomita,
Absorption-enhancing mechanism of sodium caprate and decanoylcarnitine in Caco-2 cells.
1995,
Pubmed
Turner,
'Putting the squeeze' on the tight junction: understanding cytoskeletal regulation.
2000,
Pubmed
Van Itallie,
Claudins and epithelial paracellular transport.
2006,
Pubmed
Vitzthum,
Xenopus oocytes as a heterologous expression system for analysis of tight junction proteins.
2019,
Pubmed
,
Xenbase
Wilcox,
Mutations in the gene encoding tight junction claudin-14 cause autosomal recessive deafness DFNB29.
2001,
Pubmed
Wolburg,
Localization of claudin-3 in tight junctions of the blood-brain barrier is selectively lost during experimental autoimmune encephalomyelitis and human glioblastoma multiforme.
2003,
Pubmed
Wylie,
The cytoskeleton of Xenopus oocytes and its role in development.
1985,
Pubmed
,
Xenbase
