Bicellular Localization of Tricellular Junctional Protein Angulin-3/ILDR2 Allows Detection of Podocyte Injury.

Podocytes serve as part of the renal filtration unit with slit diaphragms. Although the structure of slit diaphragms between two cells is well characterized, how the tricellular contact of podocytes is organized and how it changes in injured podocytes remains unknown. This study focused on a tricellular junction protein, angulin-3, and its localization in healthy podocytes, in developmental stages, and in pathologic conditions, using a newly established monoclonal antibody. Angulin-3 was confined at tricellular junctions of primordial podocytes, then transiently localized at bicellular junctions as foot process interdigitation developed and the intercellular junctions rearranged into slit diaphragm, and eventually distributed in a sparse punctate pattern on the foot processes of adult podocytes. In the rodent podocyte injury models, angulin-3 showed bicellular localization between the foot processes, and the localization turned from punctate to dashed linear pattern along the effaced foot processes with the progression of podocyte injury. Angulin-3 also accumulated between foot processes in a linear pattern in kidney biopsy samples of human nephrotic syndrome. Additionally, the line length of angulin-3 staining signal correlated with risk of relapse under glucocorticoid therapy in patients with minimal change nephrotic syndrome. This study proposes an image program to score the linearity of the accumulation pattern of angulin-3 to evaluate the relapse risk of patients with minimal change nephrotic syndrome.

Reconstitution of functional tight junctions with individual claudin subtypes in epithelial cells.

The claudin family of membrane proteins is responsible for the backbone structure and function of tight junctions (TJs), which regulate the paracellular permeability of epithelia. It is thought that each claudin subtype has its own unique function and the combination of expressed subtypes determines the permeability property of each epithelium. However, many issues remain unsolved in regard to claudin functions, including the detailed functional differences between claudin subtypes and the effect of the combinations of specific claudin subtypes on the structure and function of TJs. To address these issues, it would be useful to have a way of reconstituting TJs containing only the claudin subtype(s) of interest in epithelial cells. In this study, we attempted to reconstitute TJs of individual claudin subtypes in TJ-deficient MDCK cells, designated as claudin quinKO cells, which were previously established from MDCK II cells by deleting the genes of claudin-1, -2, -3, -4, and -7. Exogenous expression of each of claudin-1, -2, -3, -4, and -7 in claudin quinKO cells resulted in the reconstitution of functional TJs. These TJs did not contain claudin-12 and -16, which are endogenously expressed in claudin quinKO cells. Furthermore, overexpression of neither claudin-12 nor claudin-16 resulted in the reconstitution of TJs, demonstrating the existence of claudin subtypes lacking TJ-forming activity in epithelial cells. Exogenous expression of the channel-forming claudin-2, -10a, -10b, and -15 reconstituted TJs with reported paracellular channel properties, demonstrating that these claudin subtypes form paracellular channels by themselves without interaction with other subtypes. Thus, the reconstitution of TJs in claudin quinKO cells is advantageous for further investigation of claudin functions.Key words: tight junction, claudin, paracellular permeability, epithelial barrier.

Tight junction formation by a claudin mutant lacking the COOH-terminal PDZ domain-binding motif.

Claudin-based tight junctions (TJs) are formed at the most apical part of cell-cell contacts in epithelial cells. Previous studies suggest that scaffolding proteins ZO-1 and ZO-2 (ZO proteins) determine the location of TJs by interacting with claudins, but this idea is not conclusive. To address the role of the ZO proteins binding to claudins at TJs, a COOH-terminal PDZ domain binding motif-deleted claudin-3 mutant, which lacks the ZO protein binding, was stably expressed in claudin-deficient MDCK cells. The COOH-terminus-deleted claudin-3 was localized at the apicolateral region similar to full-length claudin-3. Consistently, freeze-fracture electron microscopy revealed that the COOH-terminus-deleted claudin-3-expressing cells reconstituted belts of TJs at the most apical region of the lateral membrane and restored functional epithelial barriers. These results suggest that the interaction of claudins with ZO proteins is not a prerequisite for TJ formation at the most apical part of cell-cell contacts.

Claudin-9 constitutes tight junctions of folliculo-stellate cells in the anterior pituitary gland.

The anterior pituitary gland regulates growth, metabolism, and reproduction by secreting hormones. Folliculo-stellate (FS) cells are non-endocrine cells located among hormone-producing cells in the anterior pituitary glands. They form follicular lumens, which are sealed by tight junctions (TJs). Although FS cells are hypothesized to contribute to fine-tuning of endocrine cells, little is known about the exact roles of FS cells. Here, we investigated the molecular composition of TJs in FS cells. We demonstrated that occludin is a good marker for TJs in the pituitary gland and examined the structure of the lumens surrounded by FS cells. We also found that claudin-9 is a major component of TJs in the FS cells. In immunoelectron microscopy, claudin-9 was specifically localized at TJs of the FS cells. The expression of claudin-9 was gradually increased in the pituitary gland after birth, suggesting that claudin-9 is developmentally regulated and performs some specific functions on the paracellular barrier of follicles in the pituitary gland. Furthermore, we found that angulin-1, angulin-2, and tricellulin are localized at the tricellular contacts of the FS cells. Our findings provide a first comprehensive molecular profile of TJs in the FS cells, and may lead us towards unveiling the FS cell functions.

Angulin-1 seals tricellular contacts independently of tricellulin and claudins.

Tricellular tight junctions (tTJs) are specialized tight junctions (TJs) that seal the intercellular space at tricellular contacts (TCs), where the vertices of three epithelial cells meet. Tricellulin and angulin family membrane proteins are known constituents of tTJs, but the molecular mechanism of tTJ formation remains elusive. Here, we investigated the roles of angulin-1 and tricellulin in tTJ formation in MDCK II cells by genome editing. Angulin-1-deficient cells lost the plasma membrane contact at TCs with impaired epithelial barrier function. The C terminus of angulin-1 bound to the TJ scaffold protein ZO-1, and disruption of their interaction influenced the localization of claudins at TCs, but not the tricellular sealing. Strikingly, the plasma membrane contact at TCs was formed in tricellulin- or claudin-deficient cells. These findings demonstrate that angulin-1 is responsible for the plasma membrane seal at TCs independently of tricellulin and claudins.

The novel membrane protein Hoka regulates septate junction organization and stem cell homeostasis in the Drosophila gut.

Smooth septate junctions (sSJs) regulate the paracellular transport in the intestinal tract in arthropods. In Drosophila, the organization and physiological function of sSJs are regulated by at least three sSJ-specific membrane proteins: Ssk, Mesh and Tsp2A. Here, we report a novel sSJ membrane protein, Hoka, which has a single membrane-spanning segment with a short extracellular region, and a cytoplasmic region with Tyr-Thr-Pro-Ala motifs. The larval midgut in hoka mutants shows a defect in sSJ structure. Hoka forms a complex with Ssk, Mesh and Tsp2A, and is required for the correct localization of these proteins to sSJs. Knockdown of hoka in the adult midgut leads to intestinal barrier dysfunction and stem cell overproliferation. In hoka-knockdown midguts, aPKC is upregulated in the cytoplasm and the apical membrane of epithelial cells. The depletion of aPKC and yki in hoka-knockdown midguts results in reduced stem cell overproliferation. These findings indicate that Hoka cooperates with the sSJ proteins Ssk, Mesh and Tsp2A to organize sSJs, and is required for maintaining intestinal stem cell homeostasis through the regulation of aPKC and Yki activities in the Drosophila midgut.

Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity.

Tight junctions (TJs) establish the epithelial barrier and are thought to form a membrane fence to regulate epithelial polarity, although the roles of TJs in epithelial polarity remain controversial. Claudins constitute TJ strands in conjunction with the cytoplasmic scaffolds ZO-1 and ZO-2 and play pivotal roles in epithelial barrier formation. However, how claudins and other TJ membrane proteins cooperate to organize TJs remains unclear. Here, we systematically knocked out TJ components by genome editing and show that while ZO-1/ZO-2-deficient cells lacked TJ structures and epithelial barriers, claudin-deficient cells lacked TJ strands and an electrolyte permeability barrier but formed membrane appositions and a macromolecule permeability barrier. Moreover, epithelial polarity was disorganized in ZO-1/ZO-2-deficient cells, but not in claudin-deficient cells. Simultaneous deletion of claudins and a TJ membrane protein JAM-A resulted in a loss of membrane appositions and a macromolecule permeability barrier and in sporadic epithelial polarity defects. These results demonstrate that claudins and JAM-A coordinately regulate TJ formation and epithelial polarity.

Septate junctions regulate gut homeostasis through regulation of stem cell proliferation and enterocyte behavior in Drosophila.

Smooth septate junctions (sSJs) contribute to the epithelial barrier, which restricts leakage of solutes through the paracellular route in epithelial cells of the Drosophila midgut. We previously identified three sSJ-associated membrane proteins, Ssk, Mesh and Tsp2A, and showed that these proteins were required for sSJ formation and intestinal barrier function in the larval midgut. Here, we investigated the roles of sSJs in the Drosophila adult midgut. Depletion of any of the sSJ proteins from enterocytes resulted in remarkably shortened lifespan and intestinal barrier dysfunction in flies. Interestingly, the sSJ-protein-deficient flies showed intestinal hypertrophy accompanied by accumulation of morphologically abnormal enterocytes. The phenotype was associated with increased stem cell proliferation and activation of the MAPK and Jak-Stat pathways in stem cells. Loss of the cytokines Unpaired 2 and Unpaired 3, which are involved in Jak-Stat pathway activation, reduced the intestinal hypertrophy, but not the increased stem cell proliferation, in flies lacking Mesh. The present findings suggest that SJs play a crucial role in maintaining tissue homeostasis through regulation of stem cell proliferation and enterocyte behavior in the Drosophila adult midgut.

A tetraspanin regulates septate junction formation in Drosophila midgut.

Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes through the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of septate junctions are observed; pleated (pSJs) and smooth (sSJs), which are present in ectodermally and endodermally derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Ssk, in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh identified a tetraspanin family protein, Tsp2A, as a newly discovered protein involved in sSJ formation in Drosophila Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised Tsp2A expression caused by RNAi or the CRISPR/Cas9 system was associated with defects in the ultrastructure of sSJs, changed localization of other sSJ proteins, and impaired barrier function of the midgut. In most Tsp2A mutant cells, Mesh failed to localize to sSJs and was distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs.

Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules.

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

Molecular characterization of water-selective AQP (EbAQP4) in hagfish: insight into ancestral origin of AQP4.

Hagfish (Eptatretus burgeri) are agnathous and are the earliest vertebrates still in existence. Pavement cells adjacent to the mitochondria-rich cells show orthogonal arrays of particles (OAPs) in the gill of hagfish, a known ultrastructural morphology of aquaporin (AQP) in mammalian freeze-replica studies, suggesting that an AQP homolog exists in pavement cells. We therefore cloned water channels from hagfish gill and examined their molecular characteristics. The cloned AQP [E. burgeri AQP4 (EbAQP4)] encodes 288 amino acids, including two NPA motifs and six transmembrane regions. The deduced amino acid sequence of EbAQP4 showed high homology to mammalian and avian AQP4 (rat, 44%; quail, 43%) and clustered with AQP4 subsets by the molecular phylogenetic tree. The osmotic water permeability of Xenopus oocytes injected with EbAQP4 cRNA increased eightfold compared with water-injected controls and was not reversibly inhibited by 0.3 mM HgCl(2). EbAQP4 mRNA expression in the gill was demonstrated by the RNase protection assay; antibody raised against the COOH terminus of EbAQP4 also detected (by Western blot analysis) a major approximately 31-kDa band in the gill. Immunohistochemistry and immunoelectron microscopy showed EbAQP4 localized along the basolateral membranes of gill pavement cells. In freeze-replica studies, OAPs were detected on the protoplasmic face of the split membrane comprising particles 5-6 nm long on the basolateral side of the pavement cells. These observations suggest that EbAQP4 is an ancestral water channel of mammalian AQP4 and plays a role in basolateral water transport in the gill pavement cells.

Normal establishment of epithelial tight junctions in mice and cultured cells lacking expression of ZO-3, a tight-junction MAGUK protein.

ZO-1, ZO-2, and ZO-3 are closely related MAGUK family proteins that localize at the cytoplasmic surface of tight junctions (TJs). ZO-1 and ZO-2 are expressed in both epithelia and endothelia, whereas ZO-3 is exclusively expressed in epithelia. In spite of intensive studies of these TJ MAGUKs, our knowledge of their functions in vivo, especially those of ZO-3, is still fragmentary. Here, we have generated mice, as well as F9 teratocarcinoma cell lines, that do not express ZO-3 by homologous recombination. Unexpectedly, ZO-3(-/-) mice were viable and fertile, and rigorous phenotypic analyses identified no significant abnormalities. Moreover, ZO-3-deficient F9 teratocarcinoma cells differentiated normally into visceral endoderm epithelium-like cells in the presence of retinoic acid. These cells had a normal epithelial appearance, and the molecular architecture of their TJs did not appear to be affected, except that TJ localization of ZO-2 was upregulated. Suppression of ZO-2 expression by RNA interference in ZO-3(-/-) cells, however, did not affect the architecture of TJs. Furthermore, the speed with which TJs formed after a Ca(2+) switch was indistinguishable between wild-type and ZO-3(-/-) cells. These findings indicate that ZO-3 is dispensable in vivo in terms of individual viability, epithelial differentiation, and the establishment of TJs, at least in the laboratory environment.

ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation.

A fundamental question in cell and developmental biology is how epithelial cells construct the diffusion barrier allowing them to separate different body compartments. Formation of tight junction (TJ) strands, which are crucial for this barrier, involves the polymerization of claudins, TJ adhesion molecules, in temporal and spatial manners. ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, yet their functional roles are poorly understood. We established cultured epithelial cells (1(ko)/2(kd)) in which the expression of ZO-1/ZO-2 was suppressed by homologous recombination and RNA interference, respectively. These cells were well polarized, except for a complete lack of TJs. When exogenously expressed in 1(ko)/2(kd) cells, ZO-1 and ZO-2 were recruited to junctional areas where claudins were polymerized, but truncated ZO-1 (NZO-1) containing only domains PDZ1-3 was not. When NZO-1 was forcibly recruited to lateral membranes and dimerized, claudins were dramatically polymerized. These findings indicate that ZO-1 and ZO-2 can independently determine whether and where claudins are polymerized.

Tricellulin constitutes a novel barrier at tricellular contacts of epithelial cells.

For epithelia to function as barriers, the intercellular space must be sealed. Sealing two adjacent cells at bicellular tight junctions (bTJs) is well described with the discovery of the claudins. Yet, there are still barrier weak points at tricellular contacts, where three cells join together. In this study, we identify tricellulin, the first integral membrane protein that is concentrated at the vertically oriented TJ strands of tricellular contacts. When tricellulin expression was suppressed with RNA interference, the epithelial barrier was compromised, and tricellular contacts and bTJs were disorganized. These findings indicate the critical function of tricellulin for formation of the epithelial barrier.

Compartmentalization established by claudin-11-based tight junctions in stria vascularis is required for hearing through generation of endocochlear potential.

Claudins are cell adhesion molecules working at tight junctions (TJs) that are directly involved in compartmentalization in multicellular organisms. The cochlea includes a rather peculiar compartment filled with endolymph. This compartment is characterized by high K+ concentration (approximately 150 mM) and a positive endocochlear potential (approximately 90 mV; EP), both indispensable conditions for cochlear hair cells to transduce acoustic stimuli to electrical signals. These conditions are thought to be generated by the stria vascularis, which is adjacent to the endolymph compartment. The stria vascularis itself constitutes an isolated compartment delineated by two epithelial barriers, marginal and basal cell layers. Because TJs of basal cells are primarily composed of claudin-11, claudin-11-deficient (Cld11-/-) mice were generated with an expectation that the compartmentalization in stria vascularis in these mice would be affected. Auditory brainstem response measurements revealed that Cld11-/- mice suffered from deafness; although no obvious gross morphological malformations were detected in Cld11-/- cochlea, freeze-fracture replica electron microscopy showed that TJs disappeared from basal cells of the stria vascularis. In good agreement with this, tracer experiments showed that the basal cell barrier was destroyed without affecting the marginal cell barrier. Importantly, in the endolymph compartment of Cld11-/- cochlea, the K+ concentration was maintained around the normal level (approximately 150 mM), whereas the EP was suppressed down to approximately 30 mV. These findings indicated that the establishment of the stria vascularis compartment, especially the basal cell barrier, is indispensable for hearing ability through the generation/maintenance of EP but not of a high K+ concentration in the endolymph.

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1.

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.

JACOP, a novel plaque protein localizing at the apical junctional complex with sequence similarity to cingulin.

The apical junctional complex is composed of various cell adhesion molecules and cytoplasmic plaque proteins. Using a monoclonal antibody that recognizes a chicken 155-kDa cytoplasmic antigen (p155) localizing at the apical junctional complex, we have cloned a cDNA of its mouse homologue. The full-length cDNA of mouse p155 encoded a 148-kDa polypeptide containing a coiled-coil domain with sequence similarity to cingulin, a tight junction (TJ)-associated plaque protein. We designated this protein JACOP (junction-associated coiled-coil protein). Immunofluorescence staining showed that JACOP was concentrated in the junctional complex in various types of epithelial and endothelial cells. Furthermore, in the liver and kidney, JACOP was also distributed along non-junctional actin filaments. Upon immunoelectron microscopy, JACOP was found to be localized to the undercoat of TJs in the liver, but in some tissues, its distribution was not restricted to TJs but extended to the area of adherens junctions. Overexpression studies have revealed that JACOP was recruited to the junctional complex in epithelial cells and to cell-cell contacts and stress fibers in fibroblasts. These findings suggest that JACOP is involved in anchoring the apical junctional complex, especially TJs, to actin-based cytoskeletons.

Expression of claudin-5 in dermal vascular endothelia.

Claudins and occludin are integral membrane proteins at tight junctions (TJs). We examined subcellular localization of claudin-5 and occludin in dermal vascular endothelia. Immunofluorescence staining showed that claudin-5 was expressed at the cell-cell border of dermal vascular endothelia in mouse skin. However, in some dermal vessels, claudin-5 expression was markedly decreased or absent in amount by double-immunofluorescence stainings with PECAM-1 and PAL-E. In contrast, occludin was not detected in dermal vessels. Freeze-fracture and immunoreplica electron microscopy on primary-cultured human dermal endothelial cells showed that claudin-5 was localized at tight junctions. These findings confirmed that TJs in dermal vascular endothelial cells are composed of claudin-5.

Dynamic behavior of paired claudin strands within apposing plasma membranes.

The tight junction (TJ) strand is a linear proteinaceous polymer spanning plasma membranes, and each TJ strand associates laterally with another TJ strand in the apposing membranes of adjacent cells to form "paired" TJ strands. Claudins have been identified as the major constituents of TJ strands, and when exogenously expressed in L fibroblasts, they polymerize into paired strands, which are morphologically similar to paired TJ strands in epithelia. Here, we show that a fusion protein of GFP with claudin-1 can also form similar paired strands in L fibroblasts, allowing us to directly observe individual paired claudin strands in live cells in real time. These paired strands showed more dynamic behavior than expected; they were occasionally broken and annealed, and dynamically associated with each other in both an end-to-side and side-to-side manner. Through this behavior of individual paired claudin strands, the network of strands was reorganized dynamically. Furthermore, fluorescence recovery after photobleaching analyses revealed that claudin molecules were not mobile within paired strands. Although these observations are not necessarily representative of TJ strands per se in epithelial cells, they provide important information on the structural and kinetic properties of TJ strands in situ with significant implications for barrier function of TJs.

Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice.

The tight junction (TJ) and its adhesion molecules, claudins, are responsible for the barrier function of simple epithelia, but TJs have not been thought to play an important role in the barrier function of mammalian stratified epithelia, including the epidermis. Here we generated claudin-1-deficient mice and found that the animals died within 1 d of birth with wrinkled skin. Dehydration assay and transepidermal water loss measurements revealed that in these mice the epidermal barrier was severely affected, although the layered organization of keratinocytes appeared to be normal. These unexpected findings prompted us to reexamine TJs in the epidermis of wild-type mice. Close inspection by immunofluorescence microscopy with an antioccludin monoclonal antibody, a TJ-specific marker, identified continuous TJs in the stratum granulosum, where claudin-1 and -4 were concentrated. The occurrence of TJs was also confirmed by ultrathin section EM. In claudin-1-deficient mice, claudin-1 appeared to have simply been removed from these TJs, leaving occludin-positive (and also claudin-4-positive) TJs. Interestingly, in the wild-type epidermis these occludin-positive TJs efficiently prevented the diffusion of subcutaneously injected tracer (approximately 600 D) toward the skin surface, whereas in the claudin-1-deficient epidermis the tracer appeared to pass through these TJs. These findings provide the first evidence that continuous claudin-based TJs occur in the epidermis and that these TJs are crucial for the barrier function of the mammalian skin.