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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

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.

ZO-1, ZO-2 and ZO-3 are tight junction-associated scaffold proteins that bind to transmembrane proteins of tight junctions and the underlying cytoskeleton. ZO-1 is involved in the regulation of cytoskeletal organization, but its detailed molecular mechanism is less well understood. Gene knockout is an ideal method to investigate the functions of proteins that might have redundant functions such as ZO proteins, when compared with methods such as RNA interference-mediated suppression of gene expression. In this study we applied transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout, and established ZO-1 knockout clones in Madin-Darby canine kidney (MDCK) cells. ZO-1 knockout induced striking changes in myosin organization at cell-cell contacts and disrupted the localization of tight junction proteins; these findings were previously unseen in studies of ZO-1 knockdown by RNA interference. Rescue experiments revealed that trace ZO-1 expression reversed these changes while excessive ZO-1 expression induced an intensive zigzag shape of cell-cell junctions. These results suggest a role for ZO-1 in the regulation of cytoskeleton and shape of cell-cell junctions in MDCK cells and indicate the advantage of knockout analysis in cultured cells.

Tight junctions (TJs) regulate the movements of substances through the paracellular pathway, and claudins are major determinants of TJ permeability. Claudin-2 forms high conductive cation pores in TJs. The suppression of claudin-2 expression by RNA interference in Madin-Darby canine kidney (MDCK) II cells (a low-resistance strain of MDCK cells) was shown to induce a three-fold increase in transepithelial electrical resistance (TER), which, however, was still lower than in high-resistance strains of MDCK cells. Because RNA interference-mediated knockdown is not complete and only reduces gene function, we considered the possibility that the remaining claudin-2 expression in the knockdown study caused the lower TER in claudin-2 knockdown cells. Therefore, we investigated the effects of claudin-2 knockout in MDCK II cells by establishing claudin-2 knockout clones using transcription activator-like effector nucleases (TALENs), a recently developed genome editing method for gene knockout. Surprisingly, claudin-2 knockout increased TER by more than 50-fold in MDCK II cells, and TER values in these cells (3000-4000 Ω·cm2) were comparable to those in the high-resistance strains of MDCK cells. Claudin-2 re-expression restored the TER of claudin-2 knockout cells dependent upon claudin-2 protein levels. In addition, we investigated the localization of claudin-1, -2, -3, -4, and -7 at TJs between control MDCK cells and their respective knockout cells using their TALENs. Claudin-2 and -7 were less efficiently localized at TJs between control and their knockout cells. Our results indicate that claudin-2 independently determines the 'leaky' property of TJs in MDCK II cells and suggest the importance of knockout analysis in cultured cells.

The paracellular passage of ions and small molecules across epithelia is controlled by tight junctions, complex meshworks of claudin polymers that form tight seals between neighboring cells. How the nanoscale architecture of tight junction meshworks enables paracellular passage of specific ions or small molecules without compromising barrier function is unknown. Here we combine super-resolution stimulated emission depletion microscopy in live and fixed cells and tissues, multivariate classification of super-resolution images and fluorescence resonance energy transfer to reveal the nanoscale organization of tight junctions formed by mammalian claudins. We show that only a subset of claudins can assemble into characteristic homotypic meshworks, whereas tight junctions formed by multiple claudins display nanoscale organization principles of intermixing, integration, induction, segregation, and exclusion of strand assemblies. Interestingly, channel-forming claudins are spatially segregated from barrier-forming claudins via determinants mainly encoded in their extracellular domains also known to harbor mutations leading to human diseases. Electrophysiological analysis of claudins in epithelial cells suggests that nanoscale segregation of distinct channel-forming claudins enables barrier function combined with specific paracellular ion flux across tight junctions. Meshworks of claudin polymers control the paracellular transport and barrier properties of epithelial tight junctions. Here, the authors show different claudin nanoscale organization principles, finding that claudin segregation enables barrier formation and paracellular ion flux across tight junctions.

In multicellular organisms, cells adopt various shapes, from flattened sheets of endothelium to dendritic neurons, that allow the cells to function effectively. Here, we elucidated the unique shape of cells in the cornified stratified epithelia of the mammalian epidermis that allows them to achieve homeostasis of the tight junction (TJ) barrier. Using intimate in vivo 3D imaging, we found that the basic shape of TJ-bearing cells is a flattened Kelvin's tetrakaidecahedron (f-TKD), an optimal shape for filling space. In vivo live imaging further elucidated the dynamic replacement of TJs on the edges of f-TKD cells that enables the TJ-bearing cells to translocate across the TJ barrier. We propose a spatiotemporal orchestration model of f-TKD cell turnover, where in the classic context of 'form follows function', cell shape provides a fundamental basis for the barrier homeostasis and physical strength of cornified stratified epithelia. The skin surface – known as the epidermis – is made up of sheets of cells that are stacked up in layers. One of the roles of the skin is to provide a protective barrier that limits what leaks into or out of the body. A particular layer of the epidermis – referred to as the stratum granulosum – is primarily responsible for forming this barrier. The cells in this layer are sealed together in a zipper-like fashion by structures known as tight junctions. New skin cells are continuously produced in the lowest cell layers of the epidermis, and move upwards to integrate into the stratum granulosum layer to replace old cells (which also move upwards to leave the layer). How stratum granulosum cells are replaced without disrupting the tight junction barrier was not well understood. Yokouchi et al. used a technique called confocal microscopy to examine the stratum granulosum cells in the ears of mice, and found that the shape of these cells forms the basis of the barrier that they form. These cells resemble a flattened version of a shape called Kelvin’s tetrakaidecahedron: a 14-sided solid with six rectangular and eight hexagonal sides. This structure was proposed by Lord Kelvin in 1887 to be the best shape for filling space. Tight junctions are present on the edges of the flattened Kelvin’s tetrakaidecahedron. Further experiments revealed that the tight junctions move from cell to cell in a spatiotemporally-coordinated manner in order to maintain a continuous barrier throughout the stratum granulosum as cells are replaced. A newly formed stratum granulosum cell appears beneath the cell that it will replace. The shape of these cells enables a new barrier of three-way tight junction contacts to form between them and the neighboring cells in the stratum granulosum. After this barrier has formed, the upper cell leaves the stratum granulosum. Future research could address how cells adopt the flattened Kelvin’s tetrakaidecahedron shape, and discover why tight junctions only form in one layer of the epidermis.

Tight junctions (TJs) form a selective barrier for ions, water, and macromolecules in simple epithelia. In keratinocytes and epidermis, TJs were shown to be involved in individual barrier functions. The absence of the TJ protein claudin-1 (Cldn1) in mice results in a skin-barrier defect characterized by lethal water loss. However, detailed molecular analyses of the various TJ barriers in keratinocytes and the contribution of distinct TJ proteins are missing. Herein, we discriminate TJ-dependent paracellular resistance from transcellular resistance in cultured keratinocytes using the two-path impedance spectroscopy. We demonstrate that keratinocyte TJs form a barrier for Na+, Cl−, and Ca2+, and contribute to barrier function for water and larger molecules of different size. In addition, knockdown of Cldn1, Cldn4, occludin, and zonula occludens-1 increased paracellular permeabilities for ions and larger molecules, demonstrating that all of these TJ proteins contribute to barrier formation. Remarkably, Cldn1 and Cldn4 are not critical for TJ barrier function for water in submerged keratinocyte cultures. However, Cldn1 influences stratum corneum (SC) proteins important for SC water barrier function, and is crucial for TJ barrier formation for allergen-sized macromolecules.

Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. Here, we discovered a previously uncharacterized, new phenomenon, which we name erebosis based on the ancient Greek word erebos (“complete darkness”), in the gut enterocytes of adult Drosophila . Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. We hypothesize that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut.

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.

The PET and LIM domain-containing protein, Prickle, plays a key role in planar cell polarity (PCP) in Drosophila . It has been reported that mutations in the PRICKLE2 gene, which encodes one of the human orthologues of Prickle, are associated with human diseases such as epilepsy and autism spectrum disorder. To develop preventive and therapeutic strategies for these intractable diseases, we studied the regulation of Prickle2 protein levels in transfected HEK293T cells. Prickle2 levels were negatively regulated by a physical interaction with another PCP protein, Van Gogh-like 2 (Vangl2). The Vangl2-mediated reduction in Prickle2 levels was, at least in part, relieved by proteasome inhibitors or by functional inhibition of the Cullin-1 E3 ubiquitin ligase. Furthermore, the expression of Vangl2 enhanced the polyubiquitination of Prickle2. This ubiquitination was partially blocked by co-expression of a ubiquitin mutant, which cannot be polymerised through their Lys48 residue to induce target proteins toward proteasomal degradation. Together, these results suggest that Prickle2 is polyubiquitinated by the Vangl2 interaction in a Cullin-1-dependent manner to limit its expression levels. This regulation may play a role in the local and temporal fine-tuning of Prickle protein levels during PCP signal-dependent cellular behaviours.