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The tight junction protein claudin‐7 is essential for tight junction function and intestinal homeostasis. Cldn7 deletion in mice leads to an inflammatory bowel disease‐like phenotype exhibiting severe intestinal epithelial damage, weight loss, inflammation, mucosal ulcerations, and epithelial hyperplasia. Claudin‐7 has also been shown to be involved in cancer metastasis and invasion. Here, we test our hypothesis that claudin‐7 plays an important role in regulating colonic intestinal stem cell function. Conditional knockout of Cldn7 in the colon led to impaired epithelial cell differentiation, hyperproliferative epithelium, a decrease in active stem cells, and dramatically altered gene expression profiles. In 3D colonoid culture, claudin‐7–deficient crypts were unable to survive and form spheroids, emphasizing the importance of claudin‐7 in stem cell survival. Inhibition of the Hippo pathway or activation of Notch signaling partially rescued the defective stem cell behavior. Concurrent Notch activation and Hippo inhibition resulted in restored colonoid survival, growth, and differentiation to the level comparable to those of wild‐type derived crypts. In this study, we highlight the essential role of claudin‐7 in regulating Notch and Hippo signaling–dependent colonic stem cell functions, including survival, self‐renewal, and differentiation. These new findings may shed light on potential avenues to explore for drug development in colorectal cancer. [ABSTRACT FROM AUTHOR]

The kidney is critical for mineral homeostasis. Calcium and magnesium reabsorption in the renal thick ascending limb (TAL) involves claudin‐16 (CLDN16) and claudin‐19 (CLDN19) and pathogenic variants in either gene lead to familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) with severe calcium and magnesium wasting. While both CLDN16 and CLDN19 localize to the TAL, varying expression patterns in the renal tubule have been reported using different antibodies. We, therefore, studied the localization of CLDN19 in the kidneys of wild‐type and Cldn19‐deleted mice using three anti‐CLDN19 antibodies and examined the role of Cldn19 deletion on CLDN16 and CLDN10 localization. We find that CLDN19 localizes to basolateral membrane domains of the medullary and cortical TAL but only to the tight junction of TALs in the outer stripe of outer medulla and cortex, where it colocalizes with CLDN16. Furthermore, in TALs from Cldn19‐deleted mice, CLDN16 is expressed in basolateral membrane domains but not at the tight junction. In contrast, Cldn19 ablation does not change CLDN10 localization. These findings directly implicate CLDN19 in regulating permeability in the TAL by allowing junctional insertion of CLDN16 and may explain the shared renal phenotypic characteristics in FHHNC patients. [ABSTRACT FROM AUTHOR]

Magnesium is the fourth most abundant cation in the body. It plays a critical role in many biological processes, including the process of energy release. Paracellular transport of magnesium is mandatory for magnesium homeostasis. In addition to intestinal absorption that occurs in part across the paracellular pathway, magnesium is reabsorbed by the kidney tubule. The bulk of magnesium is reabsorbed through the paracellular pathway in the proximal tubule and the thick ascending limb of the loop of Henle. The finding that rare genetic diseases due to pathogenic variants in genes encoding specific claudins (CLDNs), proteins located at the tight junction that determine the selectivity and the permeability of the paracellular pathway, led to an awareness of their importance in magnesium homeostasis. Familial hypomagnesemia with hypercalciuria and nephrocalcinosis is caused by a loss of function of CLDN16 or CLDN19. Pathogenic CLDN10 variants cause HELIX syndrome, which is associated with a severe renal loss of sodium chloride and hypermagnesemia. The present review summarizes the current knowledge of the mechanisms and factors involved in paracellular magnesium permeability. The review also highlights some of the unresolved questions that need to be addressed. [ABSTRACT FROM AUTHOR]

Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)‐associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy‐terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple‐gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double‐gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy‐terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy‐terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy‐terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement. [ABSTRACT FROM AUTHOR]

Members of the claudin family impart unique paracellular selectivity to tight junctions. However, the structure–function relationship between claudin's strand architecture and the paracellular charge‐ and size‐selectivity is not well‐understood. This work examines the molecular assembly of claudin‐5, a barrier‐forming protein, and claudin‐15, a channel‐forming protein, to determine their structural and functional properties. We adopt a bottom‐up approach starting from claudin monomers to build the molecular architecture of the tight junction strands. First, we investigated the cis assembly of claudin‐5 and ‐15 dimers using the Protein Association Energy Landscape method. Out of the millions of dimer conformations, we narrowed down key cis claudin‐5 and ‐15 dimer conformations that were thermodynamically and kinetically stable. Second, we performed the trans assembly of dimers to identify the tetrameric building blocks that serve as the repeat units for strand formation. Finally, the strand assembly of the tetrameric repeat units showed fundamentally distinct strand architectures. In claudin‐5, the cis and trans interactions seal the paracellular space, while in claudin‐15, the gaps in the paracellular space lead to pore formation. This detailed study suggests that each member of the claudin family is unique and requires systematic molecular‐level analysis for determining the strand architecture. [ABSTRACT FROM AUTHOR]

Tight junction (TJ) formation is vital for epidermal barrier function. We aimed to specifically manipulate TJ barriers in the reconstructed human epidermis (RHE) by claudin‐1 and ‐4 knockdown (KD) and by claudin‐binding fusion proteins of glutathione S‐transferase and modified C‐terminal fragments of Clostridium perfringens enterotoxin (GST‐cCPE). Impedance spectroscopy and tracer permeability imaging were employed for functional barrier assessment and investigation of claudin contribution. KD of claudin‐1, but not claudin‐4, impaired the paracellular barrier in vitro. Similarly, claudin‐binding GST‐cCPE variants weakened the paracellular but not the stratum corneum barrier. Combining both TJ targeting methods, we found that claudin‐1 targeting by GST‐cCPE after claudin‐4 KD led to a marked decrease in paracellular barrier properties. Conversely, after claudin‐1 KD, GST‐cCPE did not further impair the barrier. Comparison of GST‐cCPE variants with different claudin‐1/claudin‐4 affinities, NHS‐fluorescein tracer detection, and immunostaining of RHE paraffin sections showed that GST‐cCPE variants bind to extrajunctional claudin‐1 and ‐4, which are differentially distributed along the stratum basale–stratum granulosum axis. GST‐cCPE binding blocks these claudins, thereby specifically opening the paracellular barrier of RHE. The data indicate a critical role for claudin‐1 in regulating paracellular permeability for ions and small molecules in the viable epidermis. Claudin targeting is presented as a proof‐of‐concept for precise barrier modulation. [ABSTRACT FROM AUTHOR]

Claudin‐10b is an important component of the tight junction in the thick ascending limb (TAL) of Henle's loop and allows paracellular sodium transport. In immunofluorescence stainings, claudin‐10b–positive cells exhibited extensive extra staining of basolateral, column‐like structures. The precise localization and function have so far remained elusive. In isolated cortical TAL segments from C57BL/6J mice, kidney‐specific claudin‐10 knockout mice (cKO), and respective litter mates (WT), we investigated the localization and protein expression and function by fluorescence microscopy and electrophysiological measurements. Ultrastructural analysis of TAL in kidney sections was performed by electron microscopy. Claudin‐10b colocalized with the basolateral Na+‐K+ ATPase and the Cl– channel subunit barttin, but the lack of claudin‐10b did not influence the localization or abundance of these proteins. However, the accessibility of the basolateral infolded extracellular space to ouabain or fluorescein was increased by basolateral Ca2+ removal and in the absence of claudin‐10b. Ultrastructural analysis by electron microscopy revealed a widening of basolateral membrane infoldings in cKO in comparison to WT. We hypothesize that claudin‐10b shapes neighboring membrane invaginations by trans interaction to stabilize and facilitate high‐flux salt transport in a water‐tight epithelium. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Microcystin is a widespread cyanobacterial toxin that affects the intestine to produce diarrheal symptoms after ingestion of freshwater blue‐green algae. Our study aimed to characterize the mechanism by which the toxin leads to diarrhea via epithelial barrier dysfunction in a small intestine Caco‐2 cell model. Microcystin‐treated human Caco‐2 epithelial monolayers were functionally and molecularly analyzed for barrier dysfunction. Tight junctions (TJs) and cell damage were analyzed in relation to transepithelial electrical resistance (TER) changes. TER of microcystin‐treated Caco‐2 cells was reduced by 65% of the initial value after 24 h; concomitantly, permeability for fluorescein increased 2.6‐fold. Western blot analysis showed reduced claudin‐1 expression, while expression of claudin‐3 and ‐4 remained unchanged. Super‐resolution stimulated emission depletion microscopy revealed that TJ integrity was compromised by fraying and splitting of the TJ domain of the epithelial cells. Epithelial apoptosis did not significantly contribute to epithelial barrier dysfunction, while cytoskeletal actomyosin constriction was associated with TJ disintegration and the barrier defect. Our results indicate that microcystin causes intestinal barrier leakiness, which helps to explain the leak flux type of diarrhea as the main pathomechanism after ingestion of cyanobacterial toxin. [ABSTRACT FROM AUTHOR]

In epithelia, claudin proteins are important components of the tight junctions as they determine the permeability and specificity to ions of the paracellular pathway. Mutations in CLDN10 cause the rare autosomal recessive HELIX syndrome (Hypohidrosis, Electrolyte imbalance, Lacrimal gland dysfunction, Ichthyosis, and Xerostomia), in which patients display severe enamel wear. Here, we assess whether this enamel wear is caused by an innate fragility directly related to claudin‐10 deficiency in addition to xerostomia. A third molar collected from a female HELIX patient was analyzed by a combination of microanatomical and physicochemical approaches (i.e., electron microscopy, elemental mapping, Raman microspectroscopy, and synchrotron‐based X‐ray fluorescence). The enamel morphology, formation time, organization, and microstructure appeared to be within the natural variability. However, we identified accentuated strontium variations within the HELIX enamel, with alternating enrichments and depletions following the direction of the periodical striae of Retzius. These markings were also present in dentin. These data suggest that the enamel wear associated with HELIX may not be related to a disruption of enamel microstructure but rather to xerostomia. However, the occurrence of events of strontium variations within dental tissues might indicate repeated episodes of worsening of the renal dysfunction that may require further investigations. [ABSTRACT FROM AUTHOR]

Claudins (Cldns) form a large family of protein homologs that are essential for the assembly of paracellular tight junctions (TJs), where they form channels or barriers with tissue‐specific selectivity for permeants. In contrast to several family members whose physiological role has been identified, the function of claudin 4 (Cldn4) remains elusive, despite experimental evidence suggesting that it can form anion‐selective TJ channels in the renal epithelium. Computational approaches have recently been employed to elucidate the molecular basis of Cldns' function, and hence could help in clarifying the role of Cldn4. In this work, we use structural modeling and all‐atom molecular dynamics simulations to transfer two previously introduced structural models of Cldn‐based paracellular complexes to Cldn4 to reproduce a paracellular anion channel. Free energy calculations for ionic transport through the pores allow us to establish the thermodynamic properties driving the ion‐selectivity of the structures. While one model shows a cavity permeable to chloride and repulsive to cations, the other forms barrier to the passage of all the major physiological ions. Furthermore, our results confirm the charge selectivity role of the residue Lys65 in the first extracellular loop of the protein, rationalizing Cldn4 control of paracellular permeability. [ABSTRACT FROM AUTHOR]

Epithelial cells are polarized with defined apical tight junctions (TJs), lateral adherens junctions (AJs), and basal integrin–matrix interactions. However, it is increasingly recognized that resident cell junction proteins can be found in varying locations and with previously unrecognized functions. Our study here presents the nanoarchitecture and nanocolocalization of cell junction proteins in culture and tissue by stochastic optical reconstruction microscopy (STORM). The Z‐axial view of noncancerous MDCK‐II and PZ‐HPV‐7 cell–cell junctions resolved β‐catenin and p120ctn localizations to TJs and AJs, with p120ctn apical to β‐catenin and colocalizing with TJ protein claudin‐7. More basally, p120ctn and β‐catenin become colocalized. This topography was lost in isogenic Ras‐transformed MDCK cells and cancerous PC3 cells, where p120ctn becomes basally localized in relation to β‐catenin. Claudin‐7 gene conditional knockout (cKO) in mice also have altered polarity of p120ctn relative to β‐catenin, like that seen in normal‐to‐cancer cell phenotypic transformation. Additionally, claudin‐7 cKO resulted in redistribution and relocalization of other cell junction proteins, including claudin‐1, zonula occludens‐1, integrin α2, epithelial cell adhesion molecule, and focal adhesion kinase (FAK); specifically, integrin α2 and FAK were observed at the apical–lateral compartment. Our data show that STORM reveals regional cellular junction nanoarchitecture previously uncharacterized, providing new insight into potential trans‐compartmental modulation of protein functions. [ABSTRACT FROM AUTHOR]

During migration, cells invade, repair, and create barriers leading to the formation of new cellular contacts in target tissues. Cell migration requires many proteins that collectively form the cytoskeleton. The main cytoskeletal elements are actin filaments, microtubules (MTs), and intermediate filaments. These structures work in concert with a large number of accessory proteins that contribute in a variety of ways to regulate filament assembly and turnover, to alter the configuration or arrangement of filaments by bundling or crosslinking, to link the cytoskeleton to other structures in the cell, such as membranes and junctions, and to transport cargo along the filaments. Sperm flagella protein‐1 (Spef1), also designated calponin homology and microtubules‐associated protein (CLAMP), is a multifunctional protein that interacts with cytoskeletal structures, including MTs, actin filaments, and focal adhesions in epithelia. In this review, we outline Spef1/CLAMP structure and expression in several cellular models. The function of Spef1/CLAMP in flagellar and ciliary motility, MT‐binding and stability, regulation of planar cell polarity, and potential contribution to the maintenance of actin‐based structures, such as lamellipodia and filopodia during cell migration, are also discussed. [ABSTRACT FROM AUTHOR]

Phosphoinositide 3‐kinases (PI3Ks) are a family of enzymes phosphorylating phospholipids in the membrane, thereby, promoting the PI3K/AKT signaling cascade. PI3Ks are involved in a variety of fundamental cellular functions, including tumor necrosis factor α (TNFα)‐induced tight junction (TJ) impairment—a hallmark of inflammatory bowel diseases. Most of the studies analyzing the role of class I PI3K signaling in epithelial barrier maintenance did not decipher which of the isoforms are responsible for the observed effects. By using wild‐type and PI3Kγ‐deficient HT‐29/B6 cells, we characterized the functional role of PI3Kγ in these cells under inflammatory conditions. Measurement of the transepithelial electrical resistance and the paracellular flux of macromolecules revealed that monolayers of PI3Kγ‐deficient cells, compared with wild‐type cells, were protected against TNFα‐induced barrier dysfunction. This effect was independent of any PI3K activity because treatment with a pan‐PI3K inhibitor did not alter this observation. By immunostaining, we found correlative changes in the distribution of the TJ marker ZO‐1. Furthermore, the absence of PI3Kγ reduced the basal level of the pore‐forming TJ protein claudin‐2. Our study suggests a novel noncanonical, kinase‐independent scaffolding function of PI3Kγ in TNFα‐induced barrier dysfunction. [ABSTRACT FROM AUTHOR]

The integrity and functions of epithelial barriers depend on the formation of adherens junctions (AJs) and tight junctions (TJs). A characteristic feature of AJs and TJs is their association with the cortical cytoskeleton composed of actin filaments and nonmuscle myosin II (NM‐II) motors. Mechanical forces generated by the actomyosin cytoskeleton are essential for junctional assembly, stability, and remodeling. Epithelial cells express two different actin proteins and three NM‐II isoforms, all known to be associated with AJs and TJs. Despite their structural similarity, different actin and NM‐II isoforms have distinct biochemical properties, cellular distribution, and functions. The diversity of epithelial actins and myosin motors could be essential for the regulation of different steps of junctional formation, maturation, and disassembly. This review focuses on the roles of actin and NM‐II isoforms in controlling the integrity and barrier properties of various epithelia. We discuss the effects of the depletion of individual actin isoforms and NM‐II motors on the assembly and barrier function of AJs and TJs in model epithelial monolayers in vitro. We also describe the functional consequences of either total or tissue‐specific gene knockout of different actins and NM‐II motors, with a focus on the development and integrity of different epithelia in vivo. [ABSTRACT FROM AUTHOR]

Usually, duodenal barriers are investigated using intestinal cell lines like Caco‐2, which in contrast to native tissue are limited in cell‐type representation. Organoids can consist of all intestinal cell types and are supposed to better reflect the in vivo situation. Growing three‐dimensionally, with the apical side facing the lumen, application of typical physiological techniques to analyze the barrier is difficult. Organoid‐derived monolayers (ODMs) were developed to overcome this. After optimizing culturing conditions, ODMs were characterized and compared to Caco‐2 and duodenal tissue. Tight junction composition and appearance were analyzed, and electrophysiological barrier properties, like paracellular and transcellular barrier function and macromolecule permeability, were evaluated. Furthermore, transcriptomic data were analyzed. ODMs had tight junction protein expression and paracellular barrier properties much more resembling the originating tissue than Caco‐2. Transcellular barrier was similar between ODMs and native tissue but was increased in Caco‐2. Transcriptomic data showed that Caco‐2 expressed fewer solute carriers than ODMs and native tissue. In conclusion, while Caco‐2 cells differ mostly in transcellular properties, ODMs reflect trans‐ and paracellular properties of the originating tissue. If cultured under optimized conditions, ODMs possess reproducible functionality, and the variety of different cell types makes them a suitable model for human tissue‐specific investigations. [ABSTRACT FROM AUTHOR]

Although functional and structural models for paracellular channels formed by claudins have been reported, mechanisms regulating charge and size selectivity of these channels are unknown in detail. Here, claudin‐15 and claudin‐10b cation channels showing high‐sequence similarity but differing channel properties were analyzed. Mutants of pore‐lining residues were expressed in MDCK‐C7 cells. In claudin‐15, proposed ion interaction sites (D55 and E64) conserved between both claudins were neutralized. D55N and E64Q substitutions decreased ion permeabilities, and D55N/E64Q had partly additive effects. D55N increased cation dehydration capability and decreased pore diameter. Additionally, residues differing between claudin‐15 and ‐10b close to pore center were analyzed. Claudin‐10b–mimicking W63K affected neither assembly nor function of claudin‐15 channels. In contrast, in claudin‐10b, corresponding (claudin‐15b‐mimicking) K64W and K64M substitutions disturbed integration into tight junction and slightly altered relative permeabilities for differently sized monovalent cations. Removal of claudin‐10b–specific negative charge (D36A substitution) was without effect. The data suggest that a common tetra‐aspartate ring (D55/D56) in pore center of claudin‐15/‐10b channels directly attracts cations, while E64/D65 may be at least partly shielded by W63/K64. Charge at position W63/K64 affects assembly and properties for claudin‐10b but not for claudin‐15 channels. Our findings add to the mechanistic understanding of the determinants of paracellular cation permeability. [ABSTRACT FROM AUTHOR]

The blood–brain barrier is a major obstacle to the delivery of drugs to the central nervous system. In the blood–brain barrier, the spaces between adjacent brain microvascular endothelial cells are sealed by multiprotein complexes known as tight junctions. Among the many components of the tight junction, claudin‐5 has received the most attention as a target for loosening the tight‐junction seal and allowing drugs to be delivered to the brain. In mice, transient knockdown of claudin‐5 and the use of claudin‐5 binders have been shown to enhance the permeation of small molecules from the blood into the brain without apparent adverse effects. However, sustained knockdown of claudin‐5 in mice is lethal within 40 days, and administration of an anti‐claudin‐5 antibody induced convulsions in a nonhuman primate. Here, we review the safety concerns of claudin‐5–targeted technologies with respect to their clinical application. [ABSTRACT FROM AUTHOR]

Epithelial cells are the first line of mucosal defense. In the intestine, a single layer of epithelial cells must establish a selectively permeable barrier that supports nutrient absorption and waste secretion while preventing the leakage of potentially harmful luminal materials. Key to this is the tight junction, which seals the paracellular space and prevents unrestricted leakage. The tight junction is a protein complex established by interactions between members of the claudin, zonula occludens, and tight junction‐associated MARVEL protein (TAMP) families. Claudins form the characteristic tight junction strands seen by freeze‐fracture microscopy and create paracellular channels, but the functions of ZO‐1 and occludin, founding members of the zonula occludens and TAMP families, respectively, are less well defined. Recent studies have revealed that these proteins have essential noncanonical (nonbarrier) functions that allow them to regulate epithelial apoptosis and proliferation, facilitate viral entry, and organize specialized epithelial structures. Surprisingly, neither is required for intestinal barrier function or overall health in the absence of exogenous stressors. Here, we provide a brief overview of ZO‐1 and occludin canonical (barrier‐related) functions, and a more detailed examination of their noncanonical functions. [ABSTRACT FROM AUTHOR]

Short bowel syndrome can occur after extensive intestinal resection, causing intestinal insufficiency or intestinal failure, which requires long‐term parenteral nutrition. Glucagon‐like peptide‐2 (GLP‐2) pharmacotherapy is now clinically used to reduce the disease burden of intestinal failure. However, many patients still cannot be weaned off from parenteral nutrition completely. The novel dual GLP‐1 and GLP‐2 receptor agonist dapiglutide has previously been shown to be highly effective in a preclinical murine short bowel model. Here, we studied the effects of dapiglutide on intestinal epithelial barrier function. In the jejunum, dapiglutide increased claudin‐7 expression and tightened the paracellular tight junction leak pathway. At the same time, dapiglutide promoted paracellular tight junction cation size selectivity in the jejunum. This was paralleled by extension of the cation selective tight junction proteins claudin‐2 and claudin‐10b and preserved claudin‐15 expression and localization along the crypt–villus axis in the jejunum. In the colon, no barrier effects from dapiglutide were observed. In the colon, dapiglutide attenuated the short bowel–associated, compensatorily increased epithelial sodium channel activity, likely secondary, by improved volume status. Future studies are needed to address the intestinal adaptation of the colon. [ABSTRACT FROM AUTHOR]

T cell protein tyrosine phosphatase (TCPTP) dephosphorylates a number of substrates, including JAK-STAT (signal transducer and activator of transcription) signaling proteins, which are activated by interferon (IFN)-γ, a major proinflammatory cytokine involved in conditions such as inflammatory bowel disease. A critical function of the intestinal epithelium is formation of a selective barrier to luminal contents. The structural units of the epithelium that regulate barrier function are the tight junctions (TJs), and the protein composition of the TJ determines the tightness of the barrier. Claudin-2 is a TJ protein that increases permeability to cations and reduces transepithelial electrical resistance (TER). We previously showed that transient knockdown (KD) of TCPTP permits increased expression of claudin-2 by IFN-γ. Here, we demonstrate that the decreased TER in TCPTP-deficient epithelial cells is alleviated by STAT1 KD. Moreover, increased claudin-2 in TCPTP-deficient cells requires enhanced STAT1 activation and STAT1 binding to the CLDN2 promoter. We also show that mutation of this STAT-binding site prevents elevated CLDN2 promoter activity in TCPTP-deficient epithelial cells. In summary, we demonstrate that TCPTP protects the intestinal epithelial barrier by restricting STAT-induced claudin-2 expression. This is a potential mechanism by which loss-of-function mutations in the gene encoding TCPTP may contribute to barrier defects in chronic intestinal inflammatory disease.

Diseases of the esophagus, such as gastroesophageal reflux (GER), can result in changes to mucosal integrity, neurological function, and the microbiome. Although poorly understood, both age and GER can lead to changes to the enteric nervous system. In addition, the esophagus has a distinct microbiome that can be altered in GER. Mucosal integrity is also at risk due to persistent damage from acid. Diagnostic tools, such as ambulatory pH/impedance testing and esophageal mucosal impedance, can assess short‐term and longitudinal GER burden, which can also assess the risk for mucosal compromise. The quality of the mucosal barrier is determined by its intercellular spaces, tight junctions, and tight junction proteins, which are represented by claudins, occludins, and adhesion molecules. Fortunately, there are protective factors for mucosal integrity that are secreted by the esophageal submucosal mucous glands and within saliva that are augmented by mastication. These protective factors have potential as therapeutic targets for GER. In this article, we aim to review diagnostic tools used to predict mucosal integrity, aging, and microbiome changes to the esophagus and esophageal mucosal defense mechanisms. [ABSTRACT FROM AUTHOR]

The alveolar epithelium of the lung constitutes a unique interface with the outside environment. This thin barrier must maintain a surface for gas transfer while being continuously exposed to potentially hazardous environmental stimuli. Small differences in alveolar epithelial barrier properties could therefore have a large impact on disease susceptibility or outcome. Moreover, recent work has focused attention on the alveolar epithelium as central to several lung diseases, including acute lung injury and idiopathic pulmonary fibrosis. Although relatively little is known about the function and regulation of claudin tight junction proteins in the lung, new evidence suggests that environmental stimuli can influence claudin expression and alveolar barrier function in human disease. This review considers recent advances in the understanding of the role of claudins in the breakdown of the alveolar epithelial barrier in disease and in epithelial repair.

Tight and adherens junctions play critical roles in the barrier, adhesion, and signaling functions of epithelial and endothelial cells. How the molecular organization of these junctions is tuned to the widely diverse physiological requirements of each tissue type is not well understood. Here, we address this question by examining the expression, localization, and interactions of major cytoplasmic plaque proteins of tight and adherens junctions in different cultured epithelial and endothelial cell lines. Immunoblotting and immunofluorescence analyses show that the expression profiles of cingulin, paracingulin, ZO-1, ZO-2, ZO-3, PLEKHA7, afadin, PDZD11, p120-catenin, and α-catenin, as well as the transmembrane junctional proteins occludin, E-cadherin, and VE-cadherin, are significantly diverse when comparing kidney cells (MDCK, mCCD), keratinocytes (HaCaT), lung carcinoma (A427, A549), and endothelium-derived cells (bEnd.3, meEC, H5V). Proximity ligation and co-immunoprecipitation assays show that PLEKHA7 and PDZD11 are significantly more associated with the tight junction proteins cingulin and ZO-1 in aortic endothelium-derived (meEC) cells but not kidney collecting duct epithelial (mCCD) cells. These results provide evidence that the cytoplasmic plaques of tight and adherens junctions are diverse in their composition and molecular architecture and establish a conceptual framework by which we can rationally address the mechanisms of tissue-dependent junction physiology and signaling by cytoplasmic junctional proteins. [ABSTRACT FROM AUTHOR]

The majority of tight junction (TJ) proteins restrict the paracellular permeation of solutes via their extracellular loops (ECLs). Tricellulin tightens tricellular TJs (tTJs) and regulates bicellular TJ (bTJ) proteins. We demonstrate that the addition of recombinantly produced extracellular loop 2 (ECL2) of tricellulin opens cellular barriers. The peptidomimetic trictide, a synthetic peptide derived from tricellulin ECL2, increases the passage of ions, as well as of small and larger molecules up to 10 kDa, between 16 and 30 h after application to human epithelial colorectal adenocarcinoma cell line 2. Tricellulin and lipolysis-stimulated lipoprotein receptor relocate from tTJs toward bTJs, while the TJ proteins claudin-1 and occludin redistribute from bTJs to the cytosol. Analyzing the opening of the tricellular sealing tube by the peptidomimetic using super-resolution stimulated-emission depletion microscopy revealed a tricellulin-free area at the tricellular region. Cis-interactions (as measured by fluorescence resonance energy transfer) of tricellulin-tricellulin (tTJs), tricellulin-claudin-1, tricellulin-marvelD3, and occludin-occludin (bTJs) were strongly affected by trictide treatment. Circular dichroism spectroscopy and molecular modeling suggest that trictide adopts a β-sheet structure, resulting in a peculiar interaction surface for its binding to tricellulin. In conclusion, trictide is a novel and promising tool for overcoming cellular barriers at bTJs and tTJs with the potential to transiently improve drug delivery. [ABSTRACT FROM AUTHOR]

Claudins are integral components of tight junctions (TJs) in epithelia and endothelia. When expressed in cell lines devoid of TJs, claudins are able to form TJ-like strands at contacts between adjacent cells. According to a current model of TJ strand formation, claudin protomers assemble in an antiparallel double row within the plasma membrane of each cell ( cis-interaction) while binding to corresponding double rows from the neighboring cells ( trans-interaction). Cis-interaction was proposed to involve two interfaces of the protomers' first extracellular segment (extracellular loop (ECL)1). In the current study, three naturally occurring claudin-10 isoforms and two claudin-10 chimeras were used to investigate strand formation. All constructs were able to interact in cis (Förster/fluorescence resonance energy transfer (FRET)), to integrate into TJs of MDCK-C7 cells (confocal laser scanning microscopy), and to form TJ-like strands in HEK293 cells (freeze-fracture electron microscopy). Strand formation occurred despite the fact that isoform claudin-10a_i1 lacks both structural ECL1 elements reported to be crucial for cis-interaction. Furthermore, results from FRET experiments on claudin-10 chimeras indicated that identity of the first transmembrane region rather than ECL1 is decisive for claudin-10 cis-interaction. Therefore, in addition to the interaction interfaces suggested in the current model for TJ strand assembly, alternative interfaces must exist. [ABSTRACT FROM AUTHOR]

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short-term inoculation results in increased TJ barrier function-which could not be seen in HaCaT cells-hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin-4 at cell membranes. Long-term incubation resulted in decreased presence of claudin-1 and claudin-4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short-term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs. [ABSTRACT FROM AUTHOR]

Epithelial cell sheet formation is central to many aspects of vertebrate development and function. For example, it is a major principle of differentiation in embryogenesis and regeneration, enables the compartmentalization of tissues, and is the basis for the maintenance of homeostasis throughout the body. A key characteristic of biologically functional epithelial cell sheets is a clear difference between the top and bottom sides owing to the apicobasal polarity of the cells in the sheet, as seen in the simple polar epithelia. Epithelial cell sheets are formed by cell-cell adhesion conferred by junctional complexes, in particular via tight junctions (TJs), which thus create a paracellular barrier. This review focuses on the apical side of the sheet, which serves as the front line. The apical membranes and TJs of the various tissues have specific characteristics that enable them to function and adapt to their biological context: each system must be robust, but also dynamic and flexible to maintain homeostasis. Here, we describe various apical cytoskeletal structures that are critical to the integrity of epithelial cell sheets. We also discuss the association of apical cytoskeletal networks with TJs, which thus forms a combined system, tentatively termed the TJ-apical complex. [ABSTRACT FROM AUTHOR]

The blood-spinal cord barrier (BSCB) prevents leakage of molecules, such as pronociceptive mediators, into the spinal cord, but its role in the pathophysiology of neuropathic pain is not completely understood. Rats with chronic constriction injury (CCI) develop mechanical allodynia, thermal hypersensitivity, and reduced motor performance (Rota-Rod test) compared with sham-injured mice-similar to mice with spared nerve injury (SNI). The BSCB becomes permeable for small and large tracers 1 day after nerve ligation. Messenger RNA (mRNA) expression of tight junction proteins (TJPs) occludin, claudin-1, claudin-5, claudin-19, tricellulin, and ZO-1 significantly declines 7-14 days after CCI or SNI. ZO-1 and occludin are reduced in the cell membrane. In capillaries isolated from the spinal cord, immunoreactivity of claudin-5 and ZO-1 is fainter. In parallel, the number of platelet-derived growth factor receptor β (PDGF-β)+ and CD13+ pericytes in the spinal cord drops. Reduced levels of cytosolic transcription factors like β-catenin, but not SMAD4 and SLUG, could account for reduced TJP mRNA. In summary, neuropathy-induced allodynia/hypersensitivity is accompanied by a loss of pericytes in the spinal cord and a leaky BSCB. A better understanding of these pathways and mechanisms in neuropathic pain might foster the design of novel treatments to maintain spinal cord homeostasis. [ABSTRACT FROM AUTHOR]

The claudins are a family of membrane proteins with at least 27 members in humans and mice. The extracellular regions of claudin proteins play essential roles in cell-cell adhesion and the paracellular barrier functions of tight junctions (TJs) in epithelial cell sheets. Furthermore, the extracellular regions of some claudins function as paracellular channels in the paracellular barrier that allow the selective passage of water, ions, and/or small organic solutes across the TJ in the extracellular space. Structural analyses have revealed a common framework of transmembrane, cytoplasmic, and extracellular regions among the claudin-based paracellular barriers and paracellular channels; however, differences in the claudins' extracellular regions, such as their charges and conformations, determine their properties. Among the biological systems that involve fluid flow and metabolism, it is noted that hepatic bile flow, renal Na+ reabsorption, and intestinal nutrient absorption are dynamically regulated via site-specific distributions of paracellular channel-forming claudins in tissue. Here, we focus on how site-specific distributions of claudin-2- and claudin-15-based paracellular channels drive their organ-specific functions in the liver, kidney, and intestine. [ABSTRACT FROM AUTHOR]

Epithelial cells constitute a physical barrier that aids in protecting the host from microbial pathogens. Polarized epithelial cells contain distinct apical and basolateral membrane domains separated by intercellular junctions, including tight junctions (TJs), which contribute to the maintenance of apical-basal polarity. Polarity complexes also contribute to the establishment of TJ formation. Several pathogens perturb epithelial TJ barrier function and structure in addition to causing a loss of apical-basal polarity. Here, we review the impact of pathogenic bacteria on the disruption of cell-cell junctions and epithelial polarity. [ABSTRACT FROM AUTHOR]

Renal tubular transport mechanisms are optimized to be energy efficient and tailored to local gradients and transport rates. The combined transcellular action of ion channels, transporters, and pumps, together with the paracellular pathway, enables kidney function. Monogenetic diseases and mouse models indicate that both trans- and paracellular proteins can become disease-causing candidates and may be targets for future therapeutic approaches. Recent advances in tight junction research have provided new insights into their structure, function, and regulation. The thick ascending limb (TAL) is a nephron segment with specific requirements for the paracellular pathway. It has to fuel the generation of the corticomedullary concentration gradient, to be watertight, and to provide a highly selective permeability for Na+ and divalent cations. Tight junction composition and function in the TAL is organized along the corticomedullary axis. Even on the level of a seemingly homogeneous tubular epithelium like the TAL, there is a separation of tight junction protein expression in the strands between the respective tricellular nexus of the junctional network. Here, we highlight some new insights from our recent work and that of others in this context. In addition, we provide some perspectives for the further study of paracellular transport mechanisms. [ABSTRACT FROM AUTHOR]

The iron-binding glycoprotein lactoferrin (LF) is naturally present in human breast milk. Several studies suggest that LF contributes to infant health and development owing to a variety of protective effects, including antimicrobial and anti-inflammatory features. Therefore, we aimed to elucidate its protective properties on intestinal epithelial barrier dysfunction induced by infection or inflammation using the human epithelial cell culture models HT-29/B6 and T84. During barrier perturbation induced by the proinflammatory cytokine tumor necrosis factor α (TNF-α), bovine LF restored tight junction (TJ) morphometry and inhibited TNF-α-induced epithelial apoptosis. This resulted in an attenuation of the TNF-α-induced decrease in transepithelial resistance (TER) and increases in permeability of fluorescein and FITC-dextran (4 kDa) and was as effective as the apoptosis inhibitor Q-VD-Oph. The enteropathogenic bacterium Yersinia enterocolitica is a frequent cause of diarrhea in early childhood. This involves focal changes in TJ protein expression and localization. LF diminished the Y. enterocolitica-induced drop in TER in the present in vitro model, which was paralleled by an inhibition of the Yersinia-induced reduction of claudin-8 expression via c-Jun kinase signaling. In conclusion, LF exerts protective effects against inflammation- or infection-induced barrier dysfunction in human intestinal cell lines, supporting its relevance for healthy infant development. [ABSTRACT FROM AUTHOR]

Infections by Campylobacter species are the most common foodborne zoonotic disease worldwide. Campylobacter jejuni and C. coli are isolated most frequently from human stool samples, but severe infections by C. fetus (Cf), which can cause gastroenteritis, septicemia, and abortion, are also found. This study aims at the characterization of pathological changes in Cf infection using an intestinal epithelial cell model. The Cf-induced epithelial barrier defects appeared earlier than those of avian Campylobacter species like C. jejuni/C. coli. Two-path impedance spectroscopy (2PI) distinguished transcellular and paracellular resistance contributions to the overall epithelial barrier impairment. Both transcellular and paracellular resistance of Cf-infected HT-29/B6 monolayers were reduced. The latter was attributed to activation of active anion secretion. Western blot analysis showed no decrease in tight junction (TJ) protein expression (claudin-1, -2, -3, and -4) but showed redistribution of claudin-1 off the TJ domain. In addition, Cf induced epithelial cell death, cell detachment, and lesions (focal leaks), as the result of which macromolecule flux (10-kDa dextran) was increased in Cf-invaded cell monolayers. In conclusion, barrier dysfunction from Cf infection was due to TJ protein redistribution, cell death induction, and leak formation, resulting in bacterial translocation, ion leak flux, and antigen uptake (leaky gut). [ABSTRACT FROM AUTHOR]

Tricellulin is a tight junction protein localized to tricellular contacts in many epithelial tissues, where it is required for full barrier control. Here, we present crystal structures of the tricellulin C-terminal coiled-coil domain, revealing a potential dimeric arrangement. By combining structural, biochemical, functional, and mutation analyses, we gain insight into the mode of tricellulin oligomerization and suggest a model where dimerization of its cytoplasmic C-terminus may play an auxiliary role in stabilizing homophilic and potentially also heterophilic cis-interactions within tight junctions. [ABSTRACT FROM AUTHOR]

Targeted topical application promises high drug concentrations in the skin and low systemic adverse effects. To locate drugs and drug-delivery systems like nanocarriers, fluorescent dyes are commonly used as drug surrogates or nanocarrier labels in micrographs of tissue sections. Here, we investigate how labeling degree, concentration of fluorophore, and nanocarrier may affect the interpretation of these micrographs. False-negative penetration results due to inter- and intramolecular quenching effects are likely. Using tecto-dendrimers as an example, we present a detailed analysis of pitfalls in the (semi-)quantitative evaluation of skin nanocarrier penetration. Fluorescence lifetime imaging microscopy (FLIM) allows distinguishing the target fluorescence of dye-tagged nanocarriers from skin autofluorescence, providing a highly sensitive tool for clear-cut localization of the nanocarriers. Cluster-FLIM images reveal that FITC-labeled tecto-dendrimers penetrate the stratum corneum of human skin ex vivo and reconstructed human skin but do not cross the tight junction barrier. [ABSTRACT FROM AUTHOR]

Tight junctions (TJs) are key players in determining tissue-specific paracellular permeability across epithelial and endothelial membranes. Claudin proteins, the primary determinants of TJs structure and functionality, assemble in paracellular spaces to form channels and pores that are charge and size selective. Here, using molecular dynamics (MD) simulations, we elucidate the molecular assembly of claudin-3 and claudin-5 proteins of blood-brain barrier TJs. Despite having a high degree of sequence and structural similarity, these two claudins form different types of cis-interactions. Molecular docking of the observed cis-interfaces into trans-forms revealed two putative pore models that were also observed in the self-assembly simulations. The observed pore structures (pore I and II) have pore-lining residues that have been previously reported in the literature. The pore I model is consistent with a previously reported claudin-15 model. The pore II model, also consistent with biochemical results, has not been reported previously. Further analysis using in silico site-directed mutations provide convincing support for the validity of the pore II model. Using steered MD and umbrella sampling, we computed the transport properties of water and α- d-glucose through pore II. The study offers new insight into the selectivity of blood-brain barrier TJs. [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) formed by the microvascular endothelium limits cerebral drug delivery. The paraendothelial cleft is sealed by tight junctions (TJs) with a major contribution from claudin-5, which we selected as a target to modulate BBB permeability. For this purpose, drug-enhancer peptides were designed based on the first extracellular loop (ECL) of claudin-5 to allow transient BBB permeabilization. Peptidomimetics (C5C2 and derivatives, nanomolar affinity to claudin-5) size-selectively (≤40 kDa) and reversibly (12-48 h) increased the permeability of brain endothelial and claudin-5-transfected epithelial cell monolayers. Upon peptide uptake, the number of TJ strand particles diminished, claudin-5 was downregulated and redistributed from cell-cell contacts to the cytosol, and the cell shape was altered. Cellular permeability of doxorubicin (cytostatic drug, 580 Da) was enhanced after peptide administration. Mouse studies (3.5 μmol/kg i.v.) confirmed that, for both C5C2 and a d-amino acid derivative, brain uptake of Gd-diethylene-triamine penta-acetic acid (547 Da) was enhanced within 4 h of treatment. On the basis of our functional data, circular dichroism measurements, molecular modeling, and docking experiments, we suggest an association model between β-sheets flanked by α-helices, formed by claudin-5 ECLs, and the peptides. In conclusion, we identified claudin-5 peptidomimetics that improve drug delivery through endothelial and epithelial barriers expressing claudin-5. [ABSTRACT FROM AUTHOR]

Milk contains a variety of components that have been shown to affect the expression and localization of epithelial tight junction proteins and therefore the intestinal barrier. Thus, we hypothesized that milk would have an effect on intestinal barrier properties, owing to effects on the tight junction in an intraspecies porcine intestinal in vitro model. Jejunal samples of piglets derived from different age groups were analyzed. Transepithelial electrical resistance was recorded employing the Ussing chamber technique. Porcine milk or predigested milk in buffer solution was added to the apical side, and effects were compared to untreated controls. Unidirectional paracellular flux measurements were performed using sodium fluorescein. Tight junction protein expression and localization were analyzed by immunoblotting and immunofluorescence microscopy. Incubation with milk or predigested milk led to an increase in transepithelial electrical resistance, while paracellular permeability for sodium fluorescein did not result in significant changes. Densitometric analysis of immunoblot signals did not show significant alterations in claudin expression, but a reduction of claudin signals in apicolateral membrane compartments in both approaches became apparent via immunohistology. The functional effect might reflect a physiological protective mechanism, when offspring exclusively rely on their mother's milk and are exposed to a plethora of potentially barrier-perturbing factors. [ABSTRACT FROM AUTHOR]

The tricellular tight junction (tTJ) is a potential weak point of the paracellular barrier. For solving the proportional contribution of the tTJ, ion conductances and macromolecule permeabilities were analyzed in cell lines of different leakiness. MDCK II, Caco-2, and HT-29/B6 cells were subjected to two-path impedance spectroscopy and morphological analyses in order to calculate the contribution of the tTJ to paracellular and total ion conductivity. The contribution to macromolecule permeability was evaluated by tricellulin overexpression or knockdown. Tricellulin-dependent macromolecule passage was comparably regulated in leaky and tight epithelia, but relative and absolute ion permeabilities of the tTJs were different. Assuming a minimal (50 pS) and maximal (146 pS) conductivity per single tTJ, the possible range of contribution of the tTJ to paracellular ion conductance amounted to only 0.3-1.1% in the leaky cell line MDCK II, but 3-25% in the moderately tight cell line Caco-2, and not less than 29% in the tight cell line HT-29/B6. In these cells, this resulted in a contribution to total epithelial conductance of 9-32%. In conclusion, in leaky epithelia the bicellular TJ accounts for nearly the entire paracellular ion conductance, whereas in tight epithelia the low bicellular TJ conductance has large impact on the tTJ. [ABSTRACT FROM AUTHOR]

Claudins provide tight junction barrier selectivity. The human CLDN5 gene contains a high-frequency single-nucleotide polymorphism (rs885985), where the G allele codes for glutamine (Q) and the A allele codes for an amber stop codon. Thus, these different CLDN5 alleles define nested open reading frames (ORFs) encoding claudin-5 proteins that are 303 or 218 amino acids in length. Interestingly, human claudin-16 and claudin-23 also have long ORFs. The long form of claudin-5 contrasts with the majority of claudin-5 proteins in the National Center for Biotechnology Information protein database, which are less than 220 amino acids in length. Screening of genotyped human lung tissue by immunoblot revealed only the 218 amino acid form of claudin-5 protein; the long-form claudin-5 protein was not detected. Moreover, when forcibly expressed in transfected cells, the long form of human claudin-5 was retained in intracellular compartments and did not localize to the plasma membrane, in contrast to the 218 amino acid form, which localized to intercellular junctions. This suggests that the 303 amino acid claudin-5 protein is rarely expressed, and, if so, is predicted to adversely affect cell function. Potential roles for upstream ORFs in regulating claudin-5 expression are also discussed. [ABSTRACT FROM AUTHOR]

Tricellulin, a member of the tight junction-associated MAGUK protein family, preferentially localizes to tricellular junctions in confluent polarized epithelial cell layers and is downregulated during the epithelial-mesenchymal transition. Posttranslational modifications are assumed to play critical roles in the process of downregulation of tricellulin at the protein level. Here, we report that the E3 ubiquitin ligase Itch forms a complex with tricellulin and thereby enhances its ubiquitination. Pull-down assays confirmed a direct interaction between tricellulin and Itch, which is mediated by the Itch WW domain and the N-terminus of tricellulin. Experiments in the presence of the proteasome inhibitor MG-132 did not show major changes in the levels of ubiquitinated tricellulin in epithelial cells, suggesting that ubiquitination is not primarily involved in proteasomal degradation of tricellulin, but it appears to be important for endocytosis or recycling. In contrast, in HEK-293 cells, MG-132 caused polyubiquitination. Moreover, we observed that well-differentiated RT-112 and de-differentiated Cal-29 bladder cancer cells show an inverse expression of tricellulin and Itch. We postulate that ubiquitination is an important posttranslational modification involved in the determination of the intracellular fate of tricellulin deserving of more detailed further investigations into the underlying molecular mechanisms and their regulation. [ABSTRACT FROM AUTHOR]

Claudins are four-transmembrane proteins that constitute the backbone of tight junction strands via self-polymerization in the apicolateral membranes of epithelial cells. Together with their cell-cell adhesion function, claudin proteins form the paracellular barrier and/or channels through epithelial cell sheets whose permeability is primarily dependent on the claudin subtype. Recently determined crystal structures of several claudins revealed the unique claudin fold of four transmembrane helices in a left-handed helical bundle with an extracellular β-sheet domain. Here, we focus on the structural basis of the intermolecular interactions between claudin molecules and between the Clostridium perfringens enterotoxin and its receptor claudins. [ABSTRACT FROM AUTHOR]

Claudins define paracellular permeability to small molecules by forming ion-selective pores within the tight junction. We recently demonstrated that claudin-2 channels are gated and open and close on a submillisecond timescale. To determine if and how the ensemble behavior of this unique class of entirely extracellular gated ion channels could define global epithelial barrier function, we have developed an in silico model of local claudin-2 behavior. This model considers the complex anastomosing ultrastructure of tight junction strands and can be scaled to show that local behavior defines global epithelial barrier function of epithelial monolayers expressing different levels of claudin-2. This is the first mathematical model to describe global epithelial barrier function in terms of the dynamic behavior of single tight junction channels and establishes a framework to consider gating kinetics as a means to regulate barrier function. [ABSTRACT FROM AUTHOR]