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Formation of claudin-10 based tight junctions (TJs) is paramount to paracellular Na+ transport in multiple epithelia. Sequence variants in CLDN10 have been linked to HELIX syndrome, a salt-losing tubulopathy with altered handling of divalent cations accompanied by dysfunctional salivary, sweat, and lacrimal glands. Here, we investigate molecular basis and phenotypic consequences of a newly identified homozygous CLDN10 variant that translates into a single amino acid substitution within the fourth transmembrane helix of claudin-10. In addition to hypohidrosis (H), electrolyte (E) imbalance with impaired urine concentrating ability, and hypolacrimia (L), phenotypic findings include altered salivary electrolyte composition and amelogenesis imperfecta but neither ichthyosis (I) nor xerostomia (X). Employing cellular TJ reconstitution assays, we demonstrate perturbation of cis- and trans-interactions between mutant claudin-10 proteins. Ultrastructures of reconstituted TJ strands show disturbed continuity and reduced abundance in the mutant case. Throughout, both major isoforms, claudin-10a and claudin-10b, are differentially affected with claudin-10b showing more severe molecular alterations. However, expression of the mutant in renal epithelial cells with endogenous TJs results in wild-type-like ion selectivity and conductivity, indicating that aberrant claudin-10 is generally capable of forming functional paracellular channels. Thus, mutant proteins prove pathogenic by compromising claudin-10 TJ strand assembly. Additional ex vivo investigations indicate their insertion into TJs to occur in a tissue-specific manner.

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.

A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC.

Bacterial toxins gain growing attention as potential cancer treatment due to their potent cytotoxic effects. Among the very different toxins with diverse modes of action, the Clostridium perfringens enterotoxin (CPE) is in focus to treat solid cancers. This toxin targets the tight junction proteins claudin-3 and -4 (Cldn-3/4), which are frequently overexpressed in solid cancers. Binding to these claudins induces pore formation in the host cell plasma membrane leading to rapid oncoleaking cell death of tumor cells. Based on this, extending the targeting of CPE beyond Cldn-3/4 is of interest, since other claudins, such as claudin-1 or -5 are often overexpressed in various cancer entities such as non-small-cell lung cancer (NSCLC) or papillary thyroid carcinoma. In this chapter we describe the modification of a CPE-encoding vector by structure-directed mutagenesis to either preferentially target Cldn-1 and -5 or to expand targeting to Cldn1-9 for improved broadened cytotoxic targeting of claudin-overexpressing tumors such as but not limited to lung cancer via CPE gene transfer.

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.

Tight junctions regulate paracellular permeability size and charge selectively. Models have been proposed for the molecular architecture of tight junction strands and paracellular channels. However, they are not fully consistent with experimental and structural data. Here, we analysed the architecture of claudin-based tight junction strands and channels by cellular reconstitution of strands, structure-guided mutagenesis, in silico protein docking and oligomer modelling. Prototypic channel- (Cldn10b) and barrier-forming (Cldn3) claudins were analysed. Forster resonance energy transfer (FRET) assays indicated multistep claudin polymerisation, starting with cis-oligomerization specific to the claudin subtype, followed by trans-interaction-triggered cis-polymerisation. Alternative protomer interfaces were modelled in silico and tested by cysteine-mediated crosslinking, confocal- and freeze fracture EM-based analysis of strand formation. The analysed claudin mutants included also mutations causing the HELIX syndrome. The results indicated that protomers in Cldn10b and Cldn3 strands form similar antiparallel double rows, as has been suggested for Cldn15. Mutually stabilising ‐hydrophilic and hydrophobic ‐ cis- and trans-interfaces were identified that contained novel key residues of extracellular segments ECS1 and ECS2. Hydrophobic clustering of the flexible ECS1 β1β2 loops together with ECS2–ECS2 trans-interaction is suggested to be the driving force for conjunction of tetrameric building blocks into claudin polymers. Cldn10b and Cldn3 are indicated to share this polymerisation mechanism. However, in the paracellular centre of tetramers, electrostatic repulsion may lead to formation of pores (Cldn10b) and electrostatic attraction to barriers (Cldn3). Combining in vitro data and in silico modelling, this study improves mechanistic understanding of paracellular permeability regulation by elucidating claudin assembly and its pathologic alteration as in HELIX syndrome.

A change in claudin expression has been demonstrated in various tumors. The present study specifically compares claudin expression in oral squamous cell carcinoma (OSCC) with healthy oral epithelium from the same individual and analyzes the association between claudin expression and the clinically relevant course parameters. Our study includes tissue samples and clinically relevant follow-up data from 60 patients with primary and untreated OSCC. The oral mucosa was analyzed via Western blot for the expression of claudin-1, -2, -3, -4, -5, and -7. Importantly, the tumor and healthy tissues were obtained pairwise from patients, allowing for intraindividual comparisons. Both the healthy and tumor epithelium from the oral cavity did not express the claudin-3 protein. The intraindividual comparison revealed that, in OSCC, claudin-2 expression was higher, and the expression of claudin-4, -5, and -7 was lower than in healthy epithelium. An association was found between increased claudin-2 expression and shorter relapse-free survival. In addition, the reduced expression of claudin-4 had a negative impact on relapse-free survival. Furthermore, associations between the reduced expression of claudin-7 and the stage of a tumor, or the presence of lymph node metastases, were found. Thus, the expression level of claudin-2, -4, and -7 appears to be predictive of the diagnosis and prognosis of OSCC.

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.

Simple Summary Current therapies for pancreas carcinoma (PC) are of limited efficacy due to tumor aggressiveness and therapy resistance. Bacterial toxins with pore-forming (oncoleaking) potential are promising tools in cancer therapy. We have developed a novel, suicide gene therapy treatment, based on Clostridium perfringens enterotoxin (CPE)-mediated oncoleaking. This is achieved by CPE suicide gene therapy to treat PC, which overexpresses the claudin-3 and -4 (Cldn3/4) tight junction proteins, which are targets of CPE action. This targeted gene therapy causes rapid eradication of Cldn3/4 overexpressing PC cells via oncoleaking and initiation of apoptotic/necrotic signaling. We demonstrate efficacy of this approach in vitro and after nonviral in vivo gene transfer in cell lines and in patient derived xenograft PC models. This therapy approach has translational potential for treatment of pancreas carcinomas and could also be translated into new combination settings with conventional chemotherapy. Abstract Pancreatic cancer (PC) is one of the most lethal cancers worldwide, associated with poor prognosis and restricted therapeutic options. Clostridium perfringens enterotoxin (CPE), is a pore-forming (oncoleaking) toxin, which binds to claudin-3 and -4 (Cldn3/4) causing selective cytotoxicity. Cldn3/4 are highly upregulated in PC and represent an effective target for oncoleaking therapy. We utilized a translation-optimized CPE vector (optCPE) for new suicide approach of PC in vitro and in cell lines (CDX) and patient-derived pancreatic cancer xenografts (PDX) in vivo. The study demonstrates selective toxicity in Cldn3/4 overexpressing PC cells by optCPE gene transfer, mediated by pore formation, activation of apoptotic/necrotic signaling in vitro, induction of necrosis and of bystander tumor cell killing in vivo. The optCPE non-viral intratumoral in vivo jet-injection gene therapy shows targeted antitumoral efficacy in different CDX and PDX PC models, leading to reduced tumor viability and induction of tumor necrosis, which is further enhanced if combined with chemotherapy. This selective oncoleaking suicide gene therapy improves therapeutic efficacy in pancreas carcinoma and will be of value for better local control, particularly of unresectable or therapy refractory PC.

The migration of activated T cells across the blood–brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Ultrastructural analysis of T cell migration across the blood–brain barrier (BBB) under physiological flow identifies BBB tricellular junctions as sites of T cell diapedesis.

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 (Forster/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.

The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection.

Claudins (Cldn) form the backbone of tight junction (TJ) strands and thereby regulate paracellular permeability for solutes and water. Polymeric strands are formed by homo- and heterophilic cis- and trans-interactions between claudin protomers. Crystal structures of some claudins have been resolved; however, the mechanism by which claudins assemble into TJ strands remains unclear. To elucidate strand architecture, TJ-like strands were reconstituted in HEK293 cells by claudin transfection. Determinants of prototypic, classic barrier-forming claudins (Cldn1, -3, and -5) involved in strand formation were analyzed by mutagenesis. The capability of claudin constructs to interact in trans and to form strands was investigated by cell contact-enrichment assays and freeze-fracture electron microscopy. Residues in extracellular loops 1 and 2 of the claudins affecting strand formation were identified. Using homology modeling and molecular docking, we tested working concepts for the arrangement of claudin protomers within TJ strands. We show that the charge of Lys65 in Cldn1 and Glu158 in Cldn3, but not of Arg30 or Asp145 in Cldn3, and the polarity of Gln56 and Gln62 in Cldn3 and of Gln57 in Cldn5 are necessary for TJ strand formation. These residues are all conserved among barrier-forming classic claudins. The results contribute to mechanistic understanding of claudin-based regulation of paracellular permeability.

Summary In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm2 and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies., Graphical Abstract, Highlights • Establishment of a standardized human BBB co-culture model based on hiPSCs and fNSCs • Reflection of physiological BBB integrity and expression of relevant transporters/TJs • Confirmation of TJ network functionality by claudin-specific TJ modulators • Validation of physiological transcellular model tightness by permeability studies, In this article, Metzger and colleagues present the establishment of physiologically relevant human blood-brain barrier quadruple culture models based on induced pluripotent and multipotent stem cells. The novel model can be used as a powerful tool in pharmaceutical drug research and ADMET studies, which may allow a more reliable translation of promising drug candidates into clinical application.

The functional and structural concept of tight junctions has developed after discovery of claudin and TAMP proteins. Many of these proteins contribute to epi- and endothelial barrier but some, in contrast, form paracellular channels. Claudins form the backbone of tight junction (TJ) strands whereas other proteins regulate TJ dynamics. The current joined double-row model of TJ strands and channels is crucially based on the linear alignment of claudin-15 in the crystal. Molecular dynamics simulations, protein docking, mutagenesis, cellular TJ reconstitution, and electron microscopy studies largely support stability and functionality of the model. Here, we summarize in silico and in vitro data about TJ strand assembly including comparison of claudin crystal structures and alternative models. Sequence comparisons, experimental and structural data substantiate differentiation of classic and non-classic claudins differing in motifs related to strand assembly. Classic claudins seem to share a similar mechanism of strand formation. Interface variations likely contribute to TJ strand flexibility. Combined in vitro/in silico studies are expected to elucidate mechanistic keys determining TJ regulation.

The integrity of tight junctions, which regulate paracellular permeability, is challenged by many bacterial pathogens. This is caused by inflammatory responses triggered by pathogens and direct interaction of bacteria or their toxins with host epithelial cells. In some cases, tight junction proteins represent receptors for cell surface proteins or toxins of the pathogen, such as Clostridium perfringens enterotoxin (CPE). CPE causes diarrhea and cramps-the symptoms of a common foodborne illness, caused by C. perfringens type A. It uses a subgroup of the claudin family of tight junction proteins as receptors and forms pores in the membrane of intestinal epithelial cells. Ca2+ influx through these pores finally triggers cell damage. In this review, we summarize tight junction targeting and alteration by a multitude of different microorganisms such as C. perfringens, Escherichia coli, Helicobacter pylori, Salmonella typhimurium, Shigella flexneri, Vibrio cholerae, Yersinia enterocolitica, protozoan parasites, and their proteins. A focus is drawn towards CPE, the interaction with its receptors, cellular, and pathophysiological consequences for the intestinal epithelium. In addition, we portend to the use of CPE-based claudin modulators for drug delivery as well as diagnosis and therapy of cancer.

Aim Claudin-15 is mainly expressed in the small intestine and indirectly involved in glucose absorption. Similar to claudin-2 and -10b, claudin-15 is known to form a paracellular channel for small cations. Claudin-2, but not claudin-10b, also forms water channels. Here we experimentally tested whether claudin-15 also mediates water transport and if yes, whether water transport is Na+ -coupled, as seen for claudin-2. Methods MDCK C7 cells were stably transfected with claudin-15. Ion and water permeability were investigated in confluent monolayers of control and claudin-15-expressing cells. Water flux was induced by an osmotic or ionic gradient. Results Expression of claudin-15 in MDCK cells strongly increased cation permeability. The permeability ratios for monovalent cations indicated a passage of partially hydrated ions through the claudin-15 pore. Accordingly, its pore diameter was determined to be larger than that of claudin-2 and claudin-10b. Mannitol-induced water flux was elevated in claudin-15-expressing cells compared to control cells. In contrast to the Na+ -coupled water flux of claudin-2 channels, claudin-15-mediated water flux was inhibited by Na+ flux. Consequently, water flux was increased in Na+ -free solution. Likewise, Na+ flux was decreased after induction of water flux through claudin-15. Conclusion Claudin-15, similar to claudin-2, forms a paracellular cation and water channel. In functional contrast to claudin-2, water and Na+ fluxes through claudin-15 inhibit each other. Claudin-15 allows Na+ to retain part of its hydration shell within the pore. This then reduces the simultaneous passage of additional water through the pore.

The tetra-span transmembrane proteins of the claudin family are critical components of formation and function of tight junctions (TJ). Homo- and heterophilic side-by-side (cis) and intercellular head-to-head (trans) interactions of 27 claudin-subtypes regulate tissue-specifically the paracellular permeability and/or tightness between epithelial or endothelial cells. This review highlights the functional impact that has been identified for particular claudin residues by relating them to structural features and architectural characteristics in the light of structural advances, which have been contributed by homology models, cryo-electron microscopy and crystal structures. The differing contributions to the TJ functionalities by claudins are dissected for the transmembrane region, the first and the second extracellular loop of claudins separately. Their particular impact to oligomerisation and TJ strand- and pore-formation is surveyed. Detailed knowledge about structure–function relationships about claudins helps to reveal the molecular mechanisms of TJ assembly and regulation of paracellular permeability, which is yet not fully understood.

The blood-brain barrier (BBB) prevents entry of neurotoxic substances but also that of drugs into the brain. Here, the paracellular barrier is formed by tight junctions (TJs) with claudin-5 (Cldn5) being the main sealing constituent. Transient BBB opening by targeting Cldn5 could improve paracellular drug delivery. The non-toxic C-terminal domain of Clostridium perfringens enterotoxin (cCPE) binds to a subset of claudins, e.g., Cldn3, -4. Structure-based mutagenesis was used to generate Cldn5-binding variants (cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H). These cCPE-variants were tested for transient TJ opening using multiple in vitro BBB models: Primary porcine brain endothelial cells, coculture of primary rat brain endothelial cells with astrocytes and mouse cerebEND cells. cCPE-Y306W/S313H and cCPE-N218Q/Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A (not binding to claudins) decreased transendothelial electrical resistance in a concentration-dependent and reversible manner. Furthermore, permeability of carboxyfluorescein (with size of CNS drugs) was increased. cCPE-Y306W/S313H but neither cCPE-wt nor cCPE-Y306A/L315A bound to Cldn5-expressing brain endothelial cells. However, freeze-fracture EM showed that cCPE-Y306W/S313H did not cause drastic TJ breakdown. In sum, Cldn5-binding cCPE-variants enabled mild and transient opening of brain endothelial TJs. Using reliable in vitro BBB models, the results demonstrate that cCPE-based biologics designed to bind Cldn5 improve paracellular drug delivery across the BBB.

Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor α, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis-if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility.

The renal proximal tubule achieves the majority of renal water and solute reabsorption with the help of paracellular channels which lead through the tight junction. The proteins forming such channels in the proximal tubule are claudin-2, claudin-10a, and possibly claudin-17. Claudin-2 forms paracellular channels selective for small cations like Na+ and K+. Independently of each other, claudin-10a and claudin-17 form anion-selective channels. The claudins form the paracellular "pore pathway" and are integrated, together with purely sealing claudins and other tight junction proteins, in the belt of tight junction strands surrounding the tubular epithelial cells. In most species, the proximal tubular tight junction consists of only 1-2 (pars convoluta) to 3-5 (pars recta) horizontal strands. Even so, they seal the tubule very effectively against leak passage of nutrients and larger molecules. Remarkably, claudin-2 channels are also permeable to water so that 20-25% of proximal water absorption may occur paracellularly. Although the exact structure of the claudin-2 channel is still unknown, it is clear that Na+ and water share the same pore. Already solved claudin crystal structures reveal a characteristic β-sheet, comprising β-strands from both extracellular loops, which is anchored to a left-handed four-transmembrane helix bundle. This allowed homology modeling of channel-forming claudins present in the proximal tubule. The surface of cation- and anion-selective claudins differ in electrostatic potentials in the area of the proposed ion channel, resulting in the opposite charge selectivity of these claudins. Presently, while models of the molecular structure of the claudin-based oligomeric channels have been proposed, its full understanding has only started.

During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, −4 and −8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively., Graphical abstract fx1, Highlights • Simultaneous removal of Cldn3, −4 and −8 causes open neural tube defects. • Folic acid cannot rescue open NTDs caused by depletion of Cldn3, −4 and −8. • Removal of Cldn3, −4 and −8 prevents convergent extension. • Apical constriction to form the median hinge point requires Cldn3, −4 and −8. • Claudins localize polarity complex components to the apical surface.

The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl β-D-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.

Background and Purpose The blood-brain barrier (BBB) restricts drug penetration to the brain preventing effective treatment of patients suffering from brain tumours. Intra-arterial injection of short-chain alkylglycerols (AGs) opens the BBB and increases delivery of molecules to rodent brain parenchyma in vivo. The mechanism underlying AG-mediated modification of BBB permeability is still unknown. Here, we have tested the effects of AGs on barrier properties of cultured brain microvascular endothelial cells. Experimental Approach The effects of two AGs, 1-O-pentylglycerol and 2-O-hexyldiglycerol were examined using an in vitro BBB model consisting of primary cultures of rat brain endothelial cells, co-cultured with rat cerebral glial cells. Integrity of the paracellular, tight junction-based, permeation route was analysed by functional assays, immunostaining for junctional proteins, freeze-fracture electron microscopy, and analysis of claudin-claudin trans-interactions. Key Results AG treatment (5 min) reversibly reduced transendothelial electrical resistance and increased BBB permeability for fluorescein accompanied by changes in cell morphology and immunostaining for claudin-5 and β-catenin. These short-term changes were not accompanied by alterations of inter-endothelial tight junction strand complexity or the trans-interaction of claudin-5. Conclusion and Implications AG-mediated increase in brain endothelial paracellular permeability was short, reversible and did not affect tight junction strand complexity. Redistribution of junctional proteins and alterations in the cell shape indicate the involvement of the cytoskeleton in the action of AGs. These data confirm the results from in vivo studies in rodents characterizing AGs as adjuvants that transiently open the BBB.

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.

The majority of malignant tumors originate from epithelial cells, and many of them are characterized by an overexpression of claudins (Cldns) and their mislocalization out of tight junctions. We utilized the C-terminal claudin-binding domain of Clostridium perfringens enterotoxin (cCPE), with its high affinity to specific members of the claudin family, as the targeting unit for a claudin-sensitive cancer biosensor. To overcome the poor sensitivity of conventional relaxivity-based magnetic resonance imaging (MRI) contrast agents, we utilized the superior sensitivity of xenon Hyper-CEST biosensors. We labeled cCPE for both xenon MRI and fluorescence detection. As one readout module, we employed a cryptophane (CrA) monoacid and, as the second, a fluorescein molecule. Both were conjugated separately to a biotin molecule via a polyethyleneglycol chemical spacer and later via avidin linked to GST-cCPE. Nontransfected HEK293 cells and HEK293 cells stably expressing Cldn4-FLAG were incubated with the cCPE-based biosensor. Fluorescence-based flow cytometry and xenon MRI demonstrated binding of the biosensor specifically to Cldn4-expressing cells. This study provides proof of concept for the use of cCPE as a carrier for diagnostic contrast agents, a novel approach for potential detection of Cldn3/-4-overexpressing tumors for noninvasive early cancer detection.

Claudins (Cldn) form the backbone of tight junction (TJ) strands and thereby regulate paracellular permeability for solutes and water. Polymeric strands are formed by homo- and heterophilic cis- and trans-interactions between claudin protomers. Crystal structures of some claudins have been resolved; however, the mechanism by which claudins assemble into TJ strands remains unclear. To elucidate strand architecture, TJ-like strands were reconstituted in HEK293 cells by claudin transfection. Determinants of prototypic, classic barrier-forming claudins (Cldn1, -3, and -5) involved in strand formation were analyzed by mutagenesis. The capability of claudin constructs to interact in trans and to form strands was investigated by cell contact-enrichment assays and freeze-fracture electron microscopy. Residues in extracellular loops 1 and 2 of the claudins affecting strand formation were identified. Using homology modeling and molecular docking, we tested working concepts for the arrangement of claudin protomers within TJ strands. We show that the charge of Lys65 in Cldn1 and Glu158 in Cldn3, but not of Arg30 or Asp145 in Cldn3, and the polarity of Gln56 and Gln62 in Cldn3 and of Gln57 in Cldn5 are necessary for TJ strand formation. These residues are all conserved among barrier-forming classic claudins. The results contribute to mechanistic understanding of claudin-based regulation of paracellular permeability.

Summary Tight junctions seal the paracellular cleft of epithelia and endothelia, form vital barriers between tissue compartments and consist of tight-junction-associated marvel proteins (TAMPs) and claudins. The function of TAMPs and the interaction with claudins are not understood. We therefore investigated the binding between the TAMPs occludin, tricellulin, and marvelD3 and their interaction with claudins in living tight-junction-free human embryonic kidney-293 cells. In contrast to claudins and occludin, tricellulin and marvelD3 showed no enrichment at cell–cell contacts indicating lack of homophilic trans-interaction between two opposing cell membranes. However, occludin, marvelD3 and tricellulin exhibited homophilic cis-interactions, along one plasma membrane, as measured by fluorescence resonance energy transfer. MarvelD3 also cis-interacted with occludin and tricellulin heterophilically. Classic claudins, such as claudin-1 to -5 may show cis-oligomerization with TAMPs, whereas the non-classic claudin-11 did not. Claudin-1 and -5 improved enrichment of occludin and tricellulin at cell–cell contacts. The low mobile claudin-1 reduced the membrane mobility of the highly mobile occludin and tricellulin, as studied by fluorescence recovery after photobleaching. Co-transfection of claudin-1 with TAMPs led to changes of the tight junction strand network of this claudin to a more physiological morphology, depicted by freeze-fracture electron microscopy. The results demonstrate multilateral interactions between the tight junction proteins, in which claudins determine the function of TAMPs and vice versa, and provide deeper insights into the tight junction assembly.

Pore-forming properties of claudins (Cld) are likely defined by residues of their first extracellular loop (ECL1). Detailed mechanisms are unclear. MDCK cells overexpressing FLAG-Cld-1 wild-type and mutants were characterized by transepithelial resistance (TER) and ion permeability measurements. Replacing ECL1 residues of sealing Cld-1 by corresponding Cld-2 residues we aimed to identify new determinants responsible for sealing and/or pore formation. We found that E48K and S53E substitutions in human Cld-1 strongly reduced TER and increased permeability for Na(+) and Cl(-) . In contrast, K65D, D68S, and other single substitutions showed no significant change of TER and permeability for Na(+) and Cl(-) . Double substitution S53E/K65D did not change TER and ion permeability, whereas S53E/D68S decreased TER, albeit weaker than S53E. Ratio of permeabilities for Na(+) and Cl(-) revealed no clear charge specificity of the pore induced by S53E or S53E/D68S in Cld-1, suggesting that primarily S53 and potentially D68 in Cld-1 are involved in sealing of the paracellular cleft and that charge-unselective pores may be induced by substituting S53E.

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

Paracellular barrier properties of tissues are mainly determined by the composition of claudin heteropolymers. To analyze the molecular organization of tight junctions (TJ), we investigated the ability of claudins (Cld) to form homo- and heteromers. Cld1, -2, -3, -5, and -12 expressed in cerebral barriers were investigated. TJ-strands were reconstituted by claudin-transfection of HEK293-cells. cis-Interactions and/or spatial proximity were analyzed by fluorescence resonance energy transfer inside and outside of strands and ranked: Cld5/Cld5 > Cld5/Cld1 > Cld3/Cld1 > Cld3/Cld3 > Cld3/Cld5, no Cld3/Cld2. Classic Cld1, -3, and -5 but not non-classic Cld12 showed homophilic trans-interaction. Freeze-fracture electron microscopy revealed that, in contrast to classic claudins, YFP-tagged Cld12 does not form homopolymers. Heterophilic trans-interactions were analyzed in cocultures of differently monotransfected cells. trans-Interaction of Cld3/Cld5 was less pronounced than that of Cld3/Cld1, Cld5/Cld1, Cld5/Cld5 or Cld3/Cld3. The barrier function of reconstituted TJ-strands was demonstrated by a novel imaging assay. A model of the molecular organization of TJ was generated.

Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood–brain barrier. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na + ,K + -ATPase during luminal expansion of brain ventricles. These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis.

Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation.

Claudin-17 is a paracellular channel-forming tight junction protein. Unlike the cation channels claudin-2 and -15, claudin-17 forms a distinct anion-selective channel. Aim of this study was to determine the molecular basis of channel formation and charge selectivity of this protein. To achieve this, residues located in the extracellular loops (ECL) 1 and 2 of claudin-17 were substituted, preferably those whose charges differed in claudin-17 and in claudin-2 or -15. The respective mutants were stably expressed in MDCK C7 cells and their ability to form charge-selective channels was analyzed by measuring ion permeabilities and transepithelial electrical resistance. The functional data were combined with homology modeling of the claudin-17 protomer using the structure of claudin-15 as template. In ECL1, K65, R31, E48, and E44 were found to be stronger involved in Cldn17 channel function than the clustered R45, R56, R59, and R61. For K65, not only charge but also stereochemical properties were crucial for formation of the anion-selective channel. In ECL2, both Y149 and H154 were found to contribute to constitution of the anion channel in a distinct manner. In conclusion, we provide insight into the molecular mechanism of the formation of charge- and size-selective paracellular ion channels. In detail, we propose a hydrophilic furrow in the claudin-17 protomer spanning from a gap between the ends of TM2 and TM3 along R31, E48, and Y67 to a gap between K65 and S68 lining the anion channel.

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiled-coil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported

The membrane cortex has an important role in generating and maintaining spatially and functionally distinct domains in neurons. As a tool to functionally characterize molecules of the membrane cortex, we generated novel monoclonal antibodies against a fraction enriched for components of the neuronal membrane skeleton. We obtained two antibodies against the kinase-anchoring protein gravin. Gravin was strongly up-regulated during differentiation of human model neurons (NT2-N neurons) and was enriched at the inner peripheral cortex in close proximity to the plasma membrane where its localization primarily depended on association with membranes. In differentiated neurons, gravin colocalized in putative signaling complexes with protein kinase C (PKCβII) and partially with PKCα and cAMP-dependent protein kinase (PKA). Colocalization with PKCϵ was not observed. PKCβII, PKCα, and PKA but not PKCϵ coprecipitated with gravin indicating physical interaction. Binding of gravin to PKCα required the presence of Ca2+ and was increased after inhibition of PKC. In contrast, binding of PKCβII and PKA were independent of Ca2+ and PKC inhibition. Activation of PKC decreased binding of PKCα to gravin, decreased its association with the plasma membrane, and reduced the mean size of gravin particles. Taken together the data suggest that gravin provides a dynamic platform to localize kinases in an isoenzyme-specific and activation-dependent manner at specific sites in neurons.

Claudins form a large family of TJ (tight junction) proteins featuring four transmembrane segments (TM1–TM4), two extracellular loops, one intracellular loop and intracellular N- and C-termini. They form continuous and branched TJ strands by homo- or heterophilic interaction within the same membrane (cis-interaction) and with claudins of the opposing lateral cell membrane (trans-interaction). In order to clarify the molecular organization of TJ strand formation, we investigated the cis-interaction of two abundant prototypic claudins. Human claudin-1 and claudin-3, fused to ECFP or EYFP at the N- or C-terminus, were expressed in the TJ-free cell line HEK (human embryonic kidney)-293. Using FRET analysis, the proximity of claudin N- and C-termini integrated in homopolymeric strands composed of claudin-3 or of heteropolymeric strands composed of claudin-1 and claudin-3 were determined. The main results are that (i) within homo- and heteropolymers, the average distance between the cytoplasmic ends of the TM1s of cis-interacting claudin molecules is shorter than the average distance between their TM4s, and (ii) TM1 segments of neighbouring claudins are oriented towards each other as the cytoplasmic end of TM1 is in close proximity to more other TM1 segments than TM4 is to other TM4 segments. The results indicate at least two different cis-interaction interfaces within claudin-3 homopolymers as well as within claudin-1/claudin-3 heteropolymers. The data provide novel insight into the molecular TJ architecture consistent with a model with an antiparallel double-row cis-arrangement of classic claudin protomers within strands.

The development of morphological complexity of CNS neurons is thought to be regulated by extracellular factors and cellular contact. To analyze the role of contact with astroglia in this process and to determine the intraneuronal mechanisms involved, an in vitro system was developed where terminally differentiated and polar human CNS model neurons (NT2-N neurons) were cultured on a layer of mouse astrocytes or isolated membrane fractions in chemically defined medium. Morphometric analysis revealed that physical contact with living astrocytes increased the lengths of axonal and dendritic processes and lead to an increased number of branch points. Contact with astrocytes also resulted in a redistribution of the growth-associated proteins MAP1b and GAP-43 toward the growth cones of NT2-N neurons. Astrocyte-contact did not lead to a maturation of the neurons as would be detected by an increased expression of tau isoforms containing the adult-specific exons 2 and 3. Culture on immobilized membrane fractions prepared from astrocytes also increased the morphological complexity of the neurons in a qualitatively similar manner. The results indicate that physical contact with astrocyte membranes increases the morphological complexity of CNS model neurons through a mechanism that involves a redistribution of growth-associated proteins to neuronal growth cones. NT2-N neurons may provide a useful cellular model to analyze cytoskeletal mechanisms during the development of terminally differentiated and polar human neurons.

The mechanism of TJ (tight junction) assembly and the structure of TJ strand-forming Cldns (claudins) are unclear. To identify determinants of assembly of blood–brain barrier-related Cldn3 and Cldn5, chimaeric mutants were analysed by cellular reconstitution of TJ strands and live-cell imaging. On the basis of the rescue of mutants deficient for strand formation, we identified Cldn5 residues (Cys128, Ala132, Ile142, Ala163, Ile166 and Leu174) involved in Cldn folding and assembly. Experimental results were combined with structural bioinformatics approaches. Initially the experimentally validated previous model of the ECL2 (extracellular loop 2) of Cldn5 was extended to the flanking transmembrane segments (TM3/TM4). A coiled-coil interface probably caused by alternating small and large residues is supported by concomitant knob-into-hole interactions including Cldn5-specific residues identified in the present paper. To address arrangement of the TMs in a four-helix bundle, data from evolutionary sequence couplings and comparative modelling of intramolecular interfaces in the transmembrane region of Cldns led to a complete Cldn5 model. Our suggested Cldn subtype-specific intramolecular interfaces that are formed by conserved coiled-coil motifs and non-conserved residues in distinct TM positions were confirmed by the recently released crystal structure of Cldn15. The identified molecular and structural determinants essentially contribute to assembly of Cldns into TJ strands. Abbreviations: Cldn, claudin; CPE, Clostridium perfringens enterotoxin; ECL, extracellular loop; EF, enrichment factor; EMP, epithelial membrane protein; HEK, human embryonic kidney; MP20, eye-lens-specific membrane protein 20; PLM, pseudo-likelihood maximization; PMP22, peripheral myelin protein 22; TJ, tight junction; TM, transmembrane segment; wt, wild-type

Zonula occludens protein 1 (ZO-1) is a ubiquitous scaffolding protein, but it is unknown why it functions in very different cellular contacts. We hypothesized that a specific segment, the unique hinge region, can be bound by very different regulatory proteins. Using surface plasmon resonance spectroscopy and binding assays to peptide libraries, we show, for the first time, that the hinge region directly interacts with disparate signal elements such as G-proteins alpha 12 and alpha i2, the regulator of G-protein signaling 5, multifunctional signaling protein ahnak1, and L-type Ca2+-channel beta-2-subunit. The novel binding proteins specifically bound to a coiled coil-helix predicted in the hinge region of ZO-. The interactions were modulated by phosphorylation in the hinge helix. Activation of the G-proteins influenced their association to ZO-1. In colon cells, G alpha i2 and ZO-1 were associated, as shown by coimmunoprecipitation. After cotransfection in kidney cells, G alpha i2 barely colocalized with ZO-1; the colocalization coefficient was significantly increased when epinephrine activated G-protein signaling. In conclusion, proteins with different regulatory potential adhere to and influence cellular functions of ZO-proteins, and the interactions can be modulated via its hinge region and/or the binding proteins.

Tight Junctions (TJ) regulate paracellular permeability of tissue barriers. Claudins (Cld) form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity). To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy. TJ-strands were reconstituted by stable transfection of HEK293 cells with the barrier-forming Cld3 or Cld5. Strands were investigated at cell-cell contacts by Spectral Position Determination Microscopy (SPDM), a method of localization microscopy using standard fluorophores. Extended TJ-networks of Cld3-YFP and Cld5-YFP were observed. For each network, 200,000 to 1,100,000 individual molecules were detected with a mean localization accuracy of ~20 nm, yielding a mean structural resolution of ~50 nm. Compared to conventional fluorescence microscopy, this strongly improved the visualization of strand networks and enabled quantitative morphometric analysis. Two populations of elliptic meshes (mean diameter