Paracellular permeability and tight junction regulation in gut health and disease.

Epithelial tight junctions define the paracellular permeability of the intestinal barrier. Molecules can cross the tight junctions via two distinct size-selective and charge-selective paracellular pathways: the pore pathway and the leak pathway. These can be distinguished by their selectivities and differential regulation by immune cells. However, permeability increases measured in most studies are secondary to epithelial damage, which allows non-selective flux via the unrestricted pathway. Restoration of increased unrestricted pathway permeability requires mucosal healing. By contrast, tight junction barrier loss can be reversed by targeted interventions. Specific approaches are needed to restore pore pathway or leak pathway permeability increases. Recent studies have used preclinical disease models to demonstrate the potential of pore pathway or leak pathway barrier restoration in disease. In this Review, we focus on the two paracellular flux pathways that are dependent on the tight junction. We discuss the latest evidence that highlights tight junction components, structures and regulatory mechanisms, their impact on gut health and disease, and opportunities for therapeutic intervention.

Tight Junctions in the Auditory System: Structure, Distribution and Function.

Tight junctions act as a barrier between epithelial cells to limit the transport of the paracellular substance, which is a required function in various tissues to sequestrate diverse microenvironments and maintain a normal physiological state. Tight junctions are complexes that contain various proteins, like transmembrane proteins, scaffolding proteins, signaling proteins, etc. Defects in those tight junction- related proteins can lead to hearing loss in humans which is also recapitulated in many model organisms. The disruption of the barrier between the endolymph and perilymph caused by tight junction abnormalities will affect the microenvironment of hair cells; and this could be the reason for this type of hearing loss. Besides their functions as a typical barrier and channel, tight junctions are also involved in many signaling networks to regulate gene expression, cell proliferation, and differentiation. This review will summarize the structures, localization, and related signaling pathways of hearingrelated tight junction proteins and their potential contributions to the hearing disorder.

Tight-junction strand networks and tightness of the epithelial barrier.

Tight junctions (TJs) are cell-cell junction structures critical for controlling paracellular permeability. On freeze-fracture replica electron microscopy, they appear as a continuous network of fibrils (TJ strands). TJ strands function as zippers that create a physical barrier against paracellular diffusion of molecules. The morphology of the TJ strand network varies greatly between tissues, and in recent years, studies have highlighted the mechanisms regulating the morphology of TJ strand networks and on their relevance to barrier function. In this review, we discuss evidence regarding the components of the TJ strand and the mechanisms for creating the TJ strand network. Furthermore, we discuss and hypothesize how its morphology contributes to the establishment of the epithelial barrier.

Molecular mechanism of claudin-15 strand flexibility: A computational study.

Claudins are one of the major components of tight junctions that play a key role in the formation and maintenance of the epithelial barrier function. Tight junction strands are dynamic and capable of adapting their structure in response to large-scale tissue rearrangement and cellular movement. Here, we present molecular dynamics simulations of claudin-15 strands of up to 225 nm in length in two parallel lipid membranes and characterize their mechanical properties. The persistence length of claudin-15 strands is comparable with those obtained from analyses of freeze-fracture electron microscopy. Our results indicate that lateral flexibility of claudin strands is due to an interplay of three sets of interfacial interaction networks between two antiparallel double rows of claudins in the membranes. In this model, claudins are assembled into interlocking tetrameric ion channels along the strand that slide with respect to each other as the strands curve over submicrometer-length scales. These results suggest a novel molecular mechanism underlying claudin-15 strand flexibility. It also sheds light on intermolecular interactions and their role in maintaining epithelial barrier function.

Decreased ZO1 expression causes loss of time-dependent tight junction function in the liver of ob/ob mice.

Diabetes patients are at a high risk of developing complications related to angiopathy and disruption of the signal transduction system. The liver is one of the multiple organs damaged during diabetes. Few studies have evaluated the morphological effects of adhesion factors in diabetic liver. The influence of diurnal variation has been observed in the expression and functioning of adhesion molecules to maintain tissue homeostasis associated with nutrient uptake. The present study demonstrated that the rhythm-influenced functioning of tight junction was impaired in the liver of ob/ob mice. The tight junctions of hepatocytes were loosened during the dark period in control mice compared to those in ob/ob mice, where the hepatocyte gaps remained open throughout the day. The time-dependent expression of zonula occludens 1 (ZO1, encoded by Tjp1 gene) in the liver plays a vital role in the functioning of the tight junction. The time-dependent expression of ZO1 was nullified and its expression was attenuated in the liver of ob/ob mice. ZO1 expression was inhibited at the mRNA and protein levels. The expression rhythm of ZO1 was found to be regulated by heat shock factor (HSF)1/2, the expression of which was reduced in the liver of ob/ob mice. The DNA-binding ability of HSF1/2 was decreased in the liver of ob/ob mice compared to that in control mice. These findings suggest the involvement of impaired expression and functioning of adhesion factors in diabetic liver complications.

Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain Barrier Tight Junctions.

The blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between the adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for TJ formation and assemble into multiprotein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB.

Tissue Content and Pattern of Expression of Claudin-3 and Occludin in Normal and Neoplastic Tissues in Patients with Colorectal Cancer.

BACKGROUND: Metastasis is the worst prognostic variable of patients with colorectal cancer (CRC). For the development of metastases, it is necessary that cancer cells detach from the primary tumor, migrate into the angiolymphatic system, and invade the tissue where they will develop. The breakdown of the tight junctions (TJs) plays an important role in colorectal metastatic tumors. Claudin-3 and occludin are the main component proteins of TJs. AIM: To analyze the expression and tissue content of claudin-3 and occludin in normal and neoplastic tissues of patients with metastatic CRC. METHODS: Fifty-seven consecutive patients with stage III and IV CRC were included. Fragments of neoplastic tissue were collected from the tumor margins, and samples of the normal tissue were collected from the same patient in a standardized distance of 10 cm from the cranial margin of the tumor. Immunohistochemistry technique was used to identify the tissue staining of claudin-3 and occludin. To measure the content of both proteins in cellular membranes of normal and cancer cells, a validated immunoscore was used. RESULTS: Claudin-3 and occludin in normal tissues are in the apical and lateral membranes of cells, while in the neoplastic, in cytoplasm. The mean of the tissue content of claudin-3 in the normal tissue was 2.57 ± 0.16, while in the neoplastic tissue was 1.03 ± 0.13. The contents of occludin were 2.77 ± 0.1 in normal tissue, while in the neoplastic were 1.08 ± 0.14. CONCLUSION: There is a reduction in the content of the claudin-3 and occludin in the cell membranes of the neoplastic tissue in patients with CRC.

Direct inhibition of the first PDZ domain of ZO-1 by glycyrrhizin is a possible mechanism of tight junction opening of Caco-2 cells.

Glycyrrhizin (GL) is known to exhibit a variety of useful pharmacological activities, including anti-inflammation, anti-hepatotoxicity, and enhancement of intestinal drug absorption. GL has been reported to modify the assembly of actin filaments, thereby modulating tight junction (TJ) integrity, but the detailed molecular mechanisms of this remain unclear. In this study, we first found that GL binds to the first PDZ domain of zonula occludens-1 (ZO-1(PDZ1)) through NMR experiments. The structure of the GL-ZO-1(PDZ1) complex was then constructed using HADDOCK with the transferred nuclear Overhauser effect-based inter-hydrogen distance constraints as well as restrictions on the interfacial residues identified from 1H-15N HSQC spectral changes. We identified the relevant interactions between the glucuronate-2 moiety of GL and the carboxylate binding loop of the ligand binding site of ZO-1(PDZ1). We further examined the interaction of ZO-1(PDZ1) with glycyrrhetinic acid and with GA-3-monoglucuronide and observed a much lower affinity for each than for that with GL, with good agreement with the model. The other contacts found in the model were examined by using an amino acid substitution mutant of ZO-1(PDZ1). Finally, we reproduced the experiments reported by Sakai et al. in which high-dose GL prolonged the TJ-opening mediated with sodium deoxycholate as indicated by reduced transepithelial electrical resistance.

Tight Junction ZO Proteins Maintain Tissue Fluidity, Ensuring Efficient Collective Cell Migration.

Tight junctions (TJs) are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. While their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the TJ zonula occludens (ZO) proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug-of-war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward bulging apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding-induced jamming and more small cells over time. Co-cultures comprising wildtype and dKD cells migrate inefficiently due to phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation.

Chimeric Claudins: A New Tool to Study Tight Junction Structure and Function.

The tight junction (TJ) is a structure composed of multiple proteins, both cytosolic and membranal, responsible for cell-cell adhesion in polarized endothelium and epithelium. The TJ is intimately connected to the cytoskeleton and plays a role in development and homeostasis. Among the TJ's membrane proteins, claudins (CLDNs) are key to establishing blood-tissue barriers that protect organismal physiology. Recently, several crystal structures have been reported for detergent extracted recombinant CLDNs. These structural advances lack direct evidence to support quaternary structure of CLDNs. In this article, we have employed protein-engineering principles to create detergent-independent chimeric CLDNs, a combination of a 4-helix bundle soluble monomeric protein (PDB ID: 2jua) and the apical-50% of human CLDN1, the extracellular domain that is responsible for cell-cell adhesion. Maltose-binding protein-fused chimeric CLDNs (MBP-CCs) used in this study are soluble proteins that retain structural and functional aspects of native CLDNs. Here, we report the biophysical characterization of the structure and function of MBP-CCs. MBP-fused epithelial cadherin (MBP-eCAD) is used as a control and point of comparison of a well-characterized cell-adhesion molecule. Our synthetic strategy may benefit other families of 4-α-helix membrane proteins, including tetraspanins, connexins, pannexins, innexins, and more.

Observation of a tight junction structure generated in LbL-3D skin reconstructed by layer-by-layer cell coating technique.

Tissue-engineered skin equivalents are reconstructed the functions of human skin and can be used as an alternative to animal experiments in basic study or as cultured skin for regenerative medicine. Recent studies confirmed that epidermal tight junctions (TJs), which are complex intercellular junctions formed in the stratum granulosum of human skin, play an important part in the formation of the skin barrier function. In well-formed reconstructed human skin models, there are several reports on the expression of TJ proteins and their localization in epidermal layer, however, the morphological features of TJ, showing tight junctional contacts and the process of TJ formation have yet to be investigated. In this study, we systematically examined and identified TJ-related proteins and TJ structure in three-dimensional (3D) human skin equivalents reconstructed by layer-by-layer (LbL) cell coating technique (LbL-3D Skin). We demonstrate localization of TJ-related proteins and time course of formation of TJ structure with typical junctional morphology in LbL-3D Skin. These data provide evidence that the LbL-3D Skin is an in vitro model with structure and function extremely similar to living skin.

Tight Junction Structure and Function Revisited.

Tight junctions (TJs) are intercellular junctions critical for building the epithelial barrier and maintaining epithelial polarity. The claudin family of membrane proteins play central roles in TJ structure and function. However, recent findings have uncovered claudin-independent aspects of TJ structure and function, and additional players including junctional adhesion molecules (JAMs), membrane lipids, phase separation of the zonula occludens (ZO) family of scaffolding proteins, and mechanical force have been shown to play important roles in TJ structure and function. In this review, we discuss how these new findings have the potential to transform our understanding of TJ structure and function, and how the intricate network of TJ proteins and membrane lipids dynamically interact to drive TJ assembly.

Improved binding of SARS-CoV-2 Envelope protein to tight junction-associated PALS1 could play a key role in COVID-19 pathogenesis.

The Envelope (E) protein of SARS-CoV-2 is the most enigmatic protein among the four structural ones. Most of its current knowledge is based on the direct comparison to the SARS E protein, initially mistakenly undervalued and subsequently proved to be a key factor in the ER-Golgi localization and in tight junction disruption. We compared the genomic sequences of E protein of SARS-CoV-2, SARS-CoV and the closely related genomes of bats and pangolins obtained from the GISAID and GenBank databases. When compared to the known SARS E protein, we observed a significant difference in amino acid sequence in the C-terminal end of SARS-CoV-2 E protein. Subsequently, in silico modelling analyses of E proteins conformation and docking provide evidences of a strengthened binding of SARS-CoV-2 E protein with the tight junction-associated PALS1 protein. Based on our computational evidences and on data related to SARS-CoV, we believe that SARS-CoV-2 E protein interferes more stably with PALS1 leading to an enhanced epithelial barrier disruption, amplifying the inflammatory processes, and promoting tissue remodelling. These findings raise a warning on the underestimated role of the E protein in the pathogenic mechanism and open the route to detailed experimental investigations.

Channel functions of claudins in the organization of biological systems.

Claudins are tight junction proteins mostly appreciated in their function of paracellular barrier-formation. Compared to a virtual absence of any tight junctions, their paracellular sealing role certainly stands out. Yet, it was recognized immediately after the discovery of the first claudins, that some members of the claudin protein family were able to convey size and charge selectivity to the paracellular pathway. Thus, paracellular permeability can be fine-tuned according to the physiological needs of a tissue by inserting these channel-forming claudins into tight junction strands. Precise permeability adjustment is further suggested by the presence of numerous isoforms of channel-forming claudins (claudin-10b-, -15-, -16-like isoforms) in various vertebrate taxa. Moreover, their expression and localization are controlled by multiple transcriptional and posttranslational mechanisms. Consequently, mutation or dysregulation of channel-forming claudins can cause severe diseases. The present review therefore aims at providing an up-to-date report of the current research on these aspects of channel-forming claudins and their possible implications on future developments.

Ruffles and spikes: Control of tight junction morphology and permeability by claudins.

Epithelial barrier function is regulated by a family of transmembrane proteins known as claudins. Functional tight junctions are formed when claudins interact with other transmembrane proteins, cytosolic scaffold proteins and the actin cytoskeleton. The predominant scaffold protein, zonula occludens-1 (ZO-1), directly binds to most claudin C-terminal domains, crosslinking them to the actin cytoskeleton. When imaged by immunofluorescence microscopy, tight junctions most frequently are linear structures that form between tricellular junctions. However, tight junctions also adapt non-linear architectures exhibiting either a ruffled or spiked morphology, which both are responses to changes in claudin engagement of actin filaments. Other terms for ruffled tight junctions include wavy, tortuous, undulating, serpentine or zig-zag junctions. Ruffling is under the control of hypoxia induced factor (HIF) and integrin-mediated signaling, as well as direct mechanical stimulation. Tight junction ruffling is specifically enhanced by claudin-2, antagonized by claudin-1 and requires claudin binding to ZO-1. Tight junction spikes are sites of active vesicle budding and fusion that appear as perpendicular projections oriented towards the nucleus. Spikes share molecular features with focal adherens junctions and tubulobulbar complexes found in Sertoli cells. Lung epithelial cells under stress form spikes due to an increase in claudin-5 expression that directly disrupts claudin-18/ZO-1 interactions. Together this suggests that claudins are not simply passive cargoes controlled by scaffold proteins. We propose a model where claudins specifically influence tight junction scaffold proteins to control interactions with the cytoskeleton as a mechanism that regulates tight junction assembly and function.

Cell-cell junctions as sensors and transducers of mechanical forces.

Epithelial and endothelial monolayers are multicellular sheets that form barriers between the 'outside' and 'inside' of tissues. Cell-cell junctions, made by adherens junctions, tight junctions and desmosomes, hold together these monolayers. They form intercellular contacts by binding their receptor counterparts on neighboring cells and anchoring these structures intracellularly to the cytoskeleton. During tissue development, maintenance and pathogenesis, monolayers encounter a range of mechanical forces from the cells themselves and from external systemic forces, such as blood pressure or tissue stiffness. The molecular landscape of cell-cell junctions is diverse, containing transmembrane proteins that form intercellular bonds and a variety of cytoplasmic proteins that remodel the junctional connection to the cytoskeleton. Many junction-associated proteins participate in mechanotransduction cascades to confer mechanical cues into cellular responses that allow monolayers to maintain their structural integrity. We will discuss force-dependent junctional molecular events and their role in cell-cell contact organization and remodeling.

GJ-4 alleviates Aß25-35-induced memory dysfunction in mice through protecting the neurovascular unit.

Alzheimer's disease (AD) is the most common neurodegenerative disease. AD has become an important social health problem but there are few therapeutic drugs. Many researchers devote to the development of drugs for the treatment of AD. GJ-4 is crocin enrichments from Gardenia jasminoides J. Ellis, and our previous studies have shown GJ-4 had potent neuroprotective effects on several AD animal models. However, the underlying mechanisms have not been fully elucidated. The aim of the present study was to explore the mechanism of GJ-4 on a Aß25-35-intoxicated mouse model. The results demonstrated that GJ-4 treatment significantly improved spatial learning and memory abilities of the AD mice challenged by Aß25-35. Mechanistic study indicated that GJ-4 could alleviate endothelial dysfunction, as GJ-4 markedly reduced endothelial cell edema, as well as improved tight junction structures by up-regulating Zonula occludens-1 (ZO-1), Claudin-5 and Occludin expressions. Moreover, GJ-4 markedly reduced receptor for advanced glycation end products (RAGE) expression and increased low-density lipoprotein receptor-related protein-1 (LRP-1) expression, suggesting endothelial transduction and clearance of toxic species capabilities improved by GJ-4 treatment. The results also indicated that GJ-4 significantly decreased IL-6 and IL-1ß mRNA expressions, as well as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expressions, implying the inhibition of glial activation and vascular inflammation by GJ-4 treatment. Furthermore, GJ-4 treatment inhibited glial activation mediated neuroinflammation through inhibiting high-mobility group box protein 1(HMGB-1)/RAGE/NF-κB signaling pathway, which might confer to the neuroprotection. In conclusion, our present study proved GJ-4 could protect the neurovascular unit (NVU), through attenuating endothelial cell damage, enhancing tight junction function, inhibiting of glial activation and protecting of neurons. This study provided evidence that the beneficial effects of GJ-4 on AD might be owing to its protection on NVU.

Molecular architecture and assembly of the tight junction backbone.

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.

Assembly of Tight Junction Strands: Claudin-10b and Claudin-3 Form Homo-Tetrameric Building Blocks that Polymerise in a Channel-Independent Manner.

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. Förster 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 ß1ß2 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 dynamic perfusion based blood-brain barrier model for cytotoxicity testing and drug permeation.

The blood-brain barrier (BBB) serves to protect and regulate the CNS microenvironment. The development of an in-vitro mimic of the BBB requires recapitulating the correct phenotype of the in-vivo BBB, particularly for drug permeation studies. However the majority of widely used BBB models demonstrate low transendothelial electrical resistance (TEER) and poor BBB phenotype. The application of shear stress is known to enhance tight junction formation and hence improve the barrier function. We utilised a high TEER primary porcine brain microvascular endothelial cell (PBMEC) culture to assess the impact of shear stress on barrier formation using the Kirkstall QuasiVivo 600 (QV600) multi-chamber perfusion system. The application of shear stress resulted in a reorientation and enhancement of tight junction formation on both coverslip and permeable inserts, in addition to enhancing and maintaining TEER for longer, when compared to static conditions. Furthermore, the functional consequences of this was demonstrated with the reduction in flux of mitoxantrone across PBMEC monolayers. The QV600 perfusion system may service as a viable tool to enhance and maintain the high TEER PBMEC system for use in in-vitro BBB models.