A short guide to the tight junction.

Tight junctions (TJs) are specialized regions of contact between cells of epithelial and endothelial tissues that form selective semipermeable paracellular barriers that establish and maintain body compartments with different fluid compositions. As such, the formation of TJs represents a critical step in metazoan evolution, allowing the formation of multicompartmental organisms and true, barrier-forming epithelia and endothelia. In the six decades that have passed since the first observations of TJs by transmission electron microscopy, much progress has been made in understanding the structure, function, molecular composition and regulation of TJs. The goal of this Perspective is to highlight the key concepts that have emerged through this research and the future challenges that lie ahead for the field.

Dual-color live imaging unveils stepwise organization of multiple basal body arrays by cytoskeletons.

For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating.

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)-dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.

A microtubule-LUZP1 association around tight junction promotes epithelial cell apical constriction.

Apical constriction is critical for epithelial morphogenesis, including neural tube formation. Vertebrate apical constriction is induced by di-phosphorylated myosin light chain (ppMLC)-driven contraction of actomyosin-based circumferential rings (CRs), also known as perijunctional actomyosin rings, around apical junctional complexes (AJCs), mainly consisting of tight junctions (TJs) and adherens junctions (AJs). Here, we revealed a ppMLC-triggered system at TJ-associated CRs for vertebrate apical constriction involving microtubules, LUZP1, and myosin phosphatase. We first identified LUZP1 via unbiased screening of microtubule-associated proteins in the AJC-enriched fraction. In cultured epithelial cells, LUZP1 was found localized at TJ-, but not at AJ-, associated CRs, and LUZP1 knockout resulted in apical constriction defects with a significant reduction in ppMLC levels within CRs. A series of assays revealed that ppMLC promotes the recruitment of LUZP1 to TJ-associated CRs, where LUZP1 spatiotemporally inhibits myosin phosphatase in a microtubule-facilitated manner. Our results uncovered a hitherto unknown microtubule-LUZP1 association at TJ-associated CRs that inhibits myosin phosphatase, contributing significantly to the understanding of vertebrate apical constriction.

Iqgap3-Ras axis drives stem cell proliferation in the stomach corpus during homoeostasis and repair.

OBJECTIVE: Tissue stem cells are central regulators of organ homoeostasis. We looked for a protein that is exclusively expressed and functionally involved in stem cell activity in rapidly proliferating isthmus stem cells in the stomach corpus. DESIGN: We uncovered the specific expression of Iqgap3 in proliferating isthmus stem cells through immunofluorescence and in situ hybridisation. We performed lineage tracing and transcriptomic analysis of Iqgap3 +isthmus stem cells with the Iqgap3-2A-tdTomato mouse model. Depletion of Iqgap3 revealed its functional importance in maintenance and proliferation of stem cells. We further studied Iqgap3 expression and the associated gene expression changes during tissue repair after tamoxifen-induced damage. Immunohistochemistry revealed elevated expression of Iqgap3 in proliferating regions of gastric tumours from patient samples. RESULTS: Iqgap3 is a highly specific marker of proliferating isthmus stem cells during homoeostasis. Iqgap3+isthmus stem cells give rise to major cell types of the corpus unit. Iqgap3 expression is essential for the maintenance of stem potential. The Ras pathway is a critical partner of Iqgap3 in promoting strong proliferation in isthmus stem cells. The robust induction of Iqgap3 expression following tissue damage indicates an active role for Iqgap3 in tissue regeneration. CONCLUSION: IQGAP3 is a major regulator of stomach epithelial tissue homoeostasis and repair. The upregulation of IQGAP3 in gastric cancer suggests that IQGAP3 plays an important role in cancer cell proliferation.

Reciprocal Association between the Apical Junctional Complex and AMPK: A Promising Therapeutic Target for Epithelial/Endothelial Barrier Function?

Epithelial/endothelial cells adhere to each other via cell-cell junctions including tight junctions (TJs) and adherens junctions (AJs). TJs and AJs are spatiotemporally and functionally integrated, and are thus often collectively defined as apical junctional complexes (AJCs), regulating a number of spatiotemporal events including paracellular barrier, selective permeability, apicobasal cell polarity, mechano-sensing, intracellular signaling cascades, and epithelial morphogenesis. Over the past 15 years, it has been acknowledged that adenosine monophosphate (AMP)-activated protein kinase (AMPK), a well-known central regulator of energy metabolism, has a reciprocal association with AJCs. Here, we review the current knowledge of this association and show the following evidences: (1) as an upstream regulator, AJs activate the liver kinase B1 (LKB1)-AMPK axis particularly in response to applied junctional tension, and (2) TJ function and apicobasal cell polarization are downstream targets of AMPK and are promoted by AMPK activation. Although molecular mechanisms underlying these phenomena have not yet been completely elucidated, identifications of novel AMPK effectors in AJCs and AMPK-driven epithelial transcription factors have enhanced our knowledge. More intensive studies along this line would eventually lead to the development of AMPK-based therapies, enabling us to manipulate epithelial/endothelial barrier function.

Vinculin is critical for the robustness of the epithelial cell sheet paracellular barrier for ions.

The paracellular barrier function of tight junctions (TJs) in epithelial cell sheets is robustly maintained against mechanical fluctuations, by molecular mechanisms that are poorly understood. Vinculin is an adaptor of a mechanosensory complex at the adherens junction. Here, we generated vinculin KO Eph4 epithelial cells and analyzed their confluent cell-sheet properties. We found that vinculin is dispensable for the basic TJ structural integrity and the paracellular barrier function for larger solutes. However, vinculin is indispensable for the paracellular barrier function for ions. In addition, TJs stochastically showed dynamically distorted patterns in vinculin KO cell sheets. These KO phenotypes were rescued by transfecting full-length vinculin and by relaxing the actomyosin tension with blebbistatin, a myosin II ATPase activity inhibitor. Our findings indicate that vinculin resists mechanical fluctuations to maintain the TJ paracellular barrier function for ions in epithelial cell sheets.

Deficiency of Stomach-Type Claudin-18 in Mice Induces Gastric Tumor Formation Independent of H pylori Infection.

BACKGROUND & AIMS: Epithelial cells are joined by tight junctions (TJs) to form a cell sheet. In the stomach, epithelial cell sheet forms an essential barrier against gastric material, including gastric acid. Although the decreased expression of stomach-type claudin-18 (stCldn18), a TJ protein, is generally observed in human gastritis and gastric cancer, its pathological roles are not fully understood. We previously reported that mice lacking stCldn18 (stCldn18-/-) exhibit gastric acid leakage through TJs, which induces active gastritis at a young age. Here, we examined the gastric pathologies in mice after long-term stCldn18 deficiency. METHODS: The gastric pathologies in stCldn18-/- mice were sequentially analyzed from youth to old age, and compared to those in humans. To examine the relationship between stCldn18 deficiency-induced gastric pathologies and Wnt-dependent tumorigenesis, we generated Wnt1-overexpressing stCldn18-/- mice. RESULTS: StCldn18-/- mice developed chronic active gastritis at middle age, with expression of the chemoattractant CCL28. At old age, 20-30% of these mice developed gastric tumors with CXCL5 expression, indicative of EMT. In this process, spasmolytic polypeptide-expressing metaplasia (SPEM) cells appeared. Increased expressions of CD44-variants, TLR2, and CXCL5 indicated age-dependent changes in cell characteristics. Some features of the stCldn18-/- mouse gastric tumorigenesis resembled H pylori-infection-related human carcinogenesis. The gastric tumorigenesis was accelerated in Wnt1-overexpressing stCldn18-/- mice, indicating that Wnt is involved in the stCldn18-/- mouse gastric tumorigenesis. CONCLUSIONS: StCldn18 deficiency induced gastric tumorigenesis in mice without H pylori infection. Our findings revealed that several signaling networks, including the cytokine-, stemness-, and Wnt-signaling pathways, may be activated under the stCldn18-deficiency-induced chronic active gastritis to accelerate the gastric tumorigenesis.

The Claudins: From Tight Junctions to Biological Systems.

Claudins are cell-cell adhesion molecules located at the tight junctions (TJs) between cells in epithelial cell sheets. The claudin family in mammals consists of 27 four-transmembrane domain proteins. Claudins are responsible for the paracellular barrier function of TJs, and in some cases confer paracellular channel functions to the paracellular barriers of TJs. Based on recent breakthroughs in the molecular structure of claudins, the hypothetical 'antiparallel double row model' was proposed, which suggests how claudins polymerize in a linear fashion and form TJ strands with paracellular barrier and channel functions. Meanwhile, ongoing studies at the cell and tissue levels are clarifying how the paracellular barrier and/or channel functions of claudin-based TJs, which are both robust and flexible, organize various biological systems.

Site-specific distribution of claudin-based paracellular channels with roles in biological fluid flow and metabolism.

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.

Apical cytoskeletons and junctional complexes as a combined system in epithelial cell sheets.

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.

Interaction between Plasmodium Glycosylphosphatidylinositol and the Host Protein Moesin Has No Implication in Malaria Pathology.

Glycosylphosphatidylinositol (GPI) anchor of Plasmodium falciparum origin is considered an important toxin leading to severe malaria pathology through stimulation of pro-inflammatory responses from innate immune cells. Even though the GPI-induced immune response is widely described to be mediated by pattern recognition receptors such as TLR2 and TLR4, previous studies have revealed that these two receptors are dispensable for the development of severe malaria pathology. Therefore, this study aimed at the identification of potential alternative Plasmodium GPI receptors. Herein, we have identified the host protein moesin as an interaction partner of Plasmodium GPI in vitro. Given previous reports indicating the relevance of moesin especially in the LPS-mediated induction of pro-inflammatory responses, we have conducted a series of in vitro and in vivo experiments to address the physiological relevance of the moesin-Plasmodium GPI interaction in the context of malaria pathology. We report here that although moesin and Plasmodium GPI interact in vitro, moesin is not critically involved in processes leading to Plasmodium-induced pro-inflammatory immune responses or malaria-associated cerebral pathology.

Three-dimensional Organization of Layered Apical Cytoskeletal Networks Associated with Mouse Airway Tissue Development.

The cytoskeleton is an essential cellular component that enables various sophisticated functions of epithelial cells by forming specialized subcellular compartments. However, the functional and structural roles of cytoskeletons in subcellular compartmentalization are still not fully understood. Here we identified a novel network structure consisting of actin filaments, intermediate filaments, and microtubules directly beneath the apical membrane in mouse airway multiciliated cells and in cultured epithelial cells. Three-dimensional imaging by ultra-high voltage electron microscopy and immunofluorescence revealed that the morphological features of each network depended on the cell type and were spatiotemporally integrated in association with tissue development. Detailed analyses using Odf2 mutant mice, which lack ciliary basal feet and apical microtubules, suggested a novel contribution of the intermediate filaments to coordinated ciliary beating. These findings provide a new perspective for viewing epithelial cell differentiation and tissue morphogenesis through the structure and function of apical cytoskeletal networks.

Presence of anti-phosphatidylserine-prothrombin complex antibodies and anti-moesin antibodies in patients with polyarteritis nodosa.

We measured both serum anti-phosphatidylserine-prothrombin complex (anti-PSPT) antibodies and anti-moesin antibodies, as well as various cytokines (interleukin [IL]-2, IL-4, IL-5, IL-10, IL-13, IL-17, granulocyte macrophage colony-stimulating factor, γ-interferon, tumor necrosis factor-α) levels in polyarteritis nodosa (PAN) patients with cutaneous manifestations. All patients showed the presence of a histological necrotizing vasculitis in the skin specimen. They were treated with i.v. cyclophosphamide pulse therapy (IV-CY) and prednisolone therapy or steroid pulse therapy. The immunological assessments were performed on sera collected prior to and after treatment with IV-CY or steroid pulse therapy. We found a significant positive correlation between serum anti-moesin antibodies and both clinical Birmingham Vasculitis Activity Scores and Vasculitis Damage Index. Anti-PSPT antibody and IL-2 levels after treatment in PAN patients were significantly lower than before treatment. In contrast, anti-moesin antibody levels were higher following IV-CY or steroid pulse therapy compared with the pretreatment levels. In the treatment-resistant PAN patients (n = 8), anti-PSPT antibody levels after treatment were significantly lower than before treatment. In contrast, anti-moesin antibody levels after treatment in the patients were significantly higher compared with the pretreatment levels. Immunohistochemical staining revealed moesin overexpression in mainly fibrinoid necrosis of the affected arteries in the PAN patients. We suggest that measurement of serum anti-PSPT antibody levels could serve as a marker for PAN and aid in earlier diagnosis of PAN. We also propose that elevated serum anti-moesin antibodies could play some role of the exacerbation in patients with PAN.

Regulation of intestinal homeostasis by the ulcerative colitis-associated gene RNF186.

Genome-wide association studies and subsequent deep sequencing analysis have identified susceptible loci for inflammatory bowel diseases (IBDs) including ulcerative colitis (UC). A gene encoding RING finger protein 186 (RNF186) is located within UC-susceptible loci. However, it is unclear whether RNF186 is involved in IBD pathogenesis. Here, we show that RNF186 controls protein homeostasis in colonic epithelia and regulates intestinal inflammation. RNF186, which was highly expressed in colonic epithelia, acted as an E3 ligase mediating polyubiquitination of its substrates. Permeability of small organic molecules was augmented in the intestine of Rnf186-/- mice. Increased expression of several RNF186 substrates, such as occludin, was found in Rnf186-/- colonic epithelia. The disturbed protein homeostasis in Rnf186-/- mice correlated with enhanced endoplasmic reticulum (ER) stress in colonic epithelia and increased sensitivity to intestinal inflammation after dextran sulfate sodium (DSS) treatment. Introduction of an UC-associated Rnf186 mutation led to impaired E3 ligase activity and increased sensitivity to DSS-induced intestinal inflammation in mice. Thus, RNF186 maintains gut homeostasis by controlling ER stress in colonic epithelia.

Multiciliated cell basal bodies align in stereotypical patterns coordinated by the apical cytoskeleton.

Multiciliated cells (MCCs) promote fluid flow through coordinated ciliary beating, which requires properly organized basal bodies (BBs). Airway MCCs have large numbers of BBs, which are uniformly oriented and, as we show here, align linearly. The mechanism for BB alignment is unexplored. To study this mechanism, we developed a long-term and high-resolution live-imaging system and used it to observe green fluorescent protein-centrin2-labeled BBs in cultured mouse tracheal MCCs. During MCC differentiation, the BB array adopted four stereotypical patterns, from a clustering "floret" pattern to the linear "alignment." This alignment process was correlated with BB orientations, revealed by double immunostaining for BBs and their asymmetrically associated basal feet (BF). The BB alignment was disrupted by disturbing apical microtubules with nocodazole and by a BF-depleting Odf2 mutation. We constructed a theoretical model, which indicated that the apical cytoskeleton, acting like a viscoelastic fluid, provides a self-organizing mechanism in tracheal MCCs to align BBs linearly for mucociliary transport.

Directed Induction of Functional Multi-ciliated Cells in Proximal Airway Epithelial Spheroids from Human Pluripotent Stem Cells.

Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However, the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1(+)-ventralized anterior foregut endoderm cells (VAFECs), we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM(+) VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore, the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT, a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a "9 + 2" microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine.

Knockdown of ezrin causes intrahepatic cholestasis by the dysregulation of bile fluidity in the bile duct epithelium in mice.

UNLABELLED: Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2(kd/kd) ) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal cross-linker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na(+) /H(+) exchanger regulatory factor were disturbed in bile ducts of Vil2(kd/kd) mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl(-) efflux activity. Furthermore, bile flow and biliary HCO3 (-) concentration were also significantly reduced in Vil2(kd/kd) mice. CONCLUSION: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2(kd/kd) mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies.

Model for the architecture of claudin-based paracellular ion channels through tight junctions.

Claudins are main cell-cell adhesion molecules of tight junctions (TJs) between cells in epithelial sheets that form tight barriers that separate the apical from the basolateral space but also contain paracellular channels that regulate the flow of ions and solutes in between these intercellular spaces. Recently, the first crystal structure of a claudin was determined, that of claudin-15, which indicated the parts of the large extracellular domains that likely form the pore-lining surfaces of the paracellular channels. However, the crystal structure did not show how claudin molecules are arranged in the cell membrane to form the backbone of TJ strands and to mediate interactions between adjacent cells, information that is essential to understand how the paracellular channels in TJs function. Here, we propose that TJ strands consist of claudin protomers that assemble into antiparallel double rows. This model is based on cysteine crosslinking experiments that show claudin-15 to dimerize face to face through interactions between the edges of the extracellular ß-sheets. Strands observed by freeze-fracture electron microscopy of TJs also show that their width is consistent with the dimensions of a claudin dimer. Furthermore, we propose that extracellular variable regions are responsible for head-to-head interactions of TJ strands in adjoining cells, thus resulting in the formation of paracellular channels. Our model of the TJ architecture provides a basis to discuss structural mechanisms underlying the selective ion permeability and barrier properties of TJs.

Involvement of IQGAP family proteins in the regulation of mammalian cell cytokinesis.

IQGAP family proteins, comprising IQGAP1, -2, and -3 in mammals, are involved in diverse ranges of cellular processes such as adhesion and migration. IQGAP proteins in yeast also play important roles in cytokinesis. However, the involvement of IQGAP proteins in cytokinesis in mammals remains unaddressed. In this study, we showed that IQGAP3 specifically localized to the equatorial cortex at anaphase, whereas IQGAP1 localized to the cell cortex uniformly and IQGAP2 was unexpressed in HeLa cells. IQGAP3, but neither IQGAP1 nor -2, was able to interact with anillin, which was required for the localization of IQGAP3 to the contractile ring. The suppressed expression of IQGAP3 inhibited the completion of cleavage furrow ingression and led to the multinucleation of cells. The suppression of IQGAP1 also had similar inhibitory effects on cytokinesis, and the simultaneous suppression of IQGAP1 and -3 induced more severe effects. The localization of anillin and RhoA to the contractile ring was impaired by the suppression of IQGAP1 and -3, whereas their upstream regulators, the centralspindlin complex and Ect2, remained unaffected. These results suggested that mammalian IQGAP proteins may play a role in cytokinesis by regulating the localization of key cytokinesis regulatory proteins to the contractile apparatus during mitosis.