Publisher Correction: Claudin-3-deficient C57BL/6J mice display intact brain barriers.

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Claudin-3-deficient C57BL/6J mice display intact brain barriers.

The tight junction protein claudin-3 has been identified as a transcriptional target of the Wnt/ß-catenin signaling pathway regulating blood-brain barrier (BBB) maturation. In neurological disorders loss of claudin-3 immunostaining is observed at the compromised BBB and blood-cerebrospinal fluid barrier (BCSFB). Although these observations support a central role of claudin-3 in regulating brain barriers' tight junction integrity, expression of claudin-3 at the brain barriers has remained a matter of debate. This prompted us to establish claudin-3-/- C57BL/6J mice to study the role of claudin-3 in brain barrier integrity in health and neuroinflammation. Bulk and single cell RNA sequencing and direct comparative qRT-PCR analysis of brain microvascular samples from WT and claudin-3-/- mice show beyond doubt that brain endothelial cells do not express claudin-3 mRNA. Detection of claudin-3 protein at the BBB in vivo and in vitro is rather due to junctional reactivity of anti-claudin-3 antibodies to an unknown antigen still detected in claudin-3-/- brain endothelium. We confirm expression and junctional localization of claudin-3 at the BCSFB of the choroid plexus. Our study clarifies that claudin-3 is not expressed at the BBB and shows that absence of claudin-3 does not impair brain barrier function during health and neuroinflammation in C57BL/6J mice.

Deafness in occludin-deficient mice with dislocation of tricellulin and progressive apoptosis of the hair cells.

Occludin is the first identified protein in the tight junction (TJ), but its function has remained for the most part obscure. TJs have been demonstrated to play important roles in the inner ear function, and occludin is expressed in all the epithelial TJs in the inner ear. Thus, we examined the inner ears of occludin-deficient (Occ(-/-)) mice. Although inner ears initially developed normally in Occ(-/-) mice, apoptosis occurs in hair cells in the organ of Corti around day 12 after birth, and deafness develops. Since hair cell degeneration was not observed in cochlear explant cultures of Occ(-/-) mice, environmental changes were considered to be the trigger of cell death. As for the vestibular system, both the morphologies and functions are normal in Occ(-/-) mice. These phenotypes of Occ(-/-) mice are very similar with those of claudin-14 or claudin-9 deficient mice, leading us to speculate on the existence of imbalance induced by TJ abnormalities, such as localized ionic components. Moreover, the occludin deficiency led to dislocalization of tricellulin, a gene responsible for human deafness DFNB49. The deafness in Occ(-/-) mice may be due to this dislocalization of tricellulin.

Snakeskin, a membrane protein associated with smooth septate junctions, is required for intestinal barrier function in Drosophila.

Septate junctions (SJs) are the membrane specializations observed between epithelial cells in invertebrates. SJs play a crucial role in epithelial barrier function by restricting the free diffusion of solutes through the intercellular space. In arthropod species, two morphologically different types of SJs have been described: pleated septate junctions (pSJs) and smooth septate junctions (sSJs), which are specific to ectodermal and endodermal epithelia, respectively. In contrast to the recent identification of pSJ-related proteins, the molecular constituents of sSJs are mostly unknown. Here, we report the discovery of a new sSJ-specific membrane protein, designated 'Snakeskin' (Ssk). Ssk is highly concentrated in sSJs in the Drosophila midgut and Malpighian tubules. Lack of Ssk expression is embryonically lethal in Drosophila and results in defective sSJ formation accompanied by abnormal morphology of midgut epithelial cells. We also show that the barrier function of the midgut to a fluorescent tracer is impaired in ssk-knockdown larvae. These results suggest that Ssk is required for the intestinal barrier function in Drosophila.

Coordinated ciliary beating requires Odf2-mediated polarization of basal bodies via basal feet.

Coordinated beating of cilia in the trachea generates a directional flow of mucus required to clear the airways. Each cilium originates from a barrel-shaped basal body, from the side of which protrudes a structure known as the basal foot. We generated mice in which exons 6 and 7 of Odf2, encoding a basal body and centrosome-associated protein Odf2/cenexin, are disrupted. Although Odf2(ΔEx6,7/ΔEx6,7) mice form cilia, ciliary beating is uncoordinated, and the mice display a coughing/sneezing phenotype. Whereas residual expression of the C-terminal region of Odf2 in these mice is sufficient for ciliogenesis, the resulting basal bodies lack basal feet. Loss of basal feet in ciliated epithelia disrupted the polarized organization of apical microtubule lattice without affecting planar cell polarity. The requirement for Odf2 in basal foot formation, therefore, reveals a crucial role of this structure in the polarized alignment of basal bodies and coordinated ciliary beating.

Tara up-regulates E-cadherin transcription by binding to the Trio RhoGEF and inhibiting Rac signaling.

The spatiotemporal regulation of E-cadherin expression is important during body plan development and carcinogenesis. We found that Tara (Trio-associated repeat on actin) is enriched in cadherin-based adherens junctions (AJs), and its knockdown in MDCK cells (Tara-KD cells) significantly decreases the expression of E-cadherin. Tara-KD activates Rac1 through the Trio RhoGEF, which binds to E-cadherin and subsequently increases the phosphorylation of p38 and Tbx3, a transcriptional E-cadherin repressor. Accordingly, the decrease in E-cadherin expression is abrogated by ITX3 and SB203580 (specific inhibitors of Trio RhoGEF and p38MAPK, respectively), and by dephosphomimetic Tbx3. Despite the decreased E-cadherin expression, the Tara-KD cells do not undergo an epithelial-mesenchymal transition and remain as an epithelial cell sheet, presumably due to the concomitant up-regulation of cadherin-6. Tara-KD reduces the actin-belt density in the circumferential ring, and the cells form flattened cysts, suggesting that Tara functions to modulate epithelial cell sheet formation and integrity by up-regulating E-cadherin transcription.

Phosphorylation state regulates the localization of Scribble at adherens junctions and its association with E-cadherin-catenin complexes.

Mammalian ortholog of Scribble tumor suppressor has been reported to regulate cadherin-mediated epithelial cell adhesion by stabilizing the coupling of E-cadherin with catenins, but the molecular mechanism involved remains unknown. In this study, we investigated the relationship between the localization of mouse Scribble at cadherin-based adherens junctions (AJs) and its phosphorylation state. Immunofluorescence staining confirmed that Scribble was localized at AJs as well as at the basolateral plasma membrane in epithelial cells. We found that Scribble was detected as two bands by Western blotting analysis and that the band shift to the higher molecular weight was dependent on its phosphorylation at Ser 1601. Triton X-100 treatment extracted Scribble localized on the basolateral membrane but not Scribble localized at AJs in cultured epithelial cells, and the Triton X-100-resistant Scribble was the Ser 1601-unphosphorylated form. Conversely, an in-house-generated antibody that predominantly recognized Ser 1601-phosphorylated Scribble only detected Scribble protein on the lateral plasma membrane. Furthermore, Ser 1601-unphosphorylated Scribble was selectively coprecipitated with E-cadherin-catenin complexes in E-cadherin-expressing mouse L fibroblasts. Taken together, these results suggest that the phosphorylation state of Scribble regulates its complex formation with the E-cadherin-catenin system and may control cadherin-mediated cell-cell adhesion.

abLIM3 is a novel component of adherens junctions with actin-binding activity.

The interactions of adhesion molecules with dense actin filaments via cytoplasmic plaque proteins are crucial for the adhesive function of adherens junctions (AJs) in epithelial and endothelial cells. Using localization-based expression cloning, we identified abLIM3, a member of the actin-binding LIM (abLIM) protein family, as a component of the junctional complex. Immunolocalization studies revealed that abLIM3 was localized at AJs in limited cell types, including hepatocytes, bronchial epithelial cells, mesothelial cells and endothelial cells lining muscular tissues. Deletion mutant analyses in cultured cells showed that the C-terminal dematin-like domain of abLIM3, which bound to actin filaments in vitro, was colocalized with phalloidin-stained filamentous actin, whereas the N-terminal LIM domains of abLIM3 were sufficient for recruitment to cell-cell contacts. These results suggest that abLIM3 is involved in anchoring LIM domain-binding components of AJs to circumferential actin bundles in specific cell types.

Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules.

Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2(-/-)). The Cldn2(-/-) mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2(-/-) kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2(-/-) mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na(+), Cl(-), and water was significantly decreased in Cldn2(-/-) mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na(+)) selectivity and therefore relative anion (Cl(-)) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na(+) and Cl(-) excretions in Cldn2(-/-) mice were similar to those in wild types, but both were greater in Cldn2(-/-) mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na(+))-selective paracellular channels within tight junctions of mouse proximal tubules.

Roles of ZO-1 and ZO-2 in establishment of the belt-like adherens and tight junctions with paracellular permselective barrier function.

Tight junctions (TJs) create the primary permselective barrier to diffusion of solutes and ions through the paracellular pathway. The molecular architecture of TJs has gradually been unraveled in recent years, providing the basis for "barriology" (defined by Shoichiro Tsukita as the science of the barrier in multicellular organisms). Claudins are now considered to be the essential basic components of TJ strands, with which other integral membrane proteins, such as occludin, tricellulin, JAMs, and CAR, are associated. Peripherally associated scaffolding proteins are required for the organization of the integral membrane proteins. Among these, ZO-1, -2, and -3 have attracted a great deal of attention as TJ organizers, since ZO-1 (and in some cases, also ZO-2/3) was reported to be directly associated with claudins, occludin, and JAMs, as well as with AF-6/afadin and alpha-catenin. Here we summarize recent studies on ZO-1/2/3-deficiency in mice and cells, which have provided clear and important information regarding the functions of ZO-1/2/3 in vivo. In addition to the respective suppression of ZO-1/2/3 expression, simultaneous suppression of all three proteins has revealed the essential and nonessential in vivo roles of ZO-1/2 and ZO-3, respectively. ZO-3 shows an epithelial-specific TJ localization in a ZO-1/2-dependent fashion. ZO-1 and ZO-2 play pivotal roles in the final establishment of the belt-like adherens junctions (zonula adherens), followed by the formation of the belt-like TJs (zonula occludens) with paracellular barrier function, thereby providing the general basis for selective paracellular permeability in epithelial and endothelial cells.

The E3 ubiquitin ligase LNX1p80 promotes the removal of claudins from tight junctions in MDCK cells.

The structural continuity of tight junctions (TJs) is consistently maintained even when epithelial cells divide and move within the cellular sheet. This process is associated with dynamic remodeling of TJs by coordinated internalization and generation of claudin-based TJ strands, but the molecular mechanism behind the regulated turnover of TJs remains largely unknown. In this study, we identified the p80 isoform of the E3 ubiquitin ligase ligand of Numb-protein X1 (LNX1p80) as a protein binding to claudin-1. Interestingly, the concentration of claudins in TJs was remarkably reduced when LNX1p80 was overexpressed in MDCK cells, and there was a reduction not only in the number of TJ strands but also in the amount of detergent-insoluble claudins. We also found that LNX1p80 promoted polyubiquitylation of claudins. This ubiquitylation is dependent on its RING-finger domain and is not mediated by Lys48 of ubiquitin, which is used for protein degradation by the proteasome. Furthermore, LNX1p80 was often colocalized with claudins in vesicular structures containing markers for late endosomes and lysosomes. These findings suggest that LNX1p80 is involved in the ubiquitylation, endocytosis and lysosomal degradation of claudins, and that the turnover of TJs is regulated by ubiquitylation.

Similar and distinct properties of MUPP1 and Patj, two homologous PDZ domain-containing tight-junction proteins.

MUPP1 and Patj are both composed of an L27 domain and multiple PDZ domains (13 and 10 domains, respectively) and are localized to tight junctions (TJs) in epithelial cells. Although Patj is known to be responsible for the organization of TJs and epithelial polarity, characterization of MUPP1 is lacking. In this study, we found that MUPP1 and Patj share several binding partners, including JAM1, ZO-3, Pals1, Par6, and nectins (cell-cell adhesion molecules at adherens junctions). MUPP1 and Patj exhibited similar subcellular distributions, and the mechanisms with which they localize to TJs also appear to overlap. Despite these similarities, functional studies have revealed that Patj is indispensable for the establishment of TJs and epithelial polarization, whereas MUPP1 is not. Thus, although MUPP1 and Patj share several molecular properties, their functions are entirely different. We present evidence that the signaling mediated by Pals1, which has a higher affinity for Patj than for MUPP1 and is involved in the activation of the Par6-aPKC complex, is of principal importance for the function of Patj in epithelial cells.

Generation of transgenic medaka expressing claudin7-EGFP for imaging of tight junctions in living medaka embryos.

The tight junction (TJ) is a specialized cell-cell adhesion structure in epithelial and endothelial sheets unique to the chordates and functions as a barrier of fluidal diffusion across the cell sheets. In order to study the dynamics of TJ formation in vivo during embryogenesis, we have generated a transgenic medaka line that expresses claudin-7 protein fused to enhanced green fluorescent protein under the regulation of the red seabream beta-actin promoter in transparent medaka embryos. Claudins contain four transmembrane domains and have been identified as the key molecules that dictate the function of TJs. This transgenic medaka line will thus be useful for imaging of TJs in living embryos and hence in screening for mutations affecting cell-cell adhesion.

Sentan: a novel specific component of the apical structure of vertebrate motile cilia.

Human respiratory and oviductal cilia have specific apical structures characterized by a narrowed distal portion and a ciliary crown. These structures are conserved among vertebrates that have air respiration systems; however, the molecular components of these structures have not been defined, and their functions are unknown. To identify the molecular component(s) of the cilia apical structure, we screened EST libraries to identify gene(s) that are exclusively expressed in ciliated tissues, are transcriptionally up-regulated during in vitro ciliogenesis, and are not expressed in testis (because sperm flagella have no such apical structures). One of the identified gene products, named sentan, was localized to the distal tip region of motile cilia. Using anti-sentan polyclonal antibodies and electron microscopy, sentan was shown to localize exclusively to the bridging structure between the cell membrane and peripheral singlet microtubules, which specifically exists in the narrowed distal portion of cilia. Exogenously expressed sentan showed affinity for the membrane protrusions, and a protein-lipid binding assay revealed that sentan bound to phosphatidylserine. These findings suggest that sentan is the first molecular component of the ciliary tip to bridge the cell membrane and peripheral singlet microtubules, making the distal portion of the cilia narrow and stiff to allow for better airway clearance or ovum transport.

Loss of occludin affects tricellular localization of tricellulin.

The tricellular tight junction (tTJ) forms at the convergence of bicellular tight junctions (bTJs) where three epithelial cells meet in polarized epithelia, and it is required for the maintenance of the transepithelial barrier. Tricellulin is a four transmembrane domain protein recently identified as the first marker of tTJ, but little is known about how tricellulin is localized at tTJs. As for the molecular mechanism of association of tricellulin with tight junctions (TJs), we found that tricellulin was incorporated into claudin-based TJs independently of binding to zona occludens-1. Unexpectedly, exogenous expression of tricellulin increased cross-links of TJ strands in the plasma membrane. As for the molecular mechanisms for localization of tricellulin at tricellular junctions, we found that knockdown of occludin caused mislocalization of tricellulin to bTJs, implying that occludin supports tricellular localization of tricellulin by excluding tricellulin from bTJs.

IQGAP3 regulates cell proliferation through the Ras/ERK signalling cascade.

Proliferation of epithelial cells must be spatiotemporally regulated to maintain the organization of epithelial sheets. Here we show that the IQGAP family, comprising IQGAP1, 2 and 3, underlies lateral cell-cell contacts of epithelial cells. Of the three proteins, IQGAP3 is unique in that its expression is specifically confined to proliferating cells. Knockdown of IQGAP3 in cultured epithelial cells caused inhibition of proliferation and ERK activity. When exogenously expressed in quiescent cells, IQGAP3 was capable of inducing cell-cycle re-entry, which was completely inhibited by the MEK inhibitor U0126. Thus, IQGAP3 is necessary and sufficient for driving cell proliferation and ERK acts downstream of IQGAP3. Furthermore, IQGAP3 specifically interacted with the active, GTP-bound form of Ras, and in IQGAP3 knockdown cells, the activity of Ras, but not of other small GTPases, was inhibited. Thus, IQGAP3 regulates the promotion of cell proliferation through Ras-dependent ERK activation.

ZO-1- and ZO-2-dependent integration of myosin-2 to epithelial zonula adherens.

For the zonula adherens (ZA) to be established by linear arrangement of adherens junctions (AJs) in epithelial sheet cells, critical for the epithelial cell sheet formation and intercellular barrier function, myosin-2 is supposedly integrated into the ZA with the result of overlapping localization of E-cadherin/actin/myosin-2. Here, we immunofluorescently showed that myosin-2 failed to be integrated into the ZA in cultured epithelial-type ZO1(ko)/2(kd) Eph4 cells lacking ZO-1 and -2 (zonula occludens-1 and -2) by knockout and knockdown, respectively. Instead, a linearized but fragmented arrangement of AJs was formed in the way that it was positive for E-cadherin/actin, but negative for myosin-2 (designated prezonula-AJ). Transfection of full-length ZO-1 or ZO-2, or ZO-1 lacking its PDZ (PSD-95/discs large/zonula occludens-1)-1/2 domains (but not one lacking PDZ-1/2/3) into ZO1(ko)/2(kd) Eph4 cells restored the junctional integration of myosin-2 with prezonula-AJ to establish the ZA. Transfection of dominant-active RhoA or Rho-kinase (ROCK), as well as administration of lysophosphatidic acid or Y27632, which activates RhoA or inhibits ROCK, respectively, suggested that RhoA regulated the junctional integration of myosin-2 into ZA in a manner such that ROCK played a necessary but not-sufficient role. Fluorescence resonance energy transfer analyses revealed that spatiotemporal Rho-activation occurred in a ZO-1/2-dependent way to establish ZA from primordial forms in epithelial cells.

Optimized proteomic analysis on gels of cell-cell adhering junctional membrane proteins.

A high level of structural organization of functional membrane domains in very narrow regions of a plasma membrane is crucial for the functions of plasma membranes and various other cellular functions. Conventional proteomic analyses are based on total soluble cellular proteins. Thus, because of insolubility problems, they have major drawbacks for use in analyses of low-abundance proteins enriched in very limited and specific areas of cells, as well as in analyses of the membrane proteins in two-dimensional gels. We optimized proteomic analyses of cell-cell adhering junctional membrane proteins on gels. First, we increased the purity of cell-cell junctions, which are very limited and specific areas for cell-cell adhesion, from hepatic bile canaliculi. We then enriched junctional membrane proteins via a guanidine treatment; these became selectively detectable on two- dimensionally electrophoresed gels after treatment with an extremely high concentration of NP-40. The framework of major junctional integral membrane proteins was shown on gels. These included six novel junctional membrane proteins of type I, type II, and tetraspanin, which were identified by mass spectrometry and by a database sequence homology search, as well as 12 previously identified junctional membrane proteins, such as cadherins and claudins.

Deficiency of zonula occludens-1 causes embryonic lethal phenotype associated with defected yolk sac angiogenesis and apoptosis of embryonic cells.

Zonula occludens (ZO)-1/2/3 are the members of the TJ-MAGUK family of membrane-associated guanylate kinases associated with tight junctions. To investigate the role of ZO-1 (encoded by Tjp1) in vivo, ZO-1 knockout (Tjp1(-/-)) mice were generated by gene targeting. Although heterozygous mice showed normal development and fertility, delayed growth and development were evident from E8.5 onward in Tjp1(-/-) embryos, and no viable Tjp1(-/-) embryos were observed beyond E11.5. Tjp1(-/-) embryos exhibited massive apoptosis in the notochord, neural tube area, and allantois at embryonic day (E)9.5. In the yolk sac, the ZO-1 deficiency induced defects in vascular development, with impaired formation of vascular trees, along with defective chorioallantoic fusion. Immunostaining of wild-type embryos at E8.5 for ZO-1/2/3 revealed that ZO-1/2 were expressed in almost all embryonic cells, showing tight junction-localizing patterns, with or without ZO-3, which was confined to the epithelial cells. ZO-1 deficiency depleted ZO-1-expression without influence on ZO-2/3 expression. In Tjp1(+/+) yolk sac extraembryonic mesoderm, ZO-1 was dominant without ZO-2/3 expression. Thus, ZO-1 deficiency resulted in mesoderms with no ZO-1/2/3, associated with mislocalization of endothelial junctional adhesion molecules. As a result, angiogenesis was defected in Tjp1(-/-) yolk sac, although differentiation of endothelial cells seemed to be normal. In conclusion, ZO-1 may be functionally important for cell remodeling and tissue organization in both the embryonic and extraembryonic regions, thus playing an essential role in embryonic development.

Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning.

The junctional complex, including tight junctions (TJs), adherens junctions (AJs), and desmosomes, plays crucial roles in the structure and functions of epithelial cellular sheets. In this study, we evaluated the fluorescence localization-based retrovirus-mediated expression cloning (FL-REX) method as an approach to identify novel molecular components of TJs and AJs. Using an expression library of cDNA-GFP-fusions derived from mRNA of a mouse epithelial cell line, we confirmed that cDNAs for various known TJ- and AJ-components could be cloned in the FL-REX. Furthermore, cDNAs for ARHGAP12 and SPAL3, two putative GTPase activating proteins (GAPs) for small G proteins, were cloned as novel components of the junctional complex. Immunofluorescence staining using antibodies generated in-house demonstrated that these GAPs were localized at epithelial cell-cell junctions in various mouse tissues, and were specific to AJs when observed under confocal laser-scanning microscopy. These data suggest that FL-REX is a powerful tool to identify novel proteins localized at TJs and AJs.