J Cell Biol 2001 Apr 16;153(2):263-72

Conversion of Zonulae Occludentes from Tight to Leaky Strand Type by Introducing Claudin-2 into Madin-Darby Canine Kidney I Cells.

Furuse M, Furuse K, Sasaki H, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

There are two strains of MDCK cells, MDCK I and II. MDCK I cells show much higher transepithelial electric resistance (TER) than MDCK II cells, although they bear similar numbers of tight junction (TJ) strands. We examined the expression pattern of claudins, the major components of TJ strands, in these cells: claudin-1 and -4 were expressed both in MDCK I and II cells, whereas the expression of claudin-2 was restricted to MDCK II cells. The dog claudin-2 cDNA was then introduced into MDCK I cells to mimic the claudin expression pattern of MDCK II cells. Interestingly, the TER values of MDCK I clones stably expressing claudin-2 (dCL2-MDCK I) fell to the levels of MDCK II cells (>20-fold decrease). In contrast, when dog claudin-3 was introduced into MDCK I cells, no change was detected in their TER. Similar results were obtained in mouse epithelial cells, Eph4. Morphometric analyses identified no significant differences in the density of TJs or in the number of TJ strands between dCL2-MDCK I and control MDCK I cells. These findings indicated that the addition of claudin-2 markedly decreased the tightness of individual claudin-1/4-based TJ strands, leading to the speculation that the combination and mixing ratios of claudin species determine the barrier properties of individual TJ strands

Nat Rev Mol Cell Biol 2001 Apr;2(4):285-93

Multifunctional strands in tight junctions.

Tsukita S, Furuse M, Itoh M

Department of Cell Biology, Kyoto University Faculty of Medicine, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. htsukita@mfour.med.kyoto-u.ac.jp

Tight junctions are one mode of cell-cell adhesion in epithelial and endothelial cellular sheets. They act as a primary barrier to the diffusion of solutes through the intercellular space, create a boundary between the apical and the basolateral plasma membrane domains, and recruit various cytoskeletal as well as signalling molecules at their cytoplasmic surface. New insights into the molecular architecture of tight junctions allow us to now discuss the structure and functions of this unique cell-cell adhesion apparatus in molecular terms

J Cell Sci 2001 Feb;114(Pt 3):493-501

Real-time imaging of cell-cell adherens junctions reveals that Drosophila mesoderm invagination begins with two phases of apical constriction of cells.

Oda H, Tsukita S

Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Chudoji Minami-machi, Shimogyo-ku, Kyoto 600-8813, Japan. hoda@cell.tsukita.jst.go.jp

Invagination of the epithelial cell sheet of the prospective mesoderm in Drosophila gastrulation is a well-studied, relatively simple morphogenetic event that results from dynamic cell shape changes and cell movements. However, these cell behaviors have not been followed at a sufficiently short time resolution. We examined mesoderm invagination in living wild-type embryos by real-time imaging of fluorescently labeled cell-cell adherens junctions, which are located at the apical zones of cell-cell contact. Low-light fluorescence video microscopy directly visualized the onset and progression of invagination. In an initial period of approximately 2 minutes, cells around the ventral midline reduced their apical surface areas slowly in a rather synchronous manner. Next, the central and more lateral cells stochastically accelerated or initiated their apical constriction, giving rise to random arrangements of cells with small and relatively large apices. Thus, we found that mesoderm invagination began with slow synchronous and subsequent fast stochastic phases of cell apex constriction. Furthermore, we showed that the mesoderm invagination of folded gastrulation mutant embryos lacked the normal two constriction phases, and instead began with asynchronous, feeble cell shape changes. Our observations suggested that Folded gastrulation-mediated signaling enabled synchronous activation of the contractile cortex, causing competition among the individual mesodermal cells for apical constriction. Movies available on-line: http://www.biologists.com/JCS/movies/jcs2073.html

J Biol Chem 2001 Mar 30;276(13):10423-31

Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms.

Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon JM

First Department of Internal Medicine, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. hirase@med.kobe-u.ac.jp

In epithelial and endothelial cells, tight junctions regulate the paracellular permeability of ions and proteins. Disruption of tight junctions by inflammation is often associated with tissue edema, but regulatory mechanisms are not fully understood. Using ECV304 cells as a model system, lysophosphatidic acid and histamine were found to increase the paracellular permeability of the tracer horseradish peroxidase. Cytoskeletal changes induced by these agents included stimulation of stress fiber formation and myosin light chain phosphorylation. Additionally, occludin, a tight junction protein, was a target for signaling events triggered by lysophosphatidic acid and histamine, events that resulted in its phosphorylation. A dominant-negative mutant of RhoA, RhoA T19N, or a specific inhibitor of Rho-activated kinases, Y-27632, prevented stress fiber formation, myosin light chain phosphorylation, occludin phosphorylation, and the increase in tracer flux in response to lysophosphatidic acid. In contrast, although RhoA T19N and Y-27632 blocked the cytoskeletal events induced by histamine, they had no effect on the stimulation of occludin phosphorylation or increased tracer flux, indicating that occludin phosphorylation may regulate tight junction permeability independently of cytoskeletal events. Thus, occludin is a target for receptor-initiated signaling events regulating its phosphorylation, and this phosphorylation may be a key regulator of tight junction permeability.

Mol Biol Cell 2000 Dec;11(12):4131-42

Complex phenotype of mice lacking occludin, a component of tight junction strands.

Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, Takano H, Noda T, Tsukita S

Department of Cell Biology, Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606-8501, Japan.

Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the occludin gene. Occludin -/- mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin -/- males produced no litters with wild-type females, whereas occludin -/- females produced litters normally when mated with wild-type males but did not suckle them. In occludin -/- mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as occludin are more complex than previously supposed.

Development 2000 Aug;127(16):3513-22

Mechanism of glia-neuron cell-fate switch in the Drosophila thoracic neuroblast 6-4 lineage.

Akiyama-Oda Y, Hotta Y, Tsukita S, Oda H

Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. yasuko@cell.tsukita.jst.go.jp

During development of the Drosophila central nervous system, neuroblast 6-4 in the thoracic segment (NB6-4T) divides asymmetrically into a medially located glial precursor cell and a laterally located neuronal precursor cell. In this study, to understand the molecular basis for this glia-neuron cell-fate decision, we examined the effects of some known mutations on the NB6-4T lineage. First, we found that prospero (pros) mutations led to a loss of expression of Glial cells missing, which is essential to trigger glial differentiation, in the NB6-4T lineage. In wild-type embryos, Pros protein was localized at the medial cell cortex of dividing NB6-4T and segregated to the nucleus of the glial precursor cell. miranda and inscuteable mutations altered the behavior of Pros, resulting in failure to correctly switch the glial and neuronal fates. Our results suggested that NB6-4T used the same molecular machinery in the asymmetric cell division as other neuroblasts in cell divisions producing ganglion mother cells. Furthermore, we showed that outside the NB6-4T lineage most glial cells appeared independently of Pros.

FEBS Lett 2000 Jul 7;476(3):258-61

Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein.

Fujita K, Katahira J, Horiguchi Y, Sonoda N, Furuse M, Tsukita S

Department of Cell Biology, Kyoto University, Kyoto, Japan.

Claudins (claudin-1 to -18) with four transmembrane domains and two extracellular loops constitute tight junction strands. The peptide toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to claudin-3 and -4, but not to claudin-1 or -2. We constructed claudin-1/claudin-3 chimeric molecules and found that the second extracellular loop of claudin-3 conferred CPE sensitivity on L fibroblasts. Furthermore, overlay analyses revealed that the second extracellular loop of claudin-3 specifically bound to CPE at the K(a) value of 1.0x10(8) M(-1). We concluded that the second extracellular loop is the site through which claudin-3 interacts with CPE on the cell surface.

Acta Crystallogr D Biol Crystallogr 2000 Jul;56 ( Pt 7):922-3

Crystallographic characterization of the membrane-binding domain of radixin.

Hamada K, Matsui T, Tsukita S, Tsukita S, Hakoshima T

Department of Molecular Biology, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.

Radixin is a protein which cross-links plasma membranes and actin filaments and thus forms membrane-associated cytoskeleton. The radixin N-terminal domain, which is responsible for membrane association, has been purified and crystallized by vapour diffusion with polyethylene glycol 6000. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 96.36, c = 133.16 A, and diffract to a resolution of 3.0 A

EMBO J 2000 Sep 1;19(17):4449-62

Structural basis of the membrane-targeting and unmasking mechanisms of the radixin FERM domain.

Hamada K, Shimizu T, Matsui T, Tsukita S, Hakoshima T

Department of Molecular Biology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan.

Radixin is a member of the ezrin/radixin/moesin (ERM) family of proteins, which play a role in the formation of the membrane-associated cytoskeleton by linking actin filaments and adhesion proteins. This cross-linking activity is regulated by phosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP2) in the downstream of the small G protein Rho. The X-ray crystal structures of the radixin FERM domain, which is responsible for membrane binding, and its complex with inositol-(1,4, 5)-trisphosphate (IP3) have been determined. The domain consists of three subdomains featuring a ubiquitin-like fold, a four-helix bundle and a phosphotyrosine-binding-like domain, respectively. These subdomains are organized by intimate interdomain interactions to form characteristic grooves and clefts. One such groove is negatively charged and so is thought to interact with basic juxta-membrane regions of adhesion proteins. IP3 binds a basic cleft that is distinct from those of pleckstrin homology domains and is located on a positively charged flat molecular surface, suggesting an electrostatic mechanism of plasma membrane targeting. Based on the structural changes associated with IP3 binding, a possible unmasking mechanism of ERM proteins by PIP2 is proposed

J Cell Biol 2000 Sep 4;150(5):1161-76

Two cell adhesion molecules, nectin and cadherin, interact through their cytoplasmic domain-associated proteins.

Tachibana K, Nakanishi H, Mandai K, Ozaki K, Ikeda W, Yamamoto Y, Nagafuchi A, Tsukita S, Takai Y

Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.

We have found a new cell-cell adhesion system at cadherin-based cell-cell adherens junctions (AJs) consisting of at least nectin and l-afadin. Nectin is a Ca(2+)-independent homophilic immunoglobulin-like adhesion molecule, and l-afadin is an actin filament-binding protein that connects the cytoplasmic region of nectin to the actin cytoskeleton. Both the trans-interaction of nectin and the interaction of nectin with l-afadin are necessary for their colocalization with E-cadherin and catenins at AJs. Here, we examined the mechanism of interaction between these two cell-cell adhesion systems at AJs by the use of alpha-catenin-deficient F9 cell lines and cadherin-deficient L cell lines stably expressing their various components. We showed here that nectin and E-cadherin were colocalized through l-afadin and the COOH-terminal half of alpha-catenin at AJs. Nectin trans-interacted independently of E-cadherin, and the complex of E-cadherin and alpha- and beta-catenins was recruited to nectin-based cell-cell adhesion sites through l-afadin without the trans-interaction of E-cadherin. Our results indicate that nectin and cadherin interact through their cytoplasmic domain-associated proteins and suggest that these two cell-cell adhesion systems cooperatively organize cell-cell AJs

Microbiol Immunol 2000;44(8):657-68

The rabies virion-associated 100-kDa polypeptide (VAP100) is a host-derived minor component of the viral envelope.

Xiao S, Komiya K, Tochikura TS, Sagara J, Tsukita S, Kawai A

Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.

We investigated a minor polypeptide component of 100-kDa detected in the rabies virion (referred to as VAP100) by using a monoclonal antibody (mAb), #16743, which was shown to recognize the SDS-denatured VAP100 antigen by immunoblot analyses. Although the VAP100 antigen was hardly detectable in the cell by usual immunoblot methods with this mAb, we could detect the antigen by a luminescent immunoblot method as well as by immunoprecipitation from the metabolically radiolabeled cell lysates and virions. Fluorescent antibody (FA) staining with mAb #16743 detected the uniformly distributed antigen on the formalin-fixed normal BHK-21 cells, while slight accumulation of the antigen was also seen in the Golgi area when the cells were permeabilized by treatment with Triton X-100 after fixation. Rabies virus infection induced alteration of the behavior of VAP100 to show a spotted distribution pattern in virus-infected cells. Double FA staining with mAb #16743 and rabbit antibody against the rabies virus envelope antigen demonstrated colocalized distribution of the viral envelope antigens and VAP100 in the cell. From these results, we think that VAP100 is a membrane-associated component of the cell, and its colocalized distribution with the viral envelope antigens in the cell implicates an intimate association of the VAP100 with viral envelope protein(s) and a reflection of possible involvement in the efficient incorporation of VAP100 into the virion.

Biochem Biophys Res Commun 2000 Nov 2;277(3):691-8

Posttranscriptional regulation of alpha-catenin expression is required for Wnt signaling in L cells.

Takahashi N, Ishihara S, Takada S, Tsukita S, Nagafuchi A

Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto, 606-8501, Japan.

alpha-Catenin is an essential component of the cadherin-catenin cell-cell adhesion complex. An excess amount of alpha-catenin also affects the Wnt signaling pathway probably through its direct binding to beta-catenin. Here, we examined the molecular mechanisms of the posttranscriptional regulation of alpha-catenin expression. We constructed an expression vector with alpha-catenin cDNA lacking the 5'-untranslated sequence. In L cell transfectants stably expressing mRNA derived from this vector, the amount of exogenous alpha-catenin protein was about 10-fold higher than that of the endogenous protein. The expression level of the exogenously expressed alpha-catenin mRNA, however, was about 80% of that of endogenous molecule. Most of the endogenous and exogenous alpha-catenin protein in cadherin-negative cells was degraded 5 h after inhibition of protein synthesis. Although alpha-catenin contains the PEST sequence, various proteasome and calpain inhibitors did not affect the level of expression of endogenous alpha-catenin protein in L cells. Overexpressed alpha-catenin showed cytoplasmic localization, disturbed the nuclear localization of stabilized beta-catenin, and inhibited TCF-4-responsive transactivation after Wnt-3a treatment. These results suggested that the low-efficiency of translation and unidentified degradation mechanisms maintained the low levels of alpha-catenin expression in the cytoplasm as a necessary condition for the Wnt signaling pathway.

Ann N Y Acad Sci 2000;915:129-35

The structure and function of claudins, cell adhesion molecules at tight junctions.

Tsukita S, Furuse M

Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan. htsukita@mfour.med.kyoto-u.ac.jp

Tight junctions (TJs) play a pivotal role in compartmentalization in multicellular organisms by sealing the paracellular pathway in epithelial and endothelial cell sheets. Recently, novel integral membrane proteins, claudins, have been identified as major cell adhesion molecules working at TJs. Claudins comprise a multigene family, and each member of approximately 23 kDa bears four transmembrane domains. To date, 15 members of this gene family have been identified. When expression vectors of each species of claudins were transfected into fibroblasts lacking endogenous claudins or TJs, well-developed TJs were observed between adjacent transfectants. Furthermore, claudins were shown to be directly involved in the barrier function of TJs by experiments using Clostridium perfringens enterotoxin. Now that claudins have been identified, the structure and functions of TJs should be determined in detail in molecular terms.

Dev Biol 2000 Jun 15;222(2):429-39

Distinct mechanisms triggering glial differentiation in Drosophila thoracic and abdominal neuroblasts 6-4.

Akiyama-Oda Y, Hotta Y, Tsukita S, Oda H

Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.

Neurons and glia are produced in stereotyped patterns after neuroblast cell division during development of the Drosophila central nervous system. The first cell division of thoracic neuroblast 6-4 (NB6-4T) is asymmetric, giving rise to a glial precursor cell and a neuronal precursor cell. In contrast, abdominal NB6-4 (NB6-4A) divides symmetrically to produce two glial cells. To understand the relationship between cell division and glia-neuron cell fate determination, we examined and compared the effects of known cell division mutations on the NB6-4T and NB6-4A lineages. Based on observation of expression of glial fate determination and early glial differentiation genes, the onset of glial differentiation occurred in NB6-4A but not in NB6-4T when both cell cycle progression and cytokinesis were genetically arrested. On the other hand, glial differentiation started in both lineages when cytokinesis was blocked with intact cell cycle progression. These results showed that NB6-4T, but not NB6-4A, requires cell cycle progression for acquisition of glial fate, suggesting that distinct mechanisms trigger glial differentiation in the different lineages.

Curr Biol 2000 Jul 13;10(14):865-868

The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules.

Mimori-Kiyosue Y, Shiina N, Tsukita S

Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Shimogyo-ku, 600-8813, Japan

Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein . We previously showed that APC tagged with green fluorescent protein (GFP) in Xenopus A6 epithelial cells moves along a subset of microtubules and accumulates at their growing plus ends in cell extensions. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis, was also reported to be associated with microtubules. To examine the interaction between APC and EB1 within cells, we compared the dynamic behavior of EB1-GFP with that of APC-GFP in A6 transfectants. Time-lapse microscopy of live cells at interphase revealed that EB1-GFP was concentrated at all of the growing microtubule ends throughout the cytoplasm and
abruptly disappeared from the ends when microtubules began to shorten. Therefore, EB1 appeared to be co-localized and interact with APC on the growing ends of a subset of microtubules. When APC-GFP was overexpressed, endogenous EB1 was recruited to APC-GFP, which accumulated in large amounts on microtubules. On the other hand, when microtubules were disassembled by nocodazole, EB1 was not co-localized with APC-GFP, which was concentrated along the basal plasma membrane. During mitosis, APC appeared to be dissociated from microtubules, whereas EB1-GFP continued to concentrate at microtubule growing ends. These findings showed that the APC-EB1 interaction is regulated within cells and is allowed near the ends of microtubules only under
restricted conditions.

J Cell Biol 2000 Feb 7;148(3):505-18

Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells.

Mimori-Kiyosue Y, Shiina N, Tsukita S

Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan.

Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs
were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.

J Biol Chem 1999 Nov 26;274(48):34148-54

Transglutaminase type 1 and its cross-linking activity are concentrated at adherens junctions in simple epithelial cells.

Hiiragi T, Sasaki H, Nagafuchi A, Sabe H, Shen SC, Matsuki M, Yamanishi K, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

Transglutaminase type 1 was identified as a tyrosine-phosphorylated protein from the isolated junctional fraction of the mouse liver. This enzyme was reported to be involved in the covalent cross-linking of proteins in keratinocytes, but its expression and activity in other cell types have not been examined. Northern blotting revealed that transglutaminase type 1 was expressed in large amounts in epithelial tissues (lung, liver, and kidney), which was also confirmed by immunoblotting with antibodies raised against mouse recombinant protein. Immunoblotting of the isolated junctional fraction revealed that transglutaminase type 1 was concentrated in the fraction not only as a 97-kDa form but also as forms of various molecular masses cross-linked to other proteins. In agreement with this finding, endogenous transglutaminase type 1 was immunofluorescently colocalized with E-cadherin in cultured simple epithelial cells. In the liver and kidney, immunoelectron microscopy revealed that transglutaminase type 1 was concentrated, albeit not exclusively, at cadherin-based adherens junctions. Furthermore, by in vitro and in vivo labeling, transglutaminase cross-linking activity was also shown to be concentrated at intercellular junctions of simple epithelial cells. These findings suggested that the formation of covalently cross-linked multimolecular
complexes by transglutaminase type 1 is an important mechanism for maintenance of the structural integrity of simple epithelial cells, especially at cadherin-based adherens junctions.

J Cell Biol 1999 Nov 15;147(4):891-903

Manner of interaction of heterogeneous claudin species within and between tight junction strands.

Furuse M, Sasaki H, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

In tight junctions (TJs), TJ strands are associated laterally with those of adjacent cells to form paired strands to eliminate the extracellular space. Claudin-1 and -2, integral membrane proteins of TJs, reconstitute paired TJ strands when transfected into L fibroblasts. Claudins comprise a multigene family and more than two distinct claudins are coexpressed in single cells, raising the questions of whether heterogeneous claudins form heteromeric TJ strands and whether claudins interact between each of the paired strands in a heterophilic manner. To answer these questions, we cotransfected two of claudin-1, -2, and -3 into L cells, and detected their coconcentration at cell-cell borders as elaborate networks. Immunoreplica EM confirmed that distinct claudins were coincorporated into individual TJ strands. Next, two L transfectants singly expressing claudin-1, -2, or -3 were cocultured and we found that claudin-3 strands laterally associated with claudin-1 and -2 strands to form paired strands, whereas claudin-1 strands did not interact with claudin-2 strands. We concluded that distinct species of claudins can interact within and between TJ strands, except in some combinations. This mode of assembly of claudins could increase the diversity of the structure and functions of TJ strands.

J Cell Biol 1999 Dec 13;147(6):1351-63

Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.

Itoh M, Furuse M, Morita K, Kubota K, Saitou M, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ZO-1, ZO-2, and ZO-3, which contain three PDZ domains (PDZ1 to -3), are concentrated at tight junctions (TJs) in epithelial cells. TJ strands are mainly composed of two distinct types of four-transmembrane proteins, occludin, and claudins, between which occludin was reported to directly bind to ZO-1/ZO-2/ZO-3. However, in occludin-deficient intestinal epithelial cells, ZO-1/ZO-2/ZO-3 were still recruited to TJs. We then examined the possible interactions between ZO-1/ZO-2/ZO-3 and claudins. ZO-1, ZO-2, and ZO-3 bound to the COOH-terminal YV sequence of claudin-1 to -8 through their PDZ1 domains in vitro. Then, claudin-1 or -2 was transfected into L fibroblasts, which express ZO-1 but not ZO-2 or ZO-3. Claudin-1 and -2 were concentrated at cell-cell borders in an elaborate network pattern, to which endogenous ZO-1 was recruited. When ZO-2 or ZO-3 were further transfected, both were recruited to the claudin-based networks together with endogenous ZO-1. Detailed analyses showed that ZO-2 and ZO-3 are recruited to the claudin-based networks through PDZ2 (ZO-2 or ZO-3)/PDZ2 (endogenous ZO-1) and PDZ1 (ZO-2 or ZO-3)/COOH-terminal YV (claudins) interactions. In good agreement, PDZ1 and PDZ2 domains of ZO-1/ZO-2/ZO-3 were also recruited to claudin-based TJs, when introduced into cultured epithelial cells. The possible molecular architecture of TJ plaque structures is discussed.

Dev Biol 1999 Dec 1;216(1):406-22

Nonchordate classic cadherins have a structurally and functionally unique domain that is absent from chordate classic cadherins.

Oda H, Tsukita S

Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Chudoji Minami-machi, Shimogyo-ku, Kyoto, 600-8813, Japan.

Classic cadherins, which are adhesion molecules in cell-cell adherens junctions, have a large contribution to the construction of the animal body. Their molecular structures show clear differences between chordate and nonchordate metazoans. Although nonchordate classic cadherins have cadherin superfamily-specific extracellular repeats (CRs) and a highly conserved cytoplasmic domain (CP), these cadherins have a unique extracellular domain that is absent from vertebrate and ascidian classic cadherins. We called this the primitive classic cadherin domain (PCCD). To understand the roles of the PCCD, we constructed and characterized a series of mutant forms of the Drosophila classic cadherin DE-cadherin. Biochemical analyses indicated that the last two CRs and PCCD form a special structure with proteolytic cleavage. Mutations in the PCCD did not eliminate the cell-cell-binding function of DE-cadherin in cultured cells, but prevented the cadherin from efficiently translocating to the plasma membrane in epithelial cells of the developing embryo. In addition, genetic rescue assays suggested that although CP-mediated control plays a central role in tracheal fusion, the role of the PCCD in efficient recruitment of DE-cadherin to apical areas of the plasma membranes is also important for dynamic epithelial morphogenesis. We propose that there is a fundamental difference in the mode of classic cadherin-mediated cell-cell adhesion between chordate and nonchordate metazoans.

Curr Biol 1999 Nov 4;9(21):1259-62

Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases.

Matsui T, Yonemura S, Tsukita S, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606, Japan.

When activated, ERM (ezrin, radixin, moesin) proteins are recruited to the plasma membrane, with concomitant carboxy-terminal threonine phosphorylation, where they crosslink actin filaments to the plasma membrane to form microvilli . Here, we report that, when NIH3T3 or HeLa cells were transfected with a constitutively active mutant of the small GTPase RhoA (V14RhoA), microvilli were induced and the level of carboxy-terminal threonine-phosphorylated ERM proteins (CPERM) increased approximately 30-fold. This increase was not observed following transfection of constitutively active forms of two other Rho-family GTPases, Rac1 and Cdc42, or of a direct effector of Rho, Rho-kinase (also known as ROKalpha or ROCK-II) . The V14RhoA-induced phosphorylation of ERM proteins was not suppressed by Y-27632, a specific inhibitor of ROCK kinases including Rho-kinase. Overexpression of another direct effector of Rho, phosphatidylinositol 4-phosphate 5-kinase (PI4P5K) type Ialpha , but not a kinase-inactive mutant, increased approximately sixfold the level of CPERM, and induced microvilli. Together with the previous finding that the PI4P5K product phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates ERM proteins in vitro , our data suggest that PIP(2), and not ROCK kinases, is involved in the RhoA-dependent activation of ERM proteins in vivo. The active state of ERM proteins is maintained through threonine phosphorylation by as yet undetermined kinases, leading to microvillus formation.

Curr Biol 1999 Sep 23;9(18):1035-8

Ca(2+)-independent cell-adhesion activity of claudins, a family of integral membrane proteins localized at tight junctions.

Kubota K, Furuse M, Sasaki H, Sonoda N, Fujita K, Nagafuchi A, Tsukita S

Department of Cell Biology Faculty of Medicine Kyoto University Sakyo-ku, Kyoto, 606-8501, Japan.

In multicellular organisms, various compositionally distinct fluid compartments are established by epithelial and endothelial cellular sheets. For these cells to function as barriers, tight junctions (TJs)are considered to create a primary barrier for the diffusion of solutes through the paracellular pathway. In ultrathin sections viewed under electron microscopy, TJs appear as a series of apparent fusions, involving the outer leaflets of plasma membranes of adjacent cells, to form the so-called kissing points of TJs, where the intercellular space is completely obliterated. Claudins are a family of 16 proteins whose members have been identified as major integral membrane proteins localized exclusively at TJs. It remains unclear, however, whether claudins have the cell-adhesion activity that would explain the unusual intercellular adhesion at TJs. Using mouse L-fibroblast transfectants expressing various amounts of claudin-1, -2 or -3, we found that these claudins possess Ca(2+)-independent cell-adhesion activity. Using ultrathin-section electron microscopy, we observed many kissing points of TJs between adjacent transfectants. Furthermore, the cell-adhesion activity of occludin, another integral membrane protein localized at TJs , was negligible when compared with that of claudins. Thus, claudins are responsible for TJ-specific obliteration of the intercellular space.

Curr Opin Cell Biol 1999 Oct;11(5):628-33

Structural and signalling molecules come together at tight junctions.

Tsukita S, Furuse M, Itoh M

Department of Cell Biology Faculty of Medicine Kyoto University Yoshida-Konoe, Sakyo-ku, Kyoto, 606, Japan. htsukita@mfour.med. kyoto-u.ac.jp.

Tight junctions (TJs) have been suggested to act both as barriers and fences, but lack of information on the constituents of TJ strands has hampered the direct assessment of these functions. Over the
past year, our understanding of the molecular architecture of TJ strands has increased markedly and we are ready to experimentally examine how TJs are involved in their dual functions.

Expression level, subcellular distribution and rho-GDI binding affinity of merlin in comparison with Ezrin/Radixin/Moesin proteins.

Maeda-M; Matsui-T; Imamura-M; Tsukita-S; Tsukita-S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

Merlin, a neurofibromatosis type-2 tumor suppressor, shows significant sequence similarity to ERM (Ezrin/Radixin/Moesin) proteins, general actin filament/plasma membrane cross-linkers, which are regulated in a Rho-dependent manner. To understand its physiological functions, we compared merlin with ERM proteins in vivo and in vitro. Quantitative immunoblotting revealed that the molar ratio of merlin/ERM in cultured epithelial or non-epithelial cells was approximately 0.14 or approximately 0.05, respectively. After centrifugation of cell homogenate, merlin was mostly recovered in the insoluble fraction, whereas almost half of ERM proteins were found in the soluble fraction. Merlin and ERM proteins were concentrated at microvilli when introduced into fibroblasts. In contrast, in epithelial cells, introduced merlin was co-distributed with E-cadherin in lateral membranes, whereas ERM proteins were concentrated in apical microvilli. Finally, we examined the binding affinity of merlin to Rho GDP dissociation inhibitor (Rho-GDI), to which N-terminal halves of ERM proteins but not the full-length molecules specifically bind. In vitro binding assays revealed that the N-terminal halves of merlin isoform-I and -II as well as full-length merlin isoform-II bound to Rho-GDI with similar binding affinity to ERM proteins. Immunoprecipitation confirmed these findings in vivo. These findings do not favor the notion that merlin functions simply in a redundant or competitive manner to ERM proteins.

Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells.

Morita-K; Sasaki-H; Furuse-M; Tsukita-S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.

Tight junctions (TJs) in endothelial cells are thought to determine vascular permeability. Recently, claudin-1 to -15 were identified as major components of TJ strands. Among these, claudin-5 (also called transmembrane protein deleted in velo-cardio-facial syndrome [TMVCF]) was expressed ubiquitously, even in organs lacking epithelial tissues, suggesting the possible involvement of this claudin species in endothelial TJs. We then obtained a claudin-6-specific polyclonal antibody and a polyclonal antibody that recognized both claudin-5/TMVCF and claudin-6. In the brain and lung, immunofluorescence microscopy with these polyclonal antibodies showed that claudin-5/TMVCF was exclusively concentrated at cell-cell borders of endothelial cells of all segments of blood vessels, but not at those of epithelial cells. Immunoreplica electron microscopy revealed that claudin-5/TMVCF was a component of TJ strands. In contrast, in the kidney, the claudin-5/TMVCF signal was restricted to endothelial cells of arteries, but was undetectable in those of veins and capillaries. In addition, in all other tissues we examined, claudin-5/TMVCF was specifically detected in endothelial cells of some segments of blood vessels, but not in epithelial cells. Furthermore, when claudin-5/TMVCF cDNA was introduced into mouse L fibroblasts, TJ strands were reconstituted that resembled those in endothelial cells in vivo, i.e., the extracellular face-associated TJs. These findings indicated that claudin-5/TMVCF is an endothelial cell-specific component of TJ strands.

Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

Sonoda-N; Furuse-M; Sasaki-H; Yonemura-S; Katahira-J; Horiguchi-Y; Tsukita-S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

FEBS Lett 1999 Feb 26;445(2-3):395-401

Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP-actin fusion protein.

Verkhusha VV, Tsukita S, Oda H

Tsukita Cell Axis Project, ERATO, JST, Kyoto, Japan. vverkhus@cell.tsukita.jst.go.jp
Directional migration of border cells in the Drosophila egg chambers is a developmentally regulated event that requires dynamic cellular functions. In this study, the electron microscopic observation of migrating border cells revealed loose actin bundles in forepart lamellipodia and numerous microvilli extending from nurse cells and providing multiple adhesive contacts with border cells. To analyze the dynamics of actin in migrating border cells in vivo, we constructed a green fluorescent protein-actin fusion protein and induced its expression in Drosophila using the GAL4/UAS system. The green fluorescent protein-actin was incorporated into the actin bundles and it enabled visualization of the rapid cytoskeletal changes in border cell lamellipodia. During the growth of the lamellipodia, the actin bundles that increased in number and size radiated from the bundle-organizing center. Quantification of the fluorescence intensity showed that an accumulation of bundle-associated and spotted green fluorescent protein-actin signals took place during their centripetal movement. Our results favored a treadmilling model for actin behavior in border cell lamellipodia.

Dynamic features of adherens junctions during Drosophila embryonic epithelial morphogenesis revealed by a Dalpha-catenin-GFP fusion protein.

Oda H, Tsukita S

Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Chudoji Minami-machi, Shimogyo-ku, Kyoto 600-8813, Japan.
Cell-cell adherens junctions (AJs), comprised of the cadherin-catenin adhesion system, contribute to cell shape changes and cell movements in epithelial morphogenesis. However, little is known about the dynamic features of AJs in cells of the developing embryo. In this study, we constructed Dalpha-catenin fused with a green fluorescent protein (Dalpha-catenin-GFP), and found that it targeted apically located AJ-based contacts but not other lateral contacts in epithelial cells of living Drosophila embryos. Using time-lapse fluorescence microscopy, we examined the dynamic performance of AJs containing Dalpha-catenin-GFP in epithelial morphogenetic movements. In the ventral ectoderm of stage 11 embryos, concentration and deconcentration of Dalpha-catenin-GFP occurred concomitantly with changes in length of AJ contacts. In the lateral ectoderm of embryos at the same stage, dynamic behaviour of AJs was concerted with division and delamination of sensory organ precursor (SOP) cells. Moreover, changes in patterns of AJ networks during tracheal extension could be followed. Finally, we utilized Dalpha-catenin-GFP to precisely observe the defects in tracheal fusion in shotgun mutants. Thus, the Dalpha-catenin-GFP fusion protein is a helpful tool to simultaneously observe morphogenetic movements and AJ dynamics at high spatio-temporal resolution.

Direct involvement of Ezrin/Radixin/Moesin (ERM)-binding membrane proteins in the organization of microvilli in collaboration with activated ERM proteins.

Yonemura S, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.
[Medline record in process]

Ezrin/radixin/moesin (ERM) proteins have been thought to play a central role in the organization of cortical actin-based cytoskeletons including microvillar formation through cross-linking actin filaments and integral membrane proteins such as CD43, CD44, and ICAM-2. To examine the functions of these ERM-binding membrane proteins (ERMBMPs) in cortical morphogenesis, we overexpressed ERMBMPs (the extracellular domain of E-cadherin fused with the transmembrane/cytoplasmic domain of CD43, CD44, or ICAM-2) in various cultured cells. In cultured fibroblasts such as L and CV-1 cells, their overexpression significantly induced microvillar elongation, recruiting ERM proteins and actin filaments. When the ERM-binding domains were truncated from these molecules, their ability to induce microvillar elongation became undetectable. In contrast, in cultured epithelial cells such as MTD-1A and A431 cells, the overexpression of ERMBMPs did not elongate microvilli. However, in the presence of EGF, overexpression of ERMBMPs induced remarkable microvillar elongation in A431 cells. These results indicated that ERMBMPs function as organizing centers for cortical morphogenesis by organizing microvilli in collaboration with activated ERM proteins. Furthermore, immunodetection with a phosphorylated ERM-specific antibody and site-directed mutagenesis suggested that ERM proteins phosphorylated at their COOH-terminal threonine residue represent activated ERM proteins.

alpha-catenin-deficient F9 cells differentiate into signet ring cells.

Maeno Y, Moroi S, Nagashima H, Noda T, Shiozaki H, Monden M, Tsukita S, Nagafuchi A

Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto, Japan.
It has been demonstrated that alpha-catenin is frequently lost in diffuse type adenocarcinomas. We have isolated alpha-catenin-deficient mouse teratocarcinoma F9 cells by gene targeting. Wild-type F9 cell aggregates cultured in the presence of retinoic acid differentiated into embryoid bodies with an outer layer of epithelial cells. In contrast, cell aggregates of alpha-catenin-deficient cells did not develop outer layers under the same conditions. The outer surface cells of alpha-catenin-deficient cell aggregates, however, differentiated into epithelial cells as determined by their expression of epithelial marker proteins. These differentiated cells scattered from aggregates and showed signet ring cell morphology, which is frequently observed in diffuse type adenocarcinomas. We have provided clear evidence that a single mutation in the alpha-catenin gene may be a direct cause not only of the scattered properties of cells but also of signet ring cell formation in diffuse type adenocarcinoma.

Occludin and claudins in tight-junction strands: leading or supporting players?

Tsukita S, Furuse M

Dept of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
[Medline record in process]

Tight junctions have attracted much interest from cell biologists, especially electron microscopists, since on freeze-fracture electron microscopy they appear as a well-developed network of continuous, anastomosing intramembranous strands (tight-junction strands). These strands might be directly involved in the 'barrier' as well as 'fence' functions in epithelial and endothelial cell sheets, but until recently little was known of their constituents. This review discusses current understanding of the molecular architecture of tight-junction strands, focusing on the recent discovery of two distinct types of tight-junction-specific integral membrane proteins, occludin and claudins.

Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion.

Imamura Y, Itoh M, Maeno Y, Tsukita S, Nagafuchi A

Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan.
The interaction of cadherin-catenin complex with the actin-based cytoskeleton through alpha-catenin is indispensable for cadherin-based cell adhesion activity. We reported previously that E-cadherin-alpha-catenin fusion molecules showed cell adhesion and cytoskeleton binding activities when expressed in nonepithelial L cells. Here, we constructed deletion mutants of E-cadherin-alpha-catenin fusion molecules lacking various domains of alpha-catenin and introduced them into L cells. Detailed analysis identified three distinct functional domains of alpha-catenin: a vinculin/alpha-actinin-binding domain, a ZO-1-binding domain, and an adhesion-modulation domain. Furthermore, cell dissociation assay revealed that the fusion molecules containing the ZO-1-binding domain in addition to the adhesion-modulation domain conferred the strong state of cell adhesion activity on transfectants, although those lacking the ZO-1-binding domain conferred only the weak state. The disorganization of actin-based cytoskeleton by cytochalasin D treatment shifted the cadherin-based cell adhesion from the strong to the weak state. In the epithelial cells, where alpha-catenin was not precisely colocalized with ZO-1, the ZO-1-binding domain did not completely support the strong state of cell adhesion activity. Our studies showed that the interaction of alpha-catenin with the actin-based cytoskeleton through the ZO-1-binding domain is required for the strong state of E-cadherin-based cell adhesion activity.

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues.

Hayashi K, Yonemura S, Matsui T, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. yonemura@mfour.med.kyoto-u.ac.jp
[Record supplied by publisher]

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis.

Morita K, Sasaki H, Fujimoto K, Furuse M, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
Members of the newly identified claudin gene family constitute tight junction (TJ) strands, which play a pivotal role in compartmentalization in multicellular organisms. We identified oligodendrocyte-specific protein (OSP) as claudin-11, a new claudin family member, due to its sequence similarity to claudins as well as its ability to form TJ strands in transfected fibroblasts. Claudin-11/OSP mRNA was expressed in the brain and testis. Immunofluorescence microscopy with anti-claudin-11/OSP polyclonal antibody (pAb) and anti-neurofilament mAb revealed that in the brain claudin-11/OSP-positive linear structures run in a gentle spiral around neurofilament-positive axons. At the electron microscopic level, these linear structures were identified as the so-called interlamellar strands in myelin sheaths of oligodendrocytes. In testis, well-developed TJ strands of Sertoli cells were specifically labeled with anti-claudin-11/OSP pAb both at immunofluorescence and electron microscopic levels. These findings indicated that the interlamellar strands of oligodendrocyte myelin sheaths can be regarded as a variant of TJ strands found in many other epithelial cells, and that these strands share a specific claudin species, claudin-11/OSP, with those in Sertoli cells to create and maintain the repeated compartments around axons by oligodendrocytes.

Mutations at phosphorylation sites of Xenopus microtubule-associated protein 4 affect its microtubule-binding ability and chromosome movement during mitosis.

Shiina N, Tsukita S

Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto 600-8813, Japan.
Microtubule-associated proteins (MAPs) bind to and stabilize microtubules (MTs) both in vitro and in vivo and are thought to regulate MT dynamics during the cell cycle. It is known that p220, a major MAP of Xenopus, is phosphorylated by p34(cdc2) kinase as well as MAP kinase in mitotic cells, and that the phosphorylated p220 loses its MT-binding and -stabilizing abilities in vitro. We cloned a full-length cDNA encoding p220, which identified p220 as a Xenopus homologue of MAP4 (XMAP4). To examine the physiological relevance of XMAP4 phosphorylation in vivo, Xenopus A6 cells were transfected with cDNAs encoding wild-type or various XMAP4 mutants fused with a green fluorescent protein. Mutations of serine and threonine residues at p34(cdc2) kinase-specific phosphorylation sites to alanine interfered with mitosis-associated reduction in MT affinity of XMAP4, and their overexpression affected chromosome movement during anaphase A. These findings indicated that phosphorylation of XMAP4 (probably by p34(cdc2) kinase) is responsible for the decrease in its MT-binding and -stabilizing abilities during mitosis, which are important for chromosome movement during anaphase A. J Cell Physiol 1999 May;179(2):115-25

Differential behavior of E-cadherin and occludin in their colocalization with ZO-1 during the establishment of epithelial cell polarity.

Ando-Akatsuka Y, Yonemura S, Itoh M, Furuse M, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan.
At the initial stage of cell-cell contact of epithelial cells, primordial spot-like junctions are formed at the tips of thin cellular protrusions radiating from adjacent cells, where E-cadherin and ZO-1 are precisely coconcentrated (Yonemura et al., 1995, J. Cell Sci. 108:127-142). In fully polarized epithelial cells, E-cadherin and ZO-1 are completely sorted into belt-like adherens junctions (AJ) and tight junctions (TJ), respectively. Here we examined the behavior of occludin, an integral membrane protein consisting of TJ, during the establishment of epithelial cell polarity. Using confocal immunofluorescence microscopy, we quantitatively compared the spatial relationship of occludin/ZO-1 with that of E-cadherin/ZO-1 during epithelial cellular polarization by replating or wounding cultured mouse epithelial cells (MTD1-A). At the initial stage of cell-cell contact, E-cadherin and ZO-1 appeared to be simultaneously recruited to the primordial form of spot-like junctions at the tips of cellular processes which showed no concentration of occludin. Then, as cellular polarization proceeded, occludin was gradually accumulated at the ZO-1-positive spot-like junctions to form belt-like TJ, and in a complementary manner E-cadherin was sorted out from the ZO-1-positive spot-like junctions to form belt-like AJ. The molecular mechanism of TJ/AJ formation during epithelial cellular polarization is discussed with special reference to the roles of ZO-1.

Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin.

Itoh M, Morita K, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
ZO-2, a member of the MAGUK family, was thought to be specific for tight junctions (TJs) in contrast to ZO-1, another MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2 cDNA was isolated, and a specific polyclonal antibody was generated using corresponding synthetic peptides as antigens. Immunofluorescence microscopy with this polyclonal antibody revealed that, similarly to ZO-1, in addition to TJs in epithelial cells, ZO-2 was also concentrated at AJs in nonepithelial cells such as fibroblasts and cardiac muscle cells lacking TJs. When NH2-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and C-ZO-2, respectively) were separately introduced into cultured cells, N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at TJs in epithelial cells and at AJs in non-epithelial cells, whereas C-ZO-2 was distributed along actin filaments. Consistently, occludin as well as alpha catenin directly bound to N-ZO-2 as well as the NH2-terminal dlg-like portion of ZO-1 (N-ZO-1) in vitro. Furthermore, immunoprecipitation experiments revealed that the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/alpha catenin to establish TJ or AJ domains, respectively.

Normal development of mice and unimpaired cell adhesion/cell motility/actin-based cytoskeleton without compensatory up-regulation of ezrin or radixin in moesin gene knockout.

Doi Y, Itoh M, Yonemura S, Ishihara S, Takano H, Noda T, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
Ezrin/radixin/moesin (ERM) proteins are general cross-linkers between the plasma membrane and actin filaments. Because their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. On the other hand, experiments at the cellular level and in vitro have suggested their functional redundancy. To assess the possible unique functions of ERM proteins in vivo, the moesin gene located on the X chromosome was disrupted by gene targeting in embryonic stem cells. Male mice hemizygous for the mutation as well as homozygous females were completely devoid of moesin but developed normally and were fertile, with no obvious histological abnormalities in any of the tissues examined. In the tissues of the mutant mice, moesin completely disappeared without affecting the expression levels or subcellular distribution of ezrin and radixin. Also, in platelets, fibroblasts, and mast cells isolated from moesin-deficient mice, targeted disruption of the moesin gene did not affect their ERM-dependent functions, i.e. platelet aggregation, stress fiber/focal contact formation of fibroblasts, and microvillar formation of mast cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level.

Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands.

Morita K, Furuse M, Fujimoto K, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
Two related integral membrane proteins, claudin-1 and -2, recently were identified as novel components of tight junction (TJ) strands. Here, we report six more claudin-like proteins, indicating the existence of a claudin gene family. Three of these were reported previously as RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF, but their physiological functions were not determined. Through similarity searches followed by PCR, we isolated full length cDNAs of mouse RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF as well as three mouse claudin-like proteins and designated them as claudin-3 to -8, respectively. All of these claudin family members showed similar patterns on hydrophilicity plots, which predicted four transmembrane domains in each molecule. Northern blotting showed that the tissue distribution pattern varied significantly, depending on claudin species. Similarly to claudin-1 and -2, when these claudins were HA-tagged and introduced into cultured Madin-Darby canine kidney cells, all showed a tendency to concentrate at TJs. Immunofluorescence and immunoelectron microscopy with polyclonal antibodies specific for claudin-3, -4, or -8 revealed that these molecules were exclusively concentrated at TJs in the liver and/or kidney. These findings indicated that multiple claudin family members are involved in the formation of TJ strands in various tissues.

Overcoming barriers in the study of tight junction functions: from occludin to claudin.

Tsukita S, Furuse M

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan. htsukita@mfour.med.kyoto-u.ac.jp
Tight junctions (TJs) are essential structures for the physiological functions of epithelial and endothelial cells, and have been suggested to have both barrier and fence functions. Tight junctions create a primary barrier to the diffusion of solutes through the paracellular pathway, and also function as a fence between apical and basolateral membrane domains, to create and maintain cell polarity of epithelial and endothelial cells. Several peripheral membrane proteins have been shown to be concentrated at the cytoplasmic surface of TJs. However, TJ-specific integral membrane proteins had not been identified until recently, and the lack of information concerning TJ-specific integral membrane proteins has hampered a more direct assessment of the function of TJs at the molecular level. Here, we present an overview of current progress in the identification of TJ-specific integral membrane proteins.

A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts.

Furuse M, Sasaki H, Fujimoto K, Tsukita S

Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.
Three integral membrane proteins, clau- din-1, -2, and occludin, are known to be components of tight junction (TJ) strands. To examine their ability to form TJ strands, their cDNAs were introduced into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy revealed that both FLAG-tagged claudin-1 and -2 were highly concentrated at cell contact sites as planes through a homophilic interaction. In freeze-fracture replicas of these contact sites, well-developed networks of strands were identified that were similar to TJ strand networks in situ and were specifically labeled with anti-FLAG mAb. In glutaraldehyde-fixed samples, claudin-1-induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2-induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face which were occupied by chains of particles. Although occludin was also concentrated at cell contact sites as dots through its homophilic interaction, freeze-fracture replicas identified only a small number of short strands that were labeled with anti-occludin mAb. However, when occludin was cotransfected with claudin-1, it was concentrated at cell contact sites as planes to be incorporated into well- developed claudin-1-based strands. These findings suggested that claudin-1 and -2 are mainly responsible

Dynamic behavior of the cadherin-based cell-cell adhesion system during Drosophila gastrulation.

Oda H, Tsukita S, Takeichi M

Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Kyoto, Chudoji Minami-machi, Shimogyo-ku, 600-8813, Japan.
During Drosophila gastrulation, morphogenesis occurs as a series of cell shape changes and cell movements which probably involve adhesive interactions between cells. In the present study, we examined the dynamic aspects of cadherin-based cell-cell adhesion in the morphogenetic events to assess its contribution to morphogenesis. DE- and DN-cadherin show complementary expression patterns in the presumptive ectoderm and mesoderm at the mRNA level. We found that switching of cadherin expression from the DE- to the DN-type in the mesodermal germ layer occurred downstream of the mesoderm-determination genes twist and snail. However, examination of their protein expression patterns showed that considerable amounts of DE-cadherin remained on the surfaces of mesodermal cells during invagination, while DN-cadherin did not appear on the cell surfaces at this stage. Further immunocytochemical analysis of the localizations of DE-cadherin and its associated proteins Armadillo (beta-catenin) and Dalpha-catenin revealed dynamic changes in their distributions which were accompanied by changes in cell morphology in the neuroectoderm and mesoderm. Simultaneously, adherens junctions (AJs), based on the cadherin-catenin system, were shown to change their location, size, and morphology. These dynamic aspects of cadherin-based cell-cell adhesion appeared to be associated with the following: (1) initial establishment of the blastoderm epithelium, (2) acquisition of cell motility in the neuroectoderm, (3) cell sheet folding, and (4) epithelial to mesenchymal conversion of the mesoderm. These observations suggest that the behavior of the DE-cadherin-catenin adhesion system may be regulated in a stepwise manner during gastrulation to perform successive cell-morphology conversions. Moreover, the processes responsible for loss of epithelial cell polarity and elimination of preexisting DE-cadherin-based epithelial junctions during early mesodermal morphogenesis are discussed. Copyright 1998 Academic Press.

AU: Saitou-M; Fujimoto-K; Doi-Y; Itoh-M; Fujimoto-T; Furuse-M; Takano-H; Noda-T; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

SO: J-Cell-Biol. 1998 Apr 20; 141(2): 397-408

ISSN: 0021-9525

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: Occludin is the only known integral membrane protein of tight junctions (TJs), and is now believed to be directly involved in the barrier and fence functions of TJs. Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyses indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, zonula occludens (ZO)-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.

AU: Morita-K; Itoh-M; Saitou-M; Ando-Akatsuka-Y; Furuse-M; Yoneda-K; Imamura-S; Fujimoto-K; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan.

SO: J-Invest-Dermatol. 1998 Jun; 110(6): 862-6

ISSN: 0022-202X

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: Occludin is an integral membrane protein that is concentrated at tight junctions (zonulae occludentes) in simple epithelial cells. ZO-1 and ZO-2 are peripheral membrane proteins that are localized at tight junctions in simple epithelial cells and at cadherin-based adherens junctions in nonepithelial cells. In this study, we investigated the expression and subcellular distribution of occludin, ZO-1, and ZO-2 in rodent skin. Immunoblotting detected all of these molecules in isolated epidermis, but the occludin/ZO-1 (or occludin/ZO-2) ratio was significantly lower than that in cultured simple epithelial cells. In the epidermis of adult skin, occludin was concentrated at cell-cell borders only in the most superficial zone of the granular cell layer, whereas ZO-1 and ZO-2 were distributed in a much broader zone from the spinous to the granular layers. During mouse skin development, this peculiar distribution of occludin in the epidermis appeared when the periderm, a simple epithelium bearing typical occludin-based tight junctions, was sloughed off at embryonic day 16.5 of gestation. Freeze-fracture electron microscopy identified the so-called focal strands or maculae occludentes, i.e., spot tight junction-like structures, between adjacent granular cells, and anti-occludin monoclonal antibody exclusively labeled these focal strands. In hair follicles, occludin and ZO-1 were colocalized at cell-cell borders in Henle's layer and the cornifying cuticle of the inner root sheath. In addition, ZO-1 but not occludin were localized weakly at the outer root sheath and intensely at the hair cortex/matrix.

AU: Furuse-M; Fujita-K; Hiiragi-T; Fujimoto-K; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

SO: J-Cell-Biol. 1998 Jun 29; 141(7): 1539-50

ISSN: 0021-9525

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band approximately 22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as "claudin-1" and "claudin-2", respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.

AU: Moroi-S; Saitou-M; Fujimoto-K; Sakakibara-A; Furuse-M; Yoshida-O; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

SO: Am-J-Physiol. 1998 Jun; 274(6 Pt 1): C1708-17

ISSN: 0002-9513

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: Occludin is the only integral membrane protein identified to date as a component of tight junctions (TJs). Here, we examined the distribution and expression of occludin in murine testis bearing well-developed TJ. In the adult mouse testis, occludin was concentrated at TJ strands, which are located at the most basal regions of lateral membranes of Sertoli cells. In immunoblotting, occludin showed a characteristic multiple banding pattern, suggesting that occludin is highly phosphorylated in the testis. In 1-wk-old mouse testis, occludin was distributed diffusely at the lateral membranes of Sertoli cells, and even at this stage, highly phosphorylated occludin was detected. With development, occludin gradually became concentrated at the most basal regions of Sertoli cells. The same results were obtained in rat, but unexpectedly occludin was not detected in human or guinea pig Sertoli cells by immunofluorescence microscopy as well as by immunoblotting. Inasmuch as TJs are also well developed in Sertoli cells of these species, we concluded that, at least in the testes of these species, there are some Sertoli cell-specific isoforms of occludin or other TJ-associated integral membrane proteins that differ from occludin.

AU: Doi-Y; Kurita-M; Matsumoto-M; Kondo-T; Noda-T; Tsukita-S; Tsukita-S; Seya-T

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Japan.

SO: J-Virol. 1998 Feb; 72(2): 1586-92

ISSN: 0022-538X

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: The involvement of moesin in measles virus (MV) entry was investigated with moesin-positive and -negative mouse embryonic stem (ES) cells. MV infection of these cells was very ineffective and was independent of moesin expression. Furthermore, when these cells were transfected to express human CD46, a 100-fold increase in syncytium formation was observed with these cells and was independent of the expression of moesin. The only obvious difference between moesin-positive and -negative ES cells was the shape of the syncytia formed. Moesin-negative ES cells expressing or not expressing human CD46 formed separate pieces of fragmented syncytia which were torn apart during spreading, whereas ES cells expressing moesin exhibited typical syncytia. In addition, moesin was not detected on the surface of any murine cells or cell lines that we have tested by a flow cytometric assay with moesin-specific antibodies. These findings indicate that murine moesin is neither a receptor nor a CD46 coreceptor for MV entry into mouse ES cells. Moesin is involved in actin filament-plasma membrane interactions as a cross-linker, and it affects only the spreading and shape of MV-mediated syncytia.

Rho-kinase phosphorylates COOH-terminal threonines of 　　ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association.

AU: Matsui-T; Maeda-M; Doi-Y; Yonemura-S; Amano-M; Kaibuchi-K; Tsukita-S; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

SO: J-Cell-Biol. 1998 Feb 9; 140(3): 647-57

ISSN: 0021-9525

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

TI: Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2.

AU: Yonemura-S; Hirao-M; Doi-Y; Takahashi-N; Kondo-T; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan. yonemura@mfour.med.kyoto-u.ac.jp

SO: J-Cell-Biol. 1998 Feb 23; 140(4): 885-95

ISSN: 0021-9525

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20-30-amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.

TI: Immunological studies of a 21 kDa cellular component efficiently incorporated into rabies virion grown in a BHK-21 cell culture.

AU: Sagara-J; Tochikura-TS; Yamamoto-T; Tsukita-S; Tsukita-S; Kawai-A

AD: Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.

SO: Microbiol-Immunol. 1997; 41(12): 947-55

ISSN: 0385-5600

PY: 1997

LA: ENGLISH

CP: JAPAN

AB: To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components. Two of the MAbs we prepared recognized a virion-associated 21 kDa polypeptide (referred to as VAP21) from a BHK-21 cell. Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface. Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell. These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins. This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell. From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.

TI: Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.

AU: Sako-Y; Nagafuchi-A; Tsukita-S; Takeichi-M; Kusumi-A

AD: Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

SO: J-Cell-Biol. 1998 Mar 9; 140(5): 1227-40

ISSN: 0021-9525

PY: 1998

LA: ENGLISH

CP: UNITED-STATES

AB: The translational movement of E-cadherin, a calcium-dependent cell-cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and alpha-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm phi colloidal gold or 210-nm phi latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell-cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 micron by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 x 10(-10) and 2.1 x 10(-10) cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 micron2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 x 10(-10) cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/micron, respectively, indicating a difference in the skeletal structures that produce these two effects.

ERM (ezrin/radixin/moesin)-based molecular mechanism of microvillar breakdown at an early stage of apoptosis.

AU: Kondo-T; Takeuchi-K; Doi-Y; Yonemura-S; Nagata-S; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

SO: J-Cell-Biol. 1997 Nov 3; 139(3): 749-58

ISSN: 0021-9525

PY: 1997

LA: ENGLISH

CP: UNITED-STATES

AB: Breakdown of microvilli is a common early event in various types of apoptosis, but its molecular mechanism and implications remain unclear. ERM (ezrin/radixin/moesin) proteins are ubiquitously expressed microvillar proteins that are activated in the cytoplasm, translocate to the plasma membrane, and function as general actin filament/plasma membrane cross-linkers to form microvilli. Immunofluorescence microscopic and biochemical analyses revealed that, at the early phase of Fas ligand (FasL)-induced apoptosis in L cells expressing Fas (LHF), ERM proteins translocate from the plasma membranes of microvilli to the cytoplasm concomitant with dephosphorylation. When the FasL-induced dephosphorylation of ERM proteins was suppressed by calyculin A, a serine/threonine protein phosphatase inhibitor, the cytoplasmic translocation of ERM proteins was blocked. The interleukin-1beta-converting enzyme (ICE) protease inhibitors suppressed the dephosphorylation as well as the cytoplasmic translocation of ERM proteins. These findings indicate that during FasL-induced apoptosis, the ICE protease cascade was first activated, and then ERM proteins were dephosphorylated followed by their cytoplasmic translocation, i.e., microvillar breakdown. Next, to examine the subsequent events in microvillar breakdown, we prepared DiO-labeled single-layered plasma membranes with the cytoplasmic surface freely exposed from FasL-treated or nontreated LHF cells. On single-layered plasma membranes from nontreated cells, ERM proteins and actin filaments were densely detected, whereas those from FasL-treated cells were free from ERM proteins or actin filaments. We thus concluded that the cytoplasmic translocation of ERM proteins is responsible for the microvillar breakdown at an early phase of apoptosis and that the depletion of ERM proteins from plasma membranes results in the gross dissociation of actin-based cytoskeleton from plasma membranes. The physiological relevance of this ERM protein-based microvillar breakdown in apoptosis will be discussed.

TI: Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

AU: Mandai-K; Nakanishi-H; Satoh-A; Obaishi-H; Wada-M; Nishioka-H; Itoh-M; Mizoguchi-A; Aoki-T; Fujimoto-T; Matsuda-Y; Tsukita-S; Takai-Y

AD: Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.

SO: J-Cell-Biol. 1997 Oct 20; 139(2): 517-28

ISSN: 0021-9525

PY: 1997

LA: ENGLISH

CP: UNITED-STATES

AB: A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

TI: Occludin as a possible determinant of tight junction permeability in endothelial cells.

AU: Hirase-T; Staddon-JM; Saitou-M; Ando-Akatsuka-Y; Itoh-M; Furuse-M; Fujimoto-K; Tsukita-S; Rubin-LL

AD: Eisai London Research Laboratories Ltd, University College London, UK.

SO: J-Cell-Sci. 1997 Jul; 110 ( Pt 14): 1603-13

ISSN: 0021-9533

PY: 1997

LA: ENGLISH

CP: ENGLAND

AB: Endothelial cells provide a crucial interface between blood and tissue environments. Free diffusion of substances across endothelia is prevented by the endothelial tight junction, the permeability of which varies enormously depending on tissue. Endothelial cells of the blood-brain barrier possess tight junctions of severely limited permeability, whereas those of non-neural tissue are considerably leakier, but the molecular basis for this difference is not clear. Occludin is a major transmembrane protein localizing at the tight junction. In this study, we show, by immunocytochemistry, that occludin is present at high levels and is distributed continuously at cell-cell contacts in brain endothelial cells. In contrast, endothelial cells of non-neural tissue have a much lower expression of occludin, which is distributed in a discontinuous fashion at cell-cell contacts. The apparent differences in occludin expression levels were directly confirmed by immunoblotting. The differences in occludin protein were reflected at the message level, suggesting transcriptional regulation of expression. We also show that occludin expression is developmentally regulated, being low in rat brain endothelial cells at postnatal day 8 but clearly detectable at post-natal day 70. Our data indicate that regulation of occludin expression may be a crucial determinant of the tight junction permeability properties of endothelial cells in different tissues.

TI: Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein.

AU: Takahashi-K; Sasaki-T; Mammoto-A; Takaishi-K; Kameyama-T; Tsukita-S; Takai-Y

AD: Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan.

SO: J-Biol-Chem. 1997 Sep 12; 272(37): 23371-5

ISSN: 0021-9258

PY: 1997

LA: ENGLISH

CP: UNITED-STATES

AB: The Rho GDP dissociation inhibitor (GDI) forms a complex with the GDP-bound form of the Rho family small G proteins and inhibits their activation. The GDP-bound form complexed with Rho GDI is not activated by the GDP/GTP exchange factor for the Rho family members, suggesting the presence of another factor necessary for this activation. We have reported that the Rho subfamily members regulate the ezrin/radixin/moesin (ERM)-CD44 system, implicated in reorganization of actin filaments. Here we report that Rho GDI directly interacts with ERM, initiating the activation of the Rho subfamily members by reducing the Rho GDI activity. These results suggest that ERM as well as Rho GDI and the Rho GDP/GTP exchange factor are involved in the activation of the Rho subfamily members, which then regulate reorganization of actin filaments through the ERM system.

TI: Mammalian occludin in epithelial cells: its expression and subcellular distribution.

AU: Saitou-M; Ando-Akatsuka-Y; Itoh-M; Furuse-M; Inazawa-J; Fujimoto-K; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan.

SO: Eur-J-Cell-Biol. 1997 Jul; 73(3): 222-31

ISSN: 0171-9335

PY: 1997

LA: ENGLISH

CP: GERMANY

AB: Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions, and the cDNA encoding its mammalian homologue was identified very recently (Ando-Akatsuka, Y., M. Saitou, T. Hirase, M. Kishi, A. Sakakibara, M. Itoh, S. Yonemura, M. Furuse, Sh. Tsukita, J. Cell Biol. 133, 43-47 (1996)). Here we describe the basic properties of mammalian occludin in epithelial cells at the DNA, RNA, and protein levels. The human occludin gene was mapped to chromosome band 5q13.1 by fluorescent in situ hybridization. Northern blotting identified several occludin mRNA bands, suggesting the possible expression of several alternatively spliced products. Occludin mRNA was detected in cultured epithelial cells, but not in cultured fibroblasts. The mRNA level was high in the testis, kidney, liver, lung, and brain, which reportedly bear well-developed tight junctions. We then produced monoclonal and polyclonal antibodies using recombinant mouse occludin as the antigen, which reacted not only with mouse, but also human, dog and pig occludin. These antibodies recognized several bands around 60 kDa in epithelial cells but not in fibroblasts. Immunofluorescence microscopy of various tissues revealed that the staining intensity of occludin correlated well with the number of tight junction strands in epithelial cells. By contrast, the staining of ZO-1, a well-characterized tight junction-associated protein, was not specific for tight junctions. Furthermore, the exclusive concentration of occludin at tight junctions in epithelial cells was confirmed by immunoreplica electron microscopy.

TI: Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments.

AU: Itoh-M; Nagafuchi-A; Moroi-S; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.

SO: J-Cell-Biol. 1997 Jul 14; 138(1): 181-92

ISSN: 0021-9525

PY: 1997

LA: ENGLISH

CP: UNITED-STATES

AB: ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell-cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/alpha, beta catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with alpha catenin, but not to that with beta catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and alpha catenin was approximately 0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of approximately 10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with alpha catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.

TI: Expression of occludin, tight-junction-associated protein, in human digestive tract.

AU: Kimura-Y; Shiozaki-H; Hirao-M; Maeno-Y; Doki-Y; Inoue-M; Monden-T; Ando-Akatsuka-Y; Furuse-M; Tsukita-S; Monden-M

AD: Department of Surgery II, Osaka University Medical School, Japan.

SO: Am-J-Pathol. 1997 Jul; 151(1): 45-54

ISSN: 0002-9440

PY: 1997

LA: ENGLISH

CP: UNITED-STATES

AB: The tight junction seals cells together at a subapical location and functionally separates the plasma membrane into an apical and a basolateral domain. This junction is one of the most characteristic structural markers of the polarized epithelial cell. Recently, occludin has been identified as an integral transmembrane protein localizing at the tight junction and directly associated with ZO-1, an undercoat-constitutive cytoplasmic protein. We have investigated occludin expression in conjunction with ZO-1 in normal epithelia and cancers of human digestive tract by immunostaining with a new antibody raised against human occludin. In the normal simple columnar epithelium, occludin was expressed together with ZO-1 as a single line at the apical cell border. However, in the esophagus, which has a stratified squamous epithelium, no occludin expression could be detected, but ZO-1 was expressed in the spinous layer. As for cancers, both occludin and ZO-1 showed the same expression in differentiated adenocarcinoma cells as in normal epithelium, but in poorly differentiated adenocarcinomas, the expression of these two proteins was reduced. There was significant correlation between tumor differentiation and expression of these proteins. These results suggest that occludin, together with ZO-1, is involved in the formation of gland-like structures. In addition, occludin expression can serve as a histopathological indicator for differentiation in gastrointestinal adenocarcinomas.

TI: Possible involvement of phosphorylation of occludin in tight junction formation.

AU: Sakakibara-A; Furuse-M; Saitou-M; Ando-Akatsuka-Y; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan.

SO: J-Cell-Biol. 1997 Jun 16; 137(6): 1393-401

ISSN: 0021-9525

PY: 1997

LA: ENGLISH

CP: UNITED-STATES

AB: Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest Mr were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher Mr bands were selectively recovered in the NP-40-insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40-soluble and -insoluble fractions into the lowest Mr band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher Mr bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40-insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti-chicken occludin mAb, Oc-3, did not recognize the predominant lower Mr bands of occludin (non- or less phosphorylated form) but was specific to the higher Mr bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower Mr bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40-insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.

TI: A 155-kDa undercoat-constitutive protein of cell-to-cell adherens junctions.

AU: Hirase-T; Furuse-M; Tsukita-S

AD: Department of Cell Biology, Kyoto University Faculty of Medicine, Kyoto/Japan.

SO: Eur-J-Cell-Biol. 1997 Feb; 72(2): 174-81

ISSN: 0171-9335

PY: 1997

LA: ENGLISH

CP: GERMANY

AB: A fraction enriched in cell-to-cell adherens and tight junctions was isolated from the chick liver, and the undercoat-constitutive proteins were extracted from this isolated junctional fraction. Monoclonal antibodies (mAbs) were then obtained by injecting this extract into rats, and five antigens were identified to be concentrated in the isolated junctional fraction. We have characterized one mAb (E14 mAb) and its antigen (E14). By immunoblotting of the isolated junctional fraction the E14 mAb reacted strongly with a single band of approximately 155 kDa, and E14 was highly concentrated in the isolated junctional fraction. Immunofluorescence microscopy revealed that the E14 mAb exclusively stained the junctional complex region of the liver, renal epithelial cells, and the cell-cell border of endothelial cells in various tissues. The intercalated disc of the heart was also significantly stained. However, the E14 signal was hardly detected from intestinal epithelial cells. By immunoelectron microscopy using renal epithelial cells, E14 was mainly detected in the fibrous structures associated with the cell-to-cell adherens junction. We conclude that E14 is a novel undercoat-constitutive protein found in certain types of cell-to-cell adherens junctions.

TI: Dynamics of connexins, E-cadherin and alpha-catenin on cell membranes during gap junction formation.

AU: Fujimoto-K; Nagafuchi-A; Tsukita-S; Kuraoka-A; Ohokuma-A; Shibata-Y

AD: Department of Anatomy, Faculty of Medicine, Kyoto University, Japan. fujimoto@med.kyoto-u.ac.jp

SO: J-Cell-Sci. 1997 Feb; 110 ( Pt 3): 311-22

ISSN: 0021-9533

PY: 1997

LA: ENGLISH

CP: ENGLAND

AB: We examined the dynamics of connexins, E-cadherin and alpha-catenin during gap-junction disassembly and assembly in regeneration hepatocytes by immunofluorescence microscopy, and immunogold-electron microscopy using SDS-digested freeze-replicas. The present findings suggest that during the disappearance of gap junctions most of the gap junction plaques are broken up into smaller aggregates, and then the gap junction proteins may be removed from the cell membrane, but some of the connexons or connexins remain dispersed in the plane of membrane as pure morphologically indistinguishable intramembrane proteins. Double-immunogold electron microscopy using a polyclonal antibody for connexins and a monoclonal antibody for E-cadherin or alpha-catenin revealed co-localization of these molecules at cell-to-cell contact sites during the reappearance of gap junction plaques. This implies that, at least in regenerating hepatocytes, the cadherin-catenin complex-mediated cell-to-cell contact sites act as foci for gap junction formation. In addition, connexin-immunoreactivity was also observed along tight junctional strands, suggesting that the gap junction may also form along the tight junctions.

TI: ERM proteins: head-to-tail regulation of actin-plasma membrane interaction.

AU: Tsukita-S; Yonemura-S; Tsukita-S

AD: College of Medical Technology, Kyoto University, Japan. atsukita@mfour.med.kyoto-u.ac.jp

SO: Trends-Biochem-Sci. 1997 Feb; 22(2): 53-8

ISSN: 0167-7640

PY: 1997

LA: ENGLISH

CP: ENGLAND

AB: ERM (ezrin/radixin/moesin) proteins crosslink actin filaments with plasma membranes. The carboxyl termini of these proteins bind actin filaments, while the amino termini bind plasma membranes using a binding partner, such as CD44. Specific signals activate ERM proteins to bind actin filaments and the plasma membrane; these include phosphoinositides and/or phosphorylation mechanisms, which might be located downstream from the Rho-dependent pathway.

TI: ERM (ezrin/radixin/moesin) family: from cytoskeleton to signal transduction.

AU: Tsukita-S; Yonemura-S; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Yoshida-Konoe, Sakyo-ku, Kyoto 606, Japan.

SO: Curr-Opin-Cell-Biol. 1997 Feb; 9(1): 70-5

ISSN: 0955-0674

PY: 1997

LA: ENGLISH

CP: UNITED-STATES

AB: The ERM family consists of three closely related proteins, ezrin, radixin, and moesin, that are thought to work as cross-linkers between plasma membranes and actin-based cytoskeletons. Recent analyses of the structure and functions of ERM proteins have revealed that these molecules are involved not only in cytoskeletal organization but also in signal transduction. Furthermore, identification of the neurofibromatosis type 2 tumour suppressor, which shows striking sequence similarity to ERM proteins, has increased interest in this family.

TI: Occludin is a functional component of the tight junction.

AU: McCarthy-KM; Skare-IB; Stankewich-MC; Furuse-M; Tsukita-S; Rogers-RA; Lynch-RD; Schneeberger-EE

AD: Department of Pathology, Massachusetts General Hospital, Boston 02114, USA.

SO: J-Cell-Sci. 1996 Sep; 109 ( Pt 9): 2287-98

ISSN: 0021-9533

PY: 1996

LA: ENGLISH

CP: ENGLAND
AB: Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

TI: Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures.

AU: Furuse-M; Fujimoto-K; Sato-N; Hirase-T; Tsukita-S; Tsukita-S

AD: Department of Cell Biology, Kyoto University, Japan.

SO: J-Cell-Sci. 1996 Feb; 109 ( Pt 2): 429-35

ISSN: 0021-9533

PY: 1996

LA: ENGLISH

CP: ENGLAND

AB: Occludin is an integral membrane protein localizing at tight junctions with four transmembrane domains. When chicken occludin was overexpressed in insect cells by recombinant baculovirus infection, peculiar multilamellar structures accumulated in the cytoplasm. Partial isolation of these structures indicated that the introduced chicken occludin was highly enriched in these structures. Thin section electron microscopy revealed that each lamella was transformed from intracellular membranous cisternae whose luminal space was completely collapsed, and that in each lamella, outer leaflets of opposing membranes appeared to be fused with no gaps, like tight junctions. Furthermore, in the freeze-fracture replicas of these multilamellar structures, short tight junction-like intramembranous particle strands were occasionally observed, which were specifically labeled by anti-occludin mAb. These observations favor the idea that occludin plays a key role in the formation of tight junctions.

TI: Regulation mechanism of ERM (ezrin/radixin/moesin) protein/plasma membrane association: possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway.

AU: Hirao-M; Sato-N; Kondo-T; Yonemura-S; Monden-M; Sasaki-T; Takai-Y; Tsukita-S; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan.

SO: J-Cell-Biol. 1996 Oct; 135(1): 37-51

ISSN: 0021-9525

PY: 1996

LA: ENGLISH

CP: UNITED-STATES

AB: The ERM proteins, ezrin, radixin, and moesin, are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3 +/- 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4.5-bisphosphate [4,5-PIP2]), ERM proteins bound with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength: 4,5-PIP2 showed a marked effect (Kd of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP gamma S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism.

TI: Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues.

AU: Ando-Akatsuka-Y; Saitou-M; Hirase-T; Kishi-M; Sakakibara-A; Itoh-M; Yonemura-S; Furuse-M; Tsukita-S

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan.

SO: J-Cell-Biol. 1996 Apr; 133(1): 43-7

ISSN: 0021-9525

PY: 1996

LA: ENGLISH

CP: UNITED-STATES

AB: Occludin has been identified from chick liver as a novel integral membrane protein localizing at tight junctions (Furuse, M., T. Hirase, M. Itoh, A. Nagafuchi, S. Yonemura, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 123:1777-1788). To analyze and modulate the functions of tight junctions, it would be advantageous to know the mammalian homologues of occludin and their genes. Here we describe the nucleotide sequences of full length cDNAs encoding occludin of rat-kangaroo (potoroo), human, mouse, and dog. Rat-kangaroo occludin cDNA was prepared from RNA isolated from PtK2 cell culture, using a mAb against chicken occludin, whereas the others were amplified by polymerase chain reaction based on the sequence found around the human neuronal apoptosis inhibitory protein gene. The amino acid sequences of the three mammalian (human, murine, and canine) occludins were very closely related to each other (approximately 90% identity), whereas they diverged considerably from those of chicken and rat-kangaroo (approximately 50% identity). Implications of these data and novel experimental options in cell biological research are discussed.

TI: Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle.

AU: Tokai-N; Fujimoto-Nishiyama-A; Toyoshima-Y; Yonemura-S; Tsukita-S; Inoue-J; Yamamota-T

AD: The Institute of Medical Science, The University of Tokyo, Japan.

SO: EMBO-J. 1996 Feb 1; 15(3): 457-67

ISSN: 0261-4189

PY: 1996

LA: ENGLISH

CP: ENGLAND

AB: Microtubule-associated motor proteins are thought to be involved in spindle formation and chromosome movements in mitosis/meiosis. We have molecularly cloned cDNAs for a gene that codes for a novel member of the kinesin family of proteins. Nucleotide sequencing reveals that the predicted gene product is a 73 kDa protein and is related to some extent to the Drosophila node gene product, which is involved in chromosomal segregation during meiosis. A sequence similar to the microtubule binding motor domain of kinesin is present in the N-terminal half of the protein, and its ability to bind to microtubules is demonstrated. Furthermore we show that its C-terminal half contains a putative nuclear localization signal similar to that of Jun and is able to bind to DNA. Accordingly, the protein was termed Kid (kinesin-like DNA binding protein). Indirect immunofluorescence studies show that Kid colocalizes with mitotic chromosomes and that it is enriched in the kinetochore at anaphase. Thus, we propose that Kid might play a role(s) in regulating the chromosomal movement along microtubules during mitosis.

TI: Molecular dissection of tight junctions.

AU: Tsukita-S; Furuse-M; Itoh-M

AD: Department of Cell Biology, Faculty of Medicine, Kyoto University, Japan.

SO: Cell-Struct-Funct. 1996 Oct; 21(5): 381-5

ISSN: 0386-7196

PY: 1996

LA: ENGLISH

CP: JAPAN

AB: In epithelial and endothelial cells, the tight junction (TJ) seals cells to create a primary barrier to the diffusion of solutes across the cell sheet, and it also works as a boundary between the apical and basolateral membrane domains to create their polarization. An integral membrane protein working at TJ is postulated to exist, but it has remained elusive for quite some time. Most recently, using mAbs, we identified an integral membrane protein named occludin that was exclusively localized at TJ both in epithelial and endothelial cells. Here we overview our recent studies on the structure and function of occludin.