Biomedical subjects

S Tsukita

Publications and source records attributed to S Tsukita.

At least 19 recordsLinked to original sources

PubMed2005

Epithelial transport and barrier function in occludin-deficient mice.

BACKGROUND AND AIMS: This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model. METHODS: Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. RESULTS: In occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic occludin+/- or homozygotic occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin-/- mice. CONCLUSION: Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.

Animals↗

PubMed2004

Tight junction-associated proteins (occludin, ZO-1, claudin-1, claudin-4) in squamous cell carcinoma and Bowen's disease.

BACKGROUND: The epidermis, which is a typical stratified epithelium, has tight junctions (TJs) in the granular layer, as do simple epithelia. So far, abnormalities of TJs and involvement of claudin-1 have been reported in tumours of simple epithelia. OBJECTIVES: To examine the expression of TJ-associated proteins (occludin, ZO-1, claudin-1 and claudin-4) in normal human epidermis and in malignant disorders of keratinization. METHODS: Expression of the proteins in normal human epidermis, five cases of squamous cell carcinoma (SCC) of the skin and five cases of Bowen's disease (BD) was examined by immunofluorescence staining. RESULTS: In normal human epidermis, occludin, ZO-1 and claudin-4 were expressed at the cell-cell borders in the granular layer specifically or dominantly, whereas claudin-1 was expressed in the whole epithelium. In SCC, occludin, ZO-1, claudin-1 and claudin-4 were strongly expressed in tumour cells with keratinization such as cancer pearls. Claudin-1 was heterogeneously expressed in unkeratinized tumour cells, whereas expression of occludin, ZO-1 and claudin-4 was decreased or absent. In BD, aberrant expression of occludin, ZO-1, claudin-1 and claudin-4 was observed at the cell-cell borders in addition to their expression patterns observed in normal epidermis. CONCLUSIONS: Expression of occludin, ZO-1 and claudin-4 is associated with keratinization in SCC and BD. However, the heterogeneous expression of claudin-1 in SCC is not determined only by keratinization.

Aged↗

PubMed2001

Where is APC going?

Adenomatous polyposis coli (APC) protein has been thought to function as a tumor suppressor through its involvement in the Wnt/beta-catenin signaling pathway. However, its connections to the cytoskeleton and microtubules in particular are becoming apparent, and the discovery of these new functions for APC is leading to a reevaluation of its role not only in tumorigenesis, but also in normal physiology.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Junctional adhesion molecule (JAM) binds to PAR-3: a possible mechanism for the recruitment of PAR-3 to tight junctions.

At tight junctions (TJs), claudins with four transmembrane domains are incorporated into TJ strands. Junctional adhesion molecule (JAM), which belongs to the immunoglobulin superfamily, is also localized at TJs, but it remains unclear how JAM is integrated into TJs. Immunoreplica electron microscopy revealed that JAM showed an intimate spatial relationship with TJ strands in epithelial cells. In L fibroblasts expressing exogenous JAM, JAM was concentrated at cell-cell adhesion sites, where there were no strand-like structures, but rather characteristic membrane domains free of intramembranous particles were detected. These domains were specifically labeled with anti-JAM polyclonal antibody, suggesting that JAM forms planar aggregates through their lateral self-association. Immunofluorescence microscopy and in vitro binding assays revealed that ZO-1 directly binds to the COOH termini of claudins and JAM at its PDZ1 and PDZ3 domains, respectively. Furthermore, another PDZ-containing polarity-related protein, PAR-3, was directly bound to the COOH terminus of JAM, but not to that of claudins. These findings led to a molecular architectural model for TJs: small aggregates of JAM are tethered to claudin-based strands through ZO-1, and these JAM aggregates recruit PAR-3 to TJs. We also discuss the importance of this model from the perspective of the general molecular mechanisms behind the recruitment of PAR proteins to plasma membranes.

Animals↗

PubMed2001

Structural conversion between open and closed forms of radixin: low-angle shadowing electron microscopy.

The function of ERM (ezrin/radixin/moesin) proteins as general cross-linkers between actin filaments and plasma membranes is regulated downstream of Rho, through the transition between active and inactive forms. To directly examine the conformational change between the active and inactive forms of ERM proteins, we applied low-angle rotary-shadowing electron microscopy to the radixin molecules, wild-type, T564A-non-phosphorylated-type, and T564E-phosphorylated-type, since most of the active forms are reportedly stabilized in cells by the C-terminal threonine phosphorylation. As a result, the T564A- and wild-type radixin molecules yielded the globular closed forms, approximately 8-14 nm in diameter, with some striations on their surfaces. In contrast, the T564E-radixin molecules tended to take elongated open forms, in which two globular structures measuring approximately 8 nm and approximately 5 nm in diameter were associated with both ends of the filamentous structures. The filamentous structure took either a approximately 20-25 nm-long straight course or a folded course. Taken together with the biochemical and the crystal structural results obtained to date, the closed and open forms represent the inactive and active forms of radixin as cross-linkers between actin filaments and plasma membranes.

Amino Acid Substitution↗

PubMed2001

An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation.

We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.

Animals↗

PubMed2001

Crystallization and preliminary crystallographic studies of RhoGDI in complex with the radixin FERM domain.

The Rho guanine nucleotide-dissociation inhibitor (RhoGDI) is a general regulator that forms a complex with the GDP-bound form of Rho-family GTPases and suppresses their activation. The FERM domains of ERM (ezrin/radixin/moesin) proteins bind to RhoGDI and dissociate Rho from RhoGDI. The formation of a complex between RhoGDI and the FERM domain is an important step in the regulatory cycle of Rho activation. In this study, crystals of RhoGDI complexed with the FERM domain of radixin were obtained. The crystals of the binary complex belong to the space group P2(1)2(1)2, with unit-cell parameters a = 130.9 (2), b = 151.2 (2), c = 71.2 (1) A, and contain two protein complexes in the crystallographic asymmetric unit. A 2.9 A resolution data set was collected using synchrotron radiation at SPring-8.

Animals↗

PubMed2001

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of the adhesion protein ICAM-2.

Radixin is a member of the ERM proteins, which cross-link plasma membranes and actin filaments. The FERM domains located at the N-terminal regions of ERM proteins are responsible for membrane association through direct interactions with the cytoplasmic domains of integral membrane proteins. Here, crystals of the complex between the radixin FERM domain and the full-length cytoplasmic tail (28-residue peptide) of intercellular adhesion molecule 2, ICAM-2, have been obtained. The crystals were found to belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 100.44 (9), c = 99.49 (6) A, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.60 A.

Animals↗

PubMed2001

Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells.

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.

Amino Acid Sequence↗

PubMed2001

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

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.

Actins↗

PubMed2001

Dynamic changes in protein components of the tight junction during liver regeneration.

The construction of the hepatocyte tight junction is one of the most important events during liver regeneration leading to the reorganization of the bile canaliculi and the repolarization of hepatocytes after cell division. To understand this event at the molecular level, we examined the expression of tight junction proteins by Western blot analysis and their cellular localization by immunofluorescence microscopy in regenerating rat liver after two-thirds hepatectomy. The levels of tight junction components such as claudin-3, ZO-1 and atypical protein kinase C (PKC)-specific interacting protein (ASIP) increased two- to three-fold over control levels in coordination with a peak 2-3 days after partial hepatectomy, whereas occludin levels remained unchanged. The bile canaliculi outlined by tight junction components and actin filaments reveal significant morphological changes from 2-3 days after partial hepatectomy. During this period, claudin-3/ZO-1 and ASIP/ZO-1 were nearly co-localized, whereas occludin was locally reduced or almost absent on the bile canaliculi outlined by ZO-1 staining. The uncoupled localization of F-actin and tight junction components was often observed. The function of hepatocytes, as revealed by the serum bile acids level, was distorted temporally at an early stage of regeneration but mostly restored 3 days after partial hepatectomy. These observations suggest that the de novo construction of tight junctions proceeds mainly 2-3 days after partial hepatectomy in parallel with the cell polarization required for hepatocyte function. However, the complete normalization of the composition of the tight junction components, such as occludin and the association with F-actin, requires additional time, which may support the regeneration of fully polarized normal hepatocytes.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Multifunctional strands in tight junctions.

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.

Animals↗

PubMed2001

Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin.

BACKGROUND & AIMS: Recently, several members of the claudin family have been identified as integral constituents of tight junctions. Using expression profiling, we previously found claudin-4 to be overexpressed in pancreatic cancer. Because claudin-4 has been described as a receptor for the cytotoxic Clostridium perfringens enterotoxin (CPE), we investigated the effect of CPE on pancreatic cancer cells. METHODS: Expression of claudin-4 was analyzed by Northern blots. In vitro toxicity of CPE was determined by trypan blue exclusion and the (86)Rb-release assay. The in vivo effect of CPE was studied in claudin-4-expressing nude mouse xenografts of the Panc-1 cell line. RESULTS: Expression analyses showed that claudin-4 was overexpressed in most pancreatic cancer tissues and cell lines and several other gastrointestinal tumors. CPE led to an acute dose-dependent cytotoxic effect, restricted to claudin-4-expressing cells and dependent on claudin-4 expression levels. Furthermore, transforming growth factor beta was identified as a negative modulator of both claudin-4 expression and susceptibility to CPE. In vivo, intratumoral injections of CPE in Panc-1 xenografts led to large areas of tumor cell necrosis and significant reduction of tumor growth. CONCLUSIONS: Our findings suggest that targeting claudin-4-expressing tumors with CPE represents a promising new treatment modality for pancreatic cancer and other solid tumors.

Adenocarcinoma↗

PubMed2001

Outer dense fiber 2 is a widespread centrosome scaffold component preferentially associated with mother centrioles: its identification from isolated centrosomes.

Because centrosomes were enriched in the bile canaliculi fraction from the chicken liver through their association with apical membranes, we developed a procedure for isolation of centrosomes from this fraction. With the use of the centrosomes, we generated centrosome-specific monoclonal antibodies. Three of the monoclonal antibodies recognized an antigen of ~90 kDa. Cloning of its cDNA identified this antigen as a chicken homologue of outer dense fiber 2 protein (Odf2), which was initially identified as a sperm outer dense fiber-specific component. Exogenously expressed and endogenous Odf2 were shown to be concentrated at the centrosomes in a microtubule-independent manner in various types of cells at both light and electron microscopic levels. Odf2 exhibited a cell cycle-dependent pattern of localization and was preferentially associated with the mother centrioles in G0/G1-phase. Toward G1/S-phase before centrosome duplication, it became detectable in both mother and daughter centrioles. In the isolated bile canaliculi and centrosomes, Odf2, in contrast to other centrosomal components, was highly resistant to KI extraction. These findings indicate that Odf2 is a widespread KI-insoluble scaffold component of the centrosome matrix, which may be involved in the maturation event of daughter centrioles.

Amino Acid Sequence↗

PubMed2001

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

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

Animals↗

PubMed2001

Involvement of claudin-1 in the beta-catenin/Tcf signaling pathway and its frequent upregulation in human colorectal cancers.

Accumulation of beta-catenin in cytoplasm and nuclei is frequently observed in a wide variety of tumors arising, for example, in the colon, liver, uterus, or brain. In association with Tcf/LEF transcription factors, beta-catenin regulates expression of genes involved in the Wnt/wingless signaling pathway, but the precise mechanisms are unclear. Here we report evidence that the claudin-1 (CLDNI) gene is one of the genes regulated by beta-catenin. Not only did expression of CLDN1 decrease significantly in response to reduction of intracellular beta-catenin by adenovirus-mediated transfer of wild-type APC into the APC-deficient colon cancer cells, but also two putative Tcf4 binding elements in the 5' flanking region of CLDN1 were confirmed to be responsible for activating its transcription. We documented increased expression of CLDN1 in all 16 primary colorectal cancers we examined, compared with adjacent noncancerous mucosae. Furthermore, immunohistochemical staining demonstrated that claudin-1 was weakly stained at apical boarder of lateral membrane of noncancerous epithelial cells and that it was strongly stained at all cell-cell boundaries and in the cytoplasms of cancer cells. Our results imply that claudin-1 is involved in the beta-catenin-Tcf/LEF signaling pathway, and that increased expression of claudin-1 may have some role in colorectal tumorigenesis.

Claudin-1↗

PubMed2000

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

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.

5' Untranslated Regions↗

PubMed2000

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

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.

Animals↗