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S Tsukita

Publications and source records attributed to S Tsukita.

At least 19 recordsLinked to original sources
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

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

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

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↗
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↗
PubMed2000

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

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.

Amino Acid Sequence↗
PubMed2000

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

Adenomatous polyposis coli protein (APC) is a well-characterized tumor suppressor protein [1] [2] [3]. 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 [4]. EB1, which was identified as an APC-binding protein by yeast two-hybrid analysis [5], was also reported to be associated with microtubules [6] [7] [8]. 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.

Adenomatous Polyposis Coli Protein↗
PubMed2000

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

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.

Animals↗
PubMed2000

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

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.

Abdomen↗
PubMed2000

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

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.

Adenomatous Polyposis Coli Protein↗
PubMed2000

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

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.

Animals↗