445 results on '"S, Tsukita"'
Search Results
352. V-src kinase shifts the cadherin-based cell adhesion from the strong to the weak state and beta catenin is not required for the shift.
- Author
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Takeda H, Nagafuchi A, Yonemura S, Tsukita S, Behrens J, Birchmeier W, and Tsukita S
- Subjects
- Animals, Cell Aggregation physiology, Cell Line enzymology, Dogs, Kidney Tubules, Distal cytology, Mice, Phosphorylation, Precipitin Tests, Recombinant Fusion Proteins metabolism, Temperature, Tyrosine metabolism, beta Catenin, Cadherins metabolism, Cell Adhesion physiology, Cytoskeletal Proteins metabolism, Oncogene Protein pp60(v-src) metabolism, Trans-Activators
- Abstract
The elevation of tyrosine phosphorylation level is thought to induce the dysfunction of cadherin through the tyrosine phosphorylation of beta catenin. We evaluated this assumption using two cell lines. First, using temperature-sensitive v-src-transfected MDCK cells, we analyzed the modulation of cadherin-based cell adhesion by tyrosine phosphorylation. Cell aggregation and dissociation assays at nonpermissive and permissive temperatures indicated that elevation of the tyrosine phosphorylation does not totally affect the cell adhesion ability of cadherin but shifts it from a strong to a weak state. The tyrosine phosphorylation levels of beta catenin, ZO-1, ERM (ezrin/radixin/moesin), but not alpha catenin, vinculin, and alpha-actinin, were elevated in the weak state. To evaluate the involvement of the tyrosine phosphorylation of beta catenin in this shift of cadherin-based cell adhesion, we introduced v-src kinase into L fibroblasts expressing the cadherin-alpha catenin fusion protein, in which beta catenin is not involved in cell adhesion. The introduction of v-src kinase in these cells shifted their adhesion from a strong to a weak state. These findings indicated that the tyrosine phosphorylation of beta catenin is not required for the strong-to-weak state shift of cadherin-based cell adhesion, but that the tyrosine phosphorylation of other junctional proteins, ERM, ZO-1 or unidentified proteins is involved.
- Published
- 1995
- Full Text
- View/download PDF
353. Effects of tyrosine phosphorylation on tight junctions in temperature-sensitive v-src-transfected MDCK cells.
- Author
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Takeda H and Tsukita S
- Subjects
- Animals, Cell Line cytology, Cell Line ultrastructure, Dogs, Epithelial Cells, Epithelium metabolism, Epithelium ultrastructure, Microscopy, Electron, Phosphorylation, Temperature, Transfection physiology, Kidney Tubules, Distal cytology, Oncogene Protein pp60(v-src) genetics, Tight Junctions metabolism, Tyrosine metabolism, Tyrosine pharmacology
- Abstract
We analyzed the direct effects of tyrosine phosphorylation on the structure and functions of tight junctions in temperature-sensitive v-src-transfected MDCK cells. When ts-v-src MDCK cells were plated at a low density at the nonpermissive temperature, they formed small, tight colonies with a typical epithelial appearance. When these colonies were cultured at the permissive temperature, cadherin-based cell adhesion was suppressed, so that individual cells scattered and assumed a fibroblastic appearance. This resulted in the destruction of tight junctions, making it difficult to analyze the direct effects of tyrosine phosphorylation on tight junctions. To suppress cell scattering, we cultured ts-v-src MDCK cells at confluence. Under these conditions, even at the permissive temperature, the cells assumed an epithelial appearance, and the structure of tight junctions were mostly maintained both at the immunofluorescence and electron microscopy levels. The transepithelial electrical resistance (TER) dropped to about 70% of the initial value after the temperature shift from the nonpermissive to the permissive. This temperature shift facilitated the tyrosine phosphorylation of the two tight junction proteins, ZO-1 and ZO-2. We concluded that the direct effects of tyrosine phosphorylation on tight junctions are not so remarkable as those on adherens junctions.
- Published
- 1995
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354. Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows.
- Author
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Takaishi K, Sasaki T, Kameyama T, Tsukita S, Tsukita S, and Takai Y
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- 3T3 Cells, Animals, Biological Transport, Cell Adhesion, Cell Line, Cell Membrane drug effects, Cell Membrane metabolism, Complement C3 pharmacology, Cytoplasm drug effects, Cytoplasm metabolism, Dogs, Hepatocyte Growth Factor pharmacology, Mice, Microinjections, Proto-Oncogene Proteins c-myc metabolism, Tetradecanoylphorbol Acetate pharmacology, rhoA GTP-Binding Protein, GTP-Binding Proteins metabolism
- Abstract
Rho small GTP-binding protein regulates various cell functions, such as formation of stress fibers and focal adhesions, cell motility, membrane ruffling, cytokinesis and smooth muscle contraction in mammalian cells and bud formation in the yeast Saccharomyces cerevisiae. As to the functioning sites of Rho in Saccharomyces cerevisiae, we have recently shown that RHO1 protein, a homologue of mammalian RhoA, is concentrated to the growth region of the cells where cortical actin patches are clustered. However, in mammalian cells, the functioning sites of Rho have not yet been studied. In the present study, MDCK cell lines stably expressing myc-tagged RhoA (myc-RhoA) were prepared and localization of myc-RhoA was first immunohistochemically examined using an anti-myc antibody. In the resting cells, almost all of myc-RhoA was observed in the cytosol. When the cells were stimulated with phorbol ester or hepatocyte growth factor, membrane rufflings were induced and myc-RhoA was translocated to the membrane ruffling area. Moreover, myc-RhoA was translocated from the cytosol to the cell-cell adhesion sites when the cells were transferred from a low to normal Ca2+ medium. RhoA was also concentrated to the cleavage furrows during cytokinesis in Swiss 3T3 cells. Translocation of myc-RhoA to the membrane ruffling area was inhibited by prior microinjection into the cells of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, or of C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, indicating that both activation and functioning of Rho are essential for the translocation of Rho. The ERM (Ezrin, Radixin, Moesin) family members were colocalized with RhoA at all of these sites. However, RhoA was not apparently observed at the focal adhesion plaque where vinculin was localized. These results suggest that at least one of the functioning sites of Rho is the ERM family-controlled actin filament/plasma membrane association sites.
- Published
- 1995
355. [Molecular biology of intercellular junctions].
- Author
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Tsukita S, Tsukita S, Nagafuchi A, and Yonemura S
- Subjects
- Animals, Blood Proteins isolation & purification, Blood Proteins physiology, Cadherins isolation & purification, Cadherins physiology, Humans, Intercellular Junctions physiology, Membrane Proteins isolation & purification, Membrane Proteins physiology, Phosphoproteins isolation & purification, Phosphoproteins physiology, Zonula Occludens-1 Protein, Cell Adhesion, Cytoskeletal Proteins, Intercellular Junctions chemistry
- Published
- 1995
356. Cellular actin-binding ezrin-radixin-moesin (ERM) family proteins are incorporated into the rabies virion and closely associated with viral envelope proteins in the cell.
- Author
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Sagara J, Tsukita S, Yonemura S, Tsukita S, and Kawai A
- Subjects
- Animals, Antigens immunology, Blood Proteins immunology, Blood Proteins metabolism, Cell Line, Cricetinae, Fluorescent Antibody Technique, Membrane Proteins immunology, Membrane Proteins metabolism, Peptides analysis, Phosphoproteins immunology, Phosphoproteins metabolism, Protein Binding, Proteins immunology, Proteins metabolism, Virion chemistry, Actins metabolism, Cytoskeletal Proteins, Microfilament Proteins metabolism, Rabies virus metabolism, Viral Envelope Proteins metabolism, Virion metabolism
- Abstract
Cellular ezrin-radixin-moesin (ERM) family proteins, members of the actin-binding proteins of the band 4.1 superfamily, were detected in the virions of enveloped viruses, such as rabies, vesicular stomatitis, Newcastle disease, and influenza viruses. To elucidate the mechanism of ERM protein incorporation, we investigated possible association of ERM proteins with viral components in rabies virus-infected BHK-21 cells. Double immunofluorescence studies demonstrated that the ERM proteins are concentrated in the microvilli, where the colocalized viral G protein was also seen. Viral G protein expressed in the G cDNA-transfected COS-7 cells also displayed similar distributions to those seen in the virus-infected cells. Both the ERM and viral envelope proteins were coprecipitated by anti-viral G antibody from lysates of the virus-infected cells, while the anti-ERM antibody coprecipitated viral G and ERM proteins. These observations suggest that the ERM proteins are closely associated with viral envelope proteins in the cell, which would be involved in the selective incorporation of cellular actin into the virion.
- Published
- 1995
- Full Text
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357. Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells.
- Author
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Yonemura S, Itoh M, Nagafuchi A, and Tsukita S
- Subjects
- 3T3 Cells, Animals, Chlorocebus aethiops, Epithelium physiology, Epithelium ultrastructure, Fibroblasts physiology, Fibroblasts ultrastructure, Fluorescent Antibody Technique, Kidney, Mice, Microscopy, Confocal, Microscopy, Electron, Microscopy, Immunoelectron, Microscopy, Phase-Contrast, Rats, Wound Healing, Actins physiology, Actins ultrastructure, Cell Communication, Intercellular Junctions physiology, Intercellular Junctions ultrastructure
- Abstract
Cadherin has an intimate spatial relationship with actin filaments (AF) in various types of cells, forming the cell-to-cell adherens junction (AJ). We compared the AJ/AF relationship between non-polarized fibroblasts (NRK cells) and polarized epithelial cells (MTD-1A cells). E/P-cadherin, alpha-catenin, ZO-1 and vinculin were localized with reference to AF in these cells using laser scan microscopy as well as conventional light and electron microscopy. NRK cells adhered to each other at the tips of thin cellular processes, where spot-like AJ were formed, where P-cadherin, alpha-catenin, ZO-1 and vinculin were concentrated. Some stress-fiber-like AF bundles ran axially in these processes and terminated at spot-like AJ on their tips. At the electron microscopic level these spot-like AJ were seen as aggregates of small 'units' of AJ, where AF were densely and perpendicularly associated with the plasma membrane. In MTD-1A cells, the AJ/AF relationship was investigated during the cell polarization process after replating or wounding. At the early stage, the AJ/AF relationship was quite similar to that in NRK cells. As polarization proceeded, the spot-like AJs were gradually fused side by side with the concomitant shortening of the associated stress-fiber-like AF bundles. Finally, the belt-like AJ was established, which was lined with circumferential AF bundles. The similarities and differences in the AJ/AF relationship between non-polarized fibroblasts and polarized epithelial cells are discussed.
- Published
- 1995
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358. Expression of e-cadherin, alpha-catenins and Beta-catenins in human gastric carcinomas - correlation with histology and tumor progression.
- Author
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Yasui W, Kuniyasu H, Akama Y, Kitahara K, Nagafuchi A, Ishihara S, Tsukita S, and Tahara E
- Abstract
The expression of cell-cell adhesion molecule, E-cadherin and its associated proteins, alpha- and beta-catenins in human gastric carcinomas was examined by Western blotting. All the seven gastric carcinoma cell lines expressed E-cadherin except KATOIII, which was derived from pleural effusion of a scirrhous type stomach cancer or Borrmann's type-4 carcinoma. The expression of alpha-catenin was not detected in HSC43 derived from scirrhous carcinoma, while HSC39 expressed abnormal beta-catenin caused by genetic alteration. In gastric carcinoma cases, the levels of E-cadherin and alpha-catenin were significantly lower in poorly differentiated adenocarcinomas and scirrhous carcinomas when compared to other types of gastric carcinomas. Deeply invasive carcinomas expressed E-cadherin and alpha-catenin at lower levels. However, the expression level of alpha-catenin was not necessarily consistent with that of E-cadherin. One of 10 gastric carcinomas examined showed complete deletion of alpha-catenin gene in Southern blotting. beta-catenin was expressed at lower level in poorly differentiated adenocarcinomas than in well-differentiated adenocarcinomas. These findings suggest that reduction in the expression of E-cadherin and its associated molecules, catenins, is involved in the development and infiltrative growth of scirrhous type gastric carcinomas.
- Published
- 1995
359. A truncated beta-catenin disrupts the interaction between E-cadherin and alpha-catenin: a cause of loss of intercellular adhesiveness in human cancer cell lines.
- Author
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Oyama T, Kanai Y, Ochiai A, Akimoto S, Oda T, Yanagihara K, Nagafuchi A, Tsukita S, Shibamoto S, and Ito F
- Subjects
- Animals, Base Sequence, Cell Adhesion, Cytoskeletal Proteins genetics, Humans, Immunoblotting, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Neoplasm Invasiveness, Neoplasms pathology, Precipitin Tests, RNA, Messenger analysis, Tumor Cells, Cultured, beta Catenin, Cadherins physiology, Cytoskeletal Proteins physiology, Mutation, Neoplasms genetics, Trans-Activators
- Abstract
Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with catenins, a group of cytoplasmic proteins including alpha- and beta-catenins. While alpha-catenin has been demonstrated to be crucial for cadherin function, the role of beta-catenin is not yet fully understood. In this study, we analyzed the cadherin-catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of beta-catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the beta-catenin gene. On the other hand, these cells expressed E-cadherin, alpha-catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated beta-catenin but not with alpha-catenin, and antibodies against beta-catenin did not copurify alpha-catenin. However, the recombinant fusion protein containing wild-type beta-catenin precipitated alpha-catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with alpha-catenin, and that this defect results from the mutation in beta-catenin. Thus, it is most likely that the association between E-cadherin and alpha-catenin is mediated by beta-catenin, and that this process is blocked by NH2-terminal deletion in beta-catenin. These findings indicate that genetic abnormality of beta-catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers.
- Published
- 1994
360. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.
- Author
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Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S
- Subjects
- Animals, Cell Polarity, Cells, Cultured, Chickens, Cytoplasm metabolism, Fluorescent Antibody Technique, Humans, Membrane Proteins genetics, Models, Molecular, Mutation, Occludin, Protein Binding, Recombinant Fusion Proteins metabolism, Structure-Activity Relationship, Zonula Occludens-1 Protein, Zonula Occludens-2 Protein, Cell Compartmentation, Intercellular Junctions metabolism, Membrane Proteins metabolism, Phosphoproteins metabolism
- Abstract
Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.
- Published
- 1994
- Full Text
- View/download PDF
361. Induction of polarized cell-cell association and retardation of growth by activation of the E-cadherin-catenin adhesion system in a dispersed carcinoma line.
- Author
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Watabe M, Nagafuchi A, Tsukita S, and Takeichi M
- Subjects
- Cell Adhesion Molecules analysis, Cytoskeletal Proteins genetics, DNA, Complementary, Epithelial Cells, Humans, Integrins analysis, Intercellular Junctions chemistry, Intercellular Junctions ultrastructure, Laminin, Membrane Proteins analysis, Phenotype, Phosphoproteins analysis, Transfection, Tumor Cells, Cultured, Zonula Occludens-1 Protein, alpha Catenin, Cadherins analysis, Cell Adhesion, Cell Division, Cell Polarity physiology, Cytoskeletal Proteins physiology
- Abstract
PC9 lung carcinoma cells cannot tightly associate with one another, and therefore grow singly, despite their expression of E-cadherin, because of their lack of alpha-catenin, a cadherin-associated protein. However, when the E-cadherin is activated by transfection with alpha-catenin cDNA, they form spherical aggregates, each consisting of an enclosed monolayer cell sheet. In the present work, we examined whether the alpha-catenin-transfected cell layers expressed epithelial phenotypes, by determining the distribution of various cell adhesion molecules on their surfaces, including E-cadherin, ZO-1, desmoplakin, integrins, and laminin. In untransfected PC9 cells, all these molecules were randomly distributed on their cell surface. In the transfected cells, however, each of them was redistributed into a characteristic polarized pattern without a change in the amount of expression. Electron microscopic study demonstrated that the alpha-catenin-transfected cell layers acquired apical-basal polarity typical of simple epithelia; they formed microvilli only on the outer surface of the aggregates, and a junctional complex composed of tight junction adherens junction, and desmosome arranged in this order. These results indicate that the activation of E-cadherin triggered the formation of the junctional complex and the polarized distribution of cell surface proteins and structures. We also found that, in untransfected PC9 cells, ZO-1 formed condensed clusters and colocalized with E-cadherin, but that other adhesion molecules rarely showed such colocalization with E-cadherin, suggesting that there is some specific interaction between ZO-1 and E-cadherin even in the absence of cell-cell contacts. In addition, we found that the activation of E-cadherin caused a retardation of PC9 cell growth. Thus, we concluded that the E-cadherin-catenin adhesion system is essential not only for structural organization of epithelial cells but also for the control of their growth.
- Published
- 1994
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362. Expression of e-cadherin and alpha-catenin molecules in human breast-cancer tissues and association with clinicopathological features.
- Author
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Takayama T, Shiozaki H, Inoue M, Tamura S, Oka H, Kadowaki T, Takatsuka Y, Nagafuchi A, Tsukita S, and Mori T
- Abstract
E-cadherin is a transmembrane glycoprotein that is regulated by its associated proteins, including alpha-catenin. To understand intercellular adhesion of cancerous tissues more precisely, we investigated E-cadherin and alpha-catenin expression in 87 human breast cancers immunohistochemically. All of the normal mammary glands consistently showed strong alpha-catenin expression on cell-cell boundaries. In contrast, various expression patterns were observed in cancerous tissues; 22 (25%) evaluated as uniformly positive, 32 (37%) as heterogeneous and 33 (38%) as uniformly negative. We found significant correlations between reduction in alpha-catenin expression and clinicopathological features, especially tumor invasion and metastasis. Furthermore, a significant correlation was found between E-cadherin and alpha-catenin expressions. These results suggest that E-cadherin mediated adhesion system may be affected by reduction in alpha-catenin expression and that alpha-catenin may play an important role in cancer invasion and metastasis.
- Published
- 1994
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363. The roles of catenins in the cadherin-mediated cell adhesion: functional analysis of E-cadherin-alpha catenin fusion molecules.
- Author
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Nagafuchi A, Ishihara S, and Tsukita S
- Subjects
- Amino Acid Sequence, Animals, Cadherins genetics, Cell Adhesion physiology, Cell Division, Cell Movement, Cytoskeletal Proteins genetics, Cytoskeleton metabolism, DNA, Complementary, Gene Expression, L Cells, Mice, Molecular Sequence Data, Transfection, alpha Catenin, Cadherins metabolism, Cytoskeletal Proteins metabolism, Recombinant Fusion Proteins metabolism
- Abstract
The carboxyl terminus-truncated cadherin (nonfunctional cadherin) has no cell adhesion activity probably because of its failure to associate with cytoplasmic proteins called alpha and beta catenin. To rescue this nonfunctional cadherin as adhesion molecules, we constructed three cDNAs for fusion proteins between nonfunctional E-cadherin and alpha catenin, nE alpha, nE alpha N, and nE alpha C, where the intact, amino-terminal and carboxy-terminal half of alpha catenin, respectively, were directly linked to the nonfunctional E-cadherin, and introduced them into mouse L cells. The subcellular distribution and cell adhesion activity of nE alpha and nE alpha C molecules was similar to those of intact E-cadherin transfectants: they bound to cytoskeletons, were concentrated at cell-cell adhesion sites and showed strong cell adhesion activity. nE alpha N molecules, which also bound to cytoskeletons, showed very poor cell adhesion activity. Taken together, we conclude that in the formation of the cadherin-catenin complex, the mechanical association of alpha catenin, especially its carboxy-terminal half, with E-cadherin is a key step for the cadherin-mediated cell adhesion. Close comparison revealed that the behavior of nE alpha molecules during cytokinesis was quite different from that of intact E-cadherin, and that the intercellular motility, i.e., the cell movement in a confluent sheet, was significantly suppressed in nE alpha transfectants although it was facilitated in E-cadherin transfectants. Considering that nE alpha was not associated with endogenous beta catenin in transfectants, the difference in the nature of cell adhesion between nE alpha and intact E-cadherin transfectants may be explained by the function of beta catenin. The possible functions of beta catenin are discussed with a special reference to its role as a negative regulator for the cadherin-mediated cell adhesion system.
- Published
- 1994
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364. Autoimmune myocarditis induced in mice by cardiac C-protein. Cloning of complementary DNA encoding murine cardiac C-protein and partial characterization of the antigenic peptides.
- Author
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Kasahara H, Itoh M, Sugiyama T, Kido N, Hayashi H, Saito H, Tsukita S, and Kato N
- Subjects
- Adult, Aged, Amino Acid Sequence, Animals, Autoimmune Diseases chemically induced, Cardiomyopathy, Dilated blood, Carrier Proteins, Cloning, Molecular, DNA, Complementary metabolism, Female, Heart drug effects, Humans, Inflammation, Klebsiella pneumoniae, Lipopolysaccharides administration & dosage, Male, Mice, Mice, Inbred DBA, Mice, Inbred Strains, Middle Aged, Muscle Proteins biosynthesis, Myocarditis chemically induced, Recombinant Fusion Proteins biosynthesis, Recombinant Proteins toxicity, Reference Values, Restriction Mapping, Sequence Homology, Amino Acid, Autoantibodies blood, Autoimmune Diseases pathology, Cardiomyopathy, Dilated immunology, Lipopolysaccharides toxicity, Muscle Proteins toxicity, Myocarditis immunology, Myocarditis pathology, Myocardium pathology
- Abstract
Autoimmune myocarditis is considered to play a major role in the pathogenesis of dilated cardiomyopathy. A new autoimmune myocarditis model was attained by repeated immunization using murine cardiac C-protein with the immunological adjuvant, Klebsiella pneumoniae O3 lipopolysaccharide. For further analysis of a pathological epitope, the cDNA encoding C-protein was isolated; a fusion protein encoded by part of this cDNA induced myocarditis in SMA mice as well as in three other strains: DBA/1J (H-2q), O20/A (H-2pz1), and SJL (H-2s). The nucleotide sequence and its deduced amino acid analysis revealed that this protein had immunoglobulin-like and fibronectin-like repeats. This study provides a new animal model of autoimmune myocarditis which may shed light on the pathogenesis of dilated cardiomyopathy.
- Published
- 1994
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365. ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.
- Author
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Tsukita S, Oishi K, Sato N, Sagara J, Kawai A, and Tsukita S
- Subjects
- Actins metabolism, Amino Acid Sequence, Animals, Antibodies, Monoclonal, Blood Proteins analysis, Carrier Proteins genetics, Carrier Proteins immunology, Carrier Proteins isolation & purification, Cell Line, Cricetinae, Cytoskeleton chemistry, DNA, Complementary analysis, Epitopes genetics, Epitopes immunology, Hyaluronan Receptors, Membrane Proteins analysis, Mice, Molecular Sequence Data, Phosphoproteins analysis, Precipitin Tests, Proteins analysis, Rabies virus chemistry, Rabies virus immunology, Receptors, Cell Surface genetics, Receptors, Cell Surface immunology, Receptors, Cell Surface isolation & purification, Receptors, Lymphocyte Homing genetics, Receptors, Lymphocyte Homing immunology, Receptors, Lymphocyte Homing isolation & purification, Recombinant Fusion Proteins biosynthesis, Recombinant Fusion Proteins immunology, Sequence Analysis, DNA, Blood Proteins metabolism, Carrier Proteins metabolism, Cytoskeletal Proteins, Cytoskeleton metabolism, Membrane Proteins metabolism, Microfilament Proteins, Phosphoproteins metabolism, Proteins metabolism, Receptors, Cell Surface metabolism, Receptors, Lymphocyte Homing metabolism
- Abstract
The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.
- Published
- 1994
- Full Text
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366. Structural diversity of band 4.1 superfamily members.
- Author
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Takeuchi K, Kawashima A, Nagafuchi A, and Tsukita S
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Line, Cell Membrane metabolism, Cloning, Molecular, Conserved Sequence, DNA Primers, DNA, Complementary isolation & purification, Erythrocyte Membrane metabolism, Gene Expression, Membrane Proteins biosynthesis, Mice, Molecular Sequence Data, Polymerase Chain Reaction, Restriction Mapping, Sequence Homology, Amino Acid, Teratoma, Tumor Cells, Cultured, Cytoskeletal Proteins, Genetic Variation, Membrane Proteins chemistry, Membrane Proteins genetics, Multigene Family, Neuropeptides, Phylogeny
- Abstract
Several proteins contain the domain homologous to the N-terminal half of band 4.1 protein, indicating the existence of a superfamily. The members of this 'band 4.1' superfamily are thought to play crucial roles in the regulation of cytoskeleton-plasma membrane interaction just beneath plasma membranes. We examined the structural diversity of this superfamily by means of the polymerase chain reaction using synthesized mixed primers. We thus identified many members of the band 4.1 superfamily that were expressed in mouse teratocarcinoma F9 cells and mouse brain tissue. In total, 15 cDNA clones were obtained; 8 were identical to the corresponding parts of cDNAs for the known members, while 7 appeared to encode novel proteins (NBL1-7: novel band 4.1-like proteins). Sequence analyses of these clones revealed that the band 4.1 superfamily can be subdivided into 5 gene families; band 4.1 protein, ERM (ezrin/radixin/moesin/merlin/NBL6/NBL7+ ++), talin, PTPH1 (PTPH1/PTPMEG/NBL1-3), and NBL4 (NBL4/NBL5) families. The NBL4 family was first identified here, and the full-length cDNA encoding NBL4 was cloned. The deduced amino acid sequence revealed a myristoylation site, as well as phosphorylation sites for A-kinase and tyrosine kinases in its N-terminal half, suggesting its involvement in the phosphorylation-dependent regulation of cellular events just beneath the plasma membrane. In this study, we describe the initial characterization of these new members and discuss the evolution of the band 4.1 superfamily.
- Published
- 1994
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367. Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members.
- Author
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Takeuchi K, Sato N, Kasahara H, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S
- Subjects
- Animals, Base Sequence, Blood Proteins genetics, Cell Adhesion drug effects, Fluorescent Antibody Technique, Leukemia L5178, Membrane Proteins genetics, Mice, Microscopy, Electron, Scanning, Microvilli drug effects, Microvilli ultrastructure, Molecular Sequence Data, Phosphoproteins genetics, Proteins genetics, Thionucleotides pharmacology, Thymoma ultrastructure, Cell Adhesion physiology, Cytoskeletal Proteins, DNA-Binding Proteins genetics, Microfilament Proteins, Microvilli physiology, Oligonucleotides, Antisense pharmacology, Transcription Factors genetics
- Abstract
To examine the functions of ERM family members (ezrin, radixin, and moesin), mouse epithelial cells (MTD-1A cells) and thymoma cells (L5178Y), which coexpress all of them, were cultured in the presence of antisense phosphorothioate oligonucleotides (PONs) complementary to ERM sequences. Immunoblotting revealed that the antisense PONs selectively suppressed the expression of each member. Immunofluorescence microscopy of these ezrin, radixin, or moesin "single-suppressed" MTD-1A cells revealed that the ERM family members are colocalized at cell-cell adhesion sites, microvilli, and cleavage furrows, where actin filaments are densely associated with plasma membranes. The ezrin/radixin/moesin antisense PONs mixture induced the destruction of both cell-cell and cell-substrate adhesion, as well as the disappearance of microvilli. Ezrin or radixin antisense PONs individually affected the initial step of the formation of both cell-cell and cell-substrate adhesion, but did not affect the microvilli structures. In sharp contrast, moesin antisense PONs did not singly affect cell-cell and cell-substrate adhesion, whereas it partly affected the microvilli structures. These data indicate that ezrin and radixin can be functionally substituted, that moesin has some synergetic functional interaction with ezrin and radixin, and that these ERM family members are involved in cell-cell and cell-substrate adhesion, as well as microvilli formation.
- Published
- 1994
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368. Immunohistochemical detection of alpha-catenin expression in human cancers.
- Author
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Shiozaki H, Iihara K, Oka H, Kadowaki T, Matsui S, Gofuku J, Inoue M, Nagafuchi A, Tsukita S, and Mori T
- Subjects
- Adenocarcinoma metabolism, Adenocarcinoma pathology, Adult, Aged, Cadherins metabolism, Carcinoma, Squamous Cell metabolism, Carcinoma, Squamous Cell pathology, Colonic Neoplasms pathology, Down-Regulation, Esophageal Neoplasms pathology, Female, Humans, Immunoenzyme Techniques, Infant, Male, Middle Aged, Neoplasm Staging, Stomach Neoplasms pathology, alpha Catenin, Colonic Neoplasms metabolism, Cytoskeletal Proteins metabolism, Esophageal Neoplasms metabolism, Stomach Neoplasms metabolism
- Abstract
The function of E-cadherin is thought to be regulated by its associated cytoplasmic proteins including alpha-catenin. To determine whether possible downregulation of alpha-catenin expression may play a role in tumor invasion and metastasis through the dysfunction of E-cadherin, we investigated the expression of alpha-catenin in human carcinoma samples (esophagus, stomach, and colon) by immunohistochemistry using our monoclonal antibody against alpha-catenin (alpha-18). Normal epithelium expressed alpha-catenin strongly without exception. However, alpha-catenin expression was frequently reduced in primary tumors of esophagus (12 of 15:80%), stomach (14 of 20: 70%), and colon (8 of 10: 80%). Of the tumors with reduced alpha-catenin expression, alpha-catenin expression was completely negative in 70.6% of them (9 of 12 in esophagus, 9 of 14 in stomach, and 6 of 8 in colon). These results also suggested that some human cancer cells may have impaired E-cadherin-mediated cell adhesiveness through the downregulation of alpha-catenin expression.
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- 1994
369. Frequent loss of alpha catenin expression in scirrhous carcinomas with scattered cell growth.
- Author
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Ochiai A, Akimoto S, Shimoyama Y, Nagafuchi A, Tsukita S, and Hirohashi S
- Subjects
- Adenocarcinoma, Scirrhous pathology, Antibodies, Monoclonal, Breast Neoplasms pathology, Cell Division physiology, Female, Humans, Immunoblotting, Immunohistochemistry, Male, Stomach Neoplasms pathology, alpha Catenin, Adenocarcinoma, Scirrhous metabolism, Breast Neoplasms metabolism, Cadherins biosynthesis, Cytoskeletal Proteins biosynthesis, Stomach Neoplasms metabolism
- Abstract
To investigate the mechanisms of disruption of cell-cell contact in scirrhous carcinoma cells, the expression of both E-cadherin and alpha catenin, which is an intracellular cadherin-binding molecule, were determined in scirrhous-type adenocarcinomas of the stomach and breast using immunohistochemical and immunoblotting techniques. The losses of E-cadherin expression in gastric and breast scirrhous adenocarcinomas were 18.1% and 0%, respectively, and those of alpha catenin expression were 54.6% and 75%, respectively. Frequent loss of alpha catenin expression occurred in scirrhous carcinomas with scattered cell growth in the stomach and the breast and showed no organ specificity. In addition, all the infiltrating lobular carcinomas, which also infiltrate the stroma as single cells, showed no E-cadherin or alpha catenin expression. These findings suggest that down-regulation of either alpha catenin or E-cadherin plays a critical role in the disruption of cell adhesion in carcinomas with scattered cell growth.
- Published
- 1994
- Full Text
- View/download PDF
370. The Loss of the Expression of α Catenin, the 102 kD Cadherin Associated Protein, in Central Nervous Tissues during Development: (α catenin/cadherin/cell adhesion/CNS).
- Author
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Nagafuchi A and Tsukita S
- Abstract
A monoclonal antibody specific for α catenin, the 102kD cadherin-associated protein, has been characterized and used to describe the expression and distribution pattern of α catenin in adult mice and mouse embryos. This monoclonal antibody recognized an epitope in the middle part of the α catenin molecule of various vertebrate species, and bound to neither vinculin nor αN catenin, which are cytoskeletal proteins with sequence similarity to α catenin. At the early mouse embryo stage (neurulae stage) α catenin was expressed and concentrated at cell-to-cell contact sites together with various types of cadherins in all tissues. In embryos at 12.5 days of gestation, the α catenin expression was gradually diminished selectively in central nervous tissues such as brain and spinal cord, and in most of the adult central nervous tissues the α catenin expression was hardly detected. In adult non-nervous tissues most of the cells examined expressed α catenin. Especially in well-polarized tissues such as epithelial cells, α catenin appeared to be highly concentrated at cell-to-cell adherens junctions where cadherins act as adhesion molecules. This loss of α catenin expression in central nervous tissues was observed not only in mice but also in other vertebrate species such as fish and newt, suggesting that this phenomenon has important implications from the view point of nervous tissue development.
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- 1994
- Full Text
- View/download PDF
371. Possible involvement of adherens junction plaque proteins in tumorigenesis and metastasis.
- Author
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Tsukita S, Tsukita S, Nagafuchi A, and Yonemura S
- Subjects
- Animals, Blood Proteins isolation & purification, Blood Proteins physiology, Cell Adhesion, Cell Adhesion Molecules isolation & purification, Cytoskeletal Proteins isolation & purification, Cytoskeletal Proteins physiology, Drosophila melanogaster genetics, Humans, Insect Proteins genetics, Insect Proteins isolation & purification, Insect Proteins physiology, Intercellular Junctions chemistry, Liver chemistry, Membrane Proteins isolation & purification, Multigene Family, Neoplasm Proteins isolation & purification, Phosphoproteins isolation & purification, Phosphoproteins physiology, Rats, Zonula Occludens-1 Protein, alpha Catenin, src-Family Kinases isolation & purification, Cell Adhesion Molecules physiology, Cell Transformation, Neoplastic metabolism, Cell Transformation, Neoplastic pathology, Drosophila Proteins, Intercellular Junctions physiology, Liver cytology, Membrane Proteins physiology, Neoplasm Metastasis physiopathology, Neoplasm Proteins physiology, Tumor Suppressor Proteins, src-Family Kinases physiology
- Abstract
The cell-to-cell adherens junction is a site for cadherin-mediated adhesion where actin filaments are densely associated with the plasma membrane through its well developed plasmalemmal undercoat, a "plaque" structure. Recently, we succeeded in isolating the cell-to-cell adherens junctions from rat liver, and in identifying some novel AJ plaque constituents, including src-like tyrosine kinases, radixin, alpha-catenin, and a 220kD protein. The application of genetic engineering techniques to AJ plaque proteins has recently generated a wealth of novel observations, leading to the speculation that these proteins are involved in tumorigenesis and metastasis. This paper reviews these findings and discusses some functions of the AJ plaque proteins in normal cells, as well as in tumorigenesis and metastasis.
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- 1994
372. Immunohistochemical evaluation of alpha-catenin expression in human gastric cancer.
- Author
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Matsui S, Shiozaki H, Inoue M, Tamura S, Doki Y, Kadowaki T, Iwazawa T, Shimaya K, Nagafuchi A, and Tsukita S
- Subjects
- Adult, Aged, Cadherins metabolism, Humans, Immunohistochemistry, Middle Aged, Neoplasm Invasiveness, Neoplasm Metastasis, Stomach Neoplasms pathology, alpha Catenin, Cytoskeletal Proteins metabolism, Stomach Neoplasms metabolism
- Abstract
E-cadherin (E-cad) plays a major role in the maintenance of cell-cell adhesion in epithelial tissues, and impaired E-cad expression correlates with tumour invasion and metastasis. Alpha-catenin (alpha-cat), an undercoat protein of adherens junctions, binds to the cytoplasmic domain of E-cad and is essential for linking E-cad to actin-based cytoskeleton. We investigated E-cad and alpha-cat expression in 60 human gastric cancers immunohistochemically. The 60 gastric cancers were classified into 18 (30%) in which alpha-cat expression was preserved, and 42 (70%) reduced cases. The reduction of alpha-cat expression was significantly related to dedifferentiation, depth of invasion, infiltrative growth and lymph node metastasis. We also examined the co-expression of alpha-cat and E-cad. Seventeen (28%) tumours preserved both molecules [alpha-cat(+)/E-cad(+)] and 33 (55%) tumours reduced both [alpha-cat(-)/E-cad(-)], whereas 9 (15%) tumours exhibited alpha-cat(-)/E-cad(+). The frequency of lymph node metastasis in alpha-cat(-)/E-cad(+) tumour (67%) was significantly higher than that in alpha-cat(+)/E-cad(+) tumours (24%) and was close to that in alpha-cat(-)/E-cad(-) tumours (82%). The frequency of haematogenous liver metastasis in alpha-cat(-)/E-cad(+) tumours (44%) was significantly higher than that in alpha-cat(+)/E-cad(+) tumours (6%) or alpha-cat(-)/E-cad(-) tumours (9%). Thus, in all E-cad(+) tumours, the frequency of lymph node and liver metastasis was higher in alpha-cat(-) tumours than in alpha-cat(+) tumours. alpha-Cat expression is apparently better at predicting tumour invasion and metastasis than E-cad expression.
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- 1994
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373. Structure, expression and chromosome assignment of the human catenin (cadherin-associated protein) alpha 1 gene (CTNNA1).
- Author
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Furukawa Y, Nakatsuru S, Nagafuchi A, Tsukita S, Muto T, Nakamura Y, and Horii A
- Subjects
- Amino Acid Sequence, Base Sequence, Blotting, Southern, Cadherins biosynthesis, Chromosome Mapping, Cytoskeletal Proteins biosynthesis, DNA, Exons, Humans, In Situ Hybridization, Fluorescence, Molecular Sequence Data, Polymerase Chain Reaction, alpha Catenin, Cadherins genetics, Cytoskeletal Proteins genetics
- Abstract
We have isolated the human alpha-catenin gene (CTNNA1), which encodes a cadherin-associated protein, and have determined its primary structure and chromosomal localization. The transcript of CTNNA1 is 3.4 kb long and consists of 16 coding exons encoding 906 amino acids and at least one 5' noncoding exon. The 102-kDa predicted protein is the same size as the murine homolog, and the amino acid sequences of the two proteins are 99.2% homologous. Analysis by reverse transcription-PCR revealed that this gene is expressed ubiquitously in normal tissues. It was mapped to chromosome band 5q31 by fluorescent in situ hybridization.
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- 1994
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374. Assignment of the human moesin gene (MSN) to chromosome region Xq11.2-->q12.
- Author
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Kishino T, Ariga T, Soejima H, Tamura T, Ohta T, Jinno Y, Yonemura S, Sato N, Tsukita S, and Tsukita S
- Subjects
- Chromosome Mapping, DNA, Complementary, Female, Genetic Linkage, Humans, In Situ Hybridization, Fluorescence, Male, Wiskott-Aldrich Syndrome genetics, Microfilament Proteins, Proteins genetics, X Chromosome
- Abstract
The human moesin gene (MSN) was mapped to the long arm of the X chromosome. PCR products for the moesin gene cDNA were used as probes to isolate their corresponding cosmid clones. Fluorescence in situ hybridization (FISH) with two of the isolated cosmid probes showed signals at Xq11.2-->q12, whereas four other cosmids showed FISH signals on chromosome 5. Southern blot hybridization, using a PCR product corresponding to the 3' region of the moesin gene cDNA as a probe (probe-3), on one of the two cosmids that produced signals on the X chromosome gave 5.7- and 3.5-kb HindIII fragments. Further Southern hybridization of the DNA from XY, XX, and XXXXX individuals using probe-3 revealed a gene-dose effect of the X chromosome on the size of a 3.5-kb and a 3.0-kb HindIII fragment; in contrast, an invariant 9.8-kb band was present in the DNA of all individuals tested. Sequencing of an exon-intron border revealed that the two cosmids had predicted sequences. These results indicated that the two cosmids contained MSN, and it was consequently assigned to human chromosome region Xq11.2-->q12. These results strongly suggest that MSN may be removed from candidacy for Wiskott-Aldrich syndrome, which has been putatively mapped to Xp11.3-->p11.22.
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- 1994
- Full Text
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375. Assignment of the human alpha-catenin gene (CTNNA1) to chromosome 5q21-q22.
- Author
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McPherson JD, Morton RA, Ewing CM, Wasmuth JJ, Overhauser J, Nagafuchi A, Tsukita S, and Isaacs WB
- Subjects
- Animals, Base Sequence, CHO Cells, Chromosome Mapping, Cricetinae, DNA, Complementary genetics, Humans, Hybrid Cells, Molecular Sequence Data, Polymerase Chain Reaction, alpha Catenin, Chromosomes, Human, Pair 5, Cytoskeletal Proteins genetics, Genes
- Published
- 1994
- Full Text
- View/download PDF
376. Occludin: a novel integral membrane protein localizing at tight junctions.
- Author
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Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal, Base Sequence, Brain cytology, Chickens, DNA, Complementary metabolism, Epithelial Cells, Epithelium ultrastructure, Intestines ultrastructure, Liver cytology, Liver metabolism, Membrane Proteins chemistry, Molecular Sequence Data, Myocardium cytology, Occludin, Organ Specificity, Phosphoproteins analysis, Protein Conformation, Protein Structure, Secondary, Restriction Mapping, Zonula Occludens-1 Protein, Intercellular Junctions ultrastructure, Intestines cytology, Membrane Proteins analysis, Membrane Proteins biosynthesis
- Abstract
Recently, we found that ZO-1, a tight junction-associated protein, was concentrated in the so called isolated adherens junction fraction from the liver (Itoh, M., A. Nagafuchi, S. Yonemura, T. Kitani-Yasuda, Sa. Tsukita, and Sh. Tsukita. 1993. J. Cell Biol. 121:491-502). Using this fraction derived from chick liver as an antigen, we obtained three monoclonal antibodies specific for a approximately 65-kD protein in rats. This antigen was not extractable from plasma membranes without detergent, suggesting that it is an integral membrane protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this approximately 65-kD membrane protein was exclusively localized at tight junctions of both epithelial and endothelial cells: at the electron microscopic level, the labels were detected directly over the points of membrane contact in tight junctions. To further clarify the nature and structure of this membrane protein, we cloned and sequenced its cDNA. We found that the cDNA encoded a 504-amino acid polypeptide with 55.9 kDa. A search of the data base identified no proteins with significant homology to this membrane protein. A most striking feature of its primary structure was revealed by a hydrophilicity plot: four putative membrane-spanning segments were included in the NH2-terminal half. This hydrophilicity plot was very similar to that of connexin, an integral membrane protein in gap junctions. These findings revealed that an integral membrane protein localizing at tight junctions is now identified, which we designated as "occludin."
- Published
- 1993
- Full Text
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377. Submembranous junctional plaque proteins include potential tumor suppressor molecules.
- Author
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Tsukita S, Itoh M, Nagafuchi A, Yonemura S, and Tsukita S
- Subjects
- Amino Acid Sequence, Animals, Humans, Molecular Sequence Data, Cell Adhesion Molecules physiology, Cell Transformation, Neoplastic, Genes, Tumor Suppressor, Intercellular Junctions physiology
- Published
- 1993
- Full Text
- View/download PDF
378. [Morphology in molecular cellular biology].
- Author
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Tsukita S
- Subjects
- Animals, Cell Adhesion, Cell Communication, Cell Division, Humans, Molecular Biology
- Published
- 1993
379. Reduction of E-cadherin levels and deletion of the alpha-catenin gene in human prostate cancer cells.
- Author
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Morton RA, Ewing CM, Nagafuchi A, Tsukita S, and Isaacs WB
- Subjects
- Cell Adhesion, Humans, Male, Molecular Weight, Precipitin Tests, Prostatic Neoplasms genetics, RNA, Messenger analysis, Tumor Cells, Cultured, alpha Catenin, Cadherins analysis, Cytoskeletal Proteins genetics, Gene Deletion, Prostatic Neoplasms metabolism
- Abstract
The cadherins are a family of transmembrane glycoproteins responsible for calcium-dependent cell-cell adhesion. This adhesion is mediated by a group of cytoplasmic proteins, the catenins, which act inside the cell to couple the cadherin molecule to the microfilament cytoskeleton. Dysfunction of E-cadherin-dependent cell-cell adhesion has been demonstrated to contribute to the acquisition of invasive potential of malignant adenocarcinoma cells. The potential role of alterations of catenin expression in tumor cell invasion is largely unexplored. We have previously found that E-cadherin is frequently down-regulated in clinical samples of prostate cancer (Umbas, R., Schalken, J. A., Aalders, T. W., Carter, B. S., Karthaus, H. F. M., Schaafsma, H. E., Debruyne, F. M. J., and Isaacs, W. B. Cancer Res., 52: 5104-5109, 1992). In this study, we further investigate this adhesion system in both benign and malignant human prostate cells in culture. Using antibodies to E-cadherin and its cytoplasmic accessory protein, alpha-catenin, we find that 5 of 6 human prostate cancer cell lines have reduced or absent levels of one or the other or both of these molecules when compared to normal prostatic epithelial cells. Only the LNCaP prostate cancer cell line is indistinguishable from normal prostate epithelium with respect to its E-cadherin-alpha-catenin complement. Interestingly, the PC-3 line is characterized by the presence of E-cadherin, but the complete lack of alpha-catenin found at both the RNA and protein level. This lack of alpha-catenin gene expression is explained by Southern analysis, which reveals a homozygous deletion of a large portion of the alpha-catenin gene in PC-3 cells. This loss of alpha-catenin is functionally manifested by negligible Ca(2+)-dependent aggregation of these cells in vitro, when compared to LNCaP cells. These results confirm that E-cadherin-dependent cell-cell adhesion is frequently aberrant in prostate cancer cells, and suggest that in a subset of prostate cancers, this adhesion may be inactivated by loss of alpha-catenin rather than E-cadherin itself. Furthermore, these results demonstrate that mutational inactivation of the alpha-catenin gene is one mechanism responsible for the loss of normal cell-cell adhesion in prostate cancer.
- Published
- 1993
380. Cloning of the human alpha-catenin cDNA and its aberrant mRNA in a human cancer cell line.
- Author
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Oda T, Kanai Y, Shimoyama Y, Nagafuchi A, Tsukita S, and Hirohashi S
- Subjects
- Animals, Base Sequence, Blotting, Northern, Blotting, Southern, Cadherins biosynthesis, Cadherins genetics, Colonic Neoplasms, DNA Probes, DNA, Neoplasm, Gene Library, Humans, Mice, Molecular Sequence Data, Oligodeoxyribonucleotides, Polymerase Chain Reaction, RNA, Messenger biosynthesis, Sequence Homology, Nucleic Acid, Tumor Cells, Cultured, alpha Catenin, Cytoskeletal Proteins biosynthesis, Cytoskeletal Proteins genetics, RNA, Messenger metabolism, Sequence Deletion
- Abstract
Cadherin and catenin compose cell adhesion complex and are indispensable for tight cell-cell adhesion. Dysfunction of this adhesion complex causes dissociation of cancer cells from primary tumor nodules, thus possibly contributing to cancer invasion and metastasis. In this report, we present the human alpha-catenin sequence. Human alpha-catenin showed extensive homology with that of mouse, i.e., 91.8% and 99.3% at the nucleic acid and amino acid levels, respectively, indicating that this molecule has been evolutionarily conserved in mammals. Characterization of the mRNA sequence of alpha-catenin in PC9 was also carried out, and two distinct abnormal sequences, i.e., one of 957 bp deletion resulting in a 319-amino-acid deletion and another of 761 bp deletion resulting in a frameshift, were identified. These deletions were probably produced by an error of RNA splicing, presenting one possible mechanism for the loss of intact alpha-catenin expression.
- Published
- 1993
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381. Desmoyokin, a 680 kDa keratinocyte plasma membrane-associated protein, is homologous to the protein encoded by human gene AHNAK.
- Author
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Hashimoto T, Amagai M, Parry DA, Dixon TW, Tsukita S, Tsukita S, Miki K, Sakai K, Inokuchi Y, and Kudoh J
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal immunology, Base Sequence, Cattle, DNA, Complementary genetics, Desmosomes chemistry, Genes, Humans, Mice, Molecular Sequence Data, Protein Structure, Secondary, Sequence Alignment, Sequence Homology, Amino Acid, Keratinocytes metabolism, Membrane Proteins genetics, Neoplasm Proteins genetics
- Abstract
We have obtained a monoclonal antibody (33A-3D) that specifically recognize desmoyokin, a 680 kDa desmosomal plaque protein that is well characterized in bovine muzzle epidermis. A cDNA clone (DY6, 3693 bp) was isolated by immunoscreening a mouse keratinocyte expression library with 33A-3D, and it was confirmed that DY6 has a partial coding sequence for desmoyokin. DY6 consists of highly homologous repeats about 128 residues long. Furthermore, the 128-residue repeats exhibit a quasi seven-residue substructure, which we believe will adopt an antiparallel beta-sheet structure. Surprisingly, the amino acid sequence showed a significant homology with AHNAK, a newly identified human gene encoding a 700 kDa protein, which was suggested to be down-regulated in neuroblastoma. From its extensive homology, the similarity in both size and structure, and the identical patterns on Southern blot analysis of genomic DNAs, desmoyokin and AHNAK protein are thought to be identical. Although the desmoyokin/AHNAK protein is detected in a variety of cell types at both protein and mRNA levels, its distribution in keratinocytes (associated closely with cell membrane) is quite different from that in cells other than keratinocytes (distributed diffusely in the cytoplasm). These findings suggest that the desmoyokin/AHNAK protein is a ubiquitous molecule with a unique structure and appears to have different distributions (and probably different functions) among different cells.
- Published
- 1993
- Full Text
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382. Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein.
- Author
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Oda H, Uemura T, Shiomi K, Nagafuchi A, Tsukita S, and Takeichi M
- Subjects
- Amino Acid Sequence, Animals, Armadillo Domain Proteins, Base Sequence, Cell Adhesion, Cell Adhesion Molecules metabolism, Chromosome Mapping, Cloning, Molecular, DNA genetics, In Situ Hybridization, Molecular Sequence Data, Protein Binding, Proteins metabolism, Sequence Alignment, Transcription Factors, Vinculin chemistry, alpha Catenin, Cell Adhesion Molecules genetics, Cytoskeletal Proteins genetics, Drosophila Proteins, Drosophila melanogaster genetics, Genes, Insect, Proteins genetics, Trans-Activators
- Abstract
The cadherin cell adhesion system plays a central role in cell-cell adhesion in vertebrates, but its homologues are not identified in the invertebrate. alpha-Catenins are a group of proteins associated with cadherins, and this association is crucial for the cadherins' function. Here, we report the cloning of a Drosophila alpha-catenin gene by low stringent hybridization with a mouse alpha E-catenin probe. Isolated cDNAs encoded a 110-kD protein with 60% identity to mouse alpha E-catenin, and this protein was termed D alpha-catenin. The gene of this protein was located at the chromosome band 80B. Immunostaining analysis using a mAb to D alpha-catenin revealed that it was localized to cell-cell contact sites, expressed throughout development and present in a wide variety of tissues. When this protein was immunoprecipitated from detergent extracts of Drosophila embryos or cell lines, several proteins co-precipitated. These included the armadillo product which was known to be a Drosophila homologue of beta-catenin, another cadherin-associated protein in vertebrates, and a 150-kD glycoprotein. These results strongly suggest that Drosophila has a cell adhesion machinery homologous to the vertebrate cadherin-catenin system.
- Published
- 1993
- Full Text
- View/download PDF
383. Time-resolved electron microscopic analysis of the behavior of myosin heads on actin filaments after photolysis of caged ATP.
- Author
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Funatsu T, Kono E, and Tsukita S
- Subjects
- Actin Cytoskeleton ultrastructure, Adenosine Triphosphate metabolism, Animals, Computer Simulation, Freeze Etching, In Vitro Techniques, Macromolecular Substances, Microscopy, Electron, Photolysis, Protein Binding, Rabbits, Actins ultrastructure, Actomyosin ultrastructure, Myosin Subfragments metabolism, Myosins ultrastructure
- Abstract
The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P3-1-(2-nitrophenyl)ethyl ester of ATP (caged ATP) was analyzed with a newly developed freezing system using liquid helium. Actin and S1 (100 microM each) formed a ropelike double-helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the ropelike double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP, the ropelike double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 35-200 ms are so called "weakly bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly bound S1 under a rapid association-dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly developed freezing system. This enabled us to directly compare the conformation of weakly and strongly bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly and strongly bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly and strongly bound S1 have different angles of attachment to actin filaments. As compared to strongly bound S1, weakly bound S1 showed a significantly broader distribution of attachment angles. These results are discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.
- Published
- 1993
- Full Text
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384. Transmembrane control of cadherin-mediated cell-cell adhesion.
- Author
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Nagafuchi A, Tsukita S, and Takeichi M
- Subjects
- Amino Acid Sequence, Animals, Cadherins chemistry, Cytoplasm, Cytoskeletal Proteins metabolism, Molecular Sequence Data, Phosphorylation, Structure-Activity Relationship, Cadherins physiology, Cell Adhesion physiology
- Abstract
The cadherin family of cell-cell adhesion molecules plays a central role in organization of cells into multicellular structures. An important feature of the action of cadherins is that they form a complex with cytoskeletal proteins, and the formation of this complex is crucial for their adhesive function. Cadherin-mediated cell adhesion is thus controlled through the interaction with cytoplasmic proteins, and, for such control, phosphorylation of these proteins and also cadherins themselves might be involved. This regulatory mechanism of cell adhesion is perhaps fundamental to a variety of morphogenetic processes.
- Published
- 1993
- Full Text
- View/download PDF
385. [A case of adenocarcinoma presenting as a cavitary lesion with niveau formation].
- Author
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Matsuba T, Matsumoto K, Tsukita S, Kuwano K, Yamazaki H, Andou K, Fujiki T, and Matsuba K
- Subjects
- Diagnostic Errors, Female, Humans, Middle Aged, Radiography, Adenocarcinoma diagnostic imaging, Lung diagnostic imaging, Lung Neoplasms diagnostic imaging, Tuberculosis, Pulmonary diagnostic imaging
- Abstract
Primary pulmonary adenocarcinoma rarely shows cavitation with a fluid level on chest roentgenograms. Herein we describe such a case misdiagnosed as pulmonary tuberculosis. The patient was a 63-year-old, female who had never smoked. Chest roentgenograms revealed a cavitary lesion in the left lower lobe, possessing a prominent fluid level. Fiberoptic bronchoscopic aspirate was positive for acid-fast bacilli on stains. Since there was no improvement with antituberculous chemotherapy, a left lower lobectomy was performed. The present case is of interest in the light of cavity formation in pulmonary carcinoma. The diagnosis and roentgenographic features are discussed.
- Published
- 1993
386. The 220-kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a tight junction-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy.
- Author
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Itoh M, Nagafuchi A, Yonemura S, Kitani-Yasuda T, Tsukita S, and Tsukita S
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cadherins chemistry, Intercellular Junctions ultrastructure, L Cells ultrastructure, Membrane Proteins chemistry, Mice, Microscopy, Immunoelectron, Molecular Sequence Data, Molecular Weight, Phosphoproteins chemistry, Rats, Sequence Homology, Amino Acid, Tumor Cells, Cultured ultrastructure, Zonula Occludens-1 Protein, Cadherins genetics, Intercellular Junctions chemistry, Membrane Proteins genetics, Phosphoproteins genetics
- Abstract
We previously identified a 220-kD constitutive protein of the plasma membrane undercoat which colocalizes at the immunofluorescence microscopic level with cadherins and occurs not only in epithelial M., S. Yonemura, A. Nagafuchi, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). To clarify the nature and possible functions of this protein, we cloned its full-length cDNA and sequenced it. Unexpectedly, we found mouse 220-kD protein to be highly homologous to rat protein ZO-1, only a part of which had been already sequenced. This relationship was confirmed by immunoblotting with anti-ZO-1 antibody. As protein ZO-1 was originally identified as a component exclusively underlying tight junctions in epithelial cells, where cadherins are not believed to be localized, we analyzed the distribution of cadherins and the 220-kD protein by ultrathin cryosection immunoelectron microscopy. We found that in non-epithelial cells lacking tight junctions cadherins and the 220-kD protein colocalize, whereas in epithelial cells (e.g., intestinal epithelial cells) bearing well-developed tight junctions cadherins and the 220-kD protein are clearly segregated into adherens and tight junctions, respectively. Interestingly, in epithelial cells such as hepatocytes, which tight junctions are not so well developed, the 220-kD protein is detected not only in the tight junction zone but also at adherens junctions. Furthermore, we show in mouse L cells transfected with cDNAs encoding N-, P-, E-cadherins that cadherins interact directly or indirectly with the 220-kD protein. Possible functions of the 220-kD protein (ZO-1) are discussed with special reference to the molecular mechanism for adherens and tight junction formation.
- Published
- 1993
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- View/download PDF
387. Elastic filaments in situ in cardiac muscle: deep-etch replica analysis in combination with selective removal of actin and myosin filaments.
- Author
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Funatsu T, Kono E, Higuchi H, Kimura S, Ishiwata S, Yoshioka T, Maruyama K, and Tsukita S
- Subjects
- Actins isolation & purification, Animals, Antibodies, Monoclonal, Elasticity, Electrophoresis, Polyacrylamide Gel, Freeze Etching, Immunoblotting, Microscopy, Electron, Microscopy, Immunoelectron, Models, Structural, Myosins isolation & purification, Rabbits, Actin Cytoskeleton ultrastructure, Actins physiology, Myosins physiology, Papillary Muscles ultrastructure, Sarcomeres ultrastructure
- Abstract
To clarify the full picture of the connectin (titin) filament network in situ, we selectively removed actin and myosin filaments from cardiac muscle fibers by gelsolin and potassium acetate treatment, respectively, and observed the residual elastic filament network by deep-etch replica electron microscopy. In the A bands, elastic filaments of uniform diameter (6-7 nm) projecting from the M line ran parallel, and extended into the I bands. At the junction line in the I bands, which may correspond to the N2 line in skeletal muscle, individual elastic filaments branched into two or more thinner strands, which repeatedly joined and branched to reach the Z line. Considering that cardiac muscle lacks nebulin, it is very likely that these elastic filaments were composed predominantly of connectin molecules; indeed, anti-connectin monoclonal antibody specifically stained these elastic filaments. Further, striations of approximately 4 nm, characteristic of isolated connectin molecules, were also observed in the elastic filaments. Taking recent analyses of the structure of isolated connectin molecules into consideration, we concluded that individual connectin molecules stretched between the M and Z lines and that each elastic filament consisted of laterally-associated connectin molecules. Close comparison of these images with the replica images of intact and S1-decorated sarcomeres led us to conclude that, in intact sarcomeres, the elastic filaments were laterally associated with myosin and actin filaments in the A and I bands, respectively. Interestingly, it was shown that the elastic property of connectin filaments was not restricted by their lateral association with actin filaments in intact sarcomeres. Finally, we have proposed a new structural model of the cardiac muscle sarcomere that includes connectin filaments.
- Published
- 1993
- Full Text
- View/download PDF
388. [Regulation of cadherin-based cell adhesion and metastasis].
- Author
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Tsukita S
- Subjects
- Calcium physiology, Humans, Neoplasm Invasiveness, Neoplasms metabolism, Cadherins physiology, Cell Adhesion physiology, Cell Adhesion Molecules physiology, Neoplasm Metastasis pathology, Neoplasms pathology
- Abstract
Cadherins are a family of transmembrane glycoproteins which are responsible for calcium-dependent cell-cell adhesion. At least two types of proteins called alpha- and beta-catenin are known to be closely associated with the cytoplasmic domain of cadherin molecules and to play a crucial role in the regulation of cadherin cell adhesion function. Sequence analyses of cDNAs encoding these catenins have revealed that alpha- and beta-catenins have a similarity to vinculin and Drosophila armadillo protein, respectively. The possible involvement of these catenin molecules in the molecular mechanism of human cancer invasion and metastasis is discussed.
- Published
- 1993
389. Concentration of an integral membrane protein, CD43 (leukosialin, sialophorin), in the cleavage furrow through the interaction of its cytoplasmic domain with actin-based cytoskeletons.
- Author
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Yonemura S, Nagafuchi A, Sato N, and Tsukita S
- Subjects
- Actins analysis, Animals, Base Sequence, Cadherins analysis, Cadherins genetics, Cadherins metabolism, Cells, Cultured, Cytoskeleton ultrastructure, Fluorescent Antibody Technique, L Cells, Leukosialin, Lymphocytes immunology, Lymphocytes ultrastructure, Mice, Microscopy, Immunoelectron, Molecular Sequence Data, Oligodeoxyribonucleotides, Rats, Rats, Wistar, Recombinant Fusion Proteins analysis, Recombinant Fusion Proteins metabolism, Recombinant Proteins analysis, Recombinant Proteins metabolism, Sialoglycoproteins genetics, Transfection, Actins metabolism, Antigens, CD, Cytoskeleton metabolism, Lymphocytes cytology, Sialoglycoproteins analysis, Sialoglycoproteins metabolism
- Abstract
In leukocytes such as thymocytes and basophilic leukemia cells, a glycosilated integral membrane protein called CD43 (leukosialin or sialophorin), which is defective in patients with Wiskott-Aldrich syndrome, was highly concentrated in the cleavage furrow during cytokinesis. Not only at the mitotic phase but also at interphase, CD43 was precisely colocalized with ezrin-radixin-moesin family members. (ERM), which were previously reported to play an important role in the plasma membrane-actin filament association in general. At the electron microscopic level, throughout the cell cycle, both CD43 and ERM were tightly associated with microvilli, providing membrane attachment sites for actin filaments. We constructed a cDNA encoding a chimeric molecule consisting of the extracellular domain of mouse E-cadherin and the transmembrane/cytoplasmic domain of rat CD43, and introduced it into mouse L fibroblasts lacking both endogenous CD43 and E-cadherin. In dividing transfectants, the chimeric molecules were concentrated in the cleavage furrow together with ERM, and both proteins were precisely colocalized throughout the cell cycle. Furthermore, using this transfection system, we narrowed down the domain responsible for the CD43-concentration in the cleavage furrow. Based on these findings, we conclude that CD43 is concentrated in the cleavage furrow through the direct or indirect interaction of its cytoplasmic domain with ERM and actin filaments.
- Published
- 1993
- Full Text
- View/download PDF
390. Cadherin dysfunction in a human cancer cell line: possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesiveness.
- Author
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Shimoyama Y, Nagafuchi A, Fujita S, Gotoh M, Takeichi M, Tsukita S, and Hirohashi S
- Subjects
- Blotting, Northern, Cadherins analysis, Colonic Neoplasms metabolism, Colonic Neoplasms pathology, Cytoskeletal Proteins analysis, Humans, Intercellular Junctions physiology, Lung Neoplasms metabolism, Tumor Cells, Cultured, alpha Catenin, Cadherins metabolism, Cadherins physiology, Cell Adhesion physiology, Cell Communication physiology, Cytoskeletal Proteins metabolism, Lung Neoplasms pathology
- Abstract
A human lung cancer cell line, PC 9, was analyzed to elucidate the molecular mechanisms of dysfunction of cadherin-mediated cell-cell adhesion in cancer. Although PC 9 cells strongly expressed E-cadherin at the cell membrane, which was indistinguishable immunochemically from functional E-cadherin, they did not show tight cell-cell adhesion and had reduced E-cadherin-mediated aggregation activity. Immunoprecipitation with E-cadherin and Western blot analysis revealed that PC 9 cells did not express alpha-catenin, a cadherin-associated protein, suggesting that this was the cause of the cadherin dysfunction in the cell line. In addition, Northern and Southern blot analyses disclosed homozygous deletion of part of the alpha-catenin gene, which might have resulted in the loss of alpha-catenin expression in PC 9 cells.
- Published
- 1992
391. Molecular linkage between cadherins and actin filaments in cell-cell adherens junctions.
- Author
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Tsukita S, Tsukita S, Nagafuchi A, and Yonemura S
- Subjects
- Animals, Humans, Actins metabolism, Cadherins metabolism, Cell Adhesion, Intercellular Junctions metabolism
- Abstract
The cell-cell adherens junction is a site for cadherin-mediated cell adhesion where actin filaments are densely associated with the plasma membrane through its well-developed plasmalemmal undercoat. Recent research has focused on the molecular linkage between cadherins and actin filaments in the undercoat of adherens junctions in order to understand the functions of these undercoat-constitutive proteins in the regulation and signal transduction of cadherin-based cell adhesion.
- Published
- 1992
- Full Text
- View/download PDF
392. A case of subepidermal blister disease associated with autoantibody against 450 kD protein.
- Author
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Fujiwara S, Shinkai H, Takayasu S, Owaribe K, Tsukita S, and Kageshita T
- Subjects
- Aged, Aged, 80 and over, Blister blood, Blister diagnosis, Epidermis, Humans, Male, Molecular Weight, Pemphigoid, Bullous blood, Pemphigoid, Bullous diagnosis, Autoantibodies blood, Blister immunology, Pemphigoid, Bullous immunology
- Abstract
We report a case of subepidermal blister disease with eruptions distributed on the trunk and extremities. The blisters regressed after administration of corticosteroid and cyclophosphamide. An immunoblot analysis revealed that the patient's serum reacted exclusively with a 450-kD epidermal polypeptide. It did not react with a 230-kD or a 180-kD bullous pemphigoid antigen. The immunofluorescence study showed the patient's serum reacted with the basement membrane zone of human skin.
- Published
- 1992
- Full Text
- View/download PDF
393. A gene family consisting of ezrin, radixin and moesin. Its specific localization at actin filament/plasma membrane association sites.
- Author
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Sato N, Funayama N, Nagafuchi A, Yonemura S, Tsukita S, and Tsukita S
- Subjects
- Actins metabolism, Amino Acid Sequence, Animals, Base Sequence, Blood Proteins metabolism, Cell Line, Cell Membrane metabolism, DNA genetics, Humans, Immunohistochemistry, Intercellular Junctions metabolism, Membrane Proteins metabolism, Mice, Microvilli metabolism, Molecular Sequence Data, Phosphoproteins metabolism, Proteins metabolism, Restriction Mapping, Sequence Homology, Amino Acid, Blood Proteins genetics, Cytoskeletal Proteins, Membrane Proteins genetics, Microfilament Proteins, Multigene Family, Phosphoproteins genetics, Proteins genetics
- Abstract
Radixin is a barbed end-capping actin-modulating protein which was previously reported to be concentrated at cell-to-cell adherens junctions (AJ) and cleavage furrows. Recently, cDNA encoding mouse radixin was isolated, showing that radixin is highly homologous to but distinct from ezrin. From mouse teratocarcinoma cells we isolated and analyzed cDNA encoding another radixin-related protein. Sequence analysis has demonstrated that this protein is a mouse homologue of human moesin (98.3% identity) and that it shares 71.7% and 80.1% identity with ezrin and radixin, respectively. Translation experiments in vitro combined with immunoblot analyses led us to conclude that there is a gene family consisting of ezrin, radixin and moesin. These members are coexpressed in various types of cells. Then, by immunofluorescence microscopy, we closely analyzed their distribution using polyclonal and monoclonal antibodies, which could recognize all three members. In addition to cell-to-cell AJ and cleavage furrows, it was shown that they were concentrated at microvilli and ruffling membranes in various types of cells. Furthermore, the cell-to-substrate AJ (focal contacts) were clearly stained by anti-radixin pAb only after the apical/lateral membranes and cytoplasm were removed by the zinc method. We conclude that at least one of the members of the ezrin-radixin-moesin family is concentrated at specific regions where actin filaments are densely associated with plasma membranes.
- Published
- 1992
- Full Text
- View/download PDF
394. Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts.
- Author
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Matsuyoshi N, Hamaguchi M, Taniguchi S, Nagafuchi A, Tsukita S, and Takeichi M
- Subjects
- Animals, Benzoquinones, Cell Adhesion Molecules chemistry, Cell Adhesion Molecules metabolism, Cell Line, Cell Line, Transformed, Collagen, Fibroblasts cytology, Fibroblasts metabolism, Fluorescent Antibody Technique, Gels, Immunoblotting, Lactams, Macrocyclic, Oncogene Protein pp60(v-src) chemistry, Oncogene Protein pp60(v-src) genetics, Oncogenes, Phosphorylation, Rats, Rifabutin analogs & derivatives, Vanadates pharmacology, Cadherins physiology, Cell Adhesion, Neoplasm Metastasis, Oncogene Protein pp60(v-src) metabolism, Quinones pharmacology, Tyrosine metabolism
- Abstract
Rat 3Y1 cells acquire metastatic potential when transformed with v-src, and this potential is enhanced by double transformation with v-src and v-fos (Taniguchi, S., T. Kawano, T. Mitsudomi, G. Kimura, and T. Baba. 1986. Jpn. J. Cancer Res. 77:1193-1197). We compared the activity of cadherin cell adhesion molecules of normal 3Y1 cells with that of v-src transformed (SR3Y1) and v-src and v-fos double transformed (fosSR3Y1) 3Y1 cells. These cells expressed similar amounts of P-cadherin, and showed similar rates of cadherin-mediated aggregation under suspended conditions. However, the aggregates or colonies of these cells were morphologically distinct. Normal 3Y1 cells formed compacted aggregates in which cells are firmly connected with each other, whereas the transformed cells were more loosely associated, and could freely migrate out of the colonies. Overexpression of exogenous E-cadherin in these transformed cells had no significant effect on their adhesive properties. We then found that herbimycin A, a tyrosine kinase inhibitor, induced tighter cell-cell associations in the aggregates of the transformed cells. In contrast, vanadate, a tyrosine phosphatase inhibitor, inhibited the cadherin-mediated aggregation of SR3Y1 and fosSR3Y1 cells but had little effect on that of normal 3Y1 cells. These results suggest that v-src-mediated tyrosine phosphorylation perturbs cadherin function directly or indirectly, and the inhibition of tyrosine phosphorylation restores cadherin action to the normal state. We next studied tyrosine phosphorylation on cadherins and the cadherin-associated proteins, catenins. While similar amounts of catenins were expressed in all of these cells, the 98-kD catenin was strongly tyrosine phosphorylated only in SR3Y1 and fosSR3Y1 cells. Cadherins were also weakly tyrosine phosphorylated only in the transformed cells. The tyrosine phosphorylation of these proteins was enhanced by vanadate, and inhibited by herbimycin A. Thus, the tyrosine phosphorylation of the cadherin-catenin system itself might affect its function, causing instable cell-cell adhesion.
- Published
- 1992
- Full Text
- View/download PDF
395. Ca(2+)-regulated actin and phospholipid binding protein (68 kD-protein) from bovine liver: identification as a homologue for annexin VI and intracellular localization.
- Author
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Hosoya H, Kobayashi R, Tsukita S, and Matsumura F
- Subjects
- Actins drug effects, Amino Acid Sequence, Animals, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins pharmacology, Cattle, Cells, Cultured, Conserved Sequence, Immunoblotting, Immunohistochemistry, Molecular Sequence Data, Molecular Weight, Phospholipids metabolism, Actins metabolism, Annexin A6 chemistry, Calcium metabolism, Calcium-Binding Proteins isolation & purification, Liver chemistry
- Abstract
An F-actin binding protein was purified from bovine liver by means of DNase I affinity, hydroxylapatite and DEAE-cellulose column chromatographies. It consisted of a single polypeptide chain having an apparent molecular weight of 68,000 with a Stokes radius of 35 A. Electron microscopy of rotary shadowed specimens showed that the 68 kD protein is a globular protein. This protein showed a higher affinity for F-actin in the presence of Ca2+ than in its absence, which is opposite to the actin-binding property shown by nonmuscle alpha-actinin or fimbrin. The 68 kD protein had no F-actin severing and capping activity. Interestingly, the 68 kD protein was found to aggregate liposomes at micromolar Ca2+ concentrations. Immunoblot analysis and partial protein sequence data identified the 68 kD protein as an annexin VI (p68) homologue. Immunocytochemical studies showed that the 68 kD protein was localized along stress fibers as well as membrane ruffles, microspikes and focal contacts, raising the possibility that annexin VI may contribute to control membrane-microfilament interaction in the cell.
- Published
- 1992
- Full Text
- View/download PDF
396. A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites.
- Author
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Itoh M, Yonemura S, Nagafuchi A, Tsukita S, and Tsukita S
- Subjects
- 3T3 Cells, Animals, Binding Sites, Cell Line, Fibroblasts, Fluorescent Antibody Technique, Membrane Proteins immunology, Membrane Proteins metabolism, Mice, Microscopy, Immunoelectron, Rats, Spectrin metabolism, Antibodies, Monoclonal immunology, Cadherins metabolism, Cell Adhesion
- Abstract
Recently we developed an isolation procedure for the cell-to-cell adherens junctions (AJ; cadherin-based junctions) from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41). In this study, using the isolated AJ, we have obtained two mAbs specific to the 220-kD undercoat-constitutive protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this 220-kD protein was highly concentrated at the undercoat of cell-to-cell AJ in various types of tissues and that this protein was located in the immediate vicinity of the plasma membrane in the undercoat of AJ. In the cells lacking typical cell-to-cell AJ, such as fibroblasts, the 220-kD protein was immunofluorescently shown to be coconcentrated with cadherin molecules at cell-cell adhesion sites. These localization analyses appeared to indicate the possible direct or indirect association of the 220-kD protein with cadherin molecules. Furthermore, it was revealed that the 220-kD protein and alpha-spectrin were coimmunoprecipitated with the above mAbs in both the isolated AJ and the brain. The affinity-purified 220-kD protein molecule looked like a spherical particle, and its binding site on the spectrin molecule was shown to be in the position approximately 10-20 nm from the midpoint of spectrin tetramer by low-angle rotary-shadowing electron microscopy. Taking all these results together with biochemical and immunological comparisons, we are persuaded to speculate that the 220-kD protein is a novel member of the ankyrin family. However, the possibility cannot be excluded that the 220-kD protein is an isoform of beta-spectrin. The possible roles of this 220-kD protein in the association of cadherin molecules with the spectrin-based membrane skeletons at the cadherin-based cell-cell adhesion sites are discussed.
- Published
- 1991
- Full Text
- View/download PDF
397. Radixin is a novel member of the band 4.1 family.
- Author
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Funayama N, Nagafuchi A, Sato N, Tsukita S, and Tsukita S
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blood Proteins metabolism, DNA isolation & purification, Fluorescent Antibody Technique, Humans, Immunoblotting, Membrane Proteins metabolism, Mice, Molecular Sequence Data, Phosphoproteins genetics, Restriction Mapping, Sequence Alignment, Blood Proteins genetics, Cytoskeletal Proteins, Membrane Proteins genetics, Neuropeptides
- Abstract
Radixin is an actin barbed-end capping protein which is highly concentrated in the undercoat of the cell-to-cell adherens junction and the cleavage furrow in the interphase and mitotic phase, respectively (Tsukita, Sa., Y. Hieda, and Sh. Tsukita. 1989 a.J. Cell Biol. 108:2369-2382; Sato, N., S. Yonemura, T. Obinata, Sa. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 113:321-330). To further understand the structure and functions of the radixin molecule, we isolated and sequenced the cDNA clones encoding mouse radixin. Direct peptide sequencing of radixin and immunological analysis with antiserum to a fusion protein were performed to confirm that the protein encoded by these clones is identical to radixin. The composite cDNA is 4,241 nucleotides long and codes for a 583-amino acid polypeptide with a calculated molecular mass of 68.5 kD. Sequence analysis has demonstrated that mouse radixin shares 75.3% identity with human ezrin, which was reported to be a member of the band 4.1 family. We then isolated the cDNA encoding mouse ezrin. Sequence analysis and Northern blot analysis revealed that radixin and ezrin are similar but distinct (74.9% identity), leading us to conclude that radixin is a novel member of the band 4.1 family. In erythrocytes the band 4.1 protein acts as a key protein in the association of short actin filaments with a plasma membrane protein (glycophorin), together with spectrin. Therefore, the sequence similarity between radixin and band 4.1 protein described in this study favors the idea that radixin plays a crucial role in the association of the barbed ends of actin filaments with the plasma membrane in the cell-to-cell adherens junction and the cleavage furrow.
- Published
- 1991
- Full Text
- View/download PDF
398. The 102 kd cadherin-associated protein: similarity to vinculin and posttranscriptional regulation of expression.
- Author
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Nagafuchi A, Takeichi M, and Tsukita S
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Cadherins isolation & purification, Cell Line, Cytoskeletal Proteins isolation & purification, Cytoskeletal Proteins metabolism, DNA genetics, DNA isolation & purification, L Cells physiology, Mice, Molecular Sequence Data, Nucleic Acid Hybridization, Organ Specificity, RNA, Messenger genetics, Restriction Mapping, Sequence Homology, Nucleic Acid, Teratoma, Transfection, Vinculin, alpha Catenin, Cadherins genetics, Cadherins metabolism, Cytoskeletal Proteins genetics, Gene Expression Regulation, RNA Processing, Post-Transcriptional
- Abstract
The E-cadherin cell adhesion molecule is associated with cytoplasmic polypeptides, and this association is essential for its cell-binding function. Using isolated adherens junctions of the liver, we purified a 102 kd protein that can associate with E-cadherin (CAP102) and isolated cDNAs encoding this protein. Sequence analysis of the cDNAs revealed that this protein has a similarity to vinculin. L cells not expressing endogenous cadherin express the mRNA for CAP102 but have only a trace amount of CAP102 protein. Introducing exogenous E-cadherin into these cells, however, induced a high expression of CAP102 protein without affecting the amount of its mRNA, suggesting that there is a posttranscriptional regulatory mechanism for this molecule. The same effect was observed by introducing N- or P-cadherin into L cells.
- Published
- 1991
- Full Text
- View/download PDF
399. Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated.
- Author
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Tsukita S, Oishi K, Akiyama T, Yamanashi Y, Yamamoto T, and Tsukita S
- Subjects
- Age Factors, Animals, Cell Membrane metabolism, Electrophoresis, Gel, Two-Dimensional, Fluorescent Antibody Technique, Genes, src, Liver cytology, Phosphotyrosine, Proto-Oncogene Proteins c-yes, Rats, Tyrosine metabolism, Vanadates pharmacology, Cell Adhesion, Cell Communication, Protein-Tyrosine Kinases metabolism, Protein-Tyrosine Kinases physiology, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins pp60(c-src) physiology, Tyrosine analogs & derivatives, src-Family Kinases
- Abstract
To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.
- Published
- 1991
- Full Text
- View/download PDF
400. Radixin, a barbed end-capping actin-modulating protein, is concentrated at the cleavage furrow during cytokinesis.
- Author
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Sato N, Yonemura S, Obinata T, Tsukita S, and Tsukita S
- Subjects
- Animals, Antibodies, Monoclonal, Antibody Specificity, Cattle, Cell Adhesion, Cell Line, Cell Membrane metabolism, Chickens, Epitopes immunology, Fluorescent Antibody Technique, Macropodidae, Mice, Proteins immunology, Rats, Tumor Cells, Cultured, Cell Division, Cytoskeletal Proteins, Proteins metabolism
- Abstract
Radixin is a barbed end-capping actin-modulating protein which was first identified in isolated cell-to-cell adherens junctions from rat liver (Tsukita, Sa., Y. Hieda, and Sh. Tsukita, 1989. J. Cell Biol. 108:2369-2382). In the present study, we have analyzed the distribution of radixin in dividing cells. For this purpose, an mAb specific for radixin was obtained using chicken gizzard radixin as an antigen. By immunofluorescence microscopy with this mAb and a polyclonal antibody obtained previously, it was clearly shown in rat fibroblastic cells (3Y1 cells) that radixin was highly concentrated at the cleavage furrow during cytokinesis. Radixin appeared to accumulate rapidly at the cleavage furrow at the onset of furrowing, continued to be concentrated at the furrow during anaphase and telophase, and was finally enriched at the midbody. This concentration of radixin at the cleavage furrow was detected in all other cultured cells we examined: bovine epithelial cells (MDBK cells), mouse myeloma cells (P3 cells), rat kangaroo Ptk2 cells, mouse teratocarcinoma cells, and chicken fibroblasts. Furthermore, it became clear that the epitope for the mAb was immunofluorescently masked in the cell-to-cell adherens junctions. Together, these results lead us to conclude that radixin is present in the undercoat of the cell-to-cell adherens junctions and that of the cleavage furrow, although their respective molecular architectures are distinct. The possible roles of radixin at the cleavage furrow are discussed with special reference to the molecular mechanism of the actin filament-plasma membrane interaction at the furrow.
- Published
- 1991
- Full Text
- View/download PDF
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