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Cited by 677Cell Adhesion Molecules ResearchSkin and Cellular Biology ResearchBarrier Structure and Function Studies

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Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to α Catenin and Actin Filaments

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Itoh, Masahiko, Nagafuchi, Akira, Moroi, Seiji, Tsukita, Shoichiro·1997·The Journal of Cell Biology
DOI 10.1083/jcb.138.1.181PMC PMC2139940
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Abstract

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

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Figures

Figure 1 Structure of c-myc– tagged full-length ZO-1 (F-ZO-1), dlg-like amino-terminal half ZO-1 (N-ZO-1), and carboxyl-terminal half ZO-1 (C-ZO-1). A tandem array of seven c-myc epitopes (myc; EQKLISEEDL) was tagged to the carboxyl terminus of each molecule. F-ZO-1 contains three PDZ domains (PDZ1-3), one SH3 domain (SH3), one guanylate kinase domain (GUK), and a proline-rich domain (proline-rich). The α+ isotype with an insert (α) was used in this study.
Figure 1 Structure of c-myc– tagged full-length ZO-1 (F-ZO-1), dlg-like amino-terminal half ZO-1 (N-ZO-1), and carboxyl-terminal half ZO-1 (C-ZO-1). A tandem array of seven c-myc epitopes (myc; EQKLISEEDL) was tagged to the carboxyl terminus of each molecule. F-ZO-1 contains three PDZ domains (PDZ1-3), one SH3 domain (SH3), one guanylate kinase domain (GUK), and a proline-rich domain (proline-rich). The α+ isotype with an insert (α) was used in this study.CC BY-NC-SA · PubMed Central
Figure 5 Association of N-ZO-1 with α catenin in vitro. (a) GST fusion proteins with the cytoplasmic domain of occludin, the cytoplasmic domain of E-cadherin, α catenin, and β catenin, which were bound to glutathione-Sepharose beads, were incubated with the lysate of Sf9 cells expressing N-ZO-1. After washing, the proteins associated with GST fusion proteins were eluted from the beads with a buffer containing glutathione. The Sf9 cell lysate (Sf9 lysate), the eluate from GST– occludin beads (GST/Occ-eluate), the eluate from GST–E-cadherin beads (GST/Cad-eluate), the eluate from GST–α catenin beads (GST/ α cat-eluate), the eluate from GST–β catenin beads (GST/β cat-eluate), and purified GST–α catenin (GST/α cat) were separated by SDS-PAGE followed by immunoblotting with anti–ZO-1 mAb, T8-754. Although the N-ZO-1 band was not resolved from the GST–α catenin band by CBB staining, immunoblotting identified N-ZO-1 (arrowhead) in the eluate from GST–α catenin beads at the same amount as in that from GST–occludin beads (arrow), in which the N-ZO-1 band was clearly visualized by CBB staining (asterisk). N-ZO-1 was not detected in the eluates from GST–E-cadherin or GST–β catenin beads. Comparison of the CBB staining patterns between the GST–α catenin beads eluate (GST/α cat-eluate) and the purified GST–α catenin (GST/α cat) revealed that only N-ZO-1 was precipitated out from the crude Sf9 lysate by GST–α catenin, indicating the direct α catenin/N-ZO-1 interaction. 1, 2, 3, and 4 indicate the bands of GST–occludin, GST–E-cadherin, GST–α catenin, and GST–β catenin, respectively, and the other lower molecular mass bands are their degradation products. (b) GST fusion protein with α catenin, which was bound by glutathione-Sepharose beads, was incubated with the lysate of Sf9 cells expressing C-ZO-1. After washing, the proteins associated with the GST fusion protein were eluted from the beads with a buffer containing glutathione. The Sf9 cell lysate (Sf9-lysate) and the eluate from GST–α-catenin beads (GST/α cat-eluate) were separated by SDS-PAGE followed by immunoblotting with anti–C-ZO-1 mAb, T7-713. 5, the band of GST–α catenin. No specific binding was detected between GST–α-catenin and C-ZO-1 (arrow). (c) Quantitative analysis of the binding between N-ZO-1 and α catenin. Glutathione-Sepharose bead slurry containing 40 μg of GST–α catenin was incubated with the Sf9 cell lysate containing 0.012–0.6 μg of N-ZO-1. The amounts of N-ZO-1 in the Sf9 cell lysate and in each eluate (inset) were estimated as described in Materials and Methods. Each point represents the mean ± SD of triplicate determinations. The binding was saturable, and Scatchard analysis (inset) indicated that the K d was ∼0.5 nM.
Figure 5 Association of N-ZO-1 with α catenin in vitro. (a) GST fusion proteins with the cytoplasmic domain of occludin, the cytoplasmic domain of E-cadherin, α catenin, and β catenin, which were bound to glutathione-Sepharose beads, were incubated with the lysate of Sf9 cells expressing N-ZO-1. After washing, the proteins associated with GST fusion proteins were eluted from the beads with a buffer containing glutathione. The Sf9 cell lysate (Sf9 lysate), the eluate from GST– occludin beads (GST/Occ-eluate), the eluate from GST–E-cadherin beads (GST/Cad-eluate), the eluate from GST–α catenin beads (GST/ α cat-eluate), the eluate from GST–β catenin beads (GST/β cat-eluate), and purified GST–α catenin (GST/α cat) were separated by SDS-PAGE followed by immunoblotting with anti–ZO-1 mAb, T8-754. Although the N-ZO-1 band was not resolved from the GST–α catenin band by CBB staining, immunoblotting identified N-ZO-1 (arrowhead) in the eluate from GST–α catenin beads at the same amount as in that from GST–occludin beads (arrow), in which the N-ZO-1 band was clearly visualized by CBB staining (asterisk). N-ZO-1 was not detected in the eluates from GST–E-cadherin or GST–β catenin beads. Comparison of the CBB staining patterns between the GST–α catenin beads eluate (GST/α cat-eluate) and the purified GST–α catenin (GST/α cat) revealed that only N-ZO-1 was precipitated out from the crude Sf9 lysate by GST–α catenin, indicating the direct α catenin/N-ZO-1 interaction. 1, 2, 3, and 4 indicate the bands of GST–occludin, GST–E-cadherin, GST–α catenin, and GST–β catenin, respectively, and the other lower molecular mass bands are their degradation products. (b) GST fusion protein with α catenin, which was bound by glutathione-Sepharose beads, was incubated with the lysate of Sf9 cells expressing C-ZO-1. After washing, the proteins associated with the GST fusion protein were eluted from the beads with a buffer containing glutathione. The Sf9 cell lysate (Sf9-lysate) and the eluate from GST–α-catenin beads (GST/α cat-eluate) were separated by SDS-PAGE followed by immunoblotting with anti–C-ZO-1 mAb, T7-713. 5, the band of GST–α catenin. No specific binding was detected between GST–α-catenin and C-ZO-1 (arrow). (c) Quantitative analysis of the binding between N-ZO-1 and α catenin. Glutathione-Sepharose bead slurry containing 40 μg of GST–α catenin was incubated with the Sf9 cell lysate containing 0.012–0.6 μg of N-ZO-1. The amounts of N-ZO-1 in the Sf9 cell lysate and in each eluate (inset) were estimated as described in Materials and Methods. Each point represents the mean ± SD of triplicate determinations. The binding was saturable, and Scatchard analysis (inset) indicated that the K d was ∼0.5 nM.CC BY-NC-SA · PubMed Central
Figure 6 Association of C-ZO-1 with actin filaments in vitro. (a) Sf9 cell lysate containing C-ZO-1 (Sf9-lysate) was incubated with actin filaments (+Actin) or BSA (+BSA). After centrifugation, the supernatant (S) and pellet (P) were resolved by SDS-PAGE, followed by immunoblotting with anti–ZO-1 mAb, T7-713. From crude Sf9 cell lysate, only the C-ZO-1 band was precipitated out by actin filaments (arrows), but not by BSA. Arrowheads indicate actin or BSA. (b) Quantitative analysis of the binding between C-ZO-1 and actin filaments. Actin filaments were incubated with the Sf9 cell lysate containing 0.2–2 μg of C-ZO-1. The amounts of C-ZO-1 in the Sf9 cell lysate and in each pellet (inset) were estimated as described in Materials and Methods. Each point represents the mean ± SD of triplicate determinations. The binding was saturable, and Scatchard analysis (inset) indicated that the K d was ∼10 nM.
Figure 6 Association of C-ZO-1 with actin filaments in vitro. (a) Sf9 cell lysate containing C-ZO-1 (Sf9-lysate) was incubated with actin filaments (+Actin) or BSA (+BSA). After centrifugation, the supernatant (S) and pellet (P) were resolved by SDS-PAGE, followed by immunoblotting with anti–ZO-1 mAb, T7-713. From crude Sf9 cell lysate, only the C-ZO-1 band was precipitated out by actin filaments (arrows), but not by BSA. Arrowheads indicate actin or BSA. (b) Quantitative analysis of the binding between C-ZO-1 and actin filaments. Actin filaments were incubated with the Sf9 cell lysate containing 0.2–2 μg of C-ZO-1. The amounts of C-ZO-1 in the Sf9 cell lysate and in each pellet (inset) were estimated as described in Materials and Methods. Each point represents the mean ± SD of triplicate determinations. The binding was saturable, and Scatchard analysis (inset) indicated that the K d was ∼10 nM.CC BY-NC-SA · PubMed Central
Figure 2 Concentration of full-length ZO-1 at cadherin-based cell adhesion sites. When parent EL cells were doubly stained with anti– ZO-1 mAb, T8-754 (a), and anti–E-cadherin pAb (b), both signals were colocalized at the cell–cell border. Introduced myc-tagged full-length ZO-1 was similarly distributed in EL transfectants (FZ-EL cells) as revealed by anti–c-myc tag mAb, 9E10 (c). This mAb gave a signal in nuclei but not in cell–cell borders of parent EL cells (d). Bar, 10 μm.
Figure 2 Concentration of full-length ZO-1 at cadherin-based cell adhesion sites. When parent EL cells were doubly stained with anti– ZO-1 mAb, T8-754 (a), and anti–E-cadherin pAb (b), both signals were colocalized at the cell–cell border. Introduced myc-tagged full-length ZO-1 was similarly distributed in EL transfectants (FZ-EL cells) as revealed by anti–c-myc tag mAb, 9E10 (c). This mAb gave a signal in nuclei but not in cell–cell borders of parent EL cells (d). Bar, 10 μm.CC BY-NC-SA · PubMed Central
Figure 3 Subcellular distribution of N-ZO-1 and C-ZO-1 in EL transfectants (NZ-EL and CZ-EL, respectively). NZ-EL cells (a, b, and b′) were singly stained with anti–c-myc tag mAb, 9E10 (a), or doubly stained with 9E10 (b) and anti–E-cadherin pAb (b′). In addition to the diffuse cytoplasmic distribution, introduced N-ZO-1 was significantly concentrated at the cell–cell border, where E-cadherin was colocalized. N-ZO-1 and E-cadherin showed a tendency to distribute in a linear fashion, but depending on the culture conditions they showed a serrated concentration pattern (inset, 3,000×). CZ-EL cells (c, d, and d′) were singly stained with anti–c-myc tag mAb, 9E10 (c), or doubly stained with 9E10 (d) and rhodamine phalloidin (d′). Introduced C-ZO-1 was distributed along actin stress fibers visualized with rhodamine phalloidin. Bars: (a–c) 10 μm; (d and d′) 20 μm.
Figure 3 Subcellular distribution of N-ZO-1 and C-ZO-1 in EL transfectants (NZ-EL and CZ-EL, respectively). NZ-EL cells (a, b, and b′) were singly stained with anti–c-myc tag mAb, 9E10 (a), or doubly stained with 9E10 (b) and anti–E-cadherin pAb (b′). In addition to the diffuse cytoplasmic distribution, introduced N-ZO-1 was significantly concentrated at the cell–cell border, where E-cadherin was colocalized. N-ZO-1 and E-cadherin showed a tendency to distribute in a linear fashion, but depending on the culture conditions they showed a serrated concentration pattern (inset, 3,000×). CZ-EL cells (c, d, and d′) were singly stained with anti–c-myc tag mAb, 9E10 (c), or doubly stained with 9E10 (d) and rhodamine phalloidin (d′). Introduced C-ZO-1 was distributed along actin stress fibers visualized with rhodamine phalloidin. Bars: (a–c) 10 μm; (d and d′) 20 μm.CC BY-NC-SA · PubMed Central
Figure 4 Association of N-ZO-1 with the E-cadherin/ catenin complex in NZ-EL cells. (a) Silver staining pattern of E-cadherin immunoprecipitates from EL or NZ-EL cells and accompanying immunoblots with anti–ZO-1 mAb, T8-754, specific for the amino-terminal half of ZO-1. When E-cadherin (Cad) was immunoprecipitated with rat anti–E-cadherin mAb, ECCD-2 (anti-cad), from the NP-40 extract of EL or NZ-EL cells, α and β catenins were coimmunoprecipitated. (α and β, respectively). In the E-cadherin immunoprecipitates from NZ-EL cells, β catenin appeared to be degraded to some extent. Rat IgG (r-IgG) was used for control immunoprecipitation. Asterisks indicate IgG. Immunoblotting with anti–ZO-1 mAb detected N-ZO-1 (arrowhead) in the E-cadherin immunoprecipitate from NZ-EL cells but did not detect endogenous full-length ZO-1 in the E-cadherin immunoprecipitate from either NZ-EL or EL cells. (b) Immunoprecipitation with anti–c-myc tag mAb, 9E10 (anti-myc), from the NP-40 extract of NZ-EL cells. Silver staining detected the N-ZO-1 band (arrow) in addition to IgG bands (asterisks), and immunoblotting identified E-cadherin, α catenin, and β catenin in the immunoprecipitate. Mouse IgG (m-IgG) was used for control immunoprecipitation.
Figure 4 Association of N-ZO-1 with the E-cadherin/ catenin complex in NZ-EL cells. (a) Silver staining pattern of E-cadherin immunoprecipitates from EL or NZ-EL cells and accompanying immunoblots with anti–ZO-1 mAb, T8-754, specific for the amino-terminal half of ZO-1. When E-cadherin (Cad) was immunoprecipitated with rat anti–E-cadherin mAb, ECCD-2 (anti-cad), from the NP-40 extract of EL or NZ-EL cells, α and β catenins were coimmunoprecipitated. (α and β, respectively). In the E-cadherin immunoprecipitates from NZ-EL cells, β catenin appeared to be degraded to some extent. Rat IgG (r-IgG) was used for control immunoprecipitation. Asterisks indicate IgG. Immunoblotting with anti–ZO-1 mAb detected N-ZO-1 (arrowhead) in the E-cadherin immunoprecipitate from NZ-EL cells but did not detect endogenous full-length ZO-1 in the E-cadherin immunoprecipitate from either NZ-EL or EL cells. (b) Immunoprecipitation with anti–c-myc tag mAb, 9E10 (anti-myc), from the NP-40 extract of NZ-EL cells. Silver staining detected the N-ZO-1 band (arrow) in addition to IgG bands (asterisks), and immunoblotting identified E-cadherin, α catenin, and β catenin in the immunoprecipitate. Mouse IgG (m-IgG) was used for control immunoprecipitation.CC BY-NC-SA · PubMed Central
Figure 7 Cell aggregation assay of EL and NZ-EL cells. Single cells were rotated for 30 min, 2 h, or 6 h on a gyratory shaker at 80 rpm. No significant differences were detected in cell aggregation activity between EL and NZ-EL cells. Bar, 50 μm.
Figure 7 Cell aggregation assay of EL and NZ-EL cells. Single cells were rotated for 30 min, 2 h, or 6 h on a gyratory shaker at 80 rpm. No significant differences were detected in cell aggregation activity between EL and NZ-EL cells. Bar, 50 μm.CC BY-NC-SA · PubMed Central
Figure 8 Intercellular motility of L, EL, and NZ-EL cells. (a) In the intercellular motility assay (on cell sheet), the fluorescence microscopic images of four DiI-stained sister cells (white) were superimposed on the phase contrast image of a confluent cell sheet. As compared to EL/EL analysis (see Materials and Methods for details), the intercellular motility in the NZ-EL/NZ-EL analysis was suppressed down to the level of that in L/ L analysis. These cell lines showed similar scattering properties on plastic dishes in the absence of a cell sheet (on dish). (b) Quantitative analysis of the intercellular motility. Higher values on the abscissa (intercellular motility index [Dc/Dd]; see Materials and Methods for details) represent the higher degrees of intercellular motility. At least 24 independent samples were picked up to determine the intercellular motility index for each analysis. Bar, 10 μm.
Figure 8 Intercellular motility of L, EL, and NZ-EL cells. (a) In the intercellular motility assay (on cell sheet), the fluorescence microscopic images of four DiI-stained sister cells (white) were superimposed on the phase contrast image of a confluent cell sheet. As compared to EL/EL analysis (see Materials and Methods for details), the intercellular motility in the NZ-EL/NZ-EL analysis was suppressed down to the level of that in L/ L analysis. These cell lines showed similar scattering properties on plastic dishes in the absence of a cell sheet (on dish). (b) Quantitative analysis of the intercellular motility. Higher values on the abscissa (intercellular motility index [Dc/Dd]; see Materials and Methods for details) represent the higher degrees of intercellular motility. At least 24 independent samples were picked up to determine the intercellular motility index for each analysis. Bar, 10 μm.CC BY-NC-SA · PubMed Central

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Authors
Itoh, M., Nagafuchi, A., Moroi, S., & Tsukita, S.
Affiliations
  • Kyoto University (JP)
  • *Department of Cell Biology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan; and ‡Department of Urology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
1997-07-14
Volume
138
Issue
1
Pages
181-192
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.138.1.181
PMC
PMC2139940
OpenAlex
W2102090879
Cited by
677
References
71
Citations by year
2026: 10 · 2025: 13 · 2024: 15 · 2023: 14 · 2022: 20 · 2021: 18 · 2020: 21 · 2019: 17 · 2018: 21 · 2017: 21 · 2016: 13 · 2015: 9 · 2014: 15 · 2013: 30 · 2012: 22
FWCI
14.93
Type
journal article
Language
en
Open access
Locations
  • The Journal of Cell Biology (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Topics
  • Health Sciences › Medicine › Immunology and Allergy › Cell Adhesion Molecules Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Skin and Cellular Biology Research
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
ZO-1, E-cadherin, alpha-catenin, F-actin, cell-cell adhesion, stress fibers, cell aggregation, protein-protein interaction, L fibroblasts
MeSH terms
Actins, Animals, Cell Adhesion, Cell Line, Cytoskeletal Proteins, L Cells, Membrane Proteins, Phosphoproteins, Recombinant Fusion Proteins, Trans-Activators, Cadherins, Spodoptera, beta Catenin, alpha Catenin, Mice, Rats, Zonula Occludens-1 Protein

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