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Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.

Wittchen ES, Haskins J, Stevenson BR - J. Cell Biol. (2000)

Bottom Line: Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics.NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly.We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

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NZO-3 colocalizes with ZO-1 around cell borders during TJ assembly after calcium switch. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch and then costained with anti–ZO-1 and anti-VSVG (NZO-3) antibodies at 6, 12, 24, and 48 h after the re-addition of calcium. Parental MDCK cells stained with anti-VSVG antibodies show nonspecific nuclear staining. In MDCK/NZO-3 cells, NZO-3 is present at cell borders of small groups of cells where it colocalizes with ZO-1. These groups of cells showing NZO-3 and ZO-1 around their peripheries enlarge over time, with complete localization at cell borders by 48 h. Boxed areas are enlarged in B. (B) NZO-3 colocalizes with ZO-1 along extended cell peripheries, but not in intracellular aggregates or initial planes of cell–cell contact. Shown is a cell with mature TJ staining (asterisk) where ZO-1 and NZO-3 colocalize. However, NZO-3 does not colocalize with ZO-1 at initial cell–cell contact planes (arrow) or in large intracellular aggregates (arrowheads). Bars: (A) 30 μm; (B) 10 μm.
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Figure 5: NZO-3 colocalizes with ZO-1 around cell borders during TJ assembly after calcium switch. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch and then costained with anti–ZO-1 and anti-VSVG (NZO-3) antibodies at 6, 12, 24, and 48 h after the re-addition of calcium. Parental MDCK cells stained with anti-VSVG antibodies show nonspecific nuclear staining. In MDCK/NZO-3 cells, NZO-3 is present at cell borders of small groups of cells where it colocalizes with ZO-1. These groups of cells showing NZO-3 and ZO-1 around their peripheries enlarge over time, with complete localization at cell borders by 48 h. Boxed areas are enlarged in B. (B) NZO-3 colocalizes with ZO-1 along extended cell peripheries, but not in intracellular aggregates or initial planes of cell–cell contact. Shown is a cell with mature TJ staining (asterisk) where ZO-1 and NZO-3 colocalize. However, NZO-3 does not colocalize with ZO-1 at initial cell–cell contact planes (arrow) or in large intracellular aggregates (arrowheads). Bars: (A) 30 μm; (B) 10 μm.

Mentions: We first determined which region of ZO-3 encodes the information for targeting the protein to the TJ. MDCK cells were stably transfected with the VSVG-tagged FLZO-3, NZO-3, and CZO-3 constructs (Fig. 1). Subcellular localization of the constructs was detected by indirect immunofluorescence using anti-VSVG antibodies and costaining for ZO-1 as a marker for the TJ. FLZO-3 correctly targets to cell borders, colocalizing with the TJ marker ZO-1 (data not shown). The NZO-3 construct also colocalizes with endogenous ZO-1 at cell borders (Fig. 2); furthermore, its localization was also indistinguishable from endogenous ZO-1 as assayed by 0.2 μm Z-section confocal microscopy (data not shown). The nuclear staining visible with the anti-VSVG staining is nonspecific as the same staining pattern is seen in untransfected parental MDCK cells (see Fig. 5 A). Fig. 2 also shows that when expressed in MDCK cells, CZO-3 is absent from cell borders and is instead diffusely distributed in the cytoplasm. Expression of the CZO-3 construct in these cells was confirmed by immunoblot (data not shown). Combined, these data indicate that the information necessary for proper targeting of ZO-3 to the TJ is contained within the amino-terminal half of the protein.


Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.

Wittchen ES, Haskins J, Stevenson BR - J. Cell Biol. (2000)

NZO-3 colocalizes with ZO-1 around cell borders during TJ assembly after calcium switch. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch and then costained with anti–ZO-1 and anti-VSVG (NZO-3) antibodies at 6, 12, 24, and 48 h after the re-addition of calcium. Parental MDCK cells stained with anti-VSVG antibodies show nonspecific nuclear staining. In MDCK/NZO-3 cells, NZO-3 is present at cell borders of small groups of cells where it colocalizes with ZO-1. These groups of cells showing NZO-3 and ZO-1 around their peripheries enlarge over time, with complete localization at cell borders by 48 h. Boxed areas are enlarged in B. (B) NZO-3 colocalizes with ZO-1 along extended cell peripheries, but not in intracellular aggregates or initial planes of cell–cell contact. Shown is a cell with mature TJ staining (asterisk) where ZO-1 and NZO-3 colocalize. However, NZO-3 does not colocalize with ZO-1 at initial cell–cell contact planes (arrow) or in large intracellular aggregates (arrowheads). Bars: (A) 30 μm; (B) 10 μm.
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Figure 5: NZO-3 colocalizes with ZO-1 around cell borders during TJ assembly after calcium switch. (A) Parental MDCK (P) and MDCK/NZO-3 (NZO-3) cells were subjected to a calcium switch and then costained with anti–ZO-1 and anti-VSVG (NZO-3) antibodies at 6, 12, 24, and 48 h after the re-addition of calcium. Parental MDCK cells stained with anti-VSVG antibodies show nonspecific nuclear staining. In MDCK/NZO-3 cells, NZO-3 is present at cell borders of small groups of cells where it colocalizes with ZO-1. These groups of cells showing NZO-3 and ZO-1 around their peripheries enlarge over time, with complete localization at cell borders by 48 h. Boxed areas are enlarged in B. (B) NZO-3 colocalizes with ZO-1 along extended cell peripheries, but not in intracellular aggregates or initial planes of cell–cell contact. Shown is a cell with mature TJ staining (asterisk) where ZO-1 and NZO-3 colocalize. However, NZO-3 does not colocalize with ZO-1 at initial cell–cell contact planes (arrow) or in large intracellular aggregates (arrowheads). Bars: (A) 30 μm; (B) 10 μm.
Mentions: We first determined which region of ZO-3 encodes the information for targeting the protein to the TJ. MDCK cells were stably transfected with the VSVG-tagged FLZO-3, NZO-3, and CZO-3 constructs (Fig. 1). Subcellular localization of the constructs was detected by indirect immunofluorescence using anti-VSVG antibodies and costaining for ZO-1 as a marker for the TJ. FLZO-3 correctly targets to cell borders, colocalizing with the TJ marker ZO-1 (data not shown). The NZO-3 construct also colocalizes with endogenous ZO-1 at cell borders (Fig. 2); furthermore, its localization was also indistinguishable from endogenous ZO-1 as assayed by 0.2 μm Z-section confocal microscopy (data not shown). The nuclear staining visible with the anti-VSVG staining is nonspecific as the same staining pattern is seen in untransfected parental MDCK cells (see Fig. 5 A). Fig. 2 also shows that when expressed in MDCK cells, CZO-3 is absent from cell borders and is instead diffusely distributed in the cytoplasm. Expression of the CZO-3 construct in these cells was confirmed by immunoblot (data not shown). Combined, these data indicate that the information necessary for proper targeting of ZO-3 to the TJ is contained within the amino-terminal half of the protein.

Bottom Line: Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics.NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly.We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.

ABSTRACT
The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.

Show MeSH
Related in: MedlinePlus