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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 expression delays recruitment of β-catenin to cell–cell contacts. β-Catenin and ZO-1 localization in MDCK/NZO-3 cells was monitored at 6, 12, 24, and 48 h after the re-addition of calcium in a calcium switch experiment. NZO-3–expressing cells show a delayed β-catenin recruitment to intercellular junctions, which parallels the delay of E-cadherin recruitment (Fig. 7). The cells that show β-catenin localization at cell peripheries at early time points coincide with cells showing similar distribution of ZO-1, although many cells show β-catenin at cell borders preceding ZO-1. β-Catenin staining at cell borders is not complete until 24 h after the re-addition of calcium in MDCK/NZO-3 cells. Bar, 30 μm.
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Figure 8: NZO-3 expression delays recruitment of β-catenin to cell–cell contacts. β-Catenin and ZO-1 localization in MDCK/NZO-3 cells was monitored at 6, 12, 24, and 48 h after the re-addition of calcium in a calcium switch experiment. NZO-3–expressing cells show a delayed β-catenin recruitment to intercellular junctions, which parallels the delay of E-cadherin recruitment (Fig. 7). The cells that show β-catenin localization at cell peripheries at early time points coincide with cells showing similar distribution of ZO-1, although many cells show β-catenin at cell borders preceding ZO-1. β-Catenin staining at cell borders is not complete until 24 h after the re-addition of calcium in MDCK/NZO-3 cells. Bar, 30 μm.

Mentions: As the prevalent model of TJ formation involves coordinated assembly with the AJ, we were interested to determine if E-cadherin was delayed in its movement to the junctional complex in MDCK/NZO-3 cells in a calcium switch. In parental cells, E-cadherin appears at all cell–cell contacts 6 h after re-addition of calcium (Fig. 7). This time course of E-cadherin recruitment to the AJ mirrors that of TJ assembly and recruitment of ZO-1 to the TJ (Fig. 4 A). Similar to ZO-1, E-cadherin localization during the calcium switch was also delayed in NZO-3–expressing cells (Fig. 7). At 6 h, E-cadherin is localized to cell–cell contacts in small, isolated clusters of cells; these cell clusters also show ZO-1 localization to the junctional complex. An increasing number of cells show junctional E-cadherin staining at 12 and 24 h, but it is not until 48 h after calcium re-addition that E-cadherin is completely recruited to all cell borders in MDCK/NZO-3 cells. In addition, we identified cells where junctional recruitment of E-cadherin precedes that of ZO-1. The disruption of E-cadherin localization in MDCK/NZO-3 cells after calcium switch indicates that adherens and TJs are both affected by exogenous expression of the NZO-3 construct. We corroborated our E-cadherin findings by assessing the localization of β-catenin, an AJ protein peripherally associated with the membrane (Fig. 8). In parental cells, β-catenin appears at cell–cell contacts 6 h after calcium re-addition in a manner identical to the localization of E-cadherin after calcium switch (data not shown). However, in a distribution similar to E-cadherin and ZO-1, β-catenin recruitment to the lateral cell surface during junctional complex assembly was delayed in MDCK/NZO-3 cells.


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 expression delays recruitment of β-catenin to cell–cell contacts. β-Catenin and ZO-1 localization in MDCK/NZO-3 cells was monitored at 6, 12, 24, and 48 h after the re-addition of calcium in a calcium switch experiment. NZO-3–expressing cells show a delayed β-catenin recruitment to intercellular junctions, which parallels the delay of E-cadherin recruitment (Fig. 7). The cells that show β-catenin localization at cell peripheries at early time points coincide with cells showing similar distribution of ZO-1, although many cells show β-catenin at cell borders preceding ZO-1. β-Catenin staining at cell borders is not complete until 24 h after the re-addition of calcium in MDCK/NZO-3 cells. Bar, 30 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169439&req=5

Figure 8: NZO-3 expression delays recruitment of β-catenin to cell–cell contacts. β-Catenin and ZO-1 localization in MDCK/NZO-3 cells was monitored at 6, 12, 24, and 48 h after the re-addition of calcium in a calcium switch experiment. NZO-3–expressing cells show a delayed β-catenin recruitment to intercellular junctions, which parallels the delay of E-cadherin recruitment (Fig. 7). The cells that show β-catenin localization at cell peripheries at early time points coincide with cells showing similar distribution of ZO-1, although many cells show β-catenin at cell borders preceding ZO-1. β-Catenin staining at cell borders is not complete until 24 h after the re-addition of calcium in MDCK/NZO-3 cells. Bar, 30 μm.
Mentions: As the prevalent model of TJ formation involves coordinated assembly with the AJ, we were interested to determine if E-cadherin was delayed in its movement to the junctional complex in MDCK/NZO-3 cells in a calcium switch. In parental cells, E-cadherin appears at all cell–cell contacts 6 h after re-addition of calcium (Fig. 7). This time course of E-cadherin recruitment to the AJ mirrors that of TJ assembly and recruitment of ZO-1 to the TJ (Fig. 4 A). Similar to ZO-1, E-cadherin localization during the calcium switch was also delayed in NZO-3–expressing cells (Fig. 7). At 6 h, E-cadherin is localized to cell–cell contacts in small, isolated clusters of cells; these cell clusters also show ZO-1 localization to the junctional complex. An increasing number of cells show junctional E-cadherin staining at 12 and 24 h, but it is not until 48 h after calcium re-addition that E-cadherin is completely recruited to all cell borders in MDCK/NZO-3 cells. In addition, we identified cells where junctional recruitment of E-cadherin precedes that of ZO-1. The disruption of E-cadherin localization in MDCK/NZO-3 cells after calcium switch indicates that adherens and TJs are both affected by exogenous expression of the NZO-3 construct. We corroborated our E-cadherin findings by assessing the localization of β-catenin, an AJ protein peripherally associated with the membrane (Fig. 8). In parental cells, β-catenin appears at cell–cell contacts 6 h after calcium re-addition in a manner identical to the localization of E-cadherin after calcium switch (data not shown). However, in a distribution similar to E-cadherin and ZO-1, β-catenin recruitment to the lateral cell surface during junctional complex assembly was delayed in MDCK/NZO-3 cells.

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