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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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Expression of NZO-3 delays TJ formation. (A) NZO-3 expression in MDCK cells subjected to a calcium switch delays reestablishment of TER. Filter-grown confluent monolayers of untransfected parental cells or MDCK cell lines stably expressing NZO-3, CZO-3, or FLZO-3 were incubated in calcium-free media for ∼15 h to disrupt intercellular junctions and then switched to media containing 1.8 mM calcium at t = 0 to induce synchronous assembly of intercellular junctions. TER was measured over 96 h after calcium switch. CZO-3 and FLZO-3 expressing cell lines show TER recovery dynamics similar to parental cells. NZO-3–expressing cells display a delay in TER recovery, but reach the same TER as the other cell lines by 48–96 h. (B) Expression of NZO-3 delays TER recovery in MDCK cells after cD treatment. cD was added to filter-grown confluent monolayers at t = 0 and then washed out after 60 min. TER measurements were taken every 30 min to monitor TJ breakdown and reformation. TER for each cell line is expressed as percentages of the values at t = 0. After 60 min of cD treatment, all cell lines exhibit a significant drop in TER. After cD washout, MDCK/CZO-3, MDCK/FLZO-3 and parental cells recover TER to values greater than t = 0, whereas MDCK/NZO-3 cells do not fully recover TER in the duration of the experiment.
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Figure 3: Expression of NZO-3 delays TJ formation. (A) NZO-3 expression in MDCK cells subjected to a calcium switch delays reestablishment of TER. Filter-grown confluent monolayers of untransfected parental cells or MDCK cell lines stably expressing NZO-3, CZO-3, or FLZO-3 were incubated in calcium-free media for ∼15 h to disrupt intercellular junctions and then switched to media containing 1.8 mM calcium at t = 0 to induce synchronous assembly of intercellular junctions. TER was measured over 96 h after calcium switch. CZO-3 and FLZO-3 expressing cell lines show TER recovery dynamics similar to parental cells. NZO-3–expressing cells display a delay in TER recovery, but reach the same TER as the other cell lines by 48–96 h. (B) Expression of NZO-3 delays TER recovery in MDCK cells after cD treatment. cD was added to filter-grown confluent monolayers at t = 0 and then washed out after 60 min. TER measurements were taken every 30 min to monitor TJ breakdown and reformation. TER for each cell line is expressed as percentages of the values at t = 0. After 60 min of cD treatment, all cell lines exhibit a significant drop in TER. After cD washout, MDCK/CZO-3, MDCK/FLZO-3 and parental cells recover TER to values greater than t = 0, whereas MDCK/NZO-3 cells do not fully recover TER in the duration of the experiment.

Mentions: TER was measured with a Millicell-ERS apparatus (Millipore) in HBSG as previously described (Stevenson and Begg 1994). In the results depicted in Fig. 3 (A and B), duplicate filters from each cell line were measured for each time point, and the experiment was performed at least twice. Data from at least two experiments (n > 4 filters) are presented as mean ± SEM. Data from single clones of parental, MDCK/NZO-3, MDCK/CZO-3, and MDCK/FLZO-3 cells are shown. Two additional, independently selected MDCK/NZO-3 cell lines were tested in both the calcium switch and cD experiments and showed results essentially identical to the first MDCK/NZO-3 cell line (data not shown). For calcium switch experiments (see Fig. 3 A), TER is plotted in ohm × cm2. For cD experiments (see Fig. 3 B), TER is expressed as a percentage normalized to t = 0. The steady-state TER of all cell lines examined was 50–60 ohm × cm2.


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)

Expression of NZO-3 delays TJ formation. (A) NZO-3 expression in MDCK cells subjected to a calcium switch delays reestablishment of TER. Filter-grown confluent monolayers of untransfected parental cells or MDCK cell lines stably expressing NZO-3, CZO-3, or FLZO-3 were incubated in calcium-free media for ∼15 h to disrupt intercellular junctions and then switched to media containing 1.8 mM calcium at t = 0 to induce synchronous assembly of intercellular junctions. TER was measured over 96 h after calcium switch. CZO-3 and FLZO-3 expressing cell lines show TER recovery dynamics similar to parental cells. NZO-3–expressing cells display a delay in TER recovery, but reach the same TER as the other cell lines by 48–96 h. (B) Expression of NZO-3 delays TER recovery in MDCK cells after cD treatment. cD was added to filter-grown confluent monolayers at t = 0 and then washed out after 60 min. TER measurements were taken every 30 min to monitor TJ breakdown and reformation. TER for each cell line is expressed as percentages of the values at t = 0. After 60 min of cD treatment, all cell lines exhibit a significant drop in TER. After cD washout, MDCK/CZO-3, MDCK/FLZO-3 and parental cells recover TER to values greater than t = 0, whereas MDCK/NZO-3 cells do not fully recover TER in the duration of the experiment.
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Figure 3: Expression of NZO-3 delays TJ formation. (A) NZO-3 expression in MDCK cells subjected to a calcium switch delays reestablishment of TER. Filter-grown confluent monolayers of untransfected parental cells or MDCK cell lines stably expressing NZO-3, CZO-3, or FLZO-3 were incubated in calcium-free media for ∼15 h to disrupt intercellular junctions and then switched to media containing 1.8 mM calcium at t = 0 to induce synchronous assembly of intercellular junctions. TER was measured over 96 h after calcium switch. CZO-3 and FLZO-3 expressing cell lines show TER recovery dynamics similar to parental cells. NZO-3–expressing cells display a delay in TER recovery, but reach the same TER as the other cell lines by 48–96 h. (B) Expression of NZO-3 delays TER recovery in MDCK cells after cD treatment. cD was added to filter-grown confluent monolayers at t = 0 and then washed out after 60 min. TER measurements were taken every 30 min to monitor TJ breakdown and reformation. TER for each cell line is expressed as percentages of the values at t = 0. After 60 min of cD treatment, all cell lines exhibit a significant drop in TER. After cD washout, MDCK/CZO-3, MDCK/FLZO-3 and parental cells recover TER to values greater than t = 0, whereas MDCK/NZO-3 cells do not fully recover TER in the duration of the experiment.
Mentions: TER was measured with a Millicell-ERS apparatus (Millipore) in HBSG as previously described (Stevenson and Begg 1994). In the results depicted in Fig. 3 (A and B), duplicate filters from each cell line were measured for each time point, and the experiment was performed at least twice. Data from at least two experiments (n > 4 filters) are presented as mean ± SEM. Data from single clones of parental, MDCK/NZO-3, MDCK/CZO-3, and MDCK/FLZO-3 cells are shown. Two additional, independently selected MDCK/NZO-3 cell lines were tested in both the calcium switch and cD experiments and showed results essentially identical to the first MDCK/NZO-3 cell line (data not shown). For calcium switch experiments (see Fig. 3 A), TER is plotted in ohm × cm2. For cD experiments (see Fig. 3 B), TER is expressed as a percentage normalized to t = 0. The steady-state TER of all cell lines examined was 50–60 ohm × cm2.

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