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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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In vitro binding analyses show that NZO-3 binds F-actin and ZO-1 exclusively; both NZO-3 and CZO-3 bind occludin and cingulin. (A) Coomassie blue–stained gel showing the equivalent amounts of NZO-3 (N) and CZO-3 (C) fusion proteins used in all subsequent binding assays. (B) NZO-3 specifically cosediments with actin filaments; CZO-3 does not. Equivalent amounts of NZO-3 or CZO-3 were centrifuged in the presence (+) or absence (−) of F-actin. Stoichiometrically equivalent aliquots of supernatants (S) and pellets (P) were analyzed by immunoblot. NZO-3, but not CZO-3, is found in the F-actin pellet. (C) NZO-3 binds ZO-1; CZO-3 does not. 35S-labeled in vitro–transcribed/translated ZO-1 was incubated with affinity resin containing equal amounts of NZO-3 or CZO-3. The 6-histidine tag plus 36 nonspecific amino acids served as negative control (−). As detected by autoradiogram, ZO-1 is retained by the NZO-3-containing resin and does not bind to CZO-3 or the negative control peptide. (D) Both halves of ZO-3 bind occludin with similar affinity. NZO-3 and CZO-3 were incubated with affinity resin containing immobilized GST-occludin (+), or GST alone (−). Bound protein was eluted with glutathione and analyzed by immunoblot with anti–ZO-3 antibodies (first four lanes) or Coomassie blue staining (last two lanes). Both NZO-3 and CZO-3 are retained by GST-occludin and not by GST alone. The Coomassie blue–stained lanes show that approximately equal amounts of NZO-3 and CZO-3 (arrows) are bound. (E) Both halves of ZO-3 bind to the amino-terminal head region of cingulin. NZO-3 or CZO-3 were added to an affinity column containing the amino-terminal head region of cingulin (N-cing) fused to GST (+) or GST alone (−). Bound fractions were immunoblotted with antibodies specifically recognizing NZO-3 or CZO-3 (first four lanes) or stained with Coomassie blue (last two lanes). Both NZO-3 and CZO-3 are retained on the GST–N-cingulin column but not on GST alone. Coomassie blue staining of the bound fractions shows approximately equal amounts of NZO-3 or CZO-3 (arrows) are bound. The other protein bands visible in the Coomassie blue–stained samples correspond to GST–N-cingulin.
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Figure 10: In vitro binding analyses show that NZO-3 binds F-actin and ZO-1 exclusively; both NZO-3 and CZO-3 bind occludin and cingulin. (A) Coomassie blue–stained gel showing the equivalent amounts of NZO-3 (N) and CZO-3 (C) fusion proteins used in all subsequent binding assays. (B) NZO-3 specifically cosediments with actin filaments; CZO-3 does not. Equivalent amounts of NZO-3 or CZO-3 were centrifuged in the presence (+) or absence (−) of F-actin. Stoichiometrically equivalent aliquots of supernatants (S) and pellets (P) were analyzed by immunoblot. NZO-3, but not CZO-3, is found in the F-actin pellet. (C) NZO-3 binds ZO-1; CZO-3 does not. 35S-labeled in vitro–transcribed/translated ZO-1 was incubated with affinity resin containing equal amounts of NZO-3 or CZO-3. The 6-histidine tag plus 36 nonspecific amino acids served as negative control (−). As detected by autoradiogram, ZO-1 is retained by the NZO-3-containing resin and does not bind to CZO-3 or the negative control peptide. (D) Both halves of ZO-3 bind occludin with similar affinity. NZO-3 and CZO-3 were incubated with affinity resin containing immobilized GST-occludin (+), or GST alone (−). Bound protein was eluted with glutathione and analyzed by immunoblot with anti–ZO-3 antibodies (first four lanes) or Coomassie blue staining (last two lanes). Both NZO-3 and CZO-3 are retained by GST-occludin and not by GST alone. The Coomassie blue–stained lanes show that approximately equal amounts of NZO-3 and CZO-3 (arrows) are bound. (E) Both halves of ZO-3 bind to the amino-terminal head region of cingulin. NZO-3 or CZO-3 were added to an affinity column containing the amino-terminal head region of cingulin (N-cing) fused to GST (+) or GST alone (−). Bound fractions were immunoblotted with antibodies specifically recognizing NZO-3 or CZO-3 (first four lanes) or stained with Coomassie blue (last two lanes). Both NZO-3 and CZO-3 are retained on the GST–N-cingulin column but not on GST alone. Coomassie blue staining of the bound fractions shows approximately equal amounts of NZO-3 or CZO-3 (arrows) are bound. The other protein bands visible in the Coomassie blue–stained samples correspond to GST–N-cingulin.

Mentions: We determined which of the known protein–protein interactions of ZO-3 could be mapped to the amino- and carboxy-terminal halves of the molecule. It has been previously shown that ZO-3 binds directly to actin filaments in vitro (Wittchen et al. 1999). In addition, ZO-3 also binds ZO-1, occludin, and the amino terminus of cingulin, but not to ZO-2 (Haskins et al. 1998; Cordenonsi et al. 1999). Fig. 10 A shows Coomassie-stained samples of the purified NZO-3 and CZO-3 proteins, demonstrating the equal amounts of these proteins used in subsequent binding experiments. As shown by actin cosedimentation assays in Fig. 10 B, substantial NZO-3 is found in the pellet in the presence of F-actin whereas a much smaller amount is visible in the pellet in the absence of actin, likely due to a low level of fusion protein aggregation. CZO-3 remained in the supernatant fraction in both the presence and absence of actin filaments. Therefore, the actin binding site(s) of ZO-3 reside in the amino terminus of the protein. To map the binding site(s) of ZO-1 on ZO-3, we performed a binding assay using 35S-labeled in vitro–transcribed/translated human ZO-1 added to NZO-3 or CZO-3 affinity columns. As shown in Fig. 10 C, NZO-3 retains ZO-1, whereas CZO-3 and the negative control do not. This demonstrates that the amino-terminal half of ZO-3 is responsible for binding to ZO-1. This is consistent with previous studies showing that ZO-1 and ZO-2 bind each other via their second PDZ domains (Fanning et al. 1998; Itoh et al. 1999b). To define the occludin binding site on ZO-3, in vitro binding assays were performed using NZO-3 and CZO-3 added to affinity columns containing immobilized GST-occludin or GST alone, and bound proteins were detected by immunoblot (Fig. 10 D). Both NZO-3 and CZO-3 were specifically retained on the occludin column. Although the NZO-3 immunoblot reaction is stronger than that for CZO-3, the amounts of NZO-3 and CZO-3 bound to occludin appear approximately equivalent, as assayed by Coomassie blue staining of the bound fractions. This indicates that occludin binding domains of approximately equal affinity are found in both the amino- and carboxy-terminal halves of ZO-3. It has recently been reported that the amino-terminal head and to a lesser degree, the carboxy-terminal tail of cingulin bind to full-length ZO-3 in vitro (Cordenonsi et al. 1999). Here we determined which half of ZO-3 binds the amino-terminal portion of cingulin. Equivalent amounts of NZO-3 or CZO-3 were added to GST-cingulin or GST affinity columns. As shown in Fig. 10 E, both NZO-3 and CZO-3 are specifically retained by cingulin. Coomassie blue staining of the bound fractions revealed no significant difference between the amounts of NZO-3 or CZO-3 binding to cingulin, indicating that both halves of ZO-3 bind the head region of cingulin with approximately equal affinity. In summary, the binding studies shown in Fig. 10 reveal that while both halves of ZO-3 bind to occludin and cingulin equally well, F-actin and ZO-1 bind preferentially to NZO-3.


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)

In vitro binding analyses show that NZO-3 binds F-actin and ZO-1 exclusively; both NZO-3 and CZO-3 bind occludin and cingulin. (A) Coomassie blue–stained gel showing the equivalent amounts of NZO-3 (N) and CZO-3 (C) fusion proteins used in all subsequent binding assays. (B) NZO-3 specifically cosediments with actin filaments; CZO-3 does not. Equivalent amounts of NZO-3 or CZO-3 were centrifuged in the presence (+) or absence (−) of F-actin. Stoichiometrically equivalent aliquots of supernatants (S) and pellets (P) were analyzed by immunoblot. NZO-3, but not CZO-3, is found in the F-actin pellet. (C) NZO-3 binds ZO-1; CZO-3 does not. 35S-labeled in vitro–transcribed/translated ZO-1 was incubated with affinity resin containing equal amounts of NZO-3 or CZO-3. The 6-histidine tag plus 36 nonspecific amino acids served as negative control (−). As detected by autoradiogram, ZO-1 is retained by the NZO-3-containing resin and does not bind to CZO-3 or the negative control peptide. (D) Both halves of ZO-3 bind occludin with similar affinity. NZO-3 and CZO-3 were incubated with affinity resin containing immobilized GST-occludin (+), or GST alone (−). Bound protein was eluted with glutathione and analyzed by immunoblot with anti–ZO-3 antibodies (first four lanes) or Coomassie blue staining (last two lanes). Both NZO-3 and CZO-3 are retained by GST-occludin and not by GST alone. The Coomassie blue–stained lanes show that approximately equal amounts of NZO-3 and CZO-3 (arrows) are bound. (E) Both halves of ZO-3 bind to the amino-terminal head region of cingulin. NZO-3 or CZO-3 were added to an affinity column containing the amino-terminal head region of cingulin (N-cing) fused to GST (+) or GST alone (−). Bound fractions were immunoblotted with antibodies specifically recognizing NZO-3 or CZO-3 (first four lanes) or stained with Coomassie blue (last two lanes). Both NZO-3 and CZO-3 are retained on the GST–N-cingulin column but not on GST alone. Coomassie blue staining of the bound fractions shows approximately equal amounts of NZO-3 or CZO-3 (arrows) are bound. The other protein bands visible in the Coomassie blue–stained samples correspond to GST–N-cingulin.
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Figure 10: In vitro binding analyses show that NZO-3 binds F-actin and ZO-1 exclusively; both NZO-3 and CZO-3 bind occludin and cingulin. (A) Coomassie blue–stained gel showing the equivalent amounts of NZO-3 (N) and CZO-3 (C) fusion proteins used in all subsequent binding assays. (B) NZO-3 specifically cosediments with actin filaments; CZO-3 does not. Equivalent amounts of NZO-3 or CZO-3 were centrifuged in the presence (+) or absence (−) of F-actin. Stoichiometrically equivalent aliquots of supernatants (S) and pellets (P) were analyzed by immunoblot. NZO-3, but not CZO-3, is found in the F-actin pellet. (C) NZO-3 binds ZO-1; CZO-3 does not. 35S-labeled in vitro–transcribed/translated ZO-1 was incubated with affinity resin containing equal amounts of NZO-3 or CZO-3. The 6-histidine tag plus 36 nonspecific amino acids served as negative control (−). As detected by autoradiogram, ZO-1 is retained by the NZO-3-containing resin and does not bind to CZO-3 or the negative control peptide. (D) Both halves of ZO-3 bind occludin with similar affinity. NZO-3 and CZO-3 were incubated with affinity resin containing immobilized GST-occludin (+), or GST alone (−). Bound protein was eluted with glutathione and analyzed by immunoblot with anti–ZO-3 antibodies (first four lanes) or Coomassie blue staining (last two lanes). Both NZO-3 and CZO-3 are retained by GST-occludin and not by GST alone. The Coomassie blue–stained lanes show that approximately equal amounts of NZO-3 and CZO-3 (arrows) are bound. (E) Both halves of ZO-3 bind to the amino-terminal head region of cingulin. NZO-3 or CZO-3 were added to an affinity column containing the amino-terminal head region of cingulin (N-cing) fused to GST (+) or GST alone (−). Bound fractions were immunoblotted with antibodies specifically recognizing NZO-3 or CZO-3 (first four lanes) or stained with Coomassie blue (last two lanes). Both NZO-3 and CZO-3 are retained on the GST–N-cingulin column but not on GST alone. Coomassie blue staining of the bound fractions shows approximately equal amounts of NZO-3 or CZO-3 (arrows) are bound. The other protein bands visible in the Coomassie blue–stained samples correspond to GST–N-cingulin.
Mentions: We determined which of the known protein–protein interactions of ZO-3 could be mapped to the amino- and carboxy-terminal halves of the molecule. It has been previously shown that ZO-3 binds directly to actin filaments in vitro (Wittchen et al. 1999). In addition, ZO-3 also binds ZO-1, occludin, and the amino terminus of cingulin, but not to ZO-2 (Haskins et al. 1998; Cordenonsi et al. 1999). Fig. 10 A shows Coomassie-stained samples of the purified NZO-3 and CZO-3 proteins, demonstrating the equal amounts of these proteins used in subsequent binding experiments. As shown by actin cosedimentation assays in Fig. 10 B, substantial NZO-3 is found in the pellet in the presence of F-actin whereas a much smaller amount is visible in the pellet in the absence of actin, likely due to a low level of fusion protein aggregation. CZO-3 remained in the supernatant fraction in both the presence and absence of actin filaments. Therefore, the actin binding site(s) of ZO-3 reside in the amino terminus of the protein. To map the binding site(s) of ZO-1 on ZO-3, we performed a binding assay using 35S-labeled in vitro–transcribed/translated human ZO-1 added to NZO-3 or CZO-3 affinity columns. As shown in Fig. 10 C, NZO-3 retains ZO-1, whereas CZO-3 and the negative control do not. This demonstrates that the amino-terminal half of ZO-3 is responsible for binding to ZO-1. This is consistent with previous studies showing that ZO-1 and ZO-2 bind each other via their second PDZ domains (Fanning et al. 1998; Itoh et al. 1999b). To define the occludin binding site on ZO-3, in vitro binding assays were performed using NZO-3 and CZO-3 added to affinity columns containing immobilized GST-occludin or GST alone, and bound proteins were detected by immunoblot (Fig. 10 D). Both NZO-3 and CZO-3 were specifically retained on the occludin column. Although the NZO-3 immunoblot reaction is stronger than that for CZO-3, the amounts of NZO-3 and CZO-3 bound to occludin appear approximately equivalent, as assayed by Coomassie blue staining of the bound fractions. This indicates that occludin binding domains of approximately equal affinity are found in both the amino- and carboxy-terminal halves of ZO-3. It has recently been reported that the amino-terminal head and to a lesser degree, the carboxy-terminal tail of cingulin bind to full-length ZO-3 in vitro (Cordenonsi et al. 1999). Here we determined which half of ZO-3 binds the amino-terminal portion of cingulin. Equivalent amounts of NZO-3 or CZO-3 were added to GST-cingulin or GST affinity columns. As shown in Fig. 10 E, both NZO-3 and CZO-3 are specifically retained by cingulin. Coomassie blue staining of the bound fractions revealed no significant difference between the amounts of NZO-3 or CZO-3 binding to cingulin, indicating that both halves of ZO-3 bind the head region of cingulin with approximately equal affinity. In summary, the binding studies shown in Fig. 10 reveal that while both halves of ZO-3 bind to occludin and cingulin equally well, F-actin and ZO-1 bind preferentially to NZO-3.

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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Related in: MedlinePlus