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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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Schematic diagram of the ZO-3 constructs used in this study. Full-length ZO-3 (FLZO-3) contains three PDZ domains, an arginine-rich basic region (+), a proline-rich region (pro), an SH3 domain, a GUK domain, and an acidic region (−). NZO-3 comprises the amino-terminal half of ZO-3 and contains the three PDZ domains, basic region and proline-rich region. The CZO-3 construct corresponds to the carboxy-terminal half of the molecule and contains the SH3, GUK, and acidic domains. The constructs used for generating stably transfected MDCK cells have a VSVG epitope tag at their carboxy terminus.
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Figure 1: Schematic diagram of the ZO-3 constructs used in this study. Full-length ZO-3 (FLZO-3) contains three PDZ domains, an arginine-rich basic region (+), a proline-rich region (pro), an SH3 domain, a GUK domain, and an acidic region (−). NZO-3 comprises the amino-terminal half of ZO-3 and contains the three PDZ domains, basic region and proline-rich region. The CZO-3 construct corresponds to the carboxy-terminal half of the molecule and contains the SH3, GUK, and acidic domains. The constructs used for generating stably transfected MDCK cells have a VSVG epitope tag at their carboxy terminus.

Mentions: The ZO-3 constructs used in this study are shown in Fig. 1. A vesicular stomatitis virus glycoprotein (VSVG) epitope-tagged version of full-length ZO-3 (FLZO-3) in the expression vector pBK-CMV used for transfection of MDCK cells has been described (Haskins et al. 1998). The stable cell line MDCK/FLZO-3 was generated by transfection using Lipofectin (GIBCO BRL) and G418 selection as described (Haskins et al. 1998). A construct composed of the amino-terminal half of ZO-3 (NZO-3) contains the three PDZ domains, arginine-rich region, and proline-rich domain (amino acids 1–454). For stable transfection in MDCK cells, this construct was subcloned into pBK-CMV with a VSVG epitope tag inserted at the carboxy terminus. NZO-3 was also subcloned into the pFastBac HT vector series of the Baculovirus eukaryotic expression system (GIBCO BRL) to generate a 6-histidine–tagged protein used for in vitro binding experiments. A construct composed of the entire carboxy-terminal half of ZO-3 (CZO-3) containing the SH3 and GUK domains, followed by the acidic region (amino acids 455–899) was subcloned into pBK-CMV with a VSVG-epitope tag at the carboxy terminus and stably transfected into MDCK cells. CZO-3 was also inserted into the pFastBac HT vector series to generate a 6-histidine–tagged recombinant protein for binding analyses. For each stable cell transfection, multiple clones were selected and analyzed for expression of constructs by immunofluorescence and immunoblotting.


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)

Schematic diagram of the ZO-3 constructs used in this study. Full-length ZO-3 (FLZO-3) contains three PDZ domains, an arginine-rich basic region (+), a proline-rich region (pro), an SH3 domain, a GUK domain, and an acidic region (−). NZO-3 comprises the amino-terminal half of ZO-3 and contains the three PDZ domains, basic region and proline-rich region. The CZO-3 construct corresponds to the carboxy-terminal half of the molecule and contains the SH3, GUK, and acidic domains. The constructs used for generating stably transfected MDCK cells have a VSVG epitope tag at their carboxy terminus.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169439&req=5

Figure 1: Schematic diagram of the ZO-3 constructs used in this study. Full-length ZO-3 (FLZO-3) contains three PDZ domains, an arginine-rich basic region (+), a proline-rich region (pro), an SH3 domain, a GUK domain, and an acidic region (−). NZO-3 comprises the amino-terminal half of ZO-3 and contains the three PDZ domains, basic region and proline-rich region. The CZO-3 construct corresponds to the carboxy-terminal half of the molecule and contains the SH3, GUK, and acidic domains. The constructs used for generating stably transfected MDCK cells have a VSVG epitope tag at their carboxy terminus.
Mentions: The ZO-3 constructs used in this study are shown in Fig. 1. A vesicular stomatitis virus glycoprotein (VSVG) epitope-tagged version of full-length ZO-3 (FLZO-3) in the expression vector pBK-CMV used for transfection of MDCK cells has been described (Haskins et al. 1998). The stable cell line MDCK/FLZO-3 was generated by transfection using Lipofectin (GIBCO BRL) and G418 selection as described (Haskins et al. 1998). A construct composed of the amino-terminal half of ZO-3 (NZO-3) contains the three PDZ domains, arginine-rich region, and proline-rich domain (amino acids 1–454). For stable transfection in MDCK cells, this construct was subcloned into pBK-CMV with a VSVG epitope tag inserted at the carboxy terminus. NZO-3 was also subcloned into the pFastBac HT vector series of the Baculovirus eukaryotic expression system (GIBCO BRL) to generate a 6-histidine–tagged protein used for in vitro binding experiments. A construct composed of the entire carboxy-terminal half of ZO-3 (CZO-3) containing the SH3 and GUK domains, followed by the acidic region (amino acids 455–899) was subcloned into pBK-CMV with a VSVG-epitope tag at the carboxy terminus and stably transfected into MDCK cells. CZO-3 was also inserted into the pFastBac HT vector series to generate a 6-histidine–tagged recombinant protein for binding analyses. For each stable cell transfection, multiple clones were selected and analyzed for expression of constructs by immunofluorescence and immunoblotting.

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