title: Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly.
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.
authors.affiliation: Department of Cell Biology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
authors.forename: E S
authors.initials: ES
authors.lastname: Wittchen
authors.forename: J
authors.initials: J
authors.lastname: Haskins
authors.forename: B R
authors.initials: BR
authors.lastname: Stevenson
mesh_terms: D000818:Animals; D015820:Cadherins / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000032:analysis; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000502:physiology; D005786:Gene Expression Regulation; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D062793:Occludin; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D019108:Tight Junctions / Q000502:physiology* / Q000648:ultrastructure*; D015534:Trans-Activators; D014162:Transfection; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D051176:beta Catenin
chemical_list: D015820:Cadherins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D062793:Occludin; D010446:Peptide Fragments; D010750:Phosphoproteins; D011994:Recombinant Proteins; D015534:Trans-Activators; D062825:Zonula Occludens Proteins; D062826:Zonula Occludens-1 Protein; D062827:Zonula Occludens-2 Protein; D051176:beta Catenin