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© The Rockefeller University Press,
0021-9525/2000//825 $5.00
The Journal of Cell Biology, Volume 151, Number 4,
, 2000 825-836
Original Article |
Exogenous Expression of the Amino-Terminal Half of the Tight Junction Protein Zo-3 Perturbs Junctional Complex Assembly
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 β-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 β-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 β-catenin.
Key Words: zonula occludens-3 protein • tight junction • cadherins • actin cytoskeleton • β-catenin
© 2000 The Rockefeller University Press
Abbreviations used in this paper: AJ, adherens junction; cD, cytochalasin D; CZO-3, carboxy-terminal half of ZO-3; FLZO-3, full-length ZO-3; GUK, guanylate kinase; MAGUK, membrane-associated guanylate kinase; NZO-3, amino-terminal half of ZO-3; PDZ, PSD95/dlg/ZO-1; SH3, Src homology 3; TER, transepithelial resistance; TJ, tight junction; TX-100, Triton X-100; VSVG, vesicular stomatitis virus glycoprotein.
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