C‐terminal domains are required for ZO‐1 stabilization and tight junction assembly

Abstract

Zonula occludens (ZO) proteins direct assembly of epithelial tight junctions (TJs). While critical functions have been defined for N‐terminal PDZ domains, a central SH3‐GUK module, and the C‐terminal actin‐binding region (ABR), the remaining ZO‐1 C‐terminus has not been characterized. The aim of this study was to determine if the ZO‐1 C‐terminus participates in TJ assembly. Epithelial cells were stably transfected with full‐length ZO‐1 tagged with GFP at the N‐terminus (GFP‐ZO‐1) as well as a GFP‐tagged deletion mutant lacking the ABR (ZO‐1δABR) and a GFP‐tagged truncation mutant lacking the ABR and remaining C‐terminus (ZO‐11‐1152). ZO‐11‐1152 markedly delayed epithelial barrier development as well as peak transepithelial resistance (TER) achieved. In contrast, ZO‐1δABR did not delay assembly and only slightly diminished peak TER. Morphologic analysis showed that GFP‐ZO‐11‐1152, but not GFP‐ZO‐1 or GFP‐ZO‐1δABR, localized to the TJ inefficiently and interfered with junctional recruitment of endogenous ZO‐1 and β‐catenin. We have previously used fluorescence recovery after photobleaching to show that GFP‐ZO‐1 stability at the TJ increases as monolayers mature. GFP‐ZO‐11‐1152 stabilization did not occur with maturation, but GFP‐ZO‐1δABR stabilized normally. These data suggest that undefined domains within the ZO‐1 C‐terminus are essential for TJ assembly and maturation.