478 ZO-1 C-Terminal Domains Are Essential for Tight Junction Assembly and Maintenance

Abstract

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.