The role of the C-terminus of zonula occludens-1 in the structural reorganization of epithelial cells

Tight junctions are complex multi-protein systems that exist in a dynamic state of reassembly, allowing epithelial cells to respond rapidly to internal and environmental stimuli. The membrane-associated protein zonula occludens-1 (ZO-1) is a scaffolding protein that plays an important role in the regulation of tight junction protein complex dynamics. While the N-terminus of the protein has been extensively studied, the functions of the novel C-terminus of ZO-1 are still to be characterized. For this study, stable MDCK II cell lines overexpressing ZO-1 C-terminal constructs were developed. Gene expression studies, actin disruption, and fluorescence recovery after photobleaching were used to characterize the behavior of the constructs. The objective was to determine C-terminus ZO-1 function in regards to scaffolding and regulatory processes at the tight junction. Overexpression of the actin-binding region (ABR) has a dominant negative action on normal ZO-1 behavior, leading to an altered morphology of MDCK II cells and the altered expression of the mesenchymal marker vimentin. A larger construct containing ABR and the C-terminal ZU5 domain (ABR-ZU5) exhibits slowed recovery and decreased mobility after photobleaching compared to full-length ZO-1. This suggests the importance of the C-terminus in the stabilization of ZO-1 at the tight junction. In actin disruption studies, overexpression of either construct protects against the depolymerization of filamentous actin. These results indicate distinct binding and dynamic properties of the ABR and ZU5 C-terminal domains that contribute to the scaffolding and regulatory function of the ZO-1 protein