Roles of protein–protein interactions and monolayer mechanics in tricellulin localization to tricellular tight junctions

Tricellular tight junctions (tTJs) seal the space between three or more cells in epithelial monolayers. These specialized tight junctions have distinct protein components, including a transmembrane protein tricellulin. However, the mechanisms by which tricellulin localizes specifically to tTJs are incompletely understood. We demonstrate that tricellulin undergoes rapid lateral diffusion along bicellular junctions but is a very stable component of tTJs. BioID proteomics identified several proximity partners of tricellulin, and knockout studies on angulin-1/LSR, occludin and afadin provided evidence that these proteins control tricellulin accumulation to tTJs to different extents and mechanisms. Tricellulin localization was disrupted in afadin and angulin-1/LSR knockout cells, although these proteins did not display similar accumulation to tTJs, suggesting that they contribute to tricellulin localization through indirect or context-dependent mechanisms. Importantly, experiments on mixed cultures revealed that defects of tricellulin localization in occludin knockout cells were affected by the proximity of wild-type cells, and treatment of monolayers with myosin-II inhibitor resulted in displacement of tricellulin from tTJs. These results suggest that, in addition to protein-protein interactions, proper epithelial monolayer mechanics are essential for stabilizing tricellulin at tTJs.