Mechanosensitive recruitment of Vinculin maintains junction integrity and barrier function at epithelial tricellular junctions

Summary Apical cell-cell junctions, including adherens junctions (AJs) and tight junctions (TJs), adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions (BCJs) and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing gastrula-stage Xenopus laevis embryos as a model system, we describe a mechanism by which Vinculin, a mechanosensitive protein, anchors the actomyosin network at TCJs, thus maintaining TJ stability and barrier function. Using an optogenetic approach, we found that acutely increasing junctional tension results in robust recruitment of Vinculin to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that loss of Vinculin results in reduced Actin stability at TCJs. Vinculin knockdown also destabilizes Angulin-1, a key protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJs cannot maintain their proper morphology. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs. Highlights Vinculin is mechanosensitively recruited to tricellular junctions Vinculin’s actin-binding function is needed for tricellular actomyosin organization Tricellular tight junctions are unstable when Vinculin is knocked down Vinculin is required to maintain barrier function at tricellular junctions