The mechanosensitive protein Vinculin organizes actomyosin and maintains epithelial tricellular junctions

Abstract

Epithelial cells form barriers that coat and protect organs and organisms from their environments. Epithelial tissue function requires both adherens junctions to adhere cells together and tight junctions to create a selectively permeable barrier between neighboring cells. Sites of high tension along cell-cell contacts are called tricellular junctions, the points where three cells meet. Junctional integrity must be maintained when epithelia are challenged by mechanical stress, such as food passing through the intestines to mechanical stress through increasing connections to an actomyosin (F-actin and Myosin II) array.

Here, we aimed to characterize how Vinculin, actomyosin, and junctional proteins are reorganized at tricellular junctions after either Vinculin knockdown and/or the tension-increasing treatments. We began by quantifying changes in fluorescence intensity of mNeon-Vinculin and Halo-Vinculin at tricellular junctions after increasing tension in the epithelia. After ATP treatment or the optogenetic activation of RhoA, we found that Vinculin intensity is enriched at tricellular junctions and reorganizes from distinct “spots” into elongated “spokes” along the junction. To understand Vinculin’s effects in maintaining actomyosin, we performed immunofluorescence staining for F-actin and Myosin II light chain in control and Vinculin knockdown epithelia. We found that there are reduced levels of actomyosin and disrupted actomyosin organization at tricellular junctions when Vinculin is knocked down. Next, we aimed to investigate whether Vinculin supports tricellular junction integrity. To test this, we performed immunofluorescence imaging of the adherens junction protein E-cadherin and the tricellular tight junction protein Angulin-1 after Vinculin knockdown. These experiments show that Vinculin knockdown decreases E-cadherin and Angulin-1 intensity at tricellular junctions, suggesting that Vinculin plays a role in maintaining tricellular adherens and tight junctions. Finally, to explore Vinculin’s role in barrier function, we performed a barrier assay, ZnUMBA, to measure leaks in the space between cells. With this assay, we found a greater percentage of leaky tricellular tight junctions under Vinculin knockdown conditions. Taken together, these data suggest that the mechanosensitive recruitment of Vinculin to tricellular adherens or the bladder expanding. Vinculin, a mechanosensitive protein, strengthens adherens junctions in response junctions under increased tension is essential for proper actomyosin organization and maintaining junctional integrity.