Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA): a live-imaging method for detecting epithelial barrier breaches with spatiotemporal precision

Abstract

Epithelial barrier function is commonly analyzed using transepithelial electrical resistance (TER), which measures the ion flux across epithelia, or by adding traceable macromolecules to one side of the epithelium and monitoring their passage to the other side. While these methods effectively measure changes to global barrier function, they are not sensitive enough to detect local or transient disruptions in the barrier, and they do not reveal the location of barrier breaches within the context of cell or tissue morphology. Therefore, we developed a method that we named Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction (TJ) leaks with high spatial and temporal resolution (Stephenson et al., 2019; Varadarajan et al., 2021). Here, we present expanded applications for ZnUMBA. First, we show that ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin dynamics following laser injury of the junction. We also demonstrate that ZnUMBA can be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby Canine Kidney II (MDCK II) epithelial cells. ZnUMBA is a powerful and flexible method that, with some optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.

EpCAM proteolysis and release of complexed claudin-7 repair and maintain the tight junction barrier

“…A detailed protocol for ZnUMBA using MDCK II cells will be published elsewhere ( Higashi et al, 2022 Preprint ). Briefly, the cells were seeded onto the bottom surfaces of 12-well polycarbonate Transwell filters with 0.4-µm pore size (#3401; Corning) at a density of 1.0 × 10 5 cells/cm 2 and cultured until TER reached a plateau.…”

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

“…We next investigated how Vinculin-mediated changes in actomyosin organization and actin stability affect tTJ integrity. Angulin-1 is a critical tTJ component and is essential for maintaining barrier function at TCJs [29, 30]. Since Angulin-1 is the core protein at the tTJ and recruits Tricellulin [9, 12], we used Angulin-1 as a representative tTJ protein.…”