Tight junctions contribute to epithelial barrier function by selectively regulating the quantity and type of molecules that cross the paracellular barrier. Experimental approaches to evaluate the effectiveness of tight junctions are typically global, tissue-scale measures. Here, we introduce Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which we used in Xenopus laevis embryos to visualize short-lived, local breaches in epithelial barrier function. These breaches, or leaks, occur as cell boundaries elongate, correspond to visible breaks in the tight junction, and are followed by transient localized Rho activation, or Rho flares. We discovered that Rho flares restore barrier function by driving concentration of tight junction proteins through actin polymerization and ROCK-mediated localized contraction of the cell boundary. We conclude that Rho flares constitute a damage control mechanism that reinstates barrier function when tight junctions become locally compromised because of normally occurring changes in cell shape and tissue tension.
Keywords: Rho GTPase; Xenopus; ZnUMBA (Zinc-based Ultrasensitive Microscopic Barrier Assay); actin; barrier assay; epithelium; junction contraction; leak pathway; myosin II; tight junction.
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