Probing the leak pathway: Live‐cell imaging of macromolecule passage through epithelia

Abstract

Epithelia compartmentalize multicellular organisms and provide interfacing between the inside and outside. Apart from regulating the exchange of solutes, uptake of nutrients, and excretion of waste products, their major function is to prevent uncontrolled access of foreign material to immune-competent compartments. Progress in understanding this barrier function toward larger solutes and its possible defects, as can be seen in a variety of diseases, is largely hampered by a lack of methods to spatiotemporally resolve transepithelial passage of macromolecules. Using different cell culture epithelia, we applied biotinylated dextran tracers carrying an acceptor fluorophore.These bind to cell-adherent avidin carrying donor fluorophore at the basolateral membranes of single-layered epithelial sheets. Confocal fluorescence microscopy was applied to living epithelia in order to image apical-to-basolateral tracer passage as a Förster resonance energy transfer signal of the fluorescent dextran-avidin pair over time. Stimulated macromolecule passage using barrier-perturbing agents proved its effectiveness for the leak imaging method presented herein. Over hours of imaging, spontaneous leaks were rare, occurring transiently on the scale of minutes and for the most part associated with rearranging cell junctions. The discussed approach to leak imaging is expected to promote the understanding of epithelial barriers, particularly, the nature and dynamics of the epithelial cell leak pathway.

Beneficial effect of heat-killed Lactiplantibacillus plantarum L-137 on intestinal barrier function of rat small intestinal epithelial cells

“…Excessive passage of antigenic substances such as macromolecules, bacterial products, and food antigens across the epithelial barrier via the leak pathway has been suggested to underlie a variety of diseases. Furthermore, dysregulation of the leak pathway may be a contributing factor to diseases such as inflammatory bowel disease and celiac disease [54][55][56] . These findings that HK L-137 had a protective effect in a mouse model of dextran sulfate sodium-induced colitis and www.nature.com/scientificreports/ that it reduced FD-4 permeability of intestinal epithelial cells in this study suggest that HK L-137 may contribute to the treatment and prevention of various intestinal diseases by regulating the leak pathway 26 .…”