Probing the leak pathway: Live-cell imaging of macromolecule passage through epithelia.
epithelial barrier
leak pathway
live-cell imaging
macromolecule passage
permeability
transwell
Journal
Annals of the New York Academy of Sciences
ISSN: 1749-6632
Titre abrégé: Ann N Y Acad Sci
Pays: United States
ID NLM: 7506858
Informations de publication
Date de publication:
10 2022
10 2022
Historique:
pubmed:
30
6
2022
medline:
18
10
2022
entrez:
29
6
2022
Statut:
ppublish
Résumé
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.
Substances chimiques
Dextrans
0
Waste Products
0
Avidin
1405-69-2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
151-161Informations de copyright
© 2022 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals LLC on behalf of New York Academy of Sciences.
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