Abstract
Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell–cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.
Highlights
The study of epithelial and endothelial barrier permeability is critical for understanding basic physiological functions that are seminal for sustaining physiological cell and organ function
Human umbilical vein endothelial cells (HUVEC) in the same plating density were seeded on coverglasses placed in a 12-well cell culture plate in order to be used in the subsequent immunocytofluorescence steps along with the filters
While values of the blank Transwell filters differed between PE (121 ± 4 Ω) vs. PC (88 ± 5 Ω, p < 0.001) filters, the resulting baseline resistance reached by the HUVECs after correction for the blank value was similar (12.9 ± 3.5 vs. 13.2 ± 1.6 Ω cm2 ; p = 0.85 in PE vs. PC filters)
Summary
The study of epithelial and endothelial barrier permeability is critical for understanding basic physiological functions that are seminal for sustaining physiological cell and organ function. Transcellular permeability necessitates the function of the basolateral Na+ -K+ -ATPase that sustains low intracellular Na+ concentrations (around 10 mM) and maintains an electrochemical gradient between the extracellular and the intracellular compartments allowing for the function of apically located ion channels, cotransporters, and antiporters that mediate ion, water, and small molecule absorption and secretion [3] This process requires the polarization of the epithelial and endothelial monolayers that is ensured by the function of tight junctions (TJ) [4]. Through their “fence” function, TJ ensure that no lateral diffusion of transmembrane proteins, e.g., ion channels, transporters, or pumps, can occur from the apical to the basolateral side of the cell membrane (and vice versa), maintaining an asymmetry of distribution of such molecules and along with the function of Na+ -K+ -ATPase, a potential difference between the two sides of the monolayer [5] This implies that the paracellular molecular machinery is essential for proper epithelial and endothelial barrier function
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
