An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept

Autor: Damir Krunic, Felix Bestvater, Michael Hausmann, Jens H Westhoff, Jana Heigwer, Sotirios G. Zarogiannis, David Ridinger, Stefan Terjung, Iva Marinovic, Claus Peter Schmitt, Georg Hildenbrand, Eszter Levai, Conghui Zhang, Franz Schaefer, Maria Bartosova
Jazyk: angličtina
Rok vydání: 2021
Předmět:
0301 basic medicine
Zonula Occludens-1
tight junctions
QH301-705.5
Transwell
Alanyl-Glutamine
Catalysis
Umbilical vein
Article
Inorganic Chemistry
Capillary Permeability
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
automated immunofluorescence imaging
Monolayer
Human Umbilical Vein Endothelial Cells
Humans
Claudin-5
Physical and Theoretical Chemistry
Biology (General)
Claudin
Molecular Biology
QD1-999
Spectroscopy
Tight junction
Chemistry
Organic Chemistry
Dextrans
General Medicine
endothelial cells
Computer Science Applications
Endothelial stem cell
030104 developmental biology
Dextran
paracellular permeability
Permeability (electromagnetism)
030220 oncology & carcinogenesis
Paracellular transport
Biophysics
Zonula Occludens-1 Protein
cell monolayer
single molecule localization microscopy
claudins
Zdroj: International Journal of Molecular Sciences, Vol 22, Iss 8178, p 8178 (2021)
International Journal of Molecular Sciences
Volume 22
Issue 15
ISSN: 1661-6596
1422-0067
Popis: 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.
Databáze: OpenAIRE
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