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

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 lay...

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Autores principales: Bartosova, Maria, Ridinger, David, Marinovic, Iva, Heigwer, Jana, Zhang, Conghui, Levai, Eszter, Westhoff, Jens H., Schaefer, Franz, Terjung, Stefan, Hildenbrand, Georg, Krunic, Damir, Bestvater, Felix, Hausmann, Michael, Schmitt, Claus Peter, Zarogiannis, Sotirios G.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8347178/
https://www.ncbi.nlm.nih.gov/pubmed/34360944
http://dx.doi.org/10.3390/ijms22158178
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author Bartosova, Maria
Ridinger, David
Marinovic, Iva
Heigwer, Jana
Zhang, Conghui
Levai, Eszter
Westhoff, Jens H.
Schaefer, Franz
Terjung, Stefan
Hildenbrand, Georg
Krunic, Damir
Bestvater, Felix
Hausmann, Michael
Schmitt, Claus Peter
Zarogiannis, Sotirios G.
author_facet Bartosova, Maria
Ridinger, David
Marinovic, Iva
Heigwer, Jana
Zhang, Conghui
Levai, Eszter
Westhoff, Jens H.
Schaefer, Franz
Terjung, Stefan
Hildenbrand, Georg
Krunic, Damir
Bestvater, Felix
Hausmann, Michael
Schmitt, Claus Peter
Zarogiannis, Sotirios G.
author_sort Bartosova, Maria
collection PubMed
description 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.
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spelling pubmed-83471782021-08-08 An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept Bartosova, Maria Ridinger, David Marinovic, Iva Heigwer, Jana Zhang, Conghui Levai, Eszter Westhoff, Jens H. Schaefer, Franz Terjung, Stefan Hildenbrand, Georg Krunic, Damir Bestvater, Felix Hausmann, Michael Schmitt, Claus Peter Zarogiannis, Sotirios G. Int J Mol Sci Article 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. MDPI 2021-07-30 /pmc/articles/PMC8347178/ /pubmed/34360944 http://dx.doi.org/10.3390/ijms22158178 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bartosova, Maria
Ridinger, David
Marinovic, Iva
Heigwer, Jana
Zhang, Conghui
Levai, Eszter
Westhoff, Jens H.
Schaefer, Franz
Terjung, Stefan
Hildenbrand, Georg
Krunic, Damir
Bestvater, Felix
Hausmann, Michael
Schmitt, Claus Peter
Zarogiannis, Sotirios G.
An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept
title An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept
title_full An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept
title_fullStr An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept
title_full_unstemmed An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept
title_short An Experimental Workflow for Studying Barrier Integrity, Permeability, and Tight Junction Composition and Localization in a Single Endothelial Cell Monolayer: Proof of Concept
title_sort experimental workflow for studying barrier integrity, permeability, and tight junction composition and localization in a single endothelial cell monolayer: proof of concept
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8347178/
https://www.ncbi.nlm.nih.gov/pubmed/34360944
http://dx.doi.org/10.3390/ijms22158178
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