Journal Article

DZNE-2020-07584

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Visualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions.

Le Bihan, O. (First author)DZNE* ; Decossas, M. ; Gontier, E. ; Gerbod-Giannone, M.-C. ; Lambert, O. (Corresponding author)

2015
Elsevier San Diego, Calif.

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Please use a persistent id in citations: doi:10.1016/j.jsb.2015.10.009

Abstract: Cryo-electron microscopy (cryo-EM) allows the visualization of the cell architecture in its native state. We developed a robust solution to adapt cryo-electron microscopy of vitreous sections (CEMOVIS) to a monolayer of adherent cells using a functionalized polyacrylamide hydrogel growing substrate. We applied this method to reconstitute an endothelial cell monolayer to visualize the morphology of adherens junctions (AJs) which regulate permeability and integrity of the vascular barrier. The fine morphology and ultrastructure of AJs from cultured primary human coronary artery endothelial cells (HCAECs) were analyzed in their native state by using CEMOVIS. Doxycycline and sphingosine-1-phosphate (S1P) are known as efficient regulators of endothelial permeability. Doxycycline and S1P treatments both led to a drastic morphological switch from very uneven to standardized 14-17 nm wide AJs over several microns indicative of a better membrane tethering. Repetitive structures were occasionally noticed within the AJ cleft reflecting a local improved structural organization of VE-cadherin molecules. The ultrastructural stabilization of AJs observed upon treatment likely indicates a better adhesion and thus provides structural clues on the mechanism by which these treatments improve the endothelial barrier function. This method was also successfully extended to a thick epithelial barrier model. We expect our strategy to extend the reliable application of CEMOVIS to virtually any adherent cultured cell systems.

Keyword(s): Adherens Junctions: physiology (MeSH) ; Antigens, CD: metabolism (MeSH) ; Caco-2 Cells (MeSH) ; Cadherins: metabolism (MeSH) ; Cell Line, Tumor (MeSH) ; Coronary Vessels: cytology (MeSH) ; Cryoelectron Microscopy: methods (MeSH) ; Doxycycline: pharmacology (MeSH) ; Endothelial Cells: cytology (MeSH) ; Humans (MeSH) ; Lysophospholipids: pharmacology (MeSH) ; Sphingosine: analogs & derivatives (MeSH) ; Sphingosine: pharmacology (MeSH) ; Staining and Labeling (MeSH) ; Antigens, CD ; Cadherins ; Lysophospholipids ; cadherin 5 ; sphingosine 1-phosphate ; Doxycycline ; Sphingosine

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Contributing Institute(s):

1. Cryo-electron microscopy and tomography (AG Alamoudi)

Research Program(s):

1. 341 - Molecular Signaling (POF3-341) (POF3-341)

Appears in the scientific report 2015

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Database coverage:
; BIOSIS Previews ; Clarivate Analytics Master Journal List ; Current Contents - Life Sciences ; Ebsco Academic Search ; IF < 5 ; JCR ; NCBI Molecular Biology Database ; Nationallizenz ; SCOPUS ; Science Citation Index ; Science Citation Index Expanded ; Web of Science Core Collection ; Zoological Record

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