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000141262 0247_ $$2doi$$a10.1016/j.jsb.2015.10.009
000141262 0247_ $$2pmid$$apmid:26470813
000141262 0247_ $$2ISSN$$a1047-8477
000141262 0247_ $$2ISSN$$a1095-8657
000141262 037__ $$aDZNE-2020-07584
000141262 041__ $$aEnglish
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000141262 1001_ $$0P:(DE-2719)2810962$$aLe Bihan, Olivier$$b0$$eFirst author$$udzne
000141262 245__ $$aVisualization of adherent cell monolayers by cryo-electron microscopy: A snapshot of endothelial adherens junctions.
000141262 260__ $$aSan Diego, Calif.$$bElsevier$$c2015
000141262 264_1 $$2Crossref$$3print$$bElsevier BV$$c2015-12-01
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000141262 520__ $$aCryo-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.
000141262 536__ $$0G:(DE-HGF)POF3-341$$a341 - Molecular Signaling (POF3-341)$$cPOF3-341$$fPOF III$$x0
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000141262 650_7 $$2NLM Chemicals$$aAntigens, CD
000141262 650_7 $$2NLM Chemicals$$aCadherins
000141262 650_7 $$2NLM Chemicals$$aLysophospholipids
000141262 650_7 $$2NLM Chemicals$$acadherin 5
000141262 650_7 $$026993-30-6$$2NLM Chemicals$$asphingosine 1-phosphate
000141262 650_7 $$0N12000U13O$$2NLM Chemicals$$aDoxycycline
000141262 650_7 $$0NGZ37HRE42$$2NLM Chemicals$$aSphingosine
000141262 650_2 $$2MeSH$$aAdherens Junctions: physiology
000141262 650_2 $$2MeSH$$aAntigens, CD: metabolism
000141262 650_2 $$2MeSH$$aCaco-2 Cells
000141262 650_2 $$2MeSH$$aCadherins: metabolism
000141262 650_2 $$2MeSH$$aCell Line, Tumor
000141262 650_2 $$2MeSH$$aCoronary Vessels: cytology
000141262 650_2 $$2MeSH$$aCryoelectron Microscopy: methods
000141262 650_2 $$2MeSH$$aDoxycycline: pharmacology
000141262 650_2 $$2MeSH$$aEndothelial Cells: cytology
000141262 650_2 $$2MeSH$$aHumans
000141262 650_2 $$2MeSH$$aLysophospholipids: pharmacology
000141262 650_2 $$2MeSH$$aSphingosine: analogs & derivatives
000141262 650_2 $$2MeSH$$aSphingosine: pharmacology
000141262 650_2 $$2MeSH$$aStaining and Labeling
000141262 7001_ $$aDecossas, Marion$$b1
000141262 7001_ $$aGontier, Etienne$$b2
000141262 7001_ $$aGerbod-Giannone, Marie-Christine$$b3
000141262 7001_ $$0P:(DE-HGF)0$$aLambert, Olivier$$b4$$eCorresponding author
000141262 77318 $$2Crossref$$3journal-article$$a10.1016/j.jsb.2015.10.009$$b : Elsevier BV, 2015-12-01$$n3$$p470-477$$tJournal of Structural Biology$$v192$$x1047-8477$$y2015
000141262 773__ $$0PERI:(DE-600)1469822-5$$a10.1016/j.jsb.2015.10.009$$gVol. 192, no. 3, p. 470 - 477$$n3$$p470-477$$q192:3<470 - 477$$tJournal of structural biology$$v192$$x1047-8477$$y2015
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