Fiche publication
Date publication
janvier 2021
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr GASMAN Stéphane
,
Dr CHASSEROT-GOLAZ Sylvette
,
Mme ROYER Cathy
,
Dr VITALE Nicolas
Tous les auteurs :
Delavoie F, Royer C, Gasman S, Vitale N, Chasserot-Golaz S
Lien Pubmed
Résumé
To study the formation and the architecture of exocytotic site, we generated plasma membrane (PM) sheets on electron microscopy grids to visualize the membrane organization and quantitatively analyze distributions of specific proteins and lipids. This technique allows observing the cytoplasmic face of the plasma membrane by transmission electron microscope. The principle of this approach relies on application of mechanical forces to break open cells. The exposed inner membrane surface can then be visualized with different electron-dense colorations, and specific proteins or lipids can be detected with gold-conjugated probes. Moreover, the membrane sheets are sufficiently resistant to support automated acquisition of multiple-tilt projections, and thus electron tomography allows to obtain three-dimensional (3D) ultrastructural images of secretory granule docked to the plasma membrane.
Mots clés
Electron tomography, Exocytotic site, Lipid distribution, Plasma membrane, Protein distribution, Transmission electron microscopy
Référence
Methods Mol Biol. 2021 ;2233:301-309
