Protocol for electron microscopy ultrastructural localization of the fusogenic lipid phosphatidic acid on plasma membrane sheets from chromaffin cells.
Fiche publication
Date publication
juin 2021
Journal
STAR protocols
Auteurs
Membres identifiés du Cancéropôle Est :
Dr GASMAN Stéphane, Dr CHASSEROT-GOLAZ Sylvette, Mme DEMAIS Valérie, Mme ROYER Cathy, Dr VITALE Nicolas
Tous les auteurs :
Tanguy E, Thahouly T, Royer C, Demais V, Gasman S, Chasserot-Golaz S, Vitale N
Lien Pubmed
Résumé
The glycerophospholipid phosphatidic acid (PA) is a key player in regulated exocytosis, but little is known about its localization at the plasma membrane. Here, we provide a protocol for precisely determining the spatial distribution of PA at exocytotic sites by electron microscopy. Using primary bovine chromaffin cells expressing a PA sensor (Spo20p-GFP), we describe the process for cell stimulation and detergent-free preparation of plasma membrane sheets. The protocol can be applied to other cell models and to distinct membrane lipids. For complete details on the use and execution of this protocol, please refer to Tanguy et al. (2020).
Mots clés
Cell biology, Cell membrane, Microscopy, Neuroscience, Structural biology
Référence
STAR Protoc. 2021 Jun 18;2(2):100464