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

https://www.ncbi.nlm.nih.gov/pubmed/33912850

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).