SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Journal of Cell Science Année : 2018

SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry

Rafael J Argüello
Adrian G Torres
  • Fonction : Auteur
Lluis Ribas de Pouplana
  • Fonction : Auteur
Serge A van de Pavert
  • Fonction : Auteur
Evelina Gatti
  • Fonction : Auteur
  • PersonId : 915530
Philippe Pierre

Résumé

The rate at which ribosomes translate mRNAs regulates protein expression by controlling co-translational protein folding and mRNA stability. Many factors regulate translation elongation, including tRNA levels, codon usage and phosphorylation of eukaryotic elongation factor 2 (eEF2). Current methods to measure translation elongation lack single-cell resolution, require expression of multiple transgenes and have never been successfully applied ex vivo. Here, we show, by using a combination of puromycilation detection and flow cytometry (a method we call 'SunRiSE'), that translation elongation can be measured accurately in primary cells in pure or heterogenous populations isolated from blood or tissues. This method allows for the simultaneous monitoring of multiple parameters, such as mTOR or S6K1/2 signaling activity, the cell cycle stage and phosphorylation of translation factors in single cells, without elaborated, costly and lengthy purification procedures. We took advantage of SunRiSE to demonstrate that, in mouse embryonic fibroblasts, eEF2 phosphorylation by eEF2 kinase (eEF2K) mostly affects translation engagement, but has a surprisingly small effect on elongation, except after proteotoxic stress induction. This article has an associated First Person interview with the first author of the paper.

Domaines

Immunologie
Fichier principal
Vignette du fichier
jcs Arguello fulls - 1.pdf (798.52 Ko) Télécharger le fichier
Origine : Fichiers éditeurs autorisés sur une archive ouverte
Loading...

Dates et versions

hal-01982340 , version 1 (15-01-2019)

Identifiants

Citer

Rafael J Argüello, Marisa Reverendo, Andreia Mendes, Voahirana Camosseto, Adrian G Torres, et al.. SunRiSE – measuring translation elongation at single-cell resolution by means of flow cytometry. Journal of Cell Science, 2018, 131 (10), pp.jcs214346. ⟨10.1242/jcs.214346⟩. ⟨hal-01982340⟩
72 Consultations
677 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More