The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells - Université de Rennes Accéder directement au contenu
Pré-Publication, Document De Travail Année : 2022

The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells

Résumé

Although several mechanisms of autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to autophagy activation, the ATG4B protease primes the key autophagy player LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster’s Resonance Energy Transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine/tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image allows to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are small pools of unprimed LC3B upon ATG4B downregulation, and that the priming of the biosensor is completely abolished in ATG4B knockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Last, we screened for commercially-available ATG4B inhibitors, and we illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.

Dates et versions

hal-03810703 , version 1 (11-10-2022)

Identifiants

Citer

Elif Begüm Gökerküçük, Angélique Cheron, Marc Tramier, Giulia Bertolin. The LC3B FRET biosensor monitors the modes of action of ATG4B during autophagy in living cells. 2022. ⟨hal-03810703⟩
8 Consultations
1 Téléchargements

Altmetric

Partager

Gmail Facebook X LinkedIn More