The LC3B FRET biosensor monitors the modes of action of 1 ATG4B during autophagy in living cells

Although several mechanisms of autophagy have been dissected in the last decade, following 14 this pathway in real time remains challenging. Among the early events leading to autophagy 15 activation, the ATG4B protease primes the key autophagy player LC3B. Given the lack of 16 reporters to follow this event in living cells, we developed a Förster’s Resonance Energy 17 Transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was 18 generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, 19 Aquamarine/tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET 20 indicates the priming of LC3B by ATG4B and the resolution of the FRET image allows to 21 characterize the spatial heterogeneity of the priming activity. Second, quantifying the number 22 of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed 23 that there are small pools of unprimed LC3B upon ATG4B downregulation, and that the 24 priming of the biosensor is completely abolished in ATG4B quantitative monitoring of the ATG4B activity in living cells and in real time, with 31 unprecedented spatiotemporal resolution. 32 the in with show that the biosensor the main of the in terms of structures, and of its with proteins upon autophagy We also show that the biosensor can report on the changes in proLC3B and this in an Using knockout we demonstrate that the absence of maximizes the FRET response of the biosensor as a consequence of the complete lack of proLC3B priming. We then show that proLC3B priming can be rescued with the ectopic expression of the wild-type the mutant, previously By using the LC3B biosensor and performing multiple approaches to analyze FRET/FLIM, we report on the action of mechanisms of available ATG4 inhibitors. By doing so, we provide a framework of how to use the LC3B biosensor and analyze the acquired data to identify new ATG4 inhibitors with better specificity and efficacy. demonstrate the LC3B biosensor is The biosensor the they the lysosomal The autophagy-dependent changes in the number of puncta- their of the The LC3B biosensor responds to the changes in proLC3B priming in an ATG4B-dependent manner After establishing that the biosensor behaves like endogenous LC3B in cells, we sought to assess its capacity to dynamically report on LC3B processing. To this end, live cells expressing the WT or the uncleavable G120A biosensor were analyzed using FRET/FLIM. We compared the donor (Aquamarine) lifetime differences between the donor-only and the biosensor to detect FRET events, which are highlighted when the donor lifetime decreases. We then used ∆Lifetime as a readout for FRET/FLIM analyses, which was determined by calculating the net difference between the lifetime of the donor-only construct (Aquamarine- proLC3B) and that of a biosensor (either Aquamarine-proLC3B- or proLC3B G120A -tdLanYFP). We hypothesized that a positive ∆Lifetime would be indicative of a FRET event between Aquamarine and tdLanYFP, therefore corresponding to the presence of unprimed proLC3B. of inhibition of ATG4B-specific compounds. We also provide an innovative methodology where individual sets of microscopy data can be analyzed using three independent approaches. Cumulating the information obtained by the three approaches allows to spatially localize and quantify the ATG4B-dependent priming deconjugation of LC3B with unprecedented precision, and it is mandatory to characterize the mode of action of present and future ATG4B-specific inhibitors.