In vivo measurement of autophagic flux by fluorescence correlation spectroscopy

Abstract

Autophagy is a fundamental and phylogenetically conserved self-degradation process and plays a very important role in the selective degradation of deleterious proteins, organelles, and other macromolecules. Although flow cytometry and fluorescence imaging techniques have been used to assess autophagic flux, we remain less able to in vivo monitor autophagic flux in a highly sensitive, robust, and well-quantified manner. Here, we reported a new method for real-time and quantitatively monitoring autophagosomes and assessing autophagic flux in living cells based on fluorescence correlation spectroscopy (FCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) fused with an enhanced green fluorescent protein (EGFP-LC3B) was used as a biomarker to label autophagosomes in living cells, and FCS was used to monitor EGFP-LC3B labeled autophagosomes by using the characteristic diffusion time (τ_D) value and brightness per particle (BPP) value. By analyzing the distribution frequency of the τ_D values in living cells stably expressing EGFP-LC3B, mutant EGFP-LC3B (EGFP-LC3BΔG) and enhanced green fluorescent protein (EGFP), we found that the τ_D value greater than 10 ms was attributed to the signal of EGFP-LC3B labeled autophagosomes. So, we proposed a parameter P_AP as an indicator to assess the basal autophagic activity and induced autophagic flux. This new method was able to evaluate autophagy inducers, early-stage autophagy inhibitors, and late-stage autophagy inhibitors. Compared with current methods, our method shows high spatiotemporal resolution and very high sensitivity for autophagosomes in low EGFP-LC3B expressing cells and will become an attractive and alternative method for biological and medical studies, some drug screening, and disease treatment.