An Autophagic Flux Probe that Releases an Internal Control

Abstract

Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.

T-Cell Intracellular Antigens and Hu Antigen R Antagonistically Modulate Mitochondrial Activity and Dynamics by Regulating Optic Atrophy 1 Gene Expression

“…7C). We confirmed this result using the GFP-LC3-RFP-LC3ΔG fluorescent probe (44). This probe is cleaved by endogenous ATG4 protease into equimolar amounts of GFP-LC3 and RFP-LC3ΔG; GFP-LC3 is degraded by autophagy, whereas RFP-LC3ΔG remains in the cytosol, serving as an internal control.…”

Pharmacological modulation of autophagy: therapeutic potential and persisting obstacles

“…This approach can differentiate between situations of increased on-rate and decreased off-rate and can be conveniently applied to human peripheral blood mononuclear cells ex vivo 274 . As an alternative, tandem fluorescence-tagged versions of LC3 have been successfully used to identify cells in which autophagic flux is operational in vitro and in vivo 274,283 . In paraffin-fixed formalin-embedded patient samples, encouraging results have been obtained by simultaneously quantifying LC3 + puncta and p62 abundance 165 .…”

A Novel 2-Methoxyestradiol Analogue Is Responsible for Vesicle Disruption and Lysosome Aggregation in Breast Cancer Cells

“…TEM was used to assess the ultrastructural changes associated with the ESE-16 exposure [6]. Cells were seeded at a density of 750,000 per 25 cm 2 flasks and were allowed to attach overnight.…”

“…This inhibition ultimately leads to the activation of the intrinsic apoptotic pathway [2, 5]. Previous data suggests that autophagy is induced after ESE-16 exposure, but to date no conclusive data has been presented proving that ESE-16 acts via autophagy [6, 7]. 2ME2 and the analogues subsequently synthesised appear to be potent microtubule inhibitors, which would lead to the blockage of the G2/M border and subsequent apoptosis [5, 8].…”