Autophagy Flux Detection

Autophagy is an intracellular system that degrades cytosolic proteins and organelles. There are three categories of autophagy: macroautophagy, microautophagy and chaperon-mediated autophagy (CMA). Macroautophagy is most widely investigated and best known among the three pathways. Autophagy has been implicated in diverse pathological conditions including cancer, neurodegenerative and inflammatory diseases. Modulation of autophagy has become a potentially interesting therapeutic target in human diseases. Most of assays use LC3 as a model substrate to measure autophagic flux in live cells, such as mRFP-EGFP-LC3 and mCherry-EGFP-LC3. mCherry and EGFP have different sensitivity to lysosomal acidity. mCherry is more stable than GFP in acidic compartments. When the mCherry-EGFP-LC3 fusion protein localizes to autophagosomes, it emits both green and red fluorescence signals which is often shown as yellow signals in merged pictures. However, the fluorescence of GFP but not of mCherry is quenched within autolysosomes which makes autolysosomes appear red. Therefore, autophagy induction results in increased numbers of both yellow and red puncta whereas inhibition of autophagy at a late step results in an increase in the number of yellow puncta and a concurrent decrease in red puncta.

Figure 1. Detection of autophagic flux using the mRFP-EGFP-LC3 reporter system. (Lopez A, Fleming A, Rubinsztein DC)

RGBiotech offers mRFP-EGFP-LC3 and mCherry-EGFP-LC3 expression plasmid vectors that can be used for monitoring autophagic flux in live cells.

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