Schematic diagram of the tandem mRFP-EGFP-LC3 reporter to monitor autophagic flux. (a) Representation of the reporter construct mRFP-EGFP-LC3 and the behaviour of the encoded protein under different pH conditions. Under neutral pH conditions, both EGFP and RFP fluorescence is observed. Under acidic pH conditions, EGFP fluorescence is quenched and only red fluorescence is observed. (b) mRFP-EGFP-LC3 labelling during autophagosome biogenesis, maturation and degradation. Unlipidated mRFP-EGFP-LC3 remains in the cytoplasm (light yellow) whereas lipidated mRFP-EGFP-LC3 is recruited to both inner and outer membranes of phagophores and double-membrane autophagosomes. During these steps of autophagosome formation, the fluorescent signal of both fluorophores, mRFP and EGFP, is visible and vesicles appear as yellow puncta. Autophagosomes eventually fuse with lysosomes to form autolysosomes. Under these acidic conditions, the contents within the inner membrane are eventually degraded. The green fluorescent signal from EGFP is quenched in the acidic lysosomal conditions whereas the mRFP signal remains, resulting in red autolysosomes. (c) Representative images of a cell expressing mRFP-EGFP-LC3 with different levels of autophagy. The combination of green and red fluorescent signals from unlipidated mRFP-EGFP-LC3 results in a yellow background in the cytoplasm of the cells. The intensity of this yellow may change dependent upon changes in the autophagy flux. Under low autophagy conditions, most of mRFP-EGFP-LC3 remains unlipidated resulting in a yellow background and only a few yellow or red vesicles (autophagosomes and autolysosomes) are seen. After autophagy induction, many new autophagosomes form and are labelled with lipidated LC3. These rapidly fuse with lysosomes. This can be observed as an increase in the number of total vesicles and the ratio of red:yellow vesicles as well as reduced yellow background. When autophagic flux is blocked, autophagosome formation may still occur. In this scenario, autophagosomes and autolysosomes accumulate but cannot be degraded and can be observed as yellow puncta. The continuous lipidation of mRFP-EGFP-LC3 as new autophagosomes form reduces the yellow background of the cytoplasm.
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