Tracking different stages of autophagy with double-tagged LC3B. A tandem fusion of mCherry and GFP is fused to LC3B (one of the several members of the mammalian LC3 family) to make a pH-sensitive sensor that is used to monitor autophagy in live cells. The GFP tag is acid-sensitive while the mCherry tag is acid-insensitive. The double tagged LC3 can be used to label autophagosomes, amphisomes and autolysosomes. In autophagosomes both tags emit fluorescent light resulting in a yellow fluorescence. However, fusion of autophagosomes to late endosomes or lysosomes results in acidic amphisomes or autolysosomes where the green fluorescence from GFP is lost. Subsequently, the red fluorescence from mCherry is lost when the double tagged protein is degraded.
Hansen and Johansen BMC Biology 2011 9:39 doi:10.1186/1741-7007-9-39