Fig. 1: Illustration of how to use mito-SRAI to analyze mitophagic flux.
From: Seeing is believing: a novel tool for quantitating mitophagy
Mito-SRAI is a tandem construct containing a MMTS and two fluorescent protein tags, YPet and TOLLES. When mitochondria are healthy, mito-SRAI emits both yellow and cyan fluorescence in mitochondria matrix (pH ~8). When mitochondria are damaged, mitochondria are engulfed by autophagosomes and delivered to lysosomes to form mitolysosomes. Within the acidic environment of mitolysosomes (pH ~4.5), the YPet (yellow fluorescence) is irreversibly quenched and/or digested, whereas TOLLES remains completely intact and emits full cyan fluorescence. Thus, the ratio (TOLLES vs. YPet) can be used as an index of mitophagic activity.
