Tracking theTemporal Order of Mitochondrial and LysosomalDamages with a Single Functional Probe: Lysosomal Damage PrecedesMitochondrial Damage under Hyperthermia

Abstract Mitochondrial or lysosomal damage is the initial step of apoptosis, and detecting their damaging sequences is crucial for understanding programmed cell death and related diseases. However, fluorescent probes capable of reporting their damaging order are rarely reported, owing to the deficiency of valid strategies. Herein, by rationally modifying targeting groups and employing a side-chain regulation strategy, a probe Trav was designed to detect the temporal order of lysosomal and mitochondrial damage. Trav targeted both mitochondria and lysosomes in living cells and distinguished them from each other with different fluorescence colors. Upon mitochondrial damage, Trav translocated to lysosomes, accompanied by a fluorescence color change. Subsequently, after lysosomal damage, Trav was relocated to the nucleus and emitted a third color. Similarly, if lysosomal damage occurs first, Trav migrates to mitochondria, which relocated into the nucleus after subsequent mitochondrial damage. Thus, Trav enabled reporting the damaging order of mitochondria and lysosomes through subcellular translocation and fluorescence color changes. Using Trav, we successfully revealed that in hyperthermia-induced injury lysosomal damage preceded mitochondrial damage, and this process was associated with increased levels of reactive oxygen species. Reducing agents, such as cysteine, can inhibit the injury. Trav holds promise as a valuable molecular tool for advancing research in related biomedical fields.