Time-resolved simultaneous imaging of mitochondrial reactive oxygen species and lysosomal permeabilization to determine organelle-centred cell death

Mitochondria and lysosomes are pivotal in dictating cell survival or death outcomes. While mitochondrial damage and ROS production are key events in mitochondrial cell death, lysosome membrane permeabilization and cathepsin B release mark lysosomal cell death. We aimed to generate a live-cell approach to concurrently monitor mitochondrial redox alterations and lysosomal permeabilization. This would provide mechanistic insight into their dynamic interplay during cell death and enable the discovery of organelle-specific death inducers. A dual cell sensor, stably expressing tdTomato-CathepsinB and mitochondria-targeted redox GFP (mt-roGFP), was successfully engineered, and simultaneous imaging of both events by real-time confocal imaging was carried out with selected drugs. This platform faithfully reported the chronological sequence of organelle-specific events with the progression of cell death, with good temporal and spatial resolution at the single-cell level. Moreover, we have identified and categorised potential lead compounds that predominantly induce lysosomal cell death or mitochondrial cell death, as well as a subset that elicit both events concomitantly. The study provided evidence that both organelles contribute to cell death in a context-dependent manner, and the temporal analysis of both events is critical in understanding unique organelle-centred cell death.