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

Redox Report

Epub 2026 Feb 4 | Volume 31, 2026 - Issue 1 10.1080/13510002.2026.2621497

P J Jain Tiffee, Aswathy Sivasailam, Kiran S Kumar, Shine Varghese Jancy, Aparna Geetha Jayaprasad, Aman Munirpasha Halikar, Aijaz Ahmed Rather, Nithin Satheesan Sinivirgin, K G Anurup, T R Santhoshkumar

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.