Real-time simultaneous imaging of temporal alterations in cytoplasmic and mitochondrial redox in single cells during cell division and cell death

Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physio-logical and pathological cell states. This highly regulated process is crucial in life-death decisions of cells. Among organelles, the mitochondrion is the major source of intracellular-ROS and contributes to oxidation damage -induced cell death. Increase in cytosolic-redox and mitochondrial-redox is evident in cells undergoing diverse forms of cell death, such as apoptosis, necrosis, and necroptosis. The hierarchical profiling of redox signaling at the cytosol and mitochondria in a single cell is important to understand the relative contribution of each species in the initiation and shaping of cell death. Here, we demonstrate the potential application of ratiometric redox GFP (roGFP) and intensity-based redox-sensitive RFP (rxRFP) targeted to mitochondria in revealing both rapid and slow progressing changes in redox during cell division and in cells undergoing multiple modes of cell death. To generate imaging quality signal, single-cell clones stably expressing both roGFP at the cytosol and rxRFP probes targeted to mitochondria were generated. The cells provided sufficient temporal resolution with imaging -ready signal for the real-time visualization of rapidly progressing redox alterations at the cytosol and mito-chondria. The long-time imaging of the cells revealed that a moderate increase in cytosolic ROS marks the di-vision stage. Similarly, distinct forms of cell death trigger a unique and temporally regulated redox change at the cytosol and mitochondria, suggesting the potential utility of the sensor cells to dissect the nature of cell death pathways induced by specific forms of stress.