Dual-Circuit Nanopipette for Quantitative Characterization of Mitochondrial-Cytosolic Communications in Single Living...

The quantitative analysis of intricate communications linking subcellular compartments in living cells poses a significant challenge in chemistry and biology. Here, we present a dual-circuit nanopipette platform with high spatiotemporal resolution allowing for the co-tracking of reactive oxygen species (ROS) dynamics from a mitochondrion and its adjacent cytosol within the same living cell. Our findings uncover that transient ROS levels in individual mitochondrion exhibit significant fluctuations, either exceeding or falling below those measured in its surrounding cytosol. Such variations, typically obscured by averaged measurements, underscore the dynamic interplay between subcellular compartments, while simultaneously retaining functional autonomy. Importantly, our quantitative analysis indicates that ROS generated by mitochondria initiates a positive feedback mechanism that amplifies cytosolic ROS levels. Additionally, the average duration for cytosolic ROS levels to return to baseline in cyclosporin A (CsA) pretreated cells was comparable to that observed in untreated cells. This extended time is attributed to a negative feedback loop triggered by mitochondrial ROS, which enhances overall ROS clearance efficiency. Collectively, this nanopipette offers a robust platform for dissecting the complex sspatiotemporal dynamics of ROS at the single-organelle level, thereby acquiring simultaneous insights into the communication mechanisms between organelles and their immediate cytosolic surroundings.