Super-Resolution Imaging of Stress-Modulated Mitochondria-Lysosome Crosstalk in Ferroptosis

Abstract

The mechanism by which mitochondria and lysosomes coordinate at mitochondria-lysosome contacts (MLCs), particularly during ferroptosis and energetic stress, remains unclear. Classic imaging logic, which uses two separate probes targeting mitochondria and lysosomes, is unsuitable for the precise analysis of MLCs owing to spectral crosstalk, differences in photostability, inconsistent retention, and asynchronous photobleaching. In this study, we designed a fluorescent probe, SDML, that enables simultaneous, stable visualization of mitochondria and lysosomes with spectrally well-separated dual emissions. Integrating dual-fluorescence and dual-targeting capabilities into a single molecule, we addressed the aforementioned limitations and directly visualize the spatiotemporal dynamics of MLCs at a subcellular resolution, further revealing their stressmodulated remodeling. Under nutrient deprivation, SDML imaging revealed that compounded energetic stress disrupts the adaptive coordination between mitochondria and lysosomes, impairing organelle interactions during ferroptosis. This disruption establishes a previously unrecognized link between autophagy and ferroptosis via organelle crosstalk. Our study proposed a powerful tool for analyzing mitochondria-lysosome dynamics and provides insights into the impact of MLCs on cell fate under energetic stress.