Mitochondrial Electron Flow Dynamics Imaging for Assessing Mitochondrial Quality and Drug Screening

Abstract Mitochondrial quality control is paramount for cellular development, with mitochondrial electron flow (Mito‐EF) playing a central role in maintaining mitochondrial homeostasis. However, unlike visible protein entities, which can be monitored through chemical biotechnology, regulating mitochondrial quality control by invisible entities such as Mito‐EF has remained elusive. Here, a Mito‐EF tracker (Mito‐EFT) with a four‐pronged probe design is presented to elucidate the dynamic mechanisms of Mito‐EF's involvement in mitochondrial quality control. Heightened aggregation of Mito‐EF in fiber‐like healthy mitochondria compared to round‐like damaged mitochondria is demonstrated, revealed Mito‐EF aggregation correlated with mitochondrial morphological remodeling, particularly in regions undergoing mitochondrial fission and fusion, and show the Mito‐EF signal associated with mitochondrial cristae maintained by Dynamin‐Related Protein 1 (DRP1). This underscores the importance of considering Mito‐EF in assessing mitochondrial quality control parameters. A novel drug screening evaluation parameter, Mito‐EF is also introduced to screen and discover mitochondrial‐targeted therapeutic modulators. This tracker provides new avenues for investigating the role of Mito‐EF in maintaining mitochondrial homeostasis and quality control, offering a potent tool for assessing mitochondrial quality and drug screening.