Combining Fret and Super‐Resolution Microscopy Reveals Kinase Activation and Mitochondrial Activity at the Nanoscale

ABSTRACT Protein kinases are key regulators of intracellular signaling in specific subcellular compartments and in micro‐ or nano‐domains. Genetically encoded biosensors based on Förster's resonance energy transfer (FRET) are powerful tools to track kinase dynamics. Yet, they are typically limited by spatial resolution. Aurora kinase A (AURKA), a multifunctional serine/threonine kinase, has recently emerged as a critical regulator of mitochondrial physiology. However, visualising AURKA activation and activity with sub‐diffraction precision remains a challenge. Here, we introduce BioSenSRRF, a versatile approach combining conventional FRET biosensors with super‐resolution radial fluctuations (SRRF) microscopy. BioSenSRRF requires no modification of existing probes, can be implemented using standard microscopy setups, and is supported by publicly available image analysis tools . With BioSenSRRF, we uncover that mitochondrial AURKA activation and activity are compartmentalized into distinct mitochondrial domains containing the ATP synthase. These subdomains depend on AURKA catalytic activity, and they can be altered using validated AURKA inhibitors. Finally, we demonstrate that the cancer‐associated polymorphism F31I enhances AURKA activation and ATP production on ATP synthase‐enriched subdomains. Altogether, BioSenSRRF provides a broadly accessible framework to enhance the spatial resolution of genetically encoded biosensors. This strategy opens new avenues for dissecting the subcellular organization of kinases and their contribution to physiology and disease.