A modular live-cell biosensor for extracellular protease activity

Extracellular protease activity plays key roles in cell and tissue behavior, processing cell surface proteins, ligands and the extracellular matrix. Extracellular proteases can be subject to complex post-translational regulation, yet it remains challenging to quantify their activity in single cells over time. We present eNRGies (engineered neuregulin reporters as generalized indicators of extracellular shedding): modular, genetically encoded protease biosensors that translate extracellular cleavage into nuclear translocation of an intracellular (fluorescent) protein domain. We optimize the platform to report on protease activity in single cells on a timescale of minutes, and show it can be applied to soluble and cell-surface proteases including TEV protease, enterokinase, Factor Xa, MMP-9, and the sheddase ADAM17. We find ADAM17 activity can be transiently activated during mitosis and exhibit complex dynamics following EGF receptor stimulation. eNRGies biosensors enable observation of extracellular protease activity with high spatiotemporal resolution, and could be applied as synthetic biology scaffold to translate protease activity into customized cellular responses.