Engineering Organelle-Gated Reporters for Imaging Subcellular Enzyme Activity in Living Cells

Enzymes catalyze biochemical reactions in all living systems, and their subcellular localizations can profoundly influence distinct cellular functions. Yet, the physiological roles of subcellular enzyme activity remain largely unexplored, owing to the lack of tools that allow imaging of enzymatic activity with organelle-level precision in living cells. Here, we present a class of fluorogenic reporters for spatiotemporally resolved imaging of enzyme activity within specific subcellular compartments, including mitochondria (Mito), lysosomes (Lyso), endoplasmic reticulum (ER), and Golgi apparatus (Golgi). These fluorogenic reporters comprise a rhodol fluorophore whose fluorescence is quenched by an enzyme-specific substrate and a bioorthogonal cage group, with signal restoration occurring only upon sequential uncaging by an organelle-localized bioorthogonal activator and subsequent enzymatic activation. We applied this versatile strategy to visualize the activity of enzymes from different families in live cells, such as leucyl aminopeptidases (LAP), γ-glutamyl transferases (GGT), and monoamine oxidase A (MAO-A). Application to LAPs revealed distinct, compartment-dependent functions: Mito-LAP activity maintained redox homeostasis in stressed cells, favoring tumor cell survival, whereas ER-LAP activity promoted antigen presentation and immune-mediated tumor cell killing. Notably, drug profiling showed that cisplatin (CDDP) and camptothecin (CPT) concurrently amplified both Mito-LAP and ER-LAP activities, eliciting opposing influences on tumor progression. These findings also inspire the development of a mitochondria-targeted inhibitor to improve anticancer efficacy. This platform offers a genetically independent, broadly adaptable framework for dissecting compartmentalized enzymology in living cells.