Dual-responsive diazo probe for labeling of aggrephagic compartments in live cells

Aggrephagy, a selective form of autophagy pathway for degrading misfolded and aggregated proteins, plays a crucial role in maintaining cellular proteostasis. Despite its biological significance, covalent labeling strategies for aggrephagy-related aggregates remain limited, primarily due to the challenges posed by the acidic and degradative environment of lysosomes. Herein, we developed a dual-responsive diazo probe (P1, λex = 506 nm, λem = 609 nm) for labeling of aggrephagy-related aggregates in living cells. P1 integrates three functional components: an aggregation-targeting moiety, a lysosome-directing unit, and a diazo group for covalent modification. The probe selectively binds and labels aggregated proteins over their properly folded counterparts. Notably, P1 activation requires the concurrent presence of visible light (λ = 300-800 nm) and an acidic microenvironment (pH = 4.4-6.23), ensuring high spatial and conditional specificity. We demonstrate that P1 enables the visualization and enrichment of aggregated proteins involved in the aggrephagy pathway. This tool is potentially useful for capturing and profiling protein factors participating cellular aggrephagy involving in neurodegeneration and cancer progression.