Relocation of Endocytic Leaflet DNA Probes for Asymmetric and Week-Long Lysosomal Labeling

Visualization of lysosomes in living cells is essential for understanding their physiological functions; yet, most probes that target the lysosomal interior often disrupt luminal chemistry, exhibit signal leakage, and fail to support long-term imaging. To address these challenges, we developed RELAY (Relocation of Endocytic Leaflet tAg to modifY organelles), a topology-preserving labeling strategy to transfer the inner-leaflet tags on the plasma membrane to the cytosol-facing outer leaflet of lysosomes. RELAY employs liposome-cell membrane fusion to anchor fluorescent DNA probes with phosphorothioate (PS) backbones on the cytoplasmic inner leaflet of the plasma membrane, followed by endocytic trafficking that preserves the membrane topology and relocates the probes onto the lysosomal outer surface. Because this labeling occurs on the lysosomal exterior that is protected from luminal degradation and the PS backbone resists nuclease degradation, RELAY enables highly stable asymmetric labeling that sustains week-long lysosome imaging in living cells. Using this approach, we visualized lysosomal dynamics during cellular senescence and discovered random, unidirectional, intercellular lysosomal transfer in cell–cell communications via tunnelling nanotubes. Holding the capability for prolonged, high-fidelity visualization of lysosomes, RELAY facilitates the exploration of their biological functions.