Binding-Activated ¹O₂-Genic Photosensitizers for Live-Cell Control and Proteomic Mapping of Cytoskeletal Networks

Cytoskeletal filaments and their associated organelles/proteins form a system-level network that organizes cellular architecture and activity, yet chemical tools for spatiotemporal control and proteome-wide mapping of these networks in living cells remain scarce. Here we present a modular strategy to generate small-molecule, singlet-oxygen-generating (¹O₂-genic) photosensitizers for controlling and decoding cytoskeletal networks. Single-step installation of a sulfamide-PEG₂-ligand onto rhodamine photosensitizer scaffolds yields binding-activated probes that mainly exist as the non-excitable spirolactams in solution but largely switch to ¹O₂-producing zwitterions upon binding to microtubules or F-actin. Continuous illumination in confocal microscopy generates a burst of ¹O₂, driving highly localized oxidation and second-timescale collapse of filament-organelle/protein networks, revealing key roles for microtubules in lysosome transport and mitochondrial dynamics. In parallel, light-tunable mild ¹O₂ generation enables selective proteome-wide proximity labeling of microtubule- and F-actin-associated networks, unveiling previously uncharacterized dual interactors at the microtubule-F-actin interface. This modular platform provides an effective tool for genetic-manipulation-free mapping and spatiotemporally controlled, localized oxidative perturbation of endogenous networks.