Binding‐Activated ¹ O ₂ ‐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 ( 1 O 2 ‐genic) photosensitizers for controlling and decoding cytoskeletal networks. Single‐step installation of a sulfamide‐PEG 2 ‐ligand onto rhodamine photosensitizer scaffolds yields binding‐activated probes that mainly exist as the non‐excitable spirolactams in solution but largely switch to 1 O 2 ‐producing zwitterions upon binding to microtubules or F‐actin. Continuous illumination in confocal microscopy generates a burst of 1 O 2 , 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 1 O 2 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.