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<p>Fluorescent probes enable precise visualization of dynamic cellular processes, especially when combined with super-resolution imaging techniques that overcome the diffraction limit. However, traditional labeling strategies, including fluorescent protein fusions (e.g., GFP) or ligand-linked fluorophores, often perturb protein function or induce biological side effects. Here, we report a covalent fluorescent probe for endogenous tubulin, a key cytoskeletal protein governing cell division, motility, and intracellular transport. Using cabazitaxel as a tubulin targeting moiety and silicon-rhodamine as a cell permeable fluorophore, we designed and optimized probe, <b>6-SiR-</b><Emphasis Type="BoldItalic">o</Emphasis><b>-C</b><sub><b>9</b></sub><b>-CTX</b>, containing a biocompatible cleavable linker with a sulfonium center. It exhibits cell permeability, fluorogenic behavior, and efficient covalent labeling of tubulin across multiple human cell lines. Importantly, taxane targeting moiety can be removed post-labeling, preserving tubulin’s functions. This labeling strategy is compatible with STED nanoscopy in both live and fixed cells, enabling high-resolution, minimally invasive cytoskeletal imaging, and advancing the toolkit for studying dynamic cellular processes.</p>

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Biocompatible sulfonium-based covalent probes for endogenous tubulin fluorescence nanoscopy in live and fixed cells

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月11日

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  • Marie Auvray,
  • Tanja Koenen,
  • Olexandr Dybkov,
  • Henning Urlaub,
  • Gražvydas Lukinavičius

摘要

Fluorescent probes enable precise visualization of dynamic cellular processes, especially when combined with super-resolution imaging techniques that overcome the diffraction limit. However, traditional labeling strategies, including fluorescent protein fusions (e.g., GFP) or ligand-linked fluorophores, often perturb protein function or induce biological side effects. Here, we report a covalent fluorescent probe for endogenous tubulin, a key cytoskeletal protein governing cell division, motility, and intracellular transport. Using cabazitaxel as a tubulin targeting moiety and silicon-rhodamine as a cell permeable fluorophore, we designed and optimized probe, 6-SiR-o-C9-CTX, containing a biocompatible cleavable linker with a sulfonium center. It exhibits cell permeability, fluorogenic behavior, and efficient covalent labeling of tubulin across multiple human cell lines. Importantly, taxane targeting moiety can be removed post-labeling, preserving tubulin’s functions. This labeling strategy is compatible with STED nanoscopy in both live and fixed cells, enabling high-resolution, minimally invasive cytoskeletal imaging, and advancing the toolkit for studying dynamic cellular processes.