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A Highly Selective, Cell-Permeable Fluorescent Probe for Imaging Histone Deacetylase 6 in Live Cells
Văn Thắng Nguyễn1,2, Tanja Koenen3, Jonas Bucevičius1
1Chromatin Labeling and Imaging group, Department of NanoBiophotonics, Max Planck Institute for Multidisciplinary Sciences, Am Fassberg 11, Göttingen 37077, Germany.
Abstract:
Histone Deacetylase 6 (HDAC6) plays a crucial role in diverse cellular processes, including cytoskeletal regulation, protein quality control, and stress responses, and its dysregulation is linked to multiple cancers and neurodegenerative disorders, making it a key therapeutic target. However, a detailed understanding of its dynamic functions has been limited by the lack of chemical tools for its visualization in living systems. Utilizing the highly selective inhibitor Nexturastat A as a targeting scaffold, we developed 6SiR-C3-NextA probe incorporating a bright, photostable, far-red silicon-rhodamine fluorophore. Biochemical and cellular assays show that 6SiR-C3-NextA binds to HDAC6 with high affinity (Kdapp = 21 ± 4 nM) and functional selectivity over other HDAC enzymes, as validated using a developed panel of engineered cell lines expressing individual human HDACs. The probe is cell-permeable, exhibits low cytotoxicity, is compatible with super-resolution techniques, and enables the visualization of endogenous HDAC6 across multiple cell lines. We demonstrate its utility by performing imaging of HDAC6's association with the microtubule network and its dynamic recruitment to stress granules in living cells.
Insights
Researchers developed a new chemical probe, 6SiR-C3-NextA, to visualize Histone Deacetylase 6 (HDAC6) in living cells. This tool aids in understanding HDAC6
Area of Science:
- Biochemistry
- Cell Biology
- Chemical Biology
Background:
- Histone Deacetylase 6 (HDAC6) is vital for cellular functions like cytoskeletal regulation and stress responses.
- HDAC6 dysregulation is implicated in cancers and neurodegenerative diseases, highlighting its therapeutic potential.
- Limited chemical tools for HDAC6 visualization hinder understanding of its dynamic roles in living systems.
Purpose of the Study:
- To develop a novel chemical probe for visualizing HDAC6 in living cells.
- To investigate the dynamic functions of HDAC6 using advanced imaging techniques.
Main Methods:
- Development of the 6SiR-C3-NextA probe, based on the selective HDAC6 inhibitor Nexturastat A and a silicon-rhodamine fluorophore.
- Biochemical and cellular assays to determine probe affinity, selectivity, and cytotoxicity.
- Super-resolution microscopy to visualize endogenous HDAC6 localization and dynamics in various cell lines.
Main Results:
- The 6SiR-C3-NextA probe exhibits high affinity (Kdapp = 21 ± 4 nM) and functional selectivity for HDAC6.
- The probe is cell-permeable, shows low cytotoxicity, and is compatible with super-resolution imaging.
- Successful visualization of endogenous HDAC6, its association with microtubules, and recruitment to stress granules in living cells.
Conclusions:
- 6SiR-C3-NextA is a valuable chemical tool for studying HDAC6 dynamics in real-time.
- The probe enables new insights into HDAC6's role in cellular processes and disease pathogenesis.
- This development opens avenues for exploring HDAC6-targeted therapies.
