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. 2026 Jun 9;148(24):24637–24645. doi: 10.1021/jacs.5c22444

A Highly Selective, Cell-Permeable Fluorescent Probe for Imaging Histone Deacetylase 6 in Live Cells

Văn Thắng Nguyễn †,‡, Tanja Koenen §, Jonas Bucevičius †, Gražvydas Lukinavičius †,*
PMCID: PMC13307271  PMID: 42261660

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 (Kd app = 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.

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Introduction

Histone deacetylases (HDACs) are a family of enzymes that catalyze the removal of acetyl groups from lysine residues on histone and nonhistone protein substrates, playing a central role in regulating gene expression and cellular function. The 18 human HDACs are classified into four groups based on sequence homology and cofactor dependence. The zinc-dependent “classical” HDACs comprise Class I (HDAC1, 2, 3, and 8) and Class II enzymes which are further subdivided into Class IIa (HDAC4, 5, 7, and 9) and Class IIb (HDAC6 and 10). In addition, Class III HDACs (sirtuins, SIRT1–7) are NAD+-dependent enzymes, and HDAC11 is the sole member of Class IV. Among these, HDAC6 stands out due to its distinct localization and domain architecture, which includes two different catalytic domains and a C-terminal zinc finger ubiquitin-binding domain. This particular structure enables HDAC6 to regulate multiple critical cellular pathways, including cytoskeleton dynamics, protein quality control, and stress responses.

Given its central role in maintaining cellular homeostasis, the dysregulation of HDAC6 is linked to multiple human diseases. In oncology, HDAC6 promotes cellular transformation and enhances tumor cell proliferation. In the central nervous system, its activity is critical for axonal transport and synaptic plasticity, and its dysfunction is implicated in neurodegenerative diseases like Alzheimer’s and Parkinson’s disease. Consequently, the development of selective HDAC6 inhibitors has been an area of intense research, with several compounds entering clinical trials.

Despite this therapeutic interest, studying the dynamic spatial and temporal regulation of HDAC6 in its native cellular environment remains a significant challenge. Although genetic tagging with fluorescent proteins (FPs) has provided valuable insights, this approach can introduce artifacts. The large size of FPs can lead to steric hindrance and functional perturbations, and the high level overexpression can cause the fusion protein to misfold and mislocalize, failing to represent the true distribution of the native protein. Small-molecule fluorescent probes offer a compelling alternative, providing minimal perturbation and superior photophysical properties. Several fluorescent probes for HDAC6 have been developed, typically by conjugating a known inhibitor to a fluorophore. However, existing probes suffer from significant drawbacks that limit their utility (Table S1). Triphenylamine, tetraphenylethylene, naphthalimide, and fluorescein derivatives emit at shorter wavelengths, which suffer from cellular autofluorescence, limited tissue penetration for in vivo work, and potential phototoxicity. , IRDye800CW-SelSA and CyAc-RGD extend into longer wavelengths, but the embedded flexible cyanine fluorophore renders them incompatible with STED imaging. , HDAC-MB functions as a photosensitizer and is therefore not ideal for imaging. These limitations highlight the need for HDAC6 probes with red-shifted excitation/emission, high selectivity, low toxicity, and compatibility with live-cell super-resolution imaging while minimizing interference with enzyme activity.

To address this gap, we aimed to develop a fluorescent probe for HDAC6 suitable for live-cell imaging and advanced microscopy applications. We specified a probe with high affinity and functional selectivity, far-red emission to minimize autofluorescence and phototoxicity, excellent photostability, and high cell permeability. Herein, we describe the successful development of such a probe, 6SiR-C3-NextA, through a systematic design, synthesis, and imaging-based assay. We demonstrate its utility for visualizing the dynamics of endogenous HDAC6 in multiple cell lines, including its association with the microtubule network and its recruitment to stress granules during cellular stress, as well as its compatibility with STED microscopy.

Results and Discussion

Principle of Imaging-Based Assay

A critical challenge in probe development is validating selectivity within the complex environment of living cells. Conventional approaches reported for HDAC6 probes have largely relied on in vitro binding and enzymatic assays using purified HDAC proteins. While informative, such methods cannot account for cell permeability, probe stability, or potential off-target interactions with the thousands of other components inside cell. To overcome these limitations and rigorously assess probe performance in a more physiologically relevant context, we developed a robust, cell-based colocalization approach. A panel of U-2 OS cell lines was engineered to inducibly express individual HDACs (HDAC1-8) as fusions with HaloTag at N-terminus. This selection was based on the classification of human HDACs. The panel includes well-characterized members of the zinc-dependent Class I (HDAC1, 2, 3, 8) and Class II (HDAC4, 5, 6, 7) enzymes, which are commonly evaluated when studying probes based on hydroxamic acid inhibitors. The mechanistically and structurally distinct Class III HDACs (sirtuins) were excluded as they are NAD+-dependent and lack the catalytic zinc ion targeted by the class I and II inhibitors. The cell lines were constructed using a single-plasmid system containing an Epstein–Barr virus origin of replication for stable episomal maintenance. The plasmid also includes a cytomegalovirus-tetracycline operator (CMV-TetO2) promoter, which allows for tight, doxycycline-inducible control of gene expression. The coding sequence of each human HDAC was inserted into this vector upstream of a C-terminal HaloTag using Gateway cloning, with the genes of interest PCR-amplified as listed in Table S2. All final constructs were confirmed by sequencing. In this system, the HaloTag is covalently labeled with a fluorescent ligand, providing a ground-truth reference for the location of each HDAC. Crucially, we selected a HaloTag ligand that is spectrally distinct from the probe (e.g., TMR Halo-Tag ligand vs SiR probe), allowing for simultaneous, dual-color imaging without signal crosstalk. The selectivity of a candidate probe can then be quantified by measuring the Pearson’s Correlation Coefficient (PCC) between its signal and the HaloTag reference signal (Figure a). This approach provides a direct, quantitative measure of on-target engagement while simultaneously considering any significant off-target binding within the cellular environment. Furthermore, this versatile assay is not limited to HDAC6 and can be readily adapted for the discovery and validation of selective fluorescent probes targeting other HDAC enzymes.

1.

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Imaging-based assay for HDAC6 Probe selection and optimization. (a) Schematic of the assay. U-2 OS cell lines engineered to express individual HDAC-HaloTag fusions are incubated with a HaloTag ligand and the candidate probe, followed by automated widefield imaging and colocalization analysis. (b) Synthetic schemes for the SiR-conjugated probes based on (1) CAY10603, (2) Tubastatin, and (3) Nexturastat A. (c) Representative fluorescence images from the assay of probe 3. Wide-field microscopy images were acquired using Biotek Lionheart FX Automated Microscope. Scale bar: 200 μm. (d) Quantitative colocalization analysis for probes 1, 2, and 3 between the probe and HaloTag signals. Data are presented as mean ± SD from 3 independent experiments. Figure 1a was created in BioRender.

Probe Design and Structure–Activity Relationship (SAR) Study

Our strategy was based on a modular probe design tethering a recognition moiety to a fluorophore via a chemical linker. For the recognition moiety, we selected three well-characterized inhibitors known for their high selectivity for HDAC6: Nexturastat A (IC50 5.02 ± 0.06 nM), CAY10603 (IC50 0.002 nM), and Tubastatin A (IC50 15 ± 1.0 nM) , (Figure S1). The primary challenge was to ensure that this selectivity was maintained after conjugation to a bulky fluorophore.

For our probe synthesis, we chose rhodamine class as the fluorophore. Rhodamine dyes are exceptionally well-suited for demanding live-cell imaging applications and compatible with super resolution techniques due to their combination of high brightness, excellent photostability. A key feature contributing to their utility is their excellent cell permeability, which is governed by reversible equilibrium between a fluorescent, zwitterionic form and a nonfluorescent, neutral spirolactone form. The charge-neutral spirolactone readily crosses the cell membrane, after which the intracellular environment shifts the equilibrium back toward the fluorescent zwitterionic state, leading to “turn-on” of the signal. Thus, we synthesized first-generation probes, 1, 2 and 3, by conjugating these HDAC6 selective inhibitor to a cell-permeable, far-red SiR (silicon rhodamine) fluorophore (Figure b). Following the imaging-based assay, we found that there are significant differences in HDAC6 functional selectivity among the probes. The CAY10603-based probe, 1, lost its desired binding profile as shown by low correlation value toward HDAC6 cell staining (PCC = 0.29 ± 0.03) (Figures d, S2). The Tubastatin A-based probe 2 retained ability to bind HDAC6, but suffered from off-targeting (PCC = 0.66 ± 0.08) (Figures d, S2). In contrast, the Nexturastat A-based probe 3 demonstrated exceptional functional selectivity, yielding a high PCC of 0.84 ± 0.02 (Figure c, d). This finding validates our design strategy by confirming that the Nexturastat A scaffold likely maintains its high selectivity when conjugated to a large fluorophore.

With the functional selectivity of the conjugated Nexturastat A scaffold confirmed, we performed a systematic SAR study to optimize the probe’s performance by modifying the fluorophore, linker, and binding moiety (Figure , Scheme S1).

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HDAC6 fluorescent probes used for the systematic structure–activity relationship (SAR) study.

Fluorophore

The choice of fluorophore is critical as its physicochemical properties can strongly influence the overall performance of the probe. We initiated our optimization by comparing our lead SiR-based probe with analogues constructed from tetramethylrhodamine (TMR). This comparison was designed to assess the impact of the fluorophore’s core structure and polarity on cellular performance. SiR dyes are generally more hydrophobic compared to the more polar TMR, a difference illustrated by their calculated partition coefficients of their zwitterion form (cLogP of ∼−0.88 for SiR vs ∼−3.44 for TMR) and spiro-lactone form (cLogP of ∼5.86 for SiR vs ∼4.24 for TMR), properties which often correlate with probe solubility and cell permeability. Furthermore, we synthesized and evaluated SiR’s isomers-4/5/6 (probes 3, 4 and 5) and different constitutional TMR’s isomers-4/5/6 (probes 6, 7 and 8), varying the attachment point of the linker to the fluorophore’s xanthene core (Figure , Scheme S1). The specific conjugation site is known to be a critical determinant of probe performance, as it can alter the electronic structure of the fluorophore, thereby affecting its brightness and quantum yield, and can also introduce steric hindrance that impacts binding affinity or leads to nonspecific interactions. Following the imaging-based assessment, The SiR-based probes 3, 4 and 5 all showed strong colocalization with the HDAC6-Halo reference signal, as indicated by their high PCC values. In contrast, the TMR-based probes 6, 7 and 8 exhibited a significant loss of binding affinity, with dramatically lower PCCs (Figure S3a–b). Among the SiR probes, probe 3 was identified as the lead candidate due to its superior signal intensity, confirming 6SiR as the fluorophore of choice for this application (Figure S3a–c). Its far-red emission profile is particularly advantageous for live-cell imaging as it minimizes interference from cellular autofluorescence and reduces the laser power, thereby lowering phototoxicity.

Scaffold Fluorination

To investigate if modulating the inhibitor’s physicochemical properties could improve imaging performance, we synthesized and tested an analogue in which the phenyl group of the Nexturastat A core was perfluorinated (Figure , Scheme S1). Contrary to the hypothesis that this might improve cell permeability, fluorinated probe 9 proved detrimental, leading to a significant decrease in both the signal intensity and the signal-to-background ratio (Figure S4). This suggests that fluorination negatively impacts either the probe’s affinity for HDAC6 or its cellular accumulation, confirming the nonfluorinated scaffold as superior.

Zinc-Binding Group (ZBG)

We investigated alternative ZBGs, such as difluoromethyloxadiazole (DFMO), which have been reported as irrevesible HDAC6 binder with unprecedented isotype selectivity (Figure , Scheme S1). However, probes 10 and 11 incorporating DFMO failed to show better binding affinity in our imaging-based assay as shown by lower PCC values. This result confirms that the classical ZBG hydroxamic acid of Nexturastat A is essential for potent target engagement within our probe design (Figure S5).

Linker

The linker length and composition were systematically varied (Figure , Scheme S1). A series of probes 12 - 23 with alkyl chains of C2 to C10 and polyethylene glycol (PEG) linkers of 1–4 units were synthesized and tested. This analysis revealed a optimal probe, with the C3 alkyl linker 13 (6SiR-C3-NextA) providing the highest signal intensity and best signal-to-background ratio (Figure S6). This suggests an optimal distance and flexibility is required to allow the SiR fluorophore to sit outside the binding pocket without disrupting the inhibitor’s key interactions. Performance decreased with both shorter (C2) and longer (C4–C10) alkyl chains, as well as with all PEG-based linkers. In addition, the optimal probe 6SiR-C3-NextA still maintained the desired functional selectivity profile from the parent probe 3 (Figure S7).

This systematic imaging-based optimization revealed 6SiR-C3-NextA as the lead candidate, possessing the optimal combination of a SiR fluorophore, a C3 alkyl linker, and the Nexturastat A hydroxamic acid ZBG.

Biochemical and Cellular Characterization of 6SiR-C3-NextA

To quantify the binding affinity of 6SiR-C3-NextA, we performed a Homogeneous Time-Resolved Fluorescence (HTRF) saturation binding assay. This assay, which relies on Förster Resonance Energy Transfer (FRET) between a long-lifetime terbium donor on the protein and the probe’s SiR acceptor, provides a highly sensitive and robust measure of binding. The assay measured a high-affinity interaction between the probe and HDAC6, yielding an apparent dissociation constant (Kd app) of 21 ± 4 nM. In contrast, no significant HTRF signal was observed for HDAC1 or HDAC7, which represent Class I and Class IIa isozymes respectively, confirming that this probe is highlighting HDAC6 with high selectivity (Figure a). We further expanded this functional selectivity profiling to include HDAC10, the other member of the Class IIb HDACs. In a fluorescence response assay, 6SiR-C3-NextA exhibited no significant fluorescence enhancement in the presence of HDAC10, whereas HDAC6 triggered a pronounced turn-on signal (Figure S8). Notably, in agreement with literature reports, the Tubastatin A–based probe (probe 2) shows measurable fluorescence activation with both HDAC6 and HDAC10, supporting that this scaffold interacts with both Class IIb isozymes. , In this context, the absence of a detectable fluorescence response of 6SiR-C3-NextA toward HDAC10 highlights its functional selectivity within the Class IIb HDACs.

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In vitro and in cellulo characterization of 6SiR-C3-NextA. (a) Saturation binding curve of HTRF binding assay of SiR-C3-NextA against HDAC1, HDAC6 and HDAC7. Data are presented as mean values ± SD, n = 3 independent experiments. b) In vitro fluorogenicity of the 6SiR-C3-NextA probe. 6SiR-C3-NextA (100 nM) was incubated for 1 h at room temperature in U-2 OS cell lysate in either the absence or presence of recombinant HDAC6 (300 nM). Data are presented as mean values ± SD, n = 3 independent experiments c) Cell cycle perturbation induced by 6SiR-C3-NextA after 24 h incubation. d) Result of Annexin V Apoptosis Assay of SUP-M2 cells after a 24 h treatment with increasing concentrations of 6SiR-C3-NextA. Data points are mean ± SD, n = 3 independent experiments.

A highly desirable characteristic for a fluorescent probe is fluorogenicity, the ability to increase its fluorescence intensity upon binding to its target. This property significantly enhances the signal-to-background ratio by ensuring that unbound probe molecules in the cellular environment contribute minimally to the overall fluorescence. To assess this property, we measured the fluorescence emission of 6SiR-C3-NextA in the presence and absence of recombinant HDAC6 protein in U-2 OS cell lysate. Upon incubation with excess amount of HDAC6, the probe exhibited a significant, approximately 3.6-fold increase in fluorescence intensity compared to the probe alone in buffer (Figure b). This confirms that 6SiR-C3-NextA is a modestly fluorogenic probe.

Crucially for live-cell imaging, 6SiR-C3-NextA exhibited minimal cytotoxicity. Cell cycle analysis of HeLa cell showed no significant perturbations at concentrations up to 5 μM after 24 h of treatment (Figure c). The observed minimal cytotoxicity and lack of apoptosis induction are consistent with the known pharmacological profile of the parent scaffold, Nexturastat A. Furthermore, an Annexin V apoptosis assay with SUP-M2 cells revealed no significant increase in cell death at concentrations well above those required for effective imaging (≤1.25 μM) (Figure d). The minimal cytotoxicity and absence of apoptosis we observed align with the reported pharmacological profile of the parent inhibitor, Nexturastat A. Previous studies indicate that Nexturastat A induces minimal antiproliferative effects and promotes G1 cell cycle arrest rather than cell apoptosis. This suggests that the inherent biocompatibility of the Nexturastat A scaffold is responsible for the probe’s low toxicity, making it highly suitable for live-cell imaging. This low toxicity is a critical feature, ensuring that observations reflect native cellular processes rather than a response to a cytotoxic agent.

A key consideration for probes derived from inhibitors is their potential to perturb the biological system under observation. To evaluate the impact of 6SiR-C3-NextA on its target’s enzymatic activity in cells, we measured its effect on the acetylation level of Lys40 of α-tubulin, the primary substrate of HDAC6. HeLa cells were treated with increasing concentrations of the 6SiR-C3-NextA and the parent inhibitor Nexturastat A, then acetylated α-tubulin levels were quantified by immunofluorescence imaging. A dose-dependent increase in acetylated α-tubulin was observed, consistent with the inhibitory activity of the parent compound, Nexturastat A (Figures , S9). At the typically concentration used for imaging (e.g., 100 nM), 6SiR-C3-NextA did not cause a significant increase in tubulin acetylation, even after prolonged incubation. These results demonstrate that while the probe retains the inhibitory potential of its scaffold, it can be used for imaging under conditions that minimally perturb the endogenous enzymatic activity of HDAC6, ensuring that the visualized dynamics reflect the native state of the cell.

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Quantification of probe-induced HDAC6 tubulin hyperacetylation in living cells. (a) Representative immunofluorescence images of Human fibroblast cells treated for 24 h with and without 6SiR-C3-NextA. Cells were costained with antibodies for acetylated α-tubulin. Scale bar: 100 μm. Image acquisition settings are indicated in Table S3. (b) Dose–response curves plotting the normalized ratio of acetylated tubulin to β-tubulin. Data points are mean ± SD, n = 3 independent experiments.

The probe’s on-target specificity in cells was validated through colocalization experiments. In induced HDAC6 overexpressing U-2 OS cells fixed with paraformaldehyde, the signal from 6SiR-C3-NextA showed excellent correlation with that of an anti-HDAC6 antibody (Figure S10a–c) (PCC = 0.85 ± 0.04). In live cells, the probe’s signal colocalized perfectly with an orthogonally labeled HaloTag-HDAC6 fusion protein (PCC = 0.90 ± 0.03) (Figure S10b–c). This robust performance was consistent across a diverse panel of cell lines (HeLa, Huh-7, SH-SY5Y, 3T3, HEK293T, SUP-M2 and human dermal fibroblasts), where the probe signal was effectively competed away by an excess of a nonfluorescent inhibitor in live cell staining experiment (Figure S11), and further validated with immunostaining, confirming its utility for imaging endogenous HDAC6 in various cell lines (Figure S12). Notably, the probe remains functional after cell fixation with PFA, demonstrating its compatibility with immunofluorescence protocols.

Modulation of Probe Performance by Efflux Pump Inhibition

The intracellular concentration of a small-molecule probe is determined by the balance between membrane permeability and active removal by efflux pumps. To assess the impact of efflux effect on 6SiR-C3-NextA performance, we quantified its intracellular accumulation in the presence and absence of known efflux pump inhibitors across our diverse cell panel. Our results indicate that the intracellular accumulation of 6SiR-C3-NextA is significantly limited by active transport, as the probe is a substrate for efflux pumps. In six of the seven cell lines tested, coincubation with an efflux pump inhibitor resulted in a substantial, up to 9-fold increase in the intracellular fluorescence signal (Figure ). For example, 3T3 and Huh-7, showed a dramatic signal increase with the potent third-generation inhibitors Tariquidar and Zosuquidar. Interestingly, the optimal inhibitor varied, as HeLa cells and H. Fibroblasts responded more strongly to the first-generation inhibitor Verapamil. These findings confirm that 6SiR-C3-NextA is an efflux pump substrate and, more importantly, offer a practical strategy to significantly improve its performance by coincubating with an appropriate inhibitor to maximize the signal-to-noise ratio.

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Efflux of 6SiR-C3-NextA in living cells. (a) FACS analysis of efflux-inhibited staining experiments. Cells were stained at 37 °C for 1 h. Results are averages of three independent experiments (N = 3) and presented as means with standard deviations. (b) Representative fluorescence microscopy images demonstrating active efflux on 6SiR-C3-NextA staining performance. Seven cell lines from were stained with the probe alone (100 nM) or coincubated with the probe and most effective efflux inhibitor as determined from FACS analysis. Scale bar: 50 μm. Image acquisition settings are indicated in Table S3.

Microtubule Association and Stress Granule Dynamics

Having validated its performance, we applied 6SiR-C3-NextA to visualize the dynamics of HDAC6 in various cellular contexts. We aimed to monitor stress granule formation in both HDAC6-overexpressing cells and cells expressing the protein at endogenous levels. In addition, we sought to extend imaging to more complex cellular systems, such as primary neurons. As a primary tubulin deacetylase and a key component of the cellular stress response, HDAC6 is known to associate with microtubules and relocalize to stress granules (SGs) during stress. , To investigate this phenomenon, we induced osmotic stress in U-2 OS cells that were overexpressing HDAC6 labeled with our 6SiR-C3-NextA probe. This treatment triggered the rapid and dynamic recruitment of HDAC6 from a diffuse cytoplasmic pattern into distinct, punctate SGs. Using STED microscopy, we clearly observed this SG formation before and after the induction of stress. These results aligned with previous reports identifying HDAC6 as an integral SG component and additionally confirm the compatibility of our probe with super-resolution imaging techniques (Figure a and Table S3). We observed heterogeneous responses of U-2 OS cells to osmotic stress, which likely reflect inherent variability in gene expression and cellular states within cancer cell lines (Video S1).

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Fluorescence microscopy imaging of HDAC6 probe stained cells. (a) Representative STED imaging of induced HDAC6 expressing cell line stained with 100 nM of 6SiR-C3-NextA in DMEM growth medium for 1 h at 37 °C, washed with HBSS, then applied 150 mM NaCl 30 min before imaging. Scale bar: 10 μm. (b) Representative confocal image of living HeLa cell stained with 100 nM of 6SiR-C3-NextA, 10 nM of tubulin probe (4TMR-CTX), 1 μg/mL of Hoechst 33342 and 10 μM of verapamil in DMEM growth medium for 1 h at 37 °C, washed with HBSS. Signal was recorded from Cy5 channel before stress and after inducing osmotic stress with 150 mM NaCl in DMEM growth medium for 30 min. Scale bar: 10 μm. (c) Primary rat neuron cells were stain with 100 nM of probe (left) or costaining with 100 μM of Nexturastat A for 1 h at 37 °C. Representative confocal image of immunostaining experiment. Scale bar: 100 μm. d) Representative confocal image of immunostaining experiment with 6SiR-C3-NextA (magenta) and HDAC6 antibody (cyan). Numbers show the Pearson correlation between the two channels in the red-boxed region. Scale bar: 10 μm. Image acquisition settings are indicated in Table S3.

To confirm this behavior at endogenous expression levels, we repeated the experiment in HeLa cells, coincubating with the efflux pump inhibitor verapamil to enhance the signal, and live cell compatible microtubules probe (4TMR-CTX), reported previously by our lab. We again observed the robust recruitment of diffuse cytoplasmic HDAC6 into SGs upon stress induction, and also found that these granules forming along with microtubules, confirming its interaction with microtubules network, this was in line with published finding. Interestingly, we also noted that the fraction of HDAC6 located within the nucleus appeared to relocalize toward the nuclear inner membrane under these stress conditions. This nuclear retention is consistent with the known regulation of the HDAC6 nuclear localization signal (NLS). Given that acetylation of NLS inhibits its function and results in cytoplasmic retention, the observed nuclear localization implies that this fraction of HDAC6 possesses a nonacetylated, functional NLS. This state would prevent its net export, thereby promoting its accumulation at the nuclear envelope. Collectively, this real-time visualization in multiple cell models confirms the probe’s ability to track the translocation of endogenous HDAC6 in response to physiological stimuli (Figure b, Video S2).

Imaging in Primary Neurons

Given the importance of HDAC6 in neuronal health, we tested the probe in cultured primary rat neurons. The probe successfully entered the neurons and stained HDAC6; this signal was abolished by competition with excess Nexturastat A, confirming a specific binding mechanism (Figure c). However, colocalization experiments with an anti-HDAC6 antibody yielded only partial correlation (PCC ≈ 0.4–0.6). While this confirms that the probe binds to endogenous HDAC6 in neurons, it also suggests the presence of some off-target interactions in this complex cellular environment, highlighting a potential area for future probe refinement (Figure d). This observation is not entirely unexpected, as the highly complex and lipid-rich environment of neurons can sometimes lead to nonspecific partitioning of hydrophobic dyes.

Conclusions

In summary, we have developed 6SiR-C3-NextA, a novel, high-performance fluorescent probe for imaging endogenous HDAC6 in living cells. Through a rational design and systematic optimization process, we created a tool that exhibits high affinity, exceptional functional selectivity among tested HDAC1–8 overexpressing cell lines. In addition, no significant fluorescence increase was observed in the presence of HDAC10, indicating functional selectivity among the Class IIb HDACs. The probe is cell-permeable and well-suited for long-term and super-resolution imaging, as the working concentrations are over an order of magnitude lower than those found to induce cytotoxicity. We have demonstrated its utility in visualizing the enzyme’s association with microtubules and its recruitment to stress granules, highlighting its value for investigating the fundamental biology of HDAC6. This probe represents a significant addition to the chemical biology toolbox and is expected to facilitate new discoveries regarding HDAC6’s role in neurodegenerative pathologies, particularly those driven by protein aggregation and cytoskeletal dysfunction, such as Alzheimer’s and Parkinson’s disease.

Supplementary Material

ja5c22444_si_001.pdf (21.6MB, pdf)
Download video file (71.5MB, mp4)
Download video file (55.4MB, mp4)

Acknowledgments

This work was supported by the Max Planck Society. Văn Thắng Nguyễn was supported by the Ph.D. program Genome ScienceInternational Max Planck Research School. The authors are grateful to Dr. Vladimir Belov, Jan Seikowski, Jens Schimpfhauser, and Jürgen Bienert (Facility for Synthetic Chemistry, MPI-NAT), Dr. Holm Frauendorf, and the central analytics’ team (Institute for Organic and Biomolecular Chemistry, Georg-August University, Göttingen) for acquiring NMR and ESI-MS spectra, to Dr. Peter Lenart, Dr. Antonio Politi, and Jasmin Jakobi (Facility for Light Microscopy, MPINAT) for the possibility of performing live-cell spinning disk confocal microscopy and flow cytometry. The authors thank Dr. Shalini Pradhan, Dr. Kamila Kiszka, and Dr. Frederike Maaß for providing neuron samples used in the initial experiments.

Glossary

Abbreviations

FPs

Fluorescent proteins

HTRF

Homogeneous Time Resolved Fluorescence

HDAC

Histone deacetylase

SiR

Silicon Rhodamine

TMR

Carboxytetramethylrhodamine

(PCC)

Pearson’s Correlation Coefficient

SAR

Structure–Activity Relationship

(SGs)

stress granules

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c22444.

  • Supplementary Figures S1–S12; Tables S1–S3; detailed supplementary methods; sequences of used proteins; chemical synthesis details; supplementary schemes; references; copies of NMR spectra (PDF)

  • Video S1: time-lapse series of living U-2 OS induced HDAC6 expressing cells under osmotic stress (MP4)

  • Video S2: time-lapse series of living HeLa under osmotic stress (MP4)

Open access funded by Max Planck Society.

The authors declare the following competing financial interest(s): G.L. is a co-inventor on the patent (EP2748173B1 and US9346957B2, applicant EPFL) describing SiR and its derivatives.

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