【JACS】远红外荧光探针新突破！高亲和力助力活细胞HDAC6超分辨动态成像
文章标题：A Highly Selective, Cell-Permeable Fluorescent Probe for Imaging Histone Deacetylase 6 in Live Cells
通讯作者：Gražvydas Lukinavičius
文章概要
引言
组蛋白去乙酰化酶6（HDAC6）在细胞骨架调控、蛋白质质量控制和应激反应等多种细胞过程中发挥着关键作用，其功能异常与多种癌症和神经退行性疾病密切相关。然而，由于缺乏能在活系统中对其进行可视化追踪的化学工具，科学家们对HDAC6动态功能的深入研究一直受到限制。传统的荧光蛋白标签容易产生空间位阻和错误定位，而现有的活性小分子探针则普遍存在波长较短、对细胞有光毒性或不兼容超分辨率成像等缺陷。为了解决这一瓶颈，研究团队致力于开发一种具备高亲和力、强功能选择性、远红外发射且高细胞通透性的新型荧光探针，以实现对活细胞中内源性HDAC6的精准实时成像。
Figure 1. 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.
主要实验及结论
研究人员以高选择性HDAC6抑制剂Nexturastat A为靶向骨架，通过系统的结构-活性关系研究，将具备优异光稳定性的远红外硅基罗丹明（SiR）荧光团与C3烷基链连接子进行偶联，成功合成出最优探针6SiR-C3-NextA。体外生化与细胞分析表明，该探针与HDAC6表现出极高的结合亲和力，其表观解离常数低至。通过在专门构建的HaloTag融合蛋白细胞表征平台上进行共定位定量分析，证实该探针在Class I和Class II等多种HDAC同工酶中对HDAC6具有极高的功能选择性，且在细胞内表现出高达3.6倍的温和荧光增强特性。此外，细胞实验显示该探针毒性极低，在常规成像浓度下不会干扰HDAC6的内源性酶学活性。
Figure 2. HDAC6 fluorescent probes used for the systematic structure–activity relationship (SAR) study.
Figure 3. 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.
Figure 4. 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.
在实际生物学应用中，该探针成功展示了其强大的活细胞动态成像与超分辨纳米成像能力。研究团队利用受激发射损耗（STED）显微镜，清晰地观测到了在渗透压应激条件下HDAC6向应激颗粒（SGs）的动态募集全过程。活细胞共聚焦成像进一步揭示了内源性HDAC6与微管网络的紧密关联，并捕捉到了其在应激状态下从细胞质扩散模式向特定点状应激颗粒转移的异质性细胞反应。尽管探针在高度复杂的原代大鼠神经元中表现出轻微的非特异性外排作用，但通过联合使用外排泵抑制剂维拉帕米，显著提升了胞内荧光信号蓄积，从而成功实现了对多种不同细胞系中内源性HDAC6的高信噪比实时追踪。
Figure 5. 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.
Figure 6. 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.
总结及展望
这项研究成功开发出的远红外荧光探针6SiR-C3-NextA，不仅克服了传统化学探针光毒性大和分辨率低的缺陷，还以极高的亲和力和特异性为研究活细胞内源性HDAC6的生物学功能提供了强有力的工具。由于其出色的生物相容性以及与超分辨率成像技术的完美兼容，该探针成为了化学生物学工具箱中的重要新成员。在未来的研究中，这一创新工具预计将广泛应用于阐明HDAC6在诸如阿尔茨海默病和帕金森病等由蛋白质聚集或细胞骨架功能障碍驱动的神经退行性疾病中的分子机制，为相关疾病的病理研究和药物筛选开辟全新的可视化途径。