高含量成像分析研究。
Summary
在这里, 我们描述了一种高含量的成像方法来量化与视网膜色素变性相关的视紫红质突变体的转运。采用多波长评分分析法对细胞表面或整个细胞的视紫红质蛋白进行了定量。
Abstract
罗丹辛错折变导致棒光感受器死亡, 表现为常染色体显性视网膜色素变性 (rp), 一种渐进的致盲疾病, 缺乏有效的治疗。我们假设通过药理稳定突变体视紫红质蛋白, 可以缓解错折视紫红质突变体的细胞毒性。p23h 突变, 在其他 ii 类视紫红质突变中, 编码了一种结构不稳定的视紫红质突变蛋白, 该突变蛋白存在于内质网 (er) 中, 而野生型视紫红质突变蛋白则被输送到哺乳动物的质膜上细胞。我们以前进行了基于发光的高通量屏幕 (hts), 并确定了一组药理伴侣, 拯救了 p23h 视紫红素从 er 到质膜的运输。在这里, 我们使用免疫染色方法, 然后进行高含量的成像分析, 我们量化了整个细胞和质膜上的突变性视紫红素蛋白的数量。该方法信息丰富, 有效识别 hts 后误报的真实命中。此外, 高含量图像分析使我们能够量化一个单一实验的多个参数, 以评估每种化合物的药理特性。利用该方法, 我们分析了11种不同化合物对 6个 rp 相关视紫红质突变体的影响, 获得了一个二维药理谱, 对这些视紫红质突变体的结构稳定性有了定量和定性的了解以及不同化合物对这些突变体的功效。
Introduction
蛋白质错误折叠与肌肉萎缩症、神经消融以及致盲疾病有关, 包括视网膜色素变性 (rp)1。rp 是一种遗传和进行性视网膜变性, 与60多个基因的突变有关, 这些基因影响棒光感受器或视网膜色素上皮 (rpes)2,3的功能和稳态。目前没有有效的 rp 治疗方法。罗丹辛突变约占常染色体显性 (ad) rp 病例的25-30。在 150多种视紫红质突变4 (人类基因突变数据库,/www.hgmd.cf.ac.uk/) 中, ii 类突变导致导致棒光感受器死亡的视紫红质蛋白的结构不稳定,视力下降5,6,7,8。p23h 是北美最常见的视紫红质突变, 也是 ii 类视紫红质突变9,10的典型例子。由于其固有的结构不稳定性, 错折叠的视紫红质在哺乳动物细胞内质网 (er) 中积累, 而野生型视紫红质位于质膜5上。错误折叠的视紫红质 p23h 突变体表现出显性的负细胞毒性, 不是由于单倍体不足, 而是与 er 相关蛋白降解途径的激活和中断的棒外段组织有关。为了减轻棒光感受器细胞的压力, 一种策略是使用药理伴侣来稳定突变体视紫红质的原生折叠。
为了实现这一目标, 我们使用β-半乳糖苷酶片段互补试验进行了基于细胞的高通量筛选 (11,12 , 13), 以量化在血浆中运输的 p23h 视后质突变体膜。这种高温超导分析的稳健而简单的协议使我们能够探索每个屏幕大约 79, 000个小分子的活性。然而, 由于此 hts 检测读取发光信号, 包括β-gal 抑制剂、有色或细胞毒性化合物在内的误报被列入必以二次检测的命中名单。
传统的免疫染色和荧光成像方法已被用于研究哺乳动物细胞中的视紫红质转运 5,14,15,16。然而, 这些常规方法不能用来量化10种以上化合物对视紫红质运输的药理作用, 因为可靠的成像分析需要在高度一致的条件下拍摄大量图像, 即不能用传统的成像方法来修正。在这里, 我们开发了一个基于免疫染色的高含量成像协议作为二次检测, 以量化错误折叠的视紫红质突变体11,13,17的细胞表面传输。为了在质膜上贴上视紫红质的标签, 我们跳过了细胞膜渗透的步骤, 并通过单克隆 (b6-30) 反视紫红质在细胞外侧识别视紫红质 n-末端表位的免疫抑制视紫红质突变体膜18。为了想象整个细胞中的突变视紫红质, 我们将视紫红质与金星荧光蛋白融合在一起。通过对不同荧光通道中荧光强度的定量, 我们可以从一个实验中获得多个参数, 包括整个细胞、细胞表面的总视紫红质强度和视紫红质荧光在细胞表面到整个细胞。将该方法应用于表达共6个错误折叠的视紫红质突变体的稳定细胞, 我们可以生成多个小分子伴侣对这些突变体的药理特征。在该协议中, 所有细胞都在384孔板中进行免疫保留, 并在高度一致的成像条件下使用自动成像系统进行成像。对每口井进行图像分析, 包含600多个细胞的图像, 以减少因细胞形状和蛋白质表达水平不同的细胞的异质性而产生的变异。图 1总结了此协议的工作流。该方法的优点是通过基于图像的分析获得高分辨率图像和多参数量化。一般来说, 该协议可以修改和应用, 以量化任何错误折叠的膜蛋白的运输感兴趣。
Protocol
Representative Results
Discussion
在这里, 我们展示了一种高含量的成像检测方法, 用于描述从高温超导中识别的命中。这些协议中涉及的唯一自动化是高含量成像仪。视紫红质的免疫染色和荧光成像已被广泛用于表征视紫红质 5,14,15,16的定位。然而, 由于每个条件缺乏足够的细胞图像, 每次实验的图像容量较低, 以及缺乏质量控制参数,…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢 mark e. schurdak 博士和匹兹堡大学药物发现研究所提供的高含量成像仪和初步培训。krzysztof palczewski 博士 (凯斯西储大学) 慷慨地分享了1d4 和 b630 抗罗道肽抗体。nevin lambert 博士 (奥古斯塔大学) 分享了含有小鼠罗多辛-金星结构 cdna 的质粒。这项工作得到了国家卫生研究所向 EY024992 向 yc 提供的赠款和匹兹堡大学视觉研究核心赠款 p30ey008098 的支持。
Materials
| U2OS (rhodopsin-Venus) cells | NA | NA | Stable cells generated from U2OS cells |
| U2OS (T4R-rhodopsin-Venus) cells | NA | NA | Stable cells generated from U2OS cells |
| U2OS (P23H-rhodopsin-Venus) cells | NA | NA | Stable cells generated from U2OS cells |
| U2OS (P53R-rhodopsin-Venus) cells | NA | NA | Stable cells generated from U2OS cells |
| U2OS (C110Y-rhodopsin-Venus) cells | NA | NA | Stable cells generated from U2OS cells |
| U2OS (D190N-rhodopsin-Venus) cells | NA | NA | Stable cells generated from U2OS cells |
| U2OS (P267L-rhodopsin-Venus) cells | NA | NA | Stable cells generated from U2OS cells |
| DMEM high glucose | Genesee Scientific | 25-500 | With L-Glutamine, sodium pyruvate |
| Fetal bovine serum (FBS) | Gibco | 16140071 | Heat inactivated |
| Plasmocin | InvivoGen | ant-mpt | Mycoplasma elimination reagent |
| Penicillin-Streptomycin (100X) | Gibco | 15140122 | 100X concentrated antibiotic solutions to prevent bacteria contamination of cell cultures |
| Trypsin-EDTA | Genesee Scientific | 25-510 | 0.25%, 1mM EDTA in HBSS without calcium and magnesium |
| Poly-L-lysine solution | Sigma-Aldrich | P4707-50ML | Mol wt 70,000-150,000, 0.01%, sterile-filtered, BioReagent, suitable for cell culture |
| CellCarrier-384 Ultra Microplates | PerkinElmer | 6057300 | 384-well tissue culutre-treated microplates with black well walls and an optically -clear cyclic olefin bottom for imaging cells in high content analysis |
| Sterile 96-well plate | Eppendorf | 30730119 | Tissue culture treated with lid flat bottom, sterile, free of detectable pyrogens, Rnase, DNase and DNA. Non-cytotoxic |
| Phosphate Buffered Sailine (PBS) | Invitrogen | AM9625 | 10 x PBS Buffer, pH 7.4 |
| DMSO | Sigma-Aldrich | D4540 | >99.5%, cell culture tested |
| 9-cis-retinal | Sigma-Aldrich | R5754 | |
| Compounds tested | Selleckchem/Life Chemicals/Custom synthesized | NA | Compounds were purchased from different vendors or custom synthesized |
| B6-30 anti-rhodopsin antibody | Novus | NBP2-25160 | Gift from Dr. Krzysztof Palczewski |
| Cy3-conjugated goat anti-mouse secondary antibody | Jackson ImmunoResearch Laboratories, Inc | 115-165-146 | |
| 16% paraformaldehyde | Thermo Fisher Scientific | 28908 | Methanol-free |
| 10% Normal Goat Serum | Thermo Fisher Scientific | 50062Z | Blocking buffer |
| Hoechst 33342, Trihydroch | Invitrogen | H3570 | Nuclear staining solution |
| High-content imager | Molecular Devices | ImageXpress | ImageXpress® Micro Confocal High-Content Imaging System |
| MetaXpress high-content image acquisition and analysis software | Molecular Devices | MetaXpress | High-content image acquisition and analysis software |
| Multichannel pipette (0.5-10 µL) | Rainin | 17013802 | Manual 8-channel pipette, 0.5-10 µL |
| Multichannel pipette (0.5-10c | Rainin | 17013805 | Manual 8-channel pipette, 20-200 µL |
| Electronic multichannel pipette (10-200 μL) | Thermo Scientific | 14-3879-56BT | Electronic multichanenel pipette for 96- and 384-well microplate pipetting tasks |
| 50ml Reagent Reservoir | Genesee Scientific | 28-125 | Reagent reservior for multichannel pippte dispensing |
| 8-Channel aspirator | ABC Scientific | EV503 | 8-Channel stainless steel adaptor for aspirating liquids from 96- or 384-well plates |
| Excel spreadsheet software | Microsoft | Excel2016 | The spreadsheet software for data analysis and heatmap generation |
| Origin2018 scientific data analysis and graphing software | OriginLab | Origin2018 | The data analysis software for generating the dose response curves |
References
- Gregersen, N., Bross, P., Vang, S., Christensen, J. H. Protein misfolding and human disease. Annual Review of Genomics and Human Genetics. 7, 103-124 (2006).
- Daiger, S. P., Bowne, S. J., Sullivan, L. S. Perspective on genes and mutations causing retinitis pigmentosa. Archives of Ophthalmology. 125 (2), 151-158 (2007).
- Daiger, S. P., Sullivan, L. S., Bowne, S. J. Genes and mutations causing retinitis pigmentosa. Clinical Genetics. 84 (2), 132-141 (2013).
- Stenson, P. D., et al. The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Human Genetics. 136 (6), 665-677 (2017).
- Sung, C. H., Schneider, B. G., Agarwal, N., Papermaster, D. S., Nathans, J. Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa. Proceedings of the National Academy of Sciences of the United States of America. 88 (19), 8840-8844 (1991).
- Athanasiou, D., et al. The molecular and cellular basis of rhodopsin retinitis pigmentosa reveals potential strategies for therapy. Progress in Retinal and Eye Research. 62, 1-23 (2018).
- Chiang, W. C., et al. Robust Endoplasmic Reticulum-Associated Degradation of Rhodopsin Precedes Retinal Degeneration. Molecular Neurobiology. 52 (1), 679-695 (2015).
- Sakami, S., et al. Probing mechanisms of photoreceptor degeneration in a new mouse model of the common form of autosomal dominant retinitis pigmentosa due to P23H opsin mutations. Journal of Biological Chemistry. 286 (12), 10551-10567 (2011).
- Dryja, T. P., et al. Mutations within the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa. New England Journal of Medicine. 323 (19), 1302-1307 (1990).
- Sohocki, M. M., et al. Prevalence of mutations causing retinitis pigmentosa and other inherited retinopathies. Human Mutation. 17 (1), 42-51 (2001).
- Chen, Y., et al. A novel small molecule chaperone of rod opsin and its potential therapy for retinal degeneration. Nature Communications. 9 (1), (2018).
- Chen, Y., Tang, H. High-throughput screening assays to identify small molecules preventing photoreceptor degeneration caused by the rhodopsin P23H mutation. Methods in Molecular Biology. 1271, 369-390 (2015).
- Chen, Y., et al. A High-Throughput Drug Screening Strategy for Detecting Rhodopsin P23H Mutant Rescue and Degradation. Investigative Ophthalmology & Visual Science. 56 (4), 2553-2567 (2015).
- Noorwez, S. M., et al. Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa. Journal of Biological Chemistry. 278 (16), 14442-14450 (2003).
- Saliba, R. S., Munro, P. M., Luthert, P. J., Cheetham, M. E. The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation. Journal of Cell Science. 115, 2907-2918 (2002).
- Kaushal, S., Khorana, H. G. Structure and function in rhodopsin. 7. Point mutations associated with autosomal dominant retinitis pigmentosa. Biochimie. 33 (20), 6121-6128 (1994).
- Chen, Y., Brooks, M. J., Gieser, L., Swaroop, A., Palczewski, K. Transcriptome profiling of NIH3T3 cell lines expressing opsin and the P23H opsin mutant identifies candidate drugs for the treatment of retinitis pigmentosa. Pharmacological Research. 115, 1-13 (2016).
- Adamus, G., et al. Anti-rhodopsin monoclonal antibodies of defined specificity: characterization and application. Vision Research. 31 (1), 17-31 (1991).
- Goodson, H. V., Dzurisin, J. S., Wadsworth, P. Generation of stable cell lines expressing GFP-tubulin and photoactivatable-GFP-tubulin and characterization of clones. Cold Spring Harbor Protocols. 2010 (9), (2010).
- Zhang, J. H., Chung, T. D., Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of Biomolecular Screening. 4 (2), 67-73 (1999).
- Bray, M. A., Carpenter, A., Sittampalam, G. S. Advanced Assay Development Guidelines for Image-Based High Content Screening and Analysis. Assay Guidance Manual. , (2004).
- Sung, C. H., Davenport, C. M., Nathans, J. Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain. Journal of Biological Chemistry. 268 (35), 26645-26649 (1993).
- Krebs, M. P., et al. Molecular mechanisms of rhodopsin retinitis pigmentosa and the efficacy of pharmacological rescue. Journal of Molecular Biology. 395 (5), 1063-1078 (2010).
