高级共焦显微镜技术研究 DNA 损伤的蛋白质-蛋白质相互作用和动力学
Summary
激光 microirradiation 是研究活体细胞 DNA 修复的有效工具。一个方法的方法, 使用 UVA 激光诱导各种 DNA 病变显示。我们对维持正常细胞周期的局部 microirradiation 的方法进行了优化;因此, 辐照细胞通过有丝分裂进行。
Abstract
地方 microirradiation 与激光代表了一个有用的工具研究 DNA 修复相关的过程中活细胞。在这里, 我们描述了一种方法来分析蛋白质动力学在 DNA 损伤的时间或蛋白质-蛋白质相互作用的局部 microirradiated 染色质。我们还展示了如何识别细胞周期的各个阶段, 使用 Fucci 细胞系统来研究 DNA 损伤时细胞周期依赖性的蛋白质动力学。本文还介绍了使用两种紫外线激光器 (355 nm 和 405 nm) 诱导不同类型 DNA 损伤的方法。只有405纳米二极管激光器 microirradiated 的细胞进行正常的有丝分裂, 没有丁烷嘧啶聚。我们还展示了如何 microirradiated 细胞可以固定在一个特定的时间点来执行免疫内源性蛋白质的利益。对于 dna 修复研究, 我们另外描述了生物物理方法的使用, 包括 FRAP (漂白后的荧光恢复) 和电影 (荧光寿命成像显微镜) 的细胞自发发生的 dna 损伤灶。我们还展示了电影 (荧光共振能量转移) 在蛋白质-蛋白质相互作用实验研究中的应用。
Introduction
dna 损伤导致 dna 病变的出现, 包括丁烷嘧啶聚 (方案), 8-氧-78-氢-2 “-脱氧, 和单或双打破1,2。γ射线是以最高的能量和高显的电离辐射的形式, 因而辐射的来源在放射疗法被广泛使用3。另一方面, 实验性的紫外线激光引起的 DNA 损伤模仿自然暴露于紫外线。UVA microirradiation, 作为一种显微方法, 代表了一个实验工具, 研究 DNA 损伤的个体活细胞。Microirradiation 40 年前第一次使用, 以揭示染色体区域的组织4,5。该技术高度依赖于共焦显微镜的功能特性或现代 nanoscopy 的技术极限。为了诱导 DNA 损伤, 细胞可以 presensitized 5 的尿 (BrdU) 或赫斯特33342之前, 紫外线照射。Bártová et al.6以前描述了 presensitization 步骤, 最近我们优化了这种 microirradiation 技术, 以避免细胞死亡或凋亡。例如, 使用 405 nm 的 UV 激光器 (不含赫斯特 33342 presensitization) 会导致53BP1 阳性双断裂 (DSBs), 其代价是丁烷嘧啶聚。另一方面, presensitization 步骤与 UV microirradiation 相结合, 会导致高水平的方案和 DSBs 同时7,8。这种方法很难应用于单 DNA 修复通路的研究。
有了 microirradiation, 就有可能分析蛋白质的招募, 动力学, 和相互作用的 DNA 损伤活细胞。Luijsterburg et al.发布了此方法的一个示例9用于异蛋白 1β, 我们最近首次显示多因子 Oct4 和与卡哈尔体 coilin 相关的蛋白质, 被吸收到紫外线诱导的 DNA 损伤6,10。蛋白质动力学在这些脱氧核糖核酸损伤也可以研究使用 FRAP (荧光恢复在漂白以后)11,12,13或烦恼 (荧光共振能量转移) 技术14 ,15。这些方法有可能揭示蛋白质在 DNA 损伤或蛋白质-蛋白质相互作用中的简单扩散。一个有用的工具, 额外的表征蛋白质是电影 (荧光寿命成像显微镜) 或它的组合与烦恼技术 (烦恼-电影)16。这些方法能够研究在稳定或瞬时表达荧光分子标记的蛋白质的活细胞中的过程17。这里, 一个指数衰减时间 (τ) 的例子, GFP 标记的 p53 蛋白及其相互作用的伙伴, mCherry 标记 53BP1, 发挥重要作用的 DNA 损伤的响应18,19显示。参数τ, 电影计算提供的荧光的寿命, 是特定的荧光染料, 其结合能力, 及其细胞环境。因此, 这种方法可以显示我们之间的蛋白质亚群之间的区别, 他们的结合能力, 和他们的功能性质后, 例如, DNA 损伤。
这里, 在我们的实验室中使用的先进的显微技术的方法学方法的概要研究在 microirradiated 染色质部位的具体的蛋白质的招募、动力学、扩散和蛋白质-蛋白质相互作用提出。本发明提供了在活细胞中诱导局部 dna 损伤的 step-by 步骤方法, 并介绍了用于研究紫外线激光引起的局部 dna 损伤相关事件的方法。
Protocol
Representative Results
Discussion
显微镜技术代表了研究实验室的基本工具。在这里, 简要介绍了用于研究蛋白质的招募和动力学在 DNA 损伤的方法。我们特别注意到我们的实验经验, 在当地 microirradiation 的活细胞, 我们讨论的蛋白质动力学的 FRAP 和蛋白质相互作用的 DNA 病变受体漂白烦恼28及其高级修改烦恼-电影 (图 4A-d)。这里所示的方法是真正了解 DNA 修复过程的基本工具, 特?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了捷克共和国的赠款机构 P302-12-G157 项目的支持。由挪威基金监督的捷克-挪威研究方案 CZ09 和捷克共和国教育、青年和体育部 (赠款号: 7F14369) 也支持实验。
Materials
| Cell cultivation | |||
| HeLa | ATCC | CCL-2TM | |
| ES-D3 [D3] | ATCC | CRL-11632TM | |
| HeLa -Fucci cells | http://ruo.mbl.co.jp/bio/e/product/flprotein/fucci.html | ||
| DMEM | PAN-Biotech | P03-0710 | |
| DMEM high glucose | Sigma-Aldrich | D6429-500ML | |
| Fetal bovine serum (FBS) | HyClone | SV30180.03 | |
| ES Cell FBS | Gibco | 16141-079 | |
| Non-Essential Amino Acids (NEAA) | Gibco | 11140-035 | |
| mLIF (mouse Leukemia Inhibitor Factor) | Merck Millipore | ESG1107 | |
| MTG (1-Thioglycerol) | Sigma-Aldrich | M6145-25ML | |
| Penicillin-Streptomycin Solution | Biosera | XC-A4122/100 | |
| Trypsin – EDTA | Biosera | XC-T1717/100 | dilute with 1 × PBS in ratio 1:6 |
| Nunclon cell culture dishes | Sigma-Aldrich | P7866 | cultivation of mESCs D3 cells |
| µ-Dish 35m+A15:I35m Grid-500 | Ibidi GmbH | 81166 | microscopic dish |
| 0.2% Gelatine | Sigma-Aldrich | G1890-100G | dilute in destille water and autoclaved |
| Name | Company | Catalog Number | Comments |
| Cell transfection | |||
| GFP-p53 plasmid | Addgene | 12091 | |
| mCherry-PCNA plasmid | generous gift from Cristina Cardoso, Technische Universität Darmstadt | ||
| mCherry-53BP1 plasmid | Addgene | 19835 | |
| Metafecetene | Biontex Laboratories GmbH | T020–2.0 | |
| 10 × PBS | Thermo Fisher Scientific | AM9625 | for transfection use 1 × PBS diluted in nuclease-free water |
| 5-bromo-2’-deoxy-uridine | Sigma-Aldrich | 11296736001 | |
| Name | Company | Catalog Number | Comments |
| Confocal microscopy | |||
| Microscope Leica TCS SP5 | Leica Microsystems | ||
| Microscope Leica TCS SP8 | Leica Microsystems | ||
| White-light laser | Leica Microsystems | ||
| 355-nm laser | Coherent Inc. | laser power 80 mW | |
| 405-nm laser | Leica Microsystems | laser power 50 mW | |
| Name | Company | Catalog Number | Comments |
| Immunofluorescence staining | |||
| Coverslip | VWR International Ltd | 631-1580 | |
| 4% paraformaldehyde | Affymetrix | 19943 1 LT | |
| Triton X100 | MP Biomedicals | 2194854 | |
| Saponin from quillaja bark | Sigma-Aldrich | S4521 | |
| BSA | Sigma-Aldrich | A2153 | |
| 53BP1 | Abcam | ab21083 | primary antibody |
| AlexaFluore 647 | Thermo Fisher Scientific | A27040 | secondary antibody |
| Vectashield | Vector Laboratories Ltd | H-1000 | mounting medium |
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