JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생화학

原子力显微镜成像在核细胞结构和动力学研究中的应用

Published: January 31, 2019
doi:
10.3791/58820
, , ,
1Department of Pharmaceutical Sciences,University of Nebraska Medical Center

Summary

在这里, 我们提出了一个协议, 以表征核糖体粒子在单分子水平上使用静态和延时原子力显微镜 (afm) 成像技术。所描述的表面功能化方法允许在纳米尺度上以高分辨率捕获核糖体的结构和动力学。

Abstract

染色质是一个长链核糖体亚基, 是一个动态系统, 允许 dna 复制和转录等关键过程发生在真核细胞中。核糖体的动力学提供了通过复制和转录机制获得 dna 的机会, 并对染色质功能背后的分子机制做出了重要贡献。单分子研究, 如原子力显微镜 (afm) 成像, 极大地促进了我们目前对核糖体结构和动力学作用的理解。目前的协议描述了使高分辨率 afm 成像技术能够研究核糖体的结构和动态特性的步骤。该协议由获得的 fom 数据说明了集中核细胞的 h3 组蛋白被对应的丝粒蛋白 a (cenp-a) 所取代。该协议从使用连续稀释方法组装单核苷酸体开始。用氨基丙基硅膜 (aps-mica) 功能化制备云母基板, 用于核糖体成像, 对于所描述的核糖体的原子力显微镜可视化至关重要, 并提供了制备底物的程序。沉积在 aps-mica 表面的核细胞首先使用静态 afm 进行成像, 该原子力法捕获核糖体群的快照。通过对这些图像的分析, 可以测量包裹在核糖体上的 dna 大小等参数, 这一过程也是详细的。描述了液体中的延时 afm 成像过程, 描述了高速延时 afm, 它可以捕获每秒多个核糖体动力学帧。最后, 描述和说明了核糖体动力学的分析, 使动态过程的定量表征。

Introduction

在真核细胞中, dna 高度凝聚并组织成染色体。1染色体中 dna 组织的第一级是核糖体的组合, 其中 147 bp 的 dna 紧紧地包裹在一个组蛋白八分体核上。2,3核糖体颗粒聚集在一个长的 dna 分子上, 形成一个染色质阵列, 然后组织起来, 直到形成一个高度紧凑的染色体单元。4染色质的分解提供了基因转录和基因组复制等关键细胞过程所需的免费 dna, 这表明染色质是一个高度动态的系统。5,6,7了解 dna 在不同染色质水平上的动态特性对于阐明分子水平上的遗传过程至关重要, 因为在分子水平上, 错误可能导致细胞死亡或癌症等疾病的发展。8非常重要的染色质性质是核糖体的动力学。9,10,11,12这些粒子的高稳定性使其能够通过晶体学技术进行结构表征。2这些研究缺乏的是核糖体的动态细节, 如 dna 从组蛋白核解包的机制;转录和复制过程所需的动态路径。7.,9,13,14,15,16此外, 被称为重塑因子的特殊蛋白质已被证明有助于核糖体颗粒的分解17;然而, 核糖体的内在动力学是这一过程中的关键因素, 有助于整个拆卸过程。14,16,18,19

单分子荧光技术, 如单分子荧光19、20、21、光学捕获 (推子)13、18、22、23和 afm10,14,15,16,24,25,26在了解核糖体动力学方面发挥了重要作用。在这些方法中, afm 受益于几个独特而有吸引力的功能。afm 允许人们对单个核糖体以及较长的阵列27进行可视化和表征.从 afm 图像中可以测量到组蛋白核周围的 dna 长度等核糖体结构的重要特征; 一个参数, 是核成体解包动力学的表征的核心。过去的 afm 研究表明, 核糖体是高度动态的系统, dna 可以自发地从组蛋白核14中展开。当成像在水溶液14、26、29中进行时, afm 在延时模式下操作, 直接将 dna 从核糖体中解包出来。

高速延时 afm (hs-afm) 仪器的出现使得在毫秒时间尺度14、15、24的时间尺度上可视化核糖体展开过程成为可能。最近 hs-afm 16,30研究的丝粒特异性核糖体揭示了几个新的特点的核糖体相比, 规范类型。中心核细胞组成的丝粒, 染色体的一小部分对染色体分离至关重要 31。与块状染色质中的典型核糖体不同, 丝粒核糖体的组蛋白核心包含 cenp-a组蛋白, 而不是组蛋白 h332,33。由于这种组蛋白取代, dna 包裹在丝粒核糖体中的是 ~ 120 bp, 而不是规范核糖体的 ~ 147 bp;这种差异可能导致着丝粒和规范核糖体数组34的不同形态, 这表明与块状染色体相比, 丝粒染色质经历了更高的动力学。在 hs-afm16中,30项研究所显示的新动力学说明了这种单分子技术为直接可视化其结构和动态特性提供了独特的机会。核糖体。本文最后将简要讨论和说明这些特点的例子。这一进展是由于开发了新的核糖体 afm 成像方案以及对现有方法的修改。这里描述的协议的目标是使这些令人兴奋的进展, 在单分子 afm 核糖体研究的任何人谁想要利用这些技术, 在他们的染色质调查。所描述的许多技术适用于核糖体研究以外的问题, 可用于研究其他感兴趣的蛋白质和 dna 系统。这些应用的几个例子可以在出版物35、36、37、38、39、40、41、 42、43、44、45、46、47、48、49 和 afm研究的前景各种生物分子系统在评论29,50,51, 53,54给出了。

Protocol

1. 单核细胞的连续稀释组装 生成和纯化包含一个离中心的 wiom 601 核糖体定位序列的大约 400 bp dna 基板。55注: 为了限制二核苷酸的不需要的形成, 定位序列两侧的每个 “手臂” 不应超过 ~ 150 bp。 使用质粒 pgeem3z-601 与设计的引物一起, 利用 pcr 扩增底粒 dna。对于这里使用的具有122和 154 bp 臂长度的 423 bp 基板, 使用正向 (-catgattatagtatacc-3 ‘ ‘) 和反向 (5 ‘-acagcatgattactacattact…

Representative Results

首次为原子力显微镜成像实验使用连续稀释组装方法制备了单核细胞 (图 1)。然后使用不连续的 sds-page 检查制备的核糖体 (图 2)。云母表面是使用 aps 进行功能化的, 它在表面捕获核糖体, 同时保持光滑的背景, 以实现高分辨率成像 (图 3)。核细胞沉积在 aps-mica 上, 并随后使用静态 afm 成像进行了成像。作为对组装和沉积的控制, 利用…

Discussion

上述协议相当简单, 并提供了高度可重现的结果, 尽管可以强调几个重要问题。功能化的 aps-mica 是获得可靠和可重复的结果的关键基板。aps-mica 的高稳定性是该基板的重要特性之一, 它允许用户提前准备成像基板以供使用, 可在制备后至少两周内使用。59,61然而, 如果将其用于安装在金属皮球上的云母, 表面可能会被胶的蒸汽损坏。因此, 建议按照协议中的…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者贡献: yll 和 msd 设计了该项目;msd 组装核糖体。msd 和 zs 进行了 afm 实验和数据分析。所有作者都撰写和编辑了手稿。

Materials

Plasmid pGEM3Z-601 Addgene, Cambridge, MA 26656
PCR Primers IDT, Coralville, IA Custom Order (FP) 5'- CAGTGAATTGTAATACGACTC-3' (RP) 5'-ACAGCTATGACCATGATTAC-3'
DreamTaq polymerase ThermoFischer Scientific, Waltham, MA EP0701 Catalog number for 200 units
PCR purification kit Qiagen, Hilden, Germany  28104 Catalog number for 50 units
Tris base Sigma-Aldrich, St. Louis, MO 10708976001 Catalog number for 250 g
EDTA ThermoFischer Scientific, Waltham, MA 15576028 Catalog number for 500 g
(CENP-A/H4)2, recombinant human EpiCypher, Durham, NC 16-0010 Catalog number for 50 ug
H2A/H2B, recombinant human EpiCypher, Durham, NC 15-0311 Catalog number for 50 ug
H3 Octamer, recombinant human EpiCypher, Durham, NC 16-0001 Catalog number for 50 ug
Slide-A-Lyzer MINI Dialysis Device Kit, 10K MWCO, 0.1 mL ThermoFischer Scientific, Waltham, MA 69574 Catalog number for 10 devices
Sodium Chloride Sigma-Aldrich, St. Louis, MO S9888-500G Catalog number for 500 mg
Amicon Ultra-0.5 mL Centrifugal Filters  Millipore-sigma, Burlington, MO UFC501008 Catalog number for 8 devices
HCl Sigma-Aldrich, St. Louis, MO 258148-25ML Catalog number for 25 mL
Tricine Sigma-Aldrich, St. Louis, MO T0377-25G Catalog number for 25 g
SDS Sigma-Aldrich, St. Louis, MO 11667289001 Catalog number for 1 kg
Ammonium Persulfate (AmmPS)  Bio-Rad, Hercules, CA 1610700 Catalog number for 10 g
30% Acrylamide/Bis Solution, 37.5:1 Bio-Rad, Hercules, CA 1610158 Catalog number for 500 mL
TEMED Bio-Rad, Hercules, CA 1610800 Catalog number for 5 mL
4x Laemmli protein sample buffer for SDS-PAGE Bio-Rad, Hercules, CA 1610747 Catalog number for 10 mL
2-ME Sigma-Aldrich, St. Louis, MO M6250-10ML Catalog number for 10 mL
ageRuler Prestained Protein Ladder  ThermoFischer Scientific, Waltham, MA 26616 Catalog number for 500 uL
Bio-Safe™ Coomassie Stain Bio-Rad, Hercules, CA 1610786 Catalog number for 1 L
Nonwoven cleanroom wipes: TX604 TechniCloth  TexWipe, Kernersvile, NC TX604
Muscovite Block Mica AshevilleMica, Newport News, VA Grade-1
Aminopropyl silatrane (APS) Synthesized as described in 22
HEPES Sigma-Aldrich, St. Louis, MO H4034-25G Catalog number for 25 g
Scotch Tape Scotch-3M, St. Paul, MN
TESPA-V2 afm probe (for static imaging) Bruker AFM Probes, Camarillo, CA
MSNL-10 afm probe (for standard time-lapse imaing) Bruker AFM Probes, Camarillo, CA
Aron Alpha Industrial Krazy Glue Toagosei America, West Jefferson, OH AA480 Catalog number for 2 g tube
MgCl2 Sigma-Aldrich, St. Louis, MO M8266-100G Catalog number for 100 g
Millex-GP Filter, 0.22 µm Sigma-Aldrich, St. Louis, MO SLGP05010 Catalog number for 10 devices
BL-AC10DS-A2 afm probe (for HS-AFM) Olympus, Japan
Compound FG-3020C-20  FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan 
Compound FS-1010S135-0.5  FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan 
MultiMode Atomic Force Microscope Bruker-Nano/Veeco, Santa Barbara, CA
High-Speed Time-Lapse Atomic Force Microsocopy Toshio Ando, Nano-Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan
DOWNLOAD MATERIALS LIST

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Keywords: Atomic Force MicroscopyNucleosome StructureSingle-molecule ImagingProtein-DNA ComplexesAmyloid AggregatesAlzheimer’s DiseaseParkinson’s DiseaseMica Surface PreparationAPS ModificationStatic AFM Imaging
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Stumme-Diers, M. P., Stormberg, T., Sun, Z., Lyubchenko, Y. L. Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging. J. Vis. Exp. (143), e58820, doi:10.3791/58820 (2019).
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