在脑核糖凝视与原子力显微镜
Summary
While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.
Abstract
平移机械， 即核糖体或多聚核糖体是细胞中最大和最复杂的细胞质机械之一。多核糖体，核糖体，mRNA的，几种蛋白质和非编码RNA形成的，代表集成，其中平移控制发生平台。然而，尽管核糖体已被广泛研究，多核糖的组织仍然缺乏全面的了解。因而很多努力，需要以阐明多核糖体组织和可嵌入的翻译控制的任何新颖的机制。原子力显微镜（AFM）是一种类型的扫描探针显微镜的，允许在纳米级的分辨率采集3D图像。相比电子显微镜（EM）技术，原子力显微镜的主要优点之一是，它能够获得几千无论在空气中和溶液的图像，使该样本被接近生理条件下维持，而无需任何染色和固定的亲cedures。这里，对从鼠脑和它们对云母基板沉积多核糖的准确纯化的详细协议进行说明。该协议允许在空气和液体多核糖成像AFM和它们重建为三维物体。为了补充低温电子显微镜（冷冻电镜），该方法可以方便地用于系统的分析多核糖体和研究他们的组织。
Introduction
蛋白质的合成是在细胞1,2-最耗能的过程。因此，并不奇怪，蛋白质丰度主要控制在翻译，而不是转录水平3,4,5。多核糖体是转换的mRNA信息为功能蛋白质读数基波高分子组分。多聚核糖体是迄今公认的大分子复合物，几个平移控制收敛6-13。尽管数百对核糖体结构14-16研究，详细的分子见解翻译的动态和多聚核糖体的拓扑结构遇到了兴趣有限。因此，原生多聚核糖核蛋白复杂的组织结构及其在翻译的潜在影响仍然是相当模糊的问题。多聚核糖体可能隐藏还是未知数有序和职能部门，潜在的镜像什么核小体和后生CONTROLS已经代表了转录领域。事实上，这样一个有趣的假设的调查需要更多的研究和新的技术途径。在此行中，结构的技术和原子力显微镜可以卓有成效协作解开的新机制，用于控制基因表达的，类似于什么是为核小体17,18来实现的。
由于核糖体14-16的发现，它的结构已被广泛表征在原核生物19,20，酵母21以及最近在人22，在蛋白质合成的基础上提供的机制的分子描述。多核糖体进行初始确认在内质网的膜，从而形成典型的2D几何机构14。正如前面提到的，多聚核糖体装配一直没有固定利率，作为核糖体结构的对象。在过去，多聚核糖体已基本上由TR研究ansmission EM为基础的技术。只是在最近，冷冻电镜技术，使纯化多聚核糖体的三维重建体外翻译系统23-26，人类细胞裂解液27或28的细胞。这些技术提供了有关在多核糖体23，26-28和在小麦胚芽多核糖体24相邻的核糖体的接触表面进行初步分子描述的核糖体的核糖体组织更精致的信息。因此，冷冻电镜断层扫描允许与分子细节核糖体的核糖体相互作用的公开内容，但它是由大量的后处理的负担，并重建分析需要大量的计算资源进行数据处理。此外，为了得到核糖体的核糖体相互作用的分子细节，核糖体的高度解决地图是必需的，并且这种参考核糖体仅可用于几种。重要的是，冷冻电镜断层扫描是无法检测无RNA。 Theref矿石，需要新的技术来全面了解机器翻译的组织。
旁冷冻电镜，原子力显微镜也已在低等真核生物29-33和人类27用作用于多核糖体的直接成像的有用工具。相比EM，AFM不需要样品固定或标签。此外，测量结果可以在接近生理条件，并具有明确确定两个核糖体和裸RNA链27上的唯一的可能性来进行。能够相对快速地执行单个多核糖体的成像，在纳米分辨率的小的后处理工作获得成千上万的图像相比，广泛和重后处理和重建分析通过冷冻电镜显微术所需的。因此，AFM数据处理和分析，也不需要昂贵的工作站和高计算能力。因此，该技术收集多核糖体的形状信息，形态特征（如高度，长度和宽度），核糖体密度的无RNA的存在和每多核糖体27的核糖体比冷冻电镜更高的吞吐量的数量。在这样的方式，AFM代表了一个强大的和互补的方式来EM技术来描绘核糖27。
在这里，我们提出了一个完整的管道从净化到AFM应用到图像和分析小鼠大脑多聚核糖体的数据分析。该协议的重点是净化问题上用于原子力显微镜成像云母基材多聚核糖体的精确沉积。除了 可与通过AFM社区使用的通用软件容易地进行常规颗粒分析，一个ImageJ的34插件 ，称为RiboPick，提出用于计数每多核糖体27，35的核糖体的数目。
Protocol
Representative Results
Discussion
由于DNA的结构被证明是至关重要来形容转录和染色质组织提高了我们的基因表达的转录调控认识的过程，它分析多聚核糖体的组织和结构，提高真实的理解是必不可少的翻译及其调控。
所描述的协议中，轻轻地固定在一个平面上的多核糖体的最佳密度，适于原子力显微镜成像被获得。使用具有方便制备的样品这些条件下，每小时近50多核糖体可以被收购，准备作进一步的分析…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This research was supported by the AXonomIX research project financed by the Provincia Autonoma di Trento, Italy.
Materials
| Cycloheximide | Sigma | 01810 | Prepararation of lysate |
| DNAseI | Thermo Scientific | 89836 | Prepararation of lysate |
| RiboLock RNAse Inhibitor | Life technologies | EO-0381 | Prepararation of lysate |
| DEPC | Sigma | 40718 | Prepararation of lysate |
| Triton X100 | Sigma | T8532 | Prepararation of lysate |
| DTT | Sigma | 43815 | Prepararation of lysate |
| Sodium Deoxycholate | Sigma | D6750 | Prepararation of lysate |
| Microcentrifuge | Eppendorf | 5417R | Prepararation of lysate |
| Sucrose | Sigma | S5016 | Sucrose gradient preparation |
| Ultracentrifuge | Beckman Coulter | Optima LE-80K | Sucrose gradient centrifugation |
| Ultracentrifuge Rotor | Beckman Coulter | SW 41 Ti | Sucrose gradient centrifugation |
| Polyallomer tube | Beckman Coulter | 331372 | Sucrose gradient centrifugation |
| Density Gradient Fractionation System | Teledyne Isco | 67-9000-176 | Sucrose gradient fractionation |
| AFM | Asylum Research | Cypher | Polysome visualization |
Referenzen
- Buttgereit, F., Brand, M. D. A hierarchy of ATP-consuming processes in mammalian cells. Biochem J. 312, 163-167 (1995).
- Russell, J. B., Cook, G. M. Energetics of bacterial growth: balance of anabolic and catabolic reactions. Microbiol Rev. 59 (1), 48-62 (1995).
- Vogel, C., et al. Sequence signatures and mRNA concentration can explain two-thirds of protein abundance variation in a human cell line. Mol Syst Biol. 6, 400 (2010).
- Schwanhäusser, B., et al. Global quantification of mammalian gene expression control. Nature. 473, 337-342 (2011).
- Tebaldi, T., et al. Widespread uncoupling between transcriptome and translatome variations after a stimulus in mammalian cells. BMC Genomics. 13, 220 (2012).
- Kondrashov, N., et al. Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell. 145 (3), 383-397 (2011).
- Topisirovic, I., Svitkin, Y. V., Sonenberg, N., Shatkin, A. J. Cap and cap-binding proteins in the control of gene expression. Wiley Interdiscip Rev RNA. 2, 277-298 (2011).
- Darnell, J. C., et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell. 146 (2), 247-261 (2011).
- Pircher, A., et al. An mRNA-derived noncoding RNA targets and regulates the ribosome. Mol Cell. 54 (1), 147-155 (2014).
- Simms, C. L., et al. An active role for the ribosome in determining the fate of oxidized mRNA. Cell Rep. 9 (4), 1256-1264 (2014).
- Kraushar, M. L., et al. Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R. Proc Natl Acad Sci U S A. 111 (36), E3815-E3824 (2014).
- van Heesch, S., et al. Extensive localization of long noncoding RNAs to the cytosol and mono- and polyribosomal complexes. Genome Biol. 15 (1), R6 (2014).
- Preissler, S., et al. Not4-dependent translational repression is important for cellular protein homeostasis in yeast. EMBO J. 34 (14), 1905-1924 (2015).
- Palade, G. E. A small particulate component of the cytoplasm. J Biophys Biochem Cytol. 1 (1), 59-68 (1955).
- McQuillen, K., Roberts, R. B., Britten, R. J. Synthesis of nascent protein by ribosomes in Escherichia coli. Proc Natl Acad Sci U S A. 45 (9), 1437-1447 (1959).
- Wettstein, F. O., Staehelin, T., Noll, H. Ribosomal aggregate engaged in protein synthesis: characterization of the ergosome. Nature. 2 (197), 430-435 (1963).
- Dimitriadis, E. K., et al. Tetrameric organization of vertebrate centromeric nucleosomes. Proc Natl Acad Sci U S A. 107 (47), 20317-20322 (2010).
- Allen, M. J., et al. Atomic force microscope measurements of nucleosome cores assembled along defined DNA sequences. Biochemie. 32 (33), 8390-8396 (1993).
- Ban, N., Nissen, P., Hansen, J., Moore, P., Steitz, T. The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science. 289 (5481), 905-920 (2000).
- Schluenzen, F., et al. Structure of functionally activated small ribosomal subunit at 3.3 Å resolution. Cell. 102 (5), 615-623 (2000).
- Ben-Shem, A., et al. The structure of the eukaryotic ribosome at 3.0 Å resolution. Science. 334 (6062), 1524-1529 (2011).
- Anger, A. M., et al. Structures of the human and Drosophila 80S ribosome. Nature. 497 (7447), 80-85 (2013).
- Brandt, F., et al. The native 3D organization of bacterial polysomes. Cell. 136, 261-271 (2009).
- Myasnikov, A. G., et al. The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes. Nat. Commun. 5, 5294 (2014).
- Afonina, Z. A., Myasnikov, A. G., Shirokov, V. A., Klaholz, B. P., Spirin, A. S. Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study. Nucleic Acids Res. 42 (14), 9461-9469 (2014).
- Afonina, Z. A., Myasnikov, A. G., Shirokov, V. A., Klaholz, B. P., Spirin, A. S. Conformation transitions of eukaryotic polyribosomes during multi-round translation. Nucleic Acids Res. 43 (1), 618-628 (2015).
- Viero, G., et al. Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes. J Cell Biol. 208 (5), 581-596 (2015).
- Brandt, F., Carlson, L. -. A., Hartl, F. U., Baumeister, W., Grünewald, K. The three-dimensional organization of polyribosomes in intact human cells. Mol. Cell. 39, 560-569 (2010).
- Wu, X., Liu, W. Y., Xu, L., Li, M. Topography of ribosomes and initiation complexes from rat liver as revealed by atomic force microscopy. Biol Chem. 378 (5), 363-372 (1997).
- Yoshida, T., Wakiyama, M., Yazaki, K., Miura, K. Transmission electron and atomic force microscopic observation of polysomes on carbon-coated grids prepared by surface spreading. J. Electron Microsc (Tokyo). 46 (6), 503-506 (1997).
- Mikamo, E. C., et al. Native polysomes of Saccharomyces cerevisiae in liquid solution observed by atomic force microscopy. J. Struct. Biol. 151, 106-110 (2005).
- Mikamo-Satoh, E. A., et al. Profiling of gene-dependent translational progress in cellfree protein synthesis by real-space imaging. Anal. Biochem. 394, 275-280 (2009).
- Bernabò, P., et al. Studying translational control in non-model stressed organisms by polysomal profiling. J. Insect Physiol. 76, 30-35 (2015).
- Schneider, C. A., Rasband, W. S., Eliceiri, K. W. IH Image to ImageJ: 25 years of image analysis. Nat. Methods. 9, 671-675 (2012).
- Lauria, F., et al. RiboAbacus: a model trained on polyribosome images predicts ribosome density and translational efficiency from mammalian transcriptomes. Nucleic Acids Res. , (2015).
- Al Omran, A. J., et al. Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain. J Vis Exp. (100), e52853 (2015).
- Warner, J. R., Rich, A., Hall, C. E. Electron microscope studies of ribosomal clusters synthesizing hemoglobin. Science. 138, 1399-1403 (1962).
- Yazaki, K., Yoshida, T., Wakiyama, M., Miura, K. Polysomes of eukaryotic cells observed by electron microscopy. J. Electron Microsc (Tokyo). 49, 663-668 (2000).
