A Clonable High-Density for 3-D Electron Microscopy of Cellular Structures

描述（由申请人提供）：近年来，人们对包括低温电子断层扫描在内的生物电子显微镜（EM）有了强烈的兴趣。EM分析的局限性，特别是在细胞样品中，是确定感兴趣的蛋白质的位置。我们的最终目标是开发一种方法，将基于快速冷冻和玻璃化的细胞结构的可靠保存与标签技术相结合，这些技术可以提供足够的信噪比，以便这些标签很容易被EM看到，特别是在3D电子断层扫描中。我们建议开发一种金属硫蛋白基因作为“可克隆标签”，它将结合金以增强其在各种EM样品中的密度。金属硫蛋白是一种小蛋白质（~6.5 kD），是强烈的金属结合物，并且已被证明在体外形成金簇（Mercogliano & DeRosier 2006）。中华生物医学杂志，355(5):391 - 391。这种可克隆的高密度标签将彻底改变细胞断层扫描的实用性，因为蛋白质在复杂细胞结构中的三维位置可以通过纳米分辨率的断层扫描确定。本文将从两个方面探讨金属硫蛋白作为克隆标记物的应用。第一个目标是开发金属硫蛋白作为低温电子显微镜和低温电子断层扫描应用于分离或体外重组大分子组装的克隆标记。特别是，微管- eg5马达复合物将用于金属硫蛋白标记特性的定性和定量评估。金属硫蛋白作为一种直接可见的密度标记在平均和非平均结构中的用途将被分析。在完整细胞的玻璃化切片中，金属硫蛋白标记的细胞成分也将被测试，可能使用银增强。第二个目标是开发金属硫蛋白作为一种可克隆的密度标签，用于快速冷冻和冷冻替代嵌入塑料的材料中的蛋白质定位。金属硫蛋白标记的Eg5激酶将在体外使用爪蟾卵提取物组装的定位纺锤体中，以及在适合断层扫描的脊椎动物组织培养细胞中组装。最后，金属硫蛋白标签将在出芽酵母的多种蛋白质上使用，包括微管中的α -微管蛋白、Cin8激酶样马达蛋白和sp42， sp42是一种非常丰富的纺锤极成分。该金属硫蛋白可克隆密度标签有望应用于多种电子显微镜技术。

DESCRIPTION (provided by applicant): Recent years have seen a strong resurgence of interest in biological electron microscopy (EM) including cryo-electron tomography. A limitation of EM analysis, in particular in cellular samples, is determining the location of a protein of interest. Our ultimate goal is to develop methods that will combine the reliable preservation of cell structure based on rapid freezing and vitrification with labeling technologies that give sufficient signal-to-noise so that these labels are readily visible by EM, particularly in 3D electron tomograms. We propose to develop a metallothionein gene as a "clonable tag" that will bind gold to enhance its density in a variety of samples for EM. Metallothioneins are small proteins (~6.5 kD) that are avid metal binders and that have been shown to form gold clusters in vitro (Mercogliano & DeRosier 2006. J Mol Biol. 355:211-23). Such a clonable high-density tag would revolutionize the utility of cellular tomography because the 3D position of proteins in complex cellular structures could be determined by tomography at nanometer resolution. The utility of metallothionein as a clonable tag will be explored in two aims. The first aim is to develop metallothionein as a clonable label for cryo-electron microscopy and cryo-electron tomography applied to isolated or in vitro reconstituted macromolecular assemblies. In particular, the microtubule-Eg5 motor complex will be used for qualitative and quantitative assessment of the metallothionein labeling properties. The usefulness of metallothionein as a directly visible density marker in averagable and non-averagable structures will be assayed. Metallothionein-tagged cellular components in vitrified sections of intact cells, likely with the use of silver-enhancement will also be tested. The second aim is to develop metallothionein as a clonable density tag for protein localization in rapidly frozen and freeze-substituted material embedded in plastic. The metallothionein-tagged Eg5 kinesin will be localized spindles assembled in vitro using Xenopus egg extracts, as well as in vertebrate tissue culture cells which are suitable for tomography. Finally, the metallothionein tag will be used in budding yeast on a variety of proteins, including alpha-tubulin in rnicrotubules, the Cin8 kinesin-like motor protein, and Spc42, a very abundant spindle pole component. It is anticipated that the metallothionein clonable density tag will be useful for a variety of EM techniques.