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

近年来，人们对生物电子显微镜（EM）的兴趣再次高涨， 冷冻电子断层扫描EM分析的局限性，特别是在细胞样品中，是确定 目标蛋白质的位置。我们的最终目标是开发出将可靠的联合收割机 基于快速冷冻和玻璃化技术的细胞结构保存， 足够的信噪比，使得这些标记容易通过EM可见，特别是在3D电子断层摄影中。 我们建议开发一种金属硫蛋白基因作为一种“可克隆标签”，它将结合金以提高其在细胞中的密度。 EM的各种样品。金属硫蛋白是小蛋白质（~ 6.5kD），其是贪婪的金属结合剂， 已经显示在体外形成金簇（Mercogliano & DeRosier 2006. 355：211-23）。 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3D 蛋白质在复杂细胞结构中的位置可以通过纳米级的层析成像来确定 分辨率 金属硫蛋白作为可克隆标签的效用将在两个目标中探索。 金属硫蛋白作为冷冻电子显微镜和冷冻电子断层扫描的可克隆标记， 分离的或体外重构的大分子组装体。特别地，微管-Eg 5马达 复合物将用于金属硫蛋白标记性质的定性和定量评估。 金属硫蛋白作为一种直接可见的密度标记物在可平均和不可平均的 将对结构进行分析。完整细胞玻璃化切片中金属硫蛋白标记的细胞成分， 也将测试可能使用银增强的情况。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，用于蛋白质在快速冷冻和冷冻替代材料中的定位， 塑料的。金属硫蛋白标记的Eg 5驱动蛋白将在体外组装成局部纺锤体， 非洲爪蟾卵提取物，以及脊椎动物组织培养细胞，适用于断层扫描。 最后，金属硫蛋白标签将用于芽殖酵母上的各种蛋白质，包括α-微管蛋白 在微管中，Cin 8驱动蛋白样马达蛋白和Spc 42，一种非常丰富的纺锤体极组分。它 预期金属硫蛋白可克隆密度标签将可用于多种EM技术。

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 determing 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, liklely 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.