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

描述（由申请人提供）：近年来，人们对包括冷冻电子断层扫描在内的生物电子显微镜（EM）的兴趣重新强烈复苏。电磁分析（尤其是细胞样品）的局限性在于确定目标蛋白质的位置。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提供足够信噪比的标记技术相结合，以便这些标记可以通过 EM（尤其是在 3D 电子断层扫描中）轻松可见。我们建议开发一种金属硫蛋白基因作为“可克隆标签”，它将结合金以提高其在各种 EM 样品中的密度。金属硫蛋白是小蛋白质（~6.5 kD），是活跃的金属结合剂，并且已被证明可以在体外形成金簇（Mercogliano & DeRosier 2006. J Mol Biol. 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用途，因为复杂细胞结构中蛋白质的 3D 位置可以通过纳米分辨率的断层扫描来确定。金属硫蛋白作为可克隆标签的效用将在两个目标上进行探索。第一个目标是开发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，应用于分离或体外重构的大分子组装体。特别是，微管-Eg5运动复合物将用于金属硫蛋白标记特性的定性和定量评估。将测定金属硫蛋白作为可平均和不可平均结构中直接可见的密度标记的有用性。完整细胞玻璃化切片中金属硫蛋白标记的细胞成分（可能使用银增强）也将得到测试。第二个目标是开发金属硫蛋白作为可克隆的密度标签，用于嵌入塑料中的快速冷冻和冷冻替代材料中的蛋白质定位。金属硫蛋白标记的 Eg5 驱动蛋白将是使用非洲爪蟾卵提取物在体外组装的局部纺锤体，以及在适合断层扫描的脊椎动物组织培养细胞中组装的局部纺锤体。最后，金属硫蛋白标签将用于出芽酵母中的多种蛋白质，包括微管中的 α-微管蛋白、Cin8 驱动蛋白样运动蛋白和 Spc42（一种非常丰富的纺锤体极成分）。预计可克隆密度标签中的金属硫蛋白将可用于多种 EM 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.