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

用于细胞结构 3D 电子显微镜的可克隆高密度

• 批准号: 7282768
• 负责人: ANDREAS HOENGER
• 金额: $ 24.91万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282768
• 关键词: 3-Dimensional; Binding; Biological; Biological Assay; Biological Preservation; Cells; Cellular Structures; Complex; Cryoelectron Microscopy; Detection; Electron Microscopy; Electrons; Freezing; Genes; Goals; Gold; In Vitro; Kinesin; Label; Localized; Location; Metallothionein; Metals; Methods; Microtubules; Motor; Noise; Numbers; Plastics; Positioning Attribute; Property; Proteins; Resolution; Saccharomycetales; Sampling; Signal Transduction; Silver; Structure; System; Techniques; Technology; Testing; Thick; Tomogram; Tubulin; Xenopus; alpha Tubulin; base; density; egg; electron tomography; interest; intracellular protein transport; macromolecular assembly; nanometer; nanoscale; protein localization location; reconstitution; tau Proteins; tissue/cell culture; tomography; usability; 3-Dimensional; Binding; Biological; Biological Assay; Biological Preservation; Cells; Cellular Structures; Complex; Cryoelectron Microscopy; Detection; Electron Microscopy; Electrons; Freezing; Genes; Goals; Gold; In Vitro; Kinesin; Label; Localized; Location; Metallothionein; Metals; Methods; Microtubules; Motor; Noise; Numbers; Plastics; Positioning Attribute; Property; Proteins; Resolution; Saccharomycetales; Sampling; Signal Transduction; Silver; Structure; System; Techniques; Technology; Testing; Thick; Tomogram; Tubulin; Xenopus; alpha Tubulin; base; density; egg; electron tomography; interest; intracellular protein transport; macromolecular assembly; nanometer; nanoscale; protein localization location; reconstitution; tau Proteins; tissue/cell culture; tomography; usability

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.

描述（由申请人提供）：近年来，人们对生物电子显微镜（EM）（包括冷冻电子断层扫描）的兴趣浓厚。 EM分析的局限性，特别是在细胞样品中，是确定感兴趣蛋白质的位置。我们的最终目标是开发将基于快速冷冻和玻璃化的细胞结构保存的方法与标记技术相结合的方法，这些技术可提供足够的信号到信号，以便EM可以很容易地看到这些标签，尤其是在3D电子Tomogram中。我们建议将金属硫蛋白基因作为“可插座标签”开发，该基因将结合金以增强其在各种EM样品中的密度。金属霉素是小蛋白（〜6.5 kD），是狂热的金属粘合剂，已显示出在体外形成金簇（Mercogliano＆Derosier2006。JMolBiol。355：211-23）。这样的可克隆的高密度标签将彻底改变细胞断层扫描的效用，因为可以通过纳米分辨率下的断层扫描来确定蛋白质在复杂的细胞结构中的3D位置。金属硫蛋白作为可克隆标签的实用性将在两个目标中探索。第一个目的是开发金属硫蛋白作为用于隔离或体外重构的大分子分子组件的冷冻电子显微镜和冷冻电子断层扫描的可包隆标签。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。将分析金属硫硫代蛋白作为直接可见密度标记物在可见且不可验证的结构中的有用性。金属硫硫蛋白标记的细胞成分在完整细胞的玻璃化切片中，也可能会在使用银增强的情况下进行测试。第二个目的是开发金属硫蛋白作为可克隆的密度标签，用于蛋白质在快速冷冻和冻结塑料中的冻结和冻结的材料中定位。金属蛋白标记的EG5驱动蛋白将在体外使用异爪蟾鸡蛋提取物以及适合于断层扫描的脊椎动物组织培养细胞组装而成。最后，金属硫蛋白标签将用于各种蛋白质的芽酵母，包括rnicroutubules中的α-微管蛋白，CIN8驱动蛋白样运动蛋白和SPC42，SPC42是非常丰富的纺锤形杆分量。预计金属硫蛋白可克隆的密度标签将对多种EM技术有用。