Electron Tomography Of Cellular Structures

细胞结构的电子断层扫描

• 批准号: 7734363
• 负责人: Richard Leapman
• 金额: $ 3.7万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734363
• 关键词: 3-Dimensional; ATP phosphohydrolase; Actins; Bacteria; Biological; Brain; Cell Wall; Cells; Cellular Structures; Colorado; Complex; Cytoskeleton; DNA; Data; Electron Microscope; Electrons; Filament; Freeze Substitution; Freezing; Glutamate Receptor; Life; Link; Membrane; Morphologic artifacts; N-Methylaspartate; Neurons; Numbers; Plastics; Positioning Attribute; Postsynaptic Membrane; Preparation; Proteins; Range; Rattus; Ribosomes; Scaffolding Protein; Series; Site; Solvents; Specimen; Spiroplasma; Staining method; Stains; Structure; Subcellular structure; Synapses; Synaptic Transmission; System; Techniques; Temperature; Tomogram; Universities; Water; analog; base; cell motility; cell type; data acquisition; density; electron tomography; insight; interest; postsynaptic; programs; reconstruction; scaffold; size; tool; transmission process; voltage

Electron tomography (ET) is an important tool for determining three-dimensional subcellular structures. We have implemented ET in a 300 kV transmission electron microscope to determine the three-dimensional organization of supramolecular assemblies in a variety of biological systems ranging from simple cytoskeletons in bacteria to large protein complexes in neurons. It is not always feasible to obtain cryo-electron tomographic data from specimens maintained in their frozen hydrated state. In those cases, useful results can often be obtained by rapidly freezing the cells, freeze-substituting the water for solvent, embedding in plastic and sectioning at room temperature. We have collected dual axis tilt series from such freeze-substituted specimens and performed three-dimensional reconstructions using the IMOD program (University of Colorado). An advantage of this approach is that dual-axis tilt series can be acquired more easily, which reduces artifacts due to the missing wedge in the reconstruction. To gain insight into the structural basis of motility in the simplest free-living cell, the wall-less bacterium Spiroplasma, we have obtained tomographic reconstructions of Spiroplasma's cytoskeletal ribbon. We find that the cytoskeleton is composed of a major protein Fib anchored to the underlying membrane by another protein that we identify as the ATPase MreB, a bacterial analog of actin. We have also determined the number of ribosomes per unit volume of Spiroplasma and have identified a network of filaments as the cell's DNA. Electron tomography has also been applied to elucidate the structure of a highly complex supramolecular assembly, the post-synaptic density (PSD), which is the primary site for synaptic transmission. The PSD is known to contain hundreds of different proteins and has been extremely difficult to study by conventional structural techniques. Our electron tomograms recorded from suitably stained freeze-substituted neurons of cultured rat brain showed that PSDs contain vertically oriented filaments identified as the scaffolding protein PSD-95. These vertical filaments are found to intertwine with horizontally oriented filaments lying close to the postsynaptic membrane, and define an orthogonal interlinked scaffold at the core of the PSD. Vertical filaments contact two types of transmembrane structures whose sizes and positions match those of glutamate receptors and intermesh with two types of horizontally oriented filaments lying 1020 nm from the postsynaptic membrane. The longer horizontal filaments link adjacent NMDAR-type structures, whereas the smaller filaments link both NMDA- and AMPAR-type structures. The orthogonal, interlinked scaffold of filaments at the core of the PSD provides a structural basis for understanding dynamic aspects of postsynaptic function. Our results have demonstrated that ET combined with automated data acquisition in a 300 kV TEM provides useful 3-D structural information about the organization of large protein assemblies in a wide variety of cell types that are prepared by rapid freezing and freeze-substitution.

电子断层扫描（ET）是确定三维亚细胞结构的重要工具。 我们在 300 kV 透射电子显微镜中实施了 ET，以确定各种生物系统中超分子组装体的三维组织，从细菌中的简单细胞骨架到神经元中的大型蛋白质复合物。 从保持冷冻水合状态的样本中获取冷冻电子断层扫描数据并不总是可行的。 在这些情况下，通常可以通过快速冷冻细胞、冷冻替代水作为溶剂、包埋在塑料中并在室温下切片来获得有用的结果。 我们从此类冷冻替代样本中收集了双轴倾斜系列，并使用 IMOD 程序（科罗拉多大学）进行了三维重建。 这种方法的优点是可以更容易地获取双轴倾斜序列，从而减少由于重建中缺少楔块而产生的伪影。 为了深入了解最简单的自由生活细胞（无壁细菌螺原体）运动的结构基础，我们获得了螺原体细胞骨架带的断层扫描重建。我们发现细胞骨架由主要蛋白质 Fib 组成，该蛋白质通过另一种蛋白质锚定在底层膜上，我们将其称为 ATPase MreB（一种肌动蛋白的细菌类似物）。 我们还确定了螺原体每单位体积的核糖体数量，并确定了作为细胞 DNA 的细丝网络。 电子断层扫描也被用来阐明高度复杂的超分子组装体的结构，即突触后密度（PSD），它是突触传递的主要部位。 已知 PSD 含有数百种不同的蛋白质，并且很难通过传统的结构技术进行研究。 我们从培养的大鼠大脑中经过适当染色的冷冻替代神经元记录的电子断层图显示，PSD 包含垂直定向的细丝，被识别为支架蛋白 PSD-95。 这些垂直丝与靠近突触后膜的水平丝缠绕在一起，并在 PSD 的核心定义了一个正交的互连支架。 垂直丝接触两种类型的跨膜结构，其尺寸和位置与谷氨酸受体相匹配，并与距离突触后膜 1020 nm 的两种水平定向丝相互啮合。较长的水平丝连接相邻的 NMDAR 型结构，而较小的丝连接 NMDA 和 AMPAR 型结构。 PSD 核心的正交、互连的细丝支架为理解突触后功能的动态方面提供了结构基础。 我们的结果表明，ET 与 300 kV TEM 中的自动数据采集相结合，提供了有关通过快速冷冻和冷冻替代制备的各种细胞类型中大型蛋白质组装体的组织的有用 3-D 结构信息。