Electron Tomography Of Cellular Structures

细胞结构的电子断层扫描

基本信息

批准号：
8556131
负责人：
Richard Leapman
金额：
$ 10.1万
依托单位：
NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
依托单位国家：
美国
项目类别：
财政年份：
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/8556131
关键词：
3-Dimensional AMPA Receptors Accounting Appearance Area Bacteria Beryllium Brain Caliber Cells Cellular Structures Characteristics Colorado Complex Cytoskeleton Data Dimensions Electron Microscope Electrons Excitatory Synapse Family Filament Freeze Substitution Freezing Glutamate Receptor Glutamates Hippocampus (Brain)Immunoelectron Microscopy Lead Link Location Membrane Molecular Morphologic artifacts N-Methylaspartate Neurons Plastics Positioning Attribute Postsynaptic Membrane Preparation Proteins Rattus Recycling Series Shapes Signal Transduction Site Solvents Specimen Staining method Stains Structure Subcellular structure Synapses Synaptic Transmission Techniques Temperature Tomogram Universities Vertical Dimension Water base biological systems cell type computerized data processing data acquisition density electron tomography interest nervous system disorder postsynaptic programs protein complex receptor reconstruction scaffold tool transmission process virtual voltage

项目摘要

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. We have applied electron tomography to elucidate the structure of a highly complex supramolecular assembly, the post-synaptic density (PSD), which could eventually lead to a better understanding of neurological diseases. The PSD, which is embedded in the postsynaptic membrane, contains receptors, scaffold molecules, and cytoskeletal elements and is the primary postsynaptic site for signal transduction and signal processing. The PSDs at excitatory synapses contain glutamate receptors of the NMDA and AMPA type. Recycling of AMPA receptors at the PSD accounts for dynamic changes in 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, which intertwine with horizontally oriented filaments lying close to the postsynaptic membrane, and define an orthogonal interlinked scaffold at the core of the PSD. The thicket of vertical filaments gives rise to the typical dense appearance that is characteristic of PSDs in standard EM cross-sectional views. Vertical filaments are ubiquitous in reconstructions of PSDs, even in places where other structural elements are absent. Based on the observed number per unit area, it is estimated that there are approximately 400 vertical filaments in a 400-nm-diameter PSD. The dimensions of vertical filaments, and their associations with the postsynaptic membrane, suggest that they belong to the PSD-95 family of MAGUK proteins. 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 10 to 20 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. Immuno-electron microscopy helps to identify specific proteins like PSD-95 within the postsynaptic density complex. Filaments segmented in a series of virtual sections were classified on the basis of their location, shape, and dimensions. A large class of membrane-associated filaments at the PSD is nearly straight and vertically oriented with respect to the postsynaptic membrane. We refer to filaments of this type as vertical filaments. Vertical filaments are typically 5 nm in diameter and 20 nm long. Vertical filaments within the PSD are uniformly spaced, with a nearest neighbor distance of 13 nm. Our results have demonstrated that ET combined with automated data acquisition in a 300 kV TEM provides useful 3-D structuralinformation 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。我们应用电子断层扫描来阐明高度复杂的超分子组装体的结构，即突触后密度（PSD），这最终可能有助于更好地了解神经系统疾病。 PSD 嵌入突触后膜，包含受体、支架分子和细胞骨架元件，是信号转导和信号处理的主要突触后部位。兴奋性突触处的 PSD 包含 NMDA 和 AMPA 类型的谷氨酸受体。 PSD 处 AMPA 受体的循环导致了突触传递的动态变化。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，这些细丝与靠近突触后膜的水平定向的细丝交织在一起，并在 PSD 的核心定义了一个正交的互连支架。垂直细丝的丛生产生了典型的致密外观，这是标准 EM 横截面视图中 PSD 的特征。垂直丝在 PSD 的重建中无处不在，即使在缺乏其他结构元素的地方也是如此。根据观察到的每单位面积的数量，估计直径 400 nm 的 PSD 中大约有 400 条垂直细丝。垂直丝的尺寸及其与突触后膜的关联表明它们属于 MAGUK 蛋白的 PSD-95 家族。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，并与距突触后膜 10 至 20 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。免疫电子显微镜有助于识别突触后致密复合体中的 PSD-95 等特定蛋白质。分割成一系列虚拟截面的细丝根据其位置、形状和尺寸进行分类。 PSD 处的一大类与膜相关的细丝几乎是笔直的，并且相对于突触后膜垂直定向。我们将这种类型的细丝称为垂直细丝。垂直丝通常直径为 5 nm，长为 20 nm。 PSD 内的垂直细丝均匀分布，最近邻距离为 13 nm。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。