Electron Tomography Of Cellular Structures

细胞结构的电子断层扫描

• 批准号: 7967880
• 负责人: Richard Leapman
• 金额: $ 10.18万
• 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/7967880
• 关键词: 3-Dimensional; AMPA Receptors; ATP phosphohydrolase; Accounting; Actins; Appearance; Area; Bacteria; Be++ element; Beryllium; Brain; Caliber; Cell Wall; Cells; Cellular Structures; Characteristics; Colorado; Complex; Cytoskeleton; DNA; Data; Dimensions; Electron Microscope; Electrons; Elements; Excitatory Synapse; Family; Filament; Freeze Substitution; Freezing; Glutamate Receptor; Glutamates; Hippocampus (Brain); Immunoelectron Microscopy; Lead; Life; Link; Location; Membrane; Molecular; Morphologic artifacts; N-Methylaspartate; Neurons; Plastics; Positioning Attribute; Postsynaptic Membrane; Preparation; Proteins; Rattus; Recycling; Ribosomes; Series; Shapes; Signal Transduction; Site; Solvents; Specimen; Spiroplasma; Staining method; Stains; Structure; Subcellular structure; Synapses; Synaptic Transmission; Techniques; Temperature; Tomogram; Universities; Vertical Dimension; Water; analog; base; biological systems; cell motility; cell type; computerized data processing; data acquisition; density; electron tomography; insight; interest; nervous system disorder; postsynaptic; programs; protein complex; receptor; reconstruction; scaffold; tool; transmission process; virtual; 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 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 approximatley 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 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。 我们已经在300 kV的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件的三维组织，从细菌中的简单细胞骨架到神经元中的大蛋白质复合物。从保持冷冻水合状态的标本中获得冷冻电子层析成像数据并不总是可行的。 在这种情况下，通常可以通过快速冷冻细胞，冻结水以溶剂，嵌入塑料和室温下切片来获得有用的结果。我们已经从这种冻结取代的标本中收集了双轴倾斜系列，并使用IMOD计划（科罗拉多大学）进行了三维重建。 这种方法的一个优点是，双轴倾斜系列可以更轻松地获取，这可以减少由于重建中缺少的楔形而导致的伪像。 为了深入了解最简单的自由活动细胞中运动性的结构基础，无壁细菌螺旋体，我们获得了螺旋马细胞骨骼色带的层析成像重建。我们发现细胞骨架由另一个我们鉴定为ATPase MREB（肌动蛋白的细菌类似物）固定在基础膜上的主要蛋白质FIB组成。我们还确定了每单位螺旋体的核糖体数量，并确定了细丝网络为细胞的DNA。 电子断层扫描也已应用于阐明高度复杂的超分子组件的结构，即突触后密度（PSD），这最终可能导致对神经系统疾病的更好理解。 嵌入突触后膜中的PSD包含受体，支架分子和细胞骨架元素，是信号转导和信号处理的主要突触后位点。 兴奋性突触下的PSD包含NMDA和AMPA类型的谷氨酸受体。 在PSD处的AMPA受体的回收说明了突触传播的动态变化。 已知PSD包含数百种不同的蛋白质，并且通过常规结构技术很难研究。我们的电子断层图从适当染色的大鼠脑的适当染色的冻结神经元记录下来，表明PSD含有垂直定向的细丝，这些细丝与靠近突触后膜的水平方向的细丝交织在一起，并定义了PSD核心的正交交织的cackoffold。垂直丝的灌木丛产生了标准EM横截面视图中PSD的特征的典型密集外观。垂直细丝在PSD的重建中无处不在，即使在没有其他结构元素的地方也是如此。根据观察到的每单位区域的数量，估计在400 nm直径的PSD中有大约400个垂直丝。 垂直细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。 垂直细丝接触两种类型的跨膜结构，它们的大小和位置与谷氨酸受体和植物的位置与两种类型的水平定向的细丝与从突触后膜相距10至20 nm。较长的水平丝连接相邻的NMDAR型结构，而较小的细丝连接NMDA-和AMPAR型结构。 PSD核心的细丝的正交，相互关联的支架为理解突触后功能的动态方面提供了结构性基础。 免疫电子显微镜有助于鉴定突触后密度复合物中PSD-95（例如PSD-95）等特定蛋白质。 根据其位置，形状和尺寸对一系列虚拟部分分割的细丝进行了分类。相对于突触后膜，PSD处的大量膜相关丝几乎是笔直的，并且垂直定向。我们将这种类型的细丝称为垂直细丝。垂直细丝通常直径为5 nm，长20 nm。 PSD内的垂直细丝均匀间隔，最近的邻居距离为13 nm。 我们的结果表明，ET与300 kV TEM中的自动数据获取相结合，提供了有用的3D结构信息，以通过快速冷冻和冷冻固定来制备的各种细胞类型中大型蛋白质组件的组织。