Electron Tomography Of Cellular Structures

电子断层扫描（ET）是确定三维亚细胞结构的重要工具。 我们已经实现了ET在300千伏的透射电子显微镜，以确定在各种生物系统中的超分子组装的三维组织，从简单的细胞骨架在细菌中的神经元中的大蛋白质复合物。它并不总是可行的，以获得冷冻电子断层扫描数据保持在冷冻水合状态的标本。 在这些情况下，通常可以通过快速冷冻细胞，冷冻-用水代替溶剂，包埋在塑料中并在室温下切片来获得有用的结果。我们已经收集了双轴倾斜系列从这样的冷冻替代标本，并进行三维重建使用IMOD程序（科罗拉多大学）。 这种方法的优点是可以更容易地采集双轴倾斜系列，这减少了由于重建中缺少楔形而导致的伪影。 为了深入了解最简单的自由生活的细胞，无壁细菌螺原体的运动的结构基础，我们已经获得了螺旋体的细胞骨架带的断层重建。我们发现，细胞骨架是由一个主要的蛋白质纤维蛋白锚定到下面的膜由另一种蛋白质，我们确定为ATP酶MreB，肌动蛋白的细菌类似物。我们还确定了每单位体积螺原体的核糖体数量，并确定了作为细胞DNA的细丝网络。 电子断层扫描也被应用于阐明一个高度复杂的超分子组装体的结构，突触后密度（PSD），这可能最终导致更好地了解神经系统疾病。 PSD包埋在突触后膜中，包含受体、支架分子和细胞骨架元件，并且是用于信号转导和信号处理的主要突触后位点。 兴奋性突触的PSD含有NMDA和AMPA型谷氨酸受体。 AMPA受体在PSD的再循环解释了突触传递的动态变化。 已知PSD含有数百种不同的蛋白质，并且通过常规结构技术研究非常困难。我们的电子断层扫描记录从适当染色的冷冻替代神经元培养的大鼠大脑显示，PSD包含垂直取向的丝，interanimate与水平取向的丝靠近突触后膜，并定义一个正交互连支架的PSD的核心。垂直细丝的丛状物产生了典型的致密外观，这是PSD在标准EM横截面视图中的特征。垂直细丝在PSD的重建中普遍存在，即使在其他结构元素缺失的地方也是如此。根据单位面积上观察到的数目，估计在直径为400 nm的PSD中有大约400个垂直细丝。 垂直丝的尺寸，以及它们与突触后膜的关联，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，其大小和位置与谷氨酸受体相匹配，并与距离突触后膜10至20 nm的两种类型的水平取向的丝相互啮合。较长的水平纤维连接相邻的NMDAR型结构，而较小的纤维连接NMDA和AMPAR型结构。在PSD的核心的正交的，互连的支架丝提供了一个结构基础，了解动态方面的突触后功能。 免疫电子显微镜有助于识别突触后密度复合物中的特定蛋白质，如PSD-95。 在一系列的虚拟部分分割的细丝进行了分类的基础上，他们的位置，形状和尺寸。一大类膜相关的丝在PSD是近直和垂直取向的突触后膜。我们把这种类型的细丝称为垂直细丝。垂直细丝的直径通常为5 nm，长度为20 nm。 PSD内的垂直细丝均匀间隔，最近邻距离为13 nm。 我们的研究结果表明，ET结合自动数据采集在300 kV TEM提供了有用的3-D结构信息的组织中的大蛋白组装体在各种各样的细胞类型，通过快速冷冻和冷冻置换。

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.