Electron Tomography Of Cellular Structures

电子层析成像(ET)是确定三维亚细胞结构的重要工具。我们在300千伏的透射电子显微镜上实现了ET，以确定从细菌的简单细胞骨架到神经元的大型蛋白质复合体等各种生物系统中超分子组件的三维组织。从保存在冰冻水合状态的样品中获取低温电子断层扫描数据并不总是可行的。在这些情况下，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，通常可以获得有用的结果。我们从这样的冷冻替代标本中收集了双轴倾斜序列，并使用IMOD程序(科罗拉多大学)进行了三维重建。这种方法的一个优点是可以更容易地获得双轴倾斜序列，这减少了重建中由于丢失楔形而造成的伪影。 为了深入了解最简单的自由生活细胞--无壁细菌螺旋体--运动的结构基础，我们对螺旋体的细胞骨架带进行了断层重建。我们发现细胞骨架由一种主要的蛋白质Fib组成，另一种蛋白质被另一种我们确定为ATPase MreB的蛋白质锚定在下层膜上，这是一种细菌中的肌动蛋白类似物。我们还测定了单位体积螺旋体的核糖体数量，并确定了一个细丝网络就是细胞的DNA。 电子断层扫描也被应用于阐明高度复杂的超分子组装的结构，突触后密度(PSD)，这最终可能导致更好地理解神经疾病。PSD嵌入突触后膜，含有受体、支架分子和细胞骨架元素，是突触后信号转导和信号处理的主要部位。兴奋性突触的PSD含有NMDA和AMPA类型的谷氨酸受体。PSD处AMPA受体的循环可以解释突触传递的动态变化。 众所周知，PSD含有数百种不同的蛋白质，用传统的结构技术研究非常困难。我们从培养的大鼠脑内适当染色的冷冻替代神经元上记录的电子断层图像显示，PSD含有垂直取向的细丝，这些细丝与靠近突触后膜的水平取向的细丝交织在一起，并在PSD的核心形成了一个正交的相互连接的支架。在标准的EM横断面图中，垂直细丝的丛林形成了典型的密集外观，这是PSD的特征。垂直细丝普遍存在于PSD的重建中，即使在没有其他结构元素的地方也是如此。根据单位面积上观察到的数量，估计在400 nm直径的PSD中大约有400个垂直细丝。垂直细丝的尺寸，以及它们与突触后膜的关系，表明它们属于PSD-95家族的Maguk蛋白。垂直纤维接触两种大小和位置与谷氨酸受体相匹配的跨膜结构，并与位于突触后膜10~20 nm处的两种水平方向的纤维交织在一起。较长的水平细丝连接相邻的NMDAR型结构，而较小的水平细丝连接NMDAR型和AMPAR型结构。位于PSD核心的相互关联的正交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密度复合体中的特定蛋白质，如PSD-95。 根据细丝的位置、形状和尺寸对分割成一系列虚拟部分的细丝进行分类。PSD处的一大类膜相关细丝相对于突触后膜几乎是直的和垂直的。我们把这种类型的长丝称为垂直丝。垂直丝的直径通常为5 nm，长度为20 nm。PSD内的垂直丝均匀分布，最近邻距离为13 nm。 我们的结果表明，在300千伏的透射电子显微镜中，ET与自动数据采集相结合，提供了关于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组织的有用的三维结构信息。

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.