Imaging Cytoskeleton Components by Electron Microscopy.
Imaging Cytoskeleton Components by Electron Microscopy.
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DOI：
10.1007/978-1-0716-1661-1_2
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发表时间：
2022
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细胞骨架是细胞质中不溶于洗涤剂的组分的复合物，在细胞运动、形状生成和细胞的机械性质中起关键作用。纤维状聚合物-肌动蛋白丝、微管和中间体-是细胞骨架的主要组成部分，它们在细胞活动过程中不断改变其组织结构。肌动蛋白细胞骨架是特别多态的，因为肌动蛋白丝可以形成执行不同功能的多个高阶组件。关于细胞骨架组织的结构信息对于理解其功能和各种形式的细胞活动的机制至关重要。由于细胞骨架纤维的纳米级厚度，电子显微镜（EM）是确定细胞骨架结构的关键工具。本文介绍了应用旋转阴影（或铂副本）EM（PREM）的可视化细胞骨架。该程序适用于生长在玻璃盖玻片上的薄培养细胞，包括细胞的去污剂提取（或机械“去顶”）以暴露其细胞骨架，化学固定以提供稳定性，乙醇脱水和临界点干燥以保持三维性，用铂旋转阴影以产生对比度，以及碳涂层以稳定复制品。该技术提供了易于解释的三维图像，其中单个细胞骨架纤维被清楚地分辨，并且可以通过免疫金标记来识别单个蛋白质。更重要的是，PREM很容易与活细胞成像兼容，因此人们可以将细胞或其组分的动力学相关联，例如，表达荧光蛋白，在同一细胞中具有高分辨率的细胞骨架结构组织。
The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers—actin filaments, microtubules, and intermediate filaments—are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher-order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or platinum replica) EM (PREM) for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction (or mechanical “unroofing”) of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved and individual proteins can be identified by immunogold labeling. More importantly, PREM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high-resolution structural organization of the cytoskeleton in the same cell.