Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms
Improved unroofing protocols for cryo-electron microscopy, atomic force microscopy and freeze-etching electron microscopy and the associated mechanisms
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DOI：
10.1093/jmicro/dfaa028
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发表时间：
2020-12-01
期刊：
影响因子：
1.8
通讯作者：
Usukura, Jiro
中科院分区：
文献类型：
--
作者：
Morone, Nobuhiro;Usukura, Eiji;Usukura, Jiro
去顶，即对细胞进行机械剪切，暴露细胞膜的细胞质表面，是一种独特的制备方法，可以通过冷冻电镜、原子力显微镜、冷冻蚀刻电镜等方法观察膜细胞骨架。已知超声波和粘附可以机械地使细胞脱顶。在本研究中，使用这两种方法的去顶分别表示超声去顶和粘附去顶。我们阐明了在这些去顶过程中去除细胞膜的机制，并根据该机制为每个过程建立了有效的方案。在超声去顶中，超声处理产生的细小气泡以静电方式粘附到顶端细胞表面，然后在浮力和水流的帮助下去除顶端（背侧）细胞膜。通过这种方法可以观察到保留在网格上的腹侧细胞膜的细胞质表面。在粘附脱顶中，将涂有阿尔新蓝的带正电的网格压到细胞上，从而紧密吸附背侧细胞膜。随后，当网格被提起时，牢固粘附到网格上的一部分细胞膜从细胞上剥离并转移到网格上。因此，该方法允许背细胞膜的细胞质表面的可视化。本文详细描述了两种屋顶方法的稳健、改进的协议。此外，可能由纳米气泡引起的微屋顶（穿孔）被引入作为一种使细胞对电子束透明的新方法。
Unroofing, which is the mechanical shearing of a cell to expose the cytoplasmic surface of the cell membrane, is a unique preparation method that allows membrane cytoskeletons to be observed by cryo-electron microscopy, atomic force microscopy, freeze-etching electron microscopy and other methods. Ultrasound and adhesion have been known to mechanically unroof cells. In this study, unroofing using these two means was denoted sonication unroofing and adhesion unroofing, respectively. We clarified the mechanisms by which cell membranes are removed in these unroofing procedures and established efficient protocols for each based on the mechanisms. In sonication unroofing, fine bubbles generated by sonication adhered electrostatically to apical cell surfaces and then removed the apical (dorsal) cell membrane with the assistance of buoyancy and water flow. The cytoplasmic surface of the ventral cell membrane remaining on the grids became observable by this method. In adhesion unroofing, grids charged positively by coating with Alcian blue were pressed onto the cells, thereby tightly adsorbing the dorsal cell membrane. Subsequently, a part of the cell membrane strongly adhered to the grids was peeled from the cells and transferred onto the grids when the grids were lifted. This method thus allowed the visualization of the cytoplasmic surface of the dorsal cell membrane. This paper describes robust, improved protocols for the two unroofing methods in detail. In addition, micro-unroofing (perforation) likely due to nanobubbles is introduced as a new method to make cells transparent to electron beams.