A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy
A new approach for the direct visualization of the membrane cytoskeleton in cryo-electron microscopy: a comparative study with freeze-etching electron microscopy
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DOI：
10.1093/jmicro/dfw037
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发表时间：
2016-12-01
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：
Makihara, Masaki;Watanabe, Takashi;Usukura, Jiro
一种改进的去屋顶方法，包括撕下细胞膜，使用粘合剂电子显微镜（EM）网格，而不是玻璃冰切片（冷冻切片），使我们能够在其天然状态下，以极高的对比度，解剖观察膜细胞骨架。将用阿尔新蓝预处理的网格置于细胞上，并将背质膜的一部分转移到网格上，然后将其漂浮在缓冲溶液中。这些膜碎片含有足够的细胞骨架，并具有适合于冷冻电镜观察的厚度。由于细胞质中的可溶性成分流出并脱离细胞，因此在质膜的细胞质表面清楚地观察到许多肌动蛋白丝和微管，具有极高的对比度。肌动蛋白丝以光滑的轮廓向各个方向延伸，几乎没有分支。微管伸展到3 μ m或更远，同时在其天然状态下轻轻缠绕。然而，用1%戊二醛固定后，微管变得直而破碎。Cryo-EM首次揭示了天然细胞中细胞膜下的光滑内质网网络。在质膜的细胞质表面上也观察到网格蛋白涂层和小窝，类似于使用冷冻蚀刻复制EM（冷冻蚀刻EM）观察到的那些。去屋顶也是有用的免疫标记冷冻EM。抗体标记的IQGAP 1，促进肌动蛋白丝网络形成的效应蛋白之一，被定位在肌动蛋白丝旁边。冷冻蚀刻电镜证实了冷冻电镜的形态学发现。
An improved unroofing method consisting of tearing off the cell membrane using an adhesive electron microscopy (EM) grid instead of vitreous ice sectioning (cryo-sectioning) has enabled us to panoramically view the membrane cytoskeleton in its native state with extremely high contrast. Grids pre-treated with Alcian blue were placed on cells, and a portion of the dorsal plasma membrane was transferred onto the grid, which was then floated in buffer solution. These membrane fragments contained sufficient cytoskeleton and were of suitable thickness for observation by cryo-EM. Many actin filaments and microtubules were clearly observed on the cytoplasmic surface of the plasma membrane with extremely high contrast because the soluble components of the cytoplasm flowed out and broke away from the cells. Actin filaments extended in all directions in a smooth contour with little branching. Microtubules spread out as far as 3 mu m or more while winding gently in their native state. Upon fixation with 1% glutaraldehyde, however, the microtubules became straight and fragmented. Cryo-EM revealed for the first time a smooth endoplasmic reticulum network beneath the cell membrane in native cells. Clathrin coats and caveolae were also observed on the cytoplasmic surface of the plasma membrane, similar to those seen using freeze-etching replica EM (freeze-etching EM). Unroofing was also useful for immuno-labelling in cryo-EM. Antibody-labelled IQGAP1, one of the effector proteins facilitating the formation of actin filament networks, was localized alongside actin filaments. Freeze-etching EM confirmed the morphological findings of cryo-EM.