Application of spherical substrate to observe bacterial motility machineries by Quick-Freeze-Replica Electron Microscopy
Application of spherical substrate to observe bacterial motility machineries by Quick-Freeze-Replica Electron Microscopy
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DOI：
10.1038/s41598-019-51283-w
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发表时间：
2019-10-14
影响因子：
4.6
通讯作者：
Shibata, Satoshi
中科院分区：
文献类型：
--
作者：
Katayama, Eisaku;Tahara, Yuhei O.;Shibata, Satoshi
三维结构信息对于阐明各种生物机制的分子机制是必不可少的。快速冷冻深蚀刻复制电子显微镜是一种独特的技术，可以提供具有多种细胞功能的细胞外和细胞内仪器的非常高对比度的表面轮廓。虽然这些机器的全球架构主要是为了了解它们的功能特征，但由于这些物体(例如可移动的微生物)被放置在传统的平板基板上，因此用通常的测角方法很难甚至不可能描绘出相同目标的侧面或高度倾斜的视图。我们引入了硅珠作为替代底物来解决这一关键问题。细长的黄杆菌和球状支原体细胞沿着珠子表面规则地滑动，类似于平面基板上的细胞。贴在珠子上的这些细胞的快速冷冻复制品显示了不同的视图；侧视、斜视和正视，使我们不仅能够研究复杂建筑的全局形态，而且还可能研究更详细的形态。靶标与凸面的粘合可以给细胞内有趣的分子组装带来额外的好处，这与微生物的各种运动机制有关。
3-D Structural information is essential to elucidate the molecular mechanisms of various biological machineries. Quick-Freeze Deep-Etch-Replica Electron Microscopy is a unique technique to give very high-contrast surface profiles of extra- and intra-cellular apparatuses that bear numerous cellular functions. Though the global architecture of those machineries is primarily required to understand their functional features, it is difficult or even impossible to depict side- or highly-oblique views of the same targets by usual goniometry, inasmuch as the objects (e.g. motile microorganisms) are placed on conventional flat substrates. We introduced silica-beads as an alternative substrate to solve such crucial issue. Elongated Flavobacterium and globular Mycoplasmas cells glided regularly along the bead's surface, similarly to those on a flat substrate. Quick-freeze replicas of those cells attached to the beads showed various views; side-, oblique- and frontal-views, enabling us to study not only global but potentially more detailed morphology of complicated architecture. Adhesion of the targets to the convex surface could give surplus merits to visualizing intriguing molecular assemblies within the cells, which is relevant to a variety of motility machinery of microorganisms.