Visualization of cell microtubules in their native state
Visualization of cell microtubules in their native state
复制标题
DOI：
10.1042/bc20060081
复制
发表时间：
2007-01-01
影响因子：
2.7
通讯作者：
Chretien, Denis
中科院分区：
文献类型：
--
作者：
Bouchet-Marquis, Cedric;Zuber, Benoit;Chretien, Denis
背景资料。在过去的几十年里，玻璃化标本的冷冻电子显微镜已经产生了一个详细的了解，微管蛋白和微管结构的样品重新组装在体外从纯化的组件。然而，我们的知识微管结构在体内仍然是有限的化学处理通常用于观察细胞结构，使用电子显微镜。我们使用冷冻电子显微镜和冷冻电子断层扫描玻璃体切片，以探讨微管在其细胞背景下的超微结构。玻璃体切片取自大鼠海马的器官型切片和培养的中国仓鼠卵巢细胞。微管显示其原丝超微结构，极性，并在最有利的情况下，分子的细节与那些可视化的三维重建的微管重组在体外从纯化的微管蛋白。断层图像的分辨率估计约为。4nm，这使得能够检测约40nm的管腔颗粒。6 nm的直径内的微管。本研究提供了第一步的微管的描述，除了其他大分子组件，在一个未受干扰的细胞背景下，在分子水平上。由于分辨率似乎与快速冷冻样本的分辨率相似，因此它应该能够在体内识别全细胞和组织的玻璃体切片中较大的大分子组装体。
Background information. Over the past decades, cryo-electron microscopy of vitrified specimens has yielded a detailed understanding of the tubulin and microtubule structures of samples reassembled in vitro from purified components. However, our knowledge of microtubule structure in vivo remains limited by the chemical treatments commonly used to observe cellular architecture using electron microscopy.Results. We used cryo-electron microscopy and cryo-electron tomography of vitreous sections to investigate the ultrastructure of microtubules in their cellular context. Vitreous sections were obtained from organotypic slices of rat hippocampus and from Chinese-hamster ovary cells in culture. Microtubules revealed their protofilament ultrastructure, polarity and, in the most favourable cases, molecular details comparable with those visualized in three-dimensional reconstructions of microtubules reassembled in vitro from purified tubulin. The resolution of the tomograms was estimated to be approx. 4 nm, which enabled the detection of luminal particles of approx. 6 nm in diameter inside microtubules.Conclusions. The present study provides a first step towards a description of microtubules, in addition to other macromolecular assemblies, in an unperturbed cellular context at the molecular level. As the resolution appears to be similar to that obtainable with plunge-frozen samples, it should allow for the in vivo identification of larger macromolecular assemblies in vitreous sections of whole cells and tissues.