Cryo electron tomography of vitrified fibroblasts:: Microtubule plus ends in situ
Cryo electron tomography of vitrified fibroblasts:: Microtubule plus ends in situ
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DOI：
10.1016/j.jsb.2007.08.011
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发表时间：
2008-03-01
影响因子：
3
通讯作者：
Mommaas, A. Mieke
中科院分区：
文献类型：
--
作者：
Koning, Roman I.;Zovko, Sandra;Mommaas, A. Mieke
小鼠胚胎成纤维细胞(MEF)是一种具有非常适合细胞冷冻电子断层扫描的生物物理特性的细胞。MEF可以直接生长在由EM栅格支撑的碳上。它们伸展开来，在细胞的主要部分生长得比500mN更薄，在它们的皮质上达到最小厚度50纳米。这有助于通过插入冷冻和高分辨率冷冻电子断层扫描进行直接冷冻固定。通过直接冷冻电子显微镜投影成像和冷冻电子断层扫描，我们显示了各种细胞结构，如核糖体、小泡、线粒体、粗面内质网、肌动蛋白细丝、中间丝和微管。因此，它们对研究细胞骨架元素很有吸引力。在这里，我们报告了微管加末端的结构研究。在玻璃化冷冻的成纤维细胞中，我们能够用冷冻电子断层扫描观察到微管+末端的单个磨损的原细丝。此外，MEF似乎在其微管内含有密度，尽管比神经细胞中的密度少2.5-3.5倍[Garvalov，B.K.，Zuber，B.，Bouchet-Marquis，C.，Kudryashev，M.，Gruska，M.，Beek，M.，Leis，A.，Frischknecht，F.，Bradke，F.，Baumeister，W.，Dubochet，J.，和CyrCraaff，M.2006。细胞微管内的管腔颗粒。J.细胞生物学。174,759-765]。细胞微管加末端的投影成像显示，40%的微管被磨损，与MEF的微管生长和收缩速度相比，这一比例是预期的两倍。这表明磨损的末端可能在细胞皮质中稳定下来。(C)2007 Elsevier Inc.保留所有权利。
Mouse embryonic fibroblasts (MEFs) are cells that have highly suitable biophysical properties for cellular cryo electron tomography. MEFs can be grown directly on carbon supported by EM grids. They stretch out and grow thinner than 500 mn over major parts of the cell, attaining a minimal thickness of 50 nm at their cortex. This facilitates direct cryo-fixation by plunge-freezing and high resolution cryo electron tomography. Both by direct cryo electron microscopy projection imaging and cryo electron tomography of vitrified MEFs we visualized a variety of cellular structures like ribosomes, vesicles, mitochondria, rough endoplasmatic reticulum, actin filaments, intermediate filaments and microtubules.MEFs are primary cells that closely resemble native tissue and are highly motile. Therefore, they are attractive for studying cytoskeletal elements. Here we report on structural investigations of microtubule plus ends. We were able to visualize single frayed protofilaments at the microtubule plus end in vitrified fibroblasts using cryo electron tomography. Furthermore, it appeared that MEFs contain densities inside their microtubules, although 2.5-3.5 times less than in neuronal cells [Garvalov, B.K., Zuber, B., Bouchet-Marquis, C., Kudryashev, M., Gruska, M., Beek, M., Leis, A., Frischknecht, F., Bradke, F., Baumeister, W., Dubochet, J., and Cyrklaff, M. 2006. Luminal particles within cellular microtubules. J. Cell Biol. 174, 759-765]. Projection imaging of cellular microtubule plus ends showed that 40% was frayed, which is two times more than expected when compared to microtubule growth and shrinkage rates in MEFs. This suggests that frayed ends might be stabilized in the cell cortex. (C) 2007 Elsevier Inc. All rights reserved.