Determination of microtubule polarity by cryo-electron microscopy
Determination of microtubule polarity by cryo-electron microscopy
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DOI：
10.1016/s0969-2126(96)00110-4
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发表时间：
1996-09-15
期刊：
影响因子：
5.7
通讯作者：
Wade, RH
中科院分区：
文献类型：
--
作者：
Chretien, D;Kenney, JM;Wade, RH
背景资料：微管是微管蛋白二聚体的管状聚合物，其在沿着微管纵向沿着延伸的原丝中首尾相连地排列，从而赋予它们整体结构极性。微管的许多功能都依赖于这种极性，包括有丝分裂期间的定向细胞内运输和染色体分离。通过电子显微镜观察微管的纵向视图的微管极性的测定以前是不可能的。在这里，我们提出了直接确定由冷冻电子显微镜成像的单个微管的极性的方法。当用冷冻电子显微镜在玻璃冰中观察时，具有偏斜原丝的微管显示箭头状莫尔图案。我们使用中心体有核微管将莫尔图案的方向性与微管极性联系起来，我们表明，箭头指向微管的正端，原丝具有右手偏斜，并指向微管的负端，原丝具有左手偏斜，我们描述了两种方法来确定手的原丝歪斜。第一种方法使用两个或多个倾斜视图。第二种方法涉及的微管images.Conclusions的衍射图案的分析：它现在可以直接确定在体外组装的微管从冷冻电子显微镜照片的极性。这将有助于许多类型的研究，包括微管与马达蛋白相互作用的三维结构的研究，因为微管极性的知识对于理解马达方向性至关重要。
Background: Microtubules are tubular polymers of tubulin dimers, which are arranged head-to-tail in protofilaments that run lengthwise along the microtubules, giving them an overall structural polarity. Many of the functions of microtubules depend on this polarity, including directed intracellular transport and chromosome segregation during mitosis. The determination of microtubule polarity for lengthwise views of microtubules observed by electron microscopy has not previously been possible. Here, we present methods for directly determining the polarity of individual microtubules imaged by cryo-electron microscopy.Results: When observed in vitreous ice by cryo-electron microscopy, microtubules with skewed protofilaments show arrowhead moire patterns, We have used centrosome nucleated microtubules to relate the directionality of the moire patterns to microtubule polarity, We show that the arrowheads point towards the plus end of microtubules with protofilaments having a right-handed skew, and towards the minus end of microtubules with protofilaments having a left-handed skew, We describe two methods for determining the handedness of the protofilament skew. The first method uses two or more tilted views. The second method involves analysis of the diffraction patterns of the microtubule images.Conclusions: It is now possible to determine directly the polarity of in vitro assembled microtubules from cryo-electron micrographs. This will be helpful in a number of types of studies, including studies of the three-dimensional structure of microtubules interacting with motor proteins, as knowledge of the polarity of the microtubule is essential to understand motor directionality.