Tubulin hooks as probes for microtubule polarity: an analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle.
Tubulin hooks as probes for microtubule polarity: an analysis of the method and an evaluation of data on microtubule polarity in the mitotic spindle.
复制标题
微管钩作为微管极性的探针：该方法的分析和丝分裂纺锤体微管极性数据的评估。
DOI：
10.1083/jcb.98.2.525
复制
发表时间：
1984-02
影响因子：
7.8
通讯作者：
Euteneuer, U
中科院分区：
文献类型：
--
作者：
McIntosh, J R;Euteneuer, U
细胞微管的结构极性可以通过在含有微管蛋白的特殊缓冲液中裂解细胞来原位观察。在这些条件下，微管蛋白聚合形成弯曲片，其附着于内源性微管的壁上。当这种装饰的微管被切开并在电子显微镜下观察时，它们似乎带有顺时针或逆时针弯曲的钩。钩曲率的方向由修饰的微管的取向限定，因此用作微管极性的探针。在本文中，我们描述了一种方法来分析不同曲率的钩的相对频率，从而衡量钩曲率和微管极性之间的关系的保真度。该方法的假设进行了测试，并发现是有效的一个合理的精度。钩曲率和微管取向之间的相关性被证明是至少0.98的纺锤体的PtK细胞和Haemanthus胚乳在所有阶段的分裂和在所有地方的纺锤体。相关性被证明是有效的，为每个钩的形式，因此，这些微管的极性，承担多个钩的规定，甚至更好的确定性比0.98。钩装饰的这种性质被用来重新调查的可能性，一些微管的动粒纤维可能会与他们的正端远端的动粒（相反的方向先前显示占主导地位）。仔细分析无法识别这种相反方向的微管。对带有多个钩的小管的评分也表明，后期的单个区间纤维是由反平行的微管簇构成的。用于估计钩状装饰和微管极性之间的相关性的方法被证明是适用于许多结构和情况下，但我们发现，钩状装饰测定微管极性是不一致的准确性。我们建议未来使用钩装饰的研究应采用本文提供的数据分析方法来评估所获得结果的准确性。
The structural polarity of cellular microtubules can be visualized in situ by lysing cells in special buffers containing tubulin. Under these conditions, the tubulin polymerizes to form curved sheets which attach to the walls of the endogenous microtubules. When such decorated microtubules are cut in cross section and viewed in the electron microscope, they appear to bear hooks curving clockwise or counter- clockwise. The direction of hook curvature is defined by the orientation of the decorated microtubule and thus serves as a probe for microtubule polarity. In this paper we describe a way to analyze the relative frequencies of hooks of different curvatures so as to measure the fidelity of the relation between hook curvature and microtubule polarity. The assumptions of the method are tested and found to be valid to a reasonable accuracy. The correlation between hook curvature and microtubule orientation is shown to be at least 0.98 for the spindles of PtK cells and Haemanthus endosperm at all stages of division and at all places in the spindle. The correlation is shown to be valid for each hook that forms, so the polarity of those microtubules that bear multiple hooks is specified with even better certainty than 0.98. This property of hook decoration is used to reinvestigate the possibility that some of the microtubules of the kinetochore fiber might be oriented with their plus ends distal to the kinetochore (opposite to the direction previously shown to predominate). Close analysis fails to identify such oppositely oriented microtubules. The scoring of tubules bearing multiple hooks also shows that individual interzone fibers at anaphase are constructed from clusters of antiparallel microtubules. The method for estimating the correlation between hook decoration and microtubule polarity is shown to be applicable to many structures and circumstances, but we find that the hook decoration assay for microtubule polarity is not uniformly accurate. We suggest that future studies using hook decorations should employ the method of data analysis presented here to assess the accuracy of the results obtained.