Live-cell analysis of kinetochore-microtubule interaction in budding yeast.
Live-cell analysis of kinetochore-microtubule interaction in budding yeast.
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DOI：
10.1016/j.ymeth.2010.01.017
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发表时间：
2010-06
期刊：
影响因子：
4.8
通讯作者：
Tanaka, Tomoyuki U.
中科院分区：
文献类型：
--
作者：
Tanaka, Kozo;Kitamura, Etsushi;Tanaka, Tomoyuki U.
关键词：
纺锤体微管捕获和运输动粒在高保真染色体分离中起着至关重要的作用，尽管其具体机制仍不清楚。由于多个动点在很短的时间内被微管捕获，因此很难观察单个动点与微管的相互作用。我们开发了一种方法，通过调节着丝粒的活性将着丝粒中的一个与其他着丝粒分离，从而可视化酿酒酵母中单个着丝粒-微管的相互作用。我们详细介绍了这项技术，我们称之为着丝粒再激活系统，用于剖析有丝分裂纺锤体上着丝粒捕获和运输的过程。动点最初被从纺锤极延伸的微管一侧捕获，然后沿着它们向极地运输，这是从酵母到脊椎动物细胞的一个进化保守的过程。我们的系统与酵母遗传学相结合，已被证明有助于阐明动粒-微管相互作用的分子机制。我们讨论了将我们的系统应用于使用荧光显微镜的活细胞成像的实际考虑。
Kinetochore capture and transport by spindle microtubules plays a crucial role in high-fidelity chromosome segregation, although its detailed mechanism has remained elusive. It has been difficult to observe individual kinetochore–microtubule interactions because multiple kinetochores are captured by microtubules during a short period within a small space. We have developed a method to visualize individual kinetochore–microtubule interactions in Saccharomyces cerevisiae, by isolating one of the kinetochores from others through regulation of the activity of a centromere. We detail this technique, which we call ‘centromere reactivation system’, for dissection of the process of kinetochore capture and transport on mitotic spindle. Kinetochores are initially captured by the side of microtubules extending from a spindle pole, and subsequently transported poleward along them, which is an evolutionarily conserved process from yeast to vertebrate cells. Our system, in combination with amenable yeast genetics, has proved useful to elucidate the molecular mechanisms of kinetochore–microtubule interactions. We discuss practical considerations for applying our system to live cell imaging using fluorescence microscopy.
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DOI：
10.1083/jcb.149.4.863
发表时间：
2000-05-15
期刊：
The Journal of cell biology
影响因子：
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通讯作者：
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