Methods in Molecular Biology Volume 545, 2009, pp 233-242

Live Cell Imaging of Kinetochore Capture by Microtubules in Budding Yeast

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Abstract

For high-fidelity chromosome segregation, kinetochores must be properly captured by spindle microtubules, but the mechanisms of initial kinetochore capture have remained elusive. Observation of individual kinetochore–microtubule interaction has been difficult, because multiple kinetochores are captured by microtubules during a short period and within a small space. By isolating one of the kinetochores from others through regulation of the activity of a centromere, we could visualize individual kinetochore–microtubule interactions in Saccharomyces cerevisiae. This technique, which we have called the ‘centromere reactivation system’, allowed us to dissect the process of kinetochore capture and transport on the mitotic spindle into several steps, thus enabling us to identify genes involved in each step. Kinetochores are captured by the side of microtubules extending from a spindle pole, and subsequently transported poleward along them. This process is evolutionarily conserved from yeast to vertebrate cells. Therefore, our system has proved useful in elucidating the underlying mechanisms of kinetochore capture by spindle microtubules.