Protocol

Mitosis

Volume 545 of the series Methods in Molecular Biology pp 233-242

Date:

Live Cell Imaging of Kinetochore Capture by Microtubules in Budding Yeast

  • Kozo TanakaAffiliated withWellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of DundeeInstitute of Development, Aging and Cancer, Center for Research Strategy and Support, Tohoku University
  • , Tomoyuki U. TanakaAffiliated withWellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee

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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.

Key words

Saccharomyces cerevisiae kinetochore centromere spindle microtubule mitosis live cell imaging fluorescence microscopy