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The control of mitotic progression by kinetochore-microtubule attachments

  • Author(s): Kuhn, Jonathan Alexander
  • Advisor(s): Dumont, Sophie
  • et al.
Abstract

To maintain genomic integrity, the Spindle Assembly Checkpoint (SAC) prevents anaphase onset until all chromosomes attach to the mitotic spindle in a bioriented fashion. The generation of SAC signal at individual kinetochores is controlled by the amount of localization of the SAC protein Mad1. In this work, I ask how kinetochores detects and counts correct microtubule attachments to control Mad1 localization and thus prevent mitotic errors. There are two long-standing candidate signals that turn off the checkpoint – the binding interaction between kinetochores and microtubules and the tension generated on kinetochores by attached microtubule pulling forces. It has been difficult to analyze the independent contribution of these signals to Mad1 loss because they are closely associated in space and time. Using live imaging, I demonstrate that mammalian kinetochores under tension but lacking plus-end microtubule attachments remain Mad1-positive, indicating that tension is not sufficient for SAC satisfaction. I then use laser ablation to show that kinetochores that are attached to microtubules but not under tension are Mad1-negative, proving that tension is not necessary for SAC satisfaction. Finally, having shown that kinetochores detect microtubule binding to control Mad1 levels, I ask how many attachments are required to turn off the checkpoint. By limiting the number of possible kinetochore-microtubule attachments, I show that while individual kinetochores bind 15-25 microtubules at metaphase, they are capable of complete – albeit slow – Mad1 loss with four or less attachments. Taken together, my data show how kinetochores detect spindle attachments, how they process them quantitatively, and how they respond in real time. Understanding these properties of kinetochore signaling is critical for understanding kinetochore structure and biochemistry and how cells manage to divide in an accurate and robust manner.

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