Regulation of microtubule attachment at the kinetochore and cell cortex

Abstract

During mitosis, the cell divides in two, evenly distributing the genetic material and splitting the rest of cellular contents between the resulting daughter cells. This process is essential, rapid, and complex, requiring precise timing and the collaboration of many proteins. Mistakes made during mitosis can have immediate and catastrophic effects, resulting in cell death. In an organismal context, small mistakes in the relative size of the resulting daughter cells or the angle of division can have compounding consequences for development and function of complex organs, such as the brain. Mitotic chromosome segregation failures and the resulting DNA damage is also a hallmark of human cancers. There are multiple organelles that must work together to acheive a successful mitosis, including the chromosomes, kinetochores, centrosomes, and the microtubule cytoskeleton that forms the mitotic spindle. To assemble the mitotic spindle, the two centrosomes radially nucleate dynamic microtubules that make important connections with kinetochores and the cell cortex. Kinetochores mediate the connection between the chromosomes and microtubule cytoskeleton, whereas the astral microtubules contact the cell cortex and play an important role in positioning the mitotic spindle within the cell. In this thesis, I investigate the establishment and regulation of these two important microtubule-based connections. This work focuses on the kinetochore protein KNL-1, a component of the KMN (KNL-1, Mis12, Ndc80) network, and the kinetochore and microtubule-associated Astrin/SKAP complex. KNL-1 is a kinetochore specific scaffolding protein that I show to have the ability to form large oligomers in nematodes. The Astrin/SKAP complex has the unique property of localizing to diverse mitotic structures, including the kinetochore. I present data that the Astrin/SKAP complex plays multiple roles in mitosis, including kinetochore-microtubule attachment, centrosome integrity, and a previously unknown role in spindle positioning. Together, these results contribute to our understanding of kinetochore protein behavior and regulation and add a new complex to our emerging model of the microtubule connections facilitating mitotic spindle positioning.