Molecular mechanisms of kinetochore microtubule attachment

Abstract

To ensure equal chromosome segregation during mitosis, the macromolecular kinetochore must remain attached to depolymerizing microtubules, which drive poleward chromosome movement. Microtubules are highly dynamic structures that undergo dramatic structural changes during depolymerization. The results presented in this thesis define essential functions of the Astrin-SKAP-LC8 and Skal complexes at the kinetochore-microtubule interface. First, we demonstrate that the Astrin-SKAP-LC8 complex localizes preferentially to kinetochores of bioriented sister chromatids. Localization of the Astrin-SKAP-LC8 complex to kinetochores is controlled by a key regulator of kinetochore-microtubule attachments, Aurora B kinase. The Astrin-SKAP-LC8 complex is essential for mitotic progression and directly associates with microtubules. Furthermore, the microtubule polymerization factor CLASP requires the Astrin-SKAP-LC8 complex to localize to kinetochores. Second, we demonstrate that the Skal complex has many of the biochemical and biophysical properties of a molecular machine that can couple microtubule depolymerization to chromosome movement. The Skal complex diffuses on and tracks with depolymerizing microtubules and its microtubule binding activity is necessary to maintain kinetochore-fibers and power chromosome oscillations during metaphase. Importantly, we demonstrate that the Skal complex directly interacts with the peeling protofilaments present at the depolymerizing microtubule end, suggesting a unique mechanism by which the Skal complex remains attached to depolymerizing microtubules. Finally, we demonstrate that the Skal microtubule-binding domain has two conserved basic regions that are required for microtubule binding and are subject to regulation by Aurora B kinase. In total, we define essential properties of the Astrin-SKAP-LC8 and Skal complex required for the formation of kinetochore microtubule attachments.