Kinetochore-microtubule coupling and its regulation during mitosis
Tien, Jerry F.
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The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. This linkage depends on the Ndc80 complex, a conserved and essential microtubule-binding component of the kinetochore. As a member of the complex, the Ndc80 protein forms microtubule attachments through a calponin homology domain, which has been the focus of biochemical and structural characterization. However, the function of the remainder of Ndc80 is poorly understood. We coupled high-throughput sequencing to a saturating linker-scanning mutagenesis screen in <italic>Saccharomyces cerevisiae</italic> and identified essential domains in previously uncharacterized regions of Ndc80. By analyzing mutants from the screen, we found that one domain within the Ndc80 coiled-coil is involved in assembling, but not maintaining, metaphase spindles. This process likely depends on a conformational change in the complex. Furthermore, we found that a helical hairpin adjacent to the calponin homology domain influences microtubule binding by the complex <italic>in vitro</italic>, and a C-terminal segment of Ndc80 is required for tetramerization of the complex <italic>in vivo</italic>. In <italic>S. cerevisiae</italic>, the Ndc80 complex recruits the microtubule-binding Dam1 complex to kinetochores. The Ndc80 and Dam1 complexes are not redundant, but their distinct contributions are unknown. We show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to couple to microtubule tips <italic>in vitro</italic>. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by Aurora B. This provides evidence for a mechanism by which Aurora B resets aberrant kinetochore-microtubule attachments in <italic>S. cerevisiae</italic>. In higher eukaryotes, recent findings suggest that Aurora B also influences microtubule dynamics as part of its error correction mechanism. We show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). Human Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even while robustly coupled to disassembling microtubule tips. Together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the human Ndc80 complex.
- Biological chemistry