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The kinetochore is the large protein complex that assembles on centromeric DNA to mediate chromosome separation. For decades, researchers have tried to isolate whole functional kinetochores without success. Sue Biggins and colleagues now report the first purification of functional kinetochores. They also show that kinetochore particles maintain load-bearing associations with assembling and disassembling microtubules, and that tension directly increases the lifetimes of the attachments. These results provide evidence that tension selectively stabilizes kinetochore–microtubule interactions. The kinetochore is a large protein complex that assembles on centromeric DNA and captures microtubules to mediate chromosome separation. These authors report the first purification of functional kinetochores. They also show that kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules and that tension increases the lifetimes of the attachments directly. These results provide evidence that tension selectively stabilises kinetochore–microtubule interactions. Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore–microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore–microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B7,8,9,10. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore–microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation. |
