Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Nature. 2010 Nov 25;468(7323):576-9. doi: 10.1038/nature09594.
Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. 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 microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. 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 microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. 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.
着丝粒是一种大分子机器，它在细胞分裂过程中将染色体与动态微管末端连接起来，从而产生力来分离染色体。准确的分离取决于正确的“双取向”着丝粒-微管附着的选择性稳定，这些附着在微管施加的相反力的作用下产生张力。张力被认为通过抑制激酶 Aurora B 的失稳活性间接稳定这些双取向附着。然而，要全面了解张力的作用机制，需要在体外进行生化和生物物理分析来重建着丝粒-微管附着。在这里，我们表明可以从芽殖酵母中纯化保留大多数着丝粒蛋白的天然着丝粒颗粒，并用于重建动态微管附着。单个着丝粒颗粒保持与单个微管组装和拆卸端的承重关联超过 30 分钟，与体内芽殖酵母着丝粒和单个微管之间持续的偶联非常匹配。此外，张力通过一种类似捕获键的机制直接增加重建成的附着的寿命，该机制不需要 Aurora B。基于这些发现，我们提出张力通过直接机械稳定和张力依赖性磷酸化调节的组合，选择性地稳定体内正确的着丝粒-微管附着。
