Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2013 Apr 30;110(18):7282-7. doi: 10.1073/pnas.1220700110. Epub 2013 Apr 15.
During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.
在有丝分裂过程中，称为动粒的多蛋白复合物通过与动态微管尖端形成承重附着，并参与磷酸化调节错误校正，来协调染色体分离。保守的激酶 Aurora B 磷酸化主要的微管结合动粒亚复合物 Ndc80 和（在酵母中）Dam1，以促进错误附着的释放，从而为正确附着提供另一次机会。目前尚不清楚 Aurora B 磷酸化是通过增加动粒脱离的速度直接促进释放，还是通过微管尖端的不稳定性间接促进释放。此外，在整个动粒背景下，Ndc80 与 Dam1 磷酸化的相对重要性尚不清楚。为了解决这些不确定性，我们分离了携带 Ndc80 和 Dam1 磷酸化模拟突变的天然酵母动粒颗粒，并应用先进的激光捕获技术来测量它们与单个动态微管尖端的附着强度和稳定性。两个亚复合物上的磷酸化模拟突变以累加的方式降低了断裂力，表明两个亚复合物都对附着强度有独立的贡献。任一亚复合物上的磷酸化模拟突变都会在恒力下缩短附着寿命，主要是通过加速微管生长过程中的脱离。Dam1 上的磷酸化模拟突变也增加了从微管生长到缩短的转变的可能性，从而以间接的方式进一步促进释放。总之，我们的结果表明，在体内，Aurora B 通过至少两种机制释放动粒：通过削弱动粒-微管界面，以及通过使动粒附着的微管尖端不稳定。
