Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, UK.
J Cell Biol. 2021 Jun 7;220(6). doi: 10.1083/jcb.202011117.
To establish chromosome biorientation, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment, respectively). However, it is still unclear how kinetochore-microtubule interactions are exchanged during error correction. Here, we reconstituted the budding yeast kinetochore-microtubule interface in vitro by attaching the Ndc80 complexes to nanobeads. These Ndc80C nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. This in vitro assay enabled the direct comparison of lateral and end-on attachment strength and showed that Dam1 phosphorylation by Aurora B makes the end-on attachment weaker than the lateral attachment. Similar reconstitutions with purified kinetochore particles were used for comparison. We suggest the Dam1 phosphorylation weakens interaction with the Ndc80 complex, disrupts the end-on attachment, and promotes the exchange to a new lateral attachment, leading to error correction.
为了建立染色体的定向排列，有丝分裂纺锤体的动粒-微管相互作用必须被 Aurora B 激酶纠正（错误修正）。Aurora B 激酶能够区分调控动粒与微管正极和侧方的连接（分别称为端对连接和侧向连接）。然而，动粒-微管相互作用在错误修正过程中是如何被交换的，目前仍不清楚。在这里，我们通过将 Ndc80 复合物附着在纳米珠上来体外重建酿酒酵母动粒-微管界面。这些 Ndc80C 纳米珠在体外模拟了带有 Dam1 复合物的动态微管上真实动粒的侧向和端对连接。该体外实验使我们能够直接比较侧向和端对连接强度，并表明 Aurora B 对 Dam1 的磷酸化使端对连接弱于侧向连接。我们使用纯化的动粒颗粒进行了类似的重建实验以进行比较。我们提出 Dam1 的磷酸化会削弱与 Ndc80 复合物的相互作用，破坏端对连接，并促进向新的侧向连接的交换，从而导致错误修正。
