In vitro reconstitution of kinetochore-microtubule interface reveals a fundamental error correction mechanism

Abstract

For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles; this is called bi-orientation. To establish bi-orientation prior to chromosome segregation, any aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase that phosphorylates outer kinetochore components. 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 not fully understood how kinetochore-microtubule interactions are exchanged during error correction. Here we reconstituted the kinetochore-microtubule interface of budding yeast in vitro by attaching the Ndc80 complexes (Ndc80C) 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. We suggest that 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. Our study reveals a fundamental mechanism of error correction for establishment of bi-orientation.