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

Harinath Doodhi, Taciana Kasciukovic, Marek Gierlinski, Shuyu Li, Lesley Clayton, Tomoyuki U. Tanaka

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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). However, it is still unknown how kinetochore–microtubule interaction is turned over during error correction. Here we reconstituted the kinetochore–microtubule interface in vitro by attaching Ndc80 protein complexes (Ndc80Cs) to a nanobead that mimics the inner kinetochore. The Ndc80C–nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. Using this system, we provide evidence that error correction is driven by direct competition for a kinetochore between the end-on attachment to one microtubule and the lateral attachment to another. We validated this competition-based error correction, using mathematical modelling and live-cell imaging. Our study reveals a fundamental mechanism of error correction, which contributes to efficient establishment of bi-orientation.

Original languageEnglish
Number of pages31
JournalBioRxiv
DOIs
Publication statusPublished - 29 Oct 2018

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Kinetochores
Microtubules
Chromosome Segregation
Aurora Kinase B
Spindle Poles
In Vitro Techniques
Proteins

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Doodhi, Harinath ; Kasciukovic, Taciana ; Gierlinski, Marek ; Li, Shuyu ; Clayton, Lesley ; Tanaka, Tomoyuki U. / In vitro reconstitution of kinetochore–microtubule interface reveals a fundamental error correction mechanism. In: BioRxiv. 2018.
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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). However, it is still unknown how kinetochore–microtubule interaction is turned over during error correction. Here we reconstituted the kinetochore–microtubule interface in vitro by attaching Ndc80 protein complexes (Ndc80Cs) to a nanobead that mimics the inner kinetochore. The Ndc80C–nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. Using this system, we provide evidence that error correction is driven by direct competition for a kinetochore between the end-on attachment to one microtubule and the lateral attachment to another. We validated this competition-based error correction, using mathematical modelling and live-cell imaging. Our study reveals a fundamental mechanism of error correction, which contributes to efficient establishment of bi-orientation.",
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Doodhi, H, Kasciukovic, T, Gierlinski, M, Li, S, Clayton, L & Tanaka, TU 2018, 'In vitro reconstitution of kinetochore–microtubule interface reveals a fundamental error correction mechanism', BioRxiv. https://doi.org/10.1101/455873

In vitro reconstitution of kinetochore–microtubule interface reveals a fundamental error correction mechanism. / Doodhi, Harinath; Kasciukovic, Taciana; Gierlinski, Marek; Li, Shuyu; Clayton, Lesley; Tanaka, Tomoyuki U.

In: BioRxiv, 29.10.2018.

Research output: Contribution to journal › Article

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AU - Kasciukovic, Taciana

AU - Gierlinski, Marek

AU - Li, Shuyu

AU - Clayton, Lesley

AU - Tanaka, Tomoyuki U.

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Doodhi H, Kasciukovic T, Gierlinski M, Li S, Clayton L, Tanaka TU. In vitro reconstitution of kinetochore–microtubule interface reveals a fundamental error correction mechanism. BioRxiv. 2018 Oct 29. https://doi.org/10.1101/455873

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