title: Probing microtubule polymerisation state at single kinetochores during metaphase chromosome motion.
abstract: Kinetochores regulate the dynamics of attached microtubule bundles (kinetochore-fibres, K-fibres) to generate the forces necessary for chromosome movements in mitosis. Current models suggest that poleward-moving kinetochores are attached to depolymerising K-fibres and anti-poleward-moving kinetochores to polymerising K-fibres. How the dynamics of individual microtubules within the K-fibre relate to poleward and anti-poleward movements is poorly understood. To investigate this, we developed a live-cell imaging assay combined with computational image analysis that allows eGFP-tagged EB3 (also known as MAPRE3) to be quantified at thousands of individual metaphase kinetochores as they undergo poleward and anti-poleward motion. Surprisingly, we found that K-fibres are incoherent, containing both polymerising and depolymerising microtubules – with a small polymerisation bias for anti-poleward-moving kinetochores. K-fibres also display bursts of EB3 intensity, predominantly on anti-poleward-moving kinetochores, equivalent to more coherent polymerisation, and this was associated with more regular oscillations. The frequency of bursts and the polymerisation bias decreased upon loss of kinesin-13, whereas loss of kinesin-8 elevated polymerisation bias. Thus, kinetochores actively set the balance of microtubule polymerisation dynamics in the K-fibre while remaining largely robust to fluctuations in microtubule polymerisation.
authors.affiliation: Warwick Systems Biology Centre and Mathematics Institute, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Armond
authors.affiliation: Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Elina
authors.initials: E
authors.lastname: Vladimirou
authors.affiliation: Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Muriel
authors.initials: M
authors.lastname: Erent
authors.affiliation: Mechanochemical Cell Biology Building, Division of Biomedical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK a.d.mcainsh@warwick.ac.uk n.j.burroughs@warwick.ac.uk.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: McAinsh
authors.affiliation: Warwick Systems Biology Centre and Mathematics Institute, University of Warwick, Coventry CV4 7AL, UK a.d.mcainsh@warwick.ac.uk n.j.burroughs@warwick.ac.uk.
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Burroughs
mesh_terms: D000818:Animals; D002875:Chromosomes / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D016547:Kinesins / Q000378:metabolism*; D018386:Kinetochores / Q000378:metabolism*; D008677:Metaphase / Q000502:physiology*; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D058105:Polymerization
chemical_list: D016547:Kinesins
keywords: EB3; K-fibres; KIF18A; Kinesin; Kinetochores; MCAK; Mitosis