Evidence for a HURP/EB free mixed-nucleotide zone in kinetochore-microtubules

• Published in: Nature communications 2022-08, Vol.13 (1), p.4704-4704, Article 4704
• Main Authors: Castrogiovanni, Cédric, Inchingolo, Alessio V., Harrison, Jonathan U., Dudka, Damian, Sen, Onur, Burroughs, Nigel J., McAinsh, Andrew D., Meraldi, Patrick
• Format: Article
• Language: English
• Subjects: 13/109; 13/51; 14/19; 14/35; 14/63; 631/1647/328; 631/80/128/1653; 631/80/641/1656; 82/80; Antibodies; Binding; Binding sites; Cell division; Chromosomes; Fibers; Guanosine diphosphate; Guanosine triphosphate; Guanosine Triphosphate - metabolism; Humanities and Social Sciences; Kinetochores; Kinetochores - metabolism; Lattices; Localization; Microtubule-Associated Proteins - metabolism; Microtubules; Microtubules - metabolism; Mitosis; multidisciplinary; Nucleotides; Nucleotides - metabolism; Proteins; Science; Science (multidisciplinary); Spindle Apparatus - metabolism; Tubulin; Tubulin - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-32421-x

Current models infer that the microtubule-based mitotic spindle is built from GDP-tubulin with small GTP caps at microtubule plus-ends, including those that attach to kinetochores, forming the kinetochore-fibres. Here we reveal that kinetochore-fibres additionally contain a dynamic mixed-nucleotide zone that reaches several microns in length. This zone becomes visible in cells expressing fluorescently labelled end-binding proteins, a known marker for GTP-tubulin, and endogenously-labelled HURP - a protein which we show to preferentially bind the GDP microtubule lattice in vitro and in vivo. We find that in mitotic cells HURP accumulates on the kinetochore-proximal region of depolymerising kinetochore-fibres, whilst avoiding recruitment to nascent polymerising K-fibres, giving rise to a growing “HURP-gap”. The absence of end-binding proteins in the HURP-gaps leads us to postulate that they reflect a mixed-nucleotide zone. We generate a minimal quantitative model based on the preferential binding of HURP to GDP-tubulin to show that such a mixed-nucleotide zone is sufficient to recapitulate the observed in vivo dynamics of HURP-gaps. Microtubules are built from GDP-tubulin lattices with small GTP caps at their plus-ends. Here, the authors reveal that microtubules that attach to kinetochores in mitosis contain, in addition to the GTP-cap and the GDP-lattices, a dynamic micron-sized mixed-nucleotide zone.