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

Abstract

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.

Molecular interplay between HURP and Kif18A in mitotic spindle regulation

“…Consistent with previous reports, additon of HURP to dynamic microtubules reduced microtubule catastrophes and increased the rescue frequency, without affecting microtubule growth or shrinking rates (Figure 5 E-F, Supplementary Figure 12) 16 . This result led us to investigate HURP's possible effect on the capping mechanism exerted by Kif18A.…”

“…Most of the HURP residues that participate in these interactions are highly conserved among species (Supplementary Figure 7). The deep insertion in the inter-protofilament groove and the bridging interactions between adjacent tubulin subunits are consistent with HURP’s role as a microtubule stabilizing factor 14,16 . The microtubule bound structure of HURP resembles that of another spindle assembly factor, TPX2, which also contains a dual binding motif that establishes lateral and longitudinal contacts to staple tubulins together 36 (Figure 3 F).…”

“…Hepatoma upregulated protein (HURP) is a spindle assembly factor (SAF) that localizes to the chromatin-proximal region of K-fibers in a process mediated by Ran-GTP signaling [12][13][14] . Through its stabilizing and bundling activities, HURP regulates K-fiber dynamics and spindle morphology, contributing to chromosomal movements and alignment 15,16 . Both depletion and overexpression of HURP lead to defective spindles that cannot align chromosomes effectively 14,17 .…”

Mechanical coupling coordinates microtubule growth

“…These biochemical factors must work in concert with mechanical effects, and we suggest that the two classes of mechanisms probably operate over different length-and timescales. Certain tip-tracking proteins capable of influencing microtubule dynamics are enriched at growing k-fiber ends specifically near kinetochores that are moving anti-poleward (Amaro et al, 2010;Castrogiovanni et al, 2021;Tirnauer et al, 2002). Various kinesins are known to regulate the size of the metaphase spindle (Goshima et al, 2005;Neahring et al, 2021), to influence k-fiber dynamics (Maiato, Fairley, et al, 2003;Rizk et al, 2009;Stumpff et al, 2012;Wordeman et al, 2007), and to alter microtubule dynamics in vitro in a length-dependent manner (Varga et al, 2006).…”

“…Many microtubule-associated proteins that are known to regulate growth in vitro are implicated in modulating k-fiber dynamics and kinetochore movements in cells, including TOG-family proteins such as XMAP215 (Brouhard et al, 2008) and CLASP (Al-Bassam et al, 2010;Maiato, Fairley, et al, 2003;Sousa et al, 2007), mitotic kinesins such as MCAK and Kif18A (Sanhaji et al, 2011;Stumpff et al, 2012), and other plus-end-trackers such as EB1 (reviewed in (Amin et al, 2019)). The localization of some of these regulators is enriched near kinetochores specifically when a kinetochore is moving anti-poleward and its k-fiber is growing longer (Amaro et al, 2010;Castrogiovanni et al, 2021;Tirnauer et al, 2002). However, whether such selective enrichment is sufficient to explain the very tight coordination across many individual microtubule tips within a k-fiber is unclear.…”