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.

目前的模型推断，基于微管的有丝分裂纺锤体是由 GDP 微管蛋白构建的，在微管正端具有小 GTP 帽，包括那些附着在着丝粒上、形成着丝粒纤维的帽。在这里，我们揭示了着丝粒纤维还包含一个长度达到几微米的动态混合核苷酸区域。该区域在表达荧光标记的末端结合蛋白（GTP-微管蛋白的已知标记）和内源标记的 HURP（一种我们在体外和体内优先结合 GDP 微管晶格的蛋白质）的细胞中变得可见。我们发现，在有丝分裂细胞中，HURP 积聚在解聚着丝粒纤维的着丝粒近端区域，同时避免招募到新生的聚合 K 纤维，从而导致“HURP 间隙”不断增大。 HURP 缺口中末端结合蛋白的缺失使我们推测它们反映了混合核苷酸区域。我们基于 HURP 与 GDP-微管蛋白的优先结合生成了一个最小定量模型，以表明这种混合核苷酸区域足以概括观察到的 HURP-间隙的体内动态。