Doodhi Harinath, Prota Andrea E, Rodríguez-García Ruddi, Xiao Hui, Custar Daniel W, Bargsten Katja, Katrukha Eugene A, Hilbert Manuel, Hua Shasha, Jiang Kai, Grigoriev Ilya, Yang Chia-Ping H, Cox David, Horwitz Susan Band, Kapitein Lukas C, Akhmanova Anna, Steinmetz Michel O
Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, the Netherlands.
Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland.
Curr Biol. 2016 Jul 11;26(13):1713-1721. doi: 10.1016/j.cub.2016.04.053. Epub 2016 Jun 16.
Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation.
微管是由微管蛋白二聚体构成的动态聚合物，这些二聚体以首尾相连的方式连接形成原丝，原丝进一步横向结合形成微管。单个原丝的异步延长可能会导致微管末端结构改变，从而促进突然解聚，即灾变[1-4]。然而，单个原丝的动态变化与整体生长持续性之间的关系仍不清楚。在这里，我们使用微管靶向抗癌药物艾瑞布林[5-7]来探究原丝延长停滞对微管生长的影响。通过X射线晶体学，我们首先揭示了艾瑞布林与原丝正端延长所需的β-微管蛋白上的一个位点结合。基于这一结构信息，我们设计了一种荧光艾瑞布林分子。我们证明，单个艾瑞布林分子特异性地与微管正端相互作用，并且在存在EB3的情况下足以引发灾变或诱导微管缓慢且不稳定地生长。有趣的是，我们发现艾瑞布林增加了EB3彗星“分裂”的频率，即缓慢且不稳定前进的彗星之后跟着一个更快彗星的瞬态事件。这一观察结果可能反映了微管晶格的“修复”。由于在没有任何药物的对照微管中也观察到了EB3彗星分裂，我们提出艾瑞布林通过促进原丝延长的停滞来放大通向灾变的自然途径。
