Molecular Biophysics and Integrative Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, United States.
The Francis Crick Institute, London, United Kingdom.
Elife. 2017 Nov 9;6:e30959. doi: 10.7554/eLife.30959.
During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.
在有丝分裂和减数分裂过程中，微管（MT）的组装由染色质依赖的 Ran-GTP 途径局部上调。其关键靶标之一是与 MT 相关的纺锤体组装因子 TPX2。TPX2 如何刺激 MT 组装的分子机制尚不清楚，因为缺乏关于 TPX2 与 MT 相互作用的结构信息。在这里，我们确定了与 MT 表面结合的 TPX2 中心区域的冷冻电子显微镜结构。TPX2 使用两个灵活连接的元件（“脊”和“楔”）以新颖的相互作用模式同时结合在纵向和横向微管界面上。这些与 TPX2 上进口蛋白结合位点重叠的 MT 相互作用元件。基于荧光显微镜的体外重建测定表明，这种相互作用模式对于 MT 结合至关重要，并促进 MT 成核。总之，我们的结果表明了 Ran-GTP 梯度如何调节 TPX2 依赖性 MT 形成的分子机制。
