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Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.
Developmental Cell ( IF 10.7 ) Pub Date : 2020-05-19 , DOI: 10.1016/j.devcel.2020.04.019
Tanja Consolati 1 , Julia Locke 2 , Johanna Roostalu 2 , Zhuo Angel Chen 3 , Julian Gannon 2 , Jayant Asthana 1 , Wei Ming Lim 1 , Fabrizio Martino 2 , Milos A Cvetkovic 2 , Juri Rappsilber 3 , Alessandro Costa 2 , Thomas Surrey 4
Affiliation  

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a “closed” conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an “open,” nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.



中文翻译:


通过冷冻电镜结构解释单个人 γTuRC 的微管成核特性。



γ-微管蛋白环复合物(γTuRC)是细胞中主要的微管成核剂。其调节机制尚不清楚。我们纯化了人 γTuRC,并在基于全内反射荧光 (TIRF) 显微镜的实时成核测定中测量了其成核特性。我们发现 γTuRC 稳定地覆盖了随机成核的微管负端。与微管伸长相比,成核效率较低。 γTuRC 的 4 Å 分辨率冷冻电子显微镜 (cryo-EM) 结构与交联质谱分析相结合,揭示了一种不对称构象,仅部分复合物处于与微管几何形状匹配的“闭合”构象。复合物核心中的肌动蛋白和 γTuRC 封闭部分外周的 MZT2 似乎稳定了封闭构象。 γTuRC 的另一侧处于“开放”的无成核构象,导致结构不对称,这解释了纯化的人 γTuRC 的成核效率低。我们的数据表明通过 γTuRC 闭合微管成核的可能调节机制。

更新日期:2020-05-19
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