Microtubule nucleation properties of single human γTuRCs explained by their Cryo-EM Structure

Abstract

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. ; This work was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001163 and FC0010065), the UK Medical Research Council (FC001163 and FC0010065), and the Wellcome Trust (FC001163 and FC0010065) to T.S. and A.C. The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149). J. Rappsilber is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy – EXC 2008 – 390540038 – UniSysCat and 329673113. J. Roostalu. was supported by a Sir Henry Wellcome Postdoctoral Fellowship (100145/Z/12/Z) and M.A.C. is supported by a Marie Sk1odowska-Curie Postdoctoral Fellowship (agreement no. 845939). T.S. acknowledges support from the European Research Council (Advanced Grant, project 323042). A.C. receives funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement no. 820102). T.C., J.A., J.W.M., and T.S. acknowledge also the support of the Spanish Ministry of Economy, Industry and Competitiveness to the CRG-EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme of the Generalitat de Catalunya