Journal
DEVELOPMENTAL CELL
Volume 53, Issue 5, Pages 603-+Publisher
CELL PRESS
DOI: 10.1016/j.devcel.2020.04.019
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Funding
- Francis Crick Institute from Cancer Research UK [FC001163, FC0010065]
- UK Medical Research Council [FC001163, FC0010065]
- Wellcome Trust [FC001163, FC0010065, 203149]
- Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy [EXC 2008 -390540038 -UniSysCat, 329673113]
- Sir Henry Wellcome Postdoctoral Fellowship [100145/Z/12/Z]
- Marie Sk1odowskaCurie Postdoctoral Fellowship [845939]
- European Research Council [323042]
- European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [820102]
- Spanish Ministry of Economy, Industry and Competitiveness
- Centro de Excelencia Severo Ochoa
- CERCA Programme of the Generalitat de Catalunya
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The gamma-tubulin ring complex (gamma TuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human gamma TuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that gamma TuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 angstrom resolution cryoelectron microscopy (cryo-EM) structure of gamma TuRC, 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 gamma TuRC appear to stabilize the closed conformation. The opposite side of gamma TuRC is in an open, nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human gamma TuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by gamma TuRC closure.
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