Microtubule Nucleation Properties of Single Human Γturcs Investigated by TIRF Microscopy and Cryo-Electron Microscopy

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 TIRF microscopy-based real-time nucleation assay. We find that single γTuRC complexes nucleate microtubules is a cooperative and stochastic manner. They stably cap microtubule minus ends. Nucleation is inefficient compared to microtubule elongation. A 4 Å resolution 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. The opposite side of γTuRC is in an ‘open’, nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure. [published in Dev. Cell 53, 603-617, 2020; https://doi.org/10.1016/j.devcel.2020.04.019].