Developmental Cell
Volume 53, Issue 5, 8 June 2020, Pages 603-617.e8
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Article
Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure

https://doi.org/10.1016/j.devcel.2020.04.019Get rights and content
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Highlights

  • The γ-tubulin ring complex (γTuRC) nucleates microtubules and caps their minus ends

  • Microtubule nucleation from purified γTuRC is highly cooperative, yet inefficient

  • A partly open, asymmetric structure of γTuRC explains inefficient nucleation

  • Actin and MZT2 stabilize the closed part of the γTuRC structure

Summary

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.

Keywords

γ-tubulin ring complex
γTuRC structure
cryo-electron microscopy
TIRF microscopy
microtubule nucleation
CLMS
chTOG
TPX2
actin
MZT2

Cited by (0)

6

These authors contributed equally

7

Present address: The Wellcome Trust, London, United Kingdom

8

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