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Insights into the assembly and activation of the microtubule nucleator γ-TuRC

Abstract

Microtubules (MTs) are dynamic polymers of αβ-tubulin and play critical roles in cell signaling, cell migration, intracellular transport processes and chromosome segregation1. They assemble de novo from α/β-tubulin dimers in an essential process termed MT nucleation. Complexes containing the protein γ-tubulin serve as structural templates for the MT nucleation reaction2. In vertebrates, MTs are nucleated by the 2.2 MDa γ-tubulin ring complex (γ-TuRC) composed of γ-tubulin, five related γ-tubulin complex proteins (GCP2-6) and additional factors3. GCP6 is unique among the GCP proteins, because it carries an extended insertion domain of unknown function. High-resolution structural information on the γ-TuRC is not available, strongly limiting our understanding of MT formation in cells and tissue2. Here, we present the cryo-EM structure of γ-TuRC from Xenopus laevis at 4.8 Å global resolution, revealing a 14-spoked arrangement of GCPs and γ-tubulins in a partially flexible open left-handed spiral with a uniform sequence of GCP variants (Fig. 1a). Via specific interactions with other GCP proteins, the GCP6-specific insertion domain scaffolds the assembly of the γ-TuRC. Unexpectedly, we identified Actin as a bona fide structural component of γ-TuRC with functional relevance in MT nucleation. The γ-TuRC spiral geometry is suboptimal for MT nucleation and a controlled conformational rearrangement of the γ-TuRC is required for its activation. Collectively, our cryo-EM reconstruction provides unprecedented insights into the molecular organization, the assembly and the activation mechanism of vertebrate γ-TuRC and will serve as an important framework for the mechanistic understanding of fundamental biological processes associated with MT nucleation, e.g. meiotic and mitotic spindle formation and centriole biogensis4.

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Author information

Correspondence to Stefan Pfeffer or Elmar Schiebel.

Supplementary information

Supplementary Figure 1

Uncropped gels and blots with size markers

Reporting Summary

Supplementary Table 1

LFQ mass spectrometry of γ-TuRC

Supplementary Table 2

Custom made database containing additional database entries for γ-TuRC

Video 1

: Top View Conformational change required for activation of the γ-TuRC. Top view. Colouring as in Fig. 1a

Video 2

: Side view Conformational change required for activation of the γ-TuRC. Side view. Colouring as in Fig. 1a

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Liu, P., Zupa, E., Neuner, A. et al. Insights into the assembly and activation of the microtubule nucleator γ-TuRC. Nature (2019). https://doi.org/10.1038/s41586-019-1896-6

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