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Insights into the assembly and activation of the microtubule nucleator γ-TuRC
Peng Liu, Erik Zupa, Annett Neuner, Anna Böhler, Justus Loerke, Dirk Flemming, Thomas Ruppert, Till Rudack, Christoph Peter, Christian Spahn, Oliver J. Gruss, Stefan Pfeffer & Elmar Schiebel
Nature (2019)
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 biogensis 4.
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