The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

ELife
Akanksha ThawaniSabine Petry

Abstract

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

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Citations

Jul 30, 2020·Molecular Biology of the Cell·Brianna R KingTrisha N Davis

Related Concepts

Cytoskeleton
Microtubules
Protein Conformation
Tubulin
Xenopus laevis
Longitudinal
XMAP215 protein, Xenopus
Gamma-Tubulin Ring Complex
Nucleated Cell
Regulation of Biological Process

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