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Elife June 15, 2020; 9

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

Thawani A , Rale MJ , Coudray N , Bhabha G , Stone HA , Shaevitz JW , Petry S .

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.

PubMed ID: 32538784
PMC ID: PMC7338055
Article link: Elife
Grant support: [+]
Genes referenced: avd cdk5rap2 cdkn1a ckap5 kif2c nme7 pigy stmn1 tpx2
GO keywords: microtubule nucleation [+]

Article Images: [+] show captions
References [+] :
Aldaz, Insights into microtubule nucleation from the crystal structure of human gamma-tubulin. 2005, Pubmed

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