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

TitleThe transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy.
Publication TypeJournal Article
Year of Publication2020
AuthorsThawani, A, Rale, MJ, Coudray, N, Bhabha, G, Stone, HA, Shaevitz, JW, Petry, S
JournalElife
Volume9
Date Published2020 Jun 15
ISSN2050-084X
Abstract

<p>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 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.</p>

DOI10.7554/eLife.54253
Alternate JournalElife
PubMed ID32538784
PubMed Central IDPMC7338055
Grant ListGraduate Student Fellowship / / National Science Foundation /
Charlotte Elizabeth Procter Honorific Fellowship / / Princeton University /
2014-40376 / / David and Lucile Packard Foundation /
Gilliam fellowship / HHMI / Howard Hughes Medical Institute / United States
1DP2GM123493 / GM / NIGMS NIH HHS / United States
R00GM112982 / GM / NIGMS NIH HHS / United States
00027340 / / Pew Charitable Trusts /
DP2 GM123493 / GM / NIGMS NIH HHS / United States
17PRE33660328 / / American Heart Association /
PHY-1734030 / / National Science Foundation /
R00 GM112982 / GM / NIGMS NIH HHS / United States