The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy
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. Putative activation domain of CDK5RAP2, NME7 and TPX2 do not enhance γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 2, 3, 4, 6, 7 and related supplements.
Article and author information
Author details
Funding
American Heart Association (17PRE33660328)
- Akanksha Thawani
Princeton University (Charlotte Elizabeth Procter Honorific Fellowship)
- Akanksha Thawani
Howard Hughes Medical Institute (Gilliam fellowship)
- Michael J Rale
National Science Foundation (Graduate Student Fellowship)
- Michael J Rale
National Institute of General Medical Sciences (R00GM112982)
- Gira Bhabha
National Institute of General Medical Sciences (1DP2GM123493)
- Sabine Petry
Pew Charitable Trusts (00027340)
- Sabine Petry
David and Lucile Packard Foundation (2014-40376)
- Sabine Petry
National Science Foundation (PHY-1734030)
- Joshua W Shaevitz
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Jens Lüders, Institute for Research in Biomedicine, Spain
Ethics
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved Institutional Animal Care and Use Committee (IACUC) protocol # 1941-16 of Princeton University.
Version history
- Received: December 7, 2019
- Accepted: June 15, 2020
- Accepted Manuscript published: June 15, 2020 (version 1)
- Version of Record published: July 6, 2020 (version 2)
- Version of Record updated: July 9, 2020 (version 3)
Copyright
© 2020, Thawani et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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