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bioRxiv · 10.1101/853010

Molecular mechanism of microtubule nucleation from gamma-tubulin ring complex

Thawani, A.·Stone, H. A.·Shaevitz, J. W.·Petry, S.
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Abstract

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, {gamma}-tubulin ring complex ({gamma}-TuRC) has been identified, precisely how {gamma}-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 {gamma}-TuRC. We reveal a high {gamma}-/{beta}-tubulin affinity, which facilitates assembly of a MT from {gamma}-TuRC. Whereas spontaneous nucleation requires assembly of 8 {beta}-tubulins, nucleation from {gamma}-TuRC occurs efficiently with a cooperativity of 4 {beta}-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 {beta}-tubulins drive {gamma}-TuRC into a closed conformation. Putative activation domain of CDK5RAP2, NME7 and TPX2 do not enhance {gamma}-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal {gamma}-/{beta}-tubulin interaction.

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Thawani, A., Stone, H. A., Shaevitz, J. W., Petry, S.. 2019-11-23. Molecular mechanism of microtubule nucleation from gamma-tubulin ring complex. https://doi.org/10.1101/853010

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