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

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-/alpha beta-tubulin affinity, which facilitates assembly of a MT from gamma-TuRC. Whereas spontaneous nucleation requires assembly of 8 alpha beta-tubulins, nucleation from gamma-TuRC occurs efficiently with a cooperativity of 4 alpha 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 alpha beta-tubulins drive gamma-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h gamma-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal gamma-/alpha beta-tubulin interaction.