@article{183096,
  keywords = {Animals, Microscopy, Fluorescence, Xenopus Proteins, Xenopus laevis, Microtubules, Single Molecule Imaging, Tubulin, Microtubule-Associated Proteins},
  author = {Akanksha Thawani and Michael Rale and Nicolas Coudray and Gira Bhabha and Howard Stone and Joshua Shaevitz and Sabine Petry},
  title = {The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy},
  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>
},
  year = {2020},
  journal = {Elife},
  volume = {9},
  month = {06/2020},
  issn = {2050-084X},
  doi = {10.7554/eLife.54253},
  language = {eng},
}