The transition state and regulation of ?-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, ?-tubulin ring complex (?-TuRC) has been identified, precisely how ?-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 ?-TuRC. We reveal a high ?-/??-tubulin affinity, which facilitates assembly of a MT from ?-TuRC. Whereas spontaneous nucleation requires assembly of 8 ??-tubulins, nucleation from ?-TuRC occurs efficiently with a cooperativity of 4 ??-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 ??-tubulins drive ?-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 h ?-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal ?-/??-tubulin interaction.

SUBMITTER: Thawani A 

PROVIDER: S-EPMC7338055 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature