Microtubule Nucleation Properties of Single Human ?TuRCs Explained by Their Cryo-EM Structure.

ABSTRACT: The ?-tubulin ring complex (?TuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human ?TuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that ?TuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of ?TuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of ?TuRC appear to stabilize the closed conformation. The opposite side of ?TuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human ?TuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by ?TuRC closure.

SUBMITTER: Consolati T 

PROVIDER: S-EPMC7280788 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature