Structure of the native γ-Tubulin Ring Complex capping spindle microtubules

Abstract

Abstract Microtubules (MTs) are fundamental to cellular architecture, function and organismal development1. MT filaments assemble the mitotic spindle apparatus responsible for chromosome segregation, whereas the MT-based cytoskeletal network mediates dynein and kinesin-based intracellular transport. MTs are formed by the dynamic oligomerization and depolymerization of α/β-tubulin dimers in a head-to-tail fashion, with α-tubulin exposed at the ‘minus’ end of MTs and β-tubulin capping the more dynamic MT ‘plus’ end2. In cells, the large and evolutionary conserved γ-Tubulin Ring Complex (γTuRC) templates efficient MT nucleation from their ‘minus’ end at MT-organizing centres (MTOCs)3–6. Because all known γTuRC structures are devoid of MTs and exhibit an ‘open’, inactive conformation, the molecular mechanism of γTuRC-mediated MT nucleation remains unknown. Here, we used cryo-electron tomography (cryo-ET) to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched yeast mitotic spindles. In our structure, γTuRC adopts an active closed conformation to function as a perfect geometric helical template presenting a ring of g-tubulin subunits to seed nucleation of exclusively 13-protofilament microtubules. Our cryo-ET reconstruction also revealed that a novel coiled-coil protein staples the first row of α/β-tubulin molecules directly to alternating positions along the γ-tubulin ring. This positioning of α/β-tubulin onto γTuRC reveals a role for the coiled-coil protein in augmenting γTuRC-mediated microtubule nucleation. Based on our results we describe a molecular model for γTuRC activation and MT nucleation.