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

Abstract Microtubules (MTs) are fundamental to cellular architecture, function and organismal development 1 . 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’ end 2 . 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.