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

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.

Transition of human γ-tubulin ring complex into a closed conformation during microtubule nucleation

“…This arrangement of GCP subunits explains why human γTuRC serves as a perfect template for 13-protofilament microtubules—one of its key functions—despite exposing 14 γ-tubulins in its open conformation before nucleation. Our observations agree with recently reported lower-resolution cryo-EM structures of closed conformations of human and yeast γTuRC, which are, however, attached to 13-protofilament microtubules of considerably greater length than in our study ( 46 , 47 ). The perfect match of the postnucleation γTuRC and microtubule structures also provides an explanation for the exceptional stability of their interface, as observed in vitro ( 27 , 34 , 36 , 48 – 50 ), suggesting that energy-consuming destabilizing activities, such as those of severases and depolymerases, may be needed in cells to recycle the complex for additional rounds of nucleation, consistent with recent in vitro reconstitutions ( 49 ) and observations in cells ( 51 , 52 ).…”

“…The activity of budding yeast γTuRC appears to be mostly regulated by its assembly on the spindle pole body, where it adopts a structure that is already a relatively good match with the microtubule structure ( 19 , 56 ). Nevertheless, a final conformational closure step is also required in this organism to form a perfect template ( 23 , 47 ). In contrast, vertebrate γTuRC has evolved to assemble in the cytoplasm in a conformation that deviates considerably from the microtubule structure, probably to maintain a low basal level of activity.…”

Closure of the γ-tubulin ring complex by CDK5RAP2 activates microtubule nucleation

“…Our work spurs the question of whether oligomeric binding of CDK5RAP2 and/or other γ-TuRC-associated proteins could perform similar α/β-tubulin-recruitment and tethering functions at MTOCs. A recent preprint describing a subtomogram-averaged structure of S. cerevisiae spindle pole microtubules capped by the γ-TuRC demonstrated the presence of a coiled-coil protein linking alternating γ-TuRC subunits with α/β-tubulin at the minus end [61], which we note is reminiscent of the odd-numbered subunit CM1 motif-like binding in our CMG-decorated model (Figure 4A). Further structural investigations of in vitro -reconstituted as well as in situ microtubule end-capped γ-TuRC co-complex structures will undoubtedly shed light onto the regulation of microtubule nucleation at diverse MTOCs.…”