Abstract
The gamma-tubulin ring complex (γ-TuRC) is the principal microtubule nucleation template in vertebrates. Recent cryo-EM reconstructions visualized the intricate quaternary structure of the γ-TuRC, containing more than thirty subunits, raising fundamental questions about γ-TuRC assembly and the role of actin as an integral part of the complex. Here, we reveal the structural mechanism underlying modular γ-TuRC assembly and identify a functional role of actin in microtubule nucleation. During γ-TuRC assembly, a GCP6-stabilized core comprising GCP2-3-4-5-4-6 is expanded by stepwise recruitment, selective stabilization and conformational locking of four pre-formed GCP2-GCP3 units. Formation of the lumenal bridge specifies incorporation of the terminal GCP2-GCP3 unit and thereby leads to closure of the γ-TuRC ring in a left-handed spiral configuration. Actin incorporation into the complex is not relevant for γ-TuRC assembly and structural integrity, but determines γ-TuRC geometry and is required for efficient microtubule nucleation and mitotic chromosome alignment in vivo.
Highlights
The gamma-tubulin ring complex (γ-TuRC) is the principal microtubule nucleation template in vertebrates
Actin and MZT1 co-expression were observed to be essential for the structural integrity of the complex14, and the AAA+ ATPase RUVBL1/RUVBL2 was identified as an important factor for efficient γ-tubulin ring complex (γ-TuRC) reconstitution9
To structurally elucidate γ-TuRC assembly, we isolated human γ-TuRC from a recombinant insect cell expression system via gentle 2xFLAG-GCP5 affinity purification, which yields microtubule nucleationcompetent γ-TuRCs13
Summary
The gamma-tubulin ring complex (γ-TuRC) is the principal microtubule nucleation template in vertebrates. X-ray crystallography further identified the GCP5 N-terminus to be capable of forming a similar structural module in complex with MZT111, suggesting that the γ-TuRC has seven distinct binding sites for MZT1 (5 copies of GCP3 and one copy of GCP5 and GCP6, respectively), one on every other spoke. X-ray crystallography further identified the GCP5 N-terminus to be capable of forming a similar structural module in complex with MZT111, suggesting that the γ-TuRC has seven distinct binding sites for MZT1 (5 copies of GCP3 and one copy of GCP5 and GCP6, respectively), one on every other spoke12 Based on this structural molecular census of γ-TuRC components, recombinant expression systems for the human γ-TuRC were established in several independent studies, enabling direct genetic manipulation and detailed functional dissection of individual γ-TuRC components. Γ-TuRC mutants unable to integrate actin showed defects in microtubule nucleation and chromosome alignment in human cells, revealing a functional role of γ-TuRCassociated actin in cell division
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