Abstract
Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.
Highlights
Microtubules are essential cytoskeletal components with multiple important functions in chromosome segregation, cellular transport, cell organization and cell motility
The two plasmids were used for virus production: construct 1 contained 2xFLAG-GCP5, GCP4, GCP6, TUBG1 and ACTB, which were suggested to form an initial stable core complex during the assembly of γ-tubulin ring complex (γ-TuRC) [8,26]; construct 2 contained mitotic spindle organizing protein 1 (MZT1), GCP2 and GCP3, the ‘minimal set’ of additional subunits required to reconstitute the 14-spoke γ-TuRC including the luminal bridge as observed in cryo-electron microscopy (cryo-EM) studies [8,9,10,12]
This system enables fast and efficient screening of structural and functional properties of genetically modified γ-TuRC variants in follow-up experiments. The success of this strategy, which is based on FLAG-GCP5 affinity purification, indicates that the N-terminus of GCP5, which has not been completely traced in the recent cryo-EM reconstructions, is accessible for antibody binding and likely not as deeply embedded in the γ-TuRC as the GCP6 N-terminus [12]
Summary
Microtubules are essential cytoskeletal components with multiple important functions in chromosome segregation, cellular transport, cell organization and cell motility. High-resolution structural information on the γ-TuRC was limited to a few isolated components [6,7], until recent advances in cryo-electron microscopy (cryo-EM) facilitated resolving the structure of purified vertebrate γ-TuRCs to near–atomic resolution [8,9,10]. These analyses showed that the γ-TuRC contains γ-tubulin, five paralogous gamma-ring complex proteins (GCPs, GCP2 to 6), mitotic spindle organizing protein 1 (MZT1), MZT2 and actin in a defined stoichiometry [8,9,10,11]. A prominent feature of the γ-TuRC is a structural scaffold in the lumen of the complex, formed by two copies of MZT1, actin and the royalsocietypublishing.org/journal/rsob Open Biol. 11: 200325
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