The conserved centrosomal motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)

Abstract

To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules generated by γTuRCs. Here we utilize Xenopus egg extract and in vitro single molecule imaging assays to show that γTuNA activates microtubule nucleation in extract and directly activates γTuRC in vitro. Via mutation analysis, we find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA's L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, we find that γTuNA's activating effect opposes inhibitory regulation by stathmin. In sum, our study helps illuminate how γTuRC is controlled in space and time in order to build specific cytoskeletal structures.

CAMSAP-driven microtubule release from γ-TuRC and its regulation by nucleation-promoting factors

“…The percentage of CDK5RAP2-positive dots colocalizing with γ-TuRCs was rather low in these experiments (~13% with tubulin, ~41% without tubulin), likely because CDK5RAP2 was present in excess, or due to the autoinhibitory regulation of CDK5RAP2 that is controlled by its phosphorylation state 33,34 . Compared to the samples with tubulin alone or with mCherry-EB3, mCherry-CDK5RAP2 had no significant effect on microtubule growth rate or catastrophe frequency, but strongly increased the frequency of microtubule re-nucleation after depolymerization, indicating that CDK5RAP2 can maintain γ-TuRC in an active state (Fig.…”

“…Two studies showed a stimulatory effect of nanomolar γ-TuNA concentrations on microtubule nucleation by γ-TuRC purified from mammalian cells using its affinity for γ-TuNA 26, 27 , whereas, more recently, two other groups found no to little effect of even micromolar γ-TuNA concentrations on Xenopus γ-TuRC when purified using a γ-tubulin antibody 19, 51 . A recent study attributed these differences to the presence of bulkier N-terminal tags on γ-TuNA and purification methods involving different affinity tags 34 .…”

“…However, we were successful in examining the effect of CDK5RAP2. This protein is well-known for its ability to bind γ-TuRC through the domain called γ-TuNA (γ-TuRC nucleation activator), but there was some conflicting evidence about its ability to activate γ-TuRC in vitro 19, 26, 27, 34, 51 . Two studies showed a stimulatory effect of nanomolar γ-TuNA concentrations on microtubule nucleation by γ-TuRC purified from mammalian cells using its affinity for γ-TuNA 26, 27 , whereas, more recently, two other groups found no to little effect of even micromolar γ-TuNA concentrations on Xenopus γ-TuRC when purified using a γ-tubulin antibody 19, 51 .…”

Integrated Model of the Vertebrate Augmin Complex

“…Having recapitulated this purification strategy (Figure 1a), we next assessed augmin’s activity. We reconstituted branching MT nucleation in vitro by adding X. laevis augmin lacking the C-terminus of Haus6 and γ-TuRC purified using its activator, the γ-TuNA domain of CDK5RAP2 18 to a GMPCPP-stabilized MT seed attached to glass. Upon addition of tubulin and GTP, new MTs nucleated from the side of the mother MT, implying that purified X. laevis augmin recruited γ-TuRC to the MT seeds and thus stimulated branching MT nucleation.. Purified X. laevis augmin is able to stimulate branching MT nucleation, thus, by implication, recruiting γ-TuRC to the MT seeds.…”

“…X. laevis γ-TuRC was isolated from X. laevis egg extract using a purification strategy available in preprint 18 . Here we utilized magnetic beads (cat.…”