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

Abstract

1.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 {gamma}-tubulin ring complex ({gamma}TuRC). Although {gamma}TuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved {gamma}-tubulin nucleation activator ({gamma}TuNA) domain has been reported to increase the number of microtubules generated by {gamma}TuRCs. Here we utilize Xenopus egg extract and in vitro single molecule imaging assays to show that {gamma}TuNA activates microtubule nucleation in extract and directly activates {gamma}TuRC in vitro. Via mutation analysis, we find that {gamma}TuNA is an obligate dimer. Moreover, efficient dimerization as well as {gamma}TuNAs L70, F75, and L77 residues are required for binding to and activation of {gamma}TuRC. Finally, we find that {gamma}TuNAs activating effect opposes inhibitory regulation by stathmin. In sum, our study illuminates how {gamma}TuRC is controlled in space and time in order to build specific cytoskeletal structures.