Dissecting the activation of 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. However, the field to date has no real-time, direct observation of this activation effect.In this dissertation, I first present a new high-yield, high-stringency method for purifying endogenous γTuRC from Xenopus egg extract. This method builds on prior work utilizing the known interaction between the γTuNA domain and γTuRC, improving the final yield into the hundreds of nanomolar final concentration. Next, I 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. This is the first real-time, direct observation of this activation effect. Furthermore, I explore the nature of this direct interaction via mutation analysis and 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, I find that γTuNA’s activating effect opposes inhibitory regulation by stathmin. In sum, my work helps illuminates how γTuRC is controlled in space and time in order to build specific cytoskeletal structures.