Regulatory studies of microtubule nucleator [gamma]-tubulin ring complexes

Abstract

The γ-tubulin ring complex (γ-TuRC) plays a principal role in microtubule nucleation and anchoring microtubules to microtubule-organizing centers. Proper regulation of the activity and localization of γ-TuRCs is crucial for the spatial and temporal organization of microtubules. I developed a γ-TuRC purification protocol using the tandem affinity purification method. Using the γ-TuRC-binding region of the γ-TuRC-associated protein CDK5RAP2 and NEDD1, I purified γ-TuRCs and identified a number of γ-TuRC-associated protein candidates by mass spectrometry and analyzed the proteins by sorting them according to their identified abundance. Specifically, I found the co-existence of CDK5RAP2 and NEDD1 in γ-TuRCs and their cooperative effect on microtubule nucleation. New post-translational modifications (PTM) on γ-TuRC proteins were also identified through mass spectrometry. Unexpectedly, 3 cysteine sites of γ-tubulin, C26, C138 and C201 were found phosphorylated. I investigated the function of these phosphorylation events and found that the phosphor-mimetic mutant C26D cannot be integrated into γ-TuRC whereas the non-phosphorylatable mutant C26S and wide type γ-tubulin can. Further experiments showed that γ-tubulin is important for the interaction between GCPs. I also found that microtubule nucleation is inhibited when γ-TuRC contains C138D mutant. Taken together, cysteine phosphorylation on γ-tubulin participates in the microtubule organization through controlling the assembly and microtubule nucleation ability of γ-TuRC. In summary, I have purified γ-TuRC and identified several potential regulatory proteins and PTM of γ-TuRC through mass spectrometry. In addition, I found the cooperative effect of CDK5RAP2 and NEDD1 and characterized the identified cysteine phosphorylation on γ-tubulin and found their inhibitory functions for γ-TuRC activity. These findings provide novel candidates working in microtubule organization, unveil the existence and importance of the newly identified modification and demonstrate new regulation mechanism for γ-TuRC assembly and function.

Date of Award	2020
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology