Regulatory studies of the microtubule nucleator γ-tubulin ring complex

Abstract

The spatial and temporal organization of the microtubule cytoskeleton requires γ-tubulin ring complexes (γTuRCs), which initiate microtubule growth and mediate microtubule attachment at microtubule-organizing centers, such as centrosomes and the Golgi complex. In a search for γTuRC-interacting proteins, I identified the catalytic subunit of DNA polymerase δ (PolD1). Polδ is a major DNA polymerase which is responsible for DNA replication. It is also involved in DNA repair and recombination. PolD1 binds directly to γTuRCs and potently inhibits γTuRC-mediated microtubule nucleation. Whereas PolD1 depletion through RNA interference augments microtubule nucleation at the Golgi, PolD1 overexpression interferes with Golgi-based nucleation. By contrast, the depletion of PolD1 does not influence centrosome-based microtubule growth. Suppression of PolD1 expression impairs Golgi reassembly after nocodazole-induced disassembly and causes defects of Golgi reorientation during cell polarization. These results reveal a mechanism for the control of non-centrosomal γTuRC activity and a mechanism that regulates Golgi-mediated microtubule organization and functions. A body of evidence has shown that CDK5RAP2 and Nedd1 play key roles in γTuRC attachment to centrosomes, microtubule nucleation, and mitotic spindle organization. I investigated the γTuRC association with CDK5RAP2 and Nedd1, and found that most of the CDK5RAP2- and Nedd1-associated γTuRCs contain either CDK5RAP2 or Nedd1 and that their binding with γTuRCs is controlled by unknown protein factors. I found that CDK5RAP2 and Nedd1-bound γTuRCs undergo dynamic changes of the complex composition during the cell cycle. Moreover, the interphase and mitotic γTuRCs showed different microtubule-nucleating activities in an in vitro assay. Therefore, γTuRCs are regulated in a cell cycle-dependent manner. My results also suggest that CDK5RAP2 and Nedd1 may be under distinct controls for interacting with γTuRCs. In summary, I identified PolD1 to be a γTuRC inhibitor and revealed a new control mechanism of γTuRC-mediated microtubule nucleation in the Golgi complex. In addition, I showed that γTuRCs undergo cell cycle-dependent changes of subunit stoichiometry and associated proteins. My findings expand our knowledge on γTuRC regulation studies and provide insights into the organization mechanism on microtubule networks during the cell cycle.

Date of Award	2017
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology