Analysis of the function and regulation of the centrosomal protein NEDD1 during cell division and development.

Abstract

The centrosome is the major microtubule organising centre of cells and serves as a centralised location for controlling many cellular processes. A critical component of the centrosome is the γ-tubulin ring complex (γ-TuRC) which is required for the nucleation of microtubules, correct formation of the mitotic spindle and hence progression of the cell cycle. NEDD1 (mouse: Nedd1), was recently discovered as a centrosomal protein which functions primarily in targeting the γ-TuRC to the centrosome and spindle. Given the fundamental role of the centrosome in mitosis and other processes, it is no surprise that this organelle is essential during mouse development. To examine the precise role of the centrosome during development, this study analysed the expression and localisation of Nedd1 during mouse embryogenesis. This revealed a dynamic localisation of Nedd1 and the centrosome during development, and provides further evidence for their critical role in development. To investigate the regulation of NEDD1, its expression during the cell cycle was analysed. It was found that phosphorylation is the primary method of NEDD1 regulation. Additionally, it was observed that Nedd1 levels decreased upon the entry of mouse embryonic fibroblasts into cell culture-induced senescence (an irreversible state of cell cycle arrest). This correlated with a loss of centrosomal integrity. Ablation of Nedd1 in healthy cells caused premature senescence and centrosome abnormalities, suggesting that Nedd1 and the centrosome may contribute to this senescence. NEDD1 is also important in the recruitment of the γ-TuRC to the centrosome, which is essential for correct centrosome biogenesis and function. This study identified a 62 amino acid region of NEDD1 that interacts with γ-tubulin and can abrogate its function. Key residues important for this interaction were also revealed. Additional interacting proteins of NEDD1 were also identified, and the chaperone TCP-1α was characterised in more detail and shown to regulate NEDD1. Given the currently known functions of NEDD1, it was expected to be important in development. Zebrafish were chosen as a model to study this because of their many advantages for developmental studies. A zebrafish homologue of NEDD1 was identified that displayed a similar localisation and function to mammalian NEDD1. Depletion of this protein caused lethality or phenotypic abnormalities which were most obvious in the central nervous system, depending on the extent of knockdown. This demonstrates that NEDD1 is critical for development, particularly in the nervous system. The results presented in this thesis contribute to the understanding of the function and regulation of NEDD1, and thus also the centrosome, and highlights the importance of this protein during development. Additionally, this study forms the foundation for further work on centrosomes, using NEDD1 as a marker for centrosomal dynamics and function.