An essential function for the centrosomal protein NEDD1 in zebrafish development

Abstract

The centrosome is the primary microtubule organising centre of the cell. It is composed of many proteins, some of which make up the core of the centrosome, whereas others are used for specific functions. Although the cellular roles of many centrosomal proteins are well defined, much less is known about their functions and the role of the centrosome in development. In this study we investigated the function of NEDD1, a critical component of the centrosome essential for microtubule nucleation, in zebrafish (Danio rerio) development. The zebrafish homologue of NEDD1 (zNEDD1) was cloned and found to have a similar localisation and function to mammalian NEDD1. We show that zNEDD1 is essential for survival, as a high level of knockdown was embryonic lethal. Partial knockdown of zNEDD1 caused abnormalities including an increase in mitotic and apoptotic cells. Pronounced phenotypic defects were seen in the brain, with a lack of defined brain structures, incomplete neural tube formation and a disorganisation of neurons. In addition, we show that a reduction in zNEDD1 resulted in the loss of c-tubulin at the centrosome. Our data thus demonstrate that zNEDD1 is critical for the recruitment of c-tubulin to the centrosome, and is essential for the proper development of zebrafish. The centrosome is the major microtubule organising centre (MTOC) of cells and serves as a centralised location for controlling many cellular processes. The centrosomal protein neural precursor cell expressed developmentally downregulated gene 1 (NEDD1), 1 functions in the centrosomal and mitotic-spindle recruitment of the g-tubulin ring complex (g-TuRC). This complex is required for the nucleation of microtubules, correct formation of the mitotic spindle and hence progression of the cell cycle. 2,3 Because of this, depletion of NEDD1 from mammalian cells results in dispersion of the MTOC, impaired centrosomal and chromatin nucleation of microtubules, mitotic chaos and cell-cycle arrest. [2][3][4] Given the centrosomal localisation and function of NEDD1, and its dynamic expression during mouse embryogenesis, 5 it is expected that this protein has an important role during development. However, because of the early embryonic lethality of mouse knockouts of centrosomal proteins, including g-tubulin, it has not been possible to study the function of these proteins in later stages of development in this organism. 6,7 Zebrafish (Danio rerio) posses many benefits for developmental studies including the conservation of genes involved in cell growth and proliferation, 8 the rapid maturation of translucent embryos 9 and the ability to specifically deplete proteins of interest. 10 Although there has been much work analysing the cell cycle in zebrafish, 11 there has been limited analysis of the centrosome in this species. Therefore, this system was chosen as a model to study the role of NEDD1 during development. In addition, NEDD1 homologues have been described in Drosophila, Xenopus and Arabidopsis. [12][13][14] Despite the consistent centrosomal localisation o...

Prickle3 synergizes with Wtip to regulate basal body organization and cilia growth

“…4A,B ). We next assessed the localization of Nedd1 that is essential for γ-tubulin recruitment to the centrosome 54 55 56 . Endogenous Nedd1 colocalized with γ-tubulin at the basal body in GRP cells ( Supplementary Fig.…”

Divergent regulation of functionally distinct γ-tubulin complexes during differentiation

“…These proteins have been suggested to play roles in γ-tubulin recruitment to the centrosome, but these effects may be species and/or cell type dependent. For example, Nedd1 was originally shown to be necessary for γ-tubulin localization to centrosomes in human cancer cell lines but was not required for centrosomal γ-tubulin recruitment in Xenopus laevis or Drosophila melanogaster ( Liu and Wiese, 2008 ; Zeng et al, 2009 ; Manning et al, 2010a ; Reschen et al, 2012 ).…”

Dgp71WD is required for the assembly of the acentrosomal Meiosis I spindle, and is not a general targeting factor for the γ-TuRC

“…In humans, Nedd1 is essential for recruiting γ-tubulin to centrosomes (Haren et al, 2006; Lüders et al, 2006), and this is also the case in Zebrafish (Manning et al, 2010). Previous studies in Drosophila have indicated that γ-tubulin recruitment to centrosomes is partially disrupted when Dgp71WD is depleted from S2 cells or in brain cells from the original Dgp71WD GE30807 mutant (Dobbelaere et al, 2008; Verollet et al, 2006).…”

“…Dgp71WD and its human homologue Nedd1 are structurally unrelated to the γ-TuSC/γ-TuRC proteins, as they lack the conserved ‘Grip’ motifs found in these proteins (Gunawardane et al, 2003). In human cells and in Zebrafish, Nedd1/GCP-WD is essential for targeting the γ-TuRC to the centrosome (Haren et al, 2006; Lüders et al, 2006; Manning et al, 2010), and in human cells it also appears to have a role in centriole duplication (Haren et al, 2006). Nedd1 is also required to target the γ-TuRC to the spindle in human cells (Lüders et al, 2006), where it is thought to link the γ-TuRC to the Augmin complex, thus promoting MT nucleation within the spindle (Johmura et al, 2011; Uehara et al, 2009; Zhu et al, 2008b).…”