The Role of Ninein in the Regulation of Microtubule Organization at the Centrosome
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Many fundamental cellular processes, such as the transport of intracellular cargos and the segregation of mitotic chromosomes, depend on the spatiotemporal regulation of microtubule networks. Specific subcellular compartments, named Microtubule Organizing Centers (MTOCs), are involved in this crucial and delicate function by controlling microtubule nucleation and anchoring. The centrosome represents the major MTOC of eukaryotic cells, and it is composed of a pair of orthogonal centrioles surrounded by the highly dense and protein-rich pericentriolar material (PCM). The PCM proteins can interact directly and indirectly with nucleating complexes and with newly made microtubules, thus promoting nucleation and ensuring microtubule anchoring. In which way many of these proteins fulfill these functions is, however, currently poorly understood. Further studies are therefore needed in order to elucidate more precisely the dynamics of centrosomal microtubule organization. In this project, we employed CRISPR/Cas9 genome editing technology and Knock-Down via RNA interference to investigate the role of Ninein, a protein resident on the mother centriole, in the regulation of microtubule organization. Ninein is known to be essential for proper anchoring of microtubules, but the mechanism underlying its role are still not known. Whether Ninein can cover a role in microtubule nucleation remains as well to be determined. We show that Ninein localizes to the centrosome through its C-terminal domain and that its depletion severely impairs the normal microtubule density at the centrosome. Importantly, we demonstrate that Ninein and γ-tubulin do not interact, and that γ-tubulin levels at the centrosome are not affected by Ninein’s depletion. We found that the late stage of microtubule regrowth, but not the early one, is impaired in the absence of Ninein. Finally, we demonstrate that CLASP2 levels at the centrosome are dramatically decreased in Ninein Knockout cells, and that Ninein binds to the C-terminal domain of CLASP2 trough a coiled-coil domain located in the 601-1050 amino acidic region. In conclusion, by providing information about the contribution of Ninein to the processes of microtubule nucleation and anchoring, our work helped to gain new insights in the regulation of microtubule organization at the centrosome.