Spindle assembly and the control of microtubule nucleation through nedd1 phosphorylation

Resumen

During mitosis, the microtubules organize a bipolar spindle that segregates the chromosomes. In higher eukaryotes, these microtubules are nucleated through three different pathways involving the centrosomes (centrosomal pathway), the chromatin (Ran-GTP pathway), and pre-existing microtubules (Augmin-dependent pathway). These three pathways rely on the γ-tubulin ring complex (γ-TuRC) and its adaptor NEDD1. During mitosis, NEDD1 phosphorylation determines its role in microtubule nucleation through the three pathways: Nek9-dependent Ser377 for centrosomal nucleation, AuroraA-dependent Ser405 for the Ran-GTP dependent pathway and Cdk1-dependent Ser411 for the Augmin-dependent microtubule amplification. To define the specific contribution of these microtubule nucleation pathways in spindle assembly, we established several inducible stable cell lines to express phosphorylation variants of NEDD1 on Ser377, Ser405 and Ser411 individually or in combination upon NEDD1 silencing. Our data show that, in agreement with previous results, the three sites are important for spindle assembly. Moreover, they suggest that phosphorylation at Ser411 is responsible for NEDD1 mobility shift in mitosis and plays a major role in NEDD1 function in mitosis.