Abstract
During mitosis, spindle assembly relies on centrosomal and acentrosomal microtubule nucleation pathways that all require the γ-Tubulin Ring Complex (γ-TuRC) and its adaptor protein NEDD1. The activity of these different pathways needs to be coordinated to ensure bipolar spindle assembly ( Cavazza et al., 2016) but the underlying mechanism is still unclear. Previous studies have identified three sites in NEDD1 (S377, S405 and S411) that when phosphorylated drive MT nucleation at the centrosomes, around the chromosomes and on pre-existing MTs respectively ( Lüders et al., 2006; Pinyol et al., 2013; Sdelci et al., 2012). Here we aimed at getting additional insights into the mechanism that coordinates the different MT nucleation pathways in dividing cells using a collection of HeLa stable inducible cell lines expressing NEDD1 phospho-variants at these three sites and Xenopus egg extracts. Our results provide further support for the essential role of phosphorylation at the three residues. Moreover, we directly demonstrate that S411 phosphorylation is essential for MT branching using TIRF microscopy in Xenopus egg extracts and we show that it plays a crucial role in ensuring the balance between centrosome and chromosome-dependent MT nucleation required for bipolar spindle assembly in mitotic cells.