The microtubule-associated protein EML3 regulates mitotic spindle assembly by recruiting the Augmin complex to spindle microtubules

Abstract

Edited by Xiao-Fan Wang In all eukaryotes, a functional mitotic spindle is essential for distributing duplicated chromosomes into daughter cells. Mitotic spindle assembly involves highly ordered arrangement of microtubules (MTs). The Augmin protein complex recruits ␥-tubulin ring complex (␥-TuRC) to MTs and thereby promotes MT-based MT nucleation and mitotic spindle assembly. However, several factors that may promote Augmin recruitment to MTs remain unknown. Here, we show that echinoderm microtubule-associated protein-like 3 (EML3), an MT-associated protein, facilitates binding between MTs and Augmin/␥-TuRC and recruiting the latter to MTs for proper mitotic spindle assembly and kinetochore-MT connections. Using immunofluorescence microscopy, live-cell imaging, and immunoprecipitation assays, we found that EML3 recruits Augmin/␥-TuRC to the MTs to enhance MT-based MT nucleation in both spindle and small acentrosomal asters. We also noted that the EML3-mediated recruitment is controlled by cyclin-dependent kinase 1 (CDK1), which phosphorylated EML3 at Thr-881 and promoted its binding to Augmin/␥-TuRC. RNAi-mediated EML3 knockdown in HeLa cells reduced spindle localization of Augmin/␥-TuRC, which resulted in abnormal spindle assembly and caused kinetochore-MT misconnection. The introduction of exogenous WT or a Thr-881 phosphorylation mimic EML3 variant into the EML3 knockdown cells restored normal Augmin/␥-TuRC localization and spindle assembly. The EML3 knockdown also affected the spindle assembly checkpoint, delaying chromosome congression and cell division. Taken together, our results indicate that EML3 regulates mitotic spindle assembly and the kinetochore-MT connection by regulating MT-based MT nucleation and recruiting Augmin/␥-TuRC to MTs.

Spatiotemporal organization of branched microtubule networks

“…Based on our results (Fig. 4A-B), we speculate that localization of augmin to MTs in the cytoplasm is mediated by other proteins, as suggested recently for human augmin (Luo et al, 2019), and potentially by TPX2. Third, we show that the nucleator γ-TuRC cannot be recruited by TPX2 independently, but requires augmin (Fig.…”

Spatiotemporal organization of branched microtubule networks

“…Second, we show that branching MT nucleation is not a result of independent binding of TPX2 and augmin to the MT lattice, an activity that has been shown for both proteins in vitro (Brunet et al, 2004; Hsia et al, 2014; Roostalu et al, 2015; Song et al, 2018). Based on our results (Figure 4A–B), we speculate that localization of augmin to MTs in the cytoplasm is mediated by other proteins, as suggested recently for human augmin (Luo et al, 2019), and potentially by TPX2. Third, we find that the nucleator γ-TuRC cannot be recruited by TPX2 independently, but requires augmin (Figure 4C), which could not be ruled out based on previous work.…”

Biochemical reconstitution of branching microtubule nucleation

“…173 2019). This implies that TPX2 directly regulates augmin's binding to microtubules, but does not 174 rule out additional regulation that could encompass other factors such as EML3(Luo et al, 2019). 175Although microtubule nucleation effectors alone can generate microtubules in vitro 176(Roostalu et al, 2015; Woodruff et al, 2017), g-TuRC is required for physiological microtubule 177 nucleation(Kollman et al, 2011;Thawani et al, 2018), and this reconstitution highlights the 178 importance of including it when studying microtubule nucleation.…”