Abstract
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 γ-tubulin ring complex (γ-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/γ-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/γ-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/γ-TuRC. RNAi-mediated EML3 knockdown in HeLa cells reduced spindle localization of Augmin/γ-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/γ-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/γ-TuRC to MTs.
Highlights
In all eukaryotes, a functional mitotic spindle is essential for distributing duplicated chromosomes into daughter cells
The results showed that the ␥-tubulin signal on the spindle body MTs was significantly reduced in echinoderm microtubule-associated protein-like 3 (EML3) knockdown cells compared with the control (Fig. 1, A and B)
Through co-IP assays, we showed that EML3 and ␥-tubulin were co-immunoprecipitated with each other (Fig. 1, E and F)
Summary
The Augmin protein complex recruits ␥-tubulin ring complex (␥-TuRC) to MTs and thereby promotes MT-based MT nucleation and mitotic spindle assembly. 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. The distribution of duplicated chromosomes into daughter cells requires a functional mitotic spindle in all eukaryotes. In plants and animal oocytes that have no equivalent centrosomes, many small MT organization centers with unclear structural features are responsible for organizing MT arrays by regulating the assembly of small acentrosomal MT asters These small asters interact with each other, connect with the chromosomes, and gradually organize to form the bipolar spindle [17, 18]. Our data reveal a novel mechanism of how EML3 regulates mitotic spindle assembly and the kinetochore–MT connection via recruitment of Augmin and ␥-TuRC to MT for MTbased MT nucleation
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