Abstract
Proper orientation of the mitotic spindle defines the correct division plane and is essential for accurate cell division and development. In metazoans, an evolutionarily conserved complex comprising of NuMA/LGN/Gαi regulates proper orientation of the mitotic spindle by orchestrating cortical dynein levels during metaphase. However, the molecular mechanisms that modulate the spatiotemporal dynamics of this complex during mitosis remain elusive. Here, we report that acute inactivation of Polo-like kinase 1 (Plk1) during metaphase enriches cortical levels of dynein/NuMA/LGN and thus influences spindle orientation. We establish that this impact of Plk1 on cortical levels of dynein/NuMA/LGN is through NuMA, but not via dynein/LGN. Moreover, we reveal that Plk1 inhibition alters the dynamic behavior of NuMA at the cell cortex. We further show that Plk1 directly interacts and phosphorylates NuMA. Notably, NuMA-phosphorylation by Plk1 impacts its cortical localization, and this is needed for precise spindle orientation during metaphase. Overall, our finding connects spindle-pole pool of Plk1 with cortical NuMA and answers a long-standing puzzle about how spindle-pole Plk1 gradient dictates proper spindle orientation for error-free mitosis.
Highlights
The precise orientation of the mitotic spindle determines the correct placement of the cleavage furrow and maintains the relative sizes and spatial organization of the daughter cells
To study the impact of acute Pololike kinase 1 (Plk1) inactivation on the cortical levels of NuMA/ LGN/Gαi1-3 and dynein during metaphase, we adopted a multi-drug strategy that has earlier been used to study the impact of Plk1 on cytokinesis (Petronczki et al, 2007) (Fig S1A)
2536–treated cells (Fig S1B and C), suggesting that Plk1 inhibition in our setting is not provoking spindle checkpoint activation during mitosis. 12 h of BI 2536 treatment post–MG132 release resulted in substantial enrichment in the binucleated cells because of cytokinesis failure, indicating the potency of the Plk1 inhibitor in our experimental setup (Fig S1D–F) (Petronczki et al, 2007)
Summary
The precise orientation of the mitotic spindle determines the correct placement of the cleavage furrow and maintains the relative sizes and spatial organization of the daughter cells. Cortical NuMA enrichment upon Plk1 inhibition appears to stem from its decrease in the mobility at the cell cortex, and this leads to spindle orientation defects in metaphase.
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