Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes.
Bottom Line: In this study, we report that Mio, a highly conserved member of the SEACAT/GATOR2 complex necessary for the activation of mTORC1 kinase, plays a critical role in mitotic spindle formation and subsequent chromosome segregation by regulating the proper concentration of active key mitotic kinases Plk1 and Aurora A at centrosomes and spindle poles.Mio-depleted cells showed reduced activation of Plk1 and Aurora A kinase at spindle poles and an impaired localization of MCAK and HURP, two key regulators of mitotic spindle formation and known substrates of Aurora A kinase, resulting in spindle assembly and cytokinesis defects.Our results indicate that a major function of Mio in mitosis is to regulate the activation/deactivation of Plk1 and Aurora A, possibly by linking them to mTOR signaling in a pathway to promote faithful mitotic progression.
Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK email@example.com Bill.Earnshaw@ed.ac.uk.Show MeSH
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Mentions: Having previously described a requirement for Seh1 for mitotic progression (Platani et al., 2009), we next explored the role of Mio in mitotic spindle assembly and chromosome segregation. RNAi depletion of Mio resulted in an increase in the number of binucleate cells, micronucleation, and aberrant nuclear morphology with a variety of different oligos against Mio (Fig. 1, D and E; and see the following paragraphs). To better define the Mio depletion phenotype in living cells, we used time-lapse phase-contrast microscopy. Cells transfected with control siRNA underwent mitosis with normal timing, and cytokinesis was completed within 2 h followed by a period in interphase (Fig. 1 F). Apoptotic events were rarely seen (1.6%), indicating that phototoxicity is minimal under our imaging conditions. Mio-depleted cells remained in mitosis and cytokinesis for extended periods of time before either furrow ingression resulted in binucleation, a period in interphase, or apoptosis (which was observed both during mitosis and interphase; Fig. 1 F). Mio is therefore required for normal mitotic progression. Interestingly, Mio depletion did not result in an increase in the fraction of cells in mitosis (Fig. 2 A). Therefore, the increased time that cells spend in mitosis must be counterbalanced by a decrease in mitotic entry.
Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK firstname.lastname@example.org Bill.Earnshaw@ed.ac.uk.