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Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes.

Platani M, Trinkle-Mulcahy L, Porter M, Jeyaprakash AA, Earnshaw WC - J. Cell Biol. (2015)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK m.platani@ed.ac.uk Bill.Earnshaw@ed.ac.uk.

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Plk1 and Aurora A are misregulated after Mio depletion. (A) Control and Mio-depleted cells were fixed and immunostained with α–Aurora A (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (B) Control and Mio-depleted cells were fixed and immunostained with α-p–Aurora AT288 (green), α-tubulin (red), and DNA (blue). (C) Control and Mio-depleted cells were fixed and immunostained with α-Plk1 (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control and Mio-depleted cells. Depletion of Mio reduces p–Aurora AT288 levels, whereas Aurora A is mostly retained. (E) Quantification graph of PLK1 and p-PLK1T210 levels at centrosomes in control and Mio-depleted cells. Both Plk1 and p-Plk1T120 levels at centrosomes are reduced. (D and E) Fluorescence intensities are in arbitrary units (AU). Error bars represent SD. (F and G) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio from asynchronous and Monastrol-arrested cells (probed using α-PLK1, α–p-PLK1T210, α–Aurora A, and α-p–Aurora AT288) show reduction of Aurora AT288ph signal upon Mio depletion, whereas total Aurora A, Plk1, and Plk1T210ph levels appear unchanged. Tubulin serves as a loading control. n.s., not significant. Bars, 10 µm.
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fig6: Plk1 and Aurora A are misregulated after Mio depletion. (A) Control and Mio-depleted cells were fixed and immunostained with α–Aurora A (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (B) Control and Mio-depleted cells were fixed and immunostained with α-p–Aurora AT288 (green), α-tubulin (red), and DNA (blue). (C) Control and Mio-depleted cells were fixed and immunostained with α-Plk1 (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control and Mio-depleted cells. Depletion of Mio reduces p–Aurora AT288 levels, whereas Aurora A is mostly retained. (E) Quantification graph of PLK1 and p-PLK1T210 levels at centrosomes in control and Mio-depleted cells. Both Plk1 and p-Plk1T120 levels at centrosomes are reduced. (D and E) Fluorescence intensities are in arbitrary units (AU). Error bars represent SD. (F and G) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio from asynchronous and Monastrol-arrested cells (probed using α-PLK1, α–p-PLK1T210, α–Aurora A, and α-p–Aurora AT288) show reduction of Aurora AT288ph signal upon Mio depletion, whereas total Aurora A, Plk1, and Plk1T210ph levels appear unchanged. Tubulin serves as a loading control. n.s., not significant. Bars, 10 µm.

Mentions: Depletion of Mio had significant effects on the activation of Aurora A and Plk1 at centrosomes. For example, although we observed no changes in total Aurora A localization or the amount on centrosomes and spindles (Fig. 6 A), the level of active Aurora A (Aurora AT288ph, which is phosphorylated at Thr288 on the activation loop) on centrosomes was significantly lower in Mio-depleted cells than in RNAi control cells (Fig. 6, B and D). We confirmed this by immunoblotting (Fig. 6 G). Rescue experiments on HeLa si02ResGFP-Mio cells, where the endogenous Mio was depleted using si02, rescued the prometaphase delay phenotype and restored Aurora AT288ph levels at spindle poles (Fig. S1, C–E). Depletion of all Mio using si01 gave the expected prometaphase delay and reduced Aurora AT288ph signal.


Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes.

Platani M, Trinkle-Mulcahy L, Porter M, Jeyaprakash AA, Earnshaw WC - J. Cell Biol. (2015)

Plk1 and Aurora A are misregulated after Mio depletion. (A) Control and Mio-depleted cells were fixed and immunostained with α–Aurora A (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (B) Control and Mio-depleted cells were fixed and immunostained with α-p–Aurora AT288 (green), α-tubulin (red), and DNA (blue). (C) Control and Mio-depleted cells were fixed and immunostained with α-Plk1 (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control and Mio-depleted cells. Depletion of Mio reduces p–Aurora AT288 levels, whereas Aurora A is mostly retained. (E) Quantification graph of PLK1 and p-PLK1T210 levels at centrosomes in control and Mio-depleted cells. Both Plk1 and p-Plk1T120 levels at centrosomes are reduced. (D and E) Fluorescence intensities are in arbitrary units (AU). Error bars represent SD. (F and G) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio from asynchronous and Monastrol-arrested cells (probed using α-PLK1, α–p-PLK1T210, α–Aurora A, and α-p–Aurora AT288) show reduction of Aurora AT288ph signal upon Mio depletion, whereas total Aurora A, Plk1, and Plk1T210ph levels appear unchanged. Tubulin serves as a loading control. n.s., not significant. Bars, 10 µm.
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fig6: Plk1 and Aurora A are misregulated after Mio depletion. (A) Control and Mio-depleted cells were fixed and immunostained with α–Aurora A (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (B) Control and Mio-depleted cells were fixed and immunostained with α-p–Aurora AT288 (green), α-tubulin (red), and DNA (blue). (C) Control and Mio-depleted cells were fixed and immunostained with α-Plk1 (red), α-tubulin (green), and DNA (blue). Arrows point to centrosomes. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control and Mio-depleted cells. Depletion of Mio reduces p–Aurora AT288 levels, whereas Aurora A is mostly retained. (E) Quantification graph of PLK1 and p-PLK1T210 levels at centrosomes in control and Mio-depleted cells. Both Plk1 and p-Plk1T120 levels at centrosomes are reduced. (D and E) Fluorescence intensities are in arbitrary units (AU). Error bars represent SD. (F and G) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio from asynchronous and Monastrol-arrested cells (probed using α-PLK1, α–p-PLK1T210, α–Aurora A, and α-p–Aurora AT288) show reduction of Aurora AT288ph signal upon Mio depletion, whereas total Aurora A, Plk1, and Plk1T210ph levels appear unchanged. Tubulin serves as a loading control. n.s., not significant. Bars, 10 µm.
Mentions: Depletion of Mio had significant effects on the activation of Aurora A and Plk1 at centrosomes. For example, although we observed no changes in total Aurora A localization or the amount on centrosomes and spindles (Fig. 6 A), the level of active Aurora A (Aurora AT288ph, which is phosphorylated at Thr288 on the activation loop) on centrosomes was significantly lower in Mio-depleted cells than in RNAi control cells (Fig. 6, B and D). We confirmed this by immunoblotting (Fig. 6 G). Rescue experiments on HeLa si02ResGFP-Mio cells, where the endogenous Mio was depleted using si02, rescued the prometaphase delay phenotype and restored Aurora AT288ph levels at spindle poles (Fig. S1, C–E). Depletion of all Mio using si01 gave the expected prometaphase delay and reduced Aurora AT288ph signal.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK m.platani@ed.ac.uk Bill.Earnshaw@ed.ac.uk.

Show MeSH
Related in: MedlinePlus