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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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Activity of mTOR is reduced in mitotic cells after Mio depletion. (A) Quantification of p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PPP6C co-depleted cells. (B) Mitotic profile of control, Mio-depleted, and Mio/PPP6C co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (C and F) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control, Mio, Mio with PPP6C, PTEN, and Mio/PTEN from asynchronous cells (probed using α-Mio, α-PPP6C, and α-PTEN) show efficient depletion of Mio, PPP6C, and PTEN. Tubulin serves as a loading control. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PTEN co-depleted cells. PTEN co-depletion with Mio rescues the p–Aurora AT288 levels reduction. (E) Mitotic profile of control, Mio-depleted, and Mio and PTEN co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (G) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control DMSO- and rapamycin-treated cells. (A, D, and G) Fluorescence intensities are in arbitrary units (AU). (H) Control DMSO- and rapamycin-treated cells were fixed and immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue). (I) Mitotic profile of control DMSO- and rapamycin-treated cells after 4-h incubation ( n = 3). Error bars represent SD. n.s., not significant. Bar, 10 µm.
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fig10: Activity of mTOR is reduced in mitotic cells after Mio depletion. (A) Quantification of p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PPP6C co-depleted cells. (B) Mitotic profile of control, Mio-depleted, and Mio/PPP6C co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (C and F) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control, Mio, Mio with PPP6C, PTEN, and Mio/PTEN from asynchronous cells (probed using α-Mio, α-PPP6C, and α-PTEN) show efficient depletion of Mio, PPP6C, and PTEN. Tubulin serves as a loading control. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PTEN co-depleted cells. PTEN co-depletion with Mio rescues the p–Aurora AT288 levels reduction. (E) Mitotic profile of control, Mio-depleted, and Mio and PTEN co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (G) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control DMSO- and rapamycin-treated cells. (A, D, and G) Fluorescence intensities are in arbitrary units (AU). (H) Control DMSO- and rapamycin-treated cells were fixed and immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue). (I) Mitotic profile of control DMSO- and rapamycin-treated cells after 4-h incubation ( n = 3). Error bars represent SD. n.s., not significant. Bar, 10 µm.

Mentions: Protein phosphatase 6 regulates mitotic spindle formation by controlling the T loop phosphorylation of Aurora A bound to its activator TPX2 (Zeng et al., 2010). Thus, PPP6C depletion would enhance Aurora A activity and potentially restore the mitotic function of Mio-depleted cells. Indeed, co-depletion of Mio and PPP6C (catalytic subunit; see Fig. 10 C) rescued the prometaphase delay phenotype and restored Aurora AT288ph levels at spindle poles (see Fig. 10, A and B).


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)

Activity of mTOR is reduced in mitotic cells after Mio depletion. (A) Quantification of p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PPP6C co-depleted cells. (B) Mitotic profile of control, Mio-depleted, and Mio/PPP6C co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (C and F) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control, Mio, Mio with PPP6C, PTEN, and Mio/PTEN from asynchronous cells (probed using α-Mio, α-PPP6C, and α-PTEN) show efficient depletion of Mio, PPP6C, and PTEN. Tubulin serves as a loading control. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PTEN co-depleted cells. PTEN co-depletion with Mio rescues the p–Aurora AT288 levels reduction. (E) Mitotic profile of control, Mio-depleted, and Mio and PTEN co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (G) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control DMSO- and rapamycin-treated cells. (A, D, and G) Fluorescence intensities are in arbitrary units (AU). (H) Control DMSO- and rapamycin-treated cells were fixed and immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue). (I) Mitotic profile of control DMSO- and rapamycin-treated cells after 4-h incubation ( n = 3). Error bars represent SD. n.s., not significant. Bar, 10 µm.
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fig10: Activity of mTOR is reduced in mitotic cells after Mio depletion. (A) Quantification of p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PPP6C co-depleted cells. (B) Mitotic profile of control, Mio-depleted, and Mio/PPP6C co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (C and F) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control, Mio, Mio with PPP6C, PTEN, and Mio/PTEN from asynchronous cells (probed using α-Mio, α-PPP6C, and α-PTEN) show efficient depletion of Mio, PPP6C, and PTEN. Tubulin serves as a loading control. (D) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control, Mio-depleted, and Mio/PTEN co-depleted cells. PTEN co-depletion with Mio rescues the p–Aurora AT288 levels reduction. (E) Mitotic profile of control, Mio-depleted, and Mio and PTEN co-depleted cells 48 h after siRNA transfection. 300 cells per condition (n = 3). (G) Quantification of Aurora A and p–Aurora AT288 levels at spindle poles in control DMSO- and rapamycin-treated cells. (A, D, and G) Fluorescence intensities are in arbitrary units (AU). (H) Control DMSO- and rapamycin-treated cells were fixed and immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue). (I) Mitotic profile of control DMSO- and rapamycin-treated cells after 4-h incubation ( n = 3). Error bars represent SD. n.s., not significant. Bar, 10 µm.
Mentions: Protein phosphatase 6 regulates mitotic spindle formation by controlling the T loop phosphorylation of Aurora A bound to its activator TPX2 (Zeng et al., 2010). Thus, PPP6C depletion would enhance Aurora A activity and potentially restore the mitotic function of Mio-depleted cells. Indeed, co-depletion of Mio and PPP6C (catalytic subunit; see Fig. 10 C) rescued the prometaphase delay phenotype and restored Aurora AT288ph levels at spindle poles (see Fig. 10, A and B).

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