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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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Seh1 interacts with the SEACAT complex both in interphase and mitosis. (A) Seh1 complexes were purified from a cell line stably expressing GFP-Seh1, HeLaEGP-SEH1. Cells were differentially labeled with isotopic amino acids by growth in SILAC media before immunopurification of GFP-Seh1 complexes. Where indicated, cells were arrested in mitosis using 200 ng/ml nocodazole for 14 h. Immunopurified GFP-Seh1 complexes were analyzed by quantitative mass spectrometry. Plot of log ratio H/M versus relative abundance (summarized peptide intensities normalized by molecular weight) for all quantified proteins is shown. Bait protein GFP-Seh1 (green circles) and the interactors Nup107 complex (purple diamonds) and SEACAT/GATOR2 complex (blue diamonds) are highlighted. This nonbiased quantitative experiment was performed twice in asynchronous cell populations and once in mitotically arrested cells, and top hits were chosen for targeted follow-up validation by immunoprecipitation/Western blot analysis using specific antibodies. (B) Mio interacts with Seh1 in mammalian cells. Immunoprecipitates (IP) are shown of total protein extracts from HeLa cells transfected with mCherry, mCherry-Mio, and EGFP-Seh1. Immunoprecipitation was performed using α-RFP binder, and Seh1 was detected using α-Seh1 antibody. (C) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Seh1 (probed using α-Mio and α-Seh1) show that depletion of Seh1 can affect Mio stability. (D) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio (probed using α-Mio) show efficient depletion of Mio protein with different siRNA oligos 48 h after transfection. (C and D) Tubulin served as a loading control. (E) Abnormal nuclear morphology detected after Mio depletion. HeLa cells transfected with control and Mio siRNA oligos for 48 h were stained with DAPI to reveal the DNA and nuclear morphology. (F) Mio depletion increases mitotic length, binucleation, and apoptosis. HeLa cells transfected with control or Mio siRNAs were incubated for 30 h before phase-contrast imaging for a further 19 h. Images were acquired every 15 min. Image sequences were then analyzed tracking individual control (n = 132) and Mio-depleted cells (n = 289) to determine their behavior. Fate profiles of live control or Mio-depleted cells are shown as a function of time. Bars represent total time spent at different cell cycle stages for individual cells. Starting point T = 0 is NEBD. Bar, 10 µm.
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fig1: Seh1 interacts with the SEACAT complex both in interphase and mitosis. (A) Seh1 complexes were purified from a cell line stably expressing GFP-Seh1, HeLaEGP-SEH1. Cells were differentially labeled with isotopic amino acids by growth in SILAC media before immunopurification of GFP-Seh1 complexes. Where indicated, cells were arrested in mitosis using 200 ng/ml nocodazole for 14 h. Immunopurified GFP-Seh1 complexes were analyzed by quantitative mass spectrometry. Plot of log ratio H/M versus relative abundance (summarized peptide intensities normalized by molecular weight) for all quantified proteins is shown. Bait protein GFP-Seh1 (green circles) and the interactors Nup107 complex (purple diamonds) and SEACAT/GATOR2 complex (blue diamonds) are highlighted. This nonbiased quantitative experiment was performed twice in asynchronous cell populations and once in mitotically arrested cells, and top hits were chosen for targeted follow-up validation by immunoprecipitation/Western blot analysis using specific antibodies. (B) Mio interacts with Seh1 in mammalian cells. Immunoprecipitates (IP) are shown of total protein extracts from HeLa cells transfected with mCherry, mCherry-Mio, and EGFP-Seh1. Immunoprecipitation was performed using α-RFP binder, and Seh1 was detected using α-Seh1 antibody. (C) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Seh1 (probed using α-Mio and α-Seh1) show that depletion of Seh1 can affect Mio stability. (D) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio (probed using α-Mio) show efficient depletion of Mio protein with different siRNA oligos 48 h after transfection. (C and D) Tubulin served as a loading control. (E) Abnormal nuclear morphology detected after Mio depletion. HeLa cells transfected with control and Mio siRNA oligos for 48 h were stained with DAPI to reveal the DNA and nuclear morphology. (F) Mio depletion increases mitotic length, binucleation, and apoptosis. HeLa cells transfected with control or Mio siRNAs were incubated for 30 h before phase-contrast imaging for a further 19 h. Images were acquired every 15 min. Image sequences were then analyzed tracking individual control (n = 132) and Mio-depleted cells (n = 289) to determine their behavior. Fate profiles of live control or Mio-depleted cells are shown as a function of time. Bars represent total time spent at different cell cycle stages for individual cells. Starting point T = 0 is NEBD. Bar, 10 µm.

Mentions: Seh1 is a member of the Nup107 complex that functions in NPC assembly and localizes to kinetochores in mitosis (Belgareh et al., 2001). To better define its biochemical interactions in mammalian cells, we isolated GFP-Seh1 both from asynchronous and mitotically arrested HeLaGFPSeh1 cells (Platani et al., 2009) and identified interacting proteins by SILAC (stable isotope labeling by amino acids in cell culture) mass spectrometry (Ong and Mann, 2007). This analysis identified all known Seh1-interacting proteins, including components of the Nup107 complex (Nup107, 98, 133, 160, and 85) and SEACAT/GATOR2 complexes (Mios, WDR24, and WDR59; Fig. 1 A and Fig. S1 A; Dokudovskaya et al., 2011; Bar-Peled et al., 2013; Panchaud et al., 2013a). Interestingly, Seh1 interacts with the SEACAT/GATOR2 complex equally in interphase and mitosis, whereas its interaction with the rest of the Nup107 complex is stronger during mitosis.


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)

Seh1 interacts with the SEACAT complex both in interphase and mitosis. (A) Seh1 complexes were purified from a cell line stably expressing GFP-Seh1, HeLaEGP-SEH1. Cells were differentially labeled with isotopic amino acids by growth in SILAC media before immunopurification of GFP-Seh1 complexes. Where indicated, cells were arrested in mitosis using 200 ng/ml nocodazole for 14 h. Immunopurified GFP-Seh1 complexes were analyzed by quantitative mass spectrometry. Plot of log ratio H/M versus relative abundance (summarized peptide intensities normalized by molecular weight) for all quantified proteins is shown. Bait protein GFP-Seh1 (green circles) and the interactors Nup107 complex (purple diamonds) and SEACAT/GATOR2 complex (blue diamonds) are highlighted. This nonbiased quantitative experiment was performed twice in asynchronous cell populations and once in mitotically arrested cells, and top hits were chosen for targeted follow-up validation by immunoprecipitation/Western blot analysis using specific antibodies. (B) Mio interacts with Seh1 in mammalian cells. Immunoprecipitates (IP) are shown of total protein extracts from HeLa cells transfected with mCherry, mCherry-Mio, and EGFP-Seh1. Immunoprecipitation was performed using α-RFP binder, and Seh1 was detected using α-Seh1 antibody. (C) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Seh1 (probed using α-Mio and α-Seh1) show that depletion of Seh1 can affect Mio stability. (D) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio (probed using α-Mio) show efficient depletion of Mio protein with different siRNA oligos 48 h after transfection. (C and D) Tubulin served as a loading control. (E) Abnormal nuclear morphology detected after Mio depletion. HeLa cells transfected with control and Mio siRNA oligos for 48 h were stained with DAPI to reveal the DNA and nuclear morphology. (F) Mio depletion increases mitotic length, binucleation, and apoptosis. HeLa cells transfected with control or Mio siRNAs were incubated for 30 h before phase-contrast imaging for a further 19 h. Images were acquired every 15 min. Image sequences were then analyzed tracking individual control (n = 132) and Mio-depleted cells (n = 289) to determine their behavior. Fate profiles of live control or Mio-depleted cells are shown as a function of time. Bars represent total time spent at different cell cycle stages for individual cells. Starting point T = 0 is NEBD. Bar, 10 µm.
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Related In: Results  -  Collection

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fig1: Seh1 interacts with the SEACAT complex both in interphase and mitosis. (A) Seh1 complexes were purified from a cell line stably expressing GFP-Seh1, HeLaEGP-SEH1. Cells were differentially labeled with isotopic amino acids by growth in SILAC media before immunopurification of GFP-Seh1 complexes. Where indicated, cells were arrested in mitosis using 200 ng/ml nocodazole for 14 h. Immunopurified GFP-Seh1 complexes were analyzed by quantitative mass spectrometry. Plot of log ratio H/M versus relative abundance (summarized peptide intensities normalized by molecular weight) for all quantified proteins is shown. Bait protein GFP-Seh1 (green circles) and the interactors Nup107 complex (purple diamonds) and SEACAT/GATOR2 complex (blue diamonds) are highlighted. This nonbiased quantitative experiment was performed twice in asynchronous cell populations and once in mitotically arrested cells, and top hits were chosen for targeted follow-up validation by immunoprecipitation/Western blot analysis using specific antibodies. (B) Mio interacts with Seh1 in mammalian cells. Immunoprecipitates (IP) are shown of total protein extracts from HeLa cells transfected with mCherry, mCherry-Mio, and EGFP-Seh1. Immunoprecipitation was performed using α-RFP binder, and Seh1 was detected using α-Seh1 antibody. (C) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Seh1 (probed using α-Mio and α-Seh1) show that depletion of Seh1 can affect Mio stability. (D) Immunoblots of HeLa cell lysates treated with siRNAs corresponding to negative control and Mio (probed using α-Mio) show efficient depletion of Mio protein with different siRNA oligos 48 h after transfection. (C and D) Tubulin served as a loading control. (E) Abnormal nuclear morphology detected after Mio depletion. HeLa cells transfected with control and Mio siRNA oligos for 48 h were stained with DAPI to reveal the DNA and nuclear morphology. (F) Mio depletion increases mitotic length, binucleation, and apoptosis. HeLa cells transfected with control or Mio siRNAs were incubated for 30 h before phase-contrast imaging for a further 19 h. Images were acquired every 15 min. Image sequences were then analyzed tracking individual control (n = 132) and Mio-depleted cells (n = 289) to determine their behavior. Fate profiles of live control or Mio-depleted cells are shown as a function of time. Bars represent total time spent at different cell cycle stages for individual cells. Starting point T = 0 is NEBD. Bar, 10 µm.
Mentions: Seh1 is a member of the Nup107 complex that functions in NPC assembly and localizes to kinetochores in mitosis (Belgareh et al., 2001). To better define its biochemical interactions in mammalian cells, we isolated GFP-Seh1 both from asynchronous and mitotically arrested HeLaGFPSeh1 cells (Platani et al., 2009) and identified interacting proteins by SILAC (stable isotope labeling by amino acids in cell culture) mass spectrometry (Ong and Mann, 2007). This analysis identified all known Seh1-interacting proteins, including components of the Nup107 complex (Nup107, 98, 133, 160, and 85) and SEACAT/GATOR2 complexes (Mios, WDR24, and WDR59; Fig. 1 A and Fig. S1 A; Dokudovskaya et al., 2011; Bar-Peled et al., 2013; Panchaud et al., 2013a). Interestingly, Seh1 interacts with the SEACAT/GATOR2 complex equally in interphase and mitosis, whereas its interaction with the rest of the Nup107 complex is stronger during mitosis.

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