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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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Mio depletion leads to spindle misorientation and centrosomal defects. (A) Abnormal spindle morphology detected after Mio depletion. HeLa cells were transfected with control and Mio siRNA oligos for 48 h and stained with α-tubulin. (B) Quantification of metaphase spindle length between control (blue) and Mio-depleted cells (red) shows no significant change in the length of the mitotic spindle. (C) Schematic depicting the spindle angle (α) measurement relative to the fibronectin substratum. (D) Quantification of metaphase spindle angles between control (blue) and Mio-depleted cells (red) showing a significant increase of >20o of spindle angle. n = 40 from three experiments. (E) Quantification of mitotic metaphase cells with an unequal number of centrosomes and spindle poles. 3D maximum intensity projections of representative metaphase cells immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue) are shown on the right. 300 cells per condition (n = 3). Bars, 10 µm.
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fig3: Mio depletion leads to spindle misorientation and centrosomal defects. (A) Abnormal spindle morphology detected after Mio depletion. HeLa cells were transfected with control and Mio siRNA oligos for 48 h and stained with α-tubulin. (B) Quantification of metaphase spindle length between control (blue) and Mio-depleted cells (red) shows no significant change in the length of the mitotic spindle. (C) Schematic depicting the spindle angle (α) measurement relative to the fibronectin substratum. (D) Quantification of metaphase spindle angles between control (blue) and Mio-depleted cells (red) showing a significant increase of >20o of spindle angle. n = 40 from three experiments. (E) Quantification of mitotic metaphase cells with an unequal number of centrosomes and spindle poles. 3D maximum intensity projections of representative metaphase cells immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue) are shown on the right. 300 cells per condition (n = 3). Bars, 10 µm.

Mentions: Closer inspection of the distribution of α-tubulin and the centrosomal markers γ-tubulin and pericentrin in Mio-depleted cells revealed an increase in several spindle defects. These included abnormal tilt of the spindle axis, disrupted or unfocused spindle poles, monopolar spindles, displacement of the spindle from the cell center toward the cell cortex, and failure of centrosomes to localize at both spindle poles (Fig. 3 A). Most depleted cells showed one or a combination of the aforementioned phenotypes.


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)

Mio depletion leads to spindle misorientation and centrosomal defects. (A) Abnormal spindle morphology detected after Mio depletion. HeLa cells were transfected with control and Mio siRNA oligos for 48 h and stained with α-tubulin. (B) Quantification of metaphase spindle length between control (blue) and Mio-depleted cells (red) shows no significant change in the length of the mitotic spindle. (C) Schematic depicting the spindle angle (α) measurement relative to the fibronectin substratum. (D) Quantification of metaphase spindle angles between control (blue) and Mio-depleted cells (red) showing a significant increase of >20o of spindle angle. n = 40 from three experiments. (E) Quantification of mitotic metaphase cells with an unequal number of centrosomes and spindle poles. 3D maximum intensity projections of representative metaphase cells immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue) are shown on the right. 300 cells per condition (n = 3). Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494011&req=5

fig3: Mio depletion leads to spindle misorientation and centrosomal defects. (A) Abnormal spindle morphology detected after Mio depletion. HeLa cells were transfected with control and Mio siRNA oligos for 48 h and stained with α-tubulin. (B) Quantification of metaphase spindle length between control (blue) and Mio-depleted cells (red) shows no significant change in the length of the mitotic spindle. (C) Schematic depicting the spindle angle (α) measurement relative to the fibronectin substratum. (D) Quantification of metaphase spindle angles between control (blue) and Mio-depleted cells (red) showing a significant increase of >20o of spindle angle. n = 40 from three experiments. (E) Quantification of mitotic metaphase cells with an unequal number of centrosomes and spindle poles. 3D maximum intensity projections of representative metaphase cells immunostained with α-pericentrin (green), α-tubulin (red), and DNA (blue) are shown on the right. 300 cells per condition (n = 3). Bars, 10 µm.
Mentions: Closer inspection of the distribution of α-tubulin and the centrosomal markers γ-tubulin and pericentrin in Mio-depleted cells revealed an increase in several spindle defects. These included abnormal tilt of the spindle axis, disrupted or unfocused spindle poles, monopolar spindles, displacement of the spindle from the cell center toward the cell cortex, and failure of centrosomes to localize at both spindle poles (Fig. 3 A). Most depleted cells showed one or a combination of the aforementioned phenotypes.

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