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Targeting Aurora B to the equatorial cortex by MKlp2 is required for cytokinesis.

Kitagawa M, Fung SY, Onishi N, Saya H, Lee SH - PLoS ONE (2013)

Bottom Line: In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis.This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis.Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore.

ABSTRACT
Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.

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MKlp2 localizes to the growing furrow in a myosin-II-dependent manner, and it is required for cell polarization during monopolar cytokinesis.(A) Immunofluorescence analysis of HeLa cells before (panel a) or during monopolar cytokinesis (panels b-d). Asynchronously growing HeLa cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 15 min before fixation in ice-cold methanol. (B, C) HeLa cells were transfected with the indicated siRNAs for 20 h before the addition of monastrol, and monopolar cytokinesis was induced as described in panel A. For panel B, HeLa cells stably expressing GFP-α-tubulin were used. For panels A–C, images were acquired using 3D-SIM. (D) The average percentages calculated based on three independent experiments using non-polarized, polarized (determined by cortically localized Aurora B from panel C) or furrowed cells (determined by myosin-II) are shown with error bars (n>100 cells per condition). (E) Time-lapse live-cell imaging. HeLa cells were transfected with the indicated siRNAs together with the vectors encoding GFP-UtrCH to monitor cortical changes and were subjected to monopolar cytokinesis. Arrows indicate the site where a typical polarized furrow was formed and progressed. (F) Monopolar cells (from panel E) were scored as cells that completed monopolar cytokinesis (polarized MPC) or failed without polarizing the cortex (non-polarized with no MPC). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. Images were acquired using 2D-SIM. White bars represent 5 µm.
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pone-0064826-g004: MKlp2 localizes to the growing furrow in a myosin-II-dependent manner, and it is required for cell polarization during monopolar cytokinesis.(A) Immunofluorescence analysis of HeLa cells before (panel a) or during monopolar cytokinesis (panels b-d). Asynchronously growing HeLa cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 15 min before fixation in ice-cold methanol. (B, C) HeLa cells were transfected with the indicated siRNAs for 20 h before the addition of monastrol, and monopolar cytokinesis was induced as described in panel A. For panel B, HeLa cells stably expressing GFP-α-tubulin were used. For panels A–C, images were acquired using 3D-SIM. (D) The average percentages calculated based on three independent experiments using non-polarized, polarized (determined by cortically localized Aurora B from panel C) or furrowed cells (determined by myosin-II) are shown with error bars (n>100 cells per condition). (E) Time-lapse live-cell imaging. HeLa cells were transfected with the indicated siRNAs together with the vectors encoding GFP-UtrCH to monitor cortical changes and were subjected to monopolar cytokinesis. Arrows indicate the site where a typical polarized furrow was formed and progressed. (F) Monopolar cells (from panel E) were scored as cells that completed monopolar cytokinesis (polarized MPC) or failed without polarizing the cortex (non-polarized with no MPC). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. Images were acquired using 2D-SIM. White bars represent 5 µm.

Mentions: The close proximity of the spindle midzone and the equatorial cortex in the ingressing furrow during bipolar cytokinesis creates difficultly in precisely determining the role of MKlp2 within this interface. To better address the issue, we induced drug-synchronized monopolar cytokinesis [6] where Aurora B but not centralspindlin localizes to the actomyosin filaments in a gap region between the end of polarized monopolar spindles and the furrowing cortical cap [7]. In monastrol-arrested monopolar HeLa cells, the majority of MKlp2 and Aurora B was present in the cytoplasm and at the centromeres, respectively (Figure 4A, panel a). However, when these cells were forced to undergo monopolar cytokinesis caused by treatment with the potent Cdk1 inhibitor purvalanol A (Purv A), endogenous MKlp2 efficiently co-localized with Aurora B at the polarized furrowing cortex (Figure 4A, panel b). Moreover, MKlp2 co-localized with myosin-II adjacent to the polarized cortex (Figure 4A, panel d), but it was clearly distinctive from the polarized monopolar spindles (panel c). Using HeLa cells stably expressing GFP-α-tubulin and super-resolution fluorescence microscopy, MKlp2 was shown to be localized to the ends of polarized monopolar spindles and extended to the polarized actomyosin filaments at the growing furrow (Figure 4B, panel a), indicating that MKlp2 may be involved in linking actomyosin filaments at the growing furrow with polarized monopolar spindles. In contrast, siRNA-mediated depletion of myosin-II (Figure S4A) markedly suppressed the localization of MKlp2 at the cell cortex but not at the ends of monopolar spindles (Figure 4B, panel b).Of note, it is possible that since no polarized cortical structures might form in myosin-II-depleted cells, which may prevent MKlp2 from localizing to the cell cortex. Nevertheless, our results indicate that myosin-II is required for MKlp2 to localize to the actomyosin filaments at the growing furrow.


Targeting Aurora B to the equatorial cortex by MKlp2 is required for cytokinesis.

Kitagawa M, Fung SY, Onishi N, Saya H, Lee SH - PLoS ONE (2013)

MKlp2 localizes to the growing furrow in a myosin-II-dependent manner, and it is required for cell polarization during monopolar cytokinesis.(A) Immunofluorescence analysis of HeLa cells before (panel a) or during monopolar cytokinesis (panels b-d). Asynchronously growing HeLa cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 15 min before fixation in ice-cold methanol. (B, C) HeLa cells were transfected with the indicated siRNAs for 20 h before the addition of monastrol, and monopolar cytokinesis was induced as described in panel A. For panel B, HeLa cells stably expressing GFP-α-tubulin were used. For panels A–C, images were acquired using 3D-SIM. (D) The average percentages calculated based on three independent experiments using non-polarized, polarized (determined by cortically localized Aurora B from panel C) or furrowed cells (determined by myosin-II) are shown with error bars (n>100 cells per condition). (E) Time-lapse live-cell imaging. HeLa cells were transfected with the indicated siRNAs together with the vectors encoding GFP-UtrCH to monitor cortical changes and were subjected to monopolar cytokinesis. Arrows indicate the site where a typical polarized furrow was formed and progressed. (F) Monopolar cells (from panel E) were scored as cells that completed monopolar cytokinesis (polarized MPC) or failed without polarizing the cortex (non-polarized with no MPC). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. Images were acquired using 2D-SIM. White bars represent 5 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672163&req=5

pone-0064826-g004: MKlp2 localizes to the growing furrow in a myosin-II-dependent manner, and it is required for cell polarization during monopolar cytokinesis.(A) Immunofluorescence analysis of HeLa cells before (panel a) or during monopolar cytokinesis (panels b-d). Asynchronously growing HeLa cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 15 min before fixation in ice-cold methanol. (B, C) HeLa cells were transfected with the indicated siRNAs for 20 h before the addition of monastrol, and monopolar cytokinesis was induced as described in panel A. For panel B, HeLa cells stably expressing GFP-α-tubulin were used. For panels A–C, images were acquired using 3D-SIM. (D) The average percentages calculated based on three independent experiments using non-polarized, polarized (determined by cortically localized Aurora B from panel C) or furrowed cells (determined by myosin-II) are shown with error bars (n>100 cells per condition). (E) Time-lapse live-cell imaging. HeLa cells were transfected with the indicated siRNAs together with the vectors encoding GFP-UtrCH to monitor cortical changes and were subjected to monopolar cytokinesis. Arrows indicate the site where a typical polarized furrow was formed and progressed. (F) Monopolar cells (from panel E) were scored as cells that completed monopolar cytokinesis (polarized MPC) or failed without polarizing the cortex (non-polarized with no MPC). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. Images were acquired using 2D-SIM. White bars represent 5 µm.
Mentions: The close proximity of the spindle midzone and the equatorial cortex in the ingressing furrow during bipolar cytokinesis creates difficultly in precisely determining the role of MKlp2 within this interface. To better address the issue, we induced drug-synchronized monopolar cytokinesis [6] where Aurora B but not centralspindlin localizes to the actomyosin filaments in a gap region between the end of polarized monopolar spindles and the furrowing cortical cap [7]. In monastrol-arrested monopolar HeLa cells, the majority of MKlp2 and Aurora B was present in the cytoplasm and at the centromeres, respectively (Figure 4A, panel a). However, when these cells were forced to undergo monopolar cytokinesis caused by treatment with the potent Cdk1 inhibitor purvalanol A (Purv A), endogenous MKlp2 efficiently co-localized with Aurora B at the polarized furrowing cortex (Figure 4A, panel b). Moreover, MKlp2 co-localized with myosin-II adjacent to the polarized cortex (Figure 4A, panel d), but it was clearly distinctive from the polarized monopolar spindles (panel c). Using HeLa cells stably expressing GFP-α-tubulin and super-resolution fluorescence microscopy, MKlp2 was shown to be localized to the ends of polarized monopolar spindles and extended to the polarized actomyosin filaments at the growing furrow (Figure 4B, panel a), indicating that MKlp2 may be involved in linking actomyosin filaments at the growing furrow with polarized monopolar spindles. In contrast, siRNA-mediated depletion of myosin-II (Figure S4A) markedly suppressed the localization of MKlp2 at the cell cortex but not at the ends of monopolar spindles (Figure 4B, panel b).Of note, it is possible that since no polarized cortical structures might form in myosin-II-depleted cells, which may prevent MKlp2 from localizing to the cell cortex. Nevertheless, our results indicate that myosin-II is required for MKlp2 to localize to the actomyosin filaments at the growing furrow.

Bottom Line: In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis.This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis.Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Program in Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore.

ABSTRACT
Although Aurora B is important in cleavage furrow ingression and completion during cytokinesis, the mechanism by which kinase activity is targeted to the cleavage furrow and the molecule(s) responsible for this process have remained elusive. Here, we demonstrate that an essential mitotic kinesin MKlp2 requires myosin-II for its localization to the equatorial cortex, and this event is required to recruit Aurora B to the equatorial cortex in mammalian cells. This recruitment event is also required to promote the highly focused accumulation of active RhoA at the equatorial cortex and stable ingression of the cleavage furrow in bipolar cytokinesis. Specifically, in drug-induced monopolar cytokinesis, targeting Aurora B to the cell cortex by MKlp2 is essential for cell polarization and furrow formation. Once the furrow has formed, MKlp2 further recruits Aurora B to the growing furrow. This process together with continuous Aurora B kinase activity at the growing furrow is essential for stable furrow propagation and completion. In contrast, a MKlp2 mutant defective in binding myosin-II does not recruit Aurora B to the cell cortex and does not promote furrow formation during monopolar cytokinesis. This mutant is also defective in maintaining the ingressing furrow during bipolar cytokinesis. Together, these findings reveal that targeting Aurora B to the cell cortex (or the equatorial cortex) by MKlp2 is essential for the maintenance of the ingressing furrow for successful cytokinesis.

Show MeSH
Related in: MedlinePlus