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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 recruits and provides continuous Aurora B kinase activity to the growing furrow for furrow propagation and completion.(A–D) Time-lapse live-cell images. HeLa cells were transfected with expression vectors encoding mCherry-MKlp2 and GFP-UtrCH (cortical marker), and 24 h after transfection, the cells were subjected to monopolar cytokinesis. In panel A, arrows indicate where a typical polarized furrow was completed. Asterisks indicate the site where mCherry-MKlp2 accumulated at the cell cortex and the furrow. Arrow heads in panels A, C and D denote the sites of the growing furrow before adding ZM447439 or hesperadin. For panel B, Purv A and ZM447439 were added concurrently. For panels C and D, ZM447439 or hesperadin was added sequentially after purvalanol A treatment for 10 min. The fate of the same cell was continuously monitored. White bars represent 5 µm. (E) Monopolar cells (from A–D) were scored as cells that completed monopolar cytokinesis (polarized MPC), formed ectopic furrows without a polarized cortex (non-polarized with ectopic furrowing), no significant furrowing activity without a polarized cortex (non-polarized without furrowing), or formed polarized furrows but subsequently regressed (regressed after polarization). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. (F) Proposed model. (a) In monopolar cytokinesis, inhibition of Cdk1 triggers a symmetry-breaking reaction that initiates polarization of the monopolar spindles and the cell cortex. Although monopolar spindles are symmetrically positioned, they become asymmetric upon Cdk1 inhibition. Thus, the original event triggering the asymmetry of monopolar spindles remains unclear. (b) During this reaction, MKlp2 mediates Aurora B translocation from the monopolar spindles (or the spindle midzone) towards the cell cortex (or the equatorial cortex) in an interdependent manner. At the ends of monopolar spindles that contact the cell cortex, MKlp2 may act as the bridge between the asymmetrically polarizing monopolar spindles and the actomyosin filaments at the cell cortex. This event may stabilize the polarizing monopolar spindles towards the cell cortex. In turn, it facilitates a rapid accumulation of MKlp2-Aurora B to the cell cortex in a polarized manner. This cortical accumulation of MKlp2-Aurora B promotes furrow formation (or might constrict contractile ring in bipolar cytokinesis) via the kinase activity of Aurora B towards currently unidentified target(s). (c) Once the furrow is formed, MKlp2 targets Aurora B to actomyosin filaments in the gap region between the stably polarized monopolar spindles and the furrowing cortical cap. This event may continuously provide Aurora B kinase activity to the growing furrow for propagation and completion. Interestingly, the kinase activity of Aurora B at the growing furrow may also be essential for maintaining the monopolar spindles polarized towards the furrow (Movie S1, S2), indicating that positive feedback loops may exist that maintain polarization between the monopolar spindles and the growing furrow.
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pone-0064826-g006: MKlp2 recruits and provides continuous Aurora B kinase activity to the growing furrow for furrow propagation and completion.(A–D) Time-lapse live-cell images. HeLa cells were transfected with expression vectors encoding mCherry-MKlp2 and GFP-UtrCH (cortical marker), and 24 h after transfection, the cells were subjected to monopolar cytokinesis. In panel A, arrows indicate where a typical polarized furrow was completed. Asterisks indicate the site where mCherry-MKlp2 accumulated at the cell cortex and the furrow. Arrow heads in panels A, C and D denote the sites of the growing furrow before adding ZM447439 or hesperadin. For panel B, Purv A and ZM447439 were added concurrently. For panels C and D, ZM447439 or hesperadin was added sequentially after purvalanol A treatment for 10 min. The fate of the same cell was continuously monitored. White bars represent 5 µm. (E) Monopolar cells (from A–D) were scored as cells that completed monopolar cytokinesis (polarized MPC), formed ectopic furrows without a polarized cortex (non-polarized with ectopic furrowing), no significant furrowing activity without a polarized cortex (non-polarized without furrowing), or formed polarized furrows but subsequently regressed (regressed after polarization). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. (F) Proposed model. (a) In monopolar cytokinesis, inhibition of Cdk1 triggers a symmetry-breaking reaction that initiates polarization of the monopolar spindles and the cell cortex. Although monopolar spindles are symmetrically positioned, they become asymmetric upon Cdk1 inhibition. Thus, the original event triggering the asymmetry of monopolar spindles remains unclear. (b) During this reaction, MKlp2 mediates Aurora B translocation from the monopolar spindles (or the spindle midzone) towards the cell cortex (or the equatorial cortex) in an interdependent manner. At the ends of monopolar spindles that contact the cell cortex, MKlp2 may act as the bridge between the asymmetrically polarizing monopolar spindles and the actomyosin filaments at the cell cortex. This event may stabilize the polarizing monopolar spindles towards the cell cortex. In turn, it facilitates a rapid accumulation of MKlp2-Aurora B to the cell cortex in a polarized manner. This cortical accumulation of MKlp2-Aurora B promotes furrow formation (or might constrict contractile ring in bipolar cytokinesis) via the kinase activity of Aurora B towards currently unidentified target(s). (c) Once the furrow is formed, MKlp2 targets Aurora B to actomyosin filaments in the gap region between the stably polarized monopolar spindles and the furrowing cortical cap. This event may continuously provide Aurora B kinase activity to the growing furrow for propagation and completion. Interestingly, the kinase activity of Aurora B at the growing furrow may also be essential for maintaining the monopolar spindles polarized towards the furrow (Movie S1, S2), indicating that positive feedback loops may exist that maintain polarization between the monopolar spindles and the growing furrow.

Mentions: Although the kinase activity of Aurora B is required for stable furrow ingression [12], whether it functions directly at the furrow has remained elusive. MKlp2 is responsible for targeting Aurora B to the growing furrow; therefore, we tested this issue using time-lapse live-cell imaging of cells undergoing monopolar cytokinesis. First, as determined by immunofluorescence analysis, mCherry-tagged wild-type MKlp2 translocated the majority of Aurora B to the growing furrow (Figure S5B), suggesting that mCherry-MKlp2 was as functional as endogenous MKlp2. Next, using time-lapse live-cell imaging analysis, ectopically expressed mCherry-MKlp2 efficiently accumulated at the growing furrow upon Purv A treatment, and the furrow propagated and reached completion with similar kinetics compared with normal monopolar cytokinesis (Figure 6A). In contrast, when the cells were concurrently treated with Purv A and the Aurora B inhibitor ZM447439, cell polarization and furrow formation was inhibited (Figure 6B, 6E), consistent with a previous report [7]. However, this concurrent inhibition also abolished the cortical localization of mCherry-MKlp2 (Figure 6B); therefore, no cortical localization of Aurora B was observed, which suggests that MKlp2 and Aurora B are interdependently required for their relocation from anaphase chromatin to the cell cortex. This concurrent treatment also mislocalized MKlp1 from the monopolar spindles (Figure S9, panel a), creating difficulty in determining the importance of MKlp2-Aurora B at the growing furrow. Therefore, to avoid this complication, ZM447439 was sequentially added after polarizing HeLa cells with Purv A treatment (Figure 6C, 6E). Using time-lapse live-cell analysis, we first determined that mCherry-MKlp2 efficiently accumulated at the growing furrow at 10 min after Purv A treatment (Figure 6C). Then, ZM447439 was added, and the fate of the same cell was continuously monitored. Notably, upon addition of ZM447439, the furrowing activity immediately ceased, and the furrow regressed, although a considerable amount of mCherry-MKlp2 remained at the regressing furrow (Figure 6C, 6E). A similar result was also obtained using the Aurora B selective inhibitor hesperadin (Figure 6D, 6E), excluding a potential off-target effect of ZM447439. Using immunofluorescence analysis, endogenous Aurora B and MKlp2 remained at the regressing furrow under this sequential treatment condition (Figure S9, panels c & d). Also, this sequential treatment did not affect MKlp1 localization to the ends of monopolar spindles (Figure S9, panel b). Together, these results suggest that the kinase activity of Aurora B at the growing furrow is continuously required for furrow propagation and completion. Interestingly, this sequential inhibition of Aurora B also depolarized the mitotic spindle (Movie S1, S2), suggesting that the Aurora B kinase activity at the polarized cortex may be essential for maintaining the polarization of monopolar spindles. Taken together, we conclude that targeting Aurora B to the cell cortex by MKlp2 in an interdependent manner may play a role in monopolar cytokinesis by promoting polarization and efficiently maintaining the cytokinesis furrow for completion (Figure 6F). Moreover, targeting Aurora B to the equatorial cortex by MKlp2 is required for the efficient maintenance of the ingressing furrow in a partially redundant manner with MKlp1 during bipolar cytokinesis.


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 recruits and provides continuous Aurora B kinase activity to the growing furrow for furrow propagation and completion.(A–D) Time-lapse live-cell images. HeLa cells were transfected with expression vectors encoding mCherry-MKlp2 and GFP-UtrCH (cortical marker), and 24 h after transfection, the cells were subjected to monopolar cytokinesis. In panel A, arrows indicate where a typical polarized furrow was completed. Asterisks indicate the site where mCherry-MKlp2 accumulated at the cell cortex and the furrow. Arrow heads in panels A, C and D denote the sites of the growing furrow before adding ZM447439 or hesperadin. For panel B, Purv A and ZM447439 were added concurrently. For panels C and D, ZM447439 or hesperadin was added sequentially after purvalanol A treatment for 10 min. The fate of the same cell was continuously monitored. White bars represent 5 µm. (E) Monopolar cells (from A–D) were scored as cells that completed monopolar cytokinesis (polarized MPC), formed ectopic furrows without a polarized cortex (non-polarized with ectopic furrowing), no significant furrowing activity without a polarized cortex (non-polarized without furrowing), or formed polarized furrows but subsequently regressed (regressed after polarization). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. (F) Proposed model. (a) In monopolar cytokinesis, inhibition of Cdk1 triggers a symmetry-breaking reaction that initiates polarization of the monopolar spindles and the cell cortex. Although monopolar spindles are symmetrically positioned, they become asymmetric upon Cdk1 inhibition. Thus, the original event triggering the asymmetry of monopolar spindles remains unclear. (b) During this reaction, MKlp2 mediates Aurora B translocation from the monopolar spindles (or the spindle midzone) towards the cell cortex (or the equatorial cortex) in an interdependent manner. At the ends of monopolar spindles that contact the cell cortex, MKlp2 may act as the bridge between the asymmetrically polarizing monopolar spindles and the actomyosin filaments at the cell cortex. This event may stabilize the polarizing monopolar spindles towards the cell cortex. In turn, it facilitates a rapid accumulation of MKlp2-Aurora B to the cell cortex in a polarized manner. This cortical accumulation of MKlp2-Aurora B promotes furrow formation (or might constrict contractile ring in bipolar cytokinesis) via the kinase activity of Aurora B towards currently unidentified target(s). (c) Once the furrow is formed, MKlp2 targets Aurora B to actomyosin filaments in the gap region between the stably polarized monopolar spindles and the furrowing cortical cap. This event may continuously provide Aurora B kinase activity to the growing furrow for propagation and completion. Interestingly, the kinase activity of Aurora B at the growing furrow may also be essential for maintaining the monopolar spindles polarized towards the furrow (Movie S1, S2), indicating that positive feedback loops may exist that maintain polarization between the monopolar spindles and the growing furrow.
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pone-0064826-g006: MKlp2 recruits and provides continuous Aurora B kinase activity to the growing furrow for furrow propagation and completion.(A–D) Time-lapse live-cell images. HeLa cells were transfected with expression vectors encoding mCherry-MKlp2 and GFP-UtrCH (cortical marker), and 24 h after transfection, the cells were subjected to monopolar cytokinesis. In panel A, arrows indicate where a typical polarized furrow was completed. Asterisks indicate the site where mCherry-MKlp2 accumulated at the cell cortex and the furrow. Arrow heads in panels A, C and D denote the sites of the growing furrow before adding ZM447439 or hesperadin. For panel B, Purv A and ZM447439 were added concurrently. For panels C and D, ZM447439 or hesperadin was added sequentially after purvalanol A treatment for 10 min. The fate of the same cell was continuously monitored. White bars represent 5 µm. (E) Monopolar cells (from A–D) were scored as cells that completed monopolar cytokinesis (polarized MPC), formed ectopic furrows without a polarized cortex (non-polarized with ectopic furrowing), no significant furrowing activity without a polarized cortex (non-polarized without furrowing), or formed polarized furrows but subsequently regressed (regressed after polarization). The average percentages based on three independent experiments (total n>100 per condition, +/− standard deviation) are shown. (F) Proposed model. (a) In monopolar cytokinesis, inhibition of Cdk1 triggers a symmetry-breaking reaction that initiates polarization of the monopolar spindles and the cell cortex. Although monopolar spindles are symmetrically positioned, they become asymmetric upon Cdk1 inhibition. Thus, the original event triggering the asymmetry of monopolar spindles remains unclear. (b) During this reaction, MKlp2 mediates Aurora B translocation from the monopolar spindles (or the spindle midzone) towards the cell cortex (or the equatorial cortex) in an interdependent manner. At the ends of monopolar spindles that contact the cell cortex, MKlp2 may act as the bridge between the asymmetrically polarizing monopolar spindles and the actomyosin filaments at the cell cortex. This event may stabilize the polarizing monopolar spindles towards the cell cortex. In turn, it facilitates a rapid accumulation of MKlp2-Aurora B to the cell cortex in a polarized manner. This cortical accumulation of MKlp2-Aurora B promotes furrow formation (or might constrict contractile ring in bipolar cytokinesis) via the kinase activity of Aurora B towards currently unidentified target(s). (c) Once the furrow is formed, MKlp2 targets Aurora B to actomyosin filaments in the gap region between the stably polarized monopolar spindles and the furrowing cortical cap. This event may continuously provide Aurora B kinase activity to the growing furrow for propagation and completion. Interestingly, the kinase activity of Aurora B at the growing furrow may also be essential for maintaining the monopolar spindles polarized towards the furrow (Movie S1, S2), indicating that positive feedback loops may exist that maintain polarization between the monopolar spindles and the growing furrow.
Mentions: Although the kinase activity of Aurora B is required for stable furrow ingression [12], whether it functions directly at the furrow has remained elusive. MKlp2 is responsible for targeting Aurora B to the growing furrow; therefore, we tested this issue using time-lapse live-cell imaging of cells undergoing monopolar cytokinesis. First, as determined by immunofluorescence analysis, mCherry-tagged wild-type MKlp2 translocated the majority of Aurora B to the growing furrow (Figure S5B), suggesting that mCherry-MKlp2 was as functional as endogenous MKlp2. Next, using time-lapse live-cell imaging analysis, ectopically expressed mCherry-MKlp2 efficiently accumulated at the growing furrow upon Purv A treatment, and the furrow propagated and reached completion with similar kinetics compared with normal monopolar cytokinesis (Figure 6A). In contrast, when the cells were concurrently treated with Purv A and the Aurora B inhibitor ZM447439, cell polarization and furrow formation was inhibited (Figure 6B, 6E), consistent with a previous report [7]. However, this concurrent inhibition also abolished the cortical localization of mCherry-MKlp2 (Figure 6B); therefore, no cortical localization of Aurora B was observed, which suggests that MKlp2 and Aurora B are interdependently required for their relocation from anaphase chromatin to the cell cortex. This concurrent treatment also mislocalized MKlp1 from the monopolar spindles (Figure S9, panel a), creating difficulty in determining the importance of MKlp2-Aurora B at the growing furrow. Therefore, to avoid this complication, ZM447439 was sequentially added after polarizing HeLa cells with Purv A treatment (Figure 6C, 6E). Using time-lapse live-cell analysis, we first determined that mCherry-MKlp2 efficiently accumulated at the growing furrow at 10 min after Purv A treatment (Figure 6C). Then, ZM447439 was added, and the fate of the same cell was continuously monitored. Notably, upon addition of ZM447439, the furrowing activity immediately ceased, and the furrow regressed, although a considerable amount of mCherry-MKlp2 remained at the regressing furrow (Figure 6C, 6E). A similar result was also obtained using the Aurora B selective inhibitor hesperadin (Figure 6D, 6E), excluding a potential off-target effect of ZM447439. Using immunofluorescence analysis, endogenous Aurora B and MKlp2 remained at the regressing furrow under this sequential treatment condition (Figure S9, panels c & d). Also, this sequential treatment did not affect MKlp1 localization to the ends of monopolar spindles (Figure S9, panel b). Together, these results suggest that the kinase activity of Aurora B at the growing furrow is continuously required for furrow propagation and completion. Interestingly, this sequential inhibition of Aurora B also depolarized the mitotic spindle (Movie S1, S2), suggesting that the Aurora B kinase activity at the polarized cortex may be essential for maintaining the polarization of monopolar spindles. Taken together, we conclude that targeting Aurora B to the cell cortex by MKlp2 in an interdependent manner may play a role in monopolar cytokinesis by promoting polarization and efficiently maintaining the cytokinesis furrow for completion (Figure 6F). Moreover, targeting Aurora B to the equatorial cortex by MKlp2 is required for the efficient maintenance of the ingressing furrow in a partially redundant manner with MKlp1 during bipolar cytokinesis.

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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Related in: MedlinePlus