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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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Related in: MedlinePlus

Recruiting Aurora B to the cell cortex and the growing furrow by MKlp2 is required for stable polarization and furrow formation during monopolar cytokinesis.(A–C) Immunofluorescence analysis of Dox-inducible HeLa cells undergoing monopolar cytokinesis. Flag-MKlp2 expression was induced with Dox for 20 h after the transfection of MKlp2 siRNA. After Dox-treatment, cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 20 min before fixation in ice-cold methanol. (A, B) Flag-MKlp2(1-890) localized to the cell cortex and at the growing furrow together with Aurora B (A, panels b-d) and INCENP (B, panels a, b), but Flag-MKlp2(1-842) only localized to the ends of monopolar spindles together with Aurora B (A, panels f-h) and INCENP (B, panels c, d). (C) In contrast to Aurora B and INCENP in panels A and B, centralspindlin and PRC1 were efficiently localized to the ends of monopolar spindles in cells expressing Flag-MKlp2(1-890) and MKlp2(1-842). This finding indicates that Flag-MKlp2(1-842) is selectively defective in targeting Aurora B and INCENP (thus, most likely the CPC) from the monopolar spindles to the cell cortex. (D) The average percentages based on three independent experiments of non-polarized, polarized (determined by the direction of Flag-MKlp2 and the monopolar spindles towards the cell cortex) or furrowed cells (determined by the presence of Flag-MKlp2 and myosin-II in the furrow) expressing Flag-MKlp2 (n>100 per condition, +/− standard deviation) are shown.
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pone-0064826-g005: Recruiting Aurora B to the cell cortex and the growing furrow by MKlp2 is required for stable polarization and furrow formation during monopolar cytokinesis.(A–C) Immunofluorescence analysis of Dox-inducible HeLa cells undergoing monopolar cytokinesis. Flag-MKlp2 expression was induced with Dox for 20 h after the transfection of MKlp2 siRNA. After Dox-treatment, cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 20 min before fixation in ice-cold methanol. (A, B) Flag-MKlp2(1-890) localized to the cell cortex and at the growing furrow together with Aurora B (A, panels b-d) and INCENP (B, panels a, b), but Flag-MKlp2(1-842) only localized to the ends of monopolar spindles together with Aurora B (A, panels f-h) and INCENP (B, panels c, d). (C) In contrast to Aurora B and INCENP in panels A and B, centralspindlin and PRC1 were efficiently localized to the ends of monopolar spindles in cells expressing Flag-MKlp2(1-890) and MKlp2(1-842). This finding indicates that Flag-MKlp2(1-842) is selectively defective in targeting Aurora B and INCENP (thus, most likely the CPC) from the monopolar spindles to the cell cortex. (D) The average percentages based on three independent experiments of non-polarized, polarized (determined by the direction of Flag-MKlp2 and the monopolar spindles towards the cell cortex) or furrowed cells (determined by the presence of Flag-MKlp2 and myosin-II in the furrow) expressing Flag-MKlp2 (n>100 per condition, +/− standard deviation) are shown.

Mentions: Therefore, to avoid these complications, we again utilized the rescue assay system consisting of Dox-inducible siRNA-resistant Flag-MKlp2 after knockdown of endogenous MKlp2 in HeLa cells. Similar to the case of endogenous MKlp2 (Figure 4A), upon induction of monopolar cytokinesis by Purv A treatment, Dox-induced Flag-MKlp2(1-890) relocated to the polarized furrow together with Aurora B (Figure 5A, panels b-d; Figure S5A, panel a). When these cells were fixed at different time points after Purv A treatment, Flag-MKlp2(1-890) gradually moved to the cell cortex (Figure S6A, panels b, c) and subsequently to the gap region at the growing furrow (panel d). In contrast, Flag-MKlp2(1-842) defective in binding myosin-II was only able to accumulate at the ends of either non-polarized or polarized monopolar spindles in MKlp2-depleted HeLa cells and failed to accumulate at the cell cortex (Figure 5A, panels f, g; Figure S6A, panels e-h). This result is consistent with the inability of Flag-MKlp2(1-842) to localize to the equatorial cortex but not the spindle midzone in bipolar cells (Figure 3C). Moreover, Aurora B was also found at the ends of monopolar spindles together with Flag-MKlp2(1-842) but not at the cell cortex (Figure 5A, panel h; Figure S5A, panel b). Similar to Aurora B, Flag-MKlp2(1-890) moved together with INCENP to the gap region at the growing furrow (Figure 5B, panels a, b). In contrast, Flag-MKlp2(1-842) was only able to localize INCENP at the ends of monopolar spindles in MKlp2-depleted HeLa cells (Figure 5B, panels c, d), indicating that MKlp2 is responsible for localizing both Aurora B and INCENP (thus, most likely the CPC) to the cell cortex. Specifically, in our rescue assay, other essential factors for furrow formation such as centralspindlin and PRC1 were correctly localized to the ends of monopolar spindles towards the cell cortex (Figure 5C). Together, these results strongly suggest that Flag-MKlp2(1-842) is selectively defective in targeting the CPC from the monopolar spindles to the cell cortex and that this deficiency is likely specific to the CPC without affecting the localization of other essential furrow-inducing factors during monopolar 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)

Recruiting Aurora B to the cell cortex and the growing furrow by MKlp2 is required for stable polarization and furrow formation during monopolar cytokinesis.(A–C) Immunofluorescence analysis of Dox-inducible HeLa cells undergoing monopolar cytokinesis. Flag-MKlp2 expression was induced with Dox for 20 h after the transfection of MKlp2 siRNA. After Dox-treatment, cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 20 min before fixation in ice-cold methanol. (A, B) Flag-MKlp2(1-890) localized to the cell cortex and at the growing furrow together with Aurora B (A, panels b-d) and INCENP (B, panels a, b), but Flag-MKlp2(1-842) only localized to the ends of monopolar spindles together with Aurora B (A, panels f-h) and INCENP (B, panels c, d). (C) In contrast to Aurora B and INCENP in panels A and B, centralspindlin and PRC1 were efficiently localized to the ends of monopolar spindles in cells expressing Flag-MKlp2(1-890) and MKlp2(1-842). This finding indicates that Flag-MKlp2(1-842) is selectively defective in targeting Aurora B and INCENP (thus, most likely the CPC) from the monopolar spindles to the cell cortex. (D) The average percentages based on three independent experiments of non-polarized, polarized (determined by the direction of Flag-MKlp2 and the monopolar spindles towards the cell cortex) or furrowed cells (determined by the presence of Flag-MKlp2 and myosin-II in the furrow) expressing Flag-MKlp2 (n>100 per condition, +/− standard deviation) are shown.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672163&req=5

pone-0064826-g005: Recruiting Aurora B to the cell cortex and the growing furrow by MKlp2 is required for stable polarization and furrow formation during monopolar cytokinesis.(A–C) Immunofluorescence analysis of Dox-inducible HeLa cells undergoing monopolar cytokinesis. Flag-MKlp2 expression was induced with Dox for 20 h after the transfection of MKlp2 siRNA. After Dox-treatment, cells were treated with monastrol for 6 h and then treated with Purv A or DMSO for 20 min before fixation in ice-cold methanol. (A, B) Flag-MKlp2(1-890) localized to the cell cortex and at the growing furrow together with Aurora B (A, panels b-d) and INCENP (B, panels a, b), but Flag-MKlp2(1-842) only localized to the ends of monopolar spindles together with Aurora B (A, panels f-h) and INCENP (B, panels c, d). (C) In contrast to Aurora B and INCENP in panels A and B, centralspindlin and PRC1 were efficiently localized to the ends of monopolar spindles in cells expressing Flag-MKlp2(1-890) and MKlp2(1-842). This finding indicates that Flag-MKlp2(1-842) is selectively defective in targeting Aurora B and INCENP (thus, most likely the CPC) from the monopolar spindles to the cell cortex. (D) The average percentages based on three independent experiments of non-polarized, polarized (determined by the direction of Flag-MKlp2 and the monopolar spindles towards the cell cortex) or furrowed cells (determined by the presence of Flag-MKlp2 and myosin-II in the furrow) expressing Flag-MKlp2 (n>100 per condition, +/− standard deviation) are shown.
Mentions: Therefore, to avoid these complications, we again utilized the rescue assay system consisting of Dox-inducible siRNA-resistant Flag-MKlp2 after knockdown of endogenous MKlp2 in HeLa cells. Similar to the case of endogenous MKlp2 (Figure 4A), upon induction of monopolar cytokinesis by Purv A treatment, Dox-induced Flag-MKlp2(1-890) relocated to the polarized furrow together with Aurora B (Figure 5A, panels b-d; Figure S5A, panel a). When these cells were fixed at different time points after Purv A treatment, Flag-MKlp2(1-890) gradually moved to the cell cortex (Figure S6A, panels b, c) and subsequently to the gap region at the growing furrow (panel d). In contrast, Flag-MKlp2(1-842) defective in binding myosin-II was only able to accumulate at the ends of either non-polarized or polarized monopolar spindles in MKlp2-depleted HeLa cells and failed to accumulate at the cell cortex (Figure 5A, panels f, g; Figure S6A, panels e-h). This result is consistent with the inability of Flag-MKlp2(1-842) to localize to the equatorial cortex but not the spindle midzone in bipolar cells (Figure 3C). Moreover, Aurora B was also found at the ends of monopolar spindles together with Flag-MKlp2(1-842) but not at the cell cortex (Figure 5A, panel h; Figure S5A, panel b). Similar to Aurora B, Flag-MKlp2(1-890) moved together with INCENP to the gap region at the growing furrow (Figure 5B, panels a, b). In contrast, Flag-MKlp2(1-842) was only able to localize INCENP at the ends of monopolar spindles in MKlp2-depleted HeLa cells (Figure 5B, panels c, d), indicating that MKlp2 is responsible for localizing both Aurora B and INCENP (thus, most likely the CPC) to the cell cortex. Specifically, in our rescue assay, other essential factors for furrow formation such as centralspindlin and PRC1 were correctly localized to the ends of monopolar spindles towards the cell cortex (Figure 5C). Together, these results strongly suggest that Flag-MKlp2(1-842) is selectively defective in targeting the CPC from the monopolar spindles to the cell cortex and that this deficiency is likely specific to the CPC without affecting the localization of other essential furrow-inducing factors during monopolar 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.

Show MeSH
Related in: MedlinePlus