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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 promotes and maintains efficient ingression of the cleavage furrow.(A) Synchronized HeLa cells transfected with the indicated siRNAs were subjected to time-lapse live-cell imaging. Arrows indicate the ingressed cleavage furrow. (B) The cytokinesis progression of cells (n>40) that were selected at random and the time they spent in ingression before regression is plotted (top graph). All HeLa cells depleted of MKlp1 or MKlp2 formed a cleavage furrow and ingressed followed by furrow regression and cytokinesis failure, while none of the control cells showed furrow regression after ingression. The average duration of the furrow in ingression is based on three independent experiments (total n>100 per condition, +/− standard deviation) (bottom graph). To determine the statistical significance of the duration of the furrow in ingression Student’s t-test was performed. P values are indicated. (C) Immunoblot analysis of total cell lysates from panel A. C: control, M1: MKlp1, M2: MKlp2. Relative band intensities to control siRNA are shown in the bottom of each panel. (D) Immunofluorescence analysis using asynchronously grown HeLa cells was performed at 30 h after transfection with the indicated siRNAs. Cells in anaphase are shown. Images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm.
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pone-0064826-g001: MKlp2 promotes and maintains efficient ingression of the cleavage furrow.(A) Synchronized HeLa cells transfected with the indicated siRNAs were subjected to time-lapse live-cell imaging. Arrows indicate the ingressed cleavage furrow. (B) The cytokinesis progression of cells (n>40) that were selected at random and the time they spent in ingression before regression is plotted (top graph). All HeLa cells depleted of MKlp1 or MKlp2 formed a cleavage furrow and ingressed followed by furrow regression and cytokinesis failure, while none of the control cells showed furrow regression after ingression. The average duration of the furrow in ingression is based on three independent experiments (total n>100 per condition, +/− standard deviation) (bottom graph). To determine the statistical significance of the duration of the furrow in ingression Student’s t-test was performed. P values are indicated. (C) Immunoblot analysis of total cell lysates from panel A. C: control, M1: MKlp1, M2: MKlp2. Relative band intensities to control siRNA are shown in the bottom of each panel. (D) Immunofluorescence analysis using asynchronously grown HeLa cells was performed at 30 h after transfection with the indicated siRNAs. Cells in anaphase are shown. Images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm.

Mentions: Although MKlp2 is essential for cytokinesis, it is still unclear how MKlp2 contributes to cytokinesis in mammalian cells. To determine the phase(s) of cytokinesis in which MKlp2 is essential, HeLa cells transfected with either control or MKlp2 siRNAs were subjected to time-lapse live-cell imaging analysis in comparison with MKlp1-depleted cells. In control cells transfected with non-silencing siRNA, the ingressed furrow was maintained until the completion of cytokinesis (Figure 1A, panel a; Figure 1B). In contrast to control cells, the furrow formed and ingressed but subsequently regressed in MKlp1-depleted cells (Figure 1A, panel b; Figure 1B, top graph). Although the duration of ingression in MKlp2-depleted cells was longer than in MKlp1-depleted cells (Figure 1B, bottom graph), the furrow formed and ingressed but was followed by furrow regression in MKlp2-depleted cells (Figure 1A, panels c, d; Figure 1B, top graph). This finding suggests that MKlp2 is required for the maintenance of the ingressing furrow at a later stage of cytokinesis compared with MKlp1 in mammalian cells. Remarkably, however, co-depletion of MKlp1 and MKlp2 largely inhibited furrow ingression (Figure 1A, panel e; Figure 1B), suggesting that MKlp2 acts during an early stage of furrow ingression in the absence of MKlp1 in mammalian cells. This result was not due to incomplete depletion of MKlp1 or MKlp2 proteins as determined by immunoblot analysis (Figure 1C), indicating that MKlp1 and MKlp2 function in partially redundant pathways for furrow ingression.


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 promotes and maintains efficient ingression of the cleavage furrow.(A) Synchronized HeLa cells transfected with the indicated siRNAs were subjected to time-lapse live-cell imaging. Arrows indicate the ingressed cleavage furrow. (B) The cytokinesis progression of cells (n>40) that were selected at random and the time they spent in ingression before regression is plotted (top graph). All HeLa cells depleted of MKlp1 or MKlp2 formed a cleavage furrow and ingressed followed by furrow regression and cytokinesis failure, while none of the control cells showed furrow regression after ingression. The average duration of the furrow in ingression is based on three independent experiments (total n>100 per condition, +/− standard deviation) (bottom graph). To determine the statistical significance of the duration of the furrow in ingression Student’s t-test was performed. P values are indicated. (C) Immunoblot analysis of total cell lysates from panel A. C: control, M1: MKlp1, M2: MKlp2. Relative band intensities to control siRNA are shown in the bottom of each panel. (D) Immunofluorescence analysis using asynchronously grown HeLa cells was performed at 30 h after transfection with the indicated siRNAs. Cells in anaphase are shown. Images were acquired using 3D-SIM. Insets represent the boxed areas. 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-g001: MKlp2 promotes and maintains efficient ingression of the cleavage furrow.(A) Synchronized HeLa cells transfected with the indicated siRNAs were subjected to time-lapse live-cell imaging. Arrows indicate the ingressed cleavage furrow. (B) The cytokinesis progression of cells (n>40) that were selected at random and the time they spent in ingression before regression is plotted (top graph). All HeLa cells depleted of MKlp1 or MKlp2 formed a cleavage furrow and ingressed followed by furrow regression and cytokinesis failure, while none of the control cells showed furrow regression after ingression. The average duration of the furrow in ingression is based on three independent experiments (total n>100 per condition, +/− standard deviation) (bottom graph). To determine the statistical significance of the duration of the furrow in ingression Student’s t-test was performed. P values are indicated. (C) Immunoblot analysis of total cell lysates from panel A. C: control, M1: MKlp1, M2: MKlp2. Relative band intensities to control siRNA are shown in the bottom of each panel. (D) Immunofluorescence analysis using asynchronously grown HeLa cells was performed at 30 h after transfection with the indicated siRNAs. Cells in anaphase are shown. Images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm.
Mentions: Although MKlp2 is essential for cytokinesis, it is still unclear how MKlp2 contributes to cytokinesis in mammalian cells. To determine the phase(s) of cytokinesis in which MKlp2 is essential, HeLa cells transfected with either control or MKlp2 siRNAs were subjected to time-lapse live-cell imaging analysis in comparison with MKlp1-depleted cells. In control cells transfected with non-silencing siRNA, the ingressed furrow was maintained until the completion of cytokinesis (Figure 1A, panel a; Figure 1B). In contrast to control cells, the furrow formed and ingressed but subsequently regressed in MKlp1-depleted cells (Figure 1A, panel b; Figure 1B, top graph). Although the duration of ingression in MKlp2-depleted cells was longer than in MKlp1-depleted cells (Figure 1B, bottom graph), the furrow formed and ingressed but was followed by furrow regression in MKlp2-depleted cells (Figure 1A, panels c, d; Figure 1B, top graph). This finding suggests that MKlp2 is required for the maintenance of the ingressing furrow at a later stage of cytokinesis compared with MKlp1 in mammalian cells. Remarkably, however, co-depletion of MKlp1 and MKlp2 largely inhibited furrow ingression (Figure 1A, panel e; Figure 1B), suggesting that MKlp2 acts during an early stage of furrow ingression in the absence of MKlp1 in mammalian cells. This result was not due to incomplete depletion of MKlp1 or MKlp2 proteins as determined by immunoblot analysis (Figure 1C), indicating that MKlp1 and MKlp2 function in partially redundant pathways for furrow ingression.

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