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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 equatorial cortex prior to furrow ingression and promotes furrow ingression.(A, B) Immunofluorescence analysis. Asynchronously growing HeLa cells were fixed with ice-cold methanol (A, panel a; B) or 10% TCA (A, panel b) and stained with the indicated antibodies. Arrows indicate the equatorial cortex. (B) X-Z shows a cross-section at the equator (white arrow) with an intensity profile of the corresponding section (bottom). (C) Images of Dox-induced Flag-MKlp2 in MKlp2-depleted HeLa cells using siRNA transfection. Dox-inducible HeLa cells were transfected for 4 h with siRNA to depleted endogenous MKlp2. Subsequently, cells were treated with Dox for 20 h before fixing with ice-cold methanol. Flag-MKlp2(1-890) accumulated at the equatorial cortex as indicated with arrows (panel a), whereas Flag-MKlp2(1-842) only showed punctate staining patterns close to the equatorial cortex (panel b, arrow heads in inset). However, both proteins were comparably localized to the spindle midzone. For panels A and C, images were acquired using confocal microscopy. For panel B, images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm. (D) HeLa cells were transfected with non-silencing control siRNA or co-transfected with MKlp1 and MKlp2 siRNAs after the first thymidine block and were treated with Dox during the second thymidine block (see Materials and Methods). The cells were synchronously released from the G1/S boundary and subjected to time-lapse live-cell imaging. The cytokinesis progression of cells that were selected at random, and the time they spent in ingression before regression is plotted (top graph). The average duration of 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 furrow in ingression, Student’s t-test was performed. P values are indicated.
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pone-0064826-g003: MKlp2 localizes to the equatorial cortex prior to furrow ingression and promotes furrow ingression.(A, B) Immunofluorescence analysis. Asynchronously growing HeLa cells were fixed with ice-cold methanol (A, panel a; B) or 10% TCA (A, panel b) and stained with the indicated antibodies. Arrows indicate the equatorial cortex. (B) X-Z shows a cross-section at the equator (white arrow) with an intensity profile of the corresponding section (bottom). (C) Images of Dox-induced Flag-MKlp2 in MKlp2-depleted HeLa cells using siRNA transfection. Dox-inducible HeLa cells were transfected for 4 h with siRNA to depleted endogenous MKlp2. Subsequently, cells were treated with Dox for 20 h before fixing with ice-cold methanol. Flag-MKlp2(1-890) accumulated at the equatorial cortex as indicated with arrows (panel a), whereas Flag-MKlp2(1-842) only showed punctate staining patterns close to the equatorial cortex (panel b, arrow heads in inset). However, both proteins were comparably localized to the spindle midzone. For panels A and C, images were acquired using confocal microscopy. For panel B, images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm. (D) HeLa cells were transfected with non-silencing control siRNA or co-transfected with MKlp1 and MKlp2 siRNAs after the first thymidine block and were treated with Dox during the second thymidine block (see Materials and Methods). The cells were synchronously released from the G1/S boundary and subjected to time-lapse live-cell imaging. The cytokinesis progression of cells that were selected at random, and the time they spent in ingression before regression is plotted (top graph). The average duration of 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 furrow in ingression, Student’s t-test was performed. P values are indicated.

Mentions: Next, we determined whether these results obtained by the biochemical analysis explained MKlp2 localization at the equatorial cortex. Endogenous MKlp2 localized to the equatorial cortex (Figure 3A) together with RhoA (panel b) and myosin-II (Figure 3B) prior to cleavage furrow ingression as shown by immunofluorescence analysis using HeLa cells. Dox-induced Flag-MKlp2(1-890) completely co-localized with Aurora B at the cell equator (Figure S3A, S3B), supporting the hypothesis that Aurora B is the mitotic cargo of MKlp2 [9]. Moreover, similar to endogenous MKlp2, Flag-MKlp2(1-890) accumulated at the equatorial cortex and the spindle midzone (Figure 3C, panel a). In contrast, Flag-MKlp2(1-842) selectively failed to accumulate at the equatorial cortex, while it efficiently localized to the spindle midzone (Figure 3C, panel b). This result is consistent with the finding that Flag-MKlp2(1-842) was selectively defective in binding myosin-II but not other known interacting partners of MKlp2 (Figure S2). Interestingly, the RacGAP1 centralspindlin component comparably localized to the spindle midzone in cells expressing Flag-MKlp2(1-890) and Flag-MKlp2(1-842) (Figure S3C, S3D). Together, these results suggest that the MKlp2:myosin-II interaction is likely required for the accumulation of MKlp2 at the equatorial cortex.


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 equatorial cortex prior to furrow ingression and promotes furrow ingression.(A, B) Immunofluorescence analysis. Asynchronously growing HeLa cells were fixed with ice-cold methanol (A, panel a; B) or 10% TCA (A, panel b) and stained with the indicated antibodies. Arrows indicate the equatorial cortex. (B) X-Z shows a cross-section at the equator (white arrow) with an intensity profile of the corresponding section (bottom). (C) Images of Dox-induced Flag-MKlp2 in MKlp2-depleted HeLa cells using siRNA transfection. Dox-inducible HeLa cells were transfected for 4 h with siRNA to depleted endogenous MKlp2. Subsequently, cells were treated with Dox for 20 h before fixing with ice-cold methanol. Flag-MKlp2(1-890) accumulated at the equatorial cortex as indicated with arrows (panel a), whereas Flag-MKlp2(1-842) only showed punctate staining patterns close to the equatorial cortex (panel b, arrow heads in inset). However, both proteins were comparably localized to the spindle midzone. For panels A and C, images were acquired using confocal microscopy. For panel B, images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm. (D) HeLa cells were transfected with non-silencing control siRNA or co-transfected with MKlp1 and MKlp2 siRNAs after the first thymidine block and were treated with Dox during the second thymidine block (see Materials and Methods). The cells were synchronously released from the G1/S boundary and subjected to time-lapse live-cell imaging. The cytokinesis progression of cells that were selected at random, and the time they spent in ingression before regression is plotted (top graph). The average duration of 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 furrow in ingression, Student’s t-test was performed. P values are indicated.
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pone-0064826-g003: MKlp2 localizes to the equatorial cortex prior to furrow ingression and promotes furrow ingression.(A, B) Immunofluorescence analysis. Asynchronously growing HeLa cells were fixed with ice-cold methanol (A, panel a; B) or 10% TCA (A, panel b) and stained with the indicated antibodies. Arrows indicate the equatorial cortex. (B) X-Z shows a cross-section at the equator (white arrow) with an intensity profile of the corresponding section (bottom). (C) Images of Dox-induced Flag-MKlp2 in MKlp2-depleted HeLa cells using siRNA transfection. Dox-inducible HeLa cells were transfected for 4 h with siRNA to depleted endogenous MKlp2. Subsequently, cells were treated with Dox for 20 h before fixing with ice-cold methanol. Flag-MKlp2(1-890) accumulated at the equatorial cortex as indicated with arrows (panel a), whereas Flag-MKlp2(1-842) only showed punctate staining patterns close to the equatorial cortex (panel b, arrow heads in inset). However, both proteins were comparably localized to the spindle midzone. For panels A and C, images were acquired using confocal microscopy. For panel B, images were acquired using 3D-SIM. Insets represent the boxed areas. White bars represent 5 µm. (D) HeLa cells were transfected with non-silencing control siRNA or co-transfected with MKlp1 and MKlp2 siRNAs after the first thymidine block and were treated with Dox during the second thymidine block (see Materials and Methods). The cells were synchronously released from the G1/S boundary and subjected to time-lapse live-cell imaging. The cytokinesis progression of cells that were selected at random, and the time they spent in ingression before regression is plotted (top graph). The average duration of 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 furrow in ingression, Student’s t-test was performed. P values are indicated.
Mentions: Next, we determined whether these results obtained by the biochemical analysis explained MKlp2 localization at the equatorial cortex. Endogenous MKlp2 localized to the equatorial cortex (Figure 3A) together with RhoA (panel b) and myosin-II (Figure 3B) prior to cleavage furrow ingression as shown by immunofluorescence analysis using HeLa cells. Dox-induced Flag-MKlp2(1-890) completely co-localized with Aurora B at the cell equator (Figure S3A, S3B), supporting the hypothesis that Aurora B is the mitotic cargo of MKlp2 [9]. Moreover, similar to endogenous MKlp2, Flag-MKlp2(1-890) accumulated at the equatorial cortex and the spindle midzone (Figure 3C, panel a). In contrast, Flag-MKlp2(1-842) selectively failed to accumulate at the equatorial cortex, while it efficiently localized to the spindle midzone (Figure 3C, panel b). This result is consistent with the finding that Flag-MKlp2(1-842) was selectively defective in binding myosin-II but not other known interacting partners of MKlp2 (Figure S2). Interestingly, the RacGAP1 centralspindlin component comparably localized to the spindle midzone in cells expressing Flag-MKlp2(1-890) and Flag-MKlp2(1-842) (Figure S3C, S3D). Together, these results suggest that the MKlp2:myosin-II interaction is likely required for the accumulation of MKlp2 at the equatorial cortex.

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