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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 is a novel binding partner of myosin-II in vivo and in vitro.(A) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. (B–D) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. (E) Autoradiography of in vitro-translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total in vitro-translated product is shown. (F) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with in vitro-polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.
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pone-0064826-g002: MKlp2 is a novel binding partner of myosin-II in vivo and in vitro.(A) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. (B–D) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. (E) Autoradiography of in vitro-translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total in vitro-translated product is shown. (F) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with in vitro-polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.

Mentions: Endogenous (Figure 1D, panel a) and Dox-induced Flag-MKlp2 (Figure S1D) accumulated at the equatorial cortex in addition to the spindle midzone, suggesting that MKlp2 may function in furrow ingression at the equatorial cortex. To determine the potential MKlp2-mediated mechanisms(s) involved in furrow ingression at the equatorial cortex, we searched for binding partner(s) of MKlp2 by performing affinity purification of stably expressed Flag-MKlp2 using the HEK293 cell line. Using mass spectrometry analysis, non-muscle myosin-II-A (24 unique peptides) and myosin-II-B (30 unique peptides), herein referred to as myosin-II, were identified in immunoprecipitates from Flag-MKlp2 but not in control cells (data not shown). Indeed, using immunoprecipitation analysis, endogenous MKlp2 and myosin-II were precipitated together in a reciprocal manner (Figure 2A). Notably, endogenous myosin-II was co-precipitated with HA-tagged MKlp2 but not MKlp1 (Figure 2B). Moreover, compared with full-length HA-MKlp2(1-890), HA-MKlp2(1-842) failed to bind GFP-tagged myosin-II (Figure 2C). Conversely, HA-MKlp2(1-890) bound strongly to the neck domain (a.a. 779-1087) and weakly to the tail domain (a.a. 1088-1961) of myosin-II (Figure 2D). Notably, the head domain (a.a. 1-778) of myosin-I, which is responsible for binding filamentous actin, was not found to interact with MKlp2, suggesting that the interaction between MKlp2 and myosin-II was not due to the ability of myosin-II to bind filamentous actin. Specifically, HA-MKlp2(1-842) did not bind myosin-II (Figure 2D); however, the ability of HA-MKlp2(1-842) to bind microtubules, Aurora B and Plk1 was intact and comparable to HA-MKlp2(1-890) (Figure S2). Furthermore, the in vitro-translated neck domain of Myc-tagged myosin-II bound recombinant GST-MKlp2(1-890) but not GST-MKlp2(1-842) (Figure 2E), suggesting that MKlp2 is a binding partner of myosin-II in vivo and in vitro. Consistent with its ability to bind myosin-II, the majority of HA-MKlp2(1-890) and HA-MKlp2 (1-870) co-sedimented with in vitro polymerized F-actin but not MKlp2(1-842) (Figure 2F), suggesting that MKlp2 forms a complex with actomyosin filaments.


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 is a novel binding partner of myosin-II in vivo and in vitro.(A) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. (B–D) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. (E) Autoradiography of in vitro-translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total in vitro-translated product is shown. (F) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with in vitro-polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672163&req=5

pone-0064826-g002: MKlp2 is a novel binding partner of myosin-II in vivo and in vitro.(A) Asynchronously growing HeLa cells were harvested and subjected to immunoprecipitation analysis using antibodies for pre-immune control IgG (lanes 1, 4), myosin-II (lanes 2, 3), and MKlp2 with (lane 5) or without (lane 3) HeLa cell lysates. Note that the 100 kDa band is specific for MKlp2 (lane 2) and not caused by α-Myosin-II antibodies used for immunoprecipitation as it is not detected in lane 3. N.S. indicates non-specific. (B–D) Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated MKlp2 or myosin-II proteins were subjected to immunoprecipitation with the indicated antibodies. (E) Autoradiography of in vitro-translated Myc-Myosin-II precipitates with the indicated GST-MKlp2 proteins using GST-pulldown analysis (bottom, visualized with Coomassie Blue staining). Overall, 10% of the input for total in vitro-translated product is shown. (F) F-actin binding assay. Asynchronously growing HeLa cells were transfected with the indicated expression plasmids, and 24 h after transfection, HeLa cell lysates expressing the indicated HA-MKlp2 proteins were supplemented without (lanes 1-6) or with in vitro-polymerized recombinant F-actin (lanes 7-12) and subjected to ultracentrifugation. Supernatant (S) and pellet (P) fractions were subjected to immunoblot analysis.
Mentions: Endogenous (Figure 1D, panel a) and Dox-induced Flag-MKlp2 (Figure S1D) accumulated at the equatorial cortex in addition to the spindle midzone, suggesting that MKlp2 may function in furrow ingression at the equatorial cortex. To determine the potential MKlp2-mediated mechanisms(s) involved in furrow ingression at the equatorial cortex, we searched for binding partner(s) of MKlp2 by performing affinity purification of stably expressed Flag-MKlp2 using the HEK293 cell line. Using mass spectrometry analysis, non-muscle myosin-II-A (24 unique peptides) and myosin-II-B (30 unique peptides), herein referred to as myosin-II, were identified in immunoprecipitates from Flag-MKlp2 but not in control cells (data not shown). Indeed, using immunoprecipitation analysis, endogenous MKlp2 and myosin-II were precipitated together in a reciprocal manner (Figure 2A). Notably, endogenous myosin-II was co-precipitated with HA-tagged MKlp2 but not MKlp1 (Figure 2B). Moreover, compared with full-length HA-MKlp2(1-890), HA-MKlp2(1-842) failed to bind GFP-tagged myosin-II (Figure 2C). Conversely, HA-MKlp2(1-890) bound strongly to the neck domain (a.a. 779-1087) and weakly to the tail domain (a.a. 1088-1961) of myosin-II (Figure 2D). Notably, the head domain (a.a. 1-778) of myosin-I, which is responsible for binding filamentous actin, was not found to interact with MKlp2, suggesting that the interaction between MKlp2 and myosin-II was not due to the ability of myosin-II to bind filamentous actin. Specifically, HA-MKlp2(1-842) did not bind myosin-II (Figure 2D); however, the ability of HA-MKlp2(1-842) to bind microtubules, Aurora B and Plk1 was intact and comparable to HA-MKlp2(1-890) (Figure S2). Furthermore, the in vitro-translated neck domain of Myc-tagged myosin-II bound recombinant GST-MKlp2(1-890) but not GST-MKlp2(1-842) (Figure 2E), suggesting that MKlp2 is a binding partner of myosin-II in vivo and in vitro. Consistent with its ability to bind myosin-II, the majority of HA-MKlp2(1-890) and HA-MKlp2 (1-870) co-sedimented with in vitro polymerized F-actin but not MKlp2(1-842) (Figure 2F), suggesting that MKlp2 forms a complex with actomyosin filaments.

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