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Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis.

Neef R, Preisinger C, Sutcliffe J, Kopajtich R, Nigg EA, Mayer TU, Barr FA - J. Cell Biol. (2003)

Bottom Line: We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1).An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells.We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Intracellular Protein Transport, Independent Junior Research Group, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

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MKlp2 is required for normal cell division. (A) HeLa S3 cells were treated with the MKlp2 siRNA duplex for 36 h and the lamin A siRNA duplex for 72 h, arrested with 90 ng/ml nocodazole for 12 h to enrich for mitotic cells that express MKlp2, and then lysed in sample buffer. Western blot analysis of 25 μg protein was performed with antibodies to MKlp2, lamin A, MKlp1, Plk1, and α-tubulin. (B) The number of binucleated or multinucleated cells as a percentage of the total cell population was quantitated in HeLa S3 cells treated with lamin A or MKlp2 siRNA duplexes for the times indicated (n = 400 cells). (C) Live-cell imaging of HeLa S3 cells treated with control lamin A or (D) MKlp2 siRNA duplexes for 24 h. Representative frames reflecting the different stages of mitosis in these cells are shown, and times are indicated in h:min. Arrows indicate the position of the cleavage furrow; note the partial and asymmetric furrow ingression in MKlp2-depleted cells. Bar, 10 μM.
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fig1: MKlp2 is required for normal cell division. (A) HeLa S3 cells were treated with the MKlp2 siRNA duplex for 36 h and the lamin A siRNA duplex for 72 h, arrested with 90 ng/ml nocodazole for 12 h to enrich for mitotic cells that express MKlp2, and then lysed in sample buffer. Western blot analysis of 25 μg protein was performed with antibodies to MKlp2, lamin A, MKlp1, Plk1, and α-tubulin. (B) The number of binucleated or multinucleated cells as a percentage of the total cell population was quantitated in HeLa S3 cells treated with lamin A or MKlp2 siRNA duplexes for the times indicated (n = 400 cells). (C) Live-cell imaging of HeLa S3 cells treated with control lamin A or (D) MKlp2 siRNA duplexes for 24 h. Representative frames reflecting the different stages of mitosis in these cells are shown, and times are indicated in h:min. Arrows indicate the position of the cleavage furrow; note the partial and asymmetric furrow ingression in MKlp2-depleted cells. Bar, 10 μM.

Mentions: Microinjection of antibodies to MKlp2 into HeLa cells results in a cytokinesis defect and the production of binucleated cells (Hill et al., 2000). To demonstrate that this is due to an essential requirement for MKlp2 in cytokinesis, and not simply a result of the formation of a dominant-negative antibody–MKlp2 complex, depletion analyses using small interfering RNAs (siRNAs) were performed. MKlp2 and the control target lamin A can be depleted by specific siRNAs (Fig. 1 A). MKlp2-depleted cells show a highly penetrant cytokinesis defect, and after 48 h, nearly 90% of cells were binucleated (Fig. 1 B and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200306009/DC1), whereas in lamin A–depleted cells, the levels and localization of MKlp2 were normal, and no cytokinesis defect was observed (Fig. 1 and Fig. S1). Live-cell imaging revealed that MKlp2-depleted cells aligned and segregated their chromosomes normally, but had a defect in both the extent and timing of cleavage furrow ingression during anaphase and failed to perform cytokinesis, whereas control cells depleted for lamin A divided normally (Fig. 1, C and D). Therefore, MKlp2 is required for the normal cell division of human cells, specifically for normal cleavage furrow ingression and cytokinesis.


Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis.

Neef R, Preisinger C, Sutcliffe J, Kopajtich R, Nigg EA, Mayer TU, Barr FA - J. Cell Biol. (2003)

MKlp2 is required for normal cell division. (A) HeLa S3 cells were treated with the MKlp2 siRNA duplex for 36 h and the lamin A siRNA duplex for 72 h, arrested with 90 ng/ml nocodazole for 12 h to enrich for mitotic cells that express MKlp2, and then lysed in sample buffer. Western blot analysis of 25 μg protein was performed with antibodies to MKlp2, lamin A, MKlp1, Plk1, and α-tubulin. (B) The number of binucleated or multinucleated cells as a percentage of the total cell population was quantitated in HeLa S3 cells treated with lamin A or MKlp2 siRNA duplexes for the times indicated (n = 400 cells). (C) Live-cell imaging of HeLa S3 cells treated with control lamin A or (D) MKlp2 siRNA duplexes for 24 h. Representative frames reflecting the different stages of mitosis in these cells are shown, and times are indicated in h:min. Arrows indicate the position of the cleavage furrow; note the partial and asymmetric furrow ingression in MKlp2-depleted cells. Bar, 10 μM.
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Related In: Results  -  Collection

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fig1: MKlp2 is required for normal cell division. (A) HeLa S3 cells were treated with the MKlp2 siRNA duplex for 36 h and the lamin A siRNA duplex for 72 h, arrested with 90 ng/ml nocodazole for 12 h to enrich for mitotic cells that express MKlp2, and then lysed in sample buffer. Western blot analysis of 25 μg protein was performed with antibodies to MKlp2, lamin A, MKlp1, Plk1, and α-tubulin. (B) The number of binucleated or multinucleated cells as a percentage of the total cell population was quantitated in HeLa S3 cells treated with lamin A or MKlp2 siRNA duplexes for the times indicated (n = 400 cells). (C) Live-cell imaging of HeLa S3 cells treated with control lamin A or (D) MKlp2 siRNA duplexes for 24 h. Representative frames reflecting the different stages of mitosis in these cells are shown, and times are indicated in h:min. Arrows indicate the position of the cleavage furrow; note the partial and asymmetric furrow ingression in MKlp2-depleted cells. Bar, 10 μM.
Mentions: Microinjection of antibodies to MKlp2 into HeLa cells results in a cytokinesis defect and the production of binucleated cells (Hill et al., 2000). To demonstrate that this is due to an essential requirement for MKlp2 in cytokinesis, and not simply a result of the formation of a dominant-negative antibody–MKlp2 complex, depletion analyses using small interfering RNAs (siRNAs) were performed. MKlp2 and the control target lamin A can be depleted by specific siRNAs (Fig. 1 A). MKlp2-depleted cells show a highly penetrant cytokinesis defect, and after 48 h, nearly 90% of cells were binucleated (Fig. 1 B and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200306009/DC1), whereas in lamin A–depleted cells, the levels and localization of MKlp2 were normal, and no cytokinesis defect was observed (Fig. 1 and Fig. S1). Live-cell imaging revealed that MKlp2-depleted cells aligned and segregated their chromosomes normally, but had a defect in both the extent and timing of cleavage furrow ingression during anaphase and failed to perform cytokinesis, whereas control cells depleted for lamin A divided normally (Fig. 1, C and D). Therefore, MKlp2 is required for the normal cell division of human cells, specifically for normal cleavage furrow ingression and cytokinesis.

Bottom Line: We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1).An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells.We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Intracellular Protein Transport, Independent Junior Research Group, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
We have investigated the function of mitotic kinesin-like protein (MKlp) 2, a kinesin localized to the central spindle, and demonstrate that its depletion results in a failure of cleavage furrow ingression and cytokinesis, and disrupts localization of polo-like kinase 1 (Plk1). MKlp2 is a target for Plk1, and phosphorylated MKlp2 binds to the polo box domain of Plk1. Plk1 also binds directly to microtubules and targets to the central spindle via its polo box domain, and this interaction controls the activity of Plk1 toward MKlp2. An antibody to the neck region of MKlp2 that prevents phosphorylation of MKlp2 by Plk1 causes a cytokinesis defect when introduced into cells. We propose that phosphorylation of MKlp2 by Plk1 is necessary for the spatial restriction of Plk1 to the central spindle during anaphase and telophase, and the complex of these two proteins is required for cytokinesis.

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