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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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Loss of Plk1 from the central spindle in MKlp2-depleted cells. (A) HeLa cells fixed with PFA were stained with antibodies to Plk1 (red) and MKlp2 (green), and DNA was stained with DAPI (blue). Colocalization of Plk1 and MKlp2 was observed from early anaphase until cytokinesis, with the difference that MKlp2 was absent from kinetochores, unlike Plk1 (see the contrast-enhanced boxed area). (B–D) HeLa S3 cells treated with control lamin A and MKlp2 siRNA duplexes were fixed with methanol and then stained with antibodies to MKlp2, MKlp1, α-tubulin, Plk1, and anillin. Bar, 10 μM.
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fig2: Loss of Plk1 from the central spindle in MKlp2-depleted cells. (A) HeLa cells fixed with PFA were stained with antibodies to Plk1 (red) and MKlp2 (green), and DNA was stained with DAPI (blue). Colocalization of Plk1 and MKlp2 was observed from early anaphase until cytokinesis, with the difference that MKlp2 was absent from kinetochores, unlike Plk1 (see the contrast-enhanced boxed area). (B–D) HeLa S3 cells treated with control lamin A and MKlp2 siRNA duplexes were fixed with methanol and then stained with antibodies to MKlp2, MKlp1, α-tubulin, Plk1, and anillin. Bar, 10 μM.

Mentions: A survey of central spindle components revealed that MKlp2 showed extensive colocalization with Plk1 on central spindle microtubules during anaphase and telophase (Fig. 2 A), discrete from components such as PRC1 (Fig. S2 A). The effects of MKlp2 depletion on the localization of MKlp1, PRC1, and Plk1 were then examined. Control cells depleted for lamin A showed normal staining for MKlp1, MKlp2, and Plk1 (Fig. 2, B and C). Although MKlp1 and PRC1 stainings showed some abnormalities in MKlp2-depleted cells, they were still present in the central spindle region, indicating that this structure was not totally absent in these cells (Fig. 2 B and Fig. S2; summarized in Table S1). In contrast, Plk1 was absent from the central spindle in MKlp2-depleted cells during anaphase and telophase (Fig. 2 C). This was not due to the destabilization of central spindle components in general because Plk1 and MKlp1 were present at the same level in control lamin A–depleted and MKlp2-depleted cells (Fig. 1 A). Additionally, in PRC1-depleted cells, MKlp2, Plk1, and survivin still localized to the central spindle, although the staining patterns were more disorganized than in control cells (Table S1). Because cleavage furrow ingression fails on MKlp2 depletion, cells were stained for anillin and septins to find out if the cleavage furrow was disrupted (Fig. 2 D). Anillin was still localized in a cortical band around the center of the cell in MKlp2-depleted cells, although late anaphase cells did not show the typical pinched furrow staining (Fig. 2 D). Similar results were obtained for septin localization (unpublished data). Therefore, MKlp2 depletion affects the localization of a subset of central spindle components, including Plk1, but does not affect the recruitment of cleavage furrow components such as anillin.


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)

Loss of Plk1 from the central spindle in MKlp2-depleted cells. (A) HeLa cells fixed with PFA were stained with antibodies to Plk1 (red) and MKlp2 (green), and DNA was stained with DAPI (blue). Colocalization of Plk1 and MKlp2 was observed from early anaphase until cytokinesis, with the difference that MKlp2 was absent from kinetochores, unlike Plk1 (see the contrast-enhanced boxed area). (B–D) HeLa S3 cells treated with control lamin A and MKlp2 siRNA duplexes were fixed with methanol and then stained with antibodies to MKlp2, MKlp1, α-tubulin, Plk1, and anillin. Bar, 10 μM.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172827&req=5

fig2: Loss of Plk1 from the central spindle in MKlp2-depleted cells. (A) HeLa cells fixed with PFA were stained with antibodies to Plk1 (red) and MKlp2 (green), and DNA was stained with DAPI (blue). Colocalization of Plk1 and MKlp2 was observed from early anaphase until cytokinesis, with the difference that MKlp2 was absent from kinetochores, unlike Plk1 (see the contrast-enhanced boxed area). (B–D) HeLa S3 cells treated with control lamin A and MKlp2 siRNA duplexes were fixed with methanol and then stained with antibodies to MKlp2, MKlp1, α-tubulin, Plk1, and anillin. Bar, 10 μM.
Mentions: A survey of central spindle components revealed that MKlp2 showed extensive colocalization with Plk1 on central spindle microtubules during anaphase and telophase (Fig. 2 A), discrete from components such as PRC1 (Fig. S2 A). The effects of MKlp2 depletion on the localization of MKlp1, PRC1, and Plk1 were then examined. Control cells depleted for lamin A showed normal staining for MKlp1, MKlp2, and Plk1 (Fig. 2, B and C). Although MKlp1 and PRC1 stainings showed some abnormalities in MKlp2-depleted cells, they were still present in the central spindle region, indicating that this structure was not totally absent in these cells (Fig. 2 B and Fig. S2; summarized in Table S1). In contrast, Plk1 was absent from the central spindle in MKlp2-depleted cells during anaphase and telophase (Fig. 2 C). This was not due to the destabilization of central spindle components in general because Plk1 and MKlp1 were present at the same level in control lamin A–depleted and MKlp2-depleted cells (Fig. 1 A). Additionally, in PRC1-depleted cells, MKlp2, Plk1, and survivin still localized to the central spindle, although the staining patterns were more disorganized than in control cells (Table S1). Because cleavage furrow ingression fails on MKlp2 depletion, cells were stained for anillin and septins to find out if the cleavage furrow was disrupted (Fig. 2 D). Anillin was still localized in a cortical band around the center of the cell in MKlp2-depleted cells, although late anaphase cells did not show the typical pinched furrow staining (Fig. 2 D). Similar results were obtained for septin localization (unpublished data). Therefore, MKlp2 depletion affects the localization of a subset of central spindle components, including Plk1, but does not affect the recruitment of cleavage furrow components such as anillin.

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.

Show MeSH
Related in: MedlinePlus