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Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.

Smith E, Hégarat N, Vesely C, Roseboom I, Larch C, Streicher H, Straatman K, Flynn H, Skehel M, Hirota T, Kuriyama R, Hochegger H - EMBO J. (2011)

Bottom Line: Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927.Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation.Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, UK.

ABSTRACT
Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.

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Dynamics of Plk1 and Cdk1 induced centrosome separation in live cells. (A) 3D time-lapse microscopy of differentially released cdk1as cells expressing GFP-γ-tubulin (see Materials and methods). The still images are MIPs of deconvolved 3D images (scale bar, 5 μm) from a time-lapse series (see also Supplementary Movies 01, 02, 03). (B) Quantification of centrosome distance for each release experiment. Distances were measured using Imaris in the deconvolved time-lapse series. Note the difference in the time scale between the different panels. Disjunction was defined as the first time point at which a distance of >1 μm was reached. Velocities were calculated as described in Materials and methods.
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f3: Dynamics of Plk1 and Cdk1 induced centrosome separation in live cells. (A) 3D time-lapse microscopy of differentially released cdk1as cells expressing GFP-γ-tubulin (see Materials and methods). The still images are MIPs of deconvolved 3D images (scale bar, 5 μm) from a time-lapse series (see also Supplementary Movies 01, 02, 03). (B) Quantification of centrosome distance for each release experiment. Distances were measured using Imaris in the deconvolved time-lapse series. Note the difference in the time scale between the different panels. Disjunction was defined as the first time point at which a distance of >1 μm was reached. Velocities were calculated as described in Materials and methods.

Mentions: In order to observe the dynamics of disjunction and separation in single cells, we performed a parallel experiment using 3D live cell imaging of GFP-γ-tubulin-expressing cdk1as cells. Similar to the results described above, we observed a striking difference between centrosome separation triggered by Plk1 and Cdk1. If Cdk1 remained inactive, centrosome disjunction occurred on average an hour after release from BI2536 (Figure 3A and B; Supplement Movie S2). Moreover, movement of the centrosomes was not linear, but pulling and pushing forces appeared to compete with each other. We determined an average centrosome velocity of 0.04 μm/min in these cells. Activation of Cdk1 caused a dramatic change in dynamics of both disjunction and movement. The centrosomes came apart within minutes and had undergone considerable separation within the first 5 min after release (Figure 3A and B; Supplement Movie S1). The initial dynamics of Cdk1-driven centrosome separation were only modestly changed in the presence of the Plk1 inhibitor. Disjunction occurred with a brief delay at about 5 min after release, and the average velocity of separation was decreased from 1.1 μm/min when both Plk1 and Cdk1 were active to 0.5 μm/min when Plk1 was kept shut off. Similar to our observations in Figure 2, Plk1 inhibition caused a dispersal of GFP-γ-tubulin from the centrosomes after NEBD and it became progressively harder to detect the GFP signal on the spindle poles (Figure 3A; Supplement Movie S3).


Differential control of Eg5-dependent centrosome separation by Plk1 and Cdk1.

Smith E, Hégarat N, Vesely C, Roseboom I, Larch C, Streicher H, Straatman K, Flynn H, Skehel M, Hirota T, Kuriyama R, Hochegger H - EMBO J. (2011)

Dynamics of Plk1 and Cdk1 induced centrosome separation in live cells. (A) 3D time-lapse microscopy of differentially released cdk1as cells expressing GFP-γ-tubulin (see Materials and methods). The still images are MIPs of deconvolved 3D images (scale bar, 5 μm) from a time-lapse series (see also Supplementary Movies 01, 02, 03). (B) Quantification of centrosome distance for each release experiment. Distances were measured using Imaris in the deconvolved time-lapse series. Note the difference in the time scale between the different panels. Disjunction was defined as the first time point at which a distance of >1 μm was reached. Velocities were calculated as described in Materials and methods.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3117641&req=5

f3: Dynamics of Plk1 and Cdk1 induced centrosome separation in live cells. (A) 3D time-lapse microscopy of differentially released cdk1as cells expressing GFP-γ-tubulin (see Materials and methods). The still images are MIPs of deconvolved 3D images (scale bar, 5 μm) from a time-lapse series (see also Supplementary Movies 01, 02, 03). (B) Quantification of centrosome distance for each release experiment. Distances were measured using Imaris in the deconvolved time-lapse series. Note the difference in the time scale between the different panels. Disjunction was defined as the first time point at which a distance of >1 μm was reached. Velocities were calculated as described in Materials and methods.
Mentions: In order to observe the dynamics of disjunction and separation in single cells, we performed a parallel experiment using 3D live cell imaging of GFP-γ-tubulin-expressing cdk1as cells. Similar to the results described above, we observed a striking difference between centrosome separation triggered by Plk1 and Cdk1. If Cdk1 remained inactive, centrosome disjunction occurred on average an hour after release from BI2536 (Figure 3A and B; Supplement Movie S2). Moreover, movement of the centrosomes was not linear, but pulling and pushing forces appeared to compete with each other. We determined an average centrosome velocity of 0.04 μm/min in these cells. Activation of Cdk1 caused a dramatic change in dynamics of both disjunction and movement. The centrosomes came apart within minutes and had undergone considerable separation within the first 5 min after release (Figure 3A and B; Supplement Movie S1). The initial dynamics of Cdk1-driven centrosome separation were only modestly changed in the presence of the Plk1 inhibitor. Disjunction occurred with a brief delay at about 5 min after release, and the average velocity of separation was decreased from 1.1 μm/min when both Plk1 and Cdk1 were active to 0.5 μm/min when Plk1 was kept shut off. Similar to our observations in Figure 2, Plk1 inhibition caused a dispersal of GFP-γ-tubulin from the centrosomes after NEBD and it became progressively harder to detect the GFP signal on the spindle poles (Figure 3A; Supplement Movie S3).

Bottom Line: Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927.Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation.Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement.

View Article: PubMed Central - PubMed

Affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, UK.

ABSTRACT
Cyclin-dependent kinase 1 (Cdk1) is thought to trigger centrosome separation in late G2 phase by phosphorylating the motor protein Eg5 at Thr927. However, the precise control mechanism of centrosome separation remains to be understood. Here, we report that in G2 phase polo-like kinase 1 (Plk1) can trigger centrosome separation independently of Cdk1. We find that Plk1 is required for both C-Nap1 displacement and for Eg5 localization on the centrosome. Moreover, Cdk2 compensates for Cdk1, and phosphorylates Eg5 at Thr927. Nevertheless, Plk1-driven centrosome separation is slow and staggering, while Cdk1 triggers fast movement of the centrosomes. We find that actin-dependent Eg5-opposing forces slow down separation in G2 phase. Strikingly, actin depolymerization, as well as destabilization of interphase microtubules (MTs), is sufficient to remove this obstruction and to speed up Plk1-dependent separation. Conversely, MT stabilization in mitosis slows down Cdk1-dependent centrosome movement. Our findings implicate the modulation of MT stability in G2 and M phase as a regulatory element in the control of centrosome separation.

Show MeSH
Related in: MedlinePlus