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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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Plk1 triggers C-Nap1 displacement from centrosomes in Cdk1-inhibited cells. (A) Immuno-fluorescent staining of HeLa cells treated for 20 h with either 10 μM RO3306 or 10 μM RO3306 and 100 nM BI2536 with C-Nap1 and Pericentrin antibodies. Panels show MIPS of deconvolved 3D stacks (scale bar, 15 μm). The right panels show C-Nap1 staining of a magnified area around the centrosome as indicated. (B) C-Nap1 immuno-blots of HeLa cells treated as in (A). (C) Immuno-blot of C-Nap1 siRNA transfected and control cells with the indicated antibodies. (D) Confirmation of C-Nap1 knockdown by siRNA in RO3066-treated HeLa cells by immuno-fluorescence (scale bar, 5 μm). (E) Centrosome separation of C-Nap1 siRNA transfected and control cells. Separation and distances were scored from 3D stack images using Imaris. Shown are the mean values and s.d. of three independent experiments (N>100 for each condition).
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f4: Plk1 triggers C-Nap1 displacement from centrosomes in Cdk1-inhibited cells. (A) Immuno-fluorescent staining of HeLa cells treated for 20 h with either 10 μM RO3306 or 10 μM RO3306 and 100 nM BI2536 with C-Nap1 and Pericentrin antibodies. Panels show MIPS of deconvolved 3D stacks (scale bar, 15 μm). The right panels show C-Nap1 staining of a magnified area around the centrosome as indicated. (B) C-Nap1 immuno-blots of HeLa cells treated as in (A). (C) Immuno-blot of C-Nap1 siRNA transfected and control cells with the indicated antibodies. (D) Confirmation of C-Nap1 knockdown by siRNA in RO3066-treated HeLa cells by immuno-fluorescence (scale bar, 5 μm). (E) Centrosome separation of C-Nap1 siRNA transfected and control cells. Separation and distances were scored from 3D stack images using Imaris. Shown are the mean values and s.d. of three independent experiments (N>100 for each condition).

Mentions: We first investigated the role of Plk1 in centrosome disjunction. This process is initially triggered by loss of cohesive proteins that hold the duplicated centriole pairs together. C-Nap1 has been reported to form the core of this structure and depletion of this protein is sufficient to trigger centrosome splitting (Mayor et al, 2000; Bahe et al, 2005). We reasoned that if Plk1 acts upstream of C-Nap1 displacement, this protein should still be present on the centrosomes when both Cdk1 and Plk1 are inhibited. Conversely, inhibition of Cdk1 alone should result in displacement of C-Nap1 from the centrosomes. Figure 4A shows that C-Nap1 is not detectable on centrosomes in RO3306-treated HeLa cells, while it is localized at centrosomes in cells treated with both Cdk1 and Plk1 inhibitors. Previous studies reported a decrease in C-Nap1 levels in mitotic cells (Mayor et al, 2002). It was reported that this reduction is not due to proteasome-dependent degradation of C-Nap1 but may be a consequence of mitotic-specific phosphorylation. We found that C-Nap1 levels were significantly reduced in Cdk1-inhibited cells compared with an asynchronous control (Figure 4B). However, Plk1 inhibition did not alter this reduction of C-Nap1 levels. These results suggest that while Plk1 acts specifically on the displacement of C-Nap1 from the centrosomes, the changes in total protein levels occur in G2 phase independently of both Plk1 and Cdk1.


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)

Plk1 triggers C-Nap1 displacement from centrosomes in Cdk1-inhibited cells. (A) Immuno-fluorescent staining of HeLa cells treated for 20 h with either 10 μM RO3306 or 10 μM RO3306 and 100 nM BI2536 with C-Nap1 and Pericentrin antibodies. Panels show MIPS of deconvolved 3D stacks (scale bar, 15 μm). The right panels show C-Nap1 staining of a magnified area around the centrosome as indicated. (B) C-Nap1 immuno-blots of HeLa cells treated as in (A). (C) Immuno-blot of C-Nap1 siRNA transfected and control cells with the indicated antibodies. (D) Confirmation of C-Nap1 knockdown by siRNA in RO3066-treated HeLa cells by immuno-fluorescence (scale bar, 5 μm). (E) Centrosome separation of C-Nap1 siRNA transfected and control cells. Separation and distances were scored from 3D stack images using Imaris. Shown are the mean values and s.d. of three independent experiments (N>100 for each condition).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Plk1 triggers C-Nap1 displacement from centrosomes in Cdk1-inhibited cells. (A) Immuno-fluorescent staining of HeLa cells treated for 20 h with either 10 μM RO3306 or 10 μM RO3306 and 100 nM BI2536 with C-Nap1 and Pericentrin antibodies. Panels show MIPS of deconvolved 3D stacks (scale bar, 15 μm). The right panels show C-Nap1 staining of a magnified area around the centrosome as indicated. (B) C-Nap1 immuno-blots of HeLa cells treated as in (A). (C) Immuno-blot of C-Nap1 siRNA transfected and control cells with the indicated antibodies. (D) Confirmation of C-Nap1 knockdown by siRNA in RO3066-treated HeLa cells by immuno-fluorescence (scale bar, 5 μm). (E) Centrosome separation of C-Nap1 siRNA transfected and control cells. Separation and distances were scored from 3D stack images using Imaris. Shown are the mean values and s.d. of three independent experiments (N>100 for each condition).
Mentions: We first investigated the role of Plk1 in centrosome disjunction. This process is initially triggered by loss of cohesive proteins that hold the duplicated centriole pairs together. C-Nap1 has been reported to form the core of this structure and depletion of this protein is sufficient to trigger centrosome splitting (Mayor et al, 2000; Bahe et al, 2005). We reasoned that if Plk1 acts upstream of C-Nap1 displacement, this protein should still be present on the centrosomes when both Cdk1 and Plk1 are inhibited. Conversely, inhibition of Cdk1 alone should result in displacement of C-Nap1 from the centrosomes. Figure 4A shows that C-Nap1 is not detectable on centrosomes in RO3306-treated HeLa cells, while it is localized at centrosomes in cells treated with both Cdk1 and Plk1 inhibitors. Previous studies reported a decrease in C-Nap1 levels in mitotic cells (Mayor et al, 2002). It was reported that this reduction is not due to proteasome-dependent degradation of C-Nap1 but may be a consequence of mitotic-specific phosphorylation. We found that C-Nap1 levels were significantly reduced in Cdk1-inhibited cells compared with an asynchronous control (Figure 4B). However, Plk1 inhibition did not alter this reduction of C-Nap1 levels. These results suggest that while Plk1 acts specifically on the displacement of C-Nap1 from the centrosomes, the changes in total protein levels occur in G2 phase independently of both Plk1 and Cdk1.

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