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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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Cdk1-independent centrosome separation requires Plk1 and Eg5 activity. (A) DT40 cdk1as cells were analysed by immuno-fluorescence using anti-γ-tubulin and anti-centrin-2 antibodies and counterstained with DAPI. The panels display deconvolved maximum intensity projections (MIPs) of 3D images of representative samples (scale bar, 5 μm). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 6 h with 10 μM 1NMPP1 (1NM). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as 1NMPP1 (1NM+BI). To inhibit chicken Eg5, we added 33 μM trans-24 together with 1NMPP1 (1NM+Trans). (B) Quantitative analysis of centrosome separation using immuno-fluorescence and automated scanning microscope analysis (Olympus SCAN-R; see Material and methods). As., N=962; 1NM, N=1300; 1NM+BI, N=569; 1NM+Trans, N=638; error bars indicate the s.d. in three independent experiments. We scored centrosome distances in the same samples by analysing 3D images using IMARIS (N=50); distances above 0.5 μm were scored as separated; results from individual cells are plotted; the bars show the mean distances (As., 3.4 μm; 1NM, 5.25 μm; 1NM+BI, 1.8 μm; 1NM+Trans, 1.84 μm). (C) PI staining and FACS analysis of same samples as in (A). (D) HeLa cells were analysed by immuno-fluorescence using anti-γ-tubulin, anti-pericentrin antibodies and DAPI. The panels display deconvolved MIPs of 3D images of representative samples (scale bar, 10 μM). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 20 h with 7.5 μM RO3306 (RO). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as RO 3066 (RO+BI). To inhibit human Eg5, we added 5 μM STLC together with RO3306 (RO+STLC). (E) Quantitative analysis of 3D images (% separation As., N=524; RO, N=343; RO+BI, N=415; RO+STLC, N=380; error bars indicate the s.d. in three independent experiments). Distances were scored in 3D images using Imaris (N=50) as in (B); the bars indicate the mean distance; As., 5.7 μm; RO, 9.2 μm; RO+BI, 4.6 μm; RO+STLC, 4.1 μm. (F) FACS analysis of the indicated HeLa samples.
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f1: Cdk1-independent centrosome separation requires Plk1 and Eg5 activity. (A) DT40 cdk1as cells were analysed by immuno-fluorescence using anti-γ-tubulin and anti-centrin-2 antibodies and counterstained with DAPI. The panels display deconvolved maximum intensity projections (MIPs) of 3D images of representative samples (scale bar, 5 μm). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 6 h with 10 μM 1NMPP1 (1NM). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as 1NMPP1 (1NM+BI). To inhibit chicken Eg5, we added 33 μM trans-24 together with 1NMPP1 (1NM+Trans). (B) Quantitative analysis of centrosome separation using immuno-fluorescence and automated scanning microscope analysis (Olympus SCAN-R; see Material and methods). As., N=962; 1NM, N=1300; 1NM+BI, N=569; 1NM+Trans, N=638; error bars indicate the s.d. in three independent experiments. We scored centrosome distances in the same samples by analysing 3D images using IMARIS (N=50); distances above 0.5 μm were scored as separated; results from individual cells are plotted; the bars show the mean distances (As., 3.4 μm; 1NM, 5.25 μm; 1NM+BI, 1.8 μm; 1NM+Trans, 1.84 μm). (C) PI staining and FACS analysis of same samples as in (A). (D) HeLa cells were analysed by immuno-fluorescence using anti-γ-tubulin, anti-pericentrin antibodies and DAPI. The panels display deconvolved MIPs of 3D images of representative samples (scale bar, 10 μM). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 20 h with 7.5 μM RO3306 (RO). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as RO 3066 (RO+BI). To inhibit human Eg5, we added 5 μM STLC together with RO3306 (RO+STLC). (E) Quantitative analysis of 3D images (% separation As., N=524; RO, N=343; RO+BI, N=415; RO+STLC, N=380; error bars indicate the s.d. in three independent experiments). Distances were scored in 3D images using Imaris (N=50) as in (B); the bars indicate the mean distance; As., 5.7 μm; RO, 9.2 μm; RO+BI, 4.6 μm; RO+STLC, 4.1 μm. (F) FACS analysis of the indicated HeLa samples.

Mentions: To clarify the role of Cdk1 in centrosome separation, we took advantage of a cdk1as DT40 cell line that carries an analogue-sensitive mutation in Cdk1 (cdk1as cells). In these cells, the mutant Cdk1 can be inhibited with high specificity by addition of the bulky ATP analogue, 1NMPP1, resulting in a late G2 phase arrest (Figure 1C), while the ATP analogue has no effect on the cell cycle of cells expressing WT Cdk1 (Hochegger et al, 2007). We found that, despite Cdk1 inhibition, centrosomes were clearly separated in about 60% of the 1NMPP1-treated cdk1as cells (Figure 1A and B). To confirm this result in a different experimental system, we used a chemical Cdk1 inhibitor, RO3306 (Vassilev et al, 2006), in HeLa cells, and found that approximately half of the RO3306-treated, G2-arrested cells (Figure 1F) displayed widely separated centrosomes (Figure 1D and E). To compare the timing of centrosome separation in the absence or presence of Cdk1 activity in more detail, we analysed centrosome separation in cdk1as cells that were pre-synchronized in G1 by elutriation and progressed to G2/M phase in the presence or absence of Cdk1 inhibition by 1NMPP1. Supplementary Figure S1A shows that centrosomes separated while cells progressed into G2/M. However, separation was delayed by approximately 2 h in the 1NMPP1-treated cells. We conclude from these results that Cdk1 is not strictly essential for centrosome separation, but is required for timely initiation of the process.


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)

Cdk1-independent centrosome separation requires Plk1 and Eg5 activity. (A) DT40 cdk1as cells were analysed by immuno-fluorescence using anti-γ-tubulin and anti-centrin-2 antibodies and counterstained with DAPI. The panels display deconvolved maximum intensity projections (MIPs) of 3D images of representative samples (scale bar, 5 μm). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 6 h with 10 μM 1NMPP1 (1NM). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as 1NMPP1 (1NM+BI). To inhibit chicken Eg5, we added 33 μM trans-24 together with 1NMPP1 (1NM+Trans). (B) Quantitative analysis of centrosome separation using immuno-fluorescence and automated scanning microscope analysis (Olympus SCAN-R; see Material and methods). As., N=962; 1NM, N=1300; 1NM+BI, N=569; 1NM+Trans, N=638; error bars indicate the s.d. in three independent experiments. We scored centrosome distances in the same samples by analysing 3D images using IMARIS (N=50); distances above 0.5 μm were scored as separated; results from individual cells are plotted; the bars show the mean distances (As., 3.4 μm; 1NM, 5.25 μm; 1NM+BI, 1.8 μm; 1NM+Trans, 1.84 μm). (C) PI staining and FACS analysis of same samples as in (A). (D) HeLa cells were analysed by immuno-fluorescence using anti-γ-tubulin, anti-pericentrin antibodies and DAPI. The panels display deconvolved MIPs of 3D images of representative samples (scale bar, 10 μM). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 20 h with 7.5 μM RO3306 (RO). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as RO 3066 (RO+BI). To inhibit human Eg5, we added 5 μM STLC together with RO3306 (RO+STLC). (E) Quantitative analysis of 3D images (% separation As., N=524; RO, N=343; RO+BI, N=415; RO+STLC, N=380; error bars indicate the s.d. in three independent experiments). Distances were scored in 3D images using Imaris (N=50) as in (B); the bars indicate the mean distance; As., 5.7 μm; RO, 9.2 μm; RO+BI, 4.6 μm; RO+STLC, 4.1 μm. (F) FACS analysis of the indicated HeLa samples.
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Related In: Results  -  Collection

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f1: Cdk1-independent centrosome separation requires Plk1 and Eg5 activity. (A) DT40 cdk1as cells were analysed by immuno-fluorescence using anti-γ-tubulin and anti-centrin-2 antibodies and counterstained with DAPI. The panels display deconvolved maximum intensity projections (MIPs) of 3D images of representative samples (scale bar, 5 μm). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 6 h with 10 μM 1NMPP1 (1NM). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as 1NMPP1 (1NM+BI). To inhibit chicken Eg5, we added 33 μM trans-24 together with 1NMPP1 (1NM+Trans). (B) Quantitative analysis of centrosome separation using immuno-fluorescence and automated scanning microscope analysis (Olympus SCAN-R; see Material and methods). As., N=962; 1NM, N=1300; 1NM+BI, N=569; 1NM+Trans, N=638; error bars indicate the s.d. in three independent experiments. We scored centrosome distances in the same samples by analysing 3D images using IMARIS (N=50); distances above 0.5 μm were scored as separated; results from individual cells are plotted; the bars show the mean distances (As., 3.4 μm; 1NM, 5.25 μm; 1NM+BI, 1.8 μm; 1NM+Trans, 1.84 μm). (C) PI staining and FACS analysis of same samples as in (A). (D) HeLa cells were analysed by immuno-fluorescence using anti-γ-tubulin, anti-pericentrin antibodies and DAPI. The panels display deconvolved MIPs of 3D images of representative samples (scale bar, 10 μM). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 20 h with 7.5 μM RO3306 (RO). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as RO 3066 (RO+BI). To inhibit human Eg5, we added 5 μM STLC together with RO3306 (RO+STLC). (E) Quantitative analysis of 3D images (% separation As., N=524; RO, N=343; RO+BI, N=415; RO+STLC, N=380; error bars indicate the s.d. in three independent experiments). Distances were scored in 3D images using Imaris (N=50) as in (B); the bars indicate the mean distance; As., 5.7 μm; RO, 9.2 μm; RO+BI, 4.6 μm; RO+STLC, 4.1 μm. (F) FACS analysis of the indicated HeLa samples.
Mentions: To clarify the role of Cdk1 in centrosome separation, we took advantage of a cdk1as DT40 cell line that carries an analogue-sensitive mutation in Cdk1 (cdk1as cells). In these cells, the mutant Cdk1 can be inhibited with high specificity by addition of the bulky ATP analogue, 1NMPP1, resulting in a late G2 phase arrest (Figure 1C), while the ATP analogue has no effect on the cell cycle of cells expressing WT Cdk1 (Hochegger et al, 2007). We found that, despite Cdk1 inhibition, centrosomes were clearly separated in about 60% of the 1NMPP1-treated cdk1as cells (Figure 1A and B). To confirm this result in a different experimental system, we used a chemical Cdk1 inhibitor, RO3306 (Vassilev et al, 2006), in HeLa cells, and found that approximately half of the RO3306-treated, G2-arrested cells (Figure 1F) displayed widely separated centrosomes (Figure 1D and E). To compare the timing of centrosome separation in the absence or presence of Cdk1 activity in more detail, we analysed centrosome separation in cdk1as cells that were pre-synchronized in G1 by elutriation and progressed to G2/M phase in the presence or absence of Cdk1 inhibition by 1NMPP1. Supplementary Figure S1A shows that centrosomes separated while cells progressed into G2/M. However, separation was delayed by approximately 2 h in the 1NMPP1-treated cells. We conclude from these results that Cdk1 is not strictly essential for centrosome separation, but is required for timely initiation of the process.

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