In G2 phase, Cdk2 and Plk1 trigger Eg5 enrichment in the centrosome. Cdk1, and phosphorylates Eg5 at Thr927. However, Plk1-driven centrosome separation is definitely sluggish and staggering, while Cdk1 causes fast movement of the centrosomes. We find that actin-dependent Eg5-opposing causes 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 like a regulatory element in the control of centrosome separation. mutant with defective centrosomes and monopolar spindles (Sunkel and Glover, 1988). Plk1 contributes to build up of -tubulin in the centrosomes (Lane and Nigg, 1996; Casenghi et al, 2003; Oshimori et al, 2006) and stabilization of stable MT-kinetochore attachments (Sumara et al, 2004). Using Plk1 inhibitors or siRNA-mediated depletion results in collapsed spindles, with centrosomes in close proximity in the spindle equator (Sumara et al, 2004; vehicle Vugt et al, 2004; McInnes et al, 2006; Lenart et al, 2007). However, a direct part for Plk1 in centrosome disjunction and/or separation remains to be established. In this study, we targeted to investigate the part of Cdk1 and Plk1 in triggering centrosome separation. Results Centrosome separation happens in Cdk1-inhibited cells and depends on Plk1 and Eg5 activity To clarify the part of Cdk1 in centrosome separation, we took advantage of a DT40 cell collection that bears an analogue-sensitive mutation in Cdk1 (cells). In these cells, the mutant Cdk1 can be inhibited with high specificity by addition of the heavy ATP analogue, 1NMPP1, resulting in a late G2 phase arrest (Number 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 cells (Number 1A and B). To confirm this result in a different experimental system, we used a chemical Cdk1 inhibitor, RO3306 (Vassilev et al, 2006), in cells, and found that approximately half of the RO3306-treated, G2-caught cells (Number 1F) displayed widely separated centrosomes (Number 1D and E). To compare the timing of centrosome separation in the absence or presence of Cdk1 activity in more Eicosapentaenoic Acid detail, we analysed centrosome separation in cells that were pre-synchronized in G1 by elutriation and progressed to G2/M phase in the presence or absence Eicosapentaenoic Acid of Cdk1 inhibition by 1NMPP1. Supplementary Number S1A demonstrates 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 purely essential for centrosome separation, but is required for timely initiation of the process. Open in a separate windows Number 1 Cdk1-self-employed centrosome separation requires Plk1 and Eg5 activity. (A) DT40 cells were analysed by immuno-fluorescence using anti–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 pub, 5 m). Asynchronous cells are demonstrated in the much left panel (As.). Cdk1 was inhibited by treating cells for 6 h with 10 M 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 M 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., cells were analysed by immuno-fluorescence using anti–tubulin, anti-pericentrin antibodies and DAPI. The panels display deconvolved MIPs of 3D images of representative samples (scale pub, 10 M). Asynchronous cells are demonstrated in the much left panel (As.). Cdk1 was inhibited by treating cells for 20 h with 7.5 M RO3306 (RO). To inhibit Plk1, 100 nM of BI 2536 was added at the same time as RO 3066 (RO+BI). To inhibit human being Eg5, we added 5 M STLC together with RO3306 (RO+STLC). (E) Quantitative analysis of 3D images (% separation As., samples. Next, we investigated the requirement of Plk1 in Cdk1-self-employed centrosome separation. We inhibited Plk1 using the BI2536 compound (Lenart et al, 2007) in combination with Cdk1 in DT40 and cells. Plk1 inhibition clogged centrosome separation in both chicken (Number 1A and B) and human being cells (Number 1D and E). We analysed the centrioles in the BI2536/1NMPP1-treated cells by transmission electron microscopy to rule out that Plk1 inhibition blocks centrosome replication in S phase. We could readily detect four centrioles in random sections in MDA1 the Plk1-inhibited samples (Supplementary Number S1B), suggesting that in these cells, centrioles experienced replicated, but centrosomes failed to separate. We also performed a parallel.We did not find any evidence that Cdk1 further modifies Eg5 and accordingly Blangy et al (1995) showed that Thr927 was the only Cdk phosphorylation site in the protein. centrosome separation is definitely sluggish 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. mutant with defective centrosomes Eicosapentaenoic Acid and monopolar spindles (Sunkel and Glover, 1988). Plk1 contributes to accumulation of -tubulin at the centrosomes (Lane and Nigg, 1996; Casenghi et al, 2003; Oshimori et al, 2006) and stabilization of stable MT-kinetochore attachments (Sumara et al, 2004). Using Plk1 inhibitors or siRNA-mediated depletion results in collapsed spindles, with centrosomes in close proximity at the spindle equator (Sumara et al, 2004; van Vugt et al, 2004; McInnes et al, 2006; Lenart et al, 2007). However, a direct role for Plk1 in centrosome disjunction and/or separation remains to be established. In this study, we aimed to investigate the role of Cdk1 and Plk1 in triggering centrosome separation. Results Centrosome separation occurs in Cdk1-inhibited cells and depends on Plk1 and Eg5 activity To clarify the role of Cdk1 in centrosome separation, we took advantage of a DT40 cell line that carries an analogue-sensitive mutation in Cdk1 (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 (Physique 1C), while the ATP analogue has no effect on the cell cycle of cells expressing WT Cdk1 (Hochegger et Eicosapentaenoic Acid al, 2007). We found that, despite Cdk1 inhibition, centrosomes were clearly separated in about 60% of the 1NMPP1-treated cells (Physique 1A and B). To confirm this result in a different experimental system, we used a chemical Cdk1 inhibitor, RO3306 (Vassilev et al, 2006), in cells, and found that approximately half of the RO3306-treated, G2-arrested cells (Physique 1F) displayed widely separated centrosomes (Physique 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 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 Physique 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. Open in a separate window Physique 1 Cdk1-impartial centrosome separation requires Plk1 and Eg5 activity. (A) DT40 cells were analysed by immuno-fluorescence using anti–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 m). Asynchronous cells are shown in the far left panel (As.). Cdk1 was inhibited by treating cells for 6 h with 10 M 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 M 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., cells were analysed by immuno-fluorescence using anti–tubulin, anti-pericentrin antibodies and DAPI. The panels display deconvolved MIPs of.