Inhibition of Cyclin-dependent Kinase 1 Induces Cytokinesis without Chromosome Segregation in an ECT2 and MgcRacGAP-dependent Manner

Abstract

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5-or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of antiCdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.

Cyclin B2 suppresses mitotic failure and DNA re-replication in human somatic cells knocked down for both cyclins B1 and B2

“…However, DNA, even if it begins to segregate between these daughter cells, seems to be trapped in the midzone. This phenomenon is readily comparable to the phenotype described after CDK1 chemical inhibition by BMI-1026 or RO-3306 (Seong et al, 2003;Niiya et al, 2005;Vassilev et al, 2006). However, in our case, the chromosomes eventually collapse, giving rise to a single tetraploid nucleus.…”

“…Under nocodazole treatment, BMI-1026 leads to mitosis exit and cell spreading without division. Depending on the nocodazole concentration, cells can show membrane blebbing, which is assumed to represent a cortical activity of actomyosin contraction subsequent to formation of ectopic cleavage furrows (Seong et al, 2003;Niiya et al, 2005). Our observation of precocious mitosis exit and abnormal cytokinesis in proliferating cells downregulated for cyclins B1 and B2 fully reproduces these phenotypes.…”

“…It has recently been shown that chemical inhibition of CDK1 after metaphase arrest promotes a rapid mitosis exit and premature abnormal cytokinesis (Seong et al, 2003;Niiya et al, 2005;Vassilev et al, 2006).…”

Dissecting the Role of Rho-mediated Signaling in Contractile Ring Formation

“…Control cells entered anaphase 1-3 h after nocodazole release, and 88% (n ϭ 65) of the cells completed normal mitosis and cytokinesis within 4 h (Figure 10, C and D, a; and Movie S7). Like other Cdk1 inhibitors (Niiya et al, 2005), roscovitine promoted premature exit from mitosis (Movie S8). The cells formed ectopic furrows ( Figure 10C, b, arrows) and exhibited cortical contraction (81%; n ϭ 115).…”

“…Inactivation of Cdk1/cyclin B regulates the timing of mitotic exit and cytokinesis as well as the mitotic spindle array (Wheatley et al, 1997;Echard and O'Farrell, 2003;Mishima et al, 2004). Recently, it has been reported that the Cdk1 inhibitors BM-1026 and purvanalol A promote premature mitotic exit and furrow ingression (Niiya et al, 2005). To test whether the Cdk1 inhibitor roscovitine affects initiation of cytokinesis, we treated synchronized prometaphase cells with DMSO (control) or roscovitine (Figure 10A, a and b).…”