© 2006 Nature Publishing Group The reversibility of mitotic exit in vertebrate cellsTamara A. Potapova1, John R. Daum1, Bradley D. Pittman1, Joanna R. Hudson1, Tara N. Jones1,David L. Satinover2, P. Todd Stukenberg2& Gary J. Gorbsky1A guiding hypothesis for cell-cycle regulation asserts that regu-lated proteolysis constrains the directionality of certain cell-cycletransitions1,2. Here we test this hypothesis for mitotic exit, which isregulated by degradation of the cyclin-dependent kinase 1 (Cdk1)activator, cyclin B3–5. Application of chemical Cdk1 inhibitors tocells in mitosis induces cytokinesis and other normal aspects ofmitotic exit, including cyclin B degradation. However, chromatidsegregation fails, resulting in entrapment of chromatin in themidbody. If cyclin B degradation is blocked with a proteasomeinhibitor or by expression of non-degradable cyclin B, Cdkinhibitors will nonetheless induce mitotic exit and cytokinesis.However, if after mitotic exit, the Cdk1 inhibitor is washed freefrom cells in which cyclin B degradation is blocked, the cells canrevert back to M phase. This reversal is characterized by chromo-some recondensation, nuclear envelope breakdown, assembly ofmicrotubules into a mitotic spindle, and in most cases, dissolutionof the midbody, reopening of the cleavage furrow, and realignmentof chromosomesat the metaphase plate.These findings demonstratethat proteasome-dependent degradation of cyclin B providesdirectionality for the M phase to G1 transition.Cdk1, the major regulator of mitotic progression, is activatedthrough binding of cyclin A or B. Cyclin A is degraded duringprometaphase when chromosomes move to align at the metaphaseplate6,7. Cyclin B degradation begins at metaphase and continuesduring chromatid segregation in anaphase and exit from M phase5.Cytokinesis is initiated shortly after anaphase onset. Cdk1 inacti-vation and dephosphorylation of Cdk1 substrates during mitotic exitprobably serve as timing mechanisms to ensure that cytokinesisoccurs after chromatid separation8–12. For example, high Cdk1activity before anaphase blocks the accumulation of the cytokineticregulators aurora B and MKLP1 at the cleavage furrow and on themicrotubules of the spindle midzone13–15.Flavopiridol is a potent inhibitor of Cdk116. We found thattreatment of vertebrate cells in mitosis with flavopiridol resulted inpremature mitotic exit accompanied by cytokinesis (Fig. 1a andSupplementary Video 1). Similar results have recently been found forthe Cdk inhibitor BMI-1026 (ref. 17). Flavopiridol induced themicrotubule network to undergo changes characteristic of anaphaseand mitotic exit. The spindle poles moved apart, and microtubulebundles formed in the spindle midzone and at the equatorial cortex.Even though chromatid separation did not occur, cytokinetic fur-rows formed and ingressed to completion. The cleavage furrowtrapped chromosomes in the midbody, resulting in a ‘cut’ phenotype.Nevertheless, the chromosomes decondensed and nuclear envelopesreformed. Eventually, cytoplasmic contractile activity diminished ascells flattened fully onto the substratum, and the microtubule arrayestablished an interphase pattern.During normal mitotic exit, Cdk1 activity is reduced by ubiqui-tylation and proteasome-mediated degradation of cyclin B3,5.Pro-teasome inhibitors such as MG132 induce mitotic cells to arrest atmetaphase. We found that treatment with flavopiridol overridesmetaphase arrest induced with MG132, causing mitotic exit andcytokinesis accompanied by chromosome decondensation and refor-mation of the nuclear envelope (Fig. 1b and Supplementary Video 2).The proteolysis of cyclin B at mitotic exit is thought to ensure theunidirectionality of the M phase to G1 transition2. In cells withproteasome inhibition, we found that flavopiridol-induced mitoticexit was reversible. Upon flavopiridol removal, cells that had exitedmitosis could return to metaphase (Fig. 1c and SupplementaryVideos 3, 4). The microtubules, having assumed an interphaseconfiguration after flavopiridol-induced mitotic exit, reassembled amitotic spindle when flavopiridol was removed. The midbody dis-appeared and the cytokinetic furrow retracted, the newly formednuclear envelope dissolved, and the chromosomes recondensed,attached to spindle microtubules and realigned at the metaphaseplate. These findings are summarized in Supplementary Fig. 1. Wefound that cells induced to reverse back to metaphase could sub-sequently undergo a second, normal mitotic exit (including chro-matid separation and movement) as well as a second cytokinesis ifthe proteasome inhibitor was subsequently washed away (Sup-plementary Video 5). We used flavopiridol for most of our experi-ments because of its high potency as a Cdk1 inhibitor(Supplementary Fig. 2). However, some other chemical Cdkinhibitors also resulted in similar phenotypes (see SupplementaryInformation).In Xenopus S3 cells arrested at metaphase with MG132, aurora Bkinase was concentrated at centromeres and MKLP1 was localizeddiffusely in the cytoplasm (Fig. 1d). Upon treatment with flavopiridol,aurora B and MKLP1 rapidly accumulated at the equatorial cell cortex,associating with the nascent cleavage furrows and the midzone over-laying the microtubule bundles, and eventually becoming highlyconcentrated at the midbody. These translocation events were con-sistent with the typical relocation of aurora B and MKLP1 in normalanaphase, and were also reversible: in cells that were induced throughmitotic exit and then allowed to revert back to metaphase, aurora Band MKLP1 returned to their typical metaphase distributions(Fig. 1d, 60 min).Metaphase cells treated with flavopiridol in the presence ofproteasome inhibitor advanced through mitotic exit and cytokinesis,reaching the midbody stage with decondensed chromosomes andreassembled nuclear envelopes 25 min after treatment (Fig. 1b). Thereversibility of mitotic exit was dependent on the duration ofexposure to flavopiridol. Treatment with 5mM for 17–30 minresulted in most cells (81 out of 106) returning to metaphase uponflavopiridol washout (Table 1). With longer exposure to flavopiridol,the proportion of cells that underwent reversal declined. However,a few cells (3 out of 24) still reversed when flavopiridol was removed80 min after its addition. We detected no reversal among cellstreated for 90 min (n ¼ 26). Reversibility was entirely dependenton the presence of the proteasome inhibitor. In medium withoutLETTERS1Program