The reversibility of mitotic exit in vertebrate cells

Abstract

A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.

Figure 1

The Cdk inhibitor Flavopiridol induces reversible mitotic exit and cytokinesis if proteasome activity is inhibited. a, Treatment of mitotic cells with Flavopiridol induces premature mitotic exit and cytokinesis without chromatid separation. A Xenopus S3 cell expressing GFP-α-tubulin was treated with 5 μM Flavopiridol at time 0. The black arrow indicates a chromosome not yet at metaphase when Flavopiridol was added. The white arrowhead indicates the reassembled nuclear envelope. The complete video sequence is available as Supplementary Video 1. Time is indicated as hours:minutes:seconds (a.- c.). b, Flavopiridol induces mitotic exit in cells arrested at metaphase with MG132. An MG132-arrested cell was treated with 10 μM Flavopiridol at time 0. The complete video sequence is found as Supplementary Video 2. c, Flavopiridol-induced mitotic exit and cytokinesis are reversible if the proteasome is inhibited. An MG132-arrested cell was treated with 5 μM Flavopiridol at time 0. The Flavopiridol was removed at 25 min. The complete video sequence is available as Supplementary Video 3. d, Flavopiridol induces normal mitotic exit changes in the distributions of Aurora B kinase and MKLP1 that are reversible. Xenopus S3 cells were incubated in medium containing MG132 and were fixed before and after treatment with 5 μM Flavopiridol. Samples were labeled for Aurora B kinase (upper panels) or MKLP1 (lower panels). Initial and final images (0 min and 60 min) were scaled identically for brightness and contrast. Others were scaled individually. Bars = 10 μm.

Figure 2

Reversibility of mitotic exit requires preservation of Cyclin B. a, A Xenopus S3 cell treated with 100 ng/ml nocodazole at time 0. At 30 min after nuclear envelope breakdown, nocodazole was removed and the cell was arrested at metaphase with MG132. Flavopiridol at 5 μM was added at 1:04:00 and removed 25 min later after mitotic exit. The complete video sequence is available as Supplementary Video 6. Bar = 10 μm. b, Experimental protocol employed for producing samples from Hela cells at various stages of induction and reversal of mitotic exit. c, Samples were analyzed for mitotic index and blotted for the indicated proteins.

Figure 3

Cells expressing non-degradable Cyclin B1 undergo mitotic exit reversal. a, Hela cells expressing non-degradable cyclin B1 can undergo mitotic exit reversal without segregating chromosomes. Flavopiridol was added at time 0 and removed at 25 min. The complete video sequence is available Supplementary Video 7. b, Hela cells expressing non-degradable cyclin B1 can undergo mitotic exit reversal after chromatid separation. Flavopiridol added and removed as in (a). The complete video sequence is available as Supplementary Video 8. c, Hela cells expressing wild type Cyclin B1 do not undergo reversal of mitotic exit. Flavopiridol added and removed as in (a). The complete video sequence is available as Supplementary Video 9. Insets show GFP fluorescence images at time 0. Level of the wild type Cyclin B1 in (c) was approximately twice that of the non-degradable Cyclin B1 expressed in (a) and (b). d, Cells expressing high levels of non-degradable Cyclin B1 were more likely to undergo mitotic exit reversal. Cells that did not separate chromatids upon treatment with Flavopiridol are depicted by black symbols. Those that separated chromatids are depicted in red. Two cells that recondensed their chromosomes but had not opened their cleavage furrows by 90 min after Flavopiridol removal are depicted by hollow red symbols. Error bars show S.E.M. e, A mitotic Hela cell at metaphase (arrow) expressing non-degradable Cyclin B1, shows low levels of Cyclin A expression by immunofluorescence. Bars = 10μm.

Figure 4

Primary cultures of human cells can undergo mitotic exit reversal. An MG132-arrested metaphase cell was imaged by phase contrast microscopy after addition of 7.5 μM Flavopiridol at time 0. At 26 min, the Flavopiridol was removed. The complete video sequence is available as Supplementary Video 10. Bar = 10 μm.