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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 transitions1,2. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B3,4,5. 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.

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Figure 1: The Cdk inhibitor flavopiridol induces reversible mitotic exit and cytokinesis if proteasome activity is inhibited.
Figure 2: Reversibility of mitotic exit requires preservation of cyclin B.
Figure 3: Cells expressing non-degradable cyclin B1 undergo mitotic exit reversal.
Figure 4: Primary cultures of human cells can undergo mitotic exit reversal.

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Acknowledgements

We thank the staff at the OMRF Imaging Center and W. Martin for technical assistance. We thank Sanofi-Aventis Pharmaceuticals and the National Cancer Institute for providing flavopiridol. We thank J. Pines for providing cyclin B plasmids and P. Davies for TG-3 anti-phospho-nucleolin antibody. We thank J. Gannon and T. Hunt for antibodies against Cdk1 and cyclins. This work was supported by grants from the National Institutes of Health (to G.J.G. and P.T.S.), from the American Cancer Society (to P.T.S.) and from an NIH training grant (to D.L.S.).

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Correspondence to Gary J. Gorbsky.

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Supplementary Data

This file contains 3 supplementary figures with their legends. The first supplementary figure summarizes the main finding of the paper. The other two contain supplementary data. After the figures is a detailed description of the methods, a short discussion of the effectiveness of Cdk1 inhibitors other than Flavopiridol, and the Supplementary Video Legends. (PDF 5035 kb)

Supplementary Video 1

This video reveals the effects of the Cdk1 inhibitor Flavopiridol applied to a Xenopus S3 cell in prometaphase, causing premature mitotic exit and cytokinesis. This cell is depicted in Text Fig. 1a. (MOV 1237 kb)

Supplementary Video 2

This video shows that Flavopiridol induces mitotic exit and cytokinesis in a Xenopus S3 cell arrested at metaphase with the proteasome inhibitor MG132. This cell is depicted in Text Fig. 1b. (MOV 3640 kb)

Supplementary Video 3

This video shows that Flavopiridol-induced mitotic exit and cytokinesis in a Xenopus S3 cell cultured in the presence of proteasome inhibitor is reversible upon removal of the Flavopiridol. This cell is depicted in Text Fig. 1c. (QuickTime; 3.5MB) (MOV 3499 kb)

Supplementary Video 4

This video shows a second example of the reversal of mitotic exit and cytokinesis in a Xenopus S3 cell upon removal of Flavopiridol in the presence of proteasome inhibitor. (MOV 4466 kb)

Supplementary Video 5

This video shows a Xenopus S3 cultured in proteasome inhibitor and induced to go through reversal of mitotic exit by addition then removal of Flavopiridol. The proteasome inhibitor is then washed out and the cell subsequently undergoes normal anaphase and mitotic exit. (MOV 5474 kb)

Supplementary Video 6

This video shows a Xenopus S3 transiently arrested with the microtubule drug nocodazole then released to proteasome and subsequently induced to undergo mitotic exit and reversal by addition then removal of Flavopiridol. This cell is depicted in Text Fig. 2a. (MOV 5361 kb)

Supplementary Video 7

This video shows a Hela cell expressing non-degradable cyclin B1. Flavopiridol treatment causes it to exit mitosis and undergo cytokinesis without chromatid separation. Removal of Flavopiridol results in reversal of mitotic exit back to metaphase. This cell is depicted in Text Fig. 3a. (MOV 1948 kb)

Supplementary Video 8

This video shows a Hela cell expressing non-degradable cyclin B1. Flavopiridol treatment causes it to exit mitosis and undergo cytokinesis after chromatid separation. Removal of Flavopiridol results in reversal of mitotic exit back to an anaphase-like condition. This cell is depicted in Text Fig. 3b. (MOV 3054 kb)

Supplementary Video 9

This video shows a Hela cell expressing wild type cyclin B1. Flavopiridol treatment causes it to exit mitosis and undergo cytokinesis without chromatid separation. Removal of Flavopiridol does not result in reversal of mitotic exit. This cell is depicted in Text Fig. 3c. (MOV 2290 kb)

Supplementary Video 10

This video shows that Flavopiridol-induced mitotic exit and cytokinesis in a primary human keratinocyte cell cultured in the presence of proteasome inhibitor is reversible upon removal of the Flavopiridol. This cell is depicted in Text Fig. 4. (MOV 4863 kb)

Supplementary Video 11

This video shows that R0-31-8220 induces mitotic exit and cytokinesis in a Xenopus S3 cell arrested at metaphase with the proteasome inhibitor MG132. This cell is depicted in Supplementary Fig. 3a. (MOV 1298 kb)

Supplementary Video 12

This video shows the normal process of mitosis and cytokinesis in a control Xenopus S3 cell stably expressing GFP-α-tubulin. (MOV 3460 kb)

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Potapova, T., Daum, J., Pittman, B. et al. The reversibility of mitotic exit in vertebrate cells. Nature 440, 954–958 (2006). https://doi.org/10.1038/nature04652

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