Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Molecular biology of the cell (Impact Factor: 4.47). 02/2011; 22(8):1191-206. DOI: 10.1091/mbc.E10-07-0599
Source: PubMed


Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.

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Available from: Tamara A Potapova, Oct 03, 2015
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    • "Oil objective lens. Cells were then traced using Image J, and corrected total cell fluorescence (CTCF) values were calculated as described (Burgess et al., 2010; Gavet and Pines, 2010; Potapova et al., 2011; McCloy et al., 2014). At least 30 cells per coverslip were counted, and the experiment was repeated at least twice. "
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    • "To determine the total fluorescence detected in the z-stack, a region was drawn around each cell to be measured, and three regions next to the selected cell that had no fluorescence were used for background subtraction. The corrected total cell fluorescence for each cell was calculated using the following formula (Gavet and Pines, 2010; Potapova et al., 2011): "
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