Cyclin B1–Cdk1 Activation Continues after Centrosome Separation to Control Mitotic Progression

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Abstract
Activation of cyclin B1–cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome–dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1–Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1–Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1–Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1–Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1–Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1–Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1–Cdk1 activity after centrosome separation is critical to coordinate mitotic progression. When active, the enzyme cyclin B1–cyclin-dependent kinase 1 (Cdk1) commits a growing cell to the process of mitotic cell division and chromosome separation. Cyclin B1–Cdk1 activation is controlled in many ways, but once its activity rises above a certain level, further activation of cyclin B1–Cdk1 is catalyzed by a positive-feedback loop. This generates highly active cyclin B1–Cdk1 and triggers the start of mitosis, which can only be completed when cyclin B1–Cdk1 activity is properly shut off again. However, it is not clear how cyclin B1–Cdk1 activity develops in human cells or how the switch between its inactive and active states is controlled. Our work combines activation measurements with a kinetic model to study how cyclin B1–Cdk1 activity accumulates just before and during mitosis. We show that cyclin B1–Cdk1 activity develops gradually in early mitosis and that different activity levels are required for initiation of, and progression through, mitosis. We also demonstrate that once cyclin B1–Cdk1 activation is truly launched, it is bound to continue and will not lightly drop back again. We propose that the successive cyclin B1–Cdk1 activity levels by themselves may coordinate the progression through the distinct phases of mitosis.