SUMMARY Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity.
Proliferating cells must cross a point of no return before they replicate their DNA and divide. This commitment decision plays a fundamental role in cancer and degenerative diseases and has been proposed to be mediated by phosphorylation of retinoblastoma (Rb) protein. Here, we show that inactivation of the anaphase-promoting complex/cyclosome (APC(Cdh1)) has the necessary characteristics to be the point of no return for cell-cycle entry. Our study shows that APC(Cdh1) inactivation is a rapid, bistable switch initiated shortly before the start of DNA replication by cyclin E/Cdk2 and made irreversible by Emi1. Exposure to stress between Rb phosphorylation and APC(Cdh1) inactivation, but not after APC(Cdh1) inactivation, reverted cells to a mitogen-sensitive quiescent state, from which they can later re-enter the cell cycle. Thus, APC(Cdh1) inactivation is the commitment point when cells lose the ability to return to quiescence and decide to progress through the cell cycle.
Mammalian cells integrate mitogen and stress signaling prior to the end of G1 phase to decide whether or not to enter the cell cycle1–4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program, and inactivate an E3 ubiquitin ligase, the anaphase promoting complex/cyclosome (APC/CCdh1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but cannot after inactivation of APC/CCdh1, arguing that APC/CCdh1 inactivation is the point-of-no-return for cell cycle entry3. While rapid inactivation of APC/CCdh1 requires early mitotic inhibitor 1 (Emi1)3,5, the molecular mechanism controlling this cell cycle commitment step is unknown. Here we show that cell cycle commitment is mediated by an Emi1-APC/CCdh1 dual-negative feedback switch, in which Emi1 is both a substrate and an inhibitor of APC/CCdh1. The inactivation switch triggers a transition between a state with low Emi1 levels and high APC/CCdh1 activity during G1 to a state with high Emi1 levels and low APC/CCdh1 activity during S and G2. Cell-based analysis, in vitro reconstitution, and modeling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Together, our study argues that mammalian cells commit to the cell cycle by increasing CDK2 activity and Emi1 mRNA expression to trigger a one-way APC/CCdh1 inactivation switch mediated by Emi1 transitioning from a substrate to an inhibitor of APC/CCdh1.
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