Justin Moser

The Restriction Point was originally defined as the moment that cells commit to the cell cycle and was later suggested to coincide with hyperphosphorylation of the retinoblastoma protein (Rb). Current cell cycle models posit that cells exit mitosis into a pre-Restriction Point state, where they have low cyclin-dependent kinase (CDK) activity and hypophosphorylated Rb; passage through the Restriction Point then occurs in late G1. Recent single-cell studies have challenged the current paradigm, raising questions about the location of the Restriction Point and the notion that cells exit mitosis into a pre-Restriction Point state. Here, we use a variety of single-cell techniques to show that both noncancer and cancer cells bifurcate into two subpopulations after anaphase, marked by increasing vs. low CDK2 activity and hyper- vs. hypophosphorylation of Rb. Notably, subpopulations with hyper- and hypophosphorylated Rb are present within minutes after anaphase, delineating one subpopulation that never “uncrosses” the Restriction Point and continues cycling and another subpopulation that exits mitosis into an uncommitted pre-Restriction Point state. We further show that the CDK inhibitor p21 begins rising in G2 in mother cells whose daughters exit mitosis into the pre-Restriction Point, CDK2^low state. Furthermore, degradation of p21 coincides with escape from the CDK2^low state and passage through the Restriction Point. Together, these data support a model in which only a subset of cells returns to a pre-Restriction Point state after mitosis and where the Restriction Point is sensitive to not only mitogens, but also inherited DNA replication stress via p21. Significance The canonical Restriction Point model suggests that cells are born into a state in which they are uncommitted to the cell cycle, but will activate cyclin-dependent kinase 2 and cross the Restriction Point several hours later if sufficient nutrients are available. However, recent single-cell studies have challenged aspects of this model. This work examines the Restriction Point in cancerous and noncancerous cells and shows that, in six cases tested, the cell populations split such that only a subset of cells is born into a pre-Restriction Point state, while the remainder immediately commits to another cell cycle. This shows that even cancer cells can experience significant heterogeneity in this cell fate decision, which may be exploitable for therapeutic gain.

The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patterns for core cell-cycle proteins in actively proliferating (CDK2-increasing) versus spontaneously quiescent (CDK2-low) cells, including Cyclin D1, the levels of which we find to be higher in spontaneously quiescent versus proliferating cells. We also identify proteins with concentrations that steadily increase or decrease the longer cells are in quiescence, suggesting the existence of a continuum of quiescence depths. Our single-cell measurements thus provide a rich resource for the field by characterizing protein dynamics during proliferation versus quiescence. The cell cycle is by nature highly dynamic, but we lack a standardized map of how core cell-cycle regulators change over time. In this study, we used time-lapse microscopy to track the dynamics of key cell-cycle proteins in individual human cells and found several unexpected patterns, even for well-studied proteins such as Cyclin D1. Our data provide a rich resource for those focused on the cell cycle, or on any biological process that is impacted by the cell cycle, by providing a series of maps of protein dynamics during cell-cycle progression and cell-cycle exit.