A map of protein dynamics during cell-cycle progression and cell-cycle exit

Abstract

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.

Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence

“…Cells may emerge from the quiescent CDK2 low state and re-enter the cell cycle by increasing their CDK2 activity (“CDK2 emerge cells”; green in Figure 1B, top). After 24 hr of imaging, we fixed the cells, performed immunofluorescence for Ki67, and computationally matched each cell’s Ki67 signal to its cell-cycle history using previously reported techniques (Gookin et al, 2017; Spencer et al, 2013). In all four cell lines, aligning each cell’s Ki67 signal as a function of time since anaphase revealed two trajectories of Ki67 levels after mitosis that correlated with the bifurcation in CDK2 activity: cells in the CDK2 low state showed continuous decay of Ki67, while cells in the CDK2 inc state showed accumulation of Ki67 (Figure 1B, middle).…”

“…n = 200–1000. Bottom: CDk2 emerge cell traces were aligned to the time of CDK2 activity rise, and α -Ki67 signal was reconstructed as a function of time since CDK2 activity rise as in Gookin et al (2017). n = 70–200.…”

Visualizing the metazoan proliferation-terminal differentiation decisionin vivo

“…In this report we present the generation and characterization of a CDK ratiometric biosensor based on the CDK2 phosphorylation domain of human DNA Helicase B (DHB) (Hahn et al, 2009;Spencer et al, 2013) capable of distinguishing all interphase cell cycle states throughout the life cycle of C. elegans, including the G0 phase of the cell cycle. In mammalian cell culture, DHB as a CDK2 biosensor has been a useful tool to visualize the difference between proliferative and quiescent cells in asynchronous cell populations (Arora et al, 2017;Cappell et al, 2016;Gast et al, 2018;Gookin et al, 2017;Miller et al, 2018;Moser et al, 2018;Overton et al, 2014;Spencer et al, 2013;. This has prompted researchers to define a new cell cycle restriction point as a bifurcation point immediately following mitotic exit based on CDK2 activity, with proliferative cells exiting at a CDK2 increasing (CDK2 inc ) state and quiescent cells exiting at a CDK2 low (CDK2 low ) state (Spencer et al, 2013).…”

Embryo timelapses can be compiled and quantified to understand canonical histone dynamics across multiple cell cycles

“…In cell culture, where cells can be synchronized, it is possible to average quantifiable information about the cells at specific parts of the cell cycle. It is also possible to sort nonsynchronized cells through similarity in cell cycle progression (the similar kinetics of passage through the cell cycle allows postacquisition alignment of data [Gookin et al, 2017]). The C. elegans system allows the comparison of multiple differentiated lineages from as early as the 2-cell stage until hatching using either differential interference contrast (DIC) microscopy (Sulston et al, 1983) or fluorescently labeled proteins (Boyle et al, 2006).…”