Analysis of E1A domains involved in the enhancement of CDK2 activity.

E1A is an adenoviral protein which is expressed at the early phase after viral infection and contains four conserved regions (CR1, CR2, CR3 and CR4). Our previous work suggests that E1A facilitates the formation of cyclin A-CDK2 complex and thereby enhances CDK2 activity. However, the molecular function of E1A in CDK2 activation has been unclear. Here, we studied the mechanism of enhancement of CDK2 activity by E1A, using the E1A variant forms which selectively contain CR domains. We isolated four E1A variant forms, i.e. 13S (containing CR1, CR2, CR3, CR4), 12S (CR1, CR2, CR4), 10S (CR2, CR4) and 9S (CR4), derived from HEK293 cells which express E1A. 13S promoted G2/M-phase arrest, upon CDK2 hyper-activation by co-expressing a stabilized cyclin A mutant, most strongly among those E1A variant forms. Concomitantly, the specific activity of the 13S-associated CDK2 was highest among them. 10S exhibited lower affinity for CDK2 than the 13S while the affinity for CDK2 was comparable between 13S and 12S. Nonetheless, 12S did not enhance the CDK2 specific activity. On the other hand, a mutation in CR2 domain, which is essential for binding to p107, suppressed both the binding and activation of CDK2. These results suggest that CR1 domain, in addition to CR2 domain via p107 interaction, is important for binding to CycA-CDK2 complex while CR3 domain facilitates CDK2 activation. Since the function of CR3 in cell cycle regulation has been relatively unknown, we propose the enhancement of CDK2 activity as a novel function of CR3 domain.

EllipTrack: A Global-Local Cell-Tracking Pipeline for 2D Fluorescence Time-Lapse Microscopy.

Time-lapse microscopy provides an unprecedented opportunity to monitor single-cell dynamics. However, tracking cells for long periods remains a technical challenge, especially for multi-day, large-scale movies with rapid cell migration, high cell density, and drug treatments that alter cell morphology/behavior. Here, we present EllipTrack, a global-local cell-tracking pipeline optimized for tracking such movies. EllipTrack first implements a global track-linking algorithm to construct tracks that maximize the probability of cell lineages. Tracking mistakes are then corrected with a local track-correction module in which tracks generated by the global algorithm are systematically examined and amended if a more probable alternative can be found. Through benchmarking, we show that EllipTrack outperforms state-of-the-art cell trackers and generates nearly error-free cell lineages for multiple large-scale movies. In addition, EllipTrack can adapt to time- and cell-density-dependent changes in cell migration speeds and requires minimal training datasets. EllipTrack is available at https://github.com/tianchengzhe/EllipTrack.

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

Ki67 staining is widely used as a proliferation indicator in the clinic, despite poor understanding of this protein's function or dynamics. Here, we track Ki67 levels under endogenous control in single cells over time and find that Ki67 accumulation occurs only during S, G2, and M phases. Ki67 is degraded continuously in G1 and G0 phases, regardless of the cause of entry into G0/quiescence. Consequently, the level of Ki67 during G0 and G1 in individual cells is highly heterogeneous and depends on how long an individual cell has spent in G0. Thus, Ki67 is a graded rather than a binary marker both for cell-cycle progression and time since entry into quiescence.

Stochastic Endogenous Replication Stress Causes ATR-Triggered Fluctuations in CDK2 Activity that Dynamically Adjust Global DNA Synthesis Rates.

Faithful DNA replication is challenged by stalling of replication forks during S phase. Replication stress is further increased in cancer cells or in response to genotoxic insults. Using live single-cell image analysis, we found that CDK2 activity fluctuates throughout an unperturbed S phase. We show that CDK2 fluctuations result from transient ATR signals triggered by stochastic replication stress events. In turn, fluctuating endogenous CDK2 activity causes corresponding decreases and increases in DNA synthesis rates, linking changes in stochastic replication stress to fluctuating global DNA replication rates throughout S phase. Moreover, cells that re-enter the cell cycle after mitogen stimulation have increased CDK2 fluctuations and prolonged S phase resulting from increased replication stress-induced CDK2 suppression. Thus, our study reveals a dynamic control principle for DNA replication whereby CDK2 activity is suppressed and fluctuates throughout S phase to continually adjust global DNA synthesis rates in response to recurring stochastic replication stress events.