Cell proliferation and quiescence are intimately coordinated during metazoan development. Here, we adapt a cyclin-dependent kinase (CDK) sensor to uncouple these key events of the cell cycle in Caenorhabditis elegans and zebrafish through live-cell imaging. The CDK sensor consists of a fluorescently tagged CDK substrate that steadily translocates from the nucleus to the cytoplasm in response to increasing CDK activity and consequent sensor phosphorylation. We show that the CDK sensor can distinguish cycling cells in G1 from quiescent cells in G0, revealing a possible commitment point and a cryptic stochasticity in an otherwise invariant C. elegans cell lineage. Finally, we derive a predictive model of future proliferation behavior in C. elegans based on a snapshot of CDK activity in newly born cells. Thus, we introduce a live-cell imaging tool to facilitate in vivo studies of cell-cycle control in a wide-range of developmental contexts.

The current model of replication-dependent (RD) histone biosynthesis posits that RD histone gene expression is coupled to DNA replication, occurring only in S phase of the cell cycle once DNA synthesis has begun. However, several key factors in the RD histone biosynthesis pathway are up-regulated by E2F or phosphorylated by CDK2, suggesting these processes may instead begin much earlier, at the point of cell-cycle commitment. In this study, we use both fixed- and live-cell imaging of human cells to address this question, revealing a hybrid model in which RD histone biosynthesis is first initiated in G1, followed by a strong increase in histone production in S phase of the cell cycle. This suggests a mechanism by which cells that have committed to the cell cycle build up an initial small pool of RD histones to be available for the start of DNA replication, before producing most of the necessary histones required in S phase. Thus, a clear distinction exists at completion of mitosis between cells that are born with the intention of proceeding through the cell cycle and replicating their DNA and cells that have chosen to exit the cell cycle and have no immediate need for histone synthesis.

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway. In BRAF-driven melanoma cells treated with BRAF inhibitors, subpopulations of cells escape drug-induced quiescence through a nongenetic manner of adaptation and resume slow proliferation. Here, we found that this phenomenon is common to many cancer types driven by EGFR, KRAS, or BRAF mutations in response to multiple, clinically approved MAPK pathway inhibitors. In 2D cultures and 3D spheroid models of various cancer cell lines, a subset of cells escaped drug-induced quiescence within 4 days to resume proliferation. These "escapee" cells exhibited DNA replication deficits, accumulated DNA lesions, and mounted a stress response that depended on the ataxia telangiectasia and RAD3-related (ATR) kinase. We further identified that components of the Fanconi anemia (FA) DNA repair pathway are recruited to sites of mitotic DNA synthesis (MiDAS) in escapee cells, enabling successful completion of cell division. Analysis of patient tumor samples and clinical data correlated disease progression with an increase in DNA replication stress response factors. Our findings suggest that many MAPK pathway-mutant cancers rapidly escape drug action and that suppressing early stress tolerance pathways may achieve more durable clinical responses to MAPK pathway inhibitors.

Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles and then enter a CDK2-low quiescence, whereas lysine withdrawal causes immediate cell-cycle stalling. Methionine withdrawal triggers a restriction point phenotype similar to serum starvation or Mek inhibition: upon methionine withdrawal, cells complete their current cell cycle and enter a CDK2-low quiescence after mitosis. Modulation of restriction point regulators p21/p27 or cyclin D1 enables short-term rescue of proliferation under methionine and leucine withdrawal, and to a lesser extent lysine withdrawal, revealing a checkpoint connecting nutrient signaling to cell-cycle entry.

In nature, some organisms survive extreme environments by inducing a biostatic state wherein cellular contents are effectively vitrified. Recently, a synthetic biostatic state in mammalian cells is achieved via intracellular network formation using bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers. In this work, the effects of intracellular network formation on a 3D epithelial MCF10A spheroid model are explored. Macromer-transfected cells are encapsulated in Matrigel, and spheroid area is reduced by ≈50% compared to controls. The intracellular hydrogel network increases the quiescent cell population, as indicated by increased p21 expression. Additionally, bioenergetics (ATP/ADP ratio) and functional metabolic rates are reduced. To enable reversibility of the biostasis effect, a photosensitive nitrobenzyl-containing macromer is incorporated into the PEG network, allowing for light-induced degradation. Following light exposure, cell state, and proliferation return to control levels, while SPAAC-treated spheroids without light exposure (i.e., containing intact intracellular networks) remain smaller and less proliferative through this same period. These results demonstrate that photodegradable intracellular hydrogels can induce a reversible slow-growing state in 3D spheroid culture.

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. Here, we quantified ECM protein dynamics in the murine forelimb during appendicular musculoskeletal morphogenesis (embryonic days 11.5-14.5) using tissue fractionation, bioorthogonal non-canonical amino acid tagging, and mass spectrometry. Our analyses indicated that ECM protein (matrisome) composition in the embryonic forelimb changed as a function of development and growth, was distinct from other developing organs (brain), and was altered in a model of disease (osteogenesis imperfecta murine). Additionally, the tissue distribution for select matrisome was assessed via immunohistochemistry in the wild-type embryonic and postnatal musculoskeletal system. This resource will guide future research investigating the role of the matrisome during complex tissue development.

When exposed to tumor necrosis factor (TNF) or TNF-related apoptosis-inducing ligand (TRAIL), a closely related death ligand and investigational therapeutic, cells enter a protracted period of variable duration in which only upstream initiator caspases are active. A subsequent and sudden transition marks activation of the downstream effector caspases that rapidly dismantle the cell. Thus, extrinsic apoptosis is controlled by an unusual variable-delay, snap-action switch that enforces an unambiguous choice between life and death. To understand how the extrinsic apoptosis switch functions in quantitative terms, we constructed a mathematical model based on a mass-action representation of known reaction pathways. The model was trained against experimental data obtained by live-cell imaging, flow cytometry, and immunoblotting of cells perturbed by protein depletion and overexpression. The trained model accurately reproduces the behavior of normal and perturbed cells exposed to TRAIL, making it possible to study switching mechanisms in detail. Model analysis shows, and experiments confirm, that the duration of the delay prior to effector caspase activation is determined by initiator caspase-8 activity and the rates of other reactions lying immediately downstream of the TRAIL receptor. Sudden activation of effector caspases is achieved downstream by reactions involved in permeabilization of the mitochondrial membrane and relocalization of proteins such as Smac. We find that the pattern of interactions among Bcl-2 family members, the partitioning of Smac from its binding partner XIAP, and the mechanics of pore assembly are all critical for snap-action control.

Oncogene-induced senescence is a phenomenon in which aberrant oncogene expression causes non-transformed cells to enter a non-proliferative state. Cells undergoing oncogenic induction display phenotypic heterogeneity, with some cells senescing and others remaining proliferative. The causes of heterogeneity remain unclear. We studied the sources of heterogeneity in the responses of human epithelial cells to oncogenic BRAFV600E expression. We found that a narrow expression range of BRAFV600E generated a wide range of activities of its downstream effector ERK. In population-level and single-cell assays, ERK activity displayed a non-monotonic relationship to proliferation, with intermediate ERK activities leading to maximal proliferation. We profiled gene expression across a range of ERK activities over time and characterized four distinct ERK response classes, which we propose act in concert to generate the ERK-proliferation response. Altogether, our studies map the input-output relationships between ERK activity and proliferation, elucidating how heterogeneity can be generated during oncogene induction.

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.

Mathematical modeling is being applied to increasingly complex biological systems and datasets; however, the process of analyzing and calibrating against experimental data is often challenging and a rate limiting step in model development. To address this problem, we developed a systematic methodology for calibrating quantitative models of dynamic biological processes and illustrate its utility by validating a model of TRAIL (Tumor necrosis factor Related Apoptosis-Inducing Ligand)-induced cell death.

Stochastic fluctuations in gene expression give rise to cell-to-cell variability in protein levels which can potentially cause variability in cellular phenotype. For TRAIL (TNF-related apoptosis-inducing ligand) variability manifests itself as dramatic differences in the time between ligand exposure and the sudden activation of the effector caspases that kill cells. However, the contribution of individual proteins to phenotypic variability has not been explored in detail. In this paper we use feature-based sensitivity analysis as a means to estimate the impact of variation in key apoptosis regulators on variability in the dynamics of cell death. We use Monte Carlo sampling from measured protein concentration distributions in combination with a previously validated ordinary differential equation model of apoptosis to simulate the dynamics of receptor-mediated apoptosis. We find that variation in the concentrations of some proteins matters much more than variation in others and that precisely which proteins matter depends both on the concentrations of other proteins and on whether correlations in protein levels are taken into account. A prediction from simulation that we confirm experimentally is that variability in fate is sensitive to even small increases in the levels of Bcl-2. We also show that sensitivity to Bcl-2 levels is itself sensitive to the levels of interacting proteins. The contextual dependency is implicit in the mathematical formulation of sensitivity, but our data show that it is also important for biologically relevant parameter values. Our work provides a conceptual and practical means to study and understand the impact of cell-to-cell variability in protein expression levels on cell fate using deterministic models and sampling from parameter distributions.

Slow-cycling subpopulations exist in bacteria, yeast, and mammalian systems. In the case of cancer, slow-cycling subpopulations have been proposed to give rise to drug resistance. However, the origin of slow-cycling human cells is poorly studied, in large part due to lack of markers to identify these rare cells. Slow-cycling cells pass through a noncycling period marked by low CDK2 activity and high p21 levels. Here, we use this knowledge to isolate these naturally slow-cycling cells from a heterogeneous population and perform RNA sequencing to delineate the transcriptome underlying the slow-cycling state. We show that cellular stress responses-the p53 transcriptional response and the integrated stress response (ISR)-are the most salient causes of spontaneous entry into the slow-cycling state. Finally, we show that cells' ability to enter the slow-cycling state enhances their survival in stressful conditions. Thus, the slow-cycling state is hardwired to stress responses to promote cellular survival in unpredictable environments.

Faithful DNA replication requires that cells fine-tune their histone pool in coordination with cell-cycle progression. Replication-dependent histone biosynthesis is initiated at a low level upon cell-cycle commitment, followed by a burst at the G1/S transition, but it remains unclear how exactly the cell regulates this burst in histone biosynthesis as DNA replication begins. Here, we use single-cell time-lapse imaging to elucidate the mechanisms by which cells modulate histone production during different phases of the cell cycle. We find that CDK2-mediated phosphorylation of NPAT at the restriction point triggers histone transcription, which results in a burst of histone mRNA precisely at the G1/S phase boundary. Excess soluble histone protein further modulates histone abundance by promoting the degradation of histone mRNA for the duration of S phase. Thus, cells regulate their histone production in strict coordination with cell-cycle progression by two distinct mechanisms acting in concert.

The glucocorticoid receptor (GR) is a ligand-activated transcription factor that regulates a suite of genes through direct binding of GR to specific DNA promoter elements. GR also interacts with RNA, but the function of this RNA-binding activity remains elusive. Current models speculate that RNA could repress the transcriptional activity of GR. To investigate the function of the GR-RNA interaction on GR's transcriptional activity, we generated cells that stably express a mutant of GR with reduced RNA binding affinity and treated the cells with the GR agonist dexamethasone. Changes in the dexamethasone-driven transcriptome were quantified using 4-thiouridine labeling of RNAs followed by high-throughput sequencing. We find that while many genes are unaffected, GR-RNA binding is repressive for specific subsets of genes in both dexamethasone-dependent and independent contexts. Genes that are dexamethasone-dependent are activated directly by chromatin-bound GR, suggesting a competition-based repression mechanism in which increasing local concentrations of RNA may compete with DNA for binding to GR at sites of transcription. Unexpectedly, genes that are dexamethasone-independent instead display a localization to specific chromosomal regions, which points to changes in chromatin accessibility or architecture. These results show that RNA binding plays a fundamental role in regulating GR function and highlights potential functions for transcription factor-RNA interactions.

Mammalian cells have two fundamentally different states, proliferative and quiescent, but our understanding of how and why cells switch between these states is limited. We previously showed that actively proliferating populations contain a subpopulation that enters quiescence (G0) in an apparently stochastic manner. Using single-cell time-lapse imaging of CDK2 activity and DNA damage, we now show that unresolved endogenous replication stress in the previous (mother) cell cycle prompts p21-dependent entry of daughter cells into quiescence immediately after mitosis. Furthermore, the amount of time daughter cells spend in quiescence is correlated with the extent of inherited damage. Our study thus links replication errors in one cell cycle to the fate of daughter cells in the subsequent cell cycle. More broadly, this work reveals that entry into quiescence is not purely stochastic but has a strong deterministic component arising from a memory of events that occurred in the previous generation(s).

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.

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway and many targeted inhibitors now exist for clinical use, but drug resistance remains a major issue. We recently showed that BRAF-driven melanoma cells treated with BRAF inhibitors can non-genetically adapt to drug within 3-4 days to escape quiescence and resume slow proliferation. Here we show that this phenomenon is not unique to melanomas treated with BRAF inhibitors but rather is widespread across many clinical MAPK inhibitors and cancer types driven by EGFR, KRAS, and BRAF mutations. In all treatment contexts examined, a subset of cells can escape drug-induced quiescence within four days to resume proliferation. These escapee cells broadly experience aberrant DNA replication, accumulate DNA lesions, spend longer in G2-M cell cycle phases, and mount an ATR-dependent stress response. We further identify the Fanconi anemia (FA) DNA repair pathway as critical for successful mitotic completion in escapees. Long-term cultures, patient samples, and clinical data demonstrate a broad dependency on ATR- and FA-mediated stress tolerance. Together, these results highlight the pervasiveness with which MAPK-mutant cancers are able to rapidly escape drug and the importance of suppressing early stress tolerance pathways to potentially achieve more durable clinical responses to targeted MAPK pathway inhibitors.

Senescence, a state of irreversible cell-cycle withdrawal, is difficult to distinguish from quiescence, a state of reversible cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether these states are truly distinct. To address this, we use single-cell time-lapse imaging to distinguish slow-cycling cells that spend long periods in quiescence from cells that never cycle after recovery from senescence-inducing treatments, followed by staining for various senescence biomarkers. We find that the staining intensity of multiple senescence biomarkers is graded rather than binary and reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data show that quiescent and apparent senescent cells are nearly molecularly indistinguishable from each other at a snapshot in time. This suggests that cell-cycle withdrawal itself is graded rather than binary, where the intensities of senescence biomarkers integrate the duration of past cell-cycle withdrawal.

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.

When clonal populations of human cells are exposed to apoptosis-inducing agents, some cells die and others survive. This fractional killing arises not from mutation but from preexisting, stochastic differences in the levels and activities of proteins regulating apoptosis. Here we examine the properties of cells that survive treatment with agonists of two distinct death receptors, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anti-FasR antibodies. We find that "survivor" cells are highly resistant to a second ligand dose applied 1 d later. Resistance is reversible, resetting after several days of culture in the absence of death ligand. "Reset" cells appear identical to drug-naive cells with respect to death ligand sensitivity and gene expression profiles. TRAIL survivors are cross-resistant to activators of FasR and vice versa and exhibit an NF-κB-dependent inflammatory phenotype. Remarkably, reversible resistance is induced in the absence of cell death when caspase inhibitors are present and can be sustained for 1 wk or more, also without cell death, by periodic ligand exposure. Thus stochastic differences in cell state can have sustained consequences for sen-sitivity to prodeath ligands and acquisition of proinflammatory phenotypes. The important role played by periodicity in TRAIL exposure for induction of opposing apoptosis and survival mechanisms has implications for the design of optimal therapeutic agents and protocols.