Modeling a Snap-Action, Variable-Delay Switch Controlling Extrinsic Cell Death

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

Bistability in Apoptosis by Receptor Clustering

Apoptosis is a highly regulated cell death mechanism involved in many physiological processes. A key component of extrinsically activated apoptosis is the death receptor Fas, which, on binding to its cognate ligand FasL, oligomerize to form the death-inducing signaling complex. Motivated by recent experimental data, we propose a mathematical model of death ligand-receptor dynamics where FasL acts as a clustering agent for Fas, which form locally stable signaling platforms through proximity-induced receptor interactions. Significantly, the model exhibits hysteresis, providing an upstream mechanism for bistability and robustness. At low receptor concentrations, the bistability is contingent on the trimerism of FasL. Moreover, irreversible bistability, representing a committed cell death decision, emerges at high concentrations, which may be achieved through receptor pre-association or localization onto membrane lipid rafts. Thus, our model provides a novel theory for these observed biological phenomena within the unified context of bistability. Importantly, as Fas interactions initiate the extrinsic apoptotic pathway, our model also suggests a mechanism by which cells may function as bistable life/death switches independently of any such dynamics in their downstream components. Our results highlight the role of death receptors in deciding cell fate and add to the signal processing capabilities attributed to receptor clustering.Comment: Accepted by PLoS Comput Bio

Exploring the Contextual Sensitivity of Factors that Determine Cell-to-Cell Variability in Receptor-Mediated Apoptosis

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

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death’ (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death’ (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death

The regulation of oncogenic Ras/ERK signalling by dual-specificitymitogen activated protein kinase phosphatases (MKPs)

AbstractDual-specificity MAP kinase (MAPK) phosphatases (MKPs or DUSPs) are well-established negative regulators of MAPK signalling in mammalian cells and tissues. By virtue of their differential subcellular localisation and ability to specifically recognise, dephosphorylate and inactivate different MAPK isoforms, they are key spatiotemporal regulators of pathway activity. Furthermore, as they are transcriptionally regulated as downstream targets of MAPK signalling they can either act as classical negative feedback regulators or mediate cross talk between distinct MAPK pathways. Because MAPKs and particularly Ras/ERK signalling are implicated in cancer initiation and development, the observation that MKPs are abnormally regulated in human tumours has been interpreted as evidence that these enzymes can either suppress or promote carcinogenesis. However, definitive evidence of such roles has been lacking. Here we review recent work based on the use of mouse models, biochemical studies and clinical data that demonstrate key roles for MKPs in modulating the oncogenic potential of Ras/ERK signalling and also indicate that these enzymes may play a role in the response of tumours to certain anticancer drugs. Overall, this work reinforces the importance of negative regulatory mechanisms in modulating the activity of oncogenic MAPK signalling and indicates that MKPs may provide novel targets for therapeutic intervention in cancer

XIAP impairs Smac release from the mitochondria during apoptosis

X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases 3, 7 and 9, and mitochondrial Smac (second mitochondria-derived activator of caspase) release during apoptosis inhibits the activity of XIAP. In this study we show that cytosolic XIAP also feeds back to mitochondria to impair Smac release. We constructed a fluorescent XIAP-fusion protein by labelling NH2- and COOH-termini with Cerulean fluorescent protein (C-XIAP-C). Immunoprecipitation confirmed that C-XIAP-C retained the ability to interact with Smac and impaired extrinsically and intrinsically activated apoptosis in response to tumour necrosis factor-related apoptosis-inducing ligand/cycloheximide and staurosporine. In C-XIAP-C-expressing cells, cytochrome c release from mitochondria proceeded normally, whereas Smac release was significantly prolonged and incomplete. In addition, physiological expression of native XIAP prolonged or limited Smac release in HCT-116 colon cancer cells and primary mouse cortical neurons. The Smac-binding capacity of XIAP, but not caspase inhibition, was central for mitochondrial Smac retention, as evidenced in experiments using XIAP mutants that cannot bind to Smac or effector caspases. Similarly, the release of a Smac mutant that cannot bind to XIAP was not impaired by C-XIAP-C expression. Full Smac release could however be provoked by rapid cytosolic C-XIAP-C depletion upon digitonin-induced plasma membrane permeabilization. Our findings suggest that although mitochondria may already contain pores sufficient for cytochrome c release, elevated amounts of XIAP can selectively impair and limit the release of Smac

Human Cell Chips: Adapting DNA Microarray Spotting Technology to Cell-Based Imaging Assays

Here we describe human spotted cell chips, a technology for determining cellular state across arrays of cells subjected to chemical or genetic perturbation. Cells are grown and treated under standard tissue culture conditions before being fixed and printed onto replicate glass slides, effectively decoupling the experimental conditions from the assay technique. Each slide is then probed using immunofluorescence or other optical reporter and assayed by automated microscopy. We show potential applications of the cell chip by assaying HeLa and A549 samples for changes in target protein abundance (of the dsRNA-activated protein kinase PKR), subcellular localization (nuclear translocation of NFκB) and activation state (phosphorylation of STAT1 and of the p38 and JNK stress kinases) in response to treatment by several chemical effectors (anisomycin, TNFα, and interferon), and we demonstrate scalability by printing a chip with ∼4,700 discrete samples of HeLa cells. Coupling this technology to high-throughput methods for culturing and treating cell lines could enable researchers to examine the impact of exogenous effectors on the same population of experimentally treated cells across multiple reporter targets potentially representing a variety of molecular systems, thus producing a highly multiplexed dataset with minimized experimental variance and at reduced reagent cost compared to alternative techniques. The ability to prepare and store chips also allows researchers to follow up on observations gleaned from initial screens with maximal repeatability

Stochastic Competition between Mechanistically Independent Slippage and Death Pathways Determines Cell Fate during Mitotic Arrest

Variability in cell-to-cell behavior within clonal populations can be attributed to the inherent stochasticity of biochemical reactions. Most single-cell studies have examined variation in behavior due to randomness in gene transcription. Here we investigate the mechanism of cell fate choice and the origin of cell-to-cell variation during mitotic arrest, when transcription is silenced. Prolonged mitotic arrest is commonly observed in cells treated with anti-mitotic drugs. Cell fate during mitotic arrest is determined by two alternative pathways, one promoting cell death, the other promoting cyclin B1 degradation, which leads to mitotic slippage and survival. It has been unclear whether these pathways are mechanistically coupled or independent. In this study we experimentally uncoupled these two pathways using zVAD-fmk to block cell death or Cdc20 knockdown to block slippage. We then used time-lapse imaging to score the kinetics of single cells adopting the remaining fate. We also used kinetic simulation to test whether the behaviors of death versus slippage in cell populations where both pathways are active can be quantitatively recapitulated by a model that assumes stochastic competition between the pathways. Our data are well fit by a model where the two pathways are mechanistically independent, and cell fate is determined by a stochastic kinetic competition between them that results in cell-to-cell variation