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

Abstract

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. In higher eukaryotes, tissue development and homeostasis involves a subtle balance between rates of cell birth and death. Cell death (apoptosis) is triggered by activation of caspases, specialized enzymes that digest essential cellular constituents and trigger degradation of genomic DNA. Under normal circumstances receptor-dependent cell death is very tightly repressed, but it is irreversibly induced upon receipt of an appropriate signal. Mutations that interfere with this all-or-none control contribute to developmental abnormalities, autoimmune disease, and cancer. The biochemical properties of most apoptotic proteins are quite well understood, but it is unclear how these proteins work together. By combining live-cell microscopy, genetic perturbation, and mathematical modeling, we seek quantitative insight into cell death with a focus on network dynamics and control. We find that cells vary dramatically in the time between receipt of an apoptotic signal and the commitment to death. This variability arises from cell-to-cell differences in the activities of receptor-proximal biochemical reactions. Rapid all-or-none progress from commitment to actual death is achieved downstream by pro-apoptotic proteins found in the mitochondrial membrane. Our work provides a quantitative picture of apoptosis that advances understanding of oncogenic mechanisms and should eventually assist in the development of pro-apoptotic cancer therapies.