Multistability maintains redox homeostasis in human cells.

Cells metabolize nutrients through a complex metabolic and signaling network that governs redox homeostasis. At the core of this, redox regulatory network is a mutually inhibitory relationship between reduced glutathione and reactive oxygen species (ROS)-two opposing metabolites that are linked to upstream nutrient metabolic pathways (glucose, cysteine, and glutamine) and downstream feedback loops of signaling pathways (calcium and NADPH oxidase). We developed a nutrient-redox model of human cells to understand system-level properties of this network. Combining in silico modeling and ROS measurements in individual cells, we show that ROS dynamics follow a switch-like, all-or-none response upon glucose deprivation at a threshold that is approximately two orders of magnitude lower than its physiological concentration. We also confirm that this ROS switch can be irreversible and exhibits hysteresis, a hallmark of bistability. Our findings evidence that bistability modulates redox homeostasis in human cells and provide a general framework for quantitative investigations of redox regulation in humans.

Viscosity-dependent control of protein synthesis and degradation.

It has been proposed that the concentration of proteins in the cytoplasm maximizes the speed of important biochemical reactions. Here we have used Xenopus egg extracts, which can be diluted or concentrated to yield a range of cytoplasmic protein concentrations, to test the effect of cytoplasmic concentration on mRNA translation and protein degradation. We find that protein synthesis rates are maximal in ~1x cytoplasm, whereas protein degradation continues to rise to a higher optimal concentration of ~1.8x. We show that this difference in optima can be attributed to a greater sensitivity of translation to cytoplasmic viscosity. The different concentration optima could produce a negative feedback homeostatic system, where increasing the cytoplasmic protein concentration above the 1x physiological level increases the viscosity of the cytoplasm, which selectively inhibits translation and drives the system back toward the 1x set point.

Robust trigger wave speed in Xenopus cytoplasmic extracts.

Self-regenerating trigger waves can spread rapidly through the crowded cytoplasm without diminishing in amplitude or speed, providing consistent, reliable, long-range communication. The macromolecular concentration of the cytoplasm varies in response to physiological and environmental fluctuations, raising the question of how or if trigger waves can robustly operate in the face of such fluctuations. Using Xenopus extracts, we find that mitotic and apoptotic trigger wave speeds are remarkably invariant. We derive a model that accounts for this robustness and for the eventual slowing at extremely high and low cytoplasmic concentrations. The model implies that the positive and negative effects of cytoplasmic concentration (increased reactant concentration vs. increased viscosity) are nearly precisely balanced. Accordingly, artificially maintaining a constant cytoplasmic viscosity during dilution abrogates this robustness. The robustness in trigger wave speeds may contribute to the reliability of the extremely rapid embryonic cell cycle.

Protein homeostasis from diffusion-dependent control of protein synthesis and degradation.

It has been proposed that the concentration of proteins in the cytoplasm maximizes the speed of important biochemical reactions. Here we have used the Xenopus extract system, which can be diluted or concentrated to yield a range of cytoplasmic protein concentrations, to test the effect of cytoplasmic concentration on mRNA translation and protein degradation. We found that protein synthesis rates are maximal in ~1x cytoplasm, whereas protein degradation continues to rise to an optimal concentration of ~1.8x. This can be attributed to the greater sensitivity of translation to cytoplasmic viscosity, perhaps because it involves unusually large macromolecular complexes like polyribosomes. The different concentration optima sets up a negative feedback homeostatic system, where increasing the cytoplasmic protein concentration above the 1x physiological level increases the viscosity of the cytoplasm, which selectively inhibits translation and drives the system back toward the 1x set point.

Robust trigger wave speed in Xenopus cytoplasmic extracts.

Self-regenerating trigger waves can spread rapidly through the crowded cytoplasm without diminishing in amplitude or speed, providing consistent, reliable, long-range communication. The macromolecular concentration of the cytoplasm varies in response to physiological and environmental fluctuations, raising the question of how or if trigger waves can robustly operate in the face of such fluctuations. Using Xenopus extracts, we found that mitotic and apoptotic trigger wave speeds are remarkably invariant. We derived a model that accounts for this robustness and for the eventual slowing at extremely high and low cytoplasmic concentrations. The model implies that the positive and negative effects of cytoplasmic concentration (increased reactant concentration vs. increased viscosity) are nearly precisely balanced. Accordingly, artificially maintaining a constant cytoplasmic viscosity during dilution abrogates this robustness. The robustness in trigger wave speeds may contribute to the reliability of the extremely rapid embryonic cell cycle.

Dual mechanisms regulate the nucleocytoplasmic localization of human DDX6.

DDX6 is a conserved DEAD-box protein (DBP) that plays central roles in cytoplasmic RNA regulation, including processing body (P-body) assembly, mRNA decapping, and translational repression. Beyond its cytoplasmic functions, DDX6 may also have nuclear functions because its orthologues are known to localize to nuclei in several biological contexts. However, it is unclear whether DDX6 is generally present in human cell nuclei, and the molecular mechanism underlying DDX6 subcellular distribution remains elusive. In this study, we showed that DDX6 is commonly present in the nuclei of human-derived cells. Our structural and molecular analyses deviate from the current model that the shuttling of DDX6 is directly mediated by the canonical nuclear localization signal (NLS) and nuclear export signal (NES), which are recognized and transported by Importin-α/ß and CRM1, respectively. Instead, we show that DDX6 can be transported by 4E-T in a piggyback manner. Furthermore, we provide evidence for a novel nuclear targeting mechanism in which DDX6 enters the newly formed nuclei by "hitch-hiking" on mitotic chromosomes with its C-terminal domain during M phase progression. Together, our results indicate that the nucleocytoplasmic localization of DDX6 is regulated by these dual mechanisms.