Hana El-Samad

Defining Network Topologies that Can Achieve Biochemical Adaptation

Many signaling systems show adaptation-the ability to reset themselves after responding to a stimulus. We computationally searched all possible three-node enzyme network topologies to identify those that could perform adaptation. Only two major core topologies emerge as robust solutions: a negative feedback loop with a buffering node and an incoherent feedforward loop with a proportioner node. Minimal circuits containing these topologies are, within proper regions of parameter space, sufficient to achieve adaptation. More complex circuits that robustly perform adaptation all contain at least one of these topologies at their core. This analysis yields a design table highlighting a finite set of adaptive circuits. Despite the diversity of possible biochemical networks, it may be common to find that only a finite set of core topologies can execute a particular function. These design rules provide a framework for functionally classifying complex natural networks and a manual for engineering networks. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online.

Conformational biosensors reveal GPCR signalling from endosomes

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response

Computational modeling and experimentation in the unfolded protein response reveals a role for the ER-resident chaperone protein BiP in fine-tuning the systems response dynamics.

In silico feedback for in vivo regulation of a gene expression circuit

We show that difficulties in regulating cellular behavior with synthetic biological circuits may be circumvented using in silico feedback control. By tracking a circuits output in Saccharomyces cerevisiae in real time, we precisely control its behavior using an in silico feedback algorithm to compute regulatory inputs implemented through a genetically encoded light-responsive module. Moving control functions outside the cell should enable more sophisticated manipulation of cellular processes whenever real-time measurements of cellular variables are possible.

Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity

Uncoupling of Ire1’s RNAse and kinase activities reveals that its auto-phosphorylation is important for resolution of the unfolded protein response. (See also a related paper by Chawla et al. in this issue).

Bound attractant at the leading vs. the trailing edge determines chemotactic prowess

We have analyzed chemotaxis of neutrophil-differentiated HL60 cells in microfluidic devices that create exponential gradients of the chemoattractant, f-Met-Leu-Phe (fMLP). Such gradients expose each cell to a difference in fMLP concentration (ΔC) across its diameter that is directly proportional to the ambient concentration (C) at that cells position in the gradient, so the ratio ΔC/C is constant everywhere. Cells exposed to ambient fMLP concentrations near the constant of dissociation (Kd) for fMLP binding to its receptor (≈10 nM) crawl much less frequently when ΔC/C is 0.05 than when it is 0.09 or 0.13. Hence, cells can detect the gradient across their diameter without moving and, thus, without experiencing temporal changes in attractant concentration. At all ΔC/C ratios tested, the average chemotactic prowess of individual cells (indicated by the distance a cell traveled in the correct direction divided by the length of its migration path) is maximal for cells that start migrating at concentrations near the Kd and progressively decreases at higher or lower starting concentrations.

Advanced Methods and Algorithms for Biological Networks Analysis

Modeling and analysis of complex biological networks presents a number of mathematical challenges. For the models to be useful from a biological standpoint, they must be systematically compared with data. Robustness is a key to biological understanding and proper feedback to guide experiments,including both the deterministic stability and performance properties of models in the presence of parametric uncertainties and their stochastic behavior in the presence of noise. In this paper, we present mathematical and algorithmic tools to address such questions for models that may be nonlinear, hybrid,and stochastic. These tools are rooted in solid mathematical theories, primarily from robust control and dynamical systems, but with important recent developments. They also have the potential for great practical relevance, which we explore through a series of biologically motivated examples.

Tuning the Activation Threshold of a Kinase Network by Nested Feedback Loops

Interacting negative feedback loops control the sensitivity of amphibian oocytes to the hormone progesterone. Model Progesterone Response The hormone progesterone stimulates maturation of oocytes in the toad Xenopus laevis by binding to its receptor. Although receptor number and affinity for the hormone stay relatively constant, the dose of hormone required to stimulate meiotic maturation varies according to the environmental stimuli to which the animal is exposed, but little is known about how this is regulated. Justman et al. (p. 509; see the Perspective by Skotheim) combined experimental analysis and mathematical modeling to explore the role of glycogen synthase kinase—3β in desensitization, and a natural, sensitizing co-stimulus, the amino acid L-leucine. The results help to explain the layered complexity seen in signal transduction networks: If multiple stimuli act upon components of linked feedback loops, cells can tune their sensitivities dynamically to match their environment. Determining proper responsiveness to incoming signals is fundamental to all biological systems. We demonstrate that intracellular signaling nodes can tune a signaling network’s response threshold away from the basal median effective concentration established by ligand-receptor interactions. Focusing on the bistable kinase network that governs progesterone-induced meiotic entry in Xenopus oocytes, we characterized glycogen synthase kinase–3β (GSK-3β) as a dampener of progesterone responsiveness. GSK-3β engages the meiotic kinase network through a double-negative feedback loop; this specific feedback architecture raises the progesterone threshold in correspondence with the strength of double-negative signaling. We also identified a marker of nutritional status, l-leucine, which lowers the progesterone threshold, indicating that oocytes integrate additional signals into their cell-fate decisions by modulating progesterone responsiveness.

Coordinate control of gene expression noise and interchromosomal interactions in a MAP kinase pathway

In the Saccharomyces cerevisiae pheromone-response pathway, the transcription factor Ste12 is inhibited by two mitogen-activated protein (MAP)-kinase-responsive regulators, Dig1 and Dig2. These two related proteins bind to distinct regions of Ste12 but are redundant in their inhibition of Ste12-dependent gene expression. Here we describe three functions for Dig1 that are non-redundant with those of Dig2. First, the removal of Dig1 results in a specific increase in intrinsic and extrinsic noise in the transcriptional outputs of the mating pathway. Second, in dig1Δ cells, Ste12 relocalizes from the nucleoplasmic distribution seen in wild-type cells into discrete subnuclear foci. Third, genome-wide insertional chromatin immunoprecipitation studies revealed that Ste12-dependent genes have increased interchromosomal interactions in dig1Δ cells. These findings suggest that the regulation of gene expression through long-range gene interactions, a widely observed phenomenon, comes at the cost of increased noise. Consequently, cells may have evolved mechanisms to suppress noise by controlling these interactions.

Population Diversification in a Yeast Metabolic Program Promotes Anticipation of Environmental Shifts

Detailed study of the dynamic response of yeast to combinations of sugars reveals an anticipatory population diversification strategy that allows rapid adaptation to shifts in environmental carbon source availability.