Here we studied the quantitative behaviour and cell-to-cell variability of a prototypical eukaryotic cell-fate decision system, the mating pheromone response pathway in yeast. We dissected and measured sources of variation in system output, analysing thousands of individual, genetically identical cells. Only a small proportion of total cell-to-cell variation is caused by random fluctuations in gene transcription and translation during the response ('expression noise'). Instead, variation is dominated by differences in the capacity of individual cells to transmit signals through the pathway ('pathway capacity') and to express proteins from genes ('expression capacity'). Cells with high expression capacity express proteins at a higher rate and increase in volume more rapidly. Our results identify two mechanisms that regulate cell-to-cell variation in pathway capacity. First, the MAP kinase Fus3 suppresses variation at high pheromone levels, while the MAP kinase Kss1 enhances variation at low pheromone levels. Second, pathway capacity and expression capacity are negatively correlated, suggesting a compensatory mechanism that allows cells to respond more precisely to pheromone in the presence of a large variation in expression capacity.

Haploid Saccharomyces cerevisiae yeast cells use a prototypic cell signaling system to transmit information about the extracellular concentration of mating pheromone secreted by potential mating partners. The ability for cells to respond distinguishably to different pheromone concentrations depends on how much information about pheromone concentration the system can transmit. Here we show that the MAPK Fus3 mediates fast-acting negative feedback that adjusts the dose-response of downstream system response to match that of receptor-ligand binding. This “dose-response alignment”, defined by a linear relationship between receptor occupancy and downstream response, can improve the fidelity of information transmission by making downstream responses corresponding to different receptor occupancies more distinguishable and reducing amplification of stochastic noise during signal transmission. We also show that one target of the feedback is a novel signal-promoting function of the RGS protein Sst2. Our work suggests that negative feedback is a general mechanism used in signaling systems to align dose-responses and thereby increase the fidelity of information transmission.

Organismal fitness depends on the ability of gene networks to function robustly in the face of environmental and genetic perturbations. Understanding the mechanisms of this stability is one of the key aims of modern systems biology. Dissecting the basis of robustness to mutation has proven a particular challenge, with most experimental models relying on artificial DNA sequence variants engineered in the laboratory. In this work, we hypothesized that negative regulatory feedback could stabilize gene expression against the disruptions that arise from natural genetic variation. We screened yeast transcription factors for feedback and used the results to establish ROX1 (Repressor of hypOXia) as a model system for the study of feedback in circuit behaviors and its impact across genetically heterogeneous populations. Mutagenesis experiments revealed the mechanism of Rox1 as a direct transcriptional repressor at its own gene, enabling a regulatory program of rapid induction during environmental change that reached a plateau of moderate steady-state expression. Additionally, in a given environmental condition, Rox1 levels varied widely across genetically distinct strains; the ROX1 feedback loop regulated this variation, in that the range of expression levels across genetic backgrounds showed greater spread in ROX1 feedback mutants than among strains with the ROX1 feedback loop intact. Our findings indicate that the ROX1 feedback circuit is tuned to respond to perturbations arising from natural genetic variation in addition to its role in induction behavior. We suggest that regulatory feedback may be an important element of the network architectures that confer mutational robustness across biology.R obustness of organismal function in the face of perturbations is critical for fitness. Since the seminal work of Waddington (1), biologists have remarked on the stability of phenotypes against environmental and genetic variation, and understanding how organisms achieve robustness remains one of the major challenges in systems biology (2-4). Much of the search for molecular mechanisms of robustness has focused on gene regulation. Characteristics of regulatory networks that confer robustness include pathway redundancy and master regulatory organization (5), phenotypic capacitors (6-8), paired activating and inhibiting inputs (9), and cooperative and feed-forward regulation (10). Additionally, negative regulatory feedback, in which a biomolecule represses its own abundance, can buffer variation in gene expression (11,12), and negative feedback loops have been shown to underlie robustness to variable environmental conditions and stochastic intracellular change (13-15). Negative feedback may also confer network stability against the effects of mutations (3, 16), but evidence for negative feedback as a driver of mutational robustness in vivo has been at a premium (17); the relevance of this principle to natural genetic variation remains largely unknown.In this work, we focused on negative feedback in yeast hypoxia regulation motiva...

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Populations of isogenic cells often respond coherently to signals, despite differences in protein abundance and cell state. Previously, we uncovered processes in the Saccharomyces cerevisiae pheromone response system (PRS) that reduced cell‐to‐cell variability in signal strength and cellular response. Here, we screened 1,141 non‐essential genes to identify 50 “variability genes”. Most had distinct, separable effects on strength and variability of the PRS, defining these quantities as genetically distinct “axes” of system behavior. Three genes affected cytoplasmic microtubule function: BIM1, GIM2, and GIM4. We used genetic and chemical perturbations to show that, without microtubules, PRS output is reduced but variability is unaffected, while, when microtubules are present but their function is perturbed, output is sometimes lowered, but its variability is always high. The increased variability caused by microtubule perturbations required the PRS MAP kinase Fus3 and a process at or upstream of Ste5, the membrane‐localized scaffold to which Fus3 must bind to be activated. Visualization of Ste5 localization dynamics demonstrated that perturbing microtubules destabilized Ste5 at the membrane signaling site. The fact that such microtubule perturbations cause aberrant fate and polarity decisions in mammals suggests that microtubule‐dependent signal stabilization might also operate throughout metazoans.