Signaling pathways relay information about changes in the external environment so that cells can respond appropriately. How much information a pathway can carry depends on its bandwidth. We designed a microfluidic device to reliably change the environment of single cells over a range of frequencies. Using this device, we measured the bandwidth of the Saccharomyces cerevisiae signaling pathway that responds to high osmolarity. This prototypical pathway, the HOG pathway, is shown to act as a low-pass filter, integrating the signal when it changes rapidly and following it faithfully when it changes more slowly. We study the dependence of the pathway's bandwidth on its architecture. We measure previously unknown bounds on all of the in vivo reaction rates acting in this pathway. We find that the two-component Ssk1 branch of this pathway is capable of fast signal integration, whereas the kinase Ste11 branch is not. Our experimental techniques can be applied to other signaling pathways, allowing the measurement of their in vivo kinetics and the quantification of their information capacity.bandwidth ͉ HOG pathway ͉ microfluidics ͉ signal transduction F or a signaling pathway to carry accurate information about its environment, the output of the pathway must closely follow the input. Pathways measuring stimuli that change more rapidly with time need to carry more information. The information capacity of a pathway, i.e., how much information can be transmitted through the pathway per unit time, is proportional to the bandwidth of the pathway (1). The larger the bandwidth of a signaling pathway, the shorter its response time and the more accurately it can follow a rapidly varying signal. Bandwidth measurements are routinely used to characterize communications systems. In this article, we present an experimental technique combined with a microfluidic device that allows measurement of signaling-pathway bandwidth. We use this technique to measure the in vivo bandwidth of the hyperosmolar signaling pathway (HOG) in the yeast Saccharomyces cerevisiae (Fig. 1A).Signaling pathways consist of cascades of proteins where each protein activates the next (2). A signaling cascade is triggered when a receptor is activated by an external stimulus. Often, the signals are transmitted through kinase-dependent phosphorylation (Fig. 1 A). Information about the environment is then transmitted through the pathway, resulting in a response from the cell. The bandwidth of the pathway can be determined by measuring the response of the pathway to the input signal fluctuating at different frequencies. At frequencies up to the bandwidth, the pathway responds faithfully to the input stimulus. To understand how a signaling pathway responds to excitation over a range of frequencies, we numerically studied (3) the three-level branched signaling cascade shown in Fig. 1B and described in Box 1. When the input stimulus to this pathway oscillates at frequencies lower than the bandwidth b , the output of the cascade measured by the levels of the activated en...

Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.

We developed systems to rapidly express any yeast gene or to specifically degrade any protein, each with minimal untargeted disturbance of cell physiology. We illustrate applications of these new tools for elucidating the architecture and dynamics of genetic regulatory networks.

From bacteria to humans, individual cells within isogenic populations can show significant variation in stress tolerance, but the nature of this heterogeneity is not clear. To investigate this, we used single-cell RNA sequencing to quantify transcript heterogeneity in single Saccharomyces cerevisiae cells treated with and without salt stress to explore population variation and identify cellular covariates that influence the stress-responsive transcriptome. Leveraging the extensive knowledge of yeast transcriptional regulation, we uncovered significant regulatory variation in individual yeast cells, both before and after stress. We also discovered that a subset of cells appears to decouple expression of ribosomal protein genes from the environmental stress response in a manner partly correlated with the cell cycle but unrelated to the yeast ultradian metabolic cycle. Live-cell imaging of cells expressing pairs of fluorescent regulators, including the transcription factor Msn2 with Dot6, Sfp1, or MAP kinase Hog1, revealed both coordinated and decoupled nucleocytoplasmic shuttling. Together with transcriptomic analysis, our results suggest that cells maintain a cellular filter against decoupled bursts of transcription factor activation but mount a stress response upon coordinated regulation, even in a subset of unstressed cells.