Compartmentalization of a bistable switch enables memory to cross a feedback-driven transition.

Cells make accurate decisions in the face of molecular noise and environmental fluctuations by relying not only on present pathway activity, but also on their memory of past signaling dynamics. Once a decision is made, cellular transitions are often rapid and switch-like due to positive feedback loops in the regulatory network. While positive feedback loops are good at promoting switch-like transitions, they are not expected to retain information to inform subsequent decisions. However, this expectation is based on our current understanding of network motifs that accounts for temporal, but not spatial, dynamics. Here, we show how spatial organization of the feedback-driven yeast G1/S switch enables the transmission of memory of past pheromone exposure across this transition. We expect this to be one of many examples where the exquisite spatial organization of the eukaryotic cell enables previously well-characterized network motifs to perform new and unexpected signal processing functions.

CDK and MAPK Synergistically Regulate Signaling Dynamics via a Shared Multi-site Phosphorylation Region on the Scaffold Protein Ste5.

We report an unanticipated system of joint regulation by cyclin-dependent kinase (CDK) and mitogen-activated protein kinase (MAPK), involving collaborative multi-site phosphorylation of a single substrate. In budding yeast, the protein Ste5 controls signaling through a G1 arrest pathway. Upon cell-cycle entry, CDK inhibits Ste5 via multiple phosphorylation sites, disrupting its membrane association. Using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell-cycle stages. Surprisingly, in S phase, where Ste5 recruitment should be blocked, we observed an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggest that the CDK and MAPK modify shared sites, which are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Such collaborative phosphorylation can broaden regulatory inputs and diversify output dynamics of signaling pathways.

Mitotic and pheromone-specific intrinsic polarization cues interfere with gradient sensing in Saccharomyces cerevisiae.

Polarity decisions are central to many processes, including mitosis and chemotropism. In Saccharomyces cerevisiae, budding and mating projection (MP) formation use an overlapping system of cortical landmarks that converges on the small G protein Cdc42. However, pheromone-gradient sensing must override the Rsr1-dependent internal polarity cues used for budding. Using this model system, we asked what happens when intrinsic and extrinsic spatial cues are not aligned. Is there competition, or collaboration? By live-cell microscopy and microfluidics techniques, we uncovered three previously overlooked features of this signaling system. First, the cytokinesis-associated polarization patch serves as a polarity landmark independently of all known cues. Second, the Rax1-Rax2 complex functions as a pheromone-promoted polarity cue in the distal pole of the cells. Third, internal cues remain active during pheromone-gradient tracking and can interfere with this process, biasing the location of MPs. Yeast defective in internal-cue utilization align significantly better than wild type with artificially generated pheromone gradients.

Single-cell profiling screen identifies microtubule-dependent reduction of variability in signaling.

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.

Yeast GPCR signaling reflects the fraction of occupied receptors, not the number.

According to receptor theory, the effect of a ligand depends on the amount of agonist-receptor complex. Therefore, changes in receptor abundance should have quantitative effects. However, the response to pheromone in Saccharomyces cerevisiae is robust (unaltered) to increases or reductions in the abundance of the G-protein-coupled receptor (GPCR), Ste2, responding instead to the fraction of occupied receptor. We found experimentally that this robustness originates during G-protein activation. We developed a complete mathematical model of this step, which suggested the ability to compute fractional occupancy depends on the physical interaction between the inhibitory regulator of G-protein signaling (RGS), Sst2, and the receptor. Accordingly, replacing Sst2 by the heterologous hsRGS4, incapable of interacting with the receptor, abolished robustness. Conversely, forcing hsRGS4:Ste2 interaction restored robustness. Taken together with other results of our work, we conclude that this GPCR pathway computes fractional occupancy because ligand-bound GPCR-RGS complexes stimulate signaling while unoccupied complexes actively inhibit it. In eukaryotes, many RGSs bind to specific GPCRs, suggesting these complexes with opposing activities also detect fraction occupancy by a ratiometric measurement. Such complexes operate as push-pull devices, which we have recently described.

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range.

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general "systems level" mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step.

Messages do diffuse faster than messengers: reconciling disparate estimates of the morphogen bicoid diffusion coefficient.

The gradient of Bicoid (Bcd) is key for the establishment of the anterior-posterior axis in Drosophila embryos. The gradient properties are compatible with the SDD model in which Bcd is synthesized at the anterior pole and then diffuses into the embryo and is degraded with a characteristic time. Within this model, the Bcd diffusion coefficient is critical to set the timescale of gradient formation. This coefficient has been measured using two optical techniques, Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), obtaining estimates in which the FCS value is an order of magnitude larger than the FRAP one. This discrepancy raises the following questions: which estimate is "correct''; what is the reason for the disparity; and can the SDD model explain Bcd gradient formation within the experimentally observed times? In this paper, we use a simple biophysical model in which Bcd diffuses and interacts with binding sites to show that both the FRAP and the FCS estimates may be correct and compatible with the observed timescale of gradient formation. The discrepancy arises from the fact that FCS and FRAP report on different effective (concentration dependent) diffusion coefficients, one of which describes the spreading rate of the individual Bcd molecules (the messengers) and the other one that of their concentration (the message). The latter is the one that is more relevant for the gradient establishment and is compatible with its formation within the experimentally observed times.

Impact of upstream and downstream constraints on a signaling module's ultrasensitivity.

Much work has been done on the study of the biochemical mechanisms that result in ultrasensitive behavior of simple biochemical modules. However, in a living cell, such modules are embedded in a bigger network that constrains the range of inputs that the module will receive as well as the range of the module's outputs that network will be able to detect. Here, we studied how the effective ultrasensitivity of a modular system is affected by these restrictions. We use a simple setup to explore to what extent the dynamic range spanned by upstream and downstream components of an ultrasensitive module impact on the effective sensitivity of the system. Interestingly, we found for some ultrasensitive motifs that dynamic range limitations imposed by downstream components can produce effective sensitivities much larger than that of the original module when considered in isolation.

Negative feedback that improves information transmission in yeast signalling.

Haploid Saccharomyces cerevisiae yeast cells use a prototypic cell signalling system to transmit information about the extracellular concentration of mating pheromone secreted by potential mating partners. The ability of 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 mitogen-activated protein kinase Fus3 mediates fast-acting negative feedback that adjusts the dose response of the downstream system response to match the dose response 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 previously uncharacterized signal-promoting function of the regulator of G-protein signalling protein Sst2. Our work suggests that negative feedback is a general mechanism used in signalling systems to align dose responses and thereby increase the fidelity of information transmission.

Scaffold number in yeast signaling system sets tradeoff between system output and dynamic range.

Although the proteins comprising many signaling systems are known, less is known about their numbers per cell. Existing measurements often vary by more than 10-fold. Here, we devised improved quantification methods to measure protein abundances in the Saccharomyces cerevisiae pheromone response pathway, an archetypical signaling system. These methods limited variation between independent measurements of protein abundance to a factor of two. We used these measurements together with quantitative models to identify and investigate behaviors of the pheromone response system sensitive to precise abundances. The difference between the maximum and basal signaling output (dynamic range) of the pheromone response MAPK cascade was strongly sensitive to the abundance of Ste5, the MAPK scaffold protein, and absolute system output depended on the amount of Fus3, the MAPK. Additional analysis and experiment suggest that scaffold abundance sets a tradeoff between maximum system output and system dynamic range, a prediction supported by recent experiments.

Protocol for FRAP-based estimation of nuclear import and export rates in single yeast cells.

Here, we present a protocol for estimating nuclear transport parameters in single cells. We describe steps for performing four consecutive fluorescence recovery after photobleaching experiments, fitting the obtained data to an ordinary differential equations model, and statistical analysis of the fittings using a specialized R package. This protocol permits the estimation of import and export rates, nuclear or cytosolic fixed fractions, and total number of molecules. For complete details on the use and execution of this protocol, please refer to Durrieu et al.1.

Quantitative measurement of protein relocalization in live cells.

Microscope cytometry provides a powerful means to study signaling in live cells. Here we present a quantitative method to measure protein relocalization over time, which reports the absolute fraction of a tagged protein in each compartment. Using this method, we studied an essential step in the early propagation of the pheromone signal in Saccharomyces cerevisiae: recruitment to the membrane of the scaffold Ste5 by activated Gßγ dimers. We found that the dose response of Ste5 recruitment is graded (EC50 = 0.44 ± 0.08 nM, Hill coefficient = 0.8 ± 0.1). Then, we determined the effective dissociation constant (K(de)) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. K(de) changed during the first minutes from a high affinity of < 0.65 nM to a steady-state value of 17 ± 9 nM. During the same period, the total number of binding sites decreased slightly, from 1940 ± 150 to 1400 ± 200. This work shows how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system.

Modelling reveals novel roles of two parallel signalling pathways and homeostatic feedbacks in yeast.

The high osmolarity glycerol (HOG) pathway in yeast serves as a prototype signalling system for eukaryotes. We used an unprecedented amount of data to parameterise 192 models capturing different hypotheses about molecular mechanisms underlying osmo-adaptation and selected a best approximating model. This model implied novel mechanisms regulating osmo-adaptation in yeast. The model suggested that (i) the main mechanism for osmo-adaptation is a fast and transient non-transcriptional Hog1-mediated activation of glycerol production, (ii) the transcriptional response serves to maintain an increased steady-state glycerol production with low steady-state Hog1 activity, and (iii) fast negative feedbacks of activated Hog1 on upstream signalling branches serves to stabilise adaptation response. The best approximating model also indicated that homoeostatic adaptive systems with two parallel redundant signalling branches show a more robust and faster response than single-branch systems. We corroborated this notion to a large extent by dedicated measurements of volume recovery in single cells. Our study also demonstrates that systematically testing a model ensemble against data has the potential to achieve a better and unbiased understanding of molecular mechanisms.

Modulation of transcription factor dynamics allows versatile information transmission.

Cells detect changes in their environment and generate responses, often involving changes in gene expression. In this paper we use information theory and a simple transcription model to analyze whether the resulting gene expression serves to identify extracellular stimuli and assess their intensity when they are encoded in the amplitude, duration or frequency of pulses of a transcription factor's nuclear concentration (or activation state). We find, for all cases, that about three ranges of input strengths can be distinguished and that maximum information transmission occurs for fast and high activation threshold promoters. The three input modulation modes differ in the sensitivity to changes in the promoters parameters. Frequency modulation is the most sensitive and duration modulation, the least. This is key for signal identification: there are promoter parameters that yield a relatively high information transmission for duration or amplitude modulation and a much smaller value for frequency modulation. The reverse situation cannot be found with a single promoter transcription model. Thus, pulses of transcription factors can selectively activate the "frequency-tuned" promoter while prolonged nuclear accumulation would activate promoters of all three modes simultaneously. Frequency modulation is therefore highly selective and better suited than the other encoding modes for signal identification without requiring other mediators of the transduction process.

rcell2: Microscopy-Based Cytometry in R.

This article describes a method for quantifying various cellular features (e.g., volume, curvature, total and sub-cellular fluorescence localization) of individual cells from sets of microscope images, and for tracking them over time-course microscopy experiments. One purposely defocused transmission image (sometimes referred to as bright-field or BF) is used to segment the image and locate each cell. Fluorescence images (one for each of the color channels or z-stacks to be analyzed) may be acquired by conventional wide-field epifluorescence or confocal microscopy. This method uses a set of R packages called rcell2. Relative to the original release of Rcell (Bush et al., 2012), the updated version bundles, into a single software suite, the image-processing capabilities of Cell-ID, offers new data analysis tools for cytometry, and relies on the widely used data analysis and visualization tools of the statistical programming framework R. © 2023 Wiley Periodicals LLC. Basic Protocol: Extracting quantitative information from single cells Support Protocol 1: Obtaining and installing Cell-ID and R Support Protocol 2: Preparing cells for imaging.

Characterization of cell-to-cell variation in nuclear transport rates and identification of its sources.

Nuclear transport is an essential part of eukaryotic cell function. Here, we present scFRAP, a model-assisted fluorescent recovery after photobleaching (FRAP)- based method to determine nuclear import and export rates independently in individual live cells. To overcome the inherent noise of single-cell measurements, we performed sequential FRAPs on the same cell. We found large cell-to-cell variation in transport rates within isogenic yeast populations. For passive transport, the variability in NPC number might explain most of the variability. Using this approach, we studied mother-daughter cell asymmetry in the active nuclear shuttling of the transcription factor Ace2, which is specifically concentrated in daughter cell nuclei in early G1. Rather than reduced export in the daughter cell, as previously hypothesized, we found that this asymmetry is mainly due to an increased import in daughters. These results shed light on cell-to-cell variation in cellular dynamics and its sources.

PI3K/AKT pathway regulates phosphorylation of steroid receptors, hormone independence and tumor differentiation in breast cancer.

Using a model of medroxyprogesterone acetate (MPA)-induced mouse mammary tumors that transit through different stages of hormone dependence, we previously reported that the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT (protein kinase B) pathway is critical for the growth of hormone-independent (HI) mammary carcinomas but not for the growth of hormone-dependent (HD) mammary carcinomas. The objective of this work was to explore whether the activation of the PI3K/AKT pathway is responsible for the changes in tumor phenotype and for the transition to autonomous growth. We found that the inhibition of the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway suppresses HI tumor growth. In addition, we were able to induce mammary tumors in mice in the absence of MPA by inoculating HD tumor cells expressing a constitutively active form of AKT1, myristoylated AKT1 (myrAKT1). These tumors were highly differentiated and displayed a ductal phenotype with laminin-1 and cytokeratin 8 expression patterns typical of HI tumors. Furthermore, myrAKT1 increased the tumor growth of IBH-6 and IBH-7 human breast cancer cell lines. In the estrogen-dependent IBH-7 cell line, myrAKT1 induced estrogen-independent growth accompanied by the expression of the adhesion markers focal adhesion kinase and E-cadherin. Finally, we found that cells expressing myrAKT1 exhibited increased phosphorylation of the progesterone receptor at Ser190 and Ser294 and of the estrogen receptor α at Ser118 and Ser167, independently of exogenous MPA or estrogen supply. Our results indicate that the activation of the PI3K/AKT/mTOR pathway promotes tissue architecture remodeling and the activation of steroid receptors.

Regulated cell-to-cell variation in a cell-fate decision system.

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.

Akt Is S-Palmitoylated: A New Layer of Regulation for Akt.

The protein kinase Akt/PKB participates in a great variety of processes, including translation, cell proliferation and survival, as well as malignant transformation and viral infection. In the last few years, novel Akt posttranslational modifications have been found. However, how these modification patterns affect Akt subcellular localization, target specificity and, in general, function is not thoroughly understood. Here, we postulate and experimentally demonstrate by acyl-biotin exchange (ABE) assay and 3H-palmitate metabolic labeling that Akt is S-palmitoylated, a modification related to protein sorting throughout subcellular membranes. Mutating cysteine 344 into serine blocked Akt S-palmitoylation and diminished its phosphorylation at two key sites, T308 and T450. Particularly, we show that palmitoylation-deficient Akt increases its recruitment to cytoplasmic structures that colocalize with lysosomes, a process stimulated during autophagy. Finally, we found that cysteine 344 in Akt1 is important for proper its function, since Akt1-C344S was unable to support adipocyte cell differentiation in vitro. These results add an unexpected new layer to the already complex Akt molecular code, improving our understanding of cell decision-making mechanisms such as cell survival, differentiation and death.

GPCR receptor phosphorylation and endocytosis are not necessary to switch polarized growth between internal cues during pheromone response in S. cerevisiae.

Chemotactic/chemotropic cells follow accurately the direction of gradients of regulatory molecules. Many G-protein-coupled receptors (GPCR) function as chemoattractant receptors to guide polarized responses. In "a" mating type yeast, the GPCR Ste2 senses the α-cell's pheromone. Previously, phosphorylation and trafficking of this receptor have been implicated in the process of gradient sensing, where cells dynamically correct growth. Correction is often necessary since yeast have intrinsic polarity sites that interfere with a correct initial gradient decoding. We have recently showed that when actively dividing (not in G1) yeast are exposed to a uniform pheromone concentration, they initiate a pheromone-induced polarization next to the mother-daughter cytokinesis site. Then, they reorient their growth to the intrinsic polarity site. Here, to study if Ste2 phosphorylation and internalization are involved in this process, we generated receptor variants combining three types of mutated signals for the first time: phosphorylation, ubiquitylation and the NPFX1,2D Sla1-binding motif. We first characterized their effect on endocytosis and found that these processes regulate internalization in a more complex manner than previously shown. Interestingly, we showed that receptor phosphorylation can drive internalization independently of ubiquitylation and the NPFX1,2D motif. When tested in our assays, cells expressing either phosphorylation or endocytosis-deficient receptors were able to switch away from the cytokinesis site to find the intrinsic polarity site as efficiently as their WT counterparts. Thus, we conclude that these processes are not necessary for the reorientation of polarization.