TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway.

Saccharomyces cerevisiae respond to mating pheromone through the GPCRs Ste2 and Ste3, which promote growth of a mating projection in response to ligand binding. This commitment to mating is nutritionally and energetically taxing, and so we hypothesized that the cell may suppress mating signaling during starvation. We set out to investigate negative regulators of the mating pathway in nutritionally depleted environments. Here, we report that nutrient deprivation led to loss of Ste2 from the plasma membrane. Recapitulating this effect with nitrogen starvation led us to hypothesize that it was due to TORC1 signaling. Rapamycin inhibition of TORC1 impacted membrane levels of all yeast GPCRs. Inhibition of TORC1 also dampened mating pathway output. Deletion analysis revealed that TORC1 repression leads to α-arrestin-directed CME through TORC2-Ypk1 signaling. We then set out to determine whether major downstream effectors of the TOR complexes also downregulate pathway output during mating. We found that autophagy contributes to pathway downregulation through analysis of strains lacking ATG8 . We also show that Ypk1 significantly reduced pathway output. Thus, both autophagy machinery and TORC2-Ypk1 signaling serve as attenuators of pheromone signaling during mating. Altogether, we demonstrate that the stress-responsive TOR complexes coordinate GPCR endocytosis and reduce the magnitude of pheromone signaling, in ligand-independent and ligand-dependent contexts. One Sentence Summary: TOR signaling regulates the localization of all Saccharomyces cerevisiae GPCRs during starvation and suppress the mating pathway in the presence and absence of ligand.

The roles of yeast formins and their regulators Bud6 and Bil2 in the pheromone response.

In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.

The G-alpha Gpa1 directs septin localization in the mating projection of Saccharomyces cerevisiae through its Ubiquitination Domain and Endocytic Machinery.

The yeast mating response uses a G-protein coupled receptor (GPCR), Ste2, to detect mating pheromone and initiate mating projection morphogenesis. The septin cytoskeleton plays a key role in the formation of the mating projection, forming structures at the base of the projection. Desensitization of the Gα, Gpa1, by the Regulator of G-protein Signaling (RGS), Sst2, is required for proper septin organization and morphogenesis. In cells where the Gα is hyperactive, septins are mislocalized to the site of polarity, and the cells are unable to track a pheromone gradient. We set out to identify the proteins that mediate Gα control of septins during the Saccharomyces cerevisiae mating response by making mutations to rescue septin localization in cells expressing the hyperactive Gα mutant gpa1G302S. We found that single deletions of the septin chaperone Gic1, the Cdc42 GAP Bem3, and the epsins Ent1 and Ent2 rescued the polar cap accumulation of septins in the hyperactive Gα. We created an agent-based model of vesicle trafficking that predicts how changes in endocytic cargo licensing alters localization of endocytosis that mirrors the septin localization we see experimentally. We hypothesized that hyperactive Gα may increase the rate of endocytosis of a pheromone responsive cargo, thereby altering where septins are localized. Both the GPCR and the Gα are known to be internalized by clathrin-mediated endocytosis during the pheromone response. Deletion of the GPCR C-terminus to block internalization partially rescued septin organization. However, deletion of the Gpa1 ubiquitination domain required for its endocytosis completely abrogated septin accumulation at the polarity site. Our data support a model where the location of endocytosis serves as a spatial mark for septin structure assembly and that desensitization of the Gα delays its endocytosis sufficiently that septins are placed peripheral to the site of Cdc42 polarity.

Phosphorylation of RGS regulates MAP kinase localization and promotes completion of cytokinesis.

Yeast use the G-protein-coupled receptor signaling pathway to detect and track the mating pheromone. The G-protein-coupled receptor pathway is inhibited by the regulator of G-protein signaling (RGS) Sst2 which induces Gα GTPase activity and inactivation of downstream signaling. G-protein signaling activates the MAPK Fus3, which phosphorylates the RGS; however, the role of this modification is unknown. We found that pheromone-induced RGS phosphorylation peaks early; the phospho-state of RGS controls its localization and influences MAPK spatial distribution. Surprisingly, phosphorylation of the RGS promotes completion of cytokinesis before pheromone-induced growth. Completion of cytokinesis in the presence of pheromone is promoted by the kelch-repeat protein, Kel1 and antagonized by the formin Bni1. We found that RGS complexes with Kel1 and prefers the unphosphorylatable RGS mutant. We also found overexpression of unphosphorylatable RGS exacerbates cytokinetic defects, whereas they are rescued by overexpression of Kel1. These data lead us to a model where Kel1 promotes completion of cytokinesis before pheromone-induced polarity but is inhibited by unphosphorylated RGS binding.

Beneficial impacts of neuromuscular electrical stimulation on muscle structure and function in the zebrafish model of Duchenne muscular dystrophy.

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.

Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis.

Reorganization of cellular proteins into subcellular compartments, such as the concentration of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. One example of this is the induction of the yeast Nab3 transcription termination factor to rearrange from its pan-nucleoplasmic distribution to a granule at the nuclear periphery in response to nutrient limitation. Recent work in many cell types has shown that protein condensation in the nucleus is functionally important for transcription initiation, RNA processing, and termination. However, little is known about how subnuclear compartments form. Here, we have quantitatively analyzed this dynamic process in living yeast using a high-throughput computational tool and fluorescence microscopy. This analysis revealed that Nab3 granule accumulation varies in penetrance across yeast strains. A concentrated single granule is formed from at least a quarter of the nuclear Nab3 drawn from the rest of the nucleus. Levels of granule accumulation were inversely correlated with a growth defect in the absence of glucose. Importantly, the basis for some of the variation in penetrance was attributable to a defect in mitochondrial function. This publicly available computational tool provides a rigorous, reproducible, and unbiased examination of Nab3 granule accumulation that should be widely applicable to a variety of fluorescent images. Thousands of live cells can be readily examined enabling rigorous statistical verification of significance. With it, we describe a new feature of inducible subnuclear compartment formation for RNA-binding transcription factors and an important determinant of granule biogenesis.

Gradient Tracking by Yeast GPCRs in a Microfluidics Chamber.

Cells typically exist in a highly dynamic environment, which cannot easily be recreated in culture dishes or microwell plates. Microfluidic devices can provide precise control of the time, dose, and orientation of a stimulus, while simultaneously capturing quantitative single-cell data. The approach is particularly powerful when combined with the genetically tractable yeast model organism. The GPCR pathway in yeast is structurally conserved and functionally interchangeable with those in humans. We describe the implementation of a microfluidic device to investigate morphological and transcriptional responses of yeast to a gradient or pulse administration of a GPCR ligand, the peptide mating pheromone α-factor.

Cytoskeletal diversification across 1 billion years: What red algae can teach us about the cytoskeleton, and vice versa.

The cytoskeleton has a central role in eukaryotic biology, enabling cells to organize internally, polarize, and translocate. Studying cytoskeletal machinery across the tree of life can identify common elements, illuminate fundamental mechanisms, and provide insight into processes specific to less-characterized organisms. Red algae represent an ancient lineage that is diverse, ecologically significant, and biomedically relevant. Recent genomic analysis shows that red algae have a surprising paucity of cytoskeletal elements, particularly molecular motors. Here, we review the genomic and cell biological evidence and propose testable models of how red algal cells might perform processes including cell motility, cytokinesis, intracellular transport, and secretion, given their reduced cytoskeletons. In addition to enhancing understanding of red algae and lineages that evolved from red algal endosymbioses (e.g., apicomplexan parasites), these ideas may also provide insight into cytoskeletal processes in animal cells.

Temporal dynamics of viral load and false negative rate influence the levels of testing necessary to combat COVID-19 spread.

Colleges and other organizations are considering testing plans to return to operation as the COVID-19 pandemic continues. Pre-symptomatic spread and high false negative rates for testing may make it difficult to stop viral spread. Here, we develop a stochastic agent-based model of COVID-19 in a university sized population, considering the dynamics of both viral load and false negative rate of tests on the ability of testing to combat viral spread. Reported dynamics of SARS-CoV-2 can lead to an apparent false negative rate from ~ 17 to ~ 48%. Nonuniform distributions of viral load and false negative rate lead to higher requirements for frequency and fraction of population tested in order to bring the apparent Reproduction number (Rt) below 1. Thus, it is important to consider non-uniform dynamics of viral spread and false negative rate in order to model effective testing plans.

Temporal Dynamics of Viral Load and False negative Rate Influence the Levels of Testing Necessary to Combat COVID19 Spread.

Colleges and other organizations are considering testing plans to return to operation as the COVID19 pandemic continues. Pre-symptomatic spread and high false negative rates for testing may make it difficult to stop viral spread. Here, we develop a stochastic agent-based model of COVID19 in a university sized population, considering the dynamics of both viral load and false negative rate of tests on the ability of testing to combat viral spread. Reported dynamics of SARS-CoV-2 can lead to an apparent false negative rate from ~17% to ~48%. Nonuniform distributions of viral load and false negative rate lead to higher requirements for frequency and fraction of population tested in order to bring the apparent Reproduction number (Rt) below 1. Thus, it is important to consider non-uniform dynamics of viral spread and false negative rate in order to model effective testing plans.

Redundant Trojan horse and endothelial-circulatory mechanisms for host-mediated spread of Candida albicans yeast.

The host innate immune system has developed elegant processes for the detection and clearance of invasive fungal pathogens. These strategies may also aid in the spread of pathogens in vivo, although technical limitations have previously hindered our ability to view the host innate immune and endothelial cells to probe their roles in spreading disease. Here, we have leveraged zebrafish larvae as a model to view the interactions of these host processes with the fungal pathogen Candida albicans in vivo. We examined three potential host-mediated mechanisms of fungal spread: movement inside phagocytes in a "Trojan Horse" mechanism, inflammation-assisted spread, and endothelial barrier passage. Utilizing both chemical and genetic tools, we systematically tested the loss of neutrophils and macrophages and the loss of blood flow on yeast cell spread. Both neutrophils and macrophages respond to yeast-locked and wild type C. albicans in our model and time-lapse imaging revealed that macrophages can support yeast spread in a "Trojan Horse" mechanism. Surprisingly, loss of immune cells or inflammation does not alter dissemination dynamics. On the other hand, when blood flow is blocked, yeast can cross into blood vessels but they are limited in how far they travel. Blockade of both phagocytes and circulation reduces rates of dissemination and significantly limits the distance of fungal spread from the infection site. Together, this data suggests a redundant two-step process whereby (1) yeast cross the endothelium inside phagocytes or via direct uptake, and then (2) they utilize blood flow or phagocytes to travel to distant sites.

Triclosan disrupts immune cell function by depressing Ca2+ influx following acidification of the cytoplasm.

Triclosan (TCS) is an antimicrobial agent that was effectively banned by the FDA from hand soaps in 2016, hospital soaps in 2017, and hand sanitizers in 2019; however, TCS can still be found in a few products. At consumer-relevant, non-cytotoxic doses, TCS inhibits the functions of both mitochondria and mast cells, a ubiquitous cell type. Via the store-operated Ca2+ entry mechanism utilized by many immune cells, mast cells undergo antigen-stimulated Ca2+ influx into the cytosol, for proper function. Previous work showed that TCS inhibits Ca2+ dynamics in mast cells, and here we show that TCS also inhibits Ca2+ mobilization in human Jurkat T cells. However, the biochemical mechanism behind the Ca2+ dampening has yet to be elucidated. Three-dimensional super-resolution microscopy reveals that TCS induces mitochondrial swelling, in line with and extending the previous finding of TCS inhibition of mitochondrial membrane potential via its proton ionophoric activity. Inhibition of plasma membrane potential (PMP) by the canonical depolarizer gramicidin can inhibit mast cell function. However, use of the genetically encoded voltage indicators (GEVIs) ArcLight (pH-sensitive) and ASAP2 (pH-insensitive), indicates that TCS does not disrupt PMP. In conjunction with data from a plasma membrane-localized, pH-sensitive reporter, these results indicate that TCS, instead, induces cytosolic acidification in mast cells and T cells. Acidification of the cytosol likely inhibits Ca2+ influx by uncoupling the STIM1/ORAI1 interaction that is required for opening of plasma membrane Ca2+ channels. These results provide a mechanistic explanation of TCS disruption of Ca2+ influx and, thus, of immune cell function.

Systematic analysis of F-box proteins reveals a new branch of the yeast mating pathway.

The mating pathway in yeast Saccharomyces cerevisiae has long been used to reveal new mechanisms of signal transduction. The pathway comprises a pheromone receptor, a heterotrimeric G protein, and intracellular effectors of morphogenesis and transcription. Polarized cell growth, in the direction of a potential mating partner, is accomplished by the G-protein ßγ subunits and the small G-protein Cdc42. Transcription induction, needed for cell-cell fusion, is mediated by Gßγ and the mitogen-activated protein kinase (MAPK) scaffold protein Ste5. A potential third pathway is initiated by the G-protein α subunit Gpa1. Gpa1 signaling was shown previously to involve the F-box adaptor protein Dia2 and an endosomal effector protein, the phosphatidylinositol 3-kinase Vps34. Vps34 is also required for proper vacuolar sorting and autophagy. Here, using a panel of reporter assays, we demonstrate that mating pheromone stimulates vacuolar targeting of a cytoplasmic reporter protein and that this process depends on Vps34. Through a systematic analysis of F-box deletion mutants, we show that Dia2 is required to sustain pheromone-induced vacuolar targeting. We also found that other F-box proteins selectively regulate morphogenesis (Ydr306, renamed Pfu1) and transcription (Ucc1). These findings point to the existence of a new and distinct branch of the pheromone-signaling pathway, one that likely leads to vacuolar engulfment of cytoplasmic proteins and recycling of cellular contents in preparation for mating.

NAD+ improves neuromuscular development in a zebrafish model of FKRP-associated dystroglycanopathy.

BACKGROUND: Secondary dystroglycanopathies are muscular dystrophies that result from mutations in genes that participate in Dystroglycan glycosylation. Glycosylation of Dystroglycan is essential for muscle fibers to adhere to the muscle extracellular matrix (myomatrix). Although the myomatrix is disrupted in a number of secondary dystroglycanopathies, it is unknown whether improving the myomatrix is beneficial for these conditions. We previously determined that either NAD+ supplementation or overexpression of Paxillin are sufficient to improve muscle structure and the myomatrix in a zebrafish model of primary dystroglycanopathy. Here, we investigate how these modulations affect neuromuscular phenotypes in zebrafish fukutin-related protein (fkrp) morphants modeling FKRP-associated secondary dystroglycanopathy. RESULTS: We found that NAD+ supplementation prior to muscle development improved muscle structure, myotendinous junction structure, and muscle function in fkrp morphants. However, Paxillin overexpression did not improve any of these parameters in fkrp morphants. As movement also requires neuromuscular junction formation, we examined early neuromuscular junction development in fkrp morphants. The length of neuromuscular junctions was disrupted in fkrp morphants. NAD+ supplementation prior to neuromuscular junction development improved length. We investigated NMJ formation in dystroglycan (dag1) morphants and found that although NMJ morphology is disrupted in dag1 morphants, NAD+ is not sufficient to improve NMJ morphology in dag1 morphants. Ubiquitous overexpression of Fkrp rescued the fkrp morphant phenotype but muscle-specific overexpression only improved myotendinous junction structure. CONCLUSIONS: These data indicate that Fkrp plays an early and essential role in muscle, myotendinous junction, and neuromuscular junction development. These data also indicate that, at least in the zebrafish model, FKRP-associated dystroglycanopathy does not exactly phenocopy DG-deficiency. Paxillin overexpression improves muscle structure in dag1 morphants but not fkrp morphants. In contrast, NAD+ supplementation improves NMJ morphology in fkrp morphants but not dag1 morphants. Finally, these data show that muscle-specific expression of Fkrp is insufficient to rescue muscle development and homeostasis.

Fluorescence-based quantification of nucleocytoplasmic transport.

The sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software. We include discussion of the use of regions of interest and image segmentation for quantification of protein localization. Nucleocytoplasmic transport is fundamentally important for controlling protein levels and activity in the nucleus or cytoplasm, and quantitative analysis can provide insight into how biological output is achieved.

A nuclear lamina-chromatin-Ran GTPase axis modulates nuclear import and DNA damage signaling.

The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin-associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran-dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ-H2AX in response to ionizing radiation. Our data suggest a lamina-chromatin-Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.

Nab3's localization to a nuclear granule in response to nutrient deprivation is determined by its essential prion-like domain.

Ribonucleoprotein (RNP) granules are higher order assemblies of RNA, RNA-binding proteins, and other proteins, that regulate the transcriptome and protect RNAs from environmental challenge. There is a diverse range of RNP granules, many cytoplasmic, which provide various levels of regulation of RNA metabolism. Here we present evidence that the yeast transcription termination factor, Nab3, is targeted to intranuclear granules in response to glucose starvation by Nab3's proline/glutamine-rich, prion-like domain (PrLD) which can assemble into amyloid in vitro. Localization to the granule is reversible and sensitive to the chemical probe 1,6 hexanediol suggesting condensation is driven by phase separation. Nab3's RNA recognition motif is also required for localization as seen for other PrLD-containing RNA-binding proteins that phase separate. Although the PrLD is necessary, it is not sufficient to localize to the granule. A heterologous PrLD that functionally replaces Nab3's essential PrLD, directed localization to the nuclear granule, however a chimeric Nab3 molecule with a heterologous PrLD that cannot restore termination function or viability, does not form granules. The Nab3 nuclear granule shows properties similar to well characterized cytoplasmic compartments formed by phase separation, suggesting that, as seen for other elements of the transcription machinery, termination factor condensation is functionally important.

Unique Down to Our Microbes-Assessment of an Inquiry-Based Metagenomics Activity.

Metagenomics is an important method for studying microbial life. However, undergraduate exposure to metagenomics is hindered by associated software, computing demands, and dataset access. In this inquiry-based activity designed for introductory life science majors and nonmajors, students perform an investigation of the bacterial communities inhabiting the human belly button and associated metagenomics data collected through a citizen science project and visualized using an open-access bioinformatics tool. The activity is designed for attainment of the following student learning outcomes: defining terms associated with metagenomics analyses, describing the biological impact of the microbiota on human health, formulating a hypothesis, analyzing and interpreting metagenomics data to compare microbiota, evaluating a specific hypothesis, and synthesizing a conceptual model as to why bacterial populations vary. This activity was implemented in six introductory biology and biotechnology courses across five institutions. Attainment of student learning outcomes was assessed through completion of a quiz and students' presentations of their findings. In presentations, students demonstrated their ability to develop novel hypotheses and analyze and interpret metagenomic data to evaluate their hypothesis. In quizzes, students demonstrated their ability to define key terms and describe the biological impact of the microbiota on human health. Student learning gains assessment also revealed that students perceived gains for all student learning outcomes. Collectively, our assessment demonstrates achievement of the learning outcomes and supports the utility of this inquiry-based activity to engage undergraduates in the scientific process via analyses of metagenomics datasets and associated exploration of a microbial community that lives on the human body.

Modulation of receptor dynamics by the regulator of G protein signaling Sst2.

G protein-coupled receptor (GPCR) signaling is fundamental to physiological processes such as vision, the immune response, and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. After pheromone stimulation, the GPCR Ste2 is removed from the cell membrane, and new receptors are delivered to the growing edge. The regulator of G protein signaling (RGS) protein Sst2 acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 together with its interaction with the receptor function to reestablish a receptor pool at the site of polarized growth. To validate the model, we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and its induction by pheromone. Together our results reveal that a regulator of G protein signaling can also regulate the G protein-coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge.

Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK.

Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5(ND)) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or "synthetic," supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling.