The Effect of Lithium on the Budding Yeast Saccharomyces cerevisiae upon Stress Adaptation.

Lithium salts are used in the treatment of mood disorders, cancer, and Alzheimer's disease. It has been shown to prolong life span in several phyla; however, not yet in budding yeast. In our study, we investigate the influence of lithium on yeast cells' viability by characterizing protein aggregate formation, cell volume, and molecular crowding in the context of stress adaptation. While our data suggest a concentration-dependent growth inhibition caused by LiCl, we show an extended long-term survival rate as an effect of lithium addition upon glucose deprivation. We show that caloric restriction mitigates the negative impact of LiCl on cellular survival. Therefore, we suggest that lithium could affect glucose metabolism upon caloric restriction, which could explain the extended long-term survival observed in our study. We find furthermore that lithium chloride did not affect an immediate salt-induced Hsp104-dependent aggregate formation but cellular adaptation to H2O2 and acute glucose starvation. We presume that different salt types and concentrations interfere with effective Hsp104 recruitment or its ATP-dependent disaggregase activity as a response to salt stress. This work provides novel details of Li+ effect on live eukaryotic cells which may also be applicable in further research on the treatment of cancer, Alzheimer's, or other age-related diseases in humans.

A novel yeast hybrid modeling framework integrating Boolean and enzyme-constrained networks enables exploration of the interplay between signaling and metabolism.

The interplay between nutrient-induced signaling and metabolism plays an important role in maintaining homeostasis and its malfunction has been implicated in many different human diseases such as obesity, type 2 diabetes, cancer, and neurological disorders. Therefore, unraveling the role of nutrients as signaling molecules and metabolites together with their interconnectivity may provide a deeper understanding of how these conditions occur. Both signaling and metabolism have been extensively studied using various systems biology approaches. However, they are mainly studied individually and in addition, current models lack both the complexity of the dynamics and the effects of the crosstalk in the signaling system. To gain a better understanding of the interconnectivity between nutrient signaling and metabolism in yeast cells, we developed a hybrid model, combining a Boolean module, describing the main pathways of glucose and nitrogen signaling, and an enzyme-constrained model accounting for the central carbon metabolism of Saccharomyces cerevisiae, using a regulatory network as a link. The resulting hybrid model was able to capture a diverse utalization of isoenzymes and to our knowledge outperforms constraint-based models in the prediction of individual enzymes for both respiratory and mixed metabolism. The model showed that during fermentation, enzyme utilization has a major contribution in governing protein allocation, while in low glucose conditions robustness and control are prioritized. In addition, the model was capable of reproducing the regulatory effects that are associated with the Crabtree effect and glucose repression, as well as regulatory effects associated with lifespan increase during caloric restriction. Overall, we show that our hybrid model provides a comprehensive framework for the study of the non-trivial effects of the interplay between signaling and metabolism, suggesting connections between the Snf1 signaling pathways and processes that have been related to chronological lifespan of yeast cells.

Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells.

Membrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. Despite a multitude of studies on transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment. We employed super-resolved millisecond fluorescence microscopy with a single-molecule sensitivity, to track labelled molecules of interest in real time. We use as example the aquaglyceroporin Fps1 in the yeast Saccharomyces cerevisiae to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions. We show that Fps1 resides in multi tetrameric clusters while hyperosmotic and oxidative stress conditions cause Fps1 reorganization. Moreover, we demonstrate that rapid exposure to hydrogen peroxide causes Fps1 degradation. In this way we shed new light on aspects of architecture and dynamics of glycerol-permeable plasma membrane channels.

Mig1 localization exhibits biphasic behavior which is controlled by both metabolic and regulatory roles of the sugar kinases.

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.

The G2A Receptor Controls Polarization of Macrophage by Determining Their Localization Within the Inflamed Tissue.

Macrophages are highly versatile cells, which acquire, depending on their microenvironment, pro- (M1-like), or antiinflammatory (M2-like) phenotypes. Here, we studied the role of the G-protein coupled receptor G2A (GPR132), in chemotactic migration and polarization of macrophages, using the zymosan-model of acute inflammation. G2A-deficient mice showed a reduced zymosan-induced thermal hyperalgesia, which was reversed after macrophage depletion. Fittingly, the number of M1-like macrophages was reduced in the inflamed tissue in G2A-deficient mice. However, G2A activation was not sufficient to promote M1-polarization in bone marrow-derived macrophages. While the number of monocyte-derived macrophages in the inflamed paw was not altered, G2A-deficient mice had less macrophages in the direct vicinity of the origin of inflammation, an area marked by the presence of zymosan, neutrophil accumulation and proinflammatory cytokines. Fittingly neutrophil efferocytosis was decreased in G2A-deficient mice and several lipids, which are released by neutrophils and promote G2A-mediated chemotaxis, were increased in the inflamed tissue. Taken together, G2A is necessary to position macrophages in the proinflammatory microenvironment surrounding the center of inflammation. In absence of G2A the macrophages are localized in an antiinflammatory microenvironment and macrophage polarization is shifted toward M2-like macrophages.

Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae.

Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule analysis. Here we present a method to characterise maturation of GFP and mCherry genetic protein fusions in budding yeast Saccharomyces cerevisiae.

Applying Microfluidic Systems to Study Effects of Glucose at Single-Cell Level.

Microfluidic systems in combination with microscopy (e.g., fluorescence) can be a powerful tool to study, at single-cell level, the behavior and morphology of biological cells after uptake of glucose. Here, we briefly discuss the advantages of using microfluidic systems. We further describe how microfluidic systems are fabricated and how they are utilized. Finally, we discuss how the large amount of data can be analyzed in a "semi-automatic" manner using custom-made software. In summary, we provide a guide to how to use microfluidic systems in single-cell studies.

The yeast Mig1 transcriptional repressor is dephosphorylated by glucose-dependent and -independent mechanisms.

A yeast Saccharomyces cerevisiae Snf1 kinase, an analog of mammalian AMPK, regulates glucose derepression of genes required for utilization of alternative carbon sources through the transcriptional repressor Mig1. It has been suggested that the Glc7-Reg1 phosphatase dephosphorylates Mig1. Here we report that Mig1 is dephosphorylated by Glc7-Reg1 in an apparently glucose-dependent mechanism but also by a mechanism independent of glucose and Glc7-Reg1. In addition to serine/threonine phosphatases another process including tyrosine phosphorylation seems crucial for Mig1 regulation. Taken together, Mig1 dephosphorylation appears to be controlled in a complex manner, in line with the importance for rapid and sensitive regulation upon altered glucose concentrations in the growth medium.

Transcription factor clusters regulate genes in eukaryotic cells.

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

Single-cell study links metabolism with nutrient signaling and reveals sources of variability.

BACKGROUND: The yeast AMPK/SNF1 pathway is best known for its role in glucose de/repression. When glucose becomes limited, the Snf1 kinase is activated and phosphorylates the transcriptional repressor Mig1, which is then exported from the nucleus. The exact mechanism how the Snf1-Mig1 pathway is regulated is not entirely elucidated. RESULTS: Glucose uptake through the low affinity transporter Hxt1 results in nuclear accumulation of Mig1 in response to all glucose concentrations upshift, however with increasing glucose concentration the nuclear localization of Mig1 is more intense. Strains expressing Hxt7 display a constant response to all glucose concentration upshifts. We show that differences in amount of hexose transporter molecules in the cell could cause cell-to-cell variability in the Mig1-Snf1 system. We further apply mathematical modelling to our data, both general deterministic and a nonlinear mixed effect model. Our model suggests a presently unrecognized regulatory step of the Snf1-Mig1 pathway at the level of Mig1 dephosphorylation. Model predictions point to parameters involved in the transport of Mig1 in and out of the nucleus as a majorsource of cell to cell variability. CONCLUSIONS: With this modelling approach we have been able to suggest steps that contribute to the cell-to-cell variability. Our data indicate a close link between the glucose uptake rate, which determines the glycolytic rate, and the activity of the Snf1/Mig1 system. This study hence establishes a close relation between metabolism and signalling.

The yeast osmostress response is carbon source dependent.

Adaptation to altered osmotic conditions is a fundamental property of living cells and has been studied in detail in the yeast Saccharomyces cerevisiae. Yeast cells accumulate glycerol as compatible solute, controlled at different levels by the High Osmolarity Glycerol (HOG) response pathway. Up to now, essentially all osmostress studies in yeast have been performed with glucose as carbon and energy source, which is metabolised by glycolysis with glycerol as a by-product. Here we investigated the response of yeast to osmotic stress when yeast is respiring ethanol as carbon and energy source. Remarkably, yeast cells do not accumulate glycerol under these conditions and it appears that trehalose may partly take over the role as compatible solute. The HOG pathway is activated in very much the same way as during growth on glucose and is also required for osmotic adaptation. Slower volume recovery was observed in ethanol-grown cells as compared to glucose-grown cells. Dependence on key regulators as well as the global gene expression profile were similar in many ways to those previously observed in glucose-grown cells. However, there are indications that cells re-arrange redox-metabolism when respiration is hampered under osmostress, a feature that could not be observed in glucose-grown cells.

The mitogen-activated protein kinase Slt2 modulates arsenite transport through the aquaglyceroporin Fps1.

Arsenite is widely present in nature; therefore, cells have evolved mechanisms to prevent arsenite influx and promote efflux. In yeast (Saccharomyces cerevisiae), the aquaglyceroporin Fps1 mediates arsenite influx and efflux. The mitogen-activated protein kinase (MAPK) Hog1 has previously been shown to restrict arsenite influx through Fps1. In this study, we show that another MAPK, Slt2, is transiently phosphorylated in response to arsenite influx. Our findings indicate that the protein kinase activity of Slt2 is required for its role in arsenite tolerance. While Hog1 prevents arsenite influx via phosphorylation of T231 at the N-terminal domain of Fps1, Slt2 promotes arsenite efflux through phosphorylation of S537 at the C terminus. Our data suggest that Slt2 physically interacts with Fps1 and that this interaction depends on phosphorylation of S537. We hypothesize that Hog1 and Slt2 may affect each other's binding to Fps1, thereby controlling the opening and closing of the channel.

Systems Level Analysis of the Yeast Osmo-Stat.

Adaptation is an important property of living organisms enabling them to cope with environmental stress and maintaining homeostasis. Adaptation is mediated by signaling pathways responding to different stimuli. Those signaling pathways might communicate in order to orchestrate the cellular response to multiple simultaneous stimuli, a phenomenon called crosstalk. Here, we investigate possible mechanisms of crosstalk between the High Osmolarity Glycerol (HOG) and the Cell Wall Integrity (CWI) pathways in yeast, which mediate adaptation to hyper- and hypo-osmotic challenges, respectively. We combine ensemble modeling with experimental investigations to test in quantitative terms different hypotheses about the crosstalk of the HOG and the CWI pathways. Our analyses indicate that for the conditions studied i) the CWI pathway activation employs an adaptive mechanism with a variable volume-dependent threshold, in contrast to the HOG pathway, whose activation relies on a fixed volume-dependent threshold, ii) there is no or little direct crosstalk between the HOG and CWI pathways, and iii) its mainly the HOG alone mediating adaptation of cellular osmotic pressure for both hyper- as well as hypo-osmotic stress. Thus, by iteratively combining mathematical modeling with experimentation we achieved a better understanding of regulatory mechanisms of yeast osmo-homeostasis and formulated new hypotheses about osmo-sensing.

Strategies for structuring interdisciplinary education in Systems Biology: an European perspective.

Systems Biology is an approach to biology and medicine that has the potential to lead to a better understanding of how biological properties emerge from the interaction of genes, proteins, molecules, cells and organisms. The approach aims at elucidating how these interactions govern biological function by employing experimental data, mathematical models and computational simulations. As Systems Biology is inherently multidisciplinary, education within this field meets numerous hurdles including departmental barriers, availability of all required expertise locally, appropriate teaching material and example curricula. As university education at the Bachelor's level is traditionally built upon disciplinary degrees, we believe that the most effective way to implement education in Systems Biology would be at the Master's level, as it offers a more flexible framework. Our team of experts and active performers of Systems Biology education suggest here (i) a definition of the skills that students should acquire within a Master's programme in Systems Biology, (ii) a possible basic educational curriculum with flexibility to adjust to different application areas and local research strengths, (iii) a description of possible career paths for students who undergo such an education, (iv) conditions that should improve the recruitment of students to such programmes and (v) mechanisms for collaboration and excellence spreading among education professionals. With the growing interest of industry in applying Systems Biology approaches in their fields, a concerted action between academia and industry is needed to build this expertise. Here we present a reflection of the European situation and expertise, where most of the challenges we discuss are universal, anticipating that our suggestions will be useful internationally. We believe that one of the overriding goals of any Systems Biology education should be a student's ability to phrase and communicate research questions in such a manner that they can be solved by the integration of experiments and modelling, as well as to communicate and collaborate productively across different experimental and theoretical disciplines in research and development.

A fungicide-responsive kinase as a tool for synthetic cell fate regulation.

Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic 'suicide attack' system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications.

Molecular communication: crosstalk between the Snf1 and other signaling pathways.

The yeast Saccharomyces cerevisiae employs different conserved signaling pathways to adapt to altered availability of nutrient and energy sources. Crosstalk between the pathways occurs to integrate different internal and external stimuli and adjust cellular metabolism, growth and proliferation to altered environmental conditions. The main glucose repression pathway, Snf1/Mig1, plays an essential role in adaptation to glucose limitation. However, the Snf1 protein kinase is also involved in regulation of many other cellular processes. We summarize evidence that Snf1 is part of a network of communicating pathways, and we suggest research directions that may help elucidating signal flow within this network.