Bodil Nordlander

Integrative model of the response of yeast to osmotic shock

Integration of experimental studies with mathematical modeling allows insight into systems properties, prediction of perturbation effects and generation of hypotheses for further research. We present a comprehensive mathematical description of the cellular response of yeast to hyperosmotic shock. The model integrates a biochemical reaction network comprising receptor stimulation, mitogen-activated protein kinase cascade dynamics, activation of gene expression and adaptation of cellular metabolism with a thermodynamic description of volume regulation and osmotic pressure. Simulations agree well with experimental results obtained under different stress conditions or with specific mutants. The model is predictive since it suggests previously unrecognized features of the system with respect to osmolyte accumulation and feedback control, as confirmed with experiments. The mathematical description presented is a valuable tool for future studies on osmoregulation in yeast and—with appropriate modifications—other organisms. It also serves as a starting point for a comprehensive description of cellular signaling.

Chapter Two – Yeast Osmoregulation

Osmoregulation is the active control of the cellular water balance and encompasses homeostatic mechanisms crucial for life. The osmoregulatory system in the yeast Saccharomyces cerevisiae is particularly well understood. Key to yeast osmoregulation is the production and accumulation of the compatible solute glycerol, which is partly controlled by the high osmolarity glycerol (HOG) signaling system. Genetic analyses combined with studies on protein-protein interactions have revealed the wiring scheme of the HOG signaling network, a branched mitogen-activated protein (MAP) kinase (MAPK) pathway that eventually converges on the MAPK Hog1. Hog1 is activated following cell shrinking and controls posttranscriptional processes in the cytosol as well as gene expression in the nucleus. HOG pathway activity can easily and rapidly be controlled experimentally by extracellular stimuli, and signaling and adaptation can be separated by a system of forced adaptation. This makes yeast osmoregulation suitable for studies on system properties of signaling and cellular adaptation via mathematical modeling. Computational simulations and parallel quantitative time course experimentation on different levels of the regulatory system have provided a stepping stone toward a holistic understanding, revealing how the HOG pathway can combine rigorous feedback control with maintenance of signaling competence. The abundant tools make yeast a suitable model for an integrated analysis of cellular osmoregulation. Maintenance of the cellular water balance is fundamental for life. All cells, even those in multicellular organisms with an organism-wide osmoregulation, have the ability to actively control their water balance. Osmoregulation encompasses homeostatic processes that maintain an appropriate intracellular environment for biochemical processes as well as turgor of cells and organism. In the laboratory, the osmoregulatory system is studied most conveniently as a response to osmotic shock, causing rapid and dramatic changes in the extracellular water activity. Those rapid changes mediate either water efflux (hyperosmotic shock), and hence cell shrinkage, or influx (hypoosmotic shock), causing cell swelling. The yeast S. cerevisiae, as a free-living organism experiencing both slow and rapid changes in extracellular water activity, has proven a suitable and genetically tractable experimental system in studying the underlying signaling pathways and regulatory processes governing osmoregulation. Although far from complete, the present picture of yeast osmoregulation is both extensive and detailed (de Nadal et al., 2002; Hohmann, 2002; Klipp et al., 2005). Simulations using mathematical models combined with time course measurements of different molecular processes in signaling and adaptation have allowed elucidation of the first system properties on the yeast osmoregulatory network.

Network modeling of the transcriptional effects of copy number aberrations in glioblastoma

DNA copy number aberrations (CNAs) are a hallmark of cancer genomes. However, little is known about how such changes affect global gene expression. We develop a modeling framework, EPoC (Endogenous Perturbation analysis of Cancer), to (1) detect disease‐driving CNAs and their effect on target mRNA expression, and to (2) stratify cancer patients into long‐ and short‐term survivors. Our method constructs causal network models of gene expression by combining genome‐wide DNA‐ and RNA‐level data. Prognostic scores are obtained from a singular value decomposition of the networks. By applying EPoC to glioblastoma data from The Cancer Genome Atlas consortium, we demonstrate that the resulting network models contain known disease‐relevant hub genes, reveal interesting candidate hubs, and uncover predictors of patient survival. Targeted validations in four glioblastoma cell lines support selected predictions, and implicate the p53‐interacting protein Necdin in suppressing glioblastoma cell growth. We conclude that large‐scale network modeling of the effects of CNAs on gene expression may provide insights into the biology of human cancer. Free software in MATLAB and R is provided.

Anaerobicity Prepares Saccharomyces cerevisiae Cells for Faster Adaptation to Osmotic Shock

ABSTRACT Yeast cells adapt to hyperosmotic shock by accumulating glycerol and altering expression of hundreds of genes. This transcriptional response of Saccharomyces cerevisiae to osmotic shock encompasses genes whose products are implicated in protection from oxidative damage. We addressed the question of whether osmotic shock caused oxidative stress. Osmotic shock did not result in the generation of detectable levels of reactive oxygen species (ROS). To preclude any generation of ROS, osmotic shock treatments were performed in anaerobic cultures. Global gene expression response profiles were compared by employing a novel two-dimensional cluster analysis. The transcriptional profiles following osmotic shock under anaerobic and aerobic conditions were qualitatively very similar. In particular, it appeared that expression of the oxidative stress genes was stimulated upon osmotic shock even if there was no apparent need for their function. Interestingly, cells adapted to osmotic shock much more rapidly under anaerobiosis, and the signaling as well as the transcriptional response was clearly attenuated under these conditions. This more rapid adaptation is due to an enhanced glycerol production capacity in anaerobic cells, which is caused by the need for glycerol production in redox balancing. Artificially enhanced glycerol production led to an attenuated response even under aerobic conditions. These observations demonstrate the crucial role of glycerol accumulation and turgor recovery in determining the period of osmotic shock-induced signaling and the profile of cellular adaptation to osmotic shock.

Quantitative Analysis of Glycerol Accumulation, Glycolysis and Growth under Hyper Osmotic Stress

We provide an integrated dynamic view on a eukaryotic osmolyte system, linking signaling with regulation of gene expression, metabolic control and growth. Adaptation to osmotic changes enables cells to adjust cellular activity and turgor pressure to an altered environment. The yeast Saccharomyces cerevisiae adapts to hyperosmotic stress by activating the HOG signaling cascade, which controls glycerol accumulation. The Hog1 kinase stimulates transcription of genes encoding enzymes required for glycerol production (Gpd1, Gpp2) and glycerol import (Stl1) and activates a regulatory enzyme in glycolysis (Pfk26/27). In addition, glycerol outflow is prevented by closure of the Fps1 glycerol facilitator. In order to better understand the contributions to glycerol accumulation of these different mechanisms and how redox and energy metabolism as well as biomass production are maintained under such conditions we collected an extensive dataset. Over a period of 180 min after hyperosmotic shock we monitored in wild type and different mutant cells the concentrations of key metabolites and proteins relevant for osmoadaptation. The dataset was used to parameterize an ODE model that reproduces the generated data very well. A detailed computational analysis using time-dependent response coefficients showed that Pfk26/27 contributes to rerouting glycolytic flux towards lower glycolysis. The transient growth arrest following hyperosmotic shock further adds to redirecting almost all glycolytic flux from biomass towards glycerol production. Osmoadaptation is robust to loss of individual adaptation pathways because of the existence and upregulation of alternative routes of glycerol accumulation. For instance, the Stl1 glycerol importer contributes to glycerol accumulation in a mutant with diminished glycerol production capacity. In addition, our observations suggest a role for trehalose accumulation in osmoadaptation and that Hog1 probably directly contributes to the regulation of the Fps1 glycerol facilitator. Taken together, we elucidated how different metabolic adaptation mechanisms cooperate and provide hypotheses for further experimental studies.

Robustness and fragility in the yeast high osmolarity glycerol (HOG) signal‐transduction pathway

Cellular signalling networks integrate environmental stimuli with the information on cellular status. These networks must be robust against stochastic fluctuations in stimuli as well as in the amounts of signalling components. Here, we challenge the yeast HOG signal‐transduction pathway with systematic perturbations in components’ expression levels under various external conditions in search for nodes of fragility. We observe a substantially higher frequency of fragile nodes in this signal‐transduction pathway than that has been observed for other cellular processes. These fragilities disperse without any clear pattern over biochemical functions or location in pathway topology and they are largely independent of pathway activation by external stimuli. However, the strongest toxicities are caused by pathway hyperactivation. In silico analysis highlights the impact of model structure on in silico robustness, and suggests complex formation and scaffolding as important contributors to the observed fragility patterns. Thus, in vivo robustness data can be used to discriminate and improve mathematical models.

Conditional Osmotic Stress in Yeast A SYSTEM TO STUDY TRANSPORT THROUGH AQUAGLYCEROPORINS AND OSMOSTRESS SIGNALING

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Δ gpd2Δ mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Δ gpd2Δ mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-Δ1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Δ1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Δ1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Δ gpd2Δ mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Δ gpd2Δ mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our “conditional” osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.

Initiation of the transcriptional response to hyperosmotic shock correlates with the potential for volume recovery

The control of activity and localization of transcription factors is critical for appropriate transcriptional responses. In eukaryotes, signal transduction components such as mitogen‐activated protein kinase (MAPK) shuttle into the nucleus to activate transcription. It is not known in detail how different amounts of nuclear MAPK over time affect the transcriptional response. In the present study, we aimed to address this issue by studying the high osmolarity glycerol (HOG) system in Saccharomyces cerevisiae. We employed a conditional osmotic system, which changes the period of the MAPK Hog1 signal independent of the initial stress level. We determined the dynamics of the Hog1 nuclear localization and cell volume by single‐cell analysis in well‐controlled microfluidics systems and compared the responses with the global transcriptional output of cell populations. We discovered that the onset of the initial transcriptional response correlates with the potential of cells for rapid adaptation; cells that are capable of recovering quickly initiate the transcriptional responses immediately, whereas cells that require longer time to adapt also respond later. This is reflected by Hog1 nuclear localization, Hog1 promoter association and the transcriptional response, but not Hog1 phosphorylation, suggesting that a presently uncharacterized rapid adaptive mechanism precedes the Hog1 nuclear response. Furthermore, we found that the period of Hog1 nuclear residence affects the amplitude of the transcriptional response rather than the spectrum of responsive genes.

Hog1-mediated Metabolic Adjustments Following Hyperosmotic Shock in the Yeast Saccharomyces cerevisiae

Yeast cells exposed to hyperosmotic conditions adjust their metabolism in order to increase the production of the compatible solute glycerol. The MAPK Hog1 seems to affect glycerol production rapidly by stimulating flux through glycolysis and long-term via the transcriptional upregulation of genes encoding enzymes in glycerol formation. In addition, the glycerol channel Fps1 rapidly closes after a hyperosmotic shock to ensure efficient glycerol accumulation. Hog1 seems to modulate basal Fps1 activity and the MAPK is needed to allow complete closure of Fps1. Moreover, the expression of a number of metabolic genes is affected in both Hog1-dependent and independent ways. How those changes contribute to osmotic adaptation of yeast cells is not completely understood. To separate and analyze the different roles of Hog1 in the adjustment of metabolism will probably need a time-resolved holistic view on all components involved and a combination of theoretical modelling and experimentation. In addition, there is a need for more detailed analysis of direct and indirect Hog1-targets to elucidate the impact of such regulatory interactions.

Shutting the MAP off – and on again?

Signal transduction pathways are the cellular information routes with which cells monitor their surrounding as well as their own state and adjust to environmental changes or hormonal stimuli. MAP kinase pathways are one type of signalling systems in eukaryotes that control stress responses, cell growth and proliferation as well as differentiation. In this study we compare two very well studied yeast signalling systems, the pheromone response pathway and the osmosensing HOG pathway. We have recently generated mathematical models that allow in silico analysis of signalling properties for both pathways. Deactivation of signalling is as important as activation because inappropriate pathway activation causes cell cycle arrest (in the cases studied here) or uncontrolled proliferation. Both pathways are transiently activated by their stimulus, i.e. mating pheromone and osmostress, respectively, indicating rigorous feedback mechanisms. However, the HOG pathway can readily be reactivated by a subsequent stimulus and this is important for its biological role in mediating osmoadaptation. The pheromone response pathway, however, is desensitised and is unable to respond for a certain period of time. While some mechanisms of feedback control are similar in both systems (such as the downregulatory, role of protein phosphatases) the main difference seems to lie in the control of the sensors/receptors. The pheromone receptors are internalised and degraded following stimulation and hence are not available for further stimulation. The osmosensors on the other hand, seem to toggle between activated and deactivated state only controlled by osmotic changes. Together with subtle control by protein phosphatases this results in a system that is constantly receptive for stimulation.