David D. van Niekerk

Sustained glycolytic oscillations in individual isolated yeast cells

Yeast glycolytic oscillations have been studied since the 1950s in cell‐free extracts and intact cells. For intact cells, sustained oscillations have so far only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. Using optical tweezers to position yeast cells in a microfluidic chamber, we were able to observe sustained oscillations in individual isolated cells. Using a detailed kinetic model for the cellular reactions, we simulated the heterogeneity in the response of the individual cells, assuming small differences in a single internal parameter. This is the first time that sustained limit‐cycle oscillations have been demonstrated in isolated yeast cells.

From steady-state to synchronized yeast glycolytic oscillations I: Model construction

An existing detailed kinetic model for the steady‐state behavior of yeast glycolysis was tested for its ability to simulate dynamic behavior. Using a small subset of experimental data, the original model was adapted by adjusting its parameter values in three optimization steps. Only small adaptations to the original model were required for realistic simulation of experimental data for limit‐cycle oscillations. The greatest changes were required for parameter values for the phosphofructokinase reaction. The importance of ATP for the oscillatory mechanism and NAD(H) for inter‐and intra‐cellular communications and synchronization was evident in the optimization steps and simulation experiments. In an accompanying paper [du Preez F et al. (2012) FEBS J279, 2823–2836], we validate the model for a wide variety of experiments on oscillatory yeast cells. The results are important for re‐use of detailed kinetic models in modular modeling approaches and for approaches such as that used in the Silicon Cell initiative.

From steady-state to synchronized yeast glycolytic oscillations II: model validation.

In an accompanying paper [du Preez et al., (2012) FEBS J279, 2810–2822], we adapt an existing kinetic model for steady‐state yeast glycolysis to simulate limit‐cycle oscillations. Here we validate the model by testing its capacity to simulate a wide range of experiments on dynamics of yeast glycolysis. In addition to its description of the oscillations of glycolytic intermediates in intact cells and the rapid synchronization observed when mixing out‐of‐phase oscillatory cell populations (see accompanying paper), the model was able to predict the Hopf bifurcation diagram with glucose as the bifurcation parameter (and one of the bifurcation points with cyanide as the bifurcation parameter), the glucose‐ and acetaldehyde‐driven forced oscillations, glucose and acetaldehyde quenching, and cell‐free extract oscillations (including complex oscillations and mixed‐mode oscillations). Thus, the model was compliant, at least qualitatively, with the majority of available experimental data for glycolytic oscillations in yeast. To our knowledge, this is the first time that a model for yeast glycolysis has been tested against such a wide variety of independent data sets.

Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells

Oscillations are widely distributed in nature and synchronization of oscillators has been described at the cellular level (e.g. heart cells) and at the population level (e.g. fireflies). Yeast glycolysis is the best known oscillatory system, although it has been studied almost exclusively at the population level (i.e. limited to observations of average behaviour in synchronized cultures). We studied individual yeast cells that were positioned with optical tweezers in a microfluidic chamber to determine the precise conditions for autonomous glycolytic oscillations. Hopf bifurcation points were determined experimentally in individual cells as a function of glucose and cyanide concentrations. The experiments were analyzed in a detailed mathematical model and could be interpreted in terms of an oscillatory manifold in a three‐dimensional state‐space; crossing the boundaries of the manifold coincides with the onset of oscillations and positioning along the longitudinal axis of the volume sets the period. The oscillatory manifold could be approximated by allosteric control values of phosphofructokinase for ATP and AMP.

Construction and validation of a detailed kinetic model of glycolysis in Plasmodium falciparum.

The enzymes in the Embden–Meyerhof–Parnas pathway of Plasmodium falciparum trophozoites were kinetically characterized and their integrated activities analyzed in a mathematical model. For validation of the model, we compared model predictions for steady‐state fluxes and metabolite concentrations of the hexose phosphates with experimental values for intact parasites. The model, which is completely based on kinetic parameters that were measured for the individual enzymes, gives an accurate prediction of the steady‐state fluxes and intermediate concentrations. This is the first detailed kinetic model for glucose metabolism in P. falciparum, one of the most prolific malaria‐causing protozoa, and the high predictive power of the model makes it a strong tool for future drug target identification studies. The modelling workflow is transparent and reproducible, and completely documented in the SEEK platform, where all experimental data and model files are available for download.

FAIRDOMHub: a repository and collaboration environment for sharing systems biology research.

The FAIRDOMHub is a repository for publishing FAIR (Findable, Accessible, Interoperable and Reusable) Data, Operating procedures and Models (https://fairdomhub.org/) for the Systems Biology community. It is a web-accessible repository for storing and sharing systems biology research assets. It enables researchers to organize, share and publish data, models and protocols, interlink them in the context of the systems biology investigations that produced them, and to interrogate them via API interfaces. By using the FAIRDOMHub, researchers can achieve more effective exchange with geographically distributed collaborators during projects, ensure results are sustained and preserved and generate reproducible publications that adhere to the FAIR guiding principles of data stewardship.

Heterogeneity of glycolytic oscillatory behaviour in individual yeast cells.

There are many examples of oscillations in biological systems and one of the most investigated is glycolytic oscillations in yeast. These oscillations have been studied since the 1950s in dense, synchronized populations and in cell‐free extracts, but it has for long been unknown whether a high cell density is a requirement for oscillations to be induced, or if individual cells can oscillate also in isolation without synchronization. Here we present an experimental method and a detailed kinetic model for studying glycolytic oscillations in individual, isolated yeast cells and compare them to previously reported studies of single‐cell oscillations. The importance of single‐cell studies of this phenomenon and relevant future research questions are also discussed.

Targeting glycolysis in the malaria parasite Plasmodium falciparum

Glycolysis is the main pathway for ATP production in the malaria parasite Plasmodium falciparum and essential for its survival. Following a sensitivity analysis of a detailed kinetic model for glycolysis in the parasite, the glucose transport reaction was identified as the step whose activity needed to be inhibited to the least extent to result in a 50% reduction in glycolytic flux. In a subsequent inhibitor titration with cytochalasin B, we confirmed the model analysis experimentally and measured a flux control coefficient of 0.3 for the glucose transporter. In addition to the glucose transporter, the glucokinase and phosphofructokinase had high flux control coefficients, while for the ATPase a small negative flux control coefficient was predicted. In a broader comparative analysis of glycolytic models, we identified a weakness in the P. falciparum pathway design with respect to stability towards perturbations in the ATP demand.

The JWS online simulation database

Summary: JWS Online is a web‐based platform for construction, simulation and exchange of models in standard formats. We have extended the platform with a database for curated simulation experiments that can be accessed directly via a URL, allowing one‐click reproduction of published results. Users can modify the simulation experiments and export them in standard formats. The Simulation database thus lowers the bar on exploring computational models, helps users create valid simulation descriptions and improves the reproducibility of published simulation experiments. Availability and Implementation: The Simulation Database is available on line at https://jjj.bio.vu.nl/models/experiments/. Contact: jls@sun.ac.za.

Quantitative analysis of drug effects at the whole-body level: a case study for glucose metabolism in malaria patients.

We propose a hierarchical modelling approach to construct models for disease states at the whole-body level. Such models can simulate effects of drug-induced inhibition of reaction steps on the whole-body physiology. We illustrate the approach for glucose metabolism in malaria patients, by merging two detailed kinetic models for glucose metabolism in the parasite Plasmodium falciparum and the human red blood cell with a coarse-grained model for whole-body glucose metabolism. In addition we use a genome-scale metabolic model for the parasite to predict amino acid production profiles by the malaria parasite that can be used as a complex biomarker.