| 1. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Allosteric regulation of phosphofructokinase controls the emergence of glycolytic oscillations in isolated yeast cells
- 2014
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 281:12, s. 2784-2793
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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FEBS Journal Prize Lecture: Sustained glycolytic oscillations in individual isolated yeast cells
- 2013
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Ingår i: FEBS Journal. - : Wiley. - 1742-4658 .- 1742-464X. ; 280:Suppl. S1
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Yeast glycolytic oscillations have been extensively studied since the 1950s in dense populations of cells and in cell-free extracts. Until recently, sustained oscillations had only been observed at the population level, i.e. for synchronized cultures at high biomass concentrations. One question that had not been satisfactorily addressed was whether individual cells display qualitatively different behaviour from the mean behaviour of a population of cells. We were able to observe sustained oscillations in individual isolated cells using a sophisticated experimental setup in which the concentration of metabolites in glycolysis was quantified by measuring the autofluorescence intensity from NADH molecules in the individual cells, the extracellular environment was controlled both spatially and temporally using microfluidics, and the cell density and position of the cell array within the microfluidic flow chamber was varied using optical tweezers. We thus showed that a high cell density is not a requirement for induction of oscillatory behaviour. A detailed kinetic model for the cellular reactions was adjusted to describe isolated cells in a microfluidic flow chamber. It was successfully used to simulate the heterogeneity in the oscillatory response of the individual cells, assuming small differences in a single internal parameter. In further studies we have investigated the precise conditions for autonomous oscillations at the single cell level. We have also investigated how the extracellular environment affects the characteristics of the oscillations and the heterogeneity between cells. This setup also enables studies of cell-to-cell distance and flowrate dependence on cell communication and synchronization.
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| 3. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Heterogeneity of glycolytic oscillatory behaviour in individual yeast cells
- 2014
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793. ; 588:1, s. 3-7
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Forskningsöversikt (refereegranskat)abstract
- 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.
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| 4. |
- Gustavsson, Anna-Karin, 1986, et al.
(författare)
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Studying Glycolytic Oscillations in Individual Yeast Cells by Combining Fluorescence Microscopy with Microfluidics and Optical Tweezers.
- 2019
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Ingår i: Current protocols in cell biology. - : Wiley. - 1934-2616 .- 1934-2500. ; 82:1
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Tidskriftsartikel (refereegranskat)abstract
- In this unit, we provide a clear exposition of the methodology employed to study dynamic responses in individual cells, using microfluidics for controlling and adjusting the cell environment, optical tweezers for precise cell positioning, and fluorescence microscopy for detecting intracellular responses. This unit focuses on the induction and study of glycolytic oscillations in single yeast cells, but the methodology can easily be adjusted to examine other biological questions and cell types. We present a step-by-step guide for fabrication of the microfluidic device, for alignment of the optical tweezers, for cell preparation, and for time-lapse imaging of glycolytic oscillations in single cells, including a discussion of common pitfalls. A user who follows the protocols should be able to detect clear metabolite time traces over the course of up to an hour that are indicative of dynamics on the second scale in individual cells during fast and reversible environmental adjustments. © 2018 by John Wiley & Sons, Inc.
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| 5. |
- Larsson, Christer, 1958, et al.
(författare)
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Flux balance analysis for ethylene formation in genetically engineered Saccharomyces cerevisiae
- 2011
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Ingår i: IET Systems Biology. - : Institution of Engineering and Technology (IET). - 1751-8849 .- 1751-8857. ; 5:4, s. 245-251
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Tidskriftsartikel (refereegranskat)abstract
- Biosynthesis of ethylene (ethene) is mainly performed by plants and some bacteria and fungi, via two distinct metabolic routes. Plants use two steps, starting with S-adenosylmethionine, while the ethylene-forming microbes perform an oxygen dependent reaction using 2-oxoglutarate and arginine. Introduction of these systems into Saccharomyces cerevisiae was studied in silico. The reactions were added to a metabolic network of yeast and flux over the two networks was optimised for maximal ethylene formation. The maximal ethylene yields obtained for the two systems were similar in the range of 7-8-mol ethylene/10-mol glucose. The microbial metabolic network was used for testing different strategies to increase the ethylene formation. It was suggested that supplementation of exogenous proline, using a solely NAD-coupled glutamate dehydrogenase, and using glutamate as the nitrogen source, could increase the ethylene formation. Comparison of these in silico results with published experimental data for yeast expressing the microbial system confirmed an increased ethylene formation when changing nitrogen source from ammonium to glutamate. The theoretical analysis methods indicated a much higher maximal yield per glucose for ethylene than was experimentally observed. However, such high ethylene yields could only be obtained with a concomitant very high respiration (per glucose). Accordingly, when ethylene production was optimised under the additional constraint of restricted respiratory capacity (i.e. limited to experimentally measured values) the theoretical maximal ethylene yield was much lower at 0.2/10 mol glucose, and closer to the experimentally observed values.
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| 6. |
- Mojica Benavides, Martin, 1983, et al.
(författare)
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An Optical Tweezers, Epi-Fluorescence/Spinning disk confocal- and microfluidic-setup for synchronization studies of glycolytic oscillations in living yeast cells
- 2016
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Ingår i: Proceedings SPIE 9922, Optical Trapping and Optical Micromanipulation XIII. San Diego; USA. 28 August -1 September 2016. - : SPIE. - 9781510602359
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Konferensbidrag (refereegranskat)abstract
- Due to the significant importance of glycolytic oscillations studies and the recent breakthroughs on single cell analysis, a further interest arrives with intracellular and intercellular responses. Understanding cell-cell communication can give insight to oscillatory behaviors in biological systems, such as insulin secretion from pancreatic beta-cells. The aim of this work consists on the manipulation of living yeast cells to study propagation and synchronization of induced glycolytic oscillations. A setup, consisting of an optical tweezers system and microfluidic devices coupled with fluorescence imaging was designed to perform a time dependent observation during artificially induced glycolytic oscillations. Multi-channel flow devices and diffusion chambers were fabricated using soft lithography. Automatized pumps controlled specific flow rates of infused glucose and cyanide solutions, used to induce the oscillations. Flow and diffusion in the microfluidic devices were simulated to assure experimentally the desired coverage of the solutions across the yeast cells, a requirement for time dependent measurements. Using near infrared optical tweezers, yeast cells were trapped and positioned in array configurations, ranging from a single cell to clusters of various symmetries, in order to obtain information about cell-cell communications during the metabolic cycles. Confocal illumination of an entire focal plane using a spinning disk, will allow acquirement of NADH periodic fluorescence signals during glycolytic oscillations. This method permits an improvement of the 2D projection images obtained with wide field microscopy to a tomographic description of the subcellular propagation of the oscillations.
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| 7. |
- van Niekerk, David, et al.
(författare)
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Phosphofructokinase controls the acetaldehyde induced phase shift in isolated yeast glycolytic oscillators.
- 2019
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Ingår i: The Biochemical journal. - 1470-8728. ; 476:2, s. 353-363
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Tidskriftsartikel (refereegranskat)abstract
- The response of oscillatory systems to external perturbations is crucial for emergent properties such as synchronization and phase locking, and can be quantified in a phase response curve. In individual, oscillating yeast cells, we characterized experimentally the phase response of glycolytic oscillations for external acetaldehyde pulses, and followed the transduction of the perturbation through the system. Subsequently, we analyzed the control of the relevant system components in a detailed mechanistic model. The observed responses are interpreted in terms of the functional coupling and regulation in the reaction network. We find that our model quantitatively predicts the phase dependent phase shift observed in the experimental data. The phase shift is in agreement with an adaptation leading to synchronization with an external signal. Our model analysis establishes that phosphofructokinase plays a key role in the phase shift dynamics as shown in the phase response curve, and adaptation time to external perturbations. Specific mechanism-based interventions, made possible through such analyses of detailed models, can improve upon standard trial and error methods, e.g. melatonin supplementation to overcome jet-lag, which are error prone, specifically, since the effects are phase and dose dependent.
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