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- 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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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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| 3. |
- 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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| 4. |
- 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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