| 1. |
- Persson, Sebastian, 1996, et al.
(författare)
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Fine-Tuning of Energy Levels Regulates SUC2 via a SNF1-Dependent Feedback Loop
- 2020
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Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Nutrient sensing pathways are playing an important role in cellular response to different energy levels. In budding yeast, Saccharomyces cerevisiae, the sucrose non-fermenting protein kinase complex SNF1 is a master regulator of energy homeostasis. It is affected by multiple inputs, among which energy levels is the most prominent. Cells which are exposed to a switch in carbon source availability display a change in the gene expression machinery. It has been shown that the magnitude of the change varies from cell to cell. In a glucose rich environment Snf1/Mig1 pathway represses the expression of its downstream target, such as SUC2. However, upon glucose depletion SNF1 is activated which leads to an increase in SUC2 expression. Our single cell experiments indicate that upon starvation, gene expression pattern of SUC2 shows rapid increase followed by a decrease to initial state with high cell-to-cell variability. The mechanism behind this behavior is currently unknown. In this work we study the long-term behavior of the Snf1/Mig1 pathway upon glucose starvation with a microfluidics and non-linear mixed effect modeling approach. We show a negative feedback mechanism, involving Snf1 and Reg1, which reduces SUC2 expression after the initial strong activation. Snf1 kinase activity plays a key role in this feedback mechanism. Our systems biology approach proposes a negative feedback mechanism that works through the SNF1 complex and is controlled by energy levels. We further show that Reg1 likely is involved in the negative feedback mechanism.
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| 2. |
- Persson, Sebastian, 1996, et al.
(författare)
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Modelling of glucose repression signalling in yeast Saccharomyces cerevisiae
- 2022
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Ingår i: FEMS Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 22:1
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Forskningsöversikt (refereegranskat)abstract
- Saccharomyces cerevisiae has a sophisticated signalling system that plays a crucial role in cellular adaptation to changing environments. The SNF1 pathway regulates energy homeostasis upon glucose derepression; hence, it plays an important role in various processes, such as metabolism, cell cycle and autophagy. To unravel its behaviour, SNF1 signalling has been extensively studied. However, the pathway components are strongly interconnected and inconstant; therefore, elucidating its dynamic behaviour based on experimental data only is challenging. To tackle this complexity, systems biology approaches have been successfully employed. This review summarizes the progress, advantages and disadvantages of the available mathematical modelling frameworks covering Boolean, dynamic kinetic, single-cell models, which have been used to study processes and phenomena ranging from crosstalks to sources of cell-to-cell variability in the context of SNF1 signalling. Based on the lessons from existing models, we further discuss how to develop a consensus dynamic mechanistic model of the entire SNF1 pathway that can provide novel insights into the dynamics of nutrient signalling.
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| 3. |
- Persson, Sebastian, 1996, et al.
(författare)
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Scalable and flexible inference framework for stochastic dynamic single-cell models
- 2022
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Ingår i: PLoS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358.
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Tidskriftsartikel (refereegranskat)abstract
- Understanding the inherited nature of how biological processes dynamically change over time and exhibit intra- and inter-individual variability, due to the different responses to environmental stimuli and when interacting with other processes, has been a major focus of systems biology. The rise of single-cell fluorescent microscopy has enabled the study of those phenomena. The analysis of single-cell data with mechanistic models offers an invaluable tool to describe dynamic cellular processes and to rationalise cell-to-cell variability within the population. However, extracting mechanistic information from single-cell data has proven difficult. This requires statistical methods to infer unknown model parameters from dynamic, multi-individual data accounting for heterogeneity caused by both intrinsic (e.g. variations in chemical reactions) and extrinsic (e.g. variability in protein concentrations) noise. Although several inference methods exist, the availability of efficient, general and accessible methods that facilitate modelling of single-cell data, remains lacking. Here we present a scalable and flexible framework for Bayesian inference in state-space mixed-effects single-cell models with stochastic dynamic. Our approach infers model parameters when intrinsic noise is modelled by either exact or approximate stochastic simulators, and when extrinsic noise is modelled by either time-varying, or time-constant parameters that vary between cells. We demonstrate the relevance of our approach by studying how cell-to-cell variation in carbon source utilisation affects heterogeneity in the budding yeast Saccharomyces cerevisiae SNF1 nutrient sensing pathway. We identify hexokinase activity as a source of extrinsic noise and deduce that sugar availability dictates cell-to-cell variability.
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| 4. |
- Reith, Patrick Philipp, 1991, et al.
(författare)
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The Effect of Lithium on the Budding Yeast Saccharomyces cerevisiae upon Stress Adaptation
- 2022
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Schneider, Kara, et al.
(författare)
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Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular ageing research
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. While concerns that bigger protein tags, such as genetically encoded fluorescent tags, may affect protein behaviour and function have been around for quite some time, experimental evidence of how exactly the physiology of the protein of interest is altered within fluorescent protein fusions remains limited. To address this issue, we performed a comparative assessment of endogenously expressed Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on such a tag within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects may play a role in other eukaryotic systems.
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| 6. |
- Schneider, Kara, et al.
(författare)
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Growth Rate Evaluation of the Budding Yeast Saccharomyces cerevisiae Cells Carrying Endogenously Expressed Fluorescent Protein Fusions
- 2023
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Ingår i: Fluorezcent proteins. - New York, NY : Springer US. - 1064-3745.
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Bokkapitel (refereegranskat)abstract
- Fluorescent proteins within fluorescent fusions have been reported to affect cellular growth fitness via altering native protein function and intracellular localization. Here we report in detail a procedure to analyze the growth characteristics of yeast cells expressing such fusions in comparison to unmodified parental strain. This approach can serve as an initial step in fluorescent protein characterization in vivo. © 2023, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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| 7. |
- Schnitzer, Barbara Maria, 1992, et al.
(författare)
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The effect of stress on biophysical characteristics of misfolded protein aggregates in living Saccharomyces cerevisiae cells
- 2022
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Ingår i: Experimental Gerontology. - : Elsevier BV. - 1873-6815 .- 0531-5565. ; 162
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Forskningsöversikt (refereegranskat)abstract
- Aggregation of misfolded or damaged proteins is often attributed to numerous metabolic and neurodegenerative disorders. To reveal underlying mechanisms and cellular responses, it is crucial to investigate protein aggregate dynamics in cells. Here, we used super-resolution single-molecule microscopy to obtain biophysical characteristics of individual aggregates of a model misfolded protein ∆ssCPY* labelled with GFP. We demonstrated that oxidative and hyperosmotic stress lead to increased aggregate stoichiometries but not necessarily the total number of aggregates. Moreover, our data suggest the importance of the thioredoxin peroxidase Tsa1 for the controlled sequestering and clearance of aggregates upon both conditions. Our work provides novel insights into the understanding of the cellular response to stress via revealing the dynamical properties of stress-induced protein aggregates.
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| 8. |
- Shashkova, Sviatlana, 1987, et al.
(författare)
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Copy Number Analysis of the Yeast Histone Deacetylase Complex Component Cti6 Directly in Living Cells
- 2022
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Ingår i: Chromosome Architecture. - New York, NY : Springer US. - 1064-3745. ; , s. 183-190
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Bokkapitel (refereegranskat)abstract
- Proteins are one of the key components of cellular life that play a crucial role in most biological processes. Therefore, quantification of protein copy numbers is essential for revealing and better understanding of cellular behavior and functions. Here we describe a single-molecule fluorescence-based method of protein copy number quantification directly in living cells. This enables quick and reliable estimations and comparison of the protein of interest abundance without implementing large-scale studies.
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| 9. |
- Shashkova, Sviatlana, 1987, et al.
(författare)
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Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells
- 2021
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Ingår i: Methods. - : Elsevier BV. - 1095-9130 .- 1046-2023. ; 193, s. 46-53
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Shashkova, Sviatlana, 1987, et al.
(författare)
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Correlative single-molecule fluorescence barcoding of gene regulation in Saccharomyces cerevisiae
- 2021
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Ingår i: Methods. - : Elsevier BV. - 1046-2023. ; 193, s. 62-67
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Tidskriftsartikel (refereegranskat)abstract
- Most cells adapt to their environment by switching combinations of genes on and off through a complex interplay of transcription factor proteins (TFs). The mechanisms by which TFs respond to signals, move into the nucleus and find specific binding sites in target genes is still largely unknown. Single-molecule fluorescence microscopes, which can image single TFs in live cells, have begun to elucidate the problem. Here, we show that different environmental signals, in this case carbon sources, yield a unique single-molecule fluorescence pattern of foci of a key metabolic regulating transcription factor, Mig1, in the nucleus of the budding yeast, Saccharomyces cerevisiae. This pattern serves as a ‘barcode’ of the gene regulatory state of the cells which can be correlated with cell growth characteristics and other biological function. © 2020 The Authors
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