Living Saccharomyces Cerevisiae Cells Research Articles

Increasing the Efficiency of Taxifolin Encapsulation in Saccharomyces cerevisiae Yeast Cells Based on Ultrasonic Microstructuring

The aim of the present study was to investigate the possibility of encapsulating the plant antioxidant taxifolin in the living cells of the yeast Saccharomyces cerevisiae. Taxifolin is an unstable substance prone to oxidative degradation and actively enters into chemical reactions with a decrease or loss of bioactive properties. To minimize these problems, the use of encapsulation technology has been proposed. The cells of the yeast Saccharomyces cerevisiae have been chosen as a protective material for taxifolin. The encapsulation process was carried out using simple diffusion methods in living Saccharomyces cerevisiae cells in a thermostatically controlled shaker for 24 h. The aim of the study was to evaluate the effect of preliminary microstructuring of taxifolin on the efficiency of its encapsulation in yeast cells. The microstructuring process was carried out using low-frequency ultrasonic cavitation exposure for 7 min with a frequency of 22 ± 1.6 kHz and a power of 600 W/100 mL. The studies confirmed the feasibility of the proposed approach. It was found that microstructuring changes the dispersed composition of taxifolin particles and their morphology in solution and also increases the value of the antioxidant activity. Preliminary microstructuring of taxifolin increases the efficiency of its encapsulation in Saccharomyces cerevisiae yeast cells by 1.42 times compared to the initial form. A positive dependence of the growth of the encapsulation efficiency on the duration of the process was also established. Thus, the conducted studies confirmed the advantage of encapsulation of taxifolin in living cells of the yeast Saccharomyces cerevisiae in microstructured form.

The effect of stress on biophysical characteristics of misfolded protein aggregates in living Saccharomyces cerevisiae cells.

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.

Spectral artefacts induced by moving targets in live hyperspectral stimulated Raman spectroscopy: The case of lipid droplets in yeast cells

In this study, we used stimulated Raman spectroscopy (SRS) microscopy to collect Raman signatures from live Saccharomyces cerevisiae cells in the spectral range 2804−3060 cm−1 and 830−2000 cm−1 with a spectral resolution of 8 cm−1. To effect this, we tuned the pump beam to several distinct wavelengths and thus acquired a series of chemical maps in order to reconstruct SRS spectra based on the intensity of the pixels, an approach also referred as hyperspectral SRS (hsSRS). One of the advantages of hsSRS over spontaneous Raman is that it is not overtly plagued by fluorescence and so fluorescent samples like yeast can be analysed. We show however that Raman signatures acquired by this approach may be subject to spectral artefacts that manifest as drops in intensity of Raman signal due to the movement of lipid droplets (LDs) within the yeast cells. To overcome this issue, yeast cells were chemically fixed with 4% formaldehyde and no artefacts were observed in the Raman signatures acquired from ‘stationary’ samples. Our findings indicate that caution must be applied when analysing SRS signatures obtained through hsSRS from mobile LDs and/or any other moving target within a system, whether biological or not.

Chemical rescue of mutant proteins in living Saccharomyces cerevisiae cells by naturally occurring small molecules.

Intracellular proteins function in a complex milieu wherein small molecules influence protein folding and act as essential cofactors for enzymatic reactions. Thus protein function depends not only on amino acid sequence but also on the concentrations of such molecules, which are subject to wide variation between organisms, metabolic states, and environmental conditions. We previously found evidence that exogenous guanidine reverses the phenotypes of specific budding yeast septin mutants by binding to a WT septin at the former site of an Arg side chain that was lost during fungal evolution. Here, we used a combination of targeted and unbiased approaches to look for other cases of “chemical rescue” by naturally occurring small molecules. We report in vivo rescue of hundreds of Saccharomyces cerevisiae mutants representing a variety of genes, including likely examples of Arg or Lys side chain replacement by the guanidinium ion. Failed rescue of targeted mutants highlight features required for rescue, as well as key differences between the in vitro and in vivo environments. Some non-Arg mutants rescued by guanidine likely result from “off-target” effects on specific cellular processes in WT cells. Molecules isosteric to guanidine and known to influence protein folding had a range of effects, from essentially none for urea, to rescue of a few mutants by DMSO. Strikingly, the osmolyte trimethylamine-N-oxide rescued ∼20% of the mutants we tested, likely reflecting combinations of direct and indirect effects on mutant protein function. Our findings illustrate the potential of natural small molecules as therapeutic interventions and drivers of evolution.

Encapsulation of peach waste extract in Saccharomyces cerevisiae cells

As a secondary industrial product, peach waste (PW) presents an ecological problem, but is potentially a rich source of natural antioxidants. A potential and novel way to improve the phytochemical stability of waste rich in phytochemicals is encapsulation in yeast cells that possess good structural characteristics. In the present study, PW extract was encapsulated in non-plasmolyzed, plasmolyzed and living Saccharomyces cerevisiae cells using the freeze-drying method. HPLC analysis revealed that ?-carotene is the most abundant carotenoid, while epicatechin and catechin are the most abundant phenolics in PW. The highest encapsulation efficiency of carotenoids (86.59 %), as well as phenolics (66.98 %), was obtained with freeze-dried non-plasmolyzed yeast cells used as carriers. Although plasmolysis can cause some changes in the structure and properties of yeast cells, it did not enhance the encapsulation efficiency of present phytochemicals. Successful encapsulation of PW extract in yeast cells was confirmed by FTIR spectroscopy and SEM imaging. The obtained results present the encapsulation of sensitive compounds in yeast cells by freeze-drying as an excellent method for preserving valuable compounds and their potential use in the food and pharmaceutical industries.

The Effects of Antioxidants Provided with Feed on Certain Quality Parameters of Bull Semen Under Heat Stress Conditions

Abstract The aim of the current research was to assess the effects of the feed additive made of lyophilised melon juice (source of superoxide dismutase, SOD) and inactivated live Saccharomyces cerevisiae (strain R397) cells added to the feed via the product containing high levels of organically bound selenium (source of selenium-dependant glutathione peroxidase, Se-GPx) on the semen quality of bulls in heat stress conditions. The 15 bulls chosen for the experiment were assigned to three equal groups (control –group C; treated group M, given the source of SOD; and group A, treated with the source of Se-GPx). The research was conducted in summer. The activities of SOD and Se-GPx in seminal plasma were determined spectrophotometrically. Computer-assisted semen analysis was done to determine the sperm counts, motility and velocity. The temperature and humidity were recorded with a digital data logger. The average SOD activity in the control bulls was significantly lower than in M (p<0.001) and A (p<0.001), whilst the average activities in the treated groups did not differ significantly (p=0.784). Higher average SOD activity compared to the control in the treated groups showed that both feed additives increased the antioxidative capacity of the seminal fluid. The average GPx activity in the control was significantly lower than in groups A (p=0.001) and M (p=0.005), whilst the two treatments did not lead to significantly different results (p=0.701). The analysis of relations between the activity of each enzyme and sperm motility and progressive motility in each of the bulls failed to detect a significant correlation. The analysis of the relation between THI (temperature-humidity index) and the activity of the antioxidative enzymes revealed that the increase in THI coincided with the decrease in the SOD activity in the control group, but with its increase in the treated groups (p>0.05). In all of the three groups with the increase in THI there was an increase in GPx activity (p>0.05). It can be concluded that in all of the three groups of bulls there was an increase in the activity of both enzymes in the seminal plasma, but the increase was significantly lower in the control. Thus, the antioxidative capacity of the seminal plasma of untreated bulls was proven to be lower in comparison with those of the treated animals.

Thiol Peroxidases as Major Regulators of Intracellular Levels of Peroxynitrite in Live Saccharomyces cerevisiae Cells

Thiol peroxidases (TP) are ubiquitous and abundant antioxidant proteins of the peroxiredoxin and glutathione peroxidase families that can catalytically and rapidly reduce biologically relevant peroxides, such as hydrogen peroxide and peroxynitrite. However, the TP catalytic cycle is complex, depending on multiple redox reactions and partners, and is subjected to branching and competition points that may limit their peroxide reductase activity in vivo. The goals of the present study were to demonstrate peroxynitrite reductase activity of TP members in live cells in real time and to evaluate its catalytic characteristics. To these ends, we developed a simple fluorescence assay using coumarin boronic acid (CBA), exploiting that fact that TP and CBA compete for peroxynitrite, with the expectation that higher TP peroxynitrite reductase activity will lower the CBA oxidation. TP peroxynitrite reductase activity was evaluated by comparing CBA oxidation in live wild type and genetically modified Δ8 (TP-deficient strain) and Δ8+TSA1 (Δ8 strain that expresses only one TP member, the TSA1 gene) Saccharomyces cerevisiae strains. The results showed that CBA oxidation decreased with cell density and increased with increasing peroxynitrite availability. Additionally, the rate of CBA oxidation decreased in the order Δ8 > Δ8+TSA1 > WT strains both in control and glycerol-adapted (expressing higher TP levels) cells, showing that the CBA competition assay could reliably detect peroxynitrite in real time in live cells, comparing CBA oxidation in strains with reduced and increased TP expression. Finally, there were no signs of compromised TP peroxynitrite reductase activity during experimental runs, even at the highest peroxynitrite levels tested. Altogether, the results show that TP is a major component in the defense of yeast against peroxynitrite insults under basal and increasing stressful conditions.

Cadmium Causes Misfolding and Aggregation of Cytosolic Proteins in Yeast.

Cadmium is a highly poisonous metal and is classified as a human carcinogen. While its toxicity is undisputed, the underlying in vivo molecular mechanisms are not fully understood. Here, we demonstrate that cadmium induces aggregation of cytosolic proteins in living Saccharomyces cerevisiae cells. Cadmium primarily targets proteins in the process of synthesis or folding, probably by interacting with exposed thiol groups in not-yet-folded proteins. On the basis of in vitro and in vivo data, we show that cadmium-aggregated proteins form seeds that increase the misfolding of other proteins. Cells that cannot efficiently protect the proteome from cadmium-induced aggregation or clear the cytosol of protein aggregates are sensitized to cadmium. Thus, protein aggregation may contribute to cadmium toxicity. This is the first report on how cadmium causes misfolding and aggregation of cytosolic proteins in vivo The proposed mechanism might explain not only the molecular basis of the toxic effects of cadmium but also the suggested role of this poisonous metal in the pathogenesis of certain protein-folding disorders.

Boron Nitride Nanotubes and Layer‐By‐Layer Polyelectrolyte Coating for Yeast Cell Surface Engineering

AbstractThe application of living microbial cells as molecular engines for a variety of biotechnological applications including cell‐based biosensing is an ongoing research effort. However, there are significant difficulties to overcome such as the fragile structures of microbial cells and the weak efficiency of developed systems for detecting toxic agents in the environment. In this Communication, we demonstrate the interfacing of hydroxylated boron nitride nanotubes (BNNT‐OHs) with live yeast cell surfaces. BNNT‐OHs were incorporated with polyelectrolytes (PEs) using layer‐by‐layer deposition onto live Saccharomyces cerevisiae cells. The PE‐ and BNNT‐OH‐coated yeast was characterized using spectroscopic and imaging techniques (dynamic light scattering, FTIR, and SEM). Importantly, BNNT‐OH‐coated yeast cells were viable after the surface modification with nanotubes. We believe that BNNT‐OHs‐functionalized yeast will find numerous applications in biotechnology.

Sar1 localizes at the rims of COPII-coated membranes in vivo

ABSTRACTThe Sar1 GTPase controls coat assembly on coat protein complex II (COPII)-coated vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi. The GTP-bound form of Sar1, activated by the ER-localized guanine nucleotide exchange factor (GEF) Sec12, associates with the ER membrane. GTP hydrolysis by Sar1, stimulated by the COPII-vesicle-localized GTPase-activating protein (GAP) Sec23, in turn causes Sar1 to dissociate from the membrane. Thus, Sar1 is cycled between active and inactive states, and on and off vesicle membranes, but its precise spatiotemporal regulation remains unknown. Here, we examined Sar1 localization on COPII-coated membranes in living Saccharomyces cerevisiae cells. Two-dimensional (2D) observation demonstrated that Sar1 showed modest accumulation around the ER exit sites (ERES) in a manner that was dependent on Sec16 function. Detailed three-dimensional (3D) observation further demonstrated that Sar1 localized at the rims of the COPII-coated membranes, but was excluded from the rest of the COPII membranes. Additionally, a GTP-locked form of Sar1 induced abnormally enlarged COPII-coated structures and covered the entirety of these structures. These results suggested that the reversible membrane association of Sar1 GTPase leads to its localization being restricted to the rims of COPII-coated membranes in vivo.

Photobleaching of the resonance Raman lines of cytochromes in living yeast cells

The photobleaching of the resonance cytochrome Raman lines in living Saccharomyces cerevisiae cells was studied. The photobleaching rate versus the irradiation power was described by square function plus a constant in contrast to the linear dependence of the photoinjury rate. This difference distinguishes the cytochrome photooxidation from other processes of the cell photodamage. The square dependence is associated with the reaction involving two photogenerated intermediates while the constant with the dark redox balance rates. This work demonstrates a potential of Raman spectroscopy to characterize the native cytochrome reaction rates and to study the cell photodamage precursors.

Assessment of the distribution of nucleic acid intercalators in yeast cells by pseudospectral image analysis

The intracellular location of nucleic acid intercalators (NAI) in live (not fixed) Saccharomyces cerevisiae cells has been studied using fluorescence microscopy combined with computer pseudospectral image analysis. Three NAI: the anthracycline anticancer drug doxorubicin and the nucleic acid dyes ethidium bromide (E) and 4',6-diamidino-2-phenylindole (DAPI) were used. All three NAI were shown to be localized in nuclei and mitochondria. In contrast to DAPI, which interacted only with DNA, a large fraction of doxorubicin and ethidium bromide apparently bound to mitochondrial membranes. Upon combined application, a competition between these intercalators for binding sites in the nuclear and mitochondrial DNA occurred. It was concluded that this approach may be used in designing new DNA-targeted drugs and in preliminary studies of their interaction with eukaryotic cells.

Single-Molecule Atomic Force Microscopy Reveals Clustering of the Yeast Plasma-Membrane Sensor Wsc1

Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains (“rafts”) is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of ∼200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.

Polyelectrolyte-Mediated Assembly of Multiwalled Carbon Nanotubes on Living Yeast Cells

Here we report the three-dimensional assembly of carbon nanotubes on the polyelectrolyte-coated living Saccharomyces cerevisiae cells using the polyelectrolyte-mediated layer-by-layer approach. Synthetic polyelectrolytes poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) were layer-by-layer deposited on the surfaces of the yeast cells followed by the deposition of water-soluble oxidized multiwalled carbon nanotubes (MWNTs) and an additional outermost polyelectrolyte bilayer. This resulted in the fabrication of polyelectrolyte/nanotubes composite coatings on the cell walls of the yeast cells, which could be clearly seen using the conventional optical microscopy. Transmission and scanning electron microscopy was applied to further investigate the composite coatings. Viability of the encapsulated cells was confirmed using the intercellular esterase activity test. Finally, electrochemical studies using voltammetry and electrochemical impedance measurements were performed, indicating that the composite polyelectrolytes/MWNTs coatings sufficiently affect the electron mediation between the encapsulated yeast cells and the artificial electron acceptor, making it possible to distinguish between living and dead cells. The technique described here may find potential application in the development of microelectronic devices, core-shell and hollow composite microparticles, and electrochemical cell-based biosensors.

Real-time detection of cofactor availability in genetically modified living Saccharomyces cerevisiae cells — Simultaneous probing of different geno- and phenotypes

This work describes a mediated amperometric method for simultaneous real-time probing of the NAD(P)H availability in two different phenotypes, fermentative and respiratory, of the phosphoglucose isomerase deletion mutant strain of S. cerevisiae, EBY44 [ENY.WA-1A pgi1- 1D ::URA3], and its parental strain, ENY.WA-1A. The developed method is based on multichannel detection using microelectrode arrays. Its versatility was demonstrated by using four microelectrode arrays for simultaneously monitoring the NAD(P)H availability of both geno- and phenotypes under the influence of two different carbon sources, glucose and fructose, as well as the cytosolic and mitochondrial inhibitor and uncoupler, dicoumarol. The obtained results indicate that the method is capable of accurately and reproducibly (overall relative standard error of mean 3.2%) mapping the real-time responses of the cells with different genotype–phenotype combinations. The ENY.WA cells showed the same response to glucose and fructose when dicoumarol was used; fermentative cells indicated the presence of cytosolic inhibition and respiratory cells a net effect of mitochondrial uncoupling. EBY44 cells showed cytosolic inhibition with the exception of respiratory cells when fructose was used as carbon source.

Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in LivingSaccharomyces cerevisiaeCells

A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker(1)-maltose-binding protein-linker(2)-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 microM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).

Isolation of intact RNA from cytometrically sorted Saccharomyces cerevisiae for the analysis of intrapopulation diversity of gene expression

Characterizing and understanding the functional heterogeneity in a given population on the cellular and molecular level is a great challenge in microbiology. Each microorganism contributes differently to the overall performance of the community and responds differently to changing microenvironmental conditions. Here, we present a method for isolation of intact RNA out of small subpopulations of live Saccharomyces cerevisiae cells for differential gene expression analysis. The protocol includes fluorescence staining, flow cytometric analysis and sorting of live yeast cells, subsequent isolation of RNA from the resulting subpopulations and finally RNA quantification and integrity check. The isolated RNA can be transcribed into cDNA and successfully used for microarray analysis. This aids in relating molecular regulation processes within subpopulations with the dynamics and functioning of the entire population. The procedure can be accomplished in 2 d.

Single-molecule and population probing of chromatin structure using DNA methyltransferases

Probing chromatin structure with DNA methyltransferases offers advantages over more commonly used nuclease-based and chromatin immunoprecipitation methods for detection of nucleosomes and non-histone protein–DNA interactions. Here, we describe two related methods in which the readout of MTase accessibility is obtained by assaying 5-methylcytosine in DNA through the PCR-based technique of bisulfite genomic sequencing. The methyltransferase accessibility protocol (MAP) determines the relative frequency at which the enzyme accesses each of its target sites over an entire population of PCR amplified product. While MAP yields much quantitative information about relative accessibility of a region of chromatin, a complementary single-molecule view of methyltransferase accessibility, termed MAP for individual templates (MAP-IT), is provided by analysis of cloned PCR products. Absolute rather than relative methylation frequencies in a region are obtained by summing the methylation status at each site over a cohort of clones. Moreover, as the integrity of individual molecules is maintained in MAP-IT, unique information about the distribution of multiple footprints along continuous regions is gleaned. In principle, the population MAP and single-molecule MAP-IT strategies can be used to analyze chromatin structure in a variety of model systems. Here, we describe the application of MAP in living Saccharomyces cerevisiae cells and MAP-IT in the analysis of a mammalian tumor suppressor gene in nuclei. This application of MAP-IT provides the first means to simultaneously determine CpG methylation of mammalian genes and their overlying chromatin structure in the same single DNA molecule.

Analysis of living S. cerevisiae cell states—A three color approach

Biosyntheses often fluctuate with the state of the cell in the cell cycle and on the capacity of the cell to access and metabolize a carbon source. Visualization of substrate uptake by individual cells, together with the simultaneous analysis of proliferation activity and the proportion of dead cells, facilitate reliable and quasi-online process optimization. Flow cytometry and Hoechst 33342 staining were used to follow proliferation activity of living Saccharomyces cerevisiae cells, whereas 2-NBD-glucose was employed to analyze the cells' substrate affinity. Propidium iodide was used to determine the proportion of dead cells. Calibration and verification experiments were performed with cells grown batch-wise as well as in transient state regimes. A new and rapid three-color assay was developed and tested under varying microenvironmental conditions. Live/dead cell states and the affinity to 2-NBD-glucose vs. proliferation states were determined during respiratory and/or fermentative modes of metabolism.

Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of livingS. cerevisiaecells

Recently, lipid-raft-based subdomains within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using green fluorescent protein fusions, and non-overlapping subdomains containing either Pma1p or Can1p were distinguished. In this study, the long-term stability of the subdomains was investigated. Experiments with latrunculin A and nocodazole ruled out the involvement of cytoskeletal components in the stabilization of the subdomains. Also a putative role of the cell wall was excluded, because protoplasting of the cells changed neither the pattern nor the stability of the subdomains. By contrast, the expected inner dynamics of the membrane subdomains was documented by FRAP experiments. Finally, two other proteins were localized within the frame of the Can1p/Pma1p plasma-membrane partition. We show that Fur4p (another H+ symporter) and Sur7p (a protein of unknown function) occupy the Can1p subdomain.