Living Yeast Cells Research Articles

Automated Fluorescence Lifetime Imaging High-Content Analysis of Förster Resonance Energy Transfer between Endogenously Labeled Kinetochore Proteins in Live Budding Yeast Cells.

We describe an open-source automated multiwell plate fluorescence lifetime imaging (FLIM) methodology to read out Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) labeling endogenous kinetochore proteins (KPs) in live budding yeast cells. The low copy number of many KPs and their small spatial extent present significant challenges for the quantification of donor fluorescence lifetime in the presence of significant cellular autofluorescence and photobleaching. Automated FLIM data acquisition was controlled by µManager and incorporated wide-field time-gated imaging with optical sectioning to reduce background fluorescence. For data analysis, we used custom MATLAB-based software tools to perform kinetochore foci segmentation and local cellular background subtraction and fitted the fluorescence lifetime data using the open-source FLIMfit software. We validated the methodology using endogenous KPs labeled with mTurquoise2 FP and/or yellow FP and measured the donor fluorescence lifetimes for foci comprising 32 kinetochores with KP copy numbers as low as ~2 per kinetochore under an average labeling efficiency of 50%. We observed changes of median donor lifetime ≥250 ps for KPs known to form dimers. Thus, this FLIM high-content analysis platform enables the screening of relatively low-copy-number endogenous protein–protein interactions at spatially confined macromolecular complexes.

QUEEN-based Spatiotemporal ATP Imaging in Budding and Fission Yeast.

Yeasts have provided an exceptional model for studying metabolism and bioenergetics in eukaryotic cells. Among numerous metabolites, adenosine triphosphate (ATP) is a major metabolite that is essential for all living organisms. Therefore, a clearer understanding of ATP dynamics in living yeast cells is important for deciphering cellular energy metabolism. However, none of the methods currently available to measure ATP, including biochemical analyses and ATP indicators, have been suitable for close examinations of ATP concentrations in yeast cells at the single cell level. Using the recently developed ATP biosensor QUEEN, which is suitable for yeasts and bacteria, a protocol was described herein to visualize ATP concentrations in living budding and fission yeast cells. This simple method enables the easy and reliable examination of ATP dynamics in various yeast mutants, thereby providing novel molecular insights into cellular energy metabolism.

Microencapsulation of beetroot pomace extractin cells of Saccharomyces cerevisiae

Beetroot pomace is an industrial by-product and ecological problem, as well as a potential rich source of natural antioxidants and pigments. In the present study, beetroot pomace extract (BPE) was encapsulated in non-plasmolysed, plasmolysed, and living Saccharomyces cerevisiae yeast cells by a freezedrying method. Living and dead yeast cells were microscopically assessed by using Neubauer chamber in order todetermine how the freeze-drying procedure affects changes in the yeast cell number. Total and surface phenolic contents were determined using the Folin-Ciocalteu method and encapsulation efficiency (EE) was calculated. The best EE was achieved in the case when BPE was encapsulated in living yeast cells. The successful encapsulation of BPE in yeast cells was confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). These results report the encapsulation of sensitive compounds in yeast cells by freeze-drying, showing that it is a good method for valuable compound preservation and has a potential use in food and pharmaceutical industries.

The effect of bioresorbable additives and micro-bioobjects on gel formation, stabilization and thermophysical properties

The same properties of agarose gels containing neutral bioresorbable additives and living microorganisms which are important for use in additive technologies of bioreactors creation were considered. Data on the kinetics of gel formation from the solution during cooling were obtained by spectroscopic measurement by measuring the shift of the maximum spectrum of light passing through the gel, depending on the temperature. The dynamics of aging was investigated for gels of different concentrations of agarose, bioresorbable additives and living cells. The time dependences of the decrease in the optical transparency of such gels during the aging process, characterizing the changes in their structure, were obtained. Special attention was paid to the effect of liquid evaporation from gels in the process of gel formation and during long-term storage on relaxation processes leading to their spontaneous increase in density. Experiments were performed to determine the dynamics of the temperature fields simultaneously with heat flux measurements during the formation of studied gels with different concentrations. On the basis of the obtained experimental data and previously developed method, the thermophysical coefficients of agarose gels containing an admixture of starch and living yeast cells were calculated.

Microfluidic platform for dielectrophoretic separation of bio-particles using serpentine microelectrodes

Recently, there is a great concern in developing integrated microfluidic platforms for biological analysis systems that can perform general biochemistry and biological analysis. Dielectrophoresis, the movement of particles in a non-uniform electric field is widely used to separate viable and non viable cells with good efficiency. The present study aims to develop a universal bio-particle separator in a C-serpentine geometry which could separate particles by varying the operating conditions. We present the design, fabrication and testing for continuous separation structure based on dielectrophoretic forces. The C-Serpentine geometry is composed of consecutive C shaped curves to generate a gradient required for the dielectrophoretic effect. Known mixture of viable and non viable cells of Saccharomyces Cerevisiae was selectively trapped using negative dielectrophoretic force generated by microelectrodes. Through measurement of cell count, a trapping efficiency of 86% was obtained for mixed cell suspension and 93–98% trapping efficiency was obtained in case of trapping live and dead yeast cells individually. Also the results show that cell viability was not affected by the separation procedure as percentage of viable cells was calculated to be 96%.

Paracoccidioides restrepiensis B339 (PS3) and P. lutzii LDR2 yeast cells and soluble components display in vitro hemolytic and hemagglutinating activities on human erythrocytes.

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by thermodimorfic fungi of Paracoccidioides species complex. Several pathogenic fungi produce hemagglutinins and hemolysins, which are virulence factors involved in adhesion of pathogens to host tissues or cells and in destruction of erythrocytes. The present research investigated hemolytic and hemagglutinating activities of yeast cells and soluble components from P. restrepiensis (PS3; former P. brasiliensis B339) and P. lutzii (LDR2). Different concentrations of live and heat-killed yeast cells and soluble components from cell free antigen preparation (CFA) (native or heated - 56 and 100 °C, 30 min) were mixed with 1% human erythrocyte suspension. Yeast cells from both species caused hemolysis, but P. lutzii LDR2 was more hemolytic than P. restrepiensis B339, while the opposite phenomena occurred with soluble components in most conditions. Live or heat-killed yeast cells of both fungi agglutinated erythrocytes, but only heated soluble components from P. restrepiensis B339 showed hemagglutinating activity. In conclusion, yeast cells of P. restrepiensis B339 and P. lutzii LDR2 produce hemolysins and hemagglutinins, which most likely are more restricted to yeast cells in P. lutzii LDR2 and are more released in soluble form byP. restrepiensis B339, requiring further study.

Integrating triple intracellular metabolic pathways in living yeast cells for dual-functionalized cell beacons

Integrating triple intracellular metabolic pathways in living yeast cells for dual-functionalized cell beacons

Cytocompatible Fabrication of Yeast Cells/Fabrics Composite Sheet for Bioethanol Production

Entrapment of living cells into a polymer network has significant potential in various fields such as biomass conversion and tissue engineering. A crucial challenge for this strategy is to provide a mild enough condition to preserve cell viability. Here, a facile and cytocompatible method to entrap living yeast cells into a poly(ethylene glycol) (PEG) network grafting from polypropylene nonwoven fabrics via visible-light-induced surface living graft crosslinking polymerization is reported. Due to the mild reaction conditions and excellent biocompatibility of PEG, the immobilized yeast cells could maintain their viability and proliferate well. The obtained composite sheet has excellent long-term stability and shows no significant efficiency loss after 25 cycles of repeated batch bioethanol fermentation. The immobilized yeast cells exhibit 18.0% higher bioethanol fermentation efficiency than free cells. This strategy for immobilization of living cells with high viability has significant potential application.

Control of colloidal behavior of polystyrene latex nanoparticles and their cytotoxicity toward yeast cells using water-soluble polymers

Positively charged polystyrene latex (PSL) nanoparticles (NPs) dispersed in physiological saline (154 mM NaCl solution) are taken up by yeast cells. However, in low ionic strength solutions, the yeast cells are covered with the NPs, leading to cell death. The environmental conditions under which NPs are taken up are therefore limited. In this study, we attempted to control the uptake of positively charged PSL NPs by Saccharomyces cerevisiae in 5 mM NaCl solution using a water-soluble polymer. Addition of a small amount of anionic sodium carboxymethylcellulose (CMC), which has a carboxyl group, to 5 mM NaCl solution allowed the uptake of PSL NPs by living yeast cells. In contrast, non-ionic methylcellulose did not affect the NP behavior. This is because the negatively charged CMC adhered to the positively charged PSL NP surfaces and the surface charge changed from positive to negative. Atomic force microscopy using a single-NP probe consisting of one NP immobilized on the flattened end of the silicon nitride tip showed that CMC significantly reduced the interaction force between a negatively charged living yeast cell and a positively charged PSL NP.

Live-Cell Imaging of Chromatin Condensation Dynamics by CRISPR

SummaryThe spatiotemporal organization of chromatin plays central roles in cellular function. The ribosomal DNA (rDNA) chromatin undergoes dynamic structural changes during mitosis and stress. Here, we developed a CRISPR-based imaging system and tracked the condensation dynamics of rDNA chromatin in live yeast cells under glucose starvation. We found that acute glucose starvation triggers rapid condensation of rDNA. Time-lapse microscopy revealed two stages for rDNA condensation: a “primary stage,” when relaxed rDNA chromatin forms higher order loops or rings, and a “secondary stage,” when the rDNA rings further condense into compact clusters. Twisting of rDNA rings accompanies the secondary stage. The condensin complex, but not the cohesin complex, is required for efficient rDNA condensation in response to glucose starvation. Furthermore, we found that the DNA helicase Sgs1 is essential for the survival of cells expressing rDNA-bound dCas9, suggesting a role for helicases in facilitating DNA replication at dCas9-binding sites.

Quantitative imaging of chromatin decompaction in living cells.

Chromatin organization is highly dynamic and regulates transcription. Upon transcriptional activation, chromatin is remodeled and referred to as “open,” but quantitative and dynamic data of this decompaction process are lacking. Here, we have developed a quantitative high resolution–microscopy assay in living yeast cells to visualize and quantify chromatin dynamics using the GAL7-10-1 locus as a model system. Upon transcriptional activation of these three clustered genes, we detect an increase of the mean distance across this locus by >100 nm. This decompaction is linked to active transcription but is not sensitive to the histone deacetylase inhibitor trichostatin A or to deletion of the histone acetyl transferase Gcn5. In contrast, the deletion of SNF2 (encoding the ATPase of the SWI/SNF chromatin remodeling complex) or the deactivation of the histone chaperone complex FACT lead to a strongly reduced decompaction without significant effects on transcriptional induction in FACT mutants. Our findings are consistent with nucleosome remodeling and eviction activities being major contributors to chromatin reorganization during transcription but also suggest that transcription can occur in the absence of detectable decompaction.

The Development Fortified Pan Bread by Increasing Its Protein Content with High Levels of Live Yeast cells Saccharomyces Cerevisiae

The Development Fortified Pan Bread by Increasing Its Protein Content with High Levels of Live Yeast cells Saccharomyces Cerevisiae

G1/S Transcription Factor Copy Number Is a Growth-Dependent Determinant of Cell Cycle Commitment in Yeast.

To understand how commitment to cell division in late G1 phase (Start) is controlled by growth and nutrients in budding yeast, we determined the absolute concentrations of the G1/S transcription factors SBF (composed of Swi4 and Swi6) and MBF (composed of Mbp1 and Swi6), the transcriptional repressor Whi5, and the G1 cyclins, Cln1 and Cln2, in single live yeast cells using scanning number and brightness (sN&B) microscopy. In rich medium, Whi5, Mbp1, and Swi6 concentrations were independent of cell size, whereas Swi4 concentration doubled in G1 phase, leading to a size-dependent decrease in the Whi5/Swi4 ratio. In small cells, SBF and MBF copy numbers were insufficient to saturate target G1/S promoters, but this restriction diminished as cells grew in size. In poor medium, SBF and MBF subunits, as well as Cln1, were elevated, consistent with a smaller cell size at Start. A mathematical model constrained by sN&B data suggested that size- and nutrient-dependent occupancy of G1/S promoters by SBF/MBF helps set the cell size threshold for Start activation.

Mechanosensation Dynamically Coordinates Polar Growth and Cell Wall Assembly to Promote Cell Survival

How growing cells cope with size expansion while ensuring mechanical integrity is not known. In walled cells, such as those of microbes and plants, growth and viability are both supported by a thin and rigid encasing cell wall (CW). We deciphered the dynamic mechanisms controlling wall surface assembly during cell growth, using a sub-resolution microscopy approach to monitor CW thickness in live rod-shaped fission yeast cells. We found that polar cell growth yielded wall thinning and that thickness negatively influenced growth. Thickness at growing tips exhibited a fluctuating behavior with thickening phases followed by thinning phases, indicative of a delayed feedback promoting thickness homeostasis. This feedback was mediated by mechanosensing through the CW integrity pathway, which probes strain in the wall to adjust synthase localization and activity to surface growth. Mutants defective in thickness homeostasis lysed by rupturing the wall, demonstrating its pivotal role for walled cell survival.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of <100 nm in diameter in the plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane.

Enhanced cell trapping throughput using DC-biased AC electric field in a dielectrophoresis-based fluidic device with densely packed silica beads.

This paper presents the use of DC-biased AC electric field for enhancing cell trapping throughput in an insulator-based dielectrophoretic (iDEP) fluidic device with densely packed silica beads. Cell suspension is carried through the iDEP device by a pressure-driven flow. Under an applied DC-biased AC electric field, DEP trapping force is produced as a result of non-uniform electric field induced by the gap of electrically insulating silica beads packed between two mesh electrodes that allow both fluid and cells to pass through. While the AC component is mainly to control the magnitude of DEP trapping force, the DC component generates local electroosmotic (EO) flow in the cavity between the beads and the EO flow can be set to move along or against the main pressure-driven flow. Our experimental and simulation results show that desirable trapping is achieved when the EO flow direction is along (not against) the main flow direction. Using our proposed DC-biased AC field, the device can enhance the trapping throughput (in terms of the flowrate of cell suspension) up to five times while yielding almost the same cell capture rates as compared to the pure AC field case. Additionally, the device was demonstrated to selectively trap dead yeast cells from a mixture of flowing live and dead yeast cells.

Antagonistic effect of Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC on hyphal development and adhesion of Candida albicans.

The morphological transition from yeast to a hyphal form, as well as the adhesion capability to the gastrointestinal tract, are implicated virulent determinant in Candida albicans and could be potential targets for prevention of the opportunistic pathogen. Based on this rationale, two yeast strains Saccharomyces cerevisiae KTP and Issatchenkia occidentalis ApC along with reference strain Saccharomyces boulardii NCDC 363 were screened for the probiotic potential. Characters like pH, temperature, bile, simulated gastrointestinal juice tolerance tests, and Caco-2 cell line adhesion assay were determined in the present study. Further, the evaluation of its impact on C. albicans morphological transition and adhesion was assessed using microtitre germ tube test. In terms of probiotic characteristics, both the strains were tolerant to pH 2.5 and the presence of bile (0.3 to 0.6%) with an optimum growth temperature of 37°C. The strain KTP was also resistant to simulated gastric and intestinal juices as compared to control (13% and 41%, respectively) and NCDC 363 (55% and 35%, respectively). In contrast, both the yeasts had reduced adhesiveness to Caco-2 monolayer. Candida virulence in in vitro systems indicated that treatment of live probiotic yeast cells (108ml) effectively reduced the filamentation and adhesion of C. albicans. The S. cerevisiae KTP had a profound effect on the hyphal development and adhesion when compared to the ApC and NCDC 363. The strain significantly reduced (P < .05) the hyphal growth in co-cultivated (93% and 94%, respectively) and pre-existing hyphae (54% and 68%) of strains C. albicans 183 and 1151. Isolates KTP and ApC also reduced the adhesion (≈ 22% and 41%, respectively) and transition of blastoconidia at two hours of incubation in abiotic surface. This study provides knowledge on the effect of potential probiotic yeasts such as Saccharomyces and non- Saccharomyces strains against virulence characteristic of Candida albicans.

Ultrastructural Patterns of Interactions between Murine Lung Macrophages and Yeast Cells of Cryptococcus neoformans Strains with Different Virulence

This article presents the ultrastructural patterns of interactions between the murine lung macrophages and cells of low- (RKPGY-881, -1165, -1178) and high-virulence (RKPGY-1090, -1095, -1106) strains of Cryptococcus neoformans at the seventh post-experimental day. It was found that if macrophages ingest living yeast cells, the latter can: 1) become completely free from polysaccharide capsules, after that their contents undergo lysis, and cell wall debris are extruded from the macrophage (first scenario); 2) become partly free from their capsules, destroy the phagosomal plasma membrane and induce destructive processes inside the macrophage causing their death (second scenario); or 3) not lose their capsules and localize inside macrophage in latent state (third scenario). Macrophages can also ingest senescent and dead C. neoformans cells surrounded by capsules that are lost at the ingesting and phagosome stages (fourth scenario). The study revealed the dependence of cell-mediated immunity on the stage of development of ingested C. neoformans yeast cells. Here we describe a new mechanism of capsular polysaccharide elimination of C. neoformans yeast cells by murine macrophages.

Monitoring of Saccharomyces cerevisiae viability by non-Faradaic impedance spectroscopy using interdigitated screen-printed platinum electrodes

Abstract Interdigitated, screen-printed platinum microelectrodes are used for non-Faradaic impedance spectroscopy detection of viable Saccharomyces cerevisiae . The time dependence of the complex impedance of both living and heat-inactivated yeast cells immersed in plain medium or immobilized in agar is measured between 0.01 Hz and 1 MHz for 300 min. To understand the processes probed by the impedance measurement, an equivalent electrical circuit, containing a constant phase element, a charge transfer resistance and a suspension resistance, is established and analyzed. For both heat-inactivated and living yeast cells in plain medium, the suspension resistance increases over time caused by sedimentation of the yeast cells into the stray field of the electrodes. The measured increase is found to be larger for living cells than for heat-inactivated ones. The time dependence of the double-layer capacitance is rather dominated by the metabolic activity of living cells, but a possible impact of cell sedimentation cannot be completely neglected. In experiments carried out with cells immobilized in agar, cell sedimentation is suppressed, and thus, the time evolution of the double-layer capacitance is solely caused by metabolic active cells. Therefore, the time-dependent change of the double-layer capacitance can be used to directly monitor the viability of Saccharomyces cerevisiae in situ , since inactivated cells do not contribute to the signal generation in this case.

Biodegradation of phenol via meta cleavage pathway triggers de novo TAG biosynthesis pathway in oleaginous yeast

Phenol is reported to be one of the most toxic environmental pollutants present in the discharge of various industrial effluents causing a serious threat to the existing biome. Biodegradation of phenol by oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 was found to degrade 1000mg/l phenol. The pathways for phenol degradation by both ortho and meta-cleavage were proposed by the identification of metabolites and enzymatic assays of ring cleavage enzymes in the cell extracts. Results suggest that this oleaginous yeast degrade phenol via meta-cleavage pathway and accumulates a high quantity of lipid content (64.92%; wt/wt) as compared to control glucose synthetic medium (GSM). Meta-cleavage pathway of phenol degradation leads to formation of pyruvate and acetaldehyde. Both these end products feed as precursors for de novo triacylglycerols (TAG) biosynthesis pathway which causes accumulation of TAG in the lipid droplets (LD) of 6.12±0.78μm grown on phenol while 2.38±0.52μm observed on GSM. This was confirmed by fluorescence microscopic images of BODIPY505-515nm stained live yeast cells. GC–MS analysis of extracted total lipid showed enhanced amount of monounsaturated fatty acid (MUFA) which was as 51.87%, 58.33% and 62.98% in presence of 0.5, 0.75 and 1g/l of phenol.