Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: Mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

Cell Death Induced by Mild Physical Perturbations Could Be Related to Transient Plasma Membrane Modifications

An understanding of membrane destabilization induced by osmotic treatments is important to better control cell survival during biotechnological processes. The effects on the membranes of the yeast Saccharomyces cerevisiae of perturbations similar in intensity (same amount of energy) but differing in the source type (heat, compression and osmotic gradient) were investigated. The anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene was measured before and after each treatment to assess the reversibility of the membrane changes related to each treatment. Except for heat shock at 75°C, changes in membrane fluidity were reversible after the return to initial conditions, showing that two kinds of physical stress can be distinguished regarding the reversibility of membrane changes: high and mild energy stresses. With the application of osmotic gradients, anisotropy was assessed during treatment with five osmotic pressure levels from 30.7 to 95.4 MPa with two different yeast strains and related to the rate of cell death caused by each stress. The exposure of cells to increasing osmotic pressures involved a progressive lowering of membrane anisotropy during lethal perturbations. Osmotic stresses associated with reversible fluidity changes of increasing intensity in the membrane led to proportional death rates and time-dependant cell death of increasing rapidity during the application of the stress. Finally, a hypothesis relating the extent of membrane structural changes to the kinetic of cell death is proposed.     

Achievement of rapid osmotic dehydration at specific temperatures could maintain high <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> viability

Various methods have been tried to prevent cell mortality during dehydration, but the reasons why microorganisms die when submitted to dehydration and rehydration are not well understood. The aim of this study was to further investigate the reasons for yeast mortality during dehydration. Osmotic dehydration and rehydration of Saccharomyces cerevisiae W303-1A were performed at different temperatures. Two different approaches were used: isothermic treatments (dehydration and rehydration at the same temperature), and cyclic treatments (dehydration at an experimental temperature and rehydration at 25 degrees C), with significant differences in viability found between the different treatments. Dehydration at lower and higher temperatures gave higher viability results. These experiments allowed us to propose a hypothesis that relates mortality to a high water flow through an unstable membrane during phase transition.     

Anhydrobiosis in yeast: Stabilization by exogenous lactose

We have found that incubation in lactose solutions (0.75 M) of yeast culture Saccharomyces cerevisiae sensitive to dehydration damage increased the stability of the cells during dehydration. Simultaneously with this increase in viability, a decrease in plasma membrane permeability during rehydration was seen. Using Fourier transform infrared spectroscopy to measure lipid phase transitions, we observed that the lactose treatment depressed the membrane phospholipid phase transition temperature in a sensitive culture of dry yeast. As a result, it leads to the decrease in the damages of molecular organization of membranes during rehydration of dry yeast cells, thus reducing leakage from the cells.     

Extremophilic yeasts: plasma-membrane fluidity as determinant of stress tolerance

Our aim was to investigate the response of selected yeasts and yeast-like fungi from extreme?environments to various temperatures at the level of their plasma membranes, in order to elucidate the connections between their plasma-membrane fluidity (measured by electron paramagnetic resonance spectroscopy - EPR), growth temperature range, stress tolerance, and ecological distribution. Although all studied fungi can be considered mesophilic according to their growth temperature profiles, their plasma-membrane fluidity indicated otherwise. Arctic yeast Rhodosporidium diobovatum could be classified as psychrotolerant?due to its higher average membrane fluidity. Extremely halotolerant black yeast-like fungus Hortaea werneckii isolated from solar salterns, on the other hand, is not adapted to low temperature, which is reflected in the higher average rigidity of its plasma membrane and as a consequence its inability to grow at temperatures lower than 10°C. The plasma membrane of Aureobasidium sp. isolated so far exclusively from an Arctic glacier with its intermediate fluidity and high fluidity variation at different temperatures may indicate the specialization of this yeast-like fungus to the specific glacial environment. Similar behaviour of plasma membrane was detected in the reference yeast, non-extremophilic Saccharomyces cerevisiae. Its membranes of intermediate fluidity and with high fluidity?fluctuation at different temperatures may reflect the specialization of this yeast to mesophilic environments and prevent its colonization of extreme environments. Halotolerant Aureobasidium pullulans from salterns, and Arctic Cryptococcus liquefaciens and Rhodotorula?mucilaginosa with moderately fluctuating plasma membranes of intermediate fluidity are representatives of globally distributed generalistic and stress-tolerant species that can thrive in a variety of environments. Keeping the membranes stable and flexible is one of the necessities for the microorganisms to survive changes in extreme habitats. Our data suggest that plasma-membrane fluidity can be used as an indicator of fitness for survival in the extreme environments. In addition to the average fluidity of plasma membrane, the fluctuation of fluidity is an important determinant of stress tolerance: high absolute fluidity fluctuation is tied to decreased survival. The fluidity and its variation therefore reflect survival strategy and fitness in extreme environments and are good indicators?of the adaptability of microorganisms.     

Air-drying kinetics affect yeast membrane organization and survival

The plasma membrane (PM) is a key structure for the survival of cells during dehydration. In this study, we focused on the concomitant changes in survival and in the lateral organization of the PM in yeast strains during desiccation, a natural or technological environmental perturbation that involves transition from a liquid to a solid medium. To evaluate the role of the PM in survival during air-drying, a wild-type yeast strain and an osmotically fragile mutant (erg6Δ) were used. The lateral organization of the PM (microdomain distribution) was observed using a fluorescent marker related to a specific green fluorescent protein-labeled membrane protein (Sur7-GFP) after progressive or rapid desiccation. We also evaluated yeast behavior during a model dehydration experiment performed in liquid medium (osmotic stress). For both strains, we observed similar behavior after osmotic and desiccation stresses. In particular, the same lethal magnitude of dehydration and the same lethal kinetic effect were found for both dehydration methods. Thus, yeast survival after progressive air-drying was related to PM reorganization, suggesting the positive contribution of passive lateral rearrangements of the membrane components. This study also showed that the use of glycerol solutions is an efficient means to simulate air-drying desiccation.     

Anhydrobiosis in yeast: is it possible to reach anhydrobiosis for yeast grown in conditions with severe oxygen limitation?

The yeast Saccharomyces cerevisiae was shown to be extremely sensitive to dehydration–rehydration treatments when stationary phase cells were subjected to conditions of severe oxygen limitation, unlike the same cells grown in aerobic conditions. The viability of dehydrated anaerobically grown yeast cells never exceeded 2 %. It was not possible to increase this viability using gradual rehydration of dry cells in water vapour, which usually strongly reduces damage to intracellular membranes. Specific pre-dehydration treatments significantly increased the resistance of anaerobic yeast to drying. Thus, incubation of cells with trehalose (100 mM), increased the viability of dehydrated cells after slow rehydration in water vapour to 30 %. Similarly, pre-incubation of cells in 1 M xylitol or glycerol enabled up to 50–60 % of cells to successfully enter a viable state of anhydrobiosis after subsequent rehydration. We presume that trehalose and sugar alcohols function mainly according to a water replacement hypothesis, as well as initiating various protective intracellular reactions.     

The fluidity of Chinese hamster ovary cell and bull sperm membranes after cholesterol addition

Cell plasma membrane fluidity is affected by membrane lipid and protein composition as well as temperature. Altering the cholesterol content of a membrane can change membrane fluidity at different temperatures and this may affect cell survival during cryopreservation. In these experiments, we examined the effect that adding cholesterol to the membranes of Chinese hamster ovary cells (CHO) and bull sperm had on cell plasma membrane fluidity and cell survival when cells were cooled to 5 degrees C or were cryopreserved. Cells were treated with 0, 1.5 or 5.0mg cholesterol-loaded cyclodextrin (CLC), stained with N-((4-(6-phenyl-1,3,5-hexatrienyl)phenyl)propyl)trimethylammonium-p-toluenesulfonate (TMAP-DPH) to evaluate membrane fluidity and with propidium iodide to evaluate cell viability, prior to analysis by flow cytometry at 23, 5 degrees C, and after cryopreservation. CHO cells exhibited a single cell population with all cells having similar membrane fluidity. Membrane fluidity did not change when temperature had been reduced and then returned to 23 degrees C (P<0.05), however, adding cholesterol to the cells induced membranes to become more rigid (P<0.05). Bull sperm samples consisted of two cell subpopulations, one having relatively higher membrane fluidity than the other, regardless of cholesterol treatment or temperature. In addition, cells possessing the highest membrane fluidity did not survive cooling or cryopreservation efficiently. CLC treatment did not significantly alter membrane fluidity after temperature changes, but did maintain higher percentages of spermatozoa surviving cooling to 5 degrees C and cryopreservation (P<0.05). In conclusion, adding cholesterol to cell resulted in detectable membrane fluidity changes in CHO cells and increased survival of bull sperm after cooling to 5 degrees C and after cryopreservation.     

Protein dynamics in thylakoids of the desiccation-tolerant plant Boea hygroscopica during dehydration and rehydration

Plants of Boea hygroscopica F. Muell were dehydrated to 9% relative water content (RWC) by withholding water for 26 d, and afterward the plants were rehydrated. Leaves were taken from control plants after 7, 12, and 26 d from the beginning of dehydration, and after 6 and 48 h from rehydration. The RWC decreased by 80% during dehydration, but the leaves regained RWC with rehydration. Dehydrated plants showed lesser amounts of proteins, lipids, and chlorophyll, all of which increased following rewatering. The lipid-to-protein ratio, which decreased during dehydration, returned to control level after 48 h of rehydration. Thylakoid lipids were more unsaturated when RWC reached the value of 9%. EPR measurements of spin-labeled proteins showed the presence of three different groups of proteins with different mobility in thylakoid membranes. The rotational correlation time of groups 1 and 2 increased with dehydration and decreased upon rehydration, whereas group 3 showed little changes. Desiccation did not cause thylakoid swelling or breakage, but the membrane system assemblage showed changes in thylakoid stacking. After 48 h of rehydration the membrane system recovered completely the organization of the fully hydrated state, showing several well-defined and regularly distributed grana.     

Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment

In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.     

Characteristics of protectant synthesis of infective juveniles of Steinernema carpocapsae and importance of glycerol as a protectant for survival of the nematodes during osmotic dehydration

Two hypotheses on the synthesis of the protectants glycerol and trehalose of the infective juveniles (IJs) of Steinernema carpocapsae during osmotic dehydration were tested and utilised to evaluate the function and importance of glycerol on survival of the nematodes during osmotic dehydration. This was achieved by comparing the changes in survival, morphology, behaviour and levels of glycerol, trehalose and permeated compounds of the IJs dehydrated in seven hypertonic solutions at two temperature regimes: (1) 5 °C for 15 days; and (2) 23 °C for 1 day followed by 5 °C for another 14 days. The results substantiate both hypotheses tested: (1) the permeability of the IJs to various compounds, such as sucrose or ethylene glycol, when they are dehydrated in hypertonic solutions of these compounds; and (2) suppression of the synthesis of protectant glycerol but not trehalose when IJs are dehydrated at low temperature. The results also showed that: (1) although trehalose was the preferred dehydration protectant, glycerol played an important role in rapidly balancing the osmotic pressure when IJs were exposed in hypertonic solutions; (2) the presence of glycerol was essential for the IJs to survive and function properly even under moderate osmotic dehydration, especially when IJs were dehydrated in salt solutions; and (3) some exogenous compounds permeated into IJs during osmotic dehydration such as ethylene glycol, may function in the same way as glycerol and significantly improve the survival and function of the IJs. The results indicate that each of the protectants glycerol and trehalose has a specific function and neither is replaceable by the other.     

Influence of membrane fluidity modifiers on lysosomal osmotic sensitivity

Since lysosomes are prone to osmotic lysis, we have examined the correlation between their physical state and sensitivity to osmotic challenge, using agents which modify membrane fluidity. The latency loss of beta-hexosaminidase after an incubation in hypotonic sucrose medium was followed under different conditions of membrane fluidity, recorded by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3, 5-hexatriene. Increasing fluidity of the lysosomal membranes with benzyl alcohol (BA) and greater rigidity caused by cholesteryl hemisuccinate (CHS) increased and decreased the enzyme latency loss, respectively. The effects of BA and CHS treatments on osmotic sensitivity were reversible subsequently by reciprocal treatments of the lysosomes with CHS and BA, respectively. The results indicate that the physical state of the membrane does indeed affect lysosomal osmotic stability.     

Membrane fluidity and its roles in the perception of environmental signals

Poikilothermic organisms are exposed to frequent changes in environmental conditions and their survival depends on their ability to acclimate to such changes. Changes in ambient temperature and osmolarity cause fluctuations in the fluidity of cell membranes. Such fluctuations are considered to be critical to the initiation of the regulatory reactions that ultimately lead to acclimation. The mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the analysis of genome-wide gene expression using DNA microarrays has provided a powerful new approach to studies of the contribution of membrane fluidity to gene expression and to the identification of environmental sensors. In this review, we focus on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, and on the subsequent expression of genes that ensures acclimation to a new set of environmental conditions.     

Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae

Summary This paper describes the characteristics of the structural and functional organization of cellular membranes rehydrated after dehydration of the yeast Saccharomyces cerevisiae. It was noted that dehydration and subsequent rehydration of yeast cells causes a considerable increase of cytoplasmic membrane permeability. Addition of CaCl₂, glucose and polyethyleneglycol to the rehydration medium caused a decrease in cell permeability, assessed as the losses of potassium ions, nucleotides, as well as the total losses of intracellular compounds. KCl had a positive effect only at concentrations above 10%. Yeast cells, dried to residual moisture lower than 20%, showed a decrease in membrane permeability as temperatures of the rehydration medium increased up to 38°–43°C. Upon reactivation of viable dehydrated cells in a nutrient medium, a reparation of the structural damages of various intracellular membranes takes place. It was established that at cell dehydration to residual moistures of 8%–12% all the free and a part of bound water is evaporated from cells.     

Viability of Escherichia coli after combined osmotic and thermal treatment: a plasma membrane implication

This study investigates the influence of temperature (T) and osmotic pressure (Pi) on the viability of Escherichia coli K12 during an osmotic treatment. Osmotic shock (dehydration and rehydration within 1 s) in liquid media at different temperatures (4, 10, 30 and 37 degrees C) and different levels of osmotic pressure (26, 30, 35, 40, 82 and 133 MPa) were realized.Results show that a sudden dehydration, below 40 MPa, destroyed up to 80% of the bacterial population for each tested temperature, whereas viability was greater than 90% for an osmotic pressure less than 26 MPa. The influence of T and Pi on the membrane's physical structure is finally considered to explain the results in light of FTIR and electron microscopy study of the influence of temperature and osmotic pressure on E. coli membrane phospholipids conformation.     

Slow Freezing Coupled Static Magnetic Field Exposure Enhances Cryopreservative Efficiency—A Study on Human Erythrocytes

The aim of this study was to assess the cryoprotective effect of static magnetic fields (SMFs) on human erythrocytes during the slow cooling procedure. Human erythrocytes suspended in 20% glycerol were slowly frozen with a 0.4-T or 0.8-T SMF and then moved to a −80°C freezer for 24 hr. The changes in survival rate, morphology, and metabolites of the thawed erythrocytes were examined. To understand possible cryoprotective mechanisms of SMF, membrane fluidity and dehydration stability of SMF-exposed erythrocytes were tested. For each test, sham-exposed erythrocytes were used as controls. Our results showed that freezing coupled with 0.4-T or 0.8-T SMFs significantly increased the relative survival ratios of the frozen-thawed erythrocytes by 10% and 20% (p<0.001), respectively. The SMFs had no effect on erythrocyte morphology and metabolite levels. However, membrane fluidity of the samples exposed to 0.8-T SMF decreased significantly (p<0.05) in the hydrophobic regions. For the dehydration stability experiments, the samples exposed to 0.8-T SMF exhibited significantly lower (p<0.05) hemolysis. These results demonstrate that a 0.8-T SMF decreases membrane fluidity and enhances erythrocyte membrane stability to resist dehydration damage caused by slow cooling procedures.     

Biosurfactants

Abstract

Lipids are known as a part of an effective adaptation mechanism reflecting the changes in the extracellular environment. The fluidity of biological membranes is influenced by the lipid structure and the portion of saturated, unsaturated, branched, or cyclic fatty acids in individual phospholipids. For all living organisms undergoing environmental adaptation, the fluidity can be changed only to a relatively small extent. This range is genetically determined and it is specific for every microorganism. This article presents recent knowledge about the influence of some environmental parameters (temperature, osmotic pressure, pH, the presence of salt or ethanol in medium) on a microbial membrane with the emphasis on regulation aspect in fatty acid biosynthesis. The main tools for regulation of membrane fluidity, for example, fatty acid desaturation or incorporation of branched and cyclic fatty acids into phospholipids, are discussed in more detail.     

The Mycobacterium tuberculosis virulence factor trehalose dimycolate imparts desiccation resistance to model mycobacterial membranes

Mycobacteria, including persistent pathogens like Mycobacterium tuberculosis, have an unusual membrane structure in which, outside the plasma membrane, a nonfluid hydrophobic fatty acid layer supports a fluid monolayer rich in glycolipids such as trehalose 6,6′-dimycolate (TDM; cord factor). Given the abilities of mycobacteria to survive desiccation and trehalose in solution to protect biomolecules and whole organisms during freezing, drying, and other stresses, we hypothesized that TDM alone may suffice to confer dehydration resistance to the membranes of which it is a constituent. We devised an experimental model that mimics the structure of mycobacterial envelopes in which an immobile hydrophobic layer supports a TDM-rich, two-dimensionally fluid leaflet. We have found that TDM monolayers, in stark contrast to phospholipid membranes, can be dehydrated and rehydrated without loss of integrity, as assessed by fluidity and protein binding. Strikingly, this protection from dehydration extends to TDM-phospholipid mixtures with as little as 25 mol % TDM. The dependence of the recovery of membrane mobility upon rehydration on TDM fraction shows a functional form indicative of spatial percolation, implying that the connectivity of TDM plays a crucial role in membrane preservation. Our observations are the first reported instance of dehydration resistance provided by a membrane glycolipid.