A SALT-INDUCED CHLOROPLAST PROTEIN IN DUNALIELLA SALINA (CHLOROPHYTA)1

The unicellular green alga Dunaliella salina Teod, is halophilic and wall-less. The cell acclimates to osmotic stresses by accumulation or degradation of glycerol. To investigate other mechanisms involved in its physiological recovery following hyperosmotic shocks, protein profiles from cells grown in various salinities were compared. A 13-kDa protein (P13) accumulated when cells were subjected to drastic hyperosmotic shock. Front our results with antibiotic-treated cells and purified chloroplasts, we believe that this component results from de novo translation in chloroplasts. The solubility of P13 was strongly promoted by Triton X-100. Its accumulation was correlated with the recovery of photosynthesis.     

Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.     

Interrelated Effects of Cold Shock and Osmotic Pressure on the Permeability of the Escherichia coli Membrane to Permease Accumulated Substrates

Permease studies are generally carried out by incubating cells in growth medium with labeled substrate, collecting the cells on microporous membrane filters, and washing them free from extracellular radioactivity with ice-cold medium. Studies of thiomethylgalactoside, valine, and galactose accumulation indicate that in several strains of Escherichia coli the bacterial membrane is exquisitely sensitive to isosmotic cold shock. Substrate pools formed at 25 C may suffer almost total loss if the cells are rapidly chilled to approximately 0 C during sampling. In glycerol-grown cells, this rapid efflux of substrate is prevented or minimized if the cells are subjected at the moment of cold shock to a simultaneous hyperosmotic transition. Because of this protective effect, the apparent size of a permease accumulated substrate pool is extremely sensitive to the osmotic composition of the incubation medium and may appear to be increased as much as 10-fold when the osmolarity is reduced from approximately 0.3 to 0.1 osmolar. These differences vanish when sampling and washing are carried out with medium at room temperature. It is suggested that isosmotic cold shock causes crystallization of the liquid-like lipids within the membrane. The hydrophilic channels created in this process would facilitate the rapid efflux of permease accumulated substrates. The imposition of a simultaneous hyperosmotic transition by dehydrating the cell periphery would cause increased lipid interaction, thus preserving the integrity of the cells membrane.     

A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density.

The growth rates and buoyant densities of a Salmonella typhimurium mutant, TL126 (proB74A+), with enhanced osmotolerance caused by proline overproduction were measured and compared with the growth rates and buoyant densities of an isogenic (wild-type) strain, TL128 (proB+ A+), with normal control of proline production. Growth rates were determined in a rich medium (Luria broth) with added NaCl to produce various osmotic strengths ranging from 300 to 2,000 mosM. At low concentrations of NaCl, there was little variation in doubling times between the two strains. However, as the osmotic strength of the medium approached and exceeded 1,300 mosM, the doubling times of TL126 (osmotolerant) were 1.5 to 2 times faster than those of TL128 (wild type), confirming the osmotolerance of TL126. Buoyant densities were determined by equilibrium sedimentation in a Percoll gradient of osmotic strength equal to that of the growth medium. The osmolarity of the Percoll gradient was adjusted by the addition of NaCl. At low osmolarities (300 to 500 mosM), the buoyant density of TL126 (osmotolerant) was slightly but consistently lower than that of TL128 (wild type). As the osmotic strength was increased, the buoyant density of TL126 (osmotolerant) increased in proportion to the osmotic strength. In contrast, the buoyant density of strain TL128 (wild type) did not increase as much. At high osmolarities (1,600 to 2,000 mosM), the buoyant density of TL126 (osmotolerant) was consistently higher than that of TL128 (wild type). These results suggest that the intracellular accumulation of proline by TL126, the osmotolerant strain, increases both the growth rates and buoyant densities at osmolarities of 1,300 mosM and above.     

Fast, multiphase volume adaptation to hyperosmotic shock by Escherichia coli

All living cells employ an array of different mechanisms to help them survive changes in extra cellular osmotic pressure. The difference in the concentration of chemicals in a bacterium's cytoplasm and the external environment generates an osmotic pressure that inflates the cell. It is thought that the bacterium Escherichia coli use a number of interconnected systems to adapt to changes in external pressure, allowing them to maintain turgor and live in surroundings that range more than two-hundred-fold in external osmolality. Here, we use fluorescence imaging to make the first measurements of cell volume changes over time during hyperosmotic shock and subsequent adaptation on a single cell level in vivo with a time resolution on the order of seconds. We directly observe two previously unseen phases of the cytoplasmic water efflux upon hyperosmotic shock. Furthermore, we monitor cell volume changes during the post-shock recovery and observe a two-phase response that depends on the shock magnitude. The initial phase of recovery is fast, on the order of 15-20 min and shows little cell-to-cell variation. For large sucrose shocks, a secondary phase that lasts several hours adds to the recovery. We find that cells are able to recover fully from shocks as high as 1 Osmol/kg using existing systems, but that for larger shocks, protein synthesis is required for full recovery.     

Growth and buoyant density of Escherichia coli at very low osmolarities.

The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored. At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml. As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml. From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities. The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml. Both strains of E. coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9. (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM. To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly. From this study, it was determined that both Ca2+ and Mg2+ could stimulate growth below 40 mosM. With Ca2+ - and Mg2+ -supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9. Strain ML3 would only haltingly grow at 15 mosM. Four attempts were made to grow both ML3 and ML308 at 5 mosM. In three of the experiments, ML308 grew, while strain ML3 grew in one experiment. While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently. Therefore, from this study, we believe that E. coli might be defined as an euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.     

Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium

In previous studies, we had shown that the buoyant density ofEscherichia coli is determined by the osmolarity of the growth medium by varying the osmolarity of the medium with NaCl or sucrose. However, the buoyant density of the cells always exceeded that of the growth medium. Here we determined the effect of medium with a buoyant density greater than the expected buoyant density of cells by adding Nycodenz to Luria broth. Percoll gradients of cells were analyzed by laser light scattering. The buoyant density for 125- and 375-mOsM-grown cells was 0.002 g/ml and 0.003 g/ml more, respectively, for cells grown in the presence of Nycodenz than those grown without Nycodenz, while the buoyant density of 250-mOsM-grown cells was 0.005 g/ml less for cells grown in the presence of Nycodenz than those grown without Nycodenz. Cells grown in 500-mOsM medium with or without Nycodenz had the same buoyant density. the buoyant density of cultures grown in defined medium was the same as those grown in rich medium, with only the medium osmolarity correlating to buoyant density. We conclude from these experiments that neither buoyant density nor chemical make-up of the medium determines the buoyant density of cells grown in that medium. Only the medium osmolarity determines cell buoyant density, suggesting thatE. coli has no mechanisms to sense buoyant density.     

Volume regulation mechanisms in Rana castebeiana cardiac tissue under hyperosmotic stress

Volume changes of cardiac tissue under hyperosmotic stress in Rana catesbeiana were characterized by the identification of the osmolytes involved and the possible regulatory processes activated by both abrupt and gradual changes in media osmolality (from 220 to 280mosmol/kg H(2)O). Slices of R. catesbeiana cardiac tissue were subjected to hyperosmotic shock, and total tissue Na(+), K(+), Cl(-) and ninhydrin-positive substances were measured. Volume changes were also induced in the presence of transport inhibitors to identify osmolyte pathways. The results show a maximum volume loss to 90.86+/-0.73% of the original volume (measured as 9% decrease in wet weight) during abrupt hyperosmotic shock. However, during a gradual osmotic challenge the volume was never significantly different from that of the control. During both types of hyperosmotic shock, we observed an increase in Na(+) but no significant change in Cl(-) contents. Additionally, we found no change in ninhydrin-positive substances during any osmotic challenge. Pharmacological analyses suggest the involvement of the Na(+)/H(+) exchanger, and perhaps the HCO(3)(-)/Cl(-) exchanger. There is indirect evidence for decrease in Na(+)/K(+)-ATPase activity. The Na(+) fluxes seem to result from Mg(2+) signaling, as saline rich in Mg(2+) enhances the regulatory volume increase, followed by a higher intracellular Na(+) content. The volume maintenance mechanisms activated during the gradual osmotic change are similar to that activated by abrupt osmotic shock.     

Techniques for Dual Staining of DNA and Intracellular Immunoglobulins in Murine Hybridoma Cells: Applications to Cell-Cycle Analysis of Hyperosmotic Cultures

Flow cytometry was used to evaluate the effects of hyperosmotic stress on cell-cycle distribution and cell-associated immunoglobulins for murine hybridoma cells grown in batch culture. Paraformaldehyde/methanol fixation substantially increased the fluorescence signal for intracellular immunoglobulins compared to ethanol fixation. For surface immunoglobulins, similar fluorescence signals were observed regardless of fixation method. Dual staining of immunoglobulins and cellular DNA was employed to determine immunoglobulin pool size as a function of cell-cycle phase. The intracellular immunoglobulin pool sizes increased as the cells progressed through the cell cycle for both control and hyperosmotic cultures. For control cultures, the immunoglobulin pool size increased during the exponential phase of culture, followed by a decrease as the cultures entered stationary phase. In contrast, hyperosmotic cultures showed an initial decrease in immunoglobulin pool size upon the application of osmotic shock, followed by an increase to a level above that of control cultures. This behavior was observed in all phases of the cell cycle. In addition, hyperosmotic cultures exhibited an increase in cell size when compared to control cultures. When normalized for cell size, the intracellular immunoglobulin concentration in hyperosmotic cultures was initially lower than in control cultures and subsequently increased to slightly above the level of control cells. Cells in all phases of the cell cycle behaved in a similar manner. There was no apparent relationship between the intracellular antibody concentration and the rate of antibody secretion.     

Role of Glutathione in the Response of Escherichia coli to Osmotic Stress

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in -glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K⁺ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K⁺ were similarly biphasic: a rapid increase in the K⁺ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K⁺ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.     

In vivo 31P NMR study of early cellular responses to hyperosmotic shock in cultured glioma cells.

Cell volume regulation in the face of osmotic stress is a fundamental homeostatic activity, and is most critical in brain, which is spatially constrained. Despite the importance of this phenomenon, little is known about volume regulation in the brain, primarily because of the cellular heterogeneity in the tissue. We describe here simultaneous in vivo 31P nuclear magnetic resonance (NMR) measurements of cell volume, intracellular pH and phosphate metabolites during early responses to hyperosmotic stress in C6 glioma cells perfused in NMR-compatible bioreactors. Cell volume was measured using dimethyl methylphosphonate (DMMP) as a probe which has an intracellular NMR resonance shifted upfield from the extracellular resonance. The sensitivity of these measurements allowed 31P NMR spectra to be collected every 30 s. Following an increase in osmolarity from 320 to 480 mOsm by addition of NaCl to the perfusate, C6 glioma cells shrank to 67% of their original volume. We also observed a simultaneous increase of intracellular pH coincident with the decrease in cell volume. The signals from ATP decreased by 10%, but those from phosphocreatine (PCr) increased by 31% after hyperosmotic shock. However, correcting the ATP signals for the decrease in cell volume indicated that its intracellular concentrations increased after treatment. Signals from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were not changed significantly. This is the first in vivo report of early cellular responses monitored by NMR spectroscopy following hyperosmotic shock in cultured cells.     

Accumulation of glutamate by osmotically stressed Escherichia coli is dependent on pH.

In the present study, we measured the accumulation of glutamate after hyperosmotic shock in Escherichia coli growing in synthetic medium. The accumulation was high in the medium containing sucrose at a pH above 8 and decreased with decreases in the medium pH. The same results were obtained when the hyperosmotic shock was carried out with sodium chloride. The internal level of potassium ions in cells growing at a high pH was higher than that in cells growing in a neutral medium. A mutant deficient in transport systems for potassium ions accumulated glutamate upon hyperosmotic stress at a high pH without a significant increase in the internal level of potassium ions. When the medium osmolarity was moderate at a pH below 8, E. coli accumulated gamma-aminobutyrate and the accumulation of glutamate was low. These data suggest that E. coli uses different osmolytes for hyperosmotic adaptation at different environmental pHs.     

A procedure for enrichment and isolation of mutants of the salt-tolerant yeast Debaryomyces hansenii having altered glycerol metabolism

Abstract The salt-tolerant yeast Debaryomyces hansenii produces and accumulates glycerol when subjected to salt stress, whereby the buoyant density of the cells is changed. This property allows for enrichment of mutants with altered glycerol metabolism by density gradient centrifugation. Colonies derived from cells with rapidly changing density following an osmotic shock were screened for increased glycerol production by observing their ability to support growth of a glycerol-requiring strain of Escherichia coli . The glycerol overproducing phenotype of two isolates was confirmed by chemical analysis.     

Osmotic responses of unicellular blue-green algae (cyanobacteria): changes in cell volume and intracellular solute levels in response to hyperosmotic treatment

Abstract Changes in cell volume and solute content upon hyperosmotic shock have been studied for six unicellular blue-green algae (cyanobacteria): Synechococcus PCC 6301, PCC 6311; Synechocystis PCC 6702, PCC 6714, PCC 6803 and PCC 7008. The extent of change in volume was shown to be dependent upon the solute used to establish the osmotic gradient, with cells in NaCl showing a reduced shrinkage when compared to cells in media containing added sorbitol and sucrose. Uptake of extracellular solutes during hyperosmotic shock was observed in Synechocystis PCC 6714, with maximum accumulation of external solutes in NaCl and minimum solute uptake in sucrose solutions. Conversely, solute loss from the cells (K⁺ and amino acids) was greatest in sucrose-containing media and least in NaCl. The results show that these blue-green algae do not behave as ‘ideal osmometers’ in media of high osmotic strength. It is proposed that short-term changes in plasmalemma permeability in these organisms may be due to transient membrane instability resulting from osmotic imbalance between the cell and its surrounding fluid at the onset of hyperosmotic shock.     

Multiple glycerol shocks increase the calcium phosphate transfection of non-synchronized CHO cells

The exposure of CHO DG44 cells to an osmotic shock, after DNA uptake, results in a cellular volume decrease of approx. 55%. Repetitive osmotic shocks targeted different sub-populations of cells as was demonstrated using two different fluorescent reporter genes. Also the exposure of a calcium phosphate–DNA coprecipitate to high osmolarity in vitro caused the release of the DNA from the precipitate. The results demonstrate the importance of the osmotic shock on the efficient delivery of plasmid DNA to the nucleus of CHO cells following calcium phosphate-mediated transfection.     

Selection and Characterization of Dunaliella salina Mutants Defective in Haloadaptation

A technique for selection of Dunaliella mutants defective in their capacity to recover from osmotic shocks has been developed. The selection is based on physical separation of mutants on density gradients. This technique takes advantage of the fact that Dunaliella cells, when exposed to osmotic shocks, initially change volume and density due to water gain or loss and subsequently recover their volume and density by readjusting their intracellular glycerol. Eight mutants that do not recover their original density following hyperosmotic shocks have been isolated. The mutants grow similar to wild type cells in 1 molar NaCl, and recover like the wild type from hypotonic shocks but are defective in recovering from hypertonic shocks. A partial characterization of one of the mutants is described.     

Osmotic loading of articular cartilage modulates cell deformations along primary collagen fibril directions

Osmotic loading is known to modulate chondrocyte (cell) height, width and volume in articular cartilage. It is not known how cartilage architecture, especially the collagen fibril orientation, affects cell shape changes as a result of an osmotic challenge.Intact patellae of New Zealand white rabbits (n=6) were prepared for fluorescence imaging. Patellae were exposed to a hypotonic osmotic shock and cells were imaged before loading and 5–60 min after the osmotic challenge. Cell volumes and aspect ratios (height/width) were analyzed. A fibril-reinforced poroelastic swelling model with realistic primary collagen fibril orientations, i.e. horizontal, random and vertical orientation in the superficial, middle and deep zones, respectively and cells in different zones was used to estimate cell aspect ratios theoretically.As the medium osmolarity was reduced, cell aspect ratios decreased and volumes increased in the superficial zone of cartilage both experimentally (p<0.05) and theoretically. Theoretically determined aspect ratios of middle zone cells remained virtually constant, while they increased for deep zone cells as osmolarity was reduced.Findings of this study suggest that osmotic loading modulates chondrocyte shapes in accordance with the primary collagen fibril directions in articular cartilage.     

Adaptive MscS gating in the osmotic permeability response in E. coli: the question of time

Microorganisms adapt to osmotic downshifts by releasing small osmolytes through mechanosensitive (MS) channels. We want to understand how the small mechanosensitive channel's (MscS) activation and inactivation, both driven by membrane tension, optimize survival in varying hypoosmotic shock situations. By measuring light scattering with a stopped-flow device, we estimate bacterial swelling time as 30-50 ms. A partial solute equilibration follows within 150-200 ms, during which optical responses from cells with WT MscS deviate from those lacking MS channels. MscS opening rates estimated in patch clamp show the channels readily respond to tensions below the lytic limit with a time course faster than 20 ms and close promptly upon tension release. To address the role of the tension-insensitive inactivated state in vivo, we applied short, long, and two-step osmotic shock protocols to WT, noninactivating G113A, and fast-inactivating D62N mutants. WT and G113A showed a comparable survival in short 1 min 800 mOsm downshock experiments, but G113A was at a disadvantage under a long 60 min shock. Preshocking cells carrying WT MscS for 15 s to 15 min with a 200 mOsm downshift did not sensitize them to the final 500 mOsm drop in osmolarity of the second step. However, these two-step shocks induced death in D62N more than just a one-step 700 mOsm downshift. We conclude MscS is able to activate and exude osmolytes faster than lytic pressure builds inside the cell under abrupt shock. During prolonged shocks, gradual inactivation prevents continuous channel activity and assists recovery. Slow kinetics of inactivation in WT MscS ensures that mild shocks do not inactivate the entire population, leaving some protection should conditions worsen.     

Rearrangement of cortex proteins constitutes an osmoprotective mechanism in Dictyostelium.

Dictyostelium responds to hyperosmotic stress of 400 mOsm by a rapid reduction of its cell volume to 50%. The reduced cell volume is maintained as long as these osmotic conditions prevail. Dictyostelium does not accumulate compatible osmolytes to counteract the osmotic pressure applied. Using two-dimensional gel electrophoresis, we demonstrate that during the osmotic shock the protein pattern remains unaltered in whole-cell extracts. However, when cells were fractionated into membrane and cytoskeletal fractions, alterations of specific proteins could be demonstrated. In the crude membrane fraction, a 3-fold increase in the amount of protein was measured upon hyperosmotic stress. In the cytoskeletal fraction, the proteins DdLIM and the regulatory myosin light chain (RMLC) were shown to be regulated in the osmotic stress response. The elongation factors eEF1alpha (ABP50) and eEF1beta were found to increase in the cytoskeletal fraction, suggesting a translational arrest upon hyperosmotic stress. Furthermore, the two main components of the cytoskeleton, actin and myosin II, are phosphorylated as a consequence of the osmotic shock, with a tyrosine residue as the phosphorylation site on actin and three threonines in the case of the myosin II heavy chain.