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.

Equine sperm membrane phase behavior: the effects of lipid-based cryoprotectants.

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.

Lipid and protein changes due to freezing in Dunning AT-1 cells.

Defining the process of cellular injury during freezing, at the molecular level, is important for cryosurgical applications. This work shows changes to both membrane lipids and protein structures within AT-1 Dunning prostate tumor cells after a freezing stress which induced extreme injury and cell death. Cells were frozen in an uncontrolled fashion to -20 or -80 degrees C. Freezing resulted in an increase in the gel to liquid crystalline phase transition temperature (T(m)) of the cellular membranes and an increase in the temperature range over which the transition occurred, as determined by Fourier transform infrared spectroscopy (FTIR). Thin layer chromatography (TLC) analysis of total lipid extracts showed free fatty acids (FFA) in the frozen samples, indicating a change in the lipid composition. The final freezing temperature had no effect on the thermotropic response of the membranes or on the FFA content of the lipid fraction. The overall protein secondary structure as determined by FTIR showed only slight changes after freezing to -20 degrees C, in contrast to a strong and apparently irreversible denaturation after freezing to -80 degrees C. Taken together, these results suggest that the decrease in viability between control and frozen cells can be correlated with small changes in the membrane lipid composition and membrane fluidity. In addition, loss of cell viability is associated with massive protein denaturation as observed in cells frozen to -80 degrees C, which was not observed in samples frozen to -20 degrees C.

Engineering mammalian cells for solid-state sensor applications.

A fundamental advance in the development and application of cell- and tissue-based biosensors would be the ability to achieve air-dry stabilization of mammalian (especially human) cells with subsequent recovery following rehydration. The would allow for the preparation of sensors with extended shelf lives, only requiring the addition of water for activation. By understanding and subsequently employing the tactics used by desiccation-tolerant extremophiles, it may be possible to design stabilized mammalian cell-based biosensors. The approaches required to realize this goal are discussed and illustrated with several examples.

The effect of arbutin on membrane integrity during drying is mediated by stabilization of the lamellar phase in the presence of nonbilayer-forming lipids.

Arbutin (4-hydroxyphenyl-beta-glucopyranoside) is a solute accumulated to high concentrations in drought and frost resistant plants. Arbutin can inhibit membrane lysis, both free radical-mediated and enzymatic in nature, and it has been suggested that arbutin might contribute to membrane stabilization in these plants. However, we found that arbutin destabilized phosphatidylcholine vesicles during drying and rehydration, which appears to be inconsistent with the proposed protective function of arbutin for membranes. We also found, however, that arbutin stabilizes membranes containing nonbilayer-forming lipids during freezing. We now report that, in liposomes containing the nonbilayer-forming lipids monogalactosyldiacylglycerol (MGDG) or phosphatidylethanolamine (PE), arbutin served a protective function during drying, as measured by retention of carboxyfluorescein (CF) and extent of vesicle fusion. In hydrated samples containing these lipids, arbutin stabilized the lamellar liquid crystalline phase. Therefore, the interaction between arbutin and lipid membranes and the resulting effects on membrane stability depend, in a complex manner, on the lipid composition of the membrane.

Human platelets loaded with trehalose survive freeze-drying.

Human blood platelets are stored in blood banks for 5 days, after which they are discarded, by federal regulation. This short lifetime has led to a chronic shortage of platelets, a problem that is particularly acute in immunosuppressed patients, such as those with AIDS. We report here that platelets can be preserved by freeze-drying them with trehalose, a sugar found at high concentrations in organisms that naturally survive drying. We suggest that these findings will obviate the storage problem with platelets. Trehalose is rapidly taken up by human platelets at 37 degrees C, with loading efficiencies of 50% or greater. Fluid-phase endocytosis plays an important role in this efficient uptake of trehalose, but other mechanisms may also be involved. Trehalose-loaded platelets were successfully freeze-dried, with excellent recovery of intact platelets. Rehydration from the vapor phase led to a survival rate of 85%. The response of these platelets to the agonists thrombin (1 U/ml), collagen (2 microg/ml), ADP (20 micromM), and ristocetin (1.6 mg/ml) was almost identical to that of fresh, control platelets. Analysis by Fourier transform infrared spectroscopy demonstrated that the membrane and protein components of trehalose-loaded platelets after freeze-drying, prehydration, and rehydration were remarkably similar to those of fresh platelets.

Factors affecting leakage of trapped solutes from phospholipid vesicles during thermotropic phase transitions.

Liposomes are commonly used as models for chilling and freezing damage, with leakage of water-soluble contents from the aqueous interior as the most frequently used measurement of damage. In order to achieve an understanding of the mechanism of the leakage, we have conducted a study of the factors that influence the leakage from liposomes during phase transitions. While such investigations have appeared sporadically in the literature, a detailed study has not been undertaken previously, despite the fact that liposomes are widely used as models for stress injury. Thus, we suggest that these findings will be of general interest in the cryobiology community. We now report that the following variables affected leakage from liposomes during chilling: (i) increasing the rate of cooling and warming resulted in decreased leakage; (ii) maximal leakage occurred at the measured phase transition temperature; (iii) addition of defect-forming additives such as a second phospholipid or a surfactant increased leakage from the liposomes during the phase transition but not above or below that temperature; (iv) small unilamellar vesicles leaked much more rapidly than large unilamellar vesicles; and (v) increasing the pH of the external buffer decreased leakage of carboxyfluorescein, an effect that is probably particular to ionizable solutes.

Antifreeze proteins differentially affect model membranes during freezing.

Over the past decade antifreeze proteins from polar fish have been shown either to stabilize or disrupt membrane structure during low temperature and freezing stress. However, there has been no systematic study on how membrane composition affects the interaction of antifreeze proteins with membranes under stress conditions. Therefore, it is not possible at present to predict which antifreeze proteins will protect, and which will damage a particular membrane during chilling or freezing. Here, we analyze the effects of freezing on spinach thylakoid membranes and on model membranes of varying lipid composition in the presence of antifreeze protein type I (AFP I) and specific fractions of antifreeze glycoproteins (AFGP). We find that the addition of galactolipids to phospholipid model membranes changes the effect each protein has on the membrane during freezing. However, the greatest differences observed in this study are between the different types of antifreeze proteins. We find that AFP type I and the largest molecular weight fractions of AFGP induce concentration dependent leakage from, and are fusogenic to the liposomes. This is the first report that an antifreeze protein induces membrane fusion. In contrast, the smallest fraction of AFGP offers a limited degree of protection during freezing and does not induce membrane fusion at concentrations up to 10 mg/ml.

Lateral organization and domain formation in a two-component lipid membrane system.

The thermodynamic phase behavior and lateral lipid membrane organization of unilamellar vesicles made from mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) were investigated by fluorescence resonance energy transfer (FRET) as a function of temperature and composition. This was done by incorporating a headgroup-labeled lipid donor (NBD-DPPE) and acceptor (N-Rh-DPPE) in low concentrations into the binary mixtures. Two instances of increased energy transfer efficiency were observed close to the phase lines in the DMPC/DSPC phase diagram. The increase in energy transfer efficiency was attributed to a differential preference of the probes for dynamic and fluctuating gel/fluid coexisting phases. This differential preference causes the probes to segregate (S. Pedersen, K. Jørgensen, T. R. Baekmark, and O. G. Mouritsen, 1996, Biophys. J. 71:554-560). The observed increases in energy transfer match with the boundaries of the DMPC/DSPC phase diagram, as measured by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). We propose that the two instances of probe segregation are due to the presence of DMPC-rich and DSPC-rich domains, which form a dynamic structure of gel/fluid coexisting phases at two different temperatures. Monitoring the melting profile of each lipid component independently by FTIR shows that the domain structure is formed by DMPC-rich and DSPC-rich domains rather than by pure DMPC and DSPC domains.

Synechocystis HSP17 is an amphitropic protein that stabilizes heat-stressed membranes and binds denatured proteins for subsequent chaperone-mediated refolding.

The small heat shock proteins (sHSPs) are ubiquitous stress proteins proposed to act as molecular chaperones to prevent irreversible protein denaturation. We characterized the chaperone activity of Synechocystis HSP17 and found that it has not only protein-protective activity, but also a previously unrecognized ability to stabilize lipid membranes. Like other sHSPs, recombinant Synechocystis HSP17 formed stable complexes with denatured malate dehydrogenase and served as a reservoir for the unfolded substrate, transferring it to the DnaK/DnaJ/GrpE and GroEL/ES chaperone network for subsequent refolding. Large unilamellar vesicles made of synthetic and cyanobacterial lipids were found to modulate this refolding process. Investigation of HSP17-lipid interactions revealed a preference for the liquid crystalline phase and resulted in an elevated physical order in model lipid membranes. Direct evidence for the participation of HSP17 in the control of thylakoid membrane physical state in vivo was gained by examining an hsp17(-) deletion mutant compared with the isogenic wild-type hsp17(+) revertant Synechocystis cells. We suggest that, together with GroEL, HSP17 behaves as an amphitropic protein and plays a dual role. Depending on its membrane or cytosolic location, it may function as a "membrane stabilizing factor" as well as a member of a multichaperone protein-folding network. Membrane association of sHSPs could antagonize the heat-induced hyperfluidization of specific membrane domains and thereby serve to preserve structural and functional integrity of biomembranes.

The trehalose myth revisited: introduction to a symposium on stabilization of cells in the dry state.

This essay is an introduction to a series of papers arising from a symposium on stabilization of cells in the dry state. Nearly all of these investigations have utilized the sugar trehalose as a stabilizing molecule. Over the past two decades a myth has grown up about special properties of trehalose for stabilization of biomaterials. We review many of such uses here and show that under ideal conditions for drying and storage trehalose has few, if any, special properties. However, under suboptimal conditions trehalose has some distinct advantages and thus may remain the preferred excipient. We review the available mechanisms for introducing trehalose into the cytoplasm of living cells as an introduction to the papers that follow.

Membrane reorganization during chilling: implications for long-term stabilization of platelets.

This essay is a review of the various biophysical and biochemical events that make up the factors responsible for platelet cold-induced activation. It describes the formation of large membrane domains composed of raft aggregates that occur during chilling and storage. It also presents strong evidence that platelet membranes undergo lateral phase separation during prolonged storage in the cold and suggests that raft aggregation and lateral phase separation are key events which must be obviated to stabilize platelets and store them either in the frozen or in the dry state.

Non-disaccharide-based mechanisms of protection during drying.

Few tissues or organisms can survive the removal of nearly all their intra and extracellular water. These few have developed specialized adaptations to protect their cellular components from the damage caused by desiccation and rehydration. One mechanism, common to almost all such organisms, is the accumulation of disaccharides within cells and tissues at the onset of dehydration. This adaptation has been extensively studied and will not be considered in this review. It has become increasingly clear that true desiccation tolerance is likely to involve several mechanisms working in concert; thus, we will highlight several other important and complimentary adaptations found especially in the dehydration-resistant tissues of higher plants. These include the scavenging of reactive oxygen species, the down-regulation of metabolism, and the accumulation of certain amphiphilic solutes, proteins, and polysaccharides.

Comparison of the solution conformation and dynamics of antifreeze glycoproteins from Antarctic fish.

The (1)H- and (13)C-NMR spectra of antifreeze glycoprotein fractions 1-5 from Antarctic cod have been assigned, and the dynamics have been measured using (13)C relaxation at two temperatures. The chemical shifts and absence of non-sequential (1)H-(1)H NOEs are inconsistent with a folded, compact structure. (13)C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1-5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555-1563). The infrared (IR) spectra of AFGP8 and AFGP1-5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1-5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible o, psi angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops.

Animal models for studies on cold-induced platelet activation in human beings.

When human platelets are chilled below about 20 degrees C, they spontaneously activate, a phenomenon that limits their storage lifetime. We have previously shown that this activation in chilled human platelets is associated with passage through a lipid phase transition. Because animal models are necessary for Investigating methods for cold storage of platelets, it is essential to determine whether such phase transitions and chilling-induced activation are found in these models. In this study we examined platelets from some commonly used animal models-pigs, rhesus monkeys, mice, dogs, and rabbits. Using Fourier transform infrared spectroscopy (FTIR), we detected the thermotropic membrane phase transition in Intact platelets and assessed the morphologic response of the platelets to chilling. Statistical analysis of both FTIR and shape change show that of the animal models tested, pig platelets are most similar to human platelets. These studies suggest that pigs and pig platelets are the models of choice for the study of cold-induced platelet activation in human beings.

Engineering desiccation tolerance in Escherichia coli.

Recombinant sucrose-6-phosphate synthase (SpsA) was synthesized in Escherichia coli BL21DE3 by using the spsA gene of the cyanobacterium Synechocystis sp. strain PCC 6803. Transformants exhibited a 10,000-fold increase in survival compared to wild-type cells following either freeze-drying, air drying, or desiccation over phosphorus pentoxide. The phase transition temperatures and vibration frequencies (P==O stretch) in phospholipids suggested that sucrose maintained membrane fluidity during cell dehydration.

Effects of temperature on calcium-sensitive fluorescent probes.

The effect of temperature on the binding equilibria of calcium-sensing dyes has been extensively studied, but there are also important temperature-related changes in the photophysics of the dyes that have been largely ignored. We conducted a systematic study of thermal effects on five calcium-sensing dyes under calcium-saturated and calcium-free conditions. Quin-2, chlortetracycline, calcium green dextran, Indo-1, and Fura-2 all show temperature-dependent effects on fluorescence in all or part of the range tested (5-40 degrees C). Specifically, the intensity of the single-wavelength dyes increased at low temperature. The ratiometric dyes, because of variable effects at the two wavelengths, showed, in general, a reduction in the fluorescence ratio as temperature decreased. Changes in viscosity, pH, oxygen quenching, or fluorescence maxima could not fully explain the effects of temperature on fluorescence. The excited-state lifetimes of the dyes were determined, in both the presence and absence of calcium, using multifrequency phase-modulation fluorimetry. In most cases, low temperature led to prolonged fluorescence lifetimes. The increase in lifetimes at reduced temperature is probably largely responsible for the effects of temperature on the physical properties of the calcium-sensing dyes. Clearly, these temperature effects can influence reported calcium concentrations and must therefore be taken into consideration during any investigation involving variable temperatures.

Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying.

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze-drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry egg PtdCho by more than 20 degrees C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.

Lipid phase separation correlates with activation in platelets during chilling.

When human platelets are chilled below 22 degrees C, they spontaneously activate, a phenomenon that severely limits their storage life. It has previously been proposed that there is a correlation between cold-induced platelet activation and passage of the membranes through a liquid-crystalline to gel phase transition. Because animal models are essential for developing methods for cold storage of platelets, it is necessary to investigate such a correlation in animal platelets. In this work, horse platelets were used as a model, and it was found that cold-induced morphological activation is related to the lipid phase transition. Using fluorescence microscopy with the lipophilic fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil-C18), and Fourier transform infrared spectroscopy (FTIR), it was found that lipid phase separation occurs during cooling and low temperature storage. Furthermore, removal of cholesterol from the plasma membrane also induced a phase separation, possibly between specific phospholipid classes. Steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-DPH (TMA-DPH) were compared in cells and multilamellar vesicles (MLV) composed of platelet lipids. Cholesterol depletion led to a decrease in the fluorescence anisotropy of the two probes, which can be explained by changes in the order of the phospholipid molecules. In addition, the lipid composition and fatty acid profile of the cellular phospholipids were determined. Based of the similarities between horse and human platelets, it is suggested that horse platelets may be used as a model for studying cold-stored platelets. The results are discussed in relation to the possible role of phase separation during cell signalling.