Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Outer membrane protein composition of Yersinia pestis at different growth stages and incubation temperatures.

The protein composition of the outer membrane of Yersinia pestis grown at 26 and at 37 degrees C was examined. The outer membrane was isolated by isopycnic sucrose density centrifugation, and its degree of purity was determined with known inner and outer membrane components. Using two-dimensional gel electrophoresis, we identified a large number of heat-modifiable proteins in the outer membrane of cells grown at either incubation temperature. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heated preparations indicated five proteins in the outer membrane of 37 degrees C-grown cells not evident in 26 degrees C-grown cells. Differences in the protein composition of the outer membrane due to the stage of growth were evident at both 26 degrees C and 37 degrees C, although different changes were found at each temperature. When cell envelopes were examined for the presence of peptidoglycan-associated proteins, no differences were seen as a result of stage of growth. Envelopes from 26 degrees C-grown cells yielded two peptidoglycan-associated proteins, E and J. Cells grown at 37 degrees C, however, also contained an additional protein (F) which was not found in either the bound or free form 26 degrees C. The changes in outer membrane protein composition in response to incubation temperature may relate to known nutritional and antigenic changes which occur under the same conditions.     

The influence of saturated fatty acid modulation of bilayer physical state on cellular and membrane structure and function

Cultured chick fibroblasts supplemented with stearic acid in the absence of serum at 37 degrees C degenerate and die in contrast to cells grown at 41 degrees C which appear normal in comparison with controls. These degenerative effects at 37 degrees C are alleviated by addition to stearate-containing media of fatty acids known to fluidize bilayers. These observations suggest that cell degeneration at 37 degrees C may involve alterations in the physical state of the membrane. Fatty acid analysis of plasma membrane obtained from stearate-supplemented cells clearly demonstrates the enrichment of this fatty acid species into bilayer phospholipids. Moreover, the extent of enrichment is similar in cells grown at both 37 and 41 degrees C. Stearate enrichment at either temperature does not appear to alter significantly membrane cholesterol or polar lipid content. Fluorescence anisotropy measurements for perylene and diphenylhexatriene incorporated into stearate-enriched membranes reveals changes suggestive of decreased bilayer fluidity. Moreover, analysis of temperature dependence of probe anisotropy indicates that a similarity in bilayer fluidity exists between stearate-enriched membranes at 41 degrees C and control membranes at 37 degrees C. Calorimetric data from liposomes prepared from polar lipids isolated from these membranes show similar melting profiles, consistent with the above lipid and fluorescence analyses. Arrhenius plot of stearate-enriched membrane glucose transporter function reveals breaks which coincide with the main endotherm of the pure phospholipid phase transition, indicating the sensitivity of the transporter to this transition which is undetectable in these native bilayers. These data suggest the existence of regions of bilayer lipid microheterogeneity which affect integral enzyme function, cell homeostasis and viability.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385–392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15°C after the shift down of growth temperature from 39°C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15°C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39°C or 15°C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39°C or 15°C did not change the membrane fluidity (measured at 15°C) of phosphatidylcholine from whole cells grown at 39°C. On the other hand, both cardiolipins of 39°C-grown and 15°C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15°C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15°C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39°C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39°C and 15°C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39°C-grown cells and dipalmitoleoylphosphatidylcholine for 15°C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Role of membrane fluidity in pressure resistance of Escherichia coli NCTC 8164

The relationship among growth temperature, membrane fatty acid composition, and pressure resistance was examined in Escherichia coli NCTC 8164. The pressure resistance of exponential-phase cells was maximal in cells grown at 10 degrees C and decreased with increasing growth temperatures up to 45 degrees C. By contrast, the pressure resistance of stationary-phase cells was lowest in cells grown at 10 degrees C and increased with increasing growth temperature, reaching a maximum at 30 to 37 degrees C before decreasing at 45 degrees C. The proportion of unsaturated fatty acids in the membrane lipids decreased with increasing growth temperature in both exponential- and stationary-phase cells and correlated closely with the melting point of the phospholipids extracted from whole cells examined by differential scanning calorimetry. Therefore, in exponential-phase cells, pressure resistance increased with greater membrane fluidity, whereas in stationary-phase cells, there was apparently no simple relationship between membrane fluidity and pressure resistance. When exponential-phase or stationary-phase cells were pressure treated at different temperatures, resistance in both cell types increased with increasing temperatures of pressurization (between 10 and 30 degrees C). Based on the above observations, we propose that membrane fluidity affects the pressure resistance of exponential- and stationary-phase cells in a similar way, but it is the dominant factor in exponential-phase cells whereas in stationary-phase cells, its effects are superimposed on a separate but larger effect of the physiological stationary-phase response that is itself temperature dependent.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15 degrees C to 34 degrees C or decreased from 39 degrees C to 15 degrees C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34 degrees C swam at 570 microns/s. On incubation at 15 degrees C these cells swam at 100 microns/s. When the temperature was increased to 34 degrees C after a 90-min incubation at 15 degrees C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34 degrees C-grown cells decreased to 210 microns/s, and the cells ceased to move when the temperature was decreased to 15 degrees C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15 degrees C increased gradually during incubation at 15 degrees C. On the other hand, the fluidity of the heated cell decreased during incubation at 34 degrees C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 microns/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.     

Fluidity of bacterial membrane lipids monitored by intramolecular excimerization of 1.3-di(2-pyrenyl)propane

Intramolecular excimer formation of 1,3-di(2-pyrenyl)propane was used to study the fluidity of liposomes prepared from membrane polar lipids of Bacillus stearothermophilus. On the basis of spectral data, local polarity and polarizability parameters were established suggesting that the probe molecules are located well inside the membranes, but displaced towards the polar head groups of the phospholipid molecules. The excimerization rate is very sensitive to lipid phase transitions and pretransitions of synthetic pure lipid bilayers. In bacterial lipids from cultures grown at 55 and 68 degrees C, thermal profiles of excimer to monomer intensity ratios (I'/I) show a broad transition which is displaced to higher temperatures in response to the increase of the growth temperature; these results correlate well with differential scanning calorimetry data and fluorescence polarization of diphenylhexatriene. Additionally, lipid bilayers of bacteria grown at 68 degrees C exhibit a decreased membrane fluidity, as monitored by both fluorescent probes.     

Effects of temperature acclimation on Neurospora phospholipids. Fatty acid desaturation appears to be a key element in modifying phospholipid fluid properties

Experiments were conducted on the effect of growth temperature on phospholipids of Neurospora. Strains grown at high (37 degrees C) and low (15 degrees C) temperatures show large differences in the proportions of phospholipid fatty acid alpha-linolenate (18 : 3) which can vary by 10-fold over this temperature range. Changes in the phospholipid base composition are less dramatic; the most significant is an increase in phosphatidylethanolamines at low temperatures accompanied by a concomitant decrease in phosphatidylcholine. It appears that phospholipid fatty acid desaturation is closely regulated with respect to growth temperature. Over the 37 to 15 degrees C growth temperature range there appear to be at least two desaturase systems in Neurospora which are under different controls. Production of 18 : 1 and 18 : 2 species appears to occur at high levels over the entire temperature range, whereas the production of 18 : 3 seems to be inversely related to growth temperature. Shifting 37 degrees C-acclimated cultures to 15 degrees C produces a growth lag period of approximately 3 h, during which the level of 18 : 3 increases markedly. Differential scanning calorimetry of phospholipids from 37 degrees C cells shows a phase transition at -22 degrees C while lipids from 15 degrees C cultures exhibit a phase transition with reduced enthalpy at about -41 degrees C. The data are consistent with the idea that phospholipid composition in Neurospora is under strict control and suggest that membrane fluidity is regulated with respect to growth temperature through changes in membrane lipid composition.     

大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间的关系

通过对大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间关系研究发现,10℃培养,对数期细胞有最大的压力抗性,随着培养温度的升高直到4 5℃,压力抗性呈下降的趋势;相反,10℃培养,稳定期的细胞对压力最敏感,随着培养温度的升高,压力抗性呈增加趋势,30～37℃时达到最大,之后到4 5℃有下降。对数期和稳定期细胞膜脂中不饱和脂肪酸的组成随温度的上升而下降,这与从全细胞中抽提的磷脂的熔点密切相关。因此,对数期细胞压力抗性随着膜流动性的增大而升高;但稳定期细胞,膜流动性与压力抗性之间不存在简单的对应变化关系     

Role of membrane lipids and membrane fluidity in thermosensitivity and thermotolerance of mammalian cells

The role of membrane lipids and membrane fluidity in thermosensitivity of mammalian cells is not well understood. The limited experimental data in the literature have led to conflicting results. A detailed investigation of lipid composition and membrane fluidity of cellular membranes was undertaken to determine their relationship to cell survival after hyperthermia. Ehrlich ascites (EA) cells, mouse fibroblast LM cells, and HeLa S3 cells differed in thermosensitivity as expressed by a D0 of 3.1, 5.2, and 9.7 min, respectively, at 44 degrees C. No correlation with cellular thermosensitivity could be found with respect to the amount of cholesterol and to the cholesterol to phospholipid ratio in the particulate fraction of the cells. By growing the cells for some generations in different media, cholesterol and phospholipid content could be changed in the particulate fraction, but no difference in cell survival was observed. When mouse fibroblasts were grown for 24 hr in a serum-free medium supplemented with arachidonic acid (20:4), all subcellular membranes were about eight times richer in phospholipids containing polyunsaturated acyl (PUFA) chains and membrane fluidity was increased as measured by fluorescence polarization of diphenylhexatriene (DPH). The alterations resulted in a higher thermosensitivity. When mouse fibroblasts were made thermotolerant no change in cholesterol and phospholipid content could be found in the particulate fraction of the cells. The relative weights and the quality of the phospholipids as well as the fatty acid composition of the phospholipids appeared to be the same for normal and thermotolerant cells. Fluidity measurements in whole cells, isolated plasma membranes, and liposomes prepared from phospholipids extracted from the cells revealed no significant differences between normal and thermotolerant fibroblasts when assayed by fluorescence polarization (DPH) and electron spin resonance (5-nitroxystearate). It is concluded that the mechanism of thermal adaptation resulting in differences in lipid composition as reported in the literature differs from the mechanism of the acquisition of thermal tolerance. The lower heat sensitivity of thermotolerant cells, as initiated by a nonlethal triggering heat dose followed by an induction period at 37 degrees C, does not involve changes in lipid composition and membrane fluidity. However, a prompt and clear (also nonlethal) change in membrane fluidity by an increase in PUFA does result in an increased thermosensitivity, probably because of an indirect effect via the lipids in causing disfunctioning of proteins in the membrane and/or the cytoskeleton.     

A 2H NMR study on alteration of the membrane organization of mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate

In order to monitor the membrane fluidity of cells without perturbation by an introduced probe, we developed a method for large-scale preparation of 2H-labeled melanoma cells for a 2H NMR study by incubating melanoma cells with [18,18,18-2H3]stearic acid/phosphatidylcholine liposomes for 2 h at 37 degrees C. It turned out that this treatment did not significantly change the cell viability, lipid metabolism or membrane fluidity. The 2H from C-18 of stearic acid is dominantly located at the original position of the fatty acid in the 2H-labeled membrane vesicles, as studied by a tracer experiment with [1-14C]stearic acid. We found that three to four 2H-labeled species were present at 19 degrees C in 2H NMR spectra of the 2H-labeled membrane vesicles prepared from B16 melanoma cells. The extent of peak-splittings due to 2H-quadrupole interaction decreased as the temperature rose, and a definite point of phase transition was not observed. At elevated temperature, 2H-labeled lipids undergo fast exchange between the bilayer and an isotropic phase such as oil phase of triolein or inverted micelles in lipid polymorphs. We further analyzed the change of membrane organization in mouse B16 melanoma cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), which strongly inhibited melanogenesis. The magnitude of the quadrupole splitting at 19 degrees C in membranes from TPA-treated cells was significantly less (40%) than in the untreated control. This is mainly explained by decreased molecular ordering (fluidity) due to the increased amount of unsaturated fatty acids in the membranes of TPA-treated cells.     

Candida dubliniensis and Candida albicans display surface variations consistent with observed intergeneric coaggregation

Adherence of yeasts to other microorganisms and epithelial cell surfaces is important in their colonization. Comparative studies based on the coaggregation of Candida dubliniensis versus Candida albicans with Fusobacterium nucleatum and other oral bacteria suggested differences in the surfaces of these yeasts. Transmission electron microscopy was used to test the hypothesis that there are morphologic variations in the cell surface of these two species. C. dubliniensis type strain CD36 and C. albicans ATCC 18804 were grown on Sabouraud's dextrose agar at various growth temperatures. In some experiments suspensions of yeast cells were treated with dithiothreitol. Fixation for transmission electron microscopy was accomplished using dimethylsulfoxide and alcian blue added to 3% paraformaldehyde and 1% glutaraldahyde in cacodylate buffer. The cell wall of both species was predominantly electron lucent and was visibly differentiated into several layers. A thin electron dense outer layer was seen with clearly visible fibrillar structures, closely associated to the cytoplasmic membrane. The length of the fibrils of the C. albicans cells grown at 37 degrees C was approximately two times greater than those of the cells grown at 25 degrees C. The fibrils of the 37 degrees C-grown cells were thin, distinct and tightly packed whereas those of the 25 degrees C-grown cells appeared blunt, loosely spaced and aggregated. C. dubliniensis demonstrated short, blunt fibrils appearing similar to those of the 25 degrees C-grown C. albicans cells. C. dubliniensis showed no difference in the density, length and arrangement of fibrils between the 25 degrees C and 37 degrees C growth temperatures. The shortest and most aggregated fibrils seen were of the 45 degrees C-grown C. albicans cells. Dithiothreitoltreated 37 degrees C-grown C. albicans cells revealed a distorted and partially destroyed fibrillar layer. In this investigation C. dubliniensis, unlike C. albicans, displayed an outer fibrillar layer that did not vary with variations in growth temperature. In addition, the fibrils on the C. dubliniensis cells were similar to those of the 25 degrees C-grown C. albicans in that they were considerably shorter and less dense than those of the 37 degrees C-grown C. albicans cells. It can be postulated, that C. dubliniensis exhibits constant cell surface characteristics consistent with hydrophobicity and that this property may give this species an ecological advantage. Therefore, C. dubliniensis may compete well in oral environments via enhanced attachment to oral microbes and other surfaces, perhaps even more efficiently than C. albicans.     

Temperature- and surfactant-induced membrane modifications that alter Listeria monocytogenes nisin sensitivity by different mechanisms

Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation. Alterations in membrane composition might influence any of these steps. We hypothesized that cold temperatures (10 degrees C) and surfactant (0.1% Tween 20) in the growth medium would influence Listeria monocytogenes membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin. Compared to the membranes of cells grown at 30 degrees C, those of L. monocytogenes grown at 10 degrees C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity. When 0.1% Tween 20 was included in the medium and the cells were cultured at 30 degrees C, there were complex changes in lipid composition. They did not influence membrane fluidity but nonetheless increased nisin sensitivity. Further investigation found that these cells had an increased ability to bind radioactively labeled nisin. This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.     

The mechanism of membrane response to chilling. Effect of temperature on phospholipid deacylation and reacylation reactions in the cell surface membrane

The ciliary membrane of Tetrahymena pyriformis is physically and metabolically remote from the main centers of lipid metabolism. Nevertheless, it possesses an independent capacity to modify its phospholipid molecular species composition rapidly under stress. The role of ciliary phospholipid deacylating and reacylating enzymes in this phenomenon has been evaluated. Isolated cilia showed substantial phospholipase A (combined A1 and A2), acyl-CoA synthetase and acyltransferase activities. Activities of all the three enzymes of cilia from 39 degrees C-grown cells were greatly reduced when the cilia were incubated at 15 degrees C. In contrast, the phospholipase A and acyltransferase activities in cilia from 15 degrees C-grown cells were surprisingly high at 15 degrees C and twice as high at 37 degrees C as were the equivalent activities in preparations from 39 degrees C-grown cells. While the in vivo substrate specificity of phospholipase A could not be meaningfully assessed, the acyltransferases exhibited a temperature-dependent substrate specificity in vivo. Growth temperature also affected the positional distribution of fatty acids incorporated into ciliary phospholipids in vivo. The ability of acyltransferases to utilize added [14C] acyl-CoA could be markedly stimulated, and their lipid class specificity could be significantly altered in vitro by supplementing the incubation mixture with exogenous lysophospholipid acceptors. These findings suggest that the rate-limiting factor in acyl chain turnover is not the activity of acyltransferases per se but rather the availability of suitable substrates and acceptors. Therefore, we postulate that temperature alters the rate and specificity of ciliary membrane phospholipid retailoring primarily by controlling the in situ phospholipase A activity.     

Involvement of Membrane Lipids in Cold Shock-induced Autolysis of Bacillus subtilis Cells

Exponentially growing Bacillus subtilis cells autolysed when exposed to cold shock treatment in minimal medium followed by incubation at 37°C. From characteristics of the lysis, it was suggested that the cold-shock-induced cell lysis resulted from the perturbation of membrane organization that is initiated by rapid changes in temperature, lipid phase transitions. For maximum lysis induction to occur, in addition to rapid cooling to 5°C or lower, retention at temperatures lower than 10°C for at least 20 min is required. The cell sensitivity to the autolysis induction by cold shock was different between cells grown at 25°C and cells grown at 37°C. Analyses of the fatty acid composition and the phase transition temperature of membrane lipids suggested that the membrane fluidity may affect the autolysis induction. Experiments to discover the effects of cerulenin treatment and lipid addition on autolysis induction and the autolysin activity level support the hypothesis that membrane lipids are involved in cold-shock-induced cell autolysis.     

The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Temperature-induced modifications of glycosphingolipids in plasma membranes of Neurospora crassa

Plasma membranes isolated from a cell-wall-less mutant of Neurospora crassa grown at 37 and 15 degrees C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37 degrees C membranes, while 15 degrees C plasma membranes exhibited a ratio of nearly 2.0. Membranes formed under both growth conditions were found to contain glycosphingolipids. Cultures grown at the low temperature, however, were found to contain 6-fold higher levels of glycosphingolipids and a corresponding 2-fold reduction of phospholipid levels. The high glycosphingolipid content at 15 degrees C compensates for the reduced levels of phospholipids in such a way that sterol/polar lipid ratios are almost the same in plasma membranes under the two growth conditions. Temperature-dependent changes in plasma-membrane phospholipid and glycosphingolipid species were also observed. Phosphatidylethanolamine levels were sharply reduced at 15 degrees C, in addition to a moderate increase in levels of unsaturated phospholipid fatty acids. Glycosphingolipids contained high levels of long-chain hydroxy fatty acids, which constituted 75% of the total fraction at 37 degrees C, but only 50% at 15 degrees C. Compositional changes were also observed in the long-chain base component of glycosphingolipids with respect to growth temperature. Fluorescence polarization studies indicate that the observed lipid modifications in 15 degrees C plasma membranes act to modulate bulk fluidity of the plasma-membrane lipids with respect to growth temperature. These studies suggest that coordinate modulation of glycosphingolipid, phospholipid and sterol content may be involved in regulation of plasma-membrane fluid properties during temperature acclimation.     

Thermal regulation of the fatty acid composition of lipopolysaccharides and phospholipids of Proteus mirabilis.

The fatty acid composition of the lipid A moiety of the lipopolysaccharide and phospholipid fractions of Proteus mirabilis changed significantly on varying the growth temperature. A decrease in the growth temperature from 43 degrees C to 15 degrees C resulted in a decrease in the palmitic acid content of the lipopolysaccharide from 19.4% of total fatty acids at 43 degrees C to 1.4% at 15 degrees C, and by the appearance of an unsaturated fatty acid residue, hexadecenoic acid. Changes in the 3-hydroxy-myristic acid content of the lipid A were minimal. The decrease in the growth temperature also resulted in a decrease in the saturated fatty acid content of the phospholipid fraction, which was accompanied by an increase in their fluidity, as measured by the freedom of motion of spin-labeled fatty acids incorporated into dispersions made of the phospholipids. Nevertheless, the fluidity obtained with membrane phospholipids extracted from the cells grown at various temperatures were essentially the same when fluidity was determined at the growth temperature, supporting the hypothesis that variations in the fatty acid composition of membrane phospholipids serve to produce membranes having a constant fluidity at different temperatures of growth.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.