Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene

The lipid microviscosity of microsomal membranes from senescing cut carnation (Dianthus caryophyllus L. cv. White Sim) flowers rises with advancing senescence. The increase in membrane microviscosity is initiated within 3 to 4 days of cutting the flowers and coincides temporally with petal-inrolling denoting the climacteric-like rise in ethylene production. Treatment of young cut flowers with aminoethoxyvinylglycine prevented the appearance of petal-inrolling and delayed the rise in membrane microviscosity until day 9 after cutting. When freshly cut flowers or aminoethoxyvinylglycine-treated flowers were exposed to exogenous ethylene (1 microliter per liter), the microviscosity of microsomal membranes rose sharply within 24 hours, and inrolling of petals was clearly evident. Thus, treatment with ethylene accelerates membrane rigidification. Silver thiosulphate, a potent anti-ethylene agent, delayed the rise in microsomal membrane microviscosity even when the flowers were exposed to exogenous ethylene. Membrane rigidification in both naturally senescing and ethylene-treated flowers was accompanied by an increased sterol:phospholipid ratio reflecting the selective loss of membrane phospholipid that accompanies senescence. The results collectively indicate that the climacteric-like surge in ethylene production during senescence of carnation flowers facilitates physical changes in membrane lipids that presumably lead to loss of membrane function.     

Senescence and the Fluidity of Rose Petal Membranes : RELATIONSHIP TO PHOSPHOLIPID METABOLISM

In previous work, senescence of rose petal cells has been shown to be accompanied by a gradual decrease of membrane fluidity, as measured by a fluorescence polarization technique. Concomitantly, an increase in the free sterol-to-phospholipid ratio was found. Both observations were verified in this study. Further, experiments carried out on whole tissue and isolated protoplasts during senescence revealed that there was no quantitative change in the level of free sterols. The content of phospholipids decreased without any significant change in their composition. Results from experiments measuring the incorporation of [³²P]orthophosphate indicated a reduced capacity for phospholipid synthesis in senescent cells. Both young and old tissue showed phospholipase A and D activity, the former increasing with age.     

Membrane deterioration in senescing carnation flowers : coordinated effects of phospholipid degradation and the action of membranous lipoxygenase

The lipid fluidity of microsomal membranes from the petals of cut carnation flowers decreases as the flowers senesce. A comparable change in fluidity was induced by in vitro aging of microsomal membranes from young flowers under conditions in which membranous lipoxygenase-like activity was active. There was no change in fluidity when the membranes were aged in the presence of inhibitors of lipoxygenase or were heat-denatured prior to aging. Membranes from naturally senesced flowers and membranes that had been aged in vitro both sustained an increase in saturated:unsaturated fatty acid ratio that accounted for the decrease in lipid fluidity, and in both instances there was evidence for depletion of the unsaturated fatty acids, linoleic acid, and linolenic acid, which are substrates for lipoxygenase. Loss of lipid phosphate reflecting breakdown of membrane phospholipids preceded the depletion of unsaturated fatty acids attributable to the lipoxygenase-like activity. The data have been interpreted as indicating that fatty acid substrates for membrane-associated lipoxygenase-like activity are made available by the initiation of phospholipid degradation, and that the utilization of these substrates results in a selective depletion of unsaturated fatty acids from the membrane and an ensuing decrease in bulk lipid fluidity.     

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.     

Correlative changes in sucrose uptake, ATPase activity and membrane fluidity in carnation petals during senescence

Apparent sucrose uptake. ATPase activity and membrane fluidity changes were studied during the development and senescence of carnation flowers ( Dianthus caryophyllus L., cv. Cerise Royallette). Typical changes associated with senescence of a cut flower, such as respiration, ethylene production and fresh weight, were measured. Concomitant with a rise in respiration and ethylene production and a decline in fresh weight, a sharp decrease in apparent sucrose uptake was observed. Sucrose uptake was pH dependent (pH optimum, 5.5) and influenced by membrane integrity. Apparently, the activity of ATPase is related to sucrose uptake, because similar changes occurred during flower development. In addition, the activity of ATPase was well correlated with membrane fluidity.
It is suggested that sucrose uptake is controlled by ATPase activity, which in turn is modulated by membrane lipid fluidity. The decline in membrane fluidity associated with senescence leads to a corresponding reduction in ATPase activity and sucrose uptake. Further evidence supporting this view comes from experiments in which senescence was enhanced by 1-aminocyclopropane-l-carboxylic acid. It shortened the time to decline in fresh weight, rise in respiration and ethylene production. In parallel, reduction in membrane fluidity, ATPase activity and sucrose uptake were observed.     

Association between Membrane Phase Properties and Dehydration Injury in Soybean Axes

Axes of soybean seeds are tolerant to dehydration at 6 hours of imbibition, but susceptible to dehydration injury if dried at 36 hours of imbibition. Smooth microsomal membranes were isolated from axes imbibed for 6 hours (dehydration tolerant state) and 36 hours (dehydration susceptible state) before and after dehydration treatment. The phase properties and the lipid composition of the membrane fraction were investigated. Wide angle x-ray diffraction patterns of microsomal membranes from axes imbibed for 6 or 36 hours indicated a liquid-crystalline to gel phase transition at approximately 7°C. Membranes from axes dehydrated at 6 or 36 hours of imbibition and rehydrated for 2 hours exhibited a phase transition at 7°C and 47°C, respectively. Changes in fatty acid saturation did not account for the changes in phase properties. However, the increased phase transition temperature of the membranes from dehydration injured axes was associated with an increase in free fatty acid:phospholipid molar ratio and a decrease in phospholipid:sterol ratio. These results suggests that dehydration prompted a deesterification of the linkage between glycerol and fatty acid side chains of the phospholipid molecules in the membrane. The resultant increase in free fatty acid content in the membrane is thought to alter the fluidity and phase properties of the membrane and contribute to dehydration injury.     

Lectin mediated agglutination of Candida kefyr cells and their spheroplasts grown in sterol enriched growth medium and its correlation with lipid composition

Supplementation of the growth medium with erosterol, cholesterol and lanosterol enriched the Candida kefyr cells, presumably cell membranes with sterols. Sterol enriched C. kefyr cells showed a decrease in percentage of PHA and Con-A mediated agglutination. Sterol supplementation also increased the sterol: phospholipid ratio and in such cells unsaturated fatty acids predominated over saturated ones. The overall effect of these changes resulted in rigidifying the cell membranes as indicated by shift of break in Arrhenius plots of Mg2+ ATPase. This showed that lectin mediated agglutination of yeast cells may be affected by its membrane fluidity.     

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.     

Simulation of the effects of leaf senescence on membranes by treatment with paraquat

Chloroplast and microsomal membranes from the primary leaf of bean acquired increasing proportions of gel phase lipid as the tissue senesced. The lipid-phase transition temperature for microsomes rose from about 25 to 43 C and that for chloroplasts rose from below −30 C to about 52 C within 5 weeks of planting. This was accompanied by large increases (2- to 4-fold) in the sterol to phospholipid ratio of the membranes, which reflected breakdown of phospholipid. Changes in fatty acid saturation were of insufficient magnitude to account for the rise in transition temperature. All of these senescence-related changes in chloroplast and microsomal membranes were also induced by treating young, 2-week-old-plants with 10 milligrams per liter paraquat. Within 48 hours of treatment, the transition temperature rose from 25 to 57 C for microsomes and from below −30 to 24 C for chloroplasts. The membranes sustained only small changes in fatty acid saturation, comparable to those incurred during natural senescence, and there was a selective loss of phospholipid, resulting in augmented sterol to phospholipid ratios. Malondialdehyde, a product of lipid peroxidation, rose by 2- to 3-fold in both senescing and paraquat-treated leaves. Paraquat is known to form cation redicals that react with O₂ to produce O₂⁻ and has been implicated as an agent of lipid peroxidation. Accordingly, these observations suggest that membrane deterioration during natural senescence may be due in part to free radical damage.     

The effects of cotyledon senescence on the composition and physical properties of membrane lipid

The phospholipid content of rough and smooth microsomal fractions from cotyledons of germinating bean declines as the tissue becomes senescent. Both types of membrane contain comparable proportions of three major phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which collectively comprise about 90% of the total. This proportionality does not change appreciably during senescence. Only small quantities of lysophosphatides were noted at all stages of senescence. The unsaturated:saturated fatty acid ratio for total extracted lipid declined only slightly in both membrane systems, but pronounced differences in this ratio were observed among the major phospholipids of the membranes. The most striking alteration in lipid composition with advancing senescence was an increase in the sterol:phospholipid ratio; this rose by about 50% for rough microsomes and 400% for smooth microsomes. For both types of membrane the patterns of change in this ratio correlated with previously reported changes in bulk lipid transition temperature, suggesting that the increase in sterol level may contribute to changes in phase behaviour of the membranes during senescence. Arrhenius plots of rotational correlation times for the electron spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) partitioned into the membrane lipid showed an increase in viscosity with advancing senescence and a corresponding increase in activation energy for both types of membrane. These changes in activation energy and viscosity correlated closely with the increase in sterol:phospholipid ratio. However, no phase transitions were detectable between temperatures of 2 and 55 degrees C despite the fact that transitions from a lipid-crystalline to gel state are detectable within this temperature range by wide angle X-ray diffraction.     

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°C display large differences in lipid compositions. A free sterol-to-phospholipid ratio of 0.8 was found in 37°C membranes, while 15°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°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°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°C, but only 50% at 15°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°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.     

Thyrotropin regulation of membrane lipid fluidity in the FRTL-5 thyroid cell line. Its relationship to cell growth and functional activity

The mitogenic effect of thyrotropin on functional rat thyroid cells of the line FRTL-5 is correlated with membrane lipid fluidity as evaluated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Continued exposure of FRTL-5 cells to a medium lacking thyrotropin causes cessation of cell proliferation and a decrease in membrane lipid fluidity which reaches its minimum in approximately 8 days. The change in lipid fluidity is due to an absolute increase (greater than 2-fold) of membrane cholesterol, with an increased cholesterol/phospholipid ratio and an increased ratio of saturated to unsaturated fatty acids of the membrane phospholipids, contributed primarily by a nearly 4-fold increase in the ratio of saturated to unsaturated C16 fatty acids. It is also associated with a variation of the relative proportions of the major membrane phospholipids; thus, phosphatidylinositol and phosphatidylethanolamine decrease while phosphatidylcholine increases. Both membrane fluidity and lipid composition can be restored by thyrotropin to their original levels, i.e. levels measured under continuous exposure to the hormone. Complete reversal requires at least 48 h, i.e. approximately the same time required for resumption of growth when FRTL-5 cells, starved in thyrotropin, are re-exposed to the hormone. Changes in lipid composition and fluidity can be prevented or can be reversed if FRTL-5 cells are exposed to dibutyryl cAMP while being deprived of thyrotropin. Dibutyryl cAMP has only a modest direct effect on growth; however, this pretreatment eliminates the 48-h lag phase with respect to thyrotropin stimulation. It is proposed that the effects of thyrotropin on growth of FRTL-5 cells requires a modification of the molecular structure and the physical state of cell membranes, which can be mediated by cAMP, although cAMP is not sufficient by itself to promote growth.     

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.     

Altered membrane structure in transfected mouse L-cell fibroblasts expressing rat liver fatty acid-binding protein

Mouse L cell fibroblasts were transfected with cloned cDNA encoding rat liver fatty acid binding protein (L-FABP) also known as sterol carrier protein. Stable transfectant cell lines were selected and expression of L-FABP determined using Western blot analysis. The nontransfected controls and low expression cells did not differ significantly in any of the properties examined. All cell lines showed similar doubling times but cells expressing high levels of L-FABP attained 2-fold higher cell saturation density and differed significantly in their lipid metabolism as indicated by 1) higher cholesterol ester and phospholipid content, and 2) decreased sterol/phospholipid ratio. The observed changes in the lipid composition predicted a lower degree of membrane-lipid order (higher fluidity) in the plasma membranes of cells expressing high levels of L-FABP. Therefore, fluorescent molecule, 1,6-diphenyl-1,3,5-hexatriene, and multifrequency (1-250 MHz) phase and modulation fluorometry were used to probe the effect of L-FABP expression on membrane structure. Steady-state polarization and limiting anisotropy of diphenylhexatriene were significantly lower in the isolated plasma membrane vesicles from the high expression clones. The observed changes in L-cells as a result of de novo expression of L-FABP are consistent with the ability of this protein to bind sterols and fatty acids, stimulate sterol esterification, and stimulate phospholipid biosynthesis. This evidence is supportive of a physiologic role for L-FABP in modulating cellular lipid metabolism and membrane structure.     

Relationship between ethanol tolerance, lipid composition and plasma membrane fluidity in Saccharomyces cerevisiae and Kloeckera apiculata

Abstract The lipid composition of a strain of each of two yeasts, Saccharomyces csrevisiae and Kloeckera apiculata , with different ethanol tolerances, was determined for cells grown with or without added ethanol. An increase in the proportion of ergosterol, unsaturated fatty acid levels and the maintenance of phospholipid biosynthesis seemed to be responsible for ethanol tolerance. The association of ethanol tolerance of yeast cells with plasma membrane fluidity, measured by fluorescence anisotropy, is discussed. We propose that an increase in plasma membrane fluidity may be correlated with a decrease in the sterol: phospholipid and sterol: protein ratios and an increase in unsaturation index.     

Lipid composition and molecular organization in plasma membrane-enriched fractions from senescing cotyledons

Plasma membrane fractions isolated from cotyledons of Phaseolus vulgaris L. cv. Kinghorn at various stages of senescence showed no significant change in fatty acid saturation with advancing senescence. However, the steroliphospholipid ratio increased by about 400% as senescence intensified. The lipid phase transition temperature of the membranes, which was measured by wide-angle x-ray diffraction, also rose from a point well below the growing temperature for young tissue to about 50°C for membrane from extensively senescent 9-day-old tissue. This means that by day 4 of germination there was a mixture of liquid-crystalline and gel phase phospholipid in the membrane matrices. Crystallinity attributable to sterol-sterol interaction was also apparent in the diffraction patterns for senescent membranes. The co-existence of gel and liquid-crystalline phase phospholipid in the aging membranes as well as the crystalline sterol aggregates presumably render the storage cells of cotyledons leaky and may thus facilitate the translocation of hydrolyzed food reserves into the vascular network.     

Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma flocculosa

cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.     

Properties of spin labelled membranes of Fusarium oxysporum f. sp. lycopersici.

Growth temperature-induced compositional changes in membranes of Fusarium oxysporum provided a test system for study of the relationship between physical properties and composition. Growth at 15 degrees C was characterized by a decrease in phospholipid content relative to sterol content, a shift in phospholipid composition from phosphatidylcholine to phosphatidylethanolamine and a marked enhancement in the amount of polyunsaturated fatty acids in the phospholipid and triglyceride classes. Uptake of a spin labelled analog of stearic acid during growth and subsequent solution of the probe in the membranes allowed estimation of viscosity and molecular order of the membranes of live cells and of isolated membrane preparations. Less than 1/20 of the intracellular label was accessible to sodium ascorbate while none was released by sodium dodecyl sulfate. All of the label in live cells was reduced by in vivo respiratory activity above 20 degrees C but this process could be reversed or avoided by added ferricyanide. A cholestane spin probe was also incorporated into the membranes. The probes were not reduced as readily in isolated membranes and hence fluidity of the membranes could be assessed over a wide temperature range. At low temperatures (-10 degrees C) a nonlethal, liquid-solid phase transition was indicated in isolated membrane lipids while at higher (lethal) temperatures (40-45 degrees C), discontinuities appeared in Arrhenius plots of rotational correlation time. Activation energies for isotropic rotation of the stearate probes in the membranes changed markedly in this temperature range and this effect correlated closely with loss of viability of conidial cells. Correlation times for stearate probes showed little variation with growth temperature nor were any breaks in Arrhenius plots of this parameter detected in the range 0-35 degrees C in whole cells or isolated membranes. The data indicated control of membrane physical properties within close tolerances throughout the physiological temperature range regardless of growth temperature. It was concluded that this homeostatic phenomenon was due to the counteractive effects of sterol/phospholipid ratio, phospholipid composition and fatty acid polyunsaturation since the condensing and fluidizing components of the isolated total membranes vary in a reciprocal manner.     

Corresponding changes in kynurenine hydroxylase activity, membrane fluidity, and sterol composition in Saccharomyces cerevisiae mitochondria.

The effect of sterol composition on the properties of the mitochondrial membrane of Saccharomyces cerevisiae was investigated. The physical state of mitochondrial membranes from wild-type strains and sterol mutants was compared, using a fluorescence polarization technique with 1,6-diphenyl-1,3-5-hexatriene. Changes in the rate of depolarization of the probe molecule as a function of temperature suggest the occurrence of a phase transition in the mitochondrial membranes isolated from the sterol mutants but not in the membranes isolated from the wild types. Arrhenius kinetics of the mitochondrial membrane-bound enzyme L-kynurenine-3-hydroxylase exhibited changes in activation energy at temperatures similar to those observed in the fluorescence polarization study. The ratio of mitochondrial sterol to phospholipid and the phospholipid fatty acid composition of the organisms were characterized.     

Properties of spin labelled membranes of Fusarium Oxysporum f. sp. Lycopersici

Growth temperature-induced compositional changes in membranes of Fusarium oxysporum provided a test system for study of the relationship between physical properties and composition. Growth at 15 °C was characterized by a decrease in phospholipid content relative to sterol content, a shift on phospholipid composition from phosphatidylcholine to phosphatidylethanolamine and a marked enhancement in the amount of polyunsaturated fatty acids in the phospholipid and triglyceride classes.Uptake of a spin labelled analog of stearic acid during growth and subsequent solution of the probe in the membranes allowed estimation of viscosity and molecular order of the membranes of live cells and of isolated membrane preparations. Less than $\text{1}\text{20}$ of the intracellular label was accessible to sodium ascorbate while none was released by sodium dodecyl sulfate. All of the label in live cells was reduced by in vivo respiratory activity above 20 °C but this process could be reversed or avoided by added ferricyanide. A cholestane spin probe was also incorporated into the membranes. The probes were not reduced as readily in isolated membranes and hence fluidity of the membranes could be assessed over a wide temperature range. At low temperatures (?10 °C) a nonlethal, liquid-solid phase transition was indicated in isolated membrane lipids while at higher (lethal) temperatures (40–45 °C), discontinuities appeared in Arrhenius plots of rotational correlation time. Activation energies for isotropic rotation of the stearate probes in the membranes changed markedly in this temperature range and this effect correlated closely with loss of viability of conidial cells. Correlation times for stearate probes showed little variation with growth temperature nor were any breaks in Arrhenius plots of this parameter detected in the range 0–35 °C in whole cells or isolated membranes. The data indicated control of membrane physical properties within close tolerances throughout the physiological temperature range regardless of growth temperature. It was concluded that this homeostatic phenomenon was due to the counteractive effects of sterol/phospholipid ratio, phospholipid composition and fatty acid polyunsaturation since the condensing and fluidizing components of the isolated total membranes vary in a reciprocal manner.