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Disruption of the integrity of tumor cellular membranes has been proposed as an initiating event in hyperthermic cell death. Thermosensitivity measured by the shift in the harmonic mean of tumor regrowth delay of CA755 mammary adenocarcinomas grown in the hind legs of male BDF 1 mice increased 22% when the hosts were fed a diet enriched in polyunsaturated fatty acids. Although the diet elicited the anticipated increase in tumor membrane phospholipid polyunsaturated fatty acids, the proportion of total unsaturated fatty acids decreased and the proportion of membrane-rigidifying saturated fatty acids increased. Concomitantly, the concentrations of cholesterol and phospholipid phosphorus increased and the ratio of phosphatidylethanolamine to phosphatidylcholine decreased, presumably to counter the effect of the change in the fatty acid pattern. In host liver membranes, the diet-mediated increase in proportion of polyunsaturated fatty acids was not accompanied by an increase in the proportion of rigidifying saturated fatty acids. Instead, the homeoviscous adaptation consisted of decreases in monounsaturated fatty acids and cholesterol concentration and an increase in the phosphatidylethanolamine-phosphatidylcholine ratio. Addition of a natural inhibitor of cholesterol biosynthesis to the polyunsaturated fatty acid enriched-diet reversed the diet-mediated increase in the phosphatidylethanolamine-phosphatidylcholine ratio of host liver membranes. Tumor membrane lipids from hosts fed the combined dietary factors were characterized by the forementioned rigidifying increase in saturated fatty acids and compensatory decrease in the phosphatidylethanolamine-phosphatidylcholine ratio. The inhibitor reversed the compensatory increases in cholesterol and phopholipid phosphorus concentrations. As a consequence the thermosensitivity of tumors bearing this perturbed membrane was increased.

The metabolic adjustments responsible for the “homeoviscous adaptation” of membrane lipid composition in fish are examined with special reference to the rainbow trout, Salmo gairdneri. The percentage of fatty acid lipogenesis attributable to unsaturates was elevated after an acute drop in temperature but declined with continued cold exposure (i.e., cold acclimation). In contrast, selected desaturation reactions [particularly those involved in the production of polyunsaturated fatty acids (PUFA) of the n-3 and/or n-6 families] proceeded more rapidly in cold-than in warm-acclimated trout. Different time courses for the change in monoene and PUFA levels of hepatic microsomal membranes during thermal acclimation suggest that the various desaturase enzymes contribute to the acclimatory response at different times. Certain fatty acids, particularly the delta 5-desaturation products of the n-3 (20:5 delta 5,8,11,14,17) and n-6 (20:4 delta 5,8,11,14) series, were preferentially incorporated into phospholipids at cold temperatures and by cold-acclimated trout, due in part to the direct effect of temperature on the substrate preferences of the phospho- and acyltransferase enzymes of de novo phospholipid biosynthesis; however, chain length rather than degree of unsaturation per se may determine the temperature-dependent pattern of fatty acid incorporation. Both acute and chronic cold exposure elevated the incorporation of PUFA into phosphatidylserine (PS), suggesting that the conversion of PS to phosphatidylethanolamine (PE) may be activated at cold temperatures. The rate of homeoviscous adaptation appears to be limited by the rate of membrane lipid turnover, which although generally positively correlated with acclimation temperature, did vary depending on the phospholipid moiety and tissue considered. Finally the direct acylation of lysophospholipids formed during the process of membrane turnover may contribute to both rapid and acclimatory adjustments in membrane lipid composition.

Further studies on the molecular mechanisms of temperature acclimation have been carried out using the ciliate Tetrahymena pyriformis. The most prominent change in lipid metabolism during acclimation to high temperature--depression of fatty acid desaturase activity--could be simulated by supplementing the growth medium of isothermally-grown cells with polyunsaturated fatty acids. Such cells resisted the membrane-fluidizing effect of the incorporated exogenous acids by increased use of de novo synthesized saturated acids in their phospholipids. The data support the conclusions arising from earlier experiments with temperature-shifted cells (Martin, C.E., Hiramitsu, K., Kitajima, Y., Nozawa, Y., Skriver, L., and Thompson, G.A., Jr. (1976), Biochemistry 15), showing that, when membrane fluidity increased to a superoptimal level, the activity of membrane-associated fatty acid desaturases was decreased. Since the reaction is controlled by membrane fluidity, rather than temperature per se, we postulate that it is the general mechnaism employed by cells adjusting to any fluidity-modifying factor, such as cations, drugs, etc.

The thermotropic behaviour of membrane phospholipids was estimated in intact cells of Bacillus subtilis. Membrane fluidity (microviscosity) of intact cells depended markedly on the ambient temperature - increase in cultivation temperature led to an increase in membrane fluidity. Estimated as anisotropy of 1,6-diphenyl-1,3,5-hexatriene fluorescence, a 30% difference was observed when cells cultivated at 20 and 40 degrees C were compared. This lack of rigorous homeostatic control of bulk-phase lipid fluidity prompted the reevaluation of the physiological significance of the "homeoviscous adaptation" in B. subtilis.

This is a study of the molecular mechanisms employed by Tetrahymena pyriformis to change the lipid composition and thereby the fluidity of its various membranes during temperature acclimation. By quantitatively measuring the intramembrane particle aggregation using freeze-fracture electron microscopy, membrane physical properties in 39.5 degrees C grown cells shifted to 15 degrees C were found to be correlated with the degree of phospholipid fatty acid desaturation. Alteration of the phospholipid polar head group distribution from that of 39.5 degrees C-grown cells to the significantly different pattern of 15 degrees C grown cells appeared not to be of critical importance in the acclimation process. Changes in fatty acid desaturation during acclimation from high to low temperatures and vice versa were analyzed using normal cells and cells fed large amounts of polyunsaturated fatty acids. Fatty acid desaturase activity corresponded to the degree of membrane fluidity but not to the cell temperature. All evidence was compatible with the hypothesis that membrane fluidity is self-regulating, with the action of fatty acid desaturases being modulated by the physical state of their membrane environment.

Publisher Summary This chapter discusses a study to analyze homeoviscous adaptation of cellular membranes during thermal acclimation. This study examined two membranous fractions from the same tissue of differently acclimated fish to determine whether different membrane-types isolated from the same cell respond during thermal acclimation in the same way. An important aspect of all such studies is the purity of the membrane fractions. Liver microsomes were successfully prepared from green sunfish (Lepomis cyanellus) by conventional differential centrifugation techniques, although for liver mitochondria, it was necessary to develop a discontinuous Ficoll gradient centrifugation technique to remove contaminating microsomes. The acclimation of green sunfish to 5°C, 15°C, 25°C, or 34°C resulted in large and significant partial compensations of membrane fluidity as revealed by steady state fluorescence polarization techniques using 1, 6-diphenylhexatriene as probe. By contrast, liver microsomes of 5°C, 15°C, and 25°C acclimated fish were unaffected, although microsomes of 34°C acclimated fish were partially compensated with respect to 25°C acclimated fish. The analysis of phosphoglyceride fatty acid composition tended to confirm the different properties of the two membrane fractions.

The fluidity of synaptosomal membrane preparations isolated from goldfish acclimated to 5, 15 and 25 degrees C and from rat has been estimated using the fluorescence polarisation technique with 1,6-diphenyl-1,3,5-hexatriene as probe. Membranes of cold-acclimated goldfish were more fluid than those of warm-acclimated goldfish when measured at an intermediate temperature, indicating a temperature-dependent regulation of this parameter. Similarly, membranes of warm-acclimated goldfish were more fluid than those prepared from rat brain. Liposomes prepared from the purified phospholipids of goldfish and rat synaptosomal preparations showed differences similar to those of the native membranes. Increased membrane fluidity of cold-acclimated goldfish was correlated with a decrease in the proportion of saturated fatty acids of the major phospholipid classes and an increased unsaturation index in choline phosphoglycerides. Rat membranes showed a substantial reduction in unsaturation index and an increase in the proportion of saturated fatty acids compared to the membranes of 25 degrees C-acclimated goldfish. The cholesterol content of synaptosomal membranes of goldfish was unaffected by acclimation treatment. The role of homeoviscous adaptation in the compensation of the rates of membrane processes during thermal acclimation, and upon the resistance adaptation of poikilotherms to extreme temperatures is discussed.

Temperature adaptation of biological membranes was examined by comparing the fragmented sarcoplasmic reticulum preparation of goldfish acclimated to different temperatures. Membrane fluidity was estimated using the fluorescence polarization technique. There was considerable variation between preparations, but no consistent differences in fluidity were observed between 5- and 25 degrees C-acclimated goldfish, fish species adapted over an evolutionary period to arctic or desert temperatures, and rat. The fatty acid composition of the sarcoplamic reticulum preparations of differently acclimated goldfish showed differences in the proportion of mono- and polyunsaturated fatty acids while the proportion of saturated fatty acids remained relatively constant. However, the fatty acid composition of sarcoplasmic reticulum phosphoglycerides became more unsaturated in the order rat, desert pupfish, arctic sculpin, which correlates with their respective environmental or body temperature. It is concluded that differences in membrane components other than fatty acids are important in determining membrane dynamic structure. The inability to demonstrate homeoviscous adaptation in sarcoplasmic reticulum is supported by other evidence suggesting that functions of the sarcoplasmic reticulum that are measured in vitro are not affected by such modifications of their phosphoglyceride fatty acid composition as occur during thermal acclimation.

The ability of phospholipids to modify the activity of membrane-bound enzymes and change the passive permeability of artificial and naturally occurring membranes is discussed in relation to temperature adaptation. Special attention is paid to phospholipid effects on ATP generating and hydrolysing systems. A critical evaluation is made of the concept of homeoviscous adaptation.

It has been appreciated for many years that certain organisms, notably poikilotherms including bacteria, alter the fatty acyl composition of their lipids in response to changes in environmental temperature. Publication of the “fluid mosaic model” of membrane structure (Singer and Nicolson, 1972) signaled a decade in which our comprehension of the physiological significance of these fatty acyl changes has grown very considerably. The application of physical techniques, such as electron spin resonance (ESR) and nuclear magnetic resonance, to membranes and their lipids has in particular shown how membrane fluidity is modulated by lipid acyl composition. These findings are embodied in the widely accepted “mosaic” model of membrane structure in which intrinsic proteins are embedded in or traverse a lipid bilayer; thus, lipid acyl chains are in contact with polypeptide chains and may regulate the activity of enzyme active sites directly or indirectly. The nature of the phospholipid (or glycolipid) head group may also be important in lipid-protein interactions. But much less is known about the role of head groups in membrane lipid fluidity, and this aspect will not be dealt with in this review (see Keough and Davis, this volume).

Since life on earth began in an anaerobic environment, some of the present day anaerobes presumably represent a closer link to the earliest epochs than organisms that evolved after the atmosphere became aerobic. A study of the membranes of anaerobes may reveal some of the most primitive ways in which living organisms have coped with environmental changes. A wealth of information has accumulated over the past 15 years showing the importance of membrane lipids in assuring continued membrane function over a wide range of temperatures. Many anaerobes belonging to such diverse groups as the gram positive Clostridia, gram-negative bacteroides and cocci, and some spirochetes have been found to have substantial amounts of plasmalogens in addition to diacylphosphatides among their membrane lipid components (GoIdfine and Hagen, 1972). Although these lipids which are characterized by a 1-O-alk-l’-enyl 2-acyl glycerophosphate structure (Fig. la), have been known for many years as constituents of mammalian tissues (Debuch and Seng, 1972), little is known about their precise functions.

Spin-labeled aqueous dispersions of total phospholipid extracts from whole brains of hibernating hamsters and rats chronically consuming ethanol were compared with dispersions from control animals. Order parameter values and approximate rotational correlation times for the nitroxide spin labels indicated that ethanol consumption results in an adaptive decrease in bilayer membrane fluidity, while hibernation produces increases in fluidity. Since it has been proposed that changes in plasmenylethanolamine such as those seen with hibernation play a role in the homeoviscous adaptation of brain membranes, electron spin resonance studies using aqueous phospholipid dispersions containing equimolar mixtures of rat brain phosphatidylethanolamine and phosphatidylcholine, or synthetic dioleylphosphatidylcholine and dioleylphosphatidylethanolamine, and brain plasmenylethanolamine were performed. The molar amount of plasmenylethanolamine was varied within the ethanolamineglycerophospholipid fraction of each dispersion. Order parameter values of spin labels in liposomes containing brain phosphatidylcholine and phosphatidylethanolamine increased in parallel with increases in plasmenylethanolamine concentrations, indicating that fluidity was decreasing. Liposomes composed of synthetic dioleyl phospholipids exhibited biphasic changes in order parameter (S) values as plasmenylethanolamine replaced the diacyl form. Below 30% (mol%) plasmenylethanolamine, S values decreased, while above 30%, S values were seen to increase; indicating an initial fluidization, followed by a decrease in fluidity.

While adaptation of microbial membrane lipid composition is widely observed in response to changes in temperature, and in some cases to the presence of membrane-fluidizing substances, the physiological importance of such adaptations remains largely unexplored. To shed some light on this question question we have used our ability to extensively vary the membrane fatty acid composition of Acholeplasma laidalawii B to study a variety of membrane functions is membranes whose lipids contain a single fatty acyl species whose structure and physical properties can be widely varried. Our results suggest that control of the membrane lipid phase state is considerably more important to the proper functioning of the organism than is control of the ‘fluidity’ of liquid-crystalline lipids.

The validity of the principle of homeoviscous adaptation for Bacillus subtilis was tested by comparing fluorescence anisotropy (1,6-diphenyl-1,3,5-hexatriene) and electron-spin resonance (16-doxylstearate) measurements carried out in isolated plasma membranes and in phospholipid fractions. The physical measurements were supplemented by fatty-acid analysis. The results support our previous findings on intact cells. The thermoadaptive mechanism of B. subtilis manifested as an increase in relative proportion of branched anteiso-C15 and anteiso-C17 fatty acids, are not strong enough to compensate for the marked physical change of membrane fluidity induced by temperature decrease.

Publisher Summary This chapter reviews the structures of the lipids of prokaryotes and the distribution of these lipids in various groups of organisms. It discusses the arrangement of the prokaryotes in the eighth edition of Bergey's Manual of Determinative Bacteriology. The presence of acyl and alk-1-enyl chains containing double bonds, methyl branches, or small rings is critical for the maintenance of membrane fluidity that is important for the function of membrane enzymes and transport systems. Variation in polar head groups may also affect membrane fluidity and the association of certain lipid classes with these enzymes and transport systems. The chapter discusses the location of membrane lipids with respect to intracellular membranes. Lipids are often distributed asymmetrically in the two leaflets of a given membrane. Some organisms have evolved dual lipid-biosynthetic capacities that allow them to substitute glycosyl diglycerides or ornithine lipids for phosphatidylethanolamine in the absence of phosphate. Within limits of size and charge, polar lipids may replace one another in biological membranes. Exogenous fatty acyl chains and alk-1-enyl chains may also be substituted for the naturally occurring chains in bacterial auxotrophs. Much more work on membrane mutants is needed to understand the multifaceted roles of prokaryotic lipids. Work on prokaryotic organisms has not only provided an abundance of new insights into their membrane structure and functions, it is also leading the way to a more complete understanding of eukaryotic cell membranes.

Current research on cell membrane biochemical and biophysical changes which underlie tolerance is reviewed. What is known about the action of ethanol suggests that cellular tolerance to it is probably attained by normal mechanisms of membrane adaptation. The sites at which membrane properties could be regulated are discussed. Experimental studies of lipid and protein changes in membranes exposed to alcohol are reviewed. Evidence from lower organisms suggests that membrane adaptation is primarily a function of the phospholipids of the membrane lipid bilayer. In more complex mammalian membranes the possibility exists of adaptation in cholesterol or in membrane proteins. Membranes from ethanol-tolerant animals are resistant to the ‘fluidising’ effects of ethanol in vitro, but there does not seem to be a simple relationship between bulk membrane fluidity and tolerance to alcohol. The activities of several membrane-associated enzymes show tolerance to the effects of ethanol when it is chronically administered; this could be due to changes in the enzyme proteins or in the lipid surrounding them. It is suggested that the ability of mammals to perform these adaptive changes in neuronal membranes (which may be under strong genetic influence and may underlie tolerance and physical dependence) may have important implications for survival under environmental conditions which alter the milieu interieur.

In considering the various fatty acids that occur in living cells, we have questioned whether or not unsaturated fatty acids serve only to establish membrane fluidity. If this were their only role, then the wide variety of acyl structures that occurs in nature seems unnecessary since oleate (cis-9–18: 1) or palmitoleate (cis-9–16:1) appears capable of giving whatever fluidity would be required for a cellular membrane. Many membrane-related events are frequently regarded to have a good qualitative correlation with the apparent state of lipid fluidity in a way that provides little incentive to look beyond that correlative relationship. If some role for unsaturated acids beyond fluidity does occur, we will need a careful examination of the data to make it evident. This review examines quantitative evidence on the degree to which the contribution of an acyl chain to membrane fluidity influences cell function. The information is presented in a format that facilitates recognition of acyl chain effects that might be attributed to phenomena other than fluidity.

Thermophilic and psychrophilic yeasts were differentiated on the basis of their ability, in the case of thermophiles, and inability, in the case of psychrophiles, to (a) grow under strictly anaerobic conditions and (b) produce stable respiratory-deficient mutants either spontaneously or on treatment with acriflavine or ethidium bromide. A possible correlation between these properties and the polyunsaturated fatty acid composition of the membrane phospholipids was proposed. There were marked changes in the fatty acyl composition of membrane phospholipids isolated from the thermophilic yeast, Torulopsis bovina, when monitored at different stages of growth under aerobic and anaerobic conditions. During aerobic induction of anaerobically grown cells, the direct desaturation of palmitic acid residues on phosphatidylcholine to palmitoleic acid appeared to be a mechanism whereby cells adjusted their membrane fluidity.

Fluidity as a prominent feature of the phospholipid portion of biological membranes, as well as of model phospholipid bilayer systems, has been detected by numerous physical techniques 1 . However, correlation of this fluidity with biological functions of membranes is, as yet, documented in only a few cases. For example, fatty acid auxotrophs of E. coli grown on different fatty acids exhibit an abruptly increased rate of transport of metabolites across the cell wall at temperatures above the “melting” temperature of the fatty acid supplement 2,3 ). The physical properties of lipids extracted from E. coli also reflect the temperature at which the bacteria were grown 4 ). Fluidity of hydrocarbon chains has been related to the calcium dependent ATPase activity of sarcoplasmic vesicles 5 ). A number of other essential functions of biological membranes may very well be associated with fluidity 6,7 ), but such considerations are limited by lack of precise knowledge of the molecular basis of fluidity and of the rates of motions involved. The following discussion will review the use of spin labels 8–11 to determine the rates of several of the motions involved in the fluidity of phospholipid bilayers and, where possible, to provide a structural basis for these motions.

During winter insects face energetic stress driven by lack of food, and thermal stress due to sub-optimal and even lethal temperatures. To survive, most insects living in seasonal environments such as high latitudes, enter diapause, a deep resting stage characterized by a cessation of development, metabolic suppression and increased stress tolerance. The current study explores physiological adaptations related to diapause in three beetle species at high latitudes in Europe. From an ecological perspective, the comparison is interesting since one species (Leptinotarsa decemlineata) is an invasive pest that has recently expanded its range into northern Europe, where a retardation in range expansion is seen. By comparing its physiological toolkit to that of two closely related native beetles (Agelastica alni and Chrysolina polita) with similar overwintering ecology and collected from similar latitude, we can study if harsh winters might be constraining further expansion. Our results suggest all species suppress metabolism during diapause and build large lipid stores before diapause, which then are used sparingly. In all species diapause is associated with temporal shifts in storage and membrane lipid profiles, mostly in accordance with the homeoviscous adaptation hypothesis, stating that low temperatures necessitate acclimation responses that increase fluidity of storage lipids, allowing their enzymatic hydrolysis, and ensure integral protein functions. Overall, the two native species had similar lipidomic profiles when compared to the invasive species, but all species showed specific shifts in their lipid profiles after entering diapause. Taken together, all three species show adaptations that improve energy saving and storage and membrane lipid fluidity during overwintering diapause. While the three species differed in the specific strategies used to increase lipid viscosity, the two native beetle species showed a more canalized lipidomic response, than the recent invader. Since close relatives with similar winter ecology can have different winter ecophysiology, extrapolations among species should be done with care. Still, range expansion of the recent invader into high latitude habitats might indeed be retarded by lack of physiological tools to manage especially thermal stress during winter, but conversely species adapted to long cold winters may face these stressors as a consequence of ongoing climate warming.

Signal sensing and transduction is an essential biological process for cell adaptation and survival. Histidine kinases (HK) are the sensory proteins of two-component systems that control many bacterial responses to different stimuli, like environmental changes. Here, we focused on the HK DesK from Bacillus subtilis, a paradigmatic example of a transmembrane thermosensor suited to remodel membrane fluidity when the temperature drops below 30°C. DesK provides a tractable system for investigating the mechanism of transmembrane signaling, one of the majors interrogates in biology to date. Our studies demonstrate that transmembrane proline residues modulate the conformational switch of a 2-helix coiled-coil (2-HCC) structural motif that controls input-output in a variety of HK. Our results highlight the relevance of proline residues within sensor domains and could inspire investigations of their role in different signaling proteins., Environmental awareness is an essential attribute of all organisms. The homeoviscous adaptation system of Bacillus subtilis provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense the order of membrane lipids with the transmembrane (TM) protein DesK, which has an N-terminal sensor domain and an intracellular catalytic effector domain. DesK exhibits autokinase activity as well as phosphotransferase and phosphatase activities toward a cognate response regulator, DesR, that controls the expression of an enzyme that remodels membrane fluidity when the temperature drops below ∼30°C. Membrane fluidity signals are transmitted from the DesK sensor domain to the effector domain via rotational movements of a connecting 2-helix coiled coil (2-HCC). Previous molecular dynamic simulations suggested important roles for TM prolines in transducing the initial signals of membrane fluidity status to the 2-HCC. Here, we report that individual replacement of prolines in DesKs TM1 and TM5 helices by alanine (DesKPA) locked DesK in a phosphatase-ON state, abrogating membrane fluidity responses. An unbiased mutagenic screen identified the L174P replacement in the internal side of the repeated heptad of the 2-HCC structure that alleviated the signaling defects of every transmembrane DesKPA substitution. Moreover, substitutions by proline in other internal positions of the 2-HCC reestablished the kinase-ON state of the DesKPA mutants. These results imply that TM prolines are essential for finely tuned signal generation by the N-terminal sensor helices, facilitating a conformational control by the metastable 2-HCC domain of the DesK signaling state.

Temperature exerts a first-order control on microbial populations, which constantly adjust the fluidity and permeability of their cell membrane lipids to minimize loss of energy by ion diffusion across the membrane. Analytical advances in liquid chromatography coupled to mass spectrometry have allowed the detection of a stunning diversity of bacterial and archaeal lipids in extreme environments such as hot springs, hydrothermal vents and deep subsurface marine sediments. Here, we investigated a thermal gradient from 18 to 101 °C across a marine sediment field and tested the hypothesis that cell membrane lipids provide a major biochemical basis for the bioenergetics of archaea and bacteria under heat stress. This paper features a detailed lipidomics approach with the focus on membrane lipid structure-function. Membrane lipids analyzed here include polar lipids of bacteria and polar and core lipids of archaea. Reflecting the low permeability of their ether-linked isoprenoids, we found that archaeal polar lipids generally dominate over bacterial lipids in deep layers of the sediments influenced by hydrothermal fluids. A close examination of archaeal and bacterial lipids revealed a membrane quandary: not only low permeability, but also increased fluidity of membranes are required as a unified property of microbial membranes for energy conservation under heat stress. For instance, bacterial fatty acids were composed of longer chain lengths in concert with higher degree of unsaturation while archaea modified their tetraethers by incorporation of additional methyl groups at elevated sediment temperatures. It is possible that these configurations towards a more fluidized membrane at elevated temperatures are counterbalanced by the high abundance of archaeal glycolipids and bacterial sphingolipids, which could reduce membrane permeability through strong intermolecular hydrogen bonding. Our results provide a new angle for interpreting membrane lipid structure-function enabling archaea and bacteria to survive and grow in hydrothermal systems.

Dietary fatty acids can be incorporated directly into phospholipids. This poses a specific challenge to cellular membranes since their composition, hence properties, could greatly vary with different diets. That vast variations in diets are tolerated therefore implies the existence of regulatory mechanisms that monitor and regulate membrane compositions. Here we show that the adiponectin receptor AdipoR2, and its C. elegans homolog PAQR-2, are essential to counter the membrane rigidifying effects of exogenously provided saturated fatty acids. In particular, we use dietary supplements or mutated E. coli as food, together with direct measurements of membrane fluidity and composition, to show that diets containing a high ratio of saturated to monounsaturated fatty acids cause membrane rigidity and lethality in the paqr-2 mutant. We also show that mammalian cells in which AdipoR2 has been knocked-down by siRNA are unable to prevent the membrane-rigidifying effects of palmitic acid. We conclude that the PAQR-2 and AdipoR2 proteins share an evolutionarily conserved function that maintains membrane fluidity in the presence of exogenous saturated fatty acids.

Summary During cold periods ectotherms may improve low temperature tolerance via rapid cold hardening (RCH) over a period of hours and/or long-term cold acclimation (LTCA) during days, weeks or months. However, the effect of duration and the major underlying mechanisms of these processes are still not fully understood. In the present study, the molecular and biochemical responses to RCH (1-3 hours) and LTCA (1-3 days) and the corresponding benefits to survival were investigated using the chill sensitive collembolan, Folsomia candida. We investigated osmolyte accumulation, membrane restructuring and transcription of candidate genes as well as survival benefits in response to RCH and LTCA. RCH induced significant up-regulation of targeted genes encoding enzymes related to carbohydrate metabolic pathways and genes encoding small and constitutively expressed Hsps, indicating that the animals rely on protein protection from a subset of Hsps during RCH and probably also LTCA. The up-regulation of genes involved in carbohydrate metabolic processes initiated during RCH was likely responsible for a transient accumulation of myoinositol during LTCA, which may support the protection of protein and membrane function and structure. Membrane restructuring, composed especially of a significantly increased ratio of unsaturated to saturated phospholipid fatty acids seems to be a supplementary mechanism to activation of Hsps and myoinositol accumulation in LTCA. Thus, the moderate increase in cold shock tolerance conferred by RCH seems to be dominated by effects of heat shock proteins, whereas the substantially better cold tolerance achieved after LTCA is dominated by post-transcriptional processes increasing membrane fluidity and cryoprotectant concentration.