Influence of temperature, cholesterol, dipalmitoyllecithin and Ca on the rate of muscle cell fusion

The rate of muscle cell fusion increases between 28 °C and 40 °C by a factor of 15 to 20. The formal activation energy of the fusion process changes abruptly at about 35 °C. This change is discussed in terms of a phase transition of the membrane lipids at 35 °C. In the presence of cholesterol or dipalmitoyllecithin the fusion rate decreases markedly. Increasing the temperature reverses the effects of cholesterol and dipalmitoyllecithin. These results are discussed in terms of interactions between membrane lipids.     

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel,fluid or liquid-ordered phase,determined using AFM force spectroscopy

The biological membrane surrounding milk fat globules (MFGM) exhibits lateral phase separation of lipids, interpreted as gel or liquid-ordered phase sphingomyelin-rich (milk SM) domains dispersed in a fluid continuous lipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-rich domains induced by temperature (T < Tm or T > Tm) or cholesterol affected the Young modulus of the lipid membrane. Supported lipid bilayers composed of MFGM polar lipids, milk SM or milk SM/cholesterol (50:50 mol) were investigated at 20 °C and 50 °C using atomic force microscopy (AFM) and force spectroscopy. At 20 °C, gel-phase SM-rich domains and the surrounding fluid phase of the MFGM polar lipids exhibited Young modulus values of 10–20 MPa and 4–6 MPa, respectively. Upon heating at 50 °C, the milk SM-rich domains in MFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membrane with similar Young modulus values to that of a fluid phase (0–5 MPa). Upon addition of cholesterol to the milk SM to reach 50:50 mol%, membranes in the liquid-ordered phase exhibited Young modulus values of a few MPa, at either 20 or 50 °C. This indicated that the presence of cholesterol fluidized milk SM membranes and that the Young modulus was weakly affected by the temperature. These results open perspectives for the development of milk polar lipid based vesicles with modulated mechanical properties.     

Sphingolipids, cholesterol, and HIV-1: A paradigm in viral fusion

Our previous studies show that the depletion of cholesterol or sphingolipids (raft-associated lipids) from receptor-bearing adherent cell lines blocks HIV-1 entry and HIV-1 Env-mediated membrane fusion. Here we have evaluated the mechanism(s) by which these lipids contribute to the HIV-1 Env-mediated membrane fusion. We report the following: (1) GSL depletion from a suspension T lymphocyte cell line (Sup-T1) reduced subsequent fusion with HIV-1IIIB-expressing cells by 70%. (2) Cholesterol depletion from NIH3T3 cells bearing HIV-1 receptors (NIH3T3CD4R5/NIH3T3CD4X4) did not impair subsequent fusion with HeLa cells expressing the corresponding HIV-1 Envs. In contrast GSL depletion from these targets reduced fusion by 50% suggesting that GSL facilitate fusion in different ways. (3) GSL-deficient GM95 cells bearing high receptors fused with HIV-1 Env-expressing cells at 37°C with kinetics similar to that of GSL + NIH3T3 targets. Based on these observations, we propose that the plasma membrane cholesterol is required to maintain the integrity of receptor pools whereas GSLs are involved in stabilizing the coupling of inter-receptor pools.     

The status within,an Autobiography by Francois Jacob; Basic Book Inc., New York, 1988, 326 pp. $22.95

The interactions of ram spermatozoa with exogenous liposomes of varying composition were studied, with the aim of examining the mechanisms by which some lipids protect against cold-induced damage during cryostorage. Liposomes containing various preparations of phosphatidylcholine and cholesterol enhanced sperm survival during storage at 5°C, both in terms of motility and acrosomal integrity. A membrane-fluidizing agent, A₂C, was slightly deleterious in this respect. Cholesterol-containing liposomes were not superior in their effects to those prepared without cholesterol. Thus stabilization of the plasma membrane by cholesterol loading may be unimportant. When sodium vanadate was used as a functional probe of membrane integrity, the cryoprotective effects of lipids were apparent despite increased plasma membrane permeability. Incubation of spermatozoa with positively charged liposomes, containing stearyl-amine, caused considerable loss of motility and acrosomal damage, coupled with cellular aggregation. There was also some evidence that the presence of calcium lessened the effectiveness of liposomes in protecting spermatozoa against damage during cooling.     

Lipid-lipid and lipid-protein interactions in chromaffin granule membranes: A spin label ESR study

The ESR spectra of six different positional isomers of a stearic acid and three of a phosphatidylcholine spin label have been studied as a function of temperature in chromaffin granule membranes from the bovine adrenal medulla, and in bilayers formed by aqueous dispersion of the extracted membrane lipids. Only minor differences were found between the spectra of the membranes and the extracted lipid, indicating that the major portion of the membrane lipid is organized in a bilayer arrangement which is relatively unperturbed by the presence of the membrane protein. The order parameter profile of the spin label lipid chain motion is less steep over the first half of the chain than over the section toward the terminal methyl end of the chain. This ‘stiffening’ effect is attributed to the high proportion of cholesterol in the membrane and becomes less marked as the temperature is raised. The isotropic hyperfine splitting factors of the various positional isomers display a profile of decreasing polarity as one penetrates further into the interior of the membrane. No marked differences are observed between the effective polarities in the intact membranes and in bilayers of the extracted membrane lipids. The previously observed temperature-induced structural change occurring in the membranes at approx. 35°C was found also in the extracted lipid bilayers, showing this to be a result of lipid-lipid interactions and not lipid-protein interactions in the membrane. A steroid spin label indicated a second temperature-dependent structural change occurring in the lipid bilayers at lower temperatures. This corresponds to the onset of a more rapid rotation about the long axis of the lipid molecules at a temperature of approx. 10°C. The lipid bilayer regions probed by the spin labels used in this study may be involved in the fusion of the chromaffin granule membrane leading to hormone release by exocytosis.     

Effects of temperature on growth and efficiency of food utilization in fifth-instar caterpillars of the tobacco hornworm, Manduca sexta

Fifth-instar larvae of Manduca sexta were reared from hatching on artificial diet at 15, 20, 25, 30 and 35°C. Total development time decreased with increasing temperature. Very few larvae (12%) survived at 15°C, so this temperature was not considered further. There was some mortality at 30°C (11%), and at 35°C (50%).The absolute rate of growth in the fifth instar was faster at 25 than at 20°C, but was similar at 25, 30 and 35°C. This was true both for caterpillars that were chronically exposed to experimental temperatures (i.e. since hatching) and for those acutely exposed (i.e. reared up to fifth instar at 25°C).There was a progressive decrease with higher rearing temperatures in both the initial and final sizes of chronically exposed fifth-instar larvae. Acutely exposed caterpillars matched for initial size showed smaller temperature related differences in final size. Because of these size differences there were differences in relative growth rate which did not reflect true differences in absolute growth rate.Total food consumed by chronically exposed caterpillars was greatest at the lowest temperature (20°C), and decreased progressively with increasing temperature. The absolute rate of food consumption increased from 20 to 25°C, but did not vary significantly between 25 and 35°C. Differences in the sizes of the insects at the different temperatures meant that there were differences among relative measures of consumption that did not reflect absolute food consumption.For chronically exposed caterpillars, none of the three usual indices of food conversion efficiency (AD, ECI and ECD) varied significantly with temperature between 20 and 35°C. This implies that the effects of temperature on metabolic costs are closely matched to food consumption.Oxygen consumption increased with temperature between 20 and 25°C but was temperature compensated between 25 and 35°C.These findings are discussed in terms of their implications for the optimal temperature for growth in Manduca.     

Anomaly of transport of dipicrylamine anions across the central barrier of planar dipalmitoylphosphatidylcholine bilayer membranes at the phase transition

The rate of translocation of the hydrophobic ion dipicrylamine across planar lipid membranes formed from dipalmitoyllecithin in n-decane was determined by voltage jump relaxation experiments. The activation energy of the rate constant shows a change from a positive to a negative value at about 42°C near the main phase transition temperature of this lipid. Below this temperature, the rate constant was found to increase with decreasing temperature. This anomalous behaviour extends over a temperature range of at least 10 K and may be formally interpreted as an enhanced mobility of dipicrylamine in the solid state of the membrane.     

The effect of temperature on feeding,growth, and metabolism during the last larval stadium of the female house cricket, Acheta domesticus

Eighth instar female house crickets at 35°C developed faster, gained slightly more wet weight, and consumed less food, water, and oxygen than at 25°C. The duration of the 8th stadium at 25°C was 13 days (undisturbed), but was 14 days when disturbed by daily weighing. The duration of the 8th stadium at 30°C was 8 days and at 35°C was 6 days. During the first half of the 8th stadium at 25, 30, and 35°C, there was a high rate of food and water consumption resulting in statistically equal maximum dry weight achievement (124 mg). Respiratory quotients greater than one during this time indicated the conversion of ingested carbohydrate to fat. During the latter half of the 8th stadium, food and water consumption declined and the crickets lost weight. The period of weight loss was proportionally much longer at 25°C than at 30 or 35°C. Respiratory quotients lower than 1.0 during the latter half of the 8th stadium at 30 and 35°C indicated the metabolism of stored lipids. The respiratory quotient at 25°C never fell below 1.0, possibly because some food remained in the gut. The absorption efficiency was not influenced by temperature (25–35°C). Though the caloric content of the faeces was lower at 25°C than at 30 or 35°C, which correlated to the much longer time for food passage at 25°C than at 35°C, the difference in total calories egested was insufficient to alter the absorption efficiency. A longer period of reduced feeding and greater dry weight loss during the latter half of the 8th stadium at 25°C resulted in a lower metabolic efficiency at 25°C than at 30 or 35°C. Eighth instar crickets in response to a step-function transfer from 30°C–25 or 35°C showed an immediate (<1 hr) and complete metabolic adjustment which was not affected by the temperature history during the 7th stadium. House crickets did not exhibit temperature acclimation in the range 20–40°C, the metabolic rate being determined by ambient temperature. The Q₁₀ for oxygen consumption in the range 20–40°C was about 2.     

Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles

The relative kinetics of intermixing and release of liposome aqueous contents during Ca²⁺-induced membrane fusion has been investigated. Fusion was monitored by the Tb-dipicolinic acid (DPA) fluorescence assay. Release was followed by the relief of self-quenching of carboxyfluorescein or by Tb fluorescence, with essentially identical results. Fusion of large unilamellar vesicles (LUV) made of phosphatidylserine (PS) in 100 mM NaCl (pH 7.4) at 25°C was initially non-leaky, whereas the fusion of small unilamellar vesicles (SUV) was accompanied by partial release of contents. After several rounds of fusion, the internal aqueous space of the vesicles collapsed. The rate of intermixing of lipids, measured by a resonance energy transfer assay, and the rate of coalescence of aqueous contents during fusion were similar over a range of Ca²⁺ concentrations. Most of the aqueous contents were retained after the fusion of SUV (PS) in 5 mM NaCl and 1 mM Ca²⁺. LUV made of a 1:1 mixture of Bacillus subtilis cardiolipin and dioleoylphosphatidylcholine went through about two rounds of fusion in the presence of Ca²⁺ at 10°C, with complete retention of contents. Similar results were obtained with vesicles composed of phosphatidate/PS/phosphatidylethanolamine/cholesterol (1:2:3:2) in the presence of Ca²⁺ and synexin at 25°C. These results emphasize the diversity of the relative kinetics of fusion and release in different phospholipid vesicle systems under various ionic conditions, and indicate that the initial events in the fusion of LUV are in general, non-leaky.     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Temperature-induced specific lipid desaturation in the thermophilic cyanobacterium Synechococcus vulcanus

Cyanobacteria desaturate fatty acids in the membrane lipids in response to decrease in temperature. We examined the changes in lipid and fatty acid composition in the thermophilic cyanobacterium Synechococcus vulcanus, which is characterized by an optimum growth temperature of 55°C. During temperature acclimation to 45°C or 35°C, the cells synthesized oleic acid at the expense of stearic acid in the membrane lipids. Unlike mesophilic cyanobacteria, S. vulcanus did not show any significant adaptive desaturation in the galactolipids monogalactosyl diacylglycerol and digalactosyl diacylglycerol, that comprise 50% and 30% of total membrane lipids, respectively. The major changes in fatty acid unsaturation were observed in the sulfolipid sulfoquinovosyl diacylglycerol.     

Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase

Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S_A, vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S_A, was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.     

The effect of temperature on energy distribution during the last-larval stadium of the female house cricket, Acheta domesticus

The distribution of energy during the last stadium of the house cricket at two temperatures was the main theme of this study. Food consumption, growth, and oxygen consumption were greater in the first half of the stadium at both 25 and 35°C. An RQ > 1 indicated the conversion of carbohydrates to lipids during the first half of the instar at both temperatures. The duration of the stadium increased from 6 days at 35°C to 14 days at 25°C. The same maximal weight, protein content and lipid content were attained at both 25 and 35°C. A weight loss (mostly in stored lipids) after the midstadium peak weight was greater at the lower temperature. The absorption efficiency and the production of metabolic wastes were not affected by temperature, but the metabolic efficiency was much higher at 35 than at 25°C during the first half as well as the latter half of the stadium. Although during the first half of the stadium more energy was ingested, absorbed, and made available for growth at 25 than at 35°C, only slightly more growth occurred at 25°C. During the last half of the stadium less energy was ingested at 25 than at 35°C, and much more growth occurred at 35°C because of the even greater heat loss at 25 than at 35°C. Therefore at a lower temperature cricket larvae eat slightly more and reach the same maximal weight as at a higher temperature, but they end up smaller because they waste more energy during the extended duration of the stadium at the lower temperature.     

Temperature-induced vertical shift of proteins in membranes

Thermotropic changes in the transverse order of microsomal membranes isolated from Tetrahymena are revealed by low-angle X-ray diffraction. These are correlated with the lateral order of the membrane lipids by wide-angle X-ray diffraction. Upon lowering the temperature from 28 °C to 2 °C, the Bragg period of the membrane stack reveals an abrupt increase of ~3.0 nm at ~19 °C, which is reversible upon reheating to 28 °C. This is coupled with an alteration in the electron density profile, revealing a shift of mass from the hydrophobic core towards one of the two hydrophilic surfaces. Between 35 °C and 0 °C, the membrane lipids undergo a broad, thermotropic “two-stage” liquid crystalline ? crystalline phase separation with a “breakpoint” at ~18 °C. This breakpoint signals an abrupt lipid redistribution, presumably due to a change in the composition of the two coexisting liquid crystalline and crystalline lipid phases. We conclude: (1) the temperature-induced mass shift reflects a shift in the transverse partition of proteins in membranes; (2) this is triggered by an abrupt lipid redistribution occurring during a broad liquid crystalline ? crystalline phase separation.     

Phase transitions of the purple membrane and the brown holo-membrane X-ray diffraction,circular dichroism spectrum and absorption spectrum studies

The phase transition of the purple membrane observed by differential scanning calorimetry (Jackson, M.B. and Sturtevant, J.M. (1978) Biochemistry 17, 911–915) has been investigated by X-ray diffraction, circular dichroism and absorption spectrum, in comparison with the phase transition in the brown holo-membrane. The two-dimensional crystal of bacteriorhodopsin transformed into two-dimensional liquid around 74–78°C in the purple membrane and around 50–60°C in the brown holo-membrane. The X-ray diffraction patterns obtained at 78°C for the purple membrane and at 60°C for the brown holo-membrane exhibit several broad peaks. Analysis of the pattern suggests that bacteriorhodopsin molecules aggregate in trimers even above the phase transition temperature. The negative circular dichroism band in the visible region is still present at 80°C in the purple membrane and at 60°C in the brown holo-membrane, but becomes negligibly small at 70°C in the brown holo-membrane. The 560 nm absorption peak due to bacteriorhodopsin changes its position and height drastically around 80°C in the brown holo-membrane as in the purple membrane. X-ray diffraction studies have been made on membranes of total lipids extracted from the purple membrane. No indication of the phase transition has been found between ?81°C and 77°C.     

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.     

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 temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.