Shape of the fluidity gradient in the plasma membrane of living HeLa cells

The shape of the fluidity gradient of the outer hemi-leaflet of the plasma membrane of living HeLa cells was determined using a series of n-(9-anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 10, 11, 12, and 16. Fluorescence uptake and steady-state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, and of the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence were, for the n-(9-anthroyloxy) series, in the order 2 greater than 3 greater than 6 greater than 7 greater than 9 greater than 10 greater than 11 = 12 = 16. Anisotropy values were constant from 5 to 30 min after addition of the various probes, and the magnitudes were in the order 7 greater than 6 greater than 9 = 10 greater than 2 = 3 greater than 11 greater than 12 greater than 16, indicative of the shape of the fluidity gradient. No differences were noted between the values obtained with 12-(9-anthroyloxy) stearic acid and 12- (9-anthroyloxy) oleate. The kinetics of anisotropy exhibited by those probes with the anthroyloxy group in positions deeper than 9, where initially higher values declined until equilibrium was reached, were probably indicative of an energy barrier at the approximate depth sensed by 7 AS.     

Fluorescence quenching of a series of membrane probes measured in living cells by flow cytometry

The transverse location normal to the bilayer surface of a series of n-(9-anthroyloxy) fatty acid probes, where n = 2, 3, 6, 7, 9, 12, and 16, was determined by fluorescence quenching measurements with a flow cytometer. We show that the anthroyloxy moieties of the probes locate at a graded series of depths in the outer leaflet of the plasma membrane of living HeLa cells, in a manner similar to that previously observed for model membrane systems, and mitochondria. For different n, the efficiency of quenching with an aqueous phase quencher, Cu+2, was 2 greater than or equal to 3 greater than 6 greater than or equal to 7 greater than 9 greater than 12 greater than 16. Therefore, flow cytometry permits use of these probes for measurements of dynamic parameters related to membrane fluidity at different depths in the plasma membranes of living cells.     

二价金属离子对嵌有线粒体H~+-ATP酶的脂酶体不同层次脂质流动性的影响

本文报道用荧光偏振及顺磁共振两种方法研究Mg~(2+)及其它二价金属离子对嵌有H~+-ATP酶的脂酶体不同层次脂质流动性的影响。 (1)顺磁标记探剂5-、12-、16-氮氧基硬脂酸测定结果表明Mg~(2+)和其它二价金属离子都能降低膜脂双分子层表层的流动性。降低流动性的顺序为Mg~(2+)=Ca~(2+)>Sr~(2+)>Cd~(2+)。较深层脂则无明显变化。 (2)荧光探剂7-、12-(9-蒽酰)硬脂酸及16-(9-蒽酰)棕榈酸的测定结果也表明Mg~(2+)和其它二价金属离子降低了膜脂表层的流动性,尤以Mn~(2+)、Ca~(2+)降低流动性最显著,流动性降低的顺序为;Mn~(2+) Ca>Sr~(2+) Mg~(2+) Cd~(2+)。除Mn~(2+)、Ca~(2+)还能影响膜脂深层的流动性外,其它与对照无明显差异。     

Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The "bulk" membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.     

Properties and the locations of a set of fluorescent probes sensitive to the fluidity gradient of the lipid bilayer

The synthesis and properties of a set of four fluorescent probes (n-(9-anthroyloxy) fatty acids, n = 2, 6, 9, 12) sensitive to the fluidity gradient of the lipid bilayer are described. Fluorescent quenching experiments show that the probes locate at a graded series of depths in the bilayer. A fifth probe, methyl-9-anthroate, locates near the bilayer centre. As an example of their application, the probes are used to study the phase transitions of dipalmitoyl phosphatidyl-choline. Changes in the rotational relaxation times of the probes across the transitions are more pronounced at the centre of the bilayer than at the surface.     

Modulation of liposomal membrane fluidity by flavonoids and isoflavonoids

The polyphenolic structures of flavonoids and isoflavonoids confer them with the ability to scavenge free radicals and to chelate transition metals, a basis for their potent antioxidant abilities. Another possible contributory mechanism toward their antioxidant activities is their ability to stabilize membranes by decreasing membrane fluidity. In this study, the effects of representative flavonoids, isoflavonoids, and their metabolites on membrane fluidity and their preferential localization in the membrane were investigated using large unilamellar vesicles (LUVs) as the membrane models. These results were compared with those of cholesterol and alpha-tocopherol. Changes in fluorescence anisotropy values for a series of n-(9-anthroyloxy) fatty acid probes (n = 6, 12, 16) upon addition of the test compounds were used to monitor alterations in membrane fluidity at graded depths in lipid bilayer. The results of the study suggest that the flavonoids and isoflavonoids, similar to cholesterol and alpha-tocopherol, partition into the hydrophobic core of the membrane and cause a dramatic decrease in lipid fluidity in this region of the membrane. Localization of flavonoids and isoflavonoids into the membrane interiors and their resulting restrictions on fluidity of membrane components could sterically hinder diffusion of free radicals and thereby decrease the kinetics of free radical reactions.     

Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.     

13C NMR studies on fluorescent probes: 13C chemical shifts and longitudinal relaxation times of n-hydroxy-fatty (n=2,6,9,12) acids and n-(9-anthroyloxy)-stearic (n=6,12) acids

¹³C NMR has been used to confirm the structure of two fluorescent probes, n-(9-anthroyloxy)-stearic acids (n=6,12), and the series of n-hydroxy-fatty acids (n=2,6,9,12) from which the set of fluorescent fatty acids may be synthesised. ¹³C longitudinal relaxation times and correlation times of the individual carbon atoms in 12-hydroxy- and 6- and 12-(9-anthroyloxy)-stearic acids show differences in motional properties between these derivatives and the parent stearic acid in chloroform(d) solution. The correlation times of the substituted carbons in 6-, 9-, and 12-hydroxy-stearic acids are longer than the corresponding carbons in stearic acid. The change in correlation times at the substituted carbons reflects the increase in motion along the acyl chain. Attachment of the bulky anthracene ring causes greater restriction of motion at the substituted carbon atom but the gradient of motion along the chain is preserved. These results are discussed in terms of the types of motion which lead to fluorescence depolarization when the fluorescent fatty acids are used as fluidity probes in biomembranes.     

Interaction of rifampicin and isoniazid with large unilamellar liposomes: spectroscopic location studies

The location of isoniazid and rifampicin, two tuberculostatics commonly used for the treatment of Mycobacterium tuberculosis and Mycobacterium avium complex infectious diseases, in bilayers of dimyristoyl-L-alpha-phosphatidylcholine (DMPC) and dimyristoyl-L-a-phosphatidylglycerol (DMPG) have been studied by 1H NMR and fluorimetric methods. Steady-state fluorescence intensity and fluorescence energy transfer studies between rifampicin and a set of functionalized probes [n-(9-anthroyloxy)stearic acids, n=2, 12] reveal that, in both systems, isoniazid is located at the membrane surface whereas rifampicin is deeply buried inside the lipid bilayers. Steady-state fluorescence anisotropy studies performed with the probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenylhexa-triene (TMA-DPH), not only corroborate the above results, but also show that no changes in membrane fluidity were detected in either liposome. The 1H NMR results, in DMPC liposomes, confirm the location of rifampicin near the methylene group of the acyl chains of the lipid bilayers.     

Fatty acids specifically related to the anisotropic properties of plasma membrane from rat urothelium

Four different luminal surfaces of rat urothelium differing in their fatty acid composition were prepared by dietary induction. In order to induce lipid changes, each of four groups of rat received a basal diet rich in one of the unsaturated n-3, n-6 or n-9 fatty acid families and a commercial (control) diet. The effects of the dietary regime on the fatty acid composition of luminal urothelial membranes and their relation to the mobility of fluorescent probes were studied. In comparison with the control diet membrane, all three fatty acid-rich diets induced a decrease of the percentage amount of saturated fatty acid while that of the unsaturated fatty acids was increased. Accordingly, all three diets increased the unsaturation index in comparison with the control diet. The anisotropy across each membrane fraction was assessed using the n-(9-anthroyloxy) fatty acid fluorescent probes 3-AS, 7-AS and 12-AS, which locate at different depths in the membrane. Two different anisotropy profiles were observed. One profile showed the highest anisotropy at the C7 depth, whereas the other exhibited a continuous decrease of the anisotropy from the surface to the center of the bilayer. The molecular properties (isomerization) of 18:2n-9 fatty acid may account, at least in part, for the observed V-shaped profile (the ascending trend) of the membrane anisotropy values as a function of the respective 18:2n-9 fatty acid contents. Nevertheless, the minimum value of the profile did not correspond to the minimum 18:2n-9 fatty acid content, but rather to the higher amount of docosahexaenoic (22:6n-3) fatty acid. Thus, a modulating role of the 22:6n-3 fatty acid on the rigidifying effect of 18:2n-9 fatty acid is suggested, possibly mediated by relationships between fatty acid composition, saturated and unsaturated chain lengths, and freedom of motion of the phospholipid acyl chains.     

Fluorescence studies of dipalmitoylphosphatidylcholine vesicles reconstituted with the glycoprotein of vesicular stomatitis virus

The vesicular stomatitis virus glycoprotein (G) was reconstituted into dipalmitoylphosphatidylcholine (DPPC) vesicles by detergent dialysis. The DPPC gel to liquid-crystalline phase transition of the DPPC-G protein vesicles was monitored by the fluorescence anisotrophy of trans-paranaric acid, 16-(9-anthroyloxy)palmitoylglucocerebroside, 1,6-diphenyl-1,3,5-hexatriene, and 4-heptadecyl-7-hydroxycoumarin. The DPPC transition temperature measured by all four fluorescent probes was lowered in the presence of the G protein and the DPPC gel state was disordered by the G protein as evidenced by a decreased fluorescence anisotropy for all four probes below the phase-transition temperature. A possible ordering of the DPPC liquid-crystalline state by the G protein was indicated by an increased anisotropy of trans-paranaric acid and 16-(9-anthroyloxy)palmitoylglucocerebroside in the liquid-crystalline state of DPPC-G protein vesicles. The G protein in addition affected the ionization of the 4-heptadecyl-7-hydroxycoumarin in lipid vesicles, increasing the apparent pK of the probe from 9.05 to 9.45.     

Fluorescence anisotropy decays of n-(9-anthroyloxy) fatty acids in dipalmitoyl phosphatidylcholine vesicles. Localization of the effects of cholesterol addition

The localization of the effects of cholesterol addition on the dynamic structure of the fatty acyl chains of dipalmitoyl phosphatidylcholine vesicles has been investigated by the time-resolved fluorescence anisotropy technique with a set of n-(9-anthroyloxy) fatty acids probes. The major effect of cholesterol is observed in the 7–9 carbon region where both parameters of the anisotropy decays, the residual anisotropy (r_∞) and the correlation time, are greatly enhanced whatever the temperature (21, 37 and 47°C). In the 12–16 carbon region, the r_∞ values are lowered upon addition of cholesterol in the gel phase, in agreement with the effect monitored by the 1,6-diphenyl-1,3,5-hexatriene probe. Only slight perturbations on the r_∞ values are observed in the 2-carbon region whatever the temperature.     

Micro-organisation of the membrane after radiation-induced apoptosis: a flow cytometry study

The aim of this study was to detect membrane fluidity modifications in blood lymphocytes that had been exposed to γ-radiation, at a graded series of depths from the surface to the centre of the membrane bilayer and as a function of cell viability. A time course was performed to verify the contribution of the membrane to radiation-induced apoptosis. In comparison with spectrofluorimetry, flow cytometry proved to be a reliable method for measuring radiation-induced membrane alterations. Late apoptotic lymphocytes were characterised by a significant decrease of the 3-SA, 6-SA and 9-SA fluorescence anisotropy values, compared to viable lymphocytes. Moreover, a highly significant difference was observed in the early apoptotic lymphocyte subpopulation between the fluorescence anisotropy values measured 24 h (radiation-induced apoptosis) and those measured 1 h (spontaneous apoptosis) after irradiation. The simultaneous assessment of cellular viability and membrane fluidity using n-(9-anthroyloxy) fatty acid probes, may be relevant for the investigation of interactions which may exist between membrane modifications and the apoptotic process. Our observations support the specificity of radiation-induced apoptosis compared to spontaneous apoptosis in terms of biophysical modifications of membrane properties. Received: 15 January 2001 / Accepted: 1 July 2001     

Membrane fluidity and lipid composition of rat small intestinal brush-border membranes during postnatal maturation

Fluidity and lipid composition of rat small intestinal brush-border membranes (BBM) were studied during maturation in five age groups: newborns, sucklings (1-3 weeks), weaned (4-6 weeks), juveniles (8-10 weeks), and adults (12 weeks). Brush-border membrane fluidity was measured by steady-state fluorescence polarization. Fluorescent probes used were: 1,6-diphenyl-1,3,5-hexatriene, 1-(4-trimethylammonium)phenyl)-6-phenyl-1,3,5-hexatriene, and a set of n-(9-anthroyloxy) fatty acids. Fluorescence anisotropy measured with all fluorophores was increased in adult versus newborn rats (P less than 0.004). The weight ratio of saturated to cis-unsaturated fatty acids increased from birth to the suckling age (P less than 0.0004). The cholesterol to phospholipid molar ratio increased from birth to the weaned age (P less than 0.0001). Cholesterol to protein ratio and phospholipid to protein ratio decreased after the weaned age (P less than 0.004). The results not only describe maturational changes of brush-border membranes but also give a better understanding of the correlations between biophysical and biochemical data in biological membranes.     

An assessment of the fluidity gradient of the lipid bilayer as determined by a set of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12, 16).

The rotational behavior of a set of n-(9-anthroyloxy) fatty acid fluorescent probes is examined in two liquid paraffins and in liposomes composed of dipalmitoyl phosphatidylcholine. As has been observed with other membrane fluorescent probes (Hare, F., and Lussan, C. (1977) Biochim. Biophys. Acta 467, 262-272), the degree of fluorescence depolarization for a given solvent viscosity is dependent on the solvent standard employed. In addition, when the anthroyloxy group is in the terminal position of the acyl chain, it has more rotational freedom than when it is conjugated to positions 6, 9, or 12 where the rotational motion of the fluorophore is similar. When incorporated into lipid bilayers, values of fluorescence polarization reflect the gradient of "fluidity" which extends from the surface to the center of the membrane. The nature of this polarization gradient is discussed in relation to the intrinsic differences between the probes and the anisotropic rotations responsible for depolarization.     

Interaction of fluorescence quenchers with the n-(9-anthroyloxy) fatty acid membrane probes

The fluorescence quenching of the $\text{n-(9-}\text{anthroyloxy}\text{)}$ (AO) fatty acid probes has been investigated in aqueous dispersions, vesicles of egg phosphatidylcholine and vesicles formed from red cell ghosts. Negatively charged (KI), neutral (acrylamide) and positively charged (CuSO₄) quenchers were used to monitor the location of the probes. The fluorescence of the probes, with the exception of the shortest chain (11-(9-anthroyloxy)undecanoic acid) is not quenched by acrylamide when associated with vesicles. This indicates that in association with vesicles, the 9-anthroyloxy moiety of the long chain probes is buried within the hydrocarbon region and thus well shielded from the aqueous phase. Measurements with KI indicate that the probes are present in the membrane at depths corresponding to the position of the 9-anthroyloxy moiety on the fatty acid, and that the quencher itself forms a concentration gradient within the membrane. Very little or no CuSO₄ quenching was observed for $\text{n-}\text{(9-anthroyloxy)stearic}$ acid probes $\text{(n-}\text{AS)with}\text{n > 2}$, suggesting that in these vesicles Cu²⁺ does not significantly penetrate the bilayer.     

Relative location of anthroyl derivatives of stearic acid in low density lipoproteins

Effective quenching constants (K'sv) of 2-, 7- and 12-(9-anthroyloxy)stearic acid (n-AS) fluorescence in LDL were determined. Spin probes I(m, n) (n = 3, 7, 10, 14) and I- anions were used as quenchers. Quenching of 2-AS and 12-AS fluorescence by I(m, n) was the more effective, the deeper spin probe nitroxyl fragment was located (the greater n was); maximal K'sv value corresponded to I(1,14). By contrast, for 7-AS the quenching by I(12,3) was the most effective. 2-AS and 12-AS spectra maxima and fluorescence polarization were similar. We concluded that the 2-AS chromophore was located deeper in LDL phospholipid monolayer than chromophore of 7-AS (as was the case for 12-AS).     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

Plasma omega 3 polyunsaturated fatty acid status and monounsaturated fatty acids are altered by chronic social stress and predict endocrine responses to acute stress in titi monkeys

Disturbances in fatty acid (FA) metabolism may link chronic psychological stress, endocrine responsiveness, and psychopathology. Therefore, lipid metabolome-wide responses and their relationships with endocrine (cortisol, insulin, and adiponectin) responsiveness to acute stress (AS) were assessed in a primate model of chronic social stress (CS). Compared to controls (not exposed to CS), CS increased (P≤0.05) circulating triacylglycerol (TG) and phosphatidylethanolamine (PE) n-6/n-3 and reduced (P≤0.05) cholesterol ester (CE) 16:1n7 and phosphatidylcholine (PC) 18:1n7, suggesting lower omega-3 FA status and stearoyl-CoA desaturase activity, respectively. Cortisol responses to AS positively correlated with TG n-6/n-3 (r=0.93; P=0.007), but only in CS monkeys. The adiponectin response to AS inversely correlated with CE n-6/n3 (r=-0.89; P=0.045) and positively with TG 16:1n7 (r=0.98; P=0.004), only in CS monkeys. Our results are consistent with previously reported FA profiles in stress-related psychopathology and suggest that compositional changes of specific lipid FAs may form new functional markers of chronic psychological stress.     

Effects of ethanol on the Escherichia coli plasma membrane.

The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids. The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes. Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface. In comparison, hexanol decreased the polarization of probes located more deeply in the membrane. Temperature had a large effect on probes located at all depths. The effects of ethanol on E. coli membranes from cells grown with or without ethanol were also examined. Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells. In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells. These differences are explained by analyses of membrane composition. Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio. This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth. Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E. coli to growth in the presence of ethanol.