Fluorescence resonance energy transfer between lipid probes detects nanoscopic heterogeneity in the plasma membrane of live cells

Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C18 is greater with long-chain, saturated acceptor probes such as DiI-C16 than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C16 and DiI-C16 is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C16. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.     

Lateral distribution of cholesterol in dioleoylphosphatidylcholine lipid bilayers: cholesterol-phospholipid interactions at high cholesterol limit

Lateral organization of cholesterol in dioleoyl-phosphatidylcholine (DOPC) lipid bilayers at high cholesterol concentration (>45 mol%) was investigated using steady-state fluorescence anisotropy and fluorescent resonance energy transfer techniques. The recently devised Low Temperature Trap method was used to prepare compositionally uniform cholesterol/DOPC liposomes to avoid the problem of lipid demixing. The fluorescence anisotropy of diphenylhexatrience chain-labeled phosphatidylcholine (DPH-PC) in these liposomes exhibited local maxima at cholesterol mol fractions of 0.50 and 0.57, and a sharp drop at 0.67. For the liposomes labeled with both dehydroergosterol and DPH-PC, the fluorescent resonance energy transfer efficiency from dehydroergosterol to DPH-PC displayed a steep jump at cholesterol mol fraction of 0.5, and dips at 0.57 and 0.68. These results indicate the presence of highly ordered cholesterol regular distribution domains at those observed critical compositions. The observed critical mol fraction at 0.67 agreed favorably with the solubility limit of cholesterol in DOPC bilayers as independently measured by light scattering and optical microscopy. The regular distribution at 0.57 was previously predicted from a Monte Carlo simulation based on the Umbrella model. The results strongly support the hypothesis that the primary requirement for cholesterol-phospholipid mixing is that the polar phospholipid headgroups need to cover the nonpolar body of cholesterol to avoid the exposure of cholesterol to water.     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.     

Fluorescence probe partitioning between Lo/Ld phases in lipid membranes

Fluorescence microscopy imaging is an important technique for studying lipid membranes and is increasingly being used for examining lipid bilayer membranes, especially those showing macroscopic coexisting domains. Lipid phase coexistence is a phenomenon of potential biological significance. The identification of lipid membrane heterogeneity by fluorescence microscopy relies on membrane markers with well-defined partitioning behavior. While the partitioning of fluorophores between gel and liquid-disordered phases has been extensively characterized, the same is not true for coexisting liquid phases. We have used fluorescence microscopy imaging to examine a large variety of lipid membrane markers for their liquid phase partitioning in membranes with various lipid compositions. Most fluorescent lipid analogs are found to partition strongly into the liquid-disordered (L(d)) phase. In contrast, some fluorescent polycyclic aromatic hydrocarbons with a flat ring system were found to partition equally, but others partition preferentially into liquid-ordered (L(o)) phases. We have found these fluorescent markers effective for identification of coexisting macroscopic membrane phases in ternary lipid systems composed of phospholipids and cholesterol.     

Partitioning of amphiphiles between coexisting ordered and disordered phases in two-phase lipid bilayer membranes

The partition coefficients (K(P)) of a series of single-chain and double-chain fluorescent amphiphiles, between solid ordered (P(beta') and L(beta)) and liquid disordered (L(alpha) of the type l(d)) lipid phases coexisting in the same lipid bilayer, was studied using steady-state fluorescence emission anisotropy. The single-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-alkylamines, and the double-chain amphiphiles were N-(7-nitrobenzoxa-2, 3-diazol-4-yl)-phosphatidylethanolamines with chain lengths of 12-18 carbon atoms. Saturated 18-carbon alkyl/acyl chain compounds were also compared with Delta(9)-cis unsaturated chains of the same chain length. The fluorescence anisotropy of the probes was examined in lipid bilayers (multilamellar vesicles) prepared from an equimolar mixture of dilauroylphosphatidylcholine and distearoylphosphatidylcholine and studied as a function of temperature through the entire temperature range of coexistence of ordered gel phases and a disordered fluid phase in this system. The unsaturated chain amphiphiles partitioned exclusively into the fluid phase whenever this phase was present, as did the saturated chain amphiphiles with the shortest chains (C(12:0)), while K(P) ranges between 1 and 2, in favor of the L(beta) solid phase, for the amphiphiles with long saturated (C(18:0)) alkyl/acyl chains, with intermediate behavior for the intermediate chain lengths. All probes appeared to be totally excluded from P(beta') solid (gel) phases. The technique was also used to determine partitioning of some of the probes between coexisting liquid ordered (cholesterol-containing) (l(o)) and liquid disordered (l(d)) L(alpha) phases. In this case the ratio of signal amplitude to noise allowed us to obtain a qualitative, but not quantitative, measure of the phase partitioning of the probes. We conclude that the partitioning behavior of the probes examined between coexisting l(o) and l(d) phases is qualitatively similar to that observed between solid ordered and liquid disordered phases.     

Changes in the lipid and protein components of thymus cell membrane during lipid peroxidation]

Nonenzymatic lipid peroxidation in thymus cell plasma membranes was studied. The composition of lipid and protein components, intensity of fluorescence of the membrane probes (1-anilinonaphthalene-8-sulfonate, 4-dimethylaminochalcon, eosin, pyronin and rhodamine), fluorescence polarization of tryptophan residues of membrane proteins and quenching by acrylamide of intrinsic fluorescence of proteins were determined. Induction of lipid peroxidation by the Fe(2+)-ascorbate system caused changes in the composition and structure of lipids. This was paralleled with changes in the structural-dynamic organization of membrane proteins, transition of some peripheral proteins to the water phase and increased solubilization of integral proteins by Triton X-100.     

Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review).

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.     

Relationship between sterol/steroid structure and participation in ordered lipid domains (lipid rafts): implications for lipid raft structure and function

The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were studied using a series of sterols and steroids structurally similar to cholesterol. In one assay, insolubility in Triton X-100 was assessed in bilayers composed of sterol/steroid mixed with dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, or a 1:1 mixture of these phospholipids. In a second assay fluorescence quenching was used to determine the degree of ordered domain formation in bilayers containing sterol/steroid and a 1:1 mixture of DPPC and a quencher-carrying phosphatidylcholine. Both methods showed that several single modifications of the cholesterol structure weaken, but do not fully abolish, the ability of sterols and steroids to promote ordered domain formation when mixed with DPPC. Some of these modifications included a shift of the double bond from the 5-6 carbons (cholesterol) to 4-5 carbons (allocholesterol), derivatization of the 3-OH (cholesterol methyl ether, cholesteryl formate), and alteration of the 3-hydroxy to a keto group (cholestanone). An oxysterol involved in atherosclerosis, 7-ketocholesterol, formed domains with DPPC that were as thermally stable as those with cholesterol although not as tightly packed as judged by fluorescence anisotropy. It was also found that 7-ketocholesterol has fluorescence quenching properties making it a useful spectroscopic probe. Lathosterol, which has a 7-8 carbon double bond in place of the 5-6 double bond of cholesterol, formed rafts with DPPC that were at least as detergent-resistant as, and even more thermally stable than, rafts containing cholesterol. Because lathosterol is an intermediate in cholesterol biosynthesis, we conclude it is unlikely that sterol biosynthesis continues past lathosterol in order to create a raft-favoring lipid.     

Binding of alpha-synuclein affects the lipid packing in bilayers of small vesicles

The intracellular deposition of fibrillar aggregates of alpha-synuclein is a characteristic feature of Parkinson disease. Alternatively, as a result of its unusual conformational plasticity, alpha-synuclein may fold into an amphipathic helix upon contact with a lipid-water interface. Using spin label ESR and fluorescence spectroscopy, we show here that alpha-synuclein affects the lipid packing in small unilamellar vesicles. The ESR hyperfine splittings of spin-labeled phospholipid probes revealed that alpha-synuclein induces chain ordering at carbon 14 of the acyl chains below the chain melting phase transition temperature but not in the liquid crystalline state of electroneutral vesicle membranes. Binding of alpha-synuclein leads to an increase in the temperature and cooperativity of the phase transition according to the fluorescence anisotropy of the hydrophobic polyene 1,6-diphenylhexatriene and of the fluorescence emission maxima of the amphiphilic probe 6-dodecanoyl-2-dimethylaminonaphthalene. Binding parameters were obtained from the fluorescence anisotropy measurements in combination with our previous determinations by titration calorimetry (Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) J. Biol. Chem. 279, 21966-21975). We also show that alpha-synuclein interacts with vesicle membranes containing sphingomyelin and cholesterol. We propose that the protein is capable of annealing defects in curved vesicle membranes, which may prevent synaptic vesicles from premature fusion.     

Biophysical properties of ergosterol-enriched lipid rafts in yeast and tools for their study: characterization of ergosterol/phosphatidylcholine membranes with three fluorescent membrane probes

In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (l(o)) and liquid disordered (l(d)) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase. N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label l(d) domains in yeast membranes. 4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to l(o) domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol-lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.     

Triton promotes domain formation in lipid raft mixtures

Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds.     

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.     

Lipid-phase structure in epithelial cell membranes: comparison of renal brush border and basolateral membranes

The lipid-phase structures of brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rabbit renal cortex were compared by steady-state and phase-modulation measurements of diphenylhexatriene (DPH) and trans- and cis-parinaric acid (tPnA and cPnA) fluorescence. A temperature-scanning system was used which gave reproducible temperature profiles of steady-state and dynamic fluorescence parameters with a resolution of 0.1 degrees C. Steady-state anisotropy of DPH showed a triphasic dependence on temperature with slope discontinuities at 22 +/- 4 and 47 +/- 3 degrees C (BBMV) and at 23 +/- 2 and 48 +/- 1 degrees C (BLMV). At all temperatures, DPH anisotropy in BBMV was greater than that in BLMV. Ground-state heterogeneity analysis of tPnA and cPnA fluorescence lifetime data demonstrated the presence of long (greater than 12 ns) and short (less than 5 ns) lifetime components, interpreted in terms of solid-phase and fluid-phase lipid domains. The fraction of solid-phase phospholipid decreased from 0.9 to 0.1 for BBMV and from 0.7 to 0.3 in BLMV with increasing temperature (10-50 degrees C). In both membranes, tryptophan-PnA fluorescence energy-transfer measurements showed that membrane proteins were surrounded by a fluidlike phospholipid phase. These results demonstrate the inadequacy of steady-state DPH anisotropy data in defining the structural characteristics of complex biological membranes. Results obtained with the phase-sensitive parinaric acid probes demonstrate major differences in the phase structure of the two opposing cell membranes in both the bulk lipid and the lipid microenvironment around membrane proteins.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Laurdan is a fluorescent probe that detects changes in membrane phase properties through its sensitivity to the polarity of its environment in the bilayer. Variations in membrane water content cause shifts in the laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). We tested whether laurdan fluorescence could be used to distinguish differences in phospholipid order from changes in membrane fluidity by examining the temperature dependence of laurdan GP and fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) vesicles. The phase transition from the solid ordered phase to the liquid disordered phase was observed as a decrease in laurdan GP values from 0.7 to −0.14 and a reduction in anisotropy from 0.25 to 0.12. Inclusion of various amounts of cholesterol in the membranes to generate a liquid ordered phase caused an increase in the apparent melting temperature detected by laurdan GP. In contrast, cholesterol decreased the apparent melting temperature estimated from anisotropy measurements. Based on these results, it appeared that laurdan anisotropy detected changes in membrane fluidity while laurdan GP sensed changes in phospholipid order. Thus, the same fluorescent probe can be used to distinguish effects of perturbations on membrane order and fluidity by comparing the results of fluorescence emission and anisotropy measurements.     

Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)

The insolubility of lipids in detergents is a useful method for probing the structure of biological membranes. Insolubility in detergents like Triton X-100 is observed in lipid bilayers that exist in physical states in which lipid packing is tight. The Triton X-100-insoluble lipid fraction obtained after detergent extraction of eukaryotic cells is composed of detergent-insoluble membranes rich in sphingolipids and cholesterol. These insoluble membranes appear to arise from sphingolipid- and cholesterol-rich membrane domains (rafts) in the tightly packed liquid ordered state. Because the degree of lipid insolubility depends on the stability of lipid-lipid interactions relative to lipid-detergent interactions, the quantitative relationship between rafts and detergent-insoluble membranes is complex, and can depend on lipid composition, detergent and temperature. Nevertheless, when used conservatively detergent insolubility is an invaluable tool for studying cellular rafts and characterizing their composition.     

Influence of sterol structure on phospholipid phase behavior as detected by parinaric acid fluorescence spectroscopy

Phospholipid-sterol interactions were investigated using parinaric acid fluorescence spectroscopy. Cholesterol and cholesterol analogues which were modified in the sterol nucleus or side chain were added at 50 mol % to multilamellar vesicles of model phospholipids selected to be representative of major components in an LM cell plasma membrane. These included sphingomyelins and saturated and monounsaturated phosphatidylcholines and phosphatidylethanolamines. Based on the changes in cis-parinaric acid steady-state fluorescence polarization observed with addition of sterol, 50 mol % cholesterol abolished the phase transition of all the model phospholipids. Dihydrocholesterol and trans-22-dehydrocholesterol behaved like cholesterol in the two systems studied. 24-Methylcholesterols interacted well with all phospholipids except phosphatidylethanolamine which contained an unsaturated fatty acid. 24-Alkyl,trans-22-dehydrocholesterols abolished the phase transition in only two systems: sphingomyelins and phosphatidylcholines possessing relatively short saturated acyl chains. Since steady-state anisotropy is a function of fluorescence lifetime, rotational diffusion rates, and limiting anisotropy, we determined these parameters for two of the phospholipid systems. The results show that steady-state anisotropy values for phospholipid-sterol interactions correlate closely with limiting anisotropy and to a lesser extent with rotational relaxation time. The behavior of the sterols in the model phospholipids are used to interpret 1) fluorescence polarization measurements made with phospholipids extracted from LM cell plasma membranes, and 2) changes in membrane lipid composition which accompany growth of LM cells on various sterols.     

Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm³ and in the DRM fraction (1.146 g/cm³). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm³ had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm³. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.     

Differential ability of cholesterol-enriched and gel phase domains to resist benzyl alcohol-induced fluidization in multilamellar lipid vesicles

Benzyl alcohol (BA) has a well-known fluidizing effect on both artificial and cellular membranes. BA is also likely to modulate the activities of certain membrane proteins by decreasing the membrane order. This phenomenon is presumably related to the ability of BA to interrupt interactions between membrane proteins and the surrounding lipids by fluidizing the lipid bilayer. The components of biological membranes are laterally diversified into transient assemblies of varying content and order, and many proteins are suggested to be activated or inactivated by their localization in or out of membrane domains displaying different physical phases. We studied the ability of BA to fluidize artificial bilayer membranes representing liquid-disordered, cholesterol-enriched and gel phases. Multilamellar vesicles were studied by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid, which display different phase partitioning. Domains of different degree of order and thermal stability showed varying abilities to resist fluidization by BA. In bilayers composed of mixtures of an unsaturated phosphatidylcholine, a saturated high melting temperature lipid (sphingomyelin or phosphatidylcholine) and cholesterol, BA fluidized and lowered the melting temperature of the ordered and gel phase domains. In general, cholesterol-enriched domains were more resistant to BA than pure gel phase domains. In contrast, bilayers containing high melting temperature gel phase domains containing a ceramide or a galactosylceramide proved to be the most effective in resisting fluidization. The results of our study suggest that the ability of BA to affect the fluidity and lateral organization of the membranes was dependent on the characteristic features of the membrane compositions studied and related to the intermolecular cohesion in the domains.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

DHA-fluorescent probe is sensitive to membrane order and reveals molecular adaptation of DHA in ordered lipid microdomains

Docosahexaenoic acid (DHA) disrupts the size and order of plasma membrane lipid microdomains in vitro and in vivo. However, it is unknown how the highly disordered structure of DHA mechanistically adapts to increase the order of tightly packed lipid microdomains. Therefore, we studied a novel DHA-Bodipy fluorescent probe to address this issue. We first determined if the DHA-Bodipy probe localized to the plasma membrane of primary B and immortal EL4 cells. Image analysis revealed that DHA-Bodipy localized into the plasma membrane of primary B cells more efficiently than EL4 cells. We then determined if the probe detected changes in plasma membrane order. Quantitative analysis of time-lapse movies established that DHA-Bodipy was sensitive to membrane molecular order. This allowed us to investigate how DHA-Bodipy physically adapted to ordered lipid microdomains. To accomplish this, we employed steady-state and time-resolved fluorescence anisotropy measurements in lipid vesicles of varying composition. Similar to cell culture studies, the probe was highly sensitive to membrane order in lipid vesicles. Moreover, these experiments revealed, relative to controls, that upon incorporation into highly ordered microdomains, DHA-Bodipy underwent an increase in its fluorescence lifetime and molecular order. In addition, the probe displayed a significant reduction in its rotational diffusion compared to controls. Altogether, DHA-Bodipy was highly sensitive to membrane order and revealed for the first time that DHA, despite its flexibility, could become ordered with less rotational motion inside ordered lipid microdomains. Mechanistically, this explains how DHA acyl chains can increase order upon formation of lipid microdomains in vivo.