Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures

Images of giant unilamellar vesicles (GUVs) formed by different phospholipid mixtures (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1, 2-dilauroyl-sn-glycero-3-phosphocholine (DPPC/DLPC) 1:1 (mol/mol), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPE/DPPC), 7:3 and 3:7 (mol/mol) at different temperatures were obtained by exploiting the sectioning capability of a two-photon excitation fluorescence microscope. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN), 6-propionyl-2-dimethylamino-naphthalene (PRODAN), and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE) were used as fluorescent probes to reveal domain coexistence in the GUVs. We report the first characterization of the morphology of lipid domains in unsupported lipid bilayers. From the LAURDAN intensity images the excitation generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domain. On the basis of the phase diagram of each lipid mixture, we found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region in all lipid mixtures. At temperatures corresponding to the phase coexistence region we observed lipid domains of different sizes and shapes, depending on the lipid sample composition. In the case of GUVs formed by DPPE/DPPC mixture, the gel DPPE domains present different shapes, such as hexagonal, rhombic, six-cornered star, dumbbell, or dendritic. At the phase coexistence region, the gel DPPE domains are moving and growing as the temperature decreases. Separated domains remain in the GUVs at temperatures corresponding to the solid region, showing solid-solid immiscibility. A different morphology was found in GUVs composed of DLPC/DPPC 1:1 (mol/mol) mixtures. At temperatures corresponding to the phase coexistence, we observed the gel domains as line defects in the GUV surface. These lines move and become thicker as the temperature decreases. As judged by the LAURDAN GP histogram, we concluded that the lipid phase characteristics at the phase coexistence region are different between the DPPE/DPPC and DLPC/DPPC mixtures. In the DPPE/DPPC mixture the coexistence is between pure gel and pure liquid domains, while in the DLPC/DPPC 1:1 (mol/mol) mixture we observed a strong influence of one phase on the other. In all cases the domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This observation is also novel for unsupported lipid bilayers.     

Coexisting stripe- and patch-shaped domains in giant unilamellar vesicles

We report a new type of gel-liquid phase segregation in giant unilamellar vesicles (GUVs) of mixed lipids. Coexisting patch- and stripe-shaped gel domains in GUV bilayers composed of DOPC/DPPC or DLPC/DPPC are observed by confocal fluorescence microscopy. The lipids in stripe domains are shown to be tilted according to the DiIC18 fluorescence intensity dependence on the excitation polarization. The patch domains are found to be mainly composed of DPPC-d62 according to the coherent anti-Stokes Raman scattering (CARS) images of DOPC/DPPC-d62 bilayers. When cooling GUVs from above the miscibility temperature, the patch domains start to appear between the chain melting and the pretransition temperature of DPPC. In GUVs containing a high molar percentage of DPPC, the stripe domains form below the pretransition temperature. Our observations suggest that the patch and stripe domains are in the Pbeta' and Lbeta' gel phases, respectively. According to the thermoelastic properties of GUVs described by Needham and Evans [(1988) Biochemistry 27, 8261-8269], the Pbeta' and Lbeta' phases are formed at relatively low and high membrane tensions, respectively. GUVs with high DPPC percentage have high membrane surface tension and thus mainly exhibit Lbeta' domains, while GUVs with low DPPC percentage have low membrane surface tension and form Pbeta' domains accordingly. Adding negatively charged lipid to the lipid mixtures or applying an osmotic pressure to GUVs using sucrose solutions releases the surface tension and leads to the disappearance of the Lbeta' gel phase. The relationship between the observed domains in free-standing GUV bilayers and those in supported bilayers is discussed.     

A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).     

Complexation of phosphatidylcholine lipids with cholesterol

It is postulated that the specific interactions between cholesterol and lipids in biological membranes are crucial in the formation of complexes leading subsequently to membrane domains (so-called rafts). These interactions are studied in molecular dynamics simulations performed on a dipalmitoylphosphatidylcholine (DPPC)-cholesterol bilayer mixture and a dilauroylphosphatidylcholine (DLPC)-cholesterol bilayer mixture, both having a cholesterol concentration of 40 mol %. Complexation of the simulated phospholipids with cholesterol is observed and visualized, exhibiting 2:1 and 1:1 stoichiometries. The most popular complex is found to be 1:1 in the case of DLPC, whereas the DPPC system carries a larger population of 2:1 complexes. This difference in the observed populations of complexes is shown to be a result of differences in packing geometry and phospholipid conformation due to the differing tail length of the two phosphatidylcholine lipids. Furthermore, aggregation of these complexes appears to form hydrogen-bonded networks in the system containing a mixture of cholesterol and DPPC. The CH...O hydrogen bond plays a crucial role in the formation of these complexes as well as the hydrogen bonded aggregates. The aggregation and extension of such a network implies a possible means by which phospholipid:cholesterol domains form.     

Nanoscopic lipid domain dynamics revealed by atomic force microscopy

Intrinsic heterogeneities, represented as domain formations in biological membranes, are important to both the structure and function of the membranes. We observed domain formations in mixed lipid bilayers of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol) in a fluid environment using an atomic force microscope (AFM). At room temperature, we demonstrated that both microscopic and nanoscopic domains coexist and the DPPC-rich domain is approximately 1.4 nm higher than the surrounding DLPC-rich membrane areas as a consequence of intrinsic phase differences. DPPC-rich microscopic domains became larger as DPPC concentration increased. In cholesterol-free mixtures, nanoscopic DPPC-rich domain sizes ranged from 26 to 46 nm depending on phospholipid concentration. Domain size varied between 33 and 48 nm in the presence of cholesterol (0 < or = [chol] < or = 40). The nanoscopic domains were markedly fragmented near [chol] = 0.135 and appeared to fuse more readily into microscopic domains at higher and lower [chol]. By phase balance analyses we demonstrated phase behavior differences between a free-vesicle GUV system studied by confocal light microscopy and a supported membrane system studied by AFM. We propose a new three-dimensional phase diagram elucidating the effects of a solid substrate support on lipid phase behavior relevant to complex membrane phase phenomena in biological systems.     

Distribution of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine/cholesterol monolayers: a model for lipid rafts

The distribution of low concentrations of ganglioside GM1 in L-alpha-dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol monolayers supported on mica has been studied using atomic force microscopy (AFM). The monolayers studied correspond to a pure gel phase and a mixture of liquid-expanded (LE) and liquid-condensed (LC) phases for DPPC and to a single homogeneous liquid-ordered phase for 2:1 DPPC/cholesterol. The addition of 2.5-5% GM1 to phase-separated DPPC monolayers resulted in small round ganglioside-rich microdomains in the center and at the edges of the LC domains. Higher amounts of GM1 (10%) give numerous filaments in the center of the LC domains and larger patches at the edges. A gel phase DPPC monolayer containing GM1 showed large domains containing a network of GM1-rich filaments. The addition of GM1 to a liquid-ordered 2:1 DPPC/cholesterol monolayer gives small, round domains that vary in size from 50 to 150 nm for a range of surface pressures. Larger amounts of GM1 lead to coalescence of the small, round domains to give longer filaments that cover 30-40% of the monolayer surface for 10 mol % GM1. The results indicate that biologically relevant GM1 concentrations lead to submicron-sized domains in a cholesterol-rich liquid-ordered phase that is analogous to that found in detergent-insoluble membrane fractions, and are thought to be important in membrane microdomains or rafts. This demonstrates that AFM studies of model monolayers and bilayers provide a powerful method for the direct detection of microdomains that are too small for study with most other techniques.     

The phase behavior of mixed aqueous dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phases and transition sequences for aqueous dispersions of mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) have been studied by differential scanning calorimetry, dynamic x-ray diffraction, freeze-fracture electron microscopy, 31P-nuclear magnetic resonance spectroscopy, and Fourier-transform infrared spectroscopy. The results have been used to construct a dynamic phase diagram of the binary mixture as a function of temperature over the range 20 degrees-90 degrees C. It is concluded that DPPC and 1,2-DPG form two complexes in the gel phase, the first one with a DPPC/1,2-DPG molar ratio of 55:45 and the second one at a molar ratio of approximately 1:2, defining three different regions in the phase diagram. Two eutectic points are postulated to occur: one at a very low 1,2-DPG concentration and the other at a 1,2-DPG concentration slightly higher than 66 mol%. At temperatures higher than the transition temperature, lamellar phases were predominant at low 1,2-DPG concentrations, but nonlamellar phases were found to be predominant at high proportions of 1,2-DPG. A very important aspect of these DPPC/1,2-DPG mixtures was that, in the gel phase, they showed a ripple structure, as seen by freeze-fracture electron microscopy and consistent with the high lamellar repeat spacings seen by x-ray diffraction. Ripple phase characteristics were also found in the fluid lamellar phases occurring at concentrations up to 35.6 mol% of 1,2-DPG. Evidence was obtained by Fourier transform infrared spectroscopy of the dehydration of the lipid-water interface induced by the presence of 1,2-DPG. The biological significance of the presence of diacylglycerol in membrane lipid domains is discussed.     

Temperature-controlled structure and kinetics of ripple phases in one- and two-component supported lipid bilayers

Temperature-controlled atomic force microscopy (AFM) has been used to visualize and study the structure and kinetics of ripple phases in one-component dipalmitoylphosphatidylcholine (DPPC) and two-component dimyristoylphosphatidylcholine-distearoylphosphatidylcholine (DMPC-DSPC) lipid bilayers. The lipid bilayers are mica-supported double bilayers in which ripple-phase formation occurs in the top bilayer. In one-component DPPC lipid bilayers, the stable and metastable ripple phases were observed. In addition, a third ripple structure with approximately twice the wavelength of the metastable ripples was seen. From height profiles of the AFM images, estimates of the amplitudes of the different ripple phases are reported. To elucidate the processes of ripple formation and disappearance, a ripple-phase DPPC lipid bilayer was taken through the pretransition in the cooling and the heating direction and the disappearance and formation of ripples was visualized. It was found that both the disappearance and formation of ripples take place virtually one ripple at a time, thereby demonstrating the highly anisotropic nature of the ripple phase. Furthermore, when a two-component DMPC-DSPC mixture was heated from the ripple phase and into the ripple-phase/fluid-phase coexistence temperature region, the AFM images revealed that several dynamic properties of the ripple phase are important for the melting behavior of the lipid mixture. Onset of melting is observed at grain boundaries between different ripple types and different ripple orientations, and the longer-wavelength metastable ripple phase melts before the shorter-wavelength stable ripple phase. Moreover, it was observed that the ripple phase favors domain growth along the ripple direction and is responsible for creating straight-edged domains with 60 degrees and 120 degrees angles, as reported previously.     

Studies of short-wavelength collective molecular motions in lipid bilayers using high resolution inelastic X-ray scattering

We summarize a series of experimental results made with the newly developed high resolution X-ray scattering (IXS) instrument on two pure lipid bilayers, including dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) in both gel and liquid crystal phases, and lipid bilayers containing cholesterol. By analyzing the IXS data based on the generalized three effective eigenmode model (GTEE), we obtain dispersion relations of the high frequency density oscillations (phonons) of lipid molecules in these bilayers. We then compare the dispersion relations of pure lipid bilayers of different chain lengths among themselves and the dispersion relations of pure lipid bilayers with those of the cholesterol containing bilayers. We also compare our experimental results with collective dynamics data generated by computer molecular dynamics (MD) simulations for dipalmitoylphosphatidylcholine (DPPC) in gel phase and DMPC in liquid crystal phase.     

Thermodynamic characterization of interactions between ornithine transcarbamylase leader peptide and phospholipid bilayer membranes

The interactions of the targeting sequence of the mitochondrial enzyme ornithine transcarbamylase with phospholipid bilayers of different molecular compositions have been studied by high-sensitivity heating and cooling differential scanning calorimetry, high-sensitivity isothermal titration calorimetry, fluorescence spectroscopy, and electron microscopy. These studies indicate that the leader peptide interacts strongly with dipalmitoylphosphatidylcholine (DPPC) bilayer membranes containing small mole percents of the anionic phospholipids dipalmitoylphosphatidylglycerol (DPPG) or brain phosphatidylserine (brain PS) but not with pure phosphatidylcholines. For the first time, the energetics of the leader peptide-membrane interaction have been measured directly by using calorimetric techniques. At 20 degrees C, the association of the peptide with the membrane is exothermic and characterized by an association constant of 2.3 X 10(6) M-1 in the case of phosphatidylglycerol-containing and 0.35 X 10(6) M-1 in the case of phosphatidylserine-containing phospholipid bilayers. In both cases, the enthalpy of association is -60 kcal/mol of peptide. Additional experiments using fluorescence techniques suggest that the peptide does not penetrate deeply into the hydrophobic core of the membrane. The addition of the leader peptide to DPPC/DPPG (5:1) or DPPC/brain PS (5:1) small sonicated vesicles results in vesicle fusion. The fusion process is dependent on peptide concentration and is maximal at the phase transition temperature of the vesicles and minimal at temperatures below the phase transition.     

Preferential accumulation of Abeta(1-42) on gel phase domains of lipid bilayers: an AFM and fluorescence study

Peptide-membrane interactions have been implicated in both the toxicity and aggregation of beta-amyloid (Abeta) peptides. Recent studies have provided evidence for the involvement of liquid-ordered membrane domains known as lipid rafts in the formation and aggregation of Abeta. As a model, we have examined the interaction of Abeta(1-42) with phase separated DOPC/DPPC lipid bilayers using a combination of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF). AFM images show that addition of Abeta to preformed supported bilayers leads to accumulation of small peptide aggregates exclusively on the gel phase DPPC domains. Initial aggregates are observed approximately 90 min after peptide addition and increase in diameter to 45-150 nm within 24 h. TIRF studies with a mixture of Abeta and Abeta-Fl demonstrate that accumulation of the peptide on the gel phase domains occurs as early as 15 min after Abeta addition and is maintained for over 24 h. By contrast, Abeta is randomly distributed throughout both fluid and gel phases when the peptide is reconstituted into DOPC/DPPC vesicles prior to formation of a supported bilayer. The preferential accumulation of Abeta on DPPC domains suggests that rigid domains may act as platforms to concentrate peptide and enhance its aggregation and may be relevant to the postulated involvement of lipid rafts in modulating Abeta activity in vivo.     

High-resolution electron density profiles reveal influence of fatty acids on bilayer structure.

Small-angle x-ray diffraction studies were performed on gel phase-oriented bilayers of dipalmitoylphosphatidylcholine (DPPC) and DPPC containing 40 mol% of either palmitic acid (PA) or palmitic acid brominated at the 2-position (BPA). Oriented samples were prepared using a method developed by us, which is as simple as powder sample preparations while offering all the advantages of oriented samples made by traditional methods. Phases were determined using swelling experiments with structure factors plotted in reciprocal space, creating a relatively smooth curve as the amount of water between the bilayers was changed. Continuous Fourier transforms were also calculated to further test the consistency of the phase assignments. The diffraction data were used to calculate absolute electron density profiles for different bilayers to a resolution of 5-6 A. Analysis indicates the following: (a) The electron density profiles for the three preparations are virtually identical in the hydrocarbon chain region. (b) There is a decrease in the electron density of the glycerol backbone-headgroup region and d-space in DPPC-PA compared to DPPC. (c) The bromine of fatty-acid brominated at the 2-position is in the vicinity of the glycerol backbone. (d) The bilayer thickness of DPPC containing either brominated or unbrominated fatty acid remains relatively constant with increased levels of hydration, unlike DPPC bilayers.     

Experimental evidence for hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

Hydrophobic matching, in which transmembrane proteins cause the surrounding lipid bilayer to adjust its hydrocarbon thickness to match the length of the hydrophobic surface of the protein, is a commonly accepted idea in membrane biophysics. To test this idea, gramicidin (gD) was embedded in 1, 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1, 2-myristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at the peptide/lipid molar ratio of 1:10. Circular dichroism (CD) was measured to ensure that the gramicidin was in the beta6.3 helix form. The bilayer thickness (the phosphate-to-phosphate distance, or PtP) was measured by x-ray lamellar diffraction. In the Lalpha phase near full hydration, PtP is 30.8 A for pure DLPC, 32.1 A for the DLPC/gD mixture, 35.3 A for pure DMPC, and 32.7 A for the DMPC/gD mixture. Gramicidin apparently stretches DLPC and thins DMPC toward a common thickness as expected by hydrophobic matching. Concurrently, gramicidin-gramicidin correlations were measured by x-ray in-plane scattering. In the fluid phase, the gramicidin-gramicidin nearest-neighbor separation is 26.8 A in DLPC, but shortens to 23.3 A in DMPC. These experiments confirm the conjecture that when proteins are embedded in a membrane, hydrophobic matching creates a strain field in the lipid bilayer that in turn gives rise to a membrane-mediated attractive potential between proteins.     

Atomic force microscopy studies of ganglioside GM1 domains in phosphatidylcholine and phosphatidylcholine/cholesterol bilayers.

The distribution of ganglioside in supported lipid bilayers has been studied by atomic force microscopy. Hybrid dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylethanolamine (DPPE) and (2:1 DPPC/cholesterol)/DPPE bilayers were prepared using the Langmuir Blodgett technique. Egg PC and DPPC bilayers were prepared by vesicle fusion. Addition of ganglioside GM1 to each of the lipid bilayers resulted in the formation of heterogeneous surfaces that had numerous small raised domains (30--200 nm in diameter). Incubation of these bilayers with cholera toxin B subunit resulted in the detection of small protein aggregates, indicating specific binding of the protein to the GM1-rich microdomains. Similar results were obtained for DPPC, DPPC/cholesterol, and egg PC, demonstrating that the overall bilayer morphology was not dependent on the method of bilayer preparation or the fluidity of the lipid mixture. However, bilayers produced by vesicle fusion provided evidence for asymmetrically distributed GM1 domains that probably reflect the presence of ganglioside in both inner and outer monolayers of the initial vesicle. The results are discussed in relation to recent inconsistencies in the estimation of sizes of lipid rafts in model and natural membranes. It is hypothesized that small ganglioside-rich microdomains may exist within larger ordered domains in both natural and model membranes.     

Interaction of gentamicin with phosphatidylserine/phosphatidylcholine mixtures in adsorption monolayers and thin liquid films: morphology and thermodynamic properties

Gentamicin possesses strong adverse actions like oto and nephrotoxicity. The latter is a result of strong gentamicin–acid phospholipid interactions, resulting in cell fusion, fission, etc., ions as calcium interact with gentamicin and effectively deter its toxicity. In this work, the interactions of gentamicin and Ca²⁺ with phosphatidylserine/phosphatidylcholine (PS/PC) mixtures of different ratio are experimentally characterized. Special attention is paid to bridge thermodynamic and morphological properties of adsorption monolayers and thin liquid films (TLFs) composed of these lipid mixtures. Our results show that gentamicin decreases the stability of common black TLFs formed of pure PS coupled with suppression of lipid surface adsorption to the monolayers at the air–water interface; also, gentamicin reveals effects of lowering of lipid spreading on the interface and significant loss of material during monolayer cycling, increase of condensed phase, and organization of dense net-like domain monolayer texture. Gentamicin addition results in opposite effects for films formed of DPPC/PS (95:5) mixture. It increases the stability of Newton black TLFs formed by DPPC/PS correlated with faster and stronger surface adsorption and better surface spreading; also, gentamicin lowers the amount of condensed phase and organization of domains of smaller size. We also showed that Ca²⁺ itself decreases the stability of common black TLFs formed of PS accompanied with weaker surface adsorption, formation of higher amounts of condensed phase and organization of domains. In our experiments, Ca²⁺ softens, even deters, the effects of gentamicin on both PS and DPPC/PS films.     

Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.     

Phase behavior and nanoscale structure of phospholipid membranes incorporated with acylated C14-peptides

The thermotropic phase behavior and lateral structure of dipalmitoylphosphatidylcholine (DPPC) lipid bilayers containing an acylated peptide has been characterized by differential scanning calorimetry (DSC) on vesicles and atomic force microscopy (AFM) on mica-supported bilayers. The acylated peptide, which is a synthetic decapeptide N-terminally linked to a C14 acyl chain (C14-peptide), is incorporated into DPPC bilayers in amounts ranging from 0-20 mol %. The calorimetric scans of the two-component system demonstrate a distinct influence of the C14-peptide on the lipid bilayer thermodynamics. This is manifested as a concentration-dependent downshift of both the main phase transition and the pretransition. In addition, the main phase transition peak is significantly broadened, indicating phase coexistence. In the AFM imaging scans we found that the C14-peptide, when added to supported gel phase DPPC bilayers, inserts preferentially into preexisting defect regions and has a noticeable influence on the organization of the surrounding lipids. The presence of the C14-peptide gives rise to a laterally heterogeneous bilayer structure with coexisting lipid domains characterized by a 10 A height difference. The AFM images also show that the appearance of the ripple phase of the DPPC lipid bilayers is unaffected by the C14-peptide. The experimental results are supported by molecular dynamics simulations, which show that the C14-peptide has a disordering effect on the lipid acyl chains and causes a lateral expansion of the lipid bilayer. These effects are most pronounced for gel-like bilayer structures and support the observed downshift in the phase-transition temperature. Moreover, the molecular dynamics data indicate a tendency of a tryptophan residue in the peptide sequence to position itself in the bilayer headgroup region.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Characterization of complexes formed in fully hydrated dispersions of dipalmitoyl derivatives of phosphatidylcholine and diacylglycerol.

The phase diagram of fully hydrated binary mixtures of dipalmitoylphosphatidylcholine (DPPC) with 1,2-dipalmitoylglycerol (DPG) published recently by López-García et al. identifies regions where stoichiometric complexes of 1:1 and 1:2 DPPC:DPG, respectively, are formed. In this study, the structural parameters of the 1:1 complex in the presence of pure DPPC was characterized by synchrotron low angle and static x-ray diffraction methods. Structural changes upon transitions through phase boundaries were correlated with enthalpy changes observed by differential scanning calorimetry in mixtures of DPPC with 5, 7.5, 10, and 20 mol% DPG dispersed in excess water. Phase separation of a complex in gel phase could be detected by calorimetry in the mixture containing 5 mol% DPG but was not detectable by synchrotron low angle x-ray diffraction. Static x-ray measurements show evidence of phase separation, particularly in the reflections indexing chain packing. In the mixture containing 7.5 mol% DPG, two distinct lamellar repeat spacings could be seen in the temperature range from 25 to 34 degrees C. The lamellar spacing of about 6.6 nm was assigned to pure gel phase DPPC because the change in the spacing corresponds with thermal transition of the pure phospholipid, and a longer repeat spacing of about 7.2 nm was assigned to domains of the 1:1 complex of DPPC-DPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preferential accumulation of Aβ(1−42) on gel phase domains of lipid bilayers: An AFM and fluorescence study

Peptide-membrane interactions have been implicated in both the toxicity and aggregation of β-amyloid (Aβ) peptides. Recent studies have provided evidence for the involvement of liquid-ordered membrane domains known as lipid rafts in the formation and aggregation of Aβ. As a model, we have examined the interaction of Aβ(1−42) with phase separated DOPC/DPPC lipid bilayers using a combination of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF). AFM images show that addition of Aβ to preformed supported bilayers leads to accumulation of small peptide aggregates exclusively on the gel phase DPPC domains. Initial aggregates are observed approximately 90 min after peptide addition and increase in diameter to 45-150 nm within 24 h. TIRF studies with a mixture of Aβ and Aβ-Fl demonstrate that accumulation of the peptide on the gel phase domains occurs as early as 15 min after Aβ addition and is maintained for over 24 h. By contrast, Aβ is randomly distributed throughout both fluid and gel phases when the peptide is reconstituted into DOPC/DPPC vesicles prior to formation of a supported bilayer. The preferential accumulation of Aβ on DPPC domains suggests that rigid domains may act as platforms to concentrate peptide and enhance its aggregation and may be relevant to the postulated involvement of lipid rafts in modulating Aβ activity in vivo.