Cytotoxicity of bovine α-lactalbumin: Oleic acid complexes correlates with the disruption of lipid membranes

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.     

Interactions of pulmonary surfactant protein A with phospholipid monolayers change with pH.

The interaction of pulmonary surfactant protein A (SP-A) labeled with Texas Red (TR-SP-A) with monolayers containing zwitterionic and acidic phospholipids has been studied at pH 7.4 and 4.5 using epifluorescence microscopy. At pH 7.4, TR-SP-A expanded the pi-A isotherms of film of dipalmitoylphosphatidylcholine (DPPC). It interacted at high concentration at the edges of condensed-expanded phase domains, and distributed evenly at lower concentration into the fluid phase with increasing pressure. At pH 4.5, TR-SP-A expanded DPPC monolayers to a slightly lower extent than at pH 7.4. It interacted primarily at the phase boundaries but it did not distribute into the fluid phase with increasing pressure. Films of DPPC/dipalmitoylphosphatidylglycerol (DPPG) 7:3 mol/mol were somewhat expanded by TR-SP-A at pH 7.4. The protein was distributed in aggregates only at the condensed-expanded phase boundaries at all surface pressures. At pH 4.5 TR-SP-A caused no expansion of the pi-A isotherm of DPPC/DPPG, but its fluorescence was relatively homogeneously distributed throughout the expanded phase at all pressures studied. These observations can be explained by a combination of factors including the preference for SP-A aggregates to enter monolayers at packing dislocations and their disaggregation in the presence of lipid under increasing pressure, together with the influence of pH on the aggregation state of SP-A and the interaction of SP-A with zwitterionic and acidic lipid.     

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.     

Preparation of giant unilamellar vesicles from damp lipid film for better lipid compositional uniformity

Giant unilamellar vesicles (GUVs) containing cholesterol often have a wide distribution in lipid composition. In this study, GUVs of 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)/1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC)/cholesterol and 1,2-diphytanoyl-sn-glycero-3-phosphocholine(diPhyPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine(DPPC)/cholesterol were prepared from dry lipid films using the standard electroformation method as well as a modified method from damp lipid films, which are made from compositional uniform liposomes prepared using the Rapid Solvent Exchange (RSE) method. We quantified the lipid compositional distributions of GUV by measuring the miscibility transition temperature of GUVs using fluorescence microscopy, since a narrower distribution in the transition temperature should correspond to a more uniform distribution in GUV lipid composition. Cholesterol molecules can demix from other lipids in dry state and form cholesterol crystals. Using optical microscopy, micron-sized crystals were observed in some dry lipid films. Thus, a major cause of GUV lipid compositional heterogeneity is the demixing of lipids in the dry film state. By avoiding the dry film state, GUVs prepared from damp lipid films have a better uniformity in lipid composition, and the standard deviations of miscibility transition temperature are about 2.5 times smaller than that of GUVs prepared from dry lipid films. Comparing the two ternary systems, diPhyPC/DPPC/cholesterol GUVs has a larger cholesterol compositional heterogeneity, which directly correlates with the low maximum solubility of cholesterol in diPhyPC lipid bilayers (40.2±0.5mol%) measured by light scattering. Our data indicate that cholesterol interacts far less favorably with diPhyPC than it does with other PCs. The damp lipid film method also has a potential of preparing GUVs from cell membranes containing native proteins without going through a dry state.     

Lipid headgroup discrimination by antimicrobial peptide LL-37: insight into mechanism of action

Interaction of the human antimicrobial peptide LL-37 with lipid monolayers has been investigated by a range of complementary techniques including pressure-area isotherms, insertion assay, epifluorescence microscopy, and synchrotron x-ray scattering, to analyze its mechanism of action. Lipid monolayers were formed at the air-liquid interface to mimic the surface of the bacterial cell wall and the outer leaflet of erythrocyte cell membrane by using phosphatidylglycerol (DPPG), phosphatidylcholine (DPPC), and phosphatidylethanolamine (DPPE) lipids. LL-37 is found to readily insert into DPPG monolayers, disrupting their structure and thus indicating bactericidal action. In contrast, DPPC and DPPE monolayers remained virtually unaffected by LL-37, demonstrating its nonhemolytic activity and lipid discrimination. Specular x-ray reflectivity data yielded considerable differences in layer thickness and electron-density profile after addition of the peptide to DPPG monolayers, but little change was seen after peptide injection when probing monolayers composed of DPPC and DPPE. Grazing incidence x-ray diffraction demonstrated significant peptide insertion and lateral packing order disruption of the DPPG monolayer by LL-37 insertion. Epifluorescence microscopy data support these findings.     

Phospholipase D activity is regulated by product segregation and the structure formation of phosphatidic acid within model membranes

Phospholipase D from Streptomyces chromofuscus (scPLD) hydrolyzes phosphatidylcholines (PC) to produce choline and phosphatidic acid (PA), a lipid messenger molecule within biological membranes. To scrutinize the influence of membrane structure on scPLD activity, three different substrate-containing monolayers are used as model systems: pure dipalmitoylphosphatidylcholine (DPPC) as well as equimolar mixtures of DPPC/n-hexadecanol (C(16)OH) and DPPC/dipalmitoylglycerol (DPG). The activity of scPLD toward these monolayers is tested by infrared reflection-absorption spectroscopy and exhibits different dependencies on surface pressure. For pure DPPC, the catalytic turnover drastically drops above 20 mN/m. On addition of C(16)OH, this strong decrease starts at 5 mN/m. For the DPPC/DPG system, the reaction yield linearly decreases between 5 and 25 mN/m. The difference in scPLD activity is correlated to the phase state of the monolayers as examined by x-ray diffraction, Brewster angle microscopy, and atomic force microscopy. Because the additives C(16)OH and DPG mediate the miscibility of PC and PA, only a basal activity of scPLD is observed toward the mixed systems at higher surface pressures. At pure DPPC monolayers, scPLD is activated after the segregation of initially formed PA. Furthermore, scPLD is inhibited when the lipids in the PA-rich domains adopt an upright orientation. This phenomenon offers a self-regulating mechanism for the concentration of the second messenger PA within biological membranes.     

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (l(d)) domains on the surface of GUVs composed of 40:60 mol% DOPC/DPPC, over a wide range of temperatures (14-25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine(TM) rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.     

FTIR spectroscopic study of molecular organization of the antibiotic amphotericin B in aqueous solution and in DPPC lipid monolayers containing the sterols cholesterol and ergosterol

Langmuir monolayers of amphotericin B (AmB) were investigated by recording π-A isotherms under different pH conditions. To gain a better insight into antibiotic-membrane interactions they were monitored by use of the ATR-FTIR spectroscopy. It was observed for AmB monolayers that the limiting molecular area was larger at high than at neutral pH. Analysis of FTIR spectra at different pH revealed substantial differences, depending on ionic state, for different orientations of AmB molecules. These results enable better understanding of the participation of functional groups in the interactions between AmB and sterol-containing DPPC membranes. AmB molecules incorporated into two-component lipid monolayers bind strongly to the ergosterol-rich membrane (maximum penetration surface pressures ca 35?mN/m). The FTIR spectra revealed that the ionic state of AmB and the presence of sterols led to changes in membrane fluidity and molecular packing of the AmB molecules in the lipid membranes. These investigations should be further investigated to discover the molecular mechanism responsible for the mode of action AmB in biological systems.     

Aggregation of puroindoline in phospholipid monolayers spread at the air-liquid interface

Puroindolines, cationic and cystine-rich low molecular weight lipid binding proteins from wheat seeds, display unique foaming properties and antimicrobial activity. To unravel the mechanism involved in these properties, the interaction of puroindoline-a (PIN-a) with dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) monolayers was studied by coupling Langmuir-Blodgett and imaging techniques. Compression isotherms of PIN-a/phospholipid monolayers and adsorption of PIN-a to lipid monolayers showed that the protein interacted strongly with phospholipids, especially with the anionic DPPG. The electrostatic contribution led to the formation of a highly stable lipoprotein monolayer. Confocal laser scanning microscopy and atomic force microscopy showed that PIN-a was mainly inserted in the liquid-expanded phase of the DPPC, where it formed an aggregated protein network and induced the fusion of liquid-condensed domains. For DPPG, the protein partitioned in both the liquid-expanded and liquid-condensed phases, where it was aggregated. The extent of protein aggregation was related both to the physical state of phospholipids, i.e., condensed or expanded, and to the electrostatic interactions between lipids and PIN-a. Aggregation of PIN-a at air-liquid and lipid interfaces could account for the biological and technological properties of this wheat lipid binding protein.     

A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles

We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding of the enzyme to the liquid regions was also found. The lipid phase of the GUV protein binding region was verified by the introduction of 6-propionyl-2-(dimethylamino) naphthalene, which partitions quickly into the lipid fluid phase. Preferential hydrolysis of the liquid domains supported the conclusions based on the binding studies. sPLA2 hydrolyzes the liquid domains in the binary lipid mixtures DLPC:DAPC and DMPC:DMPE, indicating that the solid-phase packing of DAPC and DMPE interferes with sPLA2 binding, irrespective of the phospholipid headgroup. These studies emphasize the importance of lateral packing of the lipids in C. atrox sPLA2 enzymatic hydrolysis of a membrane surface.     

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.     

Study in mono and bilayers of the interaction of hepatitis G virus (GBV-C/HGV) synthetic antigen E2(99-118) with cell membrane phospholipids

The interaction of the hepatitis G synthetic peptide E2(99-118) with cell membrane phospholipids of different characteristics such as dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) was studied by Langmuir isotherms. Epifluorescence microscopy and Atomic force microscopy (AFM) was also used to study interactions with DPPC. Compression isotherms of DPPC/E2(99-118) and DPPG/E2(99-118) mixed monolayers showed negative deviation from ideallity consistent with the existence of attractive interactions. The incorporation of the peptide in DPPC monolayer was also confirmed in epifluorescence microscopy and AFM studies. The peptide retarded the formation of DPPC domains and did not let the phospholipid get organized. No important differences in the interactions with DPPC (neutral) or DPPG (anionic) were found, thus suggesting that electrostatics forces do not have a predominant influence in these interactions.     

Surfactant protein A forms extensive lattice-like structures on 1,2-dipalmitoylphosphatidylcholine/rough-lipopolysaccharide-mixed monolayers

Due to the inhalation of airborne particles containing bacterial lipopolysaccharide (LPS), these molecules might incorporate into the 1,2-dipalmitoylphosphatidylcholine (DPPC)-rich monolayer and interact with surfactant protein A (SP-A), the major surfactant protein component involved in host defense. In this study, epifluorescence microscopy combined with a surface balance was used to examine the interaction of SP-A with mixed monolayers of DPPC/rough LPS (Re-LPS). Binary monolayers of Re-LPS plus DPPC showed negative deviations from ideal behavior of the mean areas in the films consistent with partial miscibility and attractive interaction between the lipids. This interaction resulted in rearrangement and reduction of the size of DPPC-rich solid domains in DPPC/Re-LPS monolayers. The adsorption of SP-A to these monolayers caused expansion in the lipid molecular areas. SP-A interacted strongly with Re-LPS and promoted the formation of DPPC-rich solid domains. Fluorescently labeled Texas red-SP-A accumulated at the fluid-solid boundary regions and formed networks of interconnected filaments in the fluid phase of DPPC/Re-LPS monolayers in a Ca(2+)-independent manner. These lattice-like structures were also observed when TR-SP-A interacted with lipid A monolayers. These novel results deepen our understanding of the specific interaction of SP-A with the lipid A moiety of bacterial LPS.     

Lipid interaction of diphtheria toxin and mutants. A study with phospholipid and protein monolayers

To study the structural change of diphtheria toxin (DT) induced by low pH and its influence on the interaction with membrane lipids, protein and lipid monolayers were formed and characterized. DT at neutral and acidic pH forms stable monolayers, whose surface-pressure-increase curves allow an estimation of the apparent molecular area of 29.5 nm2/molecule at pH 7.4 (corresponding to a radius of 3.06 nm) and 34.5 nm2/molecule at pH 5.0 (corresponding to a radius of 3.32 nm). DT at pH 7.4 does not insert into phospholipid monolayers, while at pH 5.0 it penetrates into the lipid layer with a portion of apparent molecular area of 21.0 nm2/molecule (corresponding to a radius of 2.6 nm). The low-pH driven lipid interaction of the toxin is favoured by the presence of acidic phospholipids, without an apparent requirement for a particular class of negative lipids. The DT mutants crm 45 and crm 197 are capable of hydrophobic interaction already at neutral pH and cause an increase of surface pressure with a further increase upon acidification.     

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - effects beyond electrostatic

The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to study the behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151-175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7? in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP(2) in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151-175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane.     

Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks and reconstruction of 3D domain morphology using active surface models. This method permits the reconstruction of the spherical surface of GUVs and determination of the area fractions of coexisting lipid domains at the level of single vesicles. Obtaining area fractions enables the scrutiny of the lever rule along lipid phase diagram's tie lines and to test whether or not the coexistence of lipid domains in GUVs correspond to equilibrium thermodynamic phases. The analysis was applied to DLPC/DPPC GUVs displaying coexistence of lipid domains. Our results confirm the lever rule, demonstrating that the observed membrane domains correspond to equilibrium thermodynamic phases (i.e., solid ordered and liquid disordered phases). In addition, the fact that the lever rule is validated from 11 to 14 randomly selected GUVs per molar fraction indicates homogeneity in the lipid composition among the explored GUV populations. In conclusion, our study shows that GUVs are reliable model systems to perform equilibrium thermodynamic studies of membranes.     

Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles

We assayed fusion events between giant unilamellar vesicles (GUVs) and budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus), the envelopes of which have been labeled with the fluorescent dye Alexa Fluor 488. This involves observing the intensity of fluorescence emitted from the lipid bilayer of single GUVs after fusion using laser scanning microscopy. Using this assay system, we found that fusion between single GUVs and BV envelopes was significantly enhanced at around pH 5.0-6.0, which suggests that: (1) envelope glycoprotein GP64-mediated membrane fusion within the endosome of insect cells was reproduced in our artificial system; (2) acidic phospholipids in GUVs are necessary for this fusion, which are in agreement with the previous results with conventional small liposomes including large unilamellar vesicles and multilamellar vesicles; and (3) the efficiency of fusion is significantly affected by membrane properties that can be modulated by adding cholesterol to GUV lipid bilayers. In addition, the microscopic observation of BV-fused single GUVs showed that a weak interaction occurred between BVs and GUVs containing dioleoylphosphatidylserine at pH 6.0-6.5, and components of BV envelopes were unevenly distributed upon fusion with GUVs containing saturated phospholipid with cholesterol. We further demonstrated that when the recombinant membrane protein, adrenergic β₂ receptor, was expressed on recombinant BV envelopes, the protein distribution on BV-fused GUVs was also affected by their lipid contents.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.     

Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes

Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii) the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP) suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo).