Luis A. Bagatolli

Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.

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.

Two-Photon Fluorescence Microscopy Observation of Shape Changes at the Phase Transition in Phospholipid Giant Unilamellar Vesicles

Using the sectioning effect of the two-photon fluorescence microscope, we studied the behavior of phospholipid giant unilamellar vesicles (GUVs) composed of pure diacylphosphatidylcholine phospholipids during the gel-to-liquid crystalline phase transition. We used the well-characterized excitation generalized polarization function (GP(ex)) of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN), which is sensitive to the changes in water content in the lipid vesicles, to monitor the phase transition in the GUVs. Even though the vesicles do not show temperature hysteresis at the main phase transition, we observed different behaviors of the vesicle shape, depending on how the GUV sample reaches the main phase transition. During the cooling cycles, we observed an increase in the vesicle diameter at the phase transition ( approximately 0.5-1%), followed by a decrease in the diameter when the vesicle reached the gel phase. During the heating cycles and close to the phase transition temperature, a surprising behavior is observed, showing a sequence of different vesicle shapes as follows: spherical-polygonal-ellipsoidal. We attribute these changes to the effect of lipid domain coexistence on the macroscopic structure of the GUVs. The shape hysteresis in the GUVs is reversible and largely independent of the temperature scan rate. In the presence of 30 mol% of cholesterol the events observed at the phase transition in the GUVs formed by pure phospholipids were absent.

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).

Interaction of Biotin with Streptavidin THERMOSTABILITY AND CONFORMATIONAL CHANGES UPON BINDING

The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75°C in unliganded protein to 112°C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka ∼1015 reproduce the behavior observed, but the estimated values of Tm are 15-20°C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.

Giant phospholipid vesicles: Comparison among the whole lipid sample characteristics using different preparation methods - A two photon fluorescence microscopy study

Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristics--in terms of lamellarity and morphology--of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicles preparation method. Our results show that the preparation of giant unilamellar vesicles by the application of external electric fields offers several advantages among the other methods tested here. Using this method a high yield (approximately 95%) of giant unilamellar vesicles with a narrow size distribution was obtained. Independently of the preparation method, some lipid structures, which are held together by lipid tethers, were identified and resolved. These particular lipid structures show shell thickness and size heterogeneity. Labeling the lipid samples with 6-lauroyl-2-(N,N-dimethylamino)naphtalene (LAURDAN) and using the LAURDAN generalized polarization function we show that the lipid packing in these tethers or tubes is similar to those found in the phospholipid vesicles. The fact that both vesicles and tethers are found in the lipid preparations indicates similar stability between these structures.

A Model for the Interaction of 6-Lauroyl-2-(N,N- dimethylamino)naphthalene with Lipid Environments: Implications for Spectral Properties

Although 6‐lauroyl‐2‐(N,N‐dimethylamino)naphthalene (LAURDAN) is now widely used as a probe for lipid systems, most studies focus on the effect of the lipid environment on its emission properties but not on the excitation properties. The present study is intended to investigate the excitation properties of LAURDAN in diverse lipid environments. To this end, the fluorescence properties of LAURDAN were studied in synthetic ester and ether phosphatidylcholines and sphingomyelin vesicles below, at and above the corresponding lipid main phase‐transition temperature. The excitation spectra of LAURDAN in these environments always show at least two well‐resolved bands. In the different lipid vesicles the behavior of the red band in the LAURDAN excitation spectra is sensitive to the lipid chemical environment near the probe fluorescent moiety and to the packing of the different lipid phases (gel and liquid crystalline). We propose that the interaction between the LAURDAN di‐methylamino group and the ester linkage of ester phospholipids is responsible for the strong stabilization of LAURDANs red excitation band in the gel phase of ester phospholipid vesicles. We discuss the consequence of these proposed ground‐state interactions on LAURDANs emission generalized polarization function. In the context of variable excitation wavelengths, information concerning solvent dipolar relaxation through excitation generalized polarization function is also discussed.

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 GUVs, 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.

Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence.

We have characterized the fluorescence properties of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN) in pure interfaces formed by sphingomyelin and 10 chemically related glycosphingolipids (GSLs).1 The GSLs contain neutral and anionic carbohydrate residues in their oligosaccharide chain. These systems were studied at temperatures below, at, or above the main phase transition temperature of the pure lipid aggregates. The extent of solvent dipolar relaxation around the excited fluorescence probe in the GSLs series increases with the magnitude of the glycosphingolipid polar headgroup below the transition temperature. This conclusion is based on LAURDANs excitation generalized polarization (GPex) and fluorescence lifetime values found in the different interfaces. A linear dependence between the LAURDAN GPex and the intermolecular spacing among the lipid molecules was found for both neutral and anionic lipids in the GSLs series. This relationship was also followed by phospholipids. We conclude that LAURDAN in these lipid aggregates resides in sites containing different amounts of water. The dimension of these sites increases with the size of the GSLs polar headgroup. The GP function reports on the concentration and dynamics of water molecules in these sites. Upon addition of cholesterol to Gg4Cer, the fluorescence behavior of LAURDAN was similar to that of pure cerebrosides and sphingomyelin vesicles. This observation was attributed to a change in the interfacial hydration as well as changes in the shape and size of the Gg4Cer aggregates in the presence of cholesterol. After the addition of cholesterol to gangliosides, the changes in the LAURDANs spectral parameters decrease progressively as the polar headgroup of these lipids becomes more complex. This finding suggests that the dehydration effect of cholesterol depends strongly on the curvature radius and the extent of hydration of these lipid aggregates. In the gel phase of phrenosine, GalCer, Gg3Cer, sulfatide, and sphingomyelin, the excitation red band (410 nm) of LAURDAN was reduced with respect to that of LAURDAN in the gel phase of pure phospholipids. This observation indicates a local environment that interacts differently with the ground state of LAURDAN in GSLs when compared with LAURDAN in phospholipids.

Segregation of saturated chain lipids in pulmonary surfactant films and bilayers

The physical properties of organized system (bilayers and monolayers at the air water interface) composed of bovine lipid extract surfactant (BLES) were studied using correlated experimental techniques. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN)-labeled giant unilamelar vesicles (mean diameter approximately 30 microm) composed of BLES were observed at different temperatures using two-photon fluorescence microscopy. As the temperature was decreased, dark domains (gel-like) appeared at physiological temperature (37 degrees C) on the surface of BLES giant unilamelar vesicles. The LAURDAN two-photon fluorescent images show that the gel-like domains span the lipid bilayer. Quantitative analysis of the LAURDAN generalized polarization function suggests the presence of a gel/fluid phase coexistence between 37 degrees C to 20 degrees C with low compositional and energetic differences between the coexisting phases. Interestingly, the microscopic scenario of the phase coexistence observed below 20 degrees C shows different domains shape compared with that observed between 37 degrees C to 20 degrees C, suggesting the coexistence of two ordered but differently organized lipid phases on the bilayer. Epifluorescence microscopy studies of BLES monomolecular films doped with small amounts of fluorescent lipids showed the appearance and growth of dark domains (liquid condensed) dispersed in a fluorescent phase (liquid expanded) with shapes and sizes similar to those observed in BLES giant unilamelar vesicles. Our study suggests that bovine surfactant lipids can organize into discrete phases in monolayers or bilayers with equivalent temperature dependencies and may occur at physiological temperatures and surface pressures equivalent to those at the lung interface.