Transbilayer peptide sorting between raft and nonraft bilayers: comparisons of detergent extraction and confocal microscopy

Membrane microdomains ("rafts") that sequester specific proteins and lipids are often characterized by their resistance to detergent extraction. Because rafts are enriched in sphingomyelin and cholesterol, raft bilayers are thicker and have larger area compressibility moduli than nonraft bilayers. It has been postulated that rafts concentrate proteins with long transmembrane domains (TMDs) because of "hydrophobic matching" between the TMDs and the thick raft bilayers. However, previous detergent extraction experiments with bilayers containing raft and nonraft domains have shown that the peptides P-23 and P-29, designed to have single TMDs matching the hydrocarbon thicknesses of detergent soluble membranes and detergent resistant membranes, respectively, are both localized to detergent soluble membranes. Those results imply that both peptides are preferentially located in nonraft domains. However, because the detergent solubilizes part of the bilayer, it has been unclear whether or not detergent extraction experiments provide an accurate indication of the location of peptides in intact bilayers. Here we use confocal microscopy to examine the distribution of these same peptides in intact bilayers containing both raft and nonraft domains. At 20 degrees C and 37 degrees C, P-23 and P-29 were both primarily localized in fluorescently labeled nonraft domains. These confocal results validate the previous detergent extraction experiments and demonstrate the importance of bilayer cohesive properties, compared to hydrophobic mismatch, in the sorting of these peptides that contain a single TMD.     

Structure, composition, and peptide binding properties of detergent soluble bilayers and detergent resistant rafts

Lipid bilayers composed of unsaturated phosphatidylcholine (PC), sphingomyelin (SM), and cholesterol are thought to contain microdomains that have similar detergent insolubility characteristics as rafts isolated from cell plasma membranes. We chemically characterized the fractions corresponding to detergent soluble membranes (DSMs) and detergent resistant membranes (DRMs) from 1:1:1 PC:SM:cholesterol, compared the binding properties of selected peptides to bilayers with the compositions of DSMs and DRMs, used differential scanning calorimetry to identify phase transitions, and determined the structure of DRMs with x-ray diffraction. Compared with the equimolar starting material, DRMs were enriched in both SM and cholesterol. Both transmembrane and interfacial peptides bound to a greater extent to DSM bilayers than to DRM bilayers, likely because of differences in the mechanical properties of the two bilayers. Thermograms from 1:1:1 PC:SM:cholesterol from 3 to 70 degrees C showed no evidence for a liquid-ordered to liquid-disordered phase transition. Over a wide range of osmotic stresses, each x-ray pattern from equimolar PC:SM:cholesterol or DRMs contained a broad wide-angle band at 4.5 A, indicating that the bilayers were in a liquid-crystalline phase, and several sharp low-angle reflections that indexed as orders of a single lamellar repeat period. Electron density profiles showed that the total bilayer thickness was 57 A for DRMs, which was approximately 5 A greater than that of 1:1:1 PC:SM:cholesterol and 10 A greater than the thickness of bilayers with the composition of DSMs. These x-ray data provide accurate values for the widths of raft and nonraft bilayers that should be important in understanding mechanisms of protein sorting by rafts.     

The 2004 Biophysical Society-Avanti Award in Lipids address: roles of bilayer structure and elastic properties in peptide localization in membranes

This review details how bilayer structural/elastic properties impact three distinct areas of biological significance. First, the partitioning of melittin into bilayers and melittin-induced bilayer leakage depended strongly on bilayer composition. The incorporation of cholesterol into phosphatidylcholine bilayers decreased melittin-induced leakage from 73 to 3%, and bilayers composed of lipopolysaccharide (LPS), the main lipid on the surface of Gram-negative bacteria, also had low (3%) melittin-induced leakage. Second, transbilayer peptides of different hydrophobic lengths were largely excluded from bilayer microdomains (“rafts”) enriched in sphingomyelin (SM) and cholesterol, even when the length of the transbilayer peptide domain matched the hydrocarbon thickness of the raft bilayer. This is likely due to the large area compressibility modulus of SM:cholesterol bilayers. Third, the major water barrier of skin, the extracellular lamellae of the stratum corneum, was found to contain tightly packed asymmetric lipid bilayers with cholesterol located preferentially on one side of the bilayer and a unique skin ceramide containing an unsaturated acyl chain on the opposite side. We argue that, in each of these three areas, key factors are differences in lipid hydrocarbon chain packing for different lipids, particularly the tight hydrocarbon chain packing caused by cholesterol’s strong interaction with saturated chains.     

Placental alkaline phosphatase is efficiently targeted to rafts in supported lipid bilayers

Evidence is growing that biological membranes contain lipid microdomains or "rafts" that may be involved in processes such as cellular signaling and protein trafficking. In this study, we have used atomic force microscopy to examine the behavior of rafts in supported lipid bilayers. We show that bilayers composed of equimolar dioleoylphosphatidylcholine and sphingomyelin spontaneously form rafts, which are detectable as raised features. A comparison of the extents of protrusion of the rafts in monolayers and bilayers indicates that the rafts in the two leaflets of the bilayer coincide. The rafts were observed both in the absence and presence of cholesterol (33 mol %). Cholesterol reduced raft protrusion presumably by increasing the thickness of the non-raft bilayer. PLAP (glycosylphosphatidylinositol-anchored protein placental alkaline phosphatase) was purified and shown to exist as a dimer. Following its incorporation into supported lipid bilayers, PLAP was found to be targeted efficiently to rafts, both in the absence and presence of cholesterol. We suggest that atomic force microscopy provides a powerful tool for the study of raft structure and properties.     

Use of cyclodextrin for AFM monitoring of model raft formation

The lipid rafts membrane microdomains, enriched in sphingolipids and cholesterol, are implicated in numerous functions of biological membranes. Using atomic force microscopy, we have examined the effects of cholesterol-loaded methyl-beta-cyclodextrin (MbetaCD-Chl) addition to liquid disordered (l(d))-gel phase separated dioleoylphosphatidylcholine (DOPC)/sphingomyelin (SM) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SM supported bilayers. We observed that incubation with MbetaCD-Chl led to the disappearance of domains with the formation of a homogeneously flat bilayer, most likely in the liquid-ordered (l(o)) state. However, intermediate stages differed with the passage through the coexistence of l(o)-l(d) phases for DOPC/SM samples and of l(o)-gel phases for POPC/SM bilayers. Thus, gel phase SM domains surrounded by a l(o) matrix rich in cholesterol and POPC could be observed just before reaching the uniform l(o) state. This suggests that raft formation in biological membranes could occur not only via liquid-liquid but also via gel-liquid immiscibility. The data also demonstrate that MbetaCD-Chl as well as the unloaded cyclodextrin MbetaCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MbetaCD and MbetaCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution.     

Plasmon-waveguide resonance studies of lateral segregation of lipids and proteins into microdomains (rafts) in solid-supported bilayers

Plasmon-waveguide resonance (PWR) spectroscopy has been used to examine solid-supported lipid bilayers consisting of dioleoylphosphatidylcholine (DOPC), palmitoyloleoylphosphatidylcholine (POPC), sphingomyelin (SM), and phosphatidylcholine/SM binary mixtures. Spectral simulation of the resonance curves demonstrated an increase in bilayer thickness, long-range order, and molecular packing density in going from DOPC to POPC to SM single component bilayers, as expected based on the decreasing level of unsaturation in the fatty acyl chains. DOPC/SM and POPC/SM binary mixtures yielded PWR spectra that can be ascribed to a superposition of two resonances corresponding to microdomains (rafts) consisting of phosphatidylcholine- and SM-rich phases coexisting within a single bilayer. These were formed spontaneously over time as a consequence of lateral phase separation. Each microdomain contained a small proportion (<20%) of the other lipid component, which increased their kinetic and thermodynamic stability. Incorporation of a glycosylphosphatidylinositol-linked protein (placental alkaline phosphatase) occurred within each of the single component bilayers, although the insertion was less efficient into the DOPC bilayer. Incorporation of placental alkaline phosphatase into a DOPC/SM binary bilayer occurred with preferential insertion into the SM-rich phase, although the protein incorporated into both phases at higher concentrations. These results demonstrate the utility of PWR spectroscopy to provide insights into raft formation and protein sorting in model lipid membranes.     

Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation

Raft formation and enlargement was investigated in liposomes and supported bilayers prepared from sphingomyelin (SM), cholesterol, and unsaturated phospholipids; NBD-DPPE and rhodamine-(DOPE) were employed as fluorescent probes. Rafts were created by lowering temperature. Maintaining 20 mol % SM, fluorescence microscopy showed that, in the absence of photooxidation, large rafts did not form in giant unilamellar vesicles (GUVs) containing 20 or more mol % cholesterol. But if photooxidation was allowed to proceed, large rafts were readily observed. In population, cuvette experiments, small rafts formed without photooxidation at high cholesterol concentrations. Thus, photooxidation was the cause of raft enlargement during microscopy experiments. Because photooxidation results in peroxidation at lipid double bonds, photosensitization experiments were performed to explicitly produce peroxides of SM and an unsaturated phospholipid. GUVs of high cholesterol content containing the breakdown products of SM-peroxide, but not phospholipid-peroxide, resulted in large rafts after lowering temperature. In addition, GUV production by electroswelling can result in peroxides that cause large raft formation. The use of titanium electrodes eliminates this problem. In conclusion, lipid peroxides and their breakdown products are the cause of large raft formation in GUVs containing biological levels of cholesterol. It is critical that experiments investigating rafts in bilayer membranes avoid the production of peroxides.     

Brij detergents reveal new aspects of membrane microdomain in erythrocytes

Membrane microdomains enriched in cholesterol, sphingolipids (rafts), and specific proteins are involved in important physiological functions. However their structure, size and stability are still controversial. Given that detergent-resistant membranes (DRMs) are in the liquid-ordered state and are rich in raft-like components, they might correspond to rafts at least to some extent. Here we monitor the lateral order of biological membranes by characterizing DRMs from erythrocytes obtained with Brij-98, Brij-58, and TX-100 at 4?°C and 37?°C. All DRMs were enriched in cholesterol and contained the raft markers flotillin-2 and stomatin. However, sphingomyelin (SM) was only found to be enriched in TX-100-DRMs – a detergent that preferentially solubilizes the membrane inner leaflet – while Band 3 was present solely in Brij-DRMs. Electron paramagnetic resonance spectra showed that the acyl chain packing of Brij-DRMs was lower than TX-100-DRMs, providing evidence of their diverse lipid composition. Fatty acid analysis revealed that the SM fraction of the DRMs was enriched in lignoceric acid, which should specifically contribute to the resistance of SM to detergents. These results indicate that lipids from the outer leaflet, particularly SM, are essential for the formation of the liquid-ordered phase of DRMs. At last, the differential solubilization process induced by Brij-98 and TX-100 was monitored using giant unilamellar vesicles. This study suggests that Brij and TX-100-DRMs reflect different degrees of lateral order of the membrane microdomains. Additionally, Brij DRMs are composed by both inner and outer leaflet components, making them more physiologically relevant than TX-100-DRMs to the studies of membrane rafts.     

Single-Molecule Investigation of the Influence Played by Lipid Rafts on Ion Transport and Dynamic Features of the Pore-Forming Alamethicin Oligomer

In this experimental work we employed single-molecule electrical recordings on alamethicin oligomers inserted in lipid bilayers made of brain sphingomyelin (bSM), palmitoyloleoylphosphatidylcholine (POPC) and cholesterol (chol) to unravel novel aspects regarding lipid raft interactions with pore-forming peptides. We probed the effect of lipid rafts on electrical properties of inserted alamethicin oligomers, and our data convincingly prove that the single-channel electrical conductance of various subconductance states of the alamethicin oligomer (1) increases in the presence of raft-containing ternary lipid mixtures (POPC-chol-bSM) compared to cases when bilayers were made of POPC-chol and POPC and (2) decreases in the presence of raft-containing ternary lipid mixtures compared to nonraft ternary mixtures which favor the fluid and liquid ordered phases alone. Our data demonstrate that the presence of lipid rafts leads to a slower association kinetics of alamethicin oligomers, seemingly reflecting a slower lateral diffusion process of such peptide aggregates compared to the case of nonraft, binary lipid mixtures. Furthermore, we show that the electrical capacitance of ternary lipid mixtures (POPC-chol-bSM) decreases in the presence of raft domains by comparison to nonraft binary phases (POPC-chol) or POPC alone, and this could constitute an additional mechanism via which macroscopic electrical manifestations of eukaryotic cells are modulated by the coexistence of gel and fluid domains of the plasma membrane.     

Acyl-chain methyl distributions of liquid-ordered and -disordered membranes

A central feature of the lipid raft concept is the formation of cholesterol-rich lipid domains. The introduction of relatively rigid cholesterol molecules into fluid liquid-disordered (L_d) phospholipid bilayers can produce liquid-ordered (L_o) mixtures in which the rigidity of cholesterol causes partial ordering of the flexible hydrocarbon acyl chains of the phospholipids. Several lines of evidence support this concept, but direct structural information about L_o membranes is lacking. Here we present the structure of L_o membranes formed from cholesterol and dioleoylphosphatidylcholine (DOPC). Specific deuteration of the DOPC acyl-chain methyl groups and neutron diffraction measurements reveal an extraordinary disorder of the acyl chains of neat L_d DOPC bilayers. The disorder is so great that >20% of the methyl groups are in intimate contact with water in the bilayer interface. The ordering of the DOPC acyl chains by cholesterol leads to retraction of the methyl groups away from the interface. Molecular dynamics simulations based on experimental systems reveal asymmetric transbilayer distributions of the methyl groups associated with each bilayer leaflet.     

Influence of cholesterol on the biophysical properties of the sphingomyelin/DOPC binary system

The influence of cholesterol on the sphingomyelin (SM)/dioleoylphosphatidylcholine (DOPC) binary system was investigated in various respects. Electron spin resonance (ESR) measurements reveal that the order parameter of 5DS (5-doxyl stearic acid) in SM/DOPC bilayers increases notably when the concentration of cholesterol is over 30 mol%. Membrane potential measurements indicate that the K⁺ permeability of the SM/DOPC bilayer decreases steeply at 40 mol% cholesterol concentration. Both these experiments suggest that cholesterol reduces the motion amplitude of hydrocarbon chains abruptly above 30 mol%. In contrast to the ordering effects on the hydrocarbon chains, ³¹P-NMR results indicate that cholesterol slightly increases the motion of phosphate groups of the lipids. ³¹P-NMR also raises the possibility of domain formation in the presence of cholesterol. Fluorescence-quenching experiments verified that solid domains appear in the binary system when cholesterol is present, and percolation threshold occurs at 50 mol% cholesterol concentration. The solid domains bear the properties of liquid ordered phase, which is the basic structure of caveolae and functional rafts. So this work provides an artificial model for the study of rafts and caveolae on biological membranes. Received: 29 January 2001/Revised: 17 May 2001     

Control of the transmembrane orientation and interhelical interactions within membranes by hydrophobic helix length

We examined the effect of the length of the hydrophobic core of Lys-flanked poly(Leu) peptides on their behavior when inserted into model membranes. Peptide structure and membrane location were assessed by the fluorescence emission lambdamax of a Trp residue in the center of the peptide sequence, the quenching of Trp fluorescence by nitroxide-labeled lipids (parallax analysis), and circular dichroism. Peptides in which the hydrophobic core varied in length from 11 to 23 residues were found to be largely alpha-helical when inserted into the bilayer. In dioleoylphosphatidylcholine (diC18:1PC) bilayers, a peptide with a 19-residue hydrophobic core exhibited highly blue-shifted fluorescence, an indication of Trp location in a nonpolar environment, and quenching localized the Trp to the bilayer center, an indication of transmembrane structure. A peptide with an 11-residue hydrophobic core exhibited emission that was red-shifted, suggesting a more polar Trp environment, and quenching showed the Trp was significantly displaced from the bilayer center, indicating that this peptide formed a nontransmembranous structure. A peptide with a 23-residue hydrophobic core gave somewhat red-shifted fluorescence, but quenching demonstrated the Trp was still close to the bilayer center, consistent with a transmembrane structure. Analogous behavior was observed when the behavior of individual peptides was examined in model membranes with various bilayer widths. Other experiments demonstrated that in diC18:1PC bilayers the dilution of the membrane concentration of the peptide with a 23-residue hydrophobic core resulted in a blue shift of fluorescence, suggesting the red-shifted fluorescence at higher peptide concentrations was due to helix oligomerization. The intermolecular self-quenching of rhodamine observed when the peptide was rhodamine-labeled, and the concentration dependence of self-quenching, supported this conclusion. These studies indicate that the mismatch between helix length and bilayer width can control membrane location, orientation, and helix-helix interactions, and thus may mismatch control both membrane protein folding and the interactions between membrane proteins.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes.

Deuterium (2H) NMR was used to study bilayer hydrophobic thickness and mechanical properties when cholesterol and/or synthetic amphiphillic polypeptides were added to deuterated POPC lipid bilayer membranes in the liquid-crystalline (fluid) phase. Smoothed acyl chain orientational order profiles were used to calculate bilayer hydrophobic thickness. Addition of 30 mol% cholesterol to POPC at 25 degrees C increased the bilayer thickness from 2.58 to 2.99 nm. The peptides were chosen to span the bilayers with more or less mismatch between the hydrophobic peptide length and membrane hydrophobic thickness. The average thickness of the pure lipid bilayers was significantly perturbed upon addition of peptide only in cases of large mismatch, being increased (decreased) when the peptide hydrophobic length was greater (less) than that of the pure bilayer, consistent with the "mattress" model of protein lipid interactions (Mouritsen, O.G., and M. Bloom. 1984. Biophys. J. 46:141-153). The experimental results were also used to examine the combined influence of the polypeptides and cholesterol on the orientational order profile and thickness expansivity of the membranes. A detailed model for the spatial distribution of POPC and cholesterol molecules in the bilayers was proposed to reconcile the general features of these measurements with micromechanical measurements of area expansivity in closely related systems. Experiments to test the model were proposed.     

Cholesterol modulation of membrane resistance to Triton X-100 explored by atomic force microscopy

Biomembranes are not homogeneous, they present a lateral segregation of lipids and proteins which leads to the formation of detergent-resistant domains, also called "rafts". These rafts are particularly enriched in sphingolipids and cholesterol. Despite the huge body of literature on raft insolubility in non-ionic detergents, the mechanisms governing their resistance at the nanometer scale still remain poorly documented. Herein, we report a real-time atomic force microscopy (AFM) study of model lipid bilayers exposed to Triton X-100 (TX-100) at different concentrations. Different kinds of supported bilayers were prepared with dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol (Chol). The DOPC/SM 1:1 (mol/mol) membrane served as the non-resistant control, and DOPC/SM/Chol 2:1:1 (mol/mol/mol) corresponded to the raft-mimicking composition. For all the lipid compositions tested, AFM imaging revealed that TX-100 immediately solubilized the DOPC fluid phase leaving resistant patches of membrane. For the DOPC/SM bilayers, the remaining SM-enriched patches were slowly perforated leaving crumbled features reminiscent of the initial domains. For the raft model mixture, no holes appeared in the remaining SM/Chol patches and some erosion occurred. This work provides new, nanoscale information on the biomembranes' resistance to the TX-100-mediated solubilization, and especially about the influence of Chol.     

The sensitivity of lipid domains to small perturbations demonstrated by the effect of Triton

The hypothesis of lipid rafts describes functional domains in biological membranes. It is often assumed that rafts form by spontaneous de-mixing of certain lipids and that they can be isolated as detergent-resistant membrane particles (DRMs) using the detergent Triton X-100 (TX). Here, we present a model that describes the process of domain formation in membranes in the presence and in the absence of TX. We measure the interactions between TX and an equimolar mixture of sphingomyelin (SM), cholesterol (Cho), and 1-palmitoyl-2-oleoyl-3-sn-glycero-phosphatidylcholine (POPC) (1:1:1, mol) by means of isothermal titration calorimetry. Comparison with pure POPC membranes reveals a very unfavorable interaction between TX and SM/Cho, which causes a substantial tendency to segregate these molecules into separate, DRM-like (SM-rich) and fluid (TX-rich), domains. If rafts are indeed formed by spontaneous de-mixing of PC and SM/Cho, they must be very sensitive, and perturbations caused by techniques used to study rafts could lead to misleading results. If, however, rafts are much more stable than PC-SM-Cho domains, there must be an unknown raft stabilizer. Subtle changes of such a promoter could serve to modulate raft function.     

An X-ray diffraction study of model membrane raft structures

Protein sorting and assembly in membrane biogenesis and function involves the creation of ordered domains of lipids known as membrane rafts. The rafts are comprised of all the major classes of lipids, including glycerophospholipids, sphingolipids and sterol. Cholesterol is known to interact with sphingomyelin to form a liquid-ordered bilayer phase. Domains formed by sphingomyelin and cholesterol, however, represent relatively small proportions of the lipids found in membrane rafts and the properties of other raft lipids are not well characterized. We examined the structure of lipid bilayers comprised of aqueous dispersions of ternary mixtures of phosphatidylcholines and sphingomyelins from tissue extracts and cholesterol using synchrotron X-ray powder diffraction methods. Analysis of the Bragg reflections using peak-fitting methods enables the distinction of three coexisting bilayer structures: (a) a quasicrystalline structure comprised of equimolar proportions of phosphatidylcholine and sphingomyelin, (b) a liquid-ordered bilayer of phospholipid and cholesterol, and (c) fluid phospholipid bilayers. The structures have been assigned on the basis of lamellar repeat spacings, relative scattering intensities and bilayer thickness of binary and ternary lipid mixtures of varying composition subjected to thermal scans between 20 and 50 °C. The results suggest that the order created by the quasicrystalline phase may provide an appropriate scaffold for the organization and assembly of raft proteins on both sides of the membrane. Co-existing liquid-ordered structures comprised of phospholipid and cholesterol provides an additional membrane environment for assembly of different raft proteins.     

Visualization of detergent solubilization of membranes: implications for the isolation of rafts

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l_d) and raft liquid ordered (l_o) lipid phases by selectively solubilizing the l_d phase. A higher concentration of Lubrol was required, and not all the l_d phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l_d phase and then progressed to the solubilization of both l_d and l_o phases simultaneously. Octyl glucoside simultaneously solubilized both l_o and l_d phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.     

Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles

Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase.     

Probing the interaction forces between hydrophobic peptides and supported lipid bilayers using AFM

Despite the vast body of literature that has accumulated on tilted peptides in the past decade, direct information on the forces that drive their interaction with lipid membranes is lacking. Here, we attempted to use atomic force microscopy (AFM) to explore the interaction forces between the Simian immunodeficiency virus peptide and phase-separated supported bilayers composed of various lipids, i.e. dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidic acid and dipalmitoylphosphatidylethanolamine. Histidine-tagged peptides were attached onto AFM tips terminated with nitrilotriacetate and tri(ethylene glycol) groups, an approach expected to ensure optimal exposure of the C-terminal hydrophobic domain. Force-distance curves recorded between peptide-tips and the different bilayer domains always showed a long-range repulsion upon approach and a lack of adhesion upon retraction, in marked contrast with the hydrophobic nature of the peptide. To explain this unexpected behaviour, we suggest a mechanism in which lipids are pulled out from the bilayer due to strong interactions with the peptide-tip, in agreement with the very low force needed to extract lipids from supported bilayers.