Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

NBD-cholesterol probes to track cholesterol distribution in model membranes

A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L_o) and liquid-disordered (L_o) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L_o domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L_o domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.     

Direct AFM observation of saposin C-induced membrane domains in lipid bilayers: from simple to complex lipid mixtures

Saposin C (Sap C) is a small glycoprotein required by glucosylceramidase (GCase) for hydrolysis of glucosylceramide to ceramide and glucose in lysosomes. The molecular mechanism underlying Sap C stimulation of the enzyme activation is not fully understood. Here, atomic force microscopy (AFM) has been used to study Sap C-membrane interactions under physiological conditions. First, to establish how Sap C-membrane interactions affect membrane structure, lipid bilayers containing zwitterionic and anionic phospholipids were used. It was observed that Sap C induced two types of membrane restructuring effects, i.e., the formation of patch-like domains and membrane destabilization. Bilayers underwent extensive structural reorganization. To validate the biological importance of the membrane restructuring effects, interaction of Sap C with lipid bilayers composed of cholesterol, sphingomyelin, and zwitterionic and anionic phospholipids were studied. Although similar membrane restructuring effects were observed, Sap C-membrane interactions, in this case, were remarkably modulated and their effects were restricted to a limited area. As a result, nanometer-sized domains were formed. The establishment of a model membrane system will allow us to further study the dynamics, structure and mechanism of the Sap C-associated membrane domains and to examine the important role that these domains may play in enzyme activation.     

Self-assembly formation of multiple DNA-tethered lipid bilayers

Inspired by natural cell–cell junctions, where membrane-residing proteins control the separation between two or more membranes without interfering with their integrity, we report a new self-assembly route for formation of multiple highly fluid tethered lipid bilayers with the inter-membrane volume geometrically confined by membrane-anchored DNA duplexes. The formation of multiple planar membrane–membrane junctions were accomplished using disk shaped bicelles, composed of a mixture of the long-chained dimyristoyl phosphatidylcholine (DMPC) and the short-chained dihexanoyl PC further stabilized with the positively charged detergent hexadecyl-trimethyl-ammonium bromide (CTAB). Quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy and fluorescence recovery after photobleaching (FRAP) were used to monitor the formation and to characterize the integrity of the self-assembled lipid–DNA architecture.     

A theory for the effects of diffusion-reaction coupling on the carrier-mediated ionic permeability in lipid bilayers

The zero-current membrane potential and the current-voltage relations are discussed theoretically for the case in which ionic transport is mediated by carriers that form complexes with ions in the aqueous phase (‘solution complexation’ mechanism). Interest for this topic originated partly from the finding that gradients of the neutral cyclic peptide PV, cyclo (dVal-lPro-lVal-dPro)₃, commonly thought to act as a carrier via ‘solution complexation’, generate Nernstian potentials across lipid bilayers separating solutions of identical ion composition. It is shown that the general expression for the potential in a gradient of carriers reduces to the Nernst equation under any of the following conditions: slow aqueous reaction; impermeability of the membrane to the neutral carriers; high concentration of the complexing ions in solution; finite permeability of the membrane to the neutral carrier, but faster rate of movement from the membrane surface into the torus than across the middle or out of the membrane. In symmetrical solutions, the conductance is most typically characterized by a quantity that we designate by δ*, which has the dimensions of a length and is generally a complex function of ion activity. Comparing the thory with previous data on dioleoylphosphatidylcholine membranes in the presence of PV and K⁺, the order of magnitude of the rates of the aqueous reaction and of the membrane permeability to the neutral carriers is tentatively estimated.     

FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.     

Single molecule imaging of supported planar lipid bilayer--reconstituted human insulin receptors by in situ scanning probe microscopy

A 480-kDa disulfide-linked heterodimer single-pass transmembrane protein, the insulin receptor, is autophosphorylated upon insulin binding to its extracellular domain. Remarkably, the structural basis for this activation process remained largely unknown until the recent cryoelectron microscopy studies of the insulin-insulin receptor complex by Luo et al. [Science 285 (1999) 1077]. We report here the results of an in situ study by high-resolution scanning probe microscopy of the full-length insulin receptor reconstituted within supported planar lipid bilayers. Our preliminary studies confirm that (1) the intact receptor can be reconstituted constitutively within a lipid vesicle and (2) fusion of the receptor-containing vesicles to mica resulted in the formation of molecular flat 5.5-nm-thick supported planar bilayers populated by two populations of protrusions, the shape and size of which are consistent with those of the insulin receptor's intra- and extracellular domains as modeled by the cryo-EM data of Ottensmeyer et al. [Biochemistry 39 (2000) 12103]. These results establish a framework for real-time studies of insulin-insulin receptor binding by in situ SPM and single molecule force spectroscopy.     

Sphingolipid symmetry governs membrane lipid raft structure

Lipid domain formation in membranes underlies the concept of rafts but their structure is controversial because the key role of cholesterol has been challenged. The configuration of glycosphingolipid receptors for agonists, bacterial toxins and enveloped viruses in plasma membrane rafts appears to be an important factor governing ligand binding and infectivity but the details are as yet unresolved. I have used X-ray diffraction methods to examine how cholesterol affects the distribution of glycosphingolipid in aqueous dispersions of an equimolar mixture of cholesterol and egg-sphingomyelin containing different proportions of glucosylceramide from human extracts. Three coexisting liquid-ordered bilayer structures are observed at 37 °C in mixtures containing up to 20 mol% glycosphingolipid. All the cholesterol was sequestered in one bilayer with the minimum amount of sphingomyelin (33 mol%) to prevent formation of cholesterol crystals. The other two bilayers consisted of sphingomyelin and glucosylceramide. Asymmetric molecular species of glucosylceramide with N-acyl chains longer than 20 carbons form an equimolar complex with sphingomyelin in which the glycosidic residues are arranged in hexagonal array. Symmetric molecular species mix with sphingomyelin in proportions less than equimolar to form quasicrystalline bilayers. When the glycosphingolipid exceeds equimolar proportions with sphingomyelin cholesterol is incorporated into the structure and formation of a gel phase of glucosylceramide is prevented. The demonstration of particular structural features of ceramide molecular species combined with the diversity of sugar residues of glycosphingolipid classes paves the way for a rational approach to understanding the functional specificity of lipid rafts and how they are coupled across cell membranes.     

Computational investigation of pressure profiles in lipid bilayers with embedded proteins

The distribution of surface tension within a lipid bilayer, also referred to as the lateral pressure profile, has been the subject of theoretical scrutiny recently due to its potential to radically alter the function of biomedically important membrane proteins. Experimental measurements of the pressure profile are still hard to come by, leaving first-principles all-atom calculations of the profile as an important investigative tool. We describe and validate an efficient implementation of pressure profile calculations in the molecular dynamics package NAMD, capable of distinguishing between internal, bonded and nonbonded contributions as well as those of selected atom groups. The new implementation can also be used in conjunction with Ewald summation for long-range electrostatics, improving the accuracy and reproducibility of the calculated profiles. We then describe results of the calculation of a pressure profile for a simple protein–lipid system consisting of melittin embedded in a DMPC bilayer. While the lateral pressure in the protein–lipid system is nearly the same as that of the bilayer alone, partitioning of the lateral pressure by atom type revealed substantial perturbation of the pressure profile and surface tension in an asymmetric manner.     

Studies on the lack of cooperativity in the melting of lipid bilayers

The gel-to-fluid first-order melting transition of lipid bilayers is simulated by the use of a microscopic interaction model which includes a variable number of lipid-chain conformational states. The results suggest that the experimental observation of ‘continuous melting’ in pure wet lipid bilayers, rather than being ascribed to the presence of impurities, may be explained as a result of kinetically caused metastability of intermediate lipid-chain conformations.     

Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.     

Single chloride-selective channel from cardiac sarcoplasmic reticulum studied in planar lipid bilayers

Summary The behavior of single Cl^– channel was studied by fusing isolated canine cardiac sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. The channel exhibited unitary conductance of 55 pS (in 260mm Cl^–) and steady-state activation. Subconductance states were observed. Open probability was dependent on holding potentials (–60 to +60 mV) and displayed a bell-shaped relationship, with probability values ranging from 0.2 to 0.8 with a maximum at –10 mV. Channel activity was irreversibly inhibited by DIDS, a stilbene derivative. Time analysis revealed the presence of one time constant for the full open state and three time constants for the closed states. The open and the longer closed time constants were found to be voltage dependent. The behavior of the channel was not affected by changing Ca²⁺ and Mg²⁺ concentrations in both chambers, nor by adding millimolar adenosine triphosphate, or by changing the pH from 7.4 to 6.8. The presence of sulfate anions decreased the unit current amplitude, but did not affect the open probability. These results reveal that at the unitary level the cardiac SR anion-selective channel has distinctive as well as similar electrical properties characteristic of other types of Cl^– channels.     

Phase transition and lateral phase separation in planar lipid membranes as sensed by the lipophilic ion dipicrylamine

The permeation of the lipophilic ion dipicrylamine through planar lipid membranes formed from dipalmitoylphosphatidylcholine in n-decane shows an anomaly near the main phase transition of this system. Both the rate constant, k_i, of ion translocation across the membrane interior and the interfacial concentration, N, of this ion have a maximum at about 36°C. Analogous experiments were performed with tetraphenylborate. A considerably lesser effect of the phase transition was found. The addition of cholesterol leads to a broadening of the maxima for k_i and N. The time course of the current following a voltage jump shows a characteristic change below a temperature of about 45°C, if the molar ratio cholesterol/ phosphatidylcholine in the membrane forming solution exceeds 1. While the current transient decays exponentially above 45°C, a sum of two exponential terms yields an adequate fit below that temperature. This is regarded as evidence for a lateral phase separation below 45°C into structurally different domains, which provide two different pathways for dipicrylamine.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

Gravimetric antigen detection utilizing antibody-modified lipid bilayers

Lipid bilayers containing 5% nitrilotriacetic acid (NTA) lipids supported on SiO₂ have been used as a template for immobilization of oligohistidine-tagged single-chained antibody fragments (scFvs) directed against cholera toxin. It was demonstrated that histidine-tagged scFvs could be equally efficiently coupled to an NTA-Ni²⁺-containing lipid bilayer from a purified sample as from an expression supernatant, thereby providing a coupling method that eliminates time-consuming protein prepurification steps. Irrespective of whether the coupling was made from the unpurified or purified antibody preparation, the template proved to be efficient for antigen (cholera toxin) detection, verified using quartz crystal microbalance with dissipation monitoring. In addition, via a secondary amplification step using lipid vesicles containing G_M1 (the natural membrane receptor for cholera toxin), the detection limit of cholera toxin was less than 750 pM. To further strengthen the coupling of scFvs to the lipid bilayer, scFvs containing two histidine tags, instead of just one tag, were also evaluated. The increased coupling strength provided via the bivalent anchoring significantly reduced scFv displacement in complex solutions containing large amounts of histidine-containing proteins, verified via cholera toxin detection in serum.     

AFM for structure and dynamics of biomembranes

We review structure and dynamic measurements of biomembranes by atomic force microscopy (AFM). We focus mainly on studies involving supported lipid bilayers (SLBs), particularly formation by vesicle rupture on flat and corrugated surfaces, nucleation and growth of domains in phase-separated systems, anesthetic-lipid interactions, and protein/peptide interactions in multicomponent systems. We show that carefully designed experiments along with real-time AFM imaging with superior lateral and z resolution (0.1 nm) have revealed quantitative details of the mechanisms and factors controlling vesicle rupture, domain shape and size, phase transformations, and some model biological interactions. The AFM tip can also be used as a mechanical transducer and incorporated in electrochemical measurements of membrane components; therefore, we touch on these important applications in both model and cell membranes.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L_α-L_β') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.     

Cryopreservation disrupts lipid rafts and heat shock proteins in yellow catfish sperm

Lipid rafts and associated membrane proteins (flotillin, caveolin) play important roles in cell signaling and sperm fertilization while heat shock proteins (Hsp) ensure properly protein folding to fulfill their physiological functions. The markedly reduced fertility in thawed sperm after cryopreservation could result from disrupted membrane lipid rafts and these proteins. To explore the effect of sperm cryopreservation on lipid rafts and heat shock proteins, we compared lipid raft integrity, and the expression levels of lipid raft associated proteins (Flot-1, Flot-2, Cav-1) as well as heat shock proteins (Hsp90, Hsp70) in fresh and thawed sperm cryopreserved under different scenarios in yellow catfish. We found higher lipid raft integrity, higher protein expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 in fresh sperm samples than in thawed sperm samples, in thawed sperm samples cryopreserved with optimal cooling rate than those cryopreserved with sub-optimal cooling rate, and in thawed sperm samples cryopreserved with extenders supplemented with cholesterol than those supplemented with methyl-β-cyclodextrin (for cholesterol removal). Our findings indicate that lipid raft integrity, and expression levels of Flot-1, Flot-2, Cav-1, Hsp90, and Hsp70 are clearly associated with sperm quality, and together they may play a cumulative role in reduced fertility associated with thawed sperm in aquatic species.     

Nanoscale analysis of supported lipid bilayers using atomic force microscopy

During the past 15 years, atomic force microscopy (AFM) has opened new opportunities for imaging supported lipid bilayers (SLBs) on the nanoscale. AFM offers a means to visualize the nanoscale structure of SLBs in physiological conditions. A unique feature of AFM is its ability to monitor dynamic events, like bilayer alteration, remodelling or digestion, upon incubation with various external agents such as drugs, detergents, proteins, peptides, nanoparticles, and solvents. Here, we survey recent progress made in the area.