Two-dimensional microelectrophoresis in supported lipid bilayers

We report the application of supported bilayers for two-dimensional microelectrophoresis. This method allows the lateral separation and accumulation of charged amphiphilic molecular probes in bilayers by application of an electric field parallel to the bilayer surface. Diffusion coefficient and mobility of the fluorescent probes are determined by observation of the fluorescence recovery after photobleaching (pattern bleaching). The diffusion coefficients and the mobilities of oppositely charged fluorescent probes in one bilayer can be determined independently from a single measurement. By analysis of the motion of charged and uncharged probes in one membrane one can distinguish between the motion caused by the electric field acting on the charge of individual probes and that caused by frictional forces due to electroosmosis.     

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.     

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.     

Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin

Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, DeltaPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that DeltaPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40 nm, and their helical pitch decreases from approximately 13 to 9.4 nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.     

Formation of three-dimensional structures in supported lipid bilayers

The creation of three-dimensional structures in supported lipid bilayers has been examined. In bilayers, shape transformations can be triggered by adjusting a variety of parameters. Here, it is shown that bilayers composed of phosphatidylcholine and phosphatidic acid can be induced to reversibly form cap structures when exposed to an asymmetry in ionic strength. The structures that form depend on the asymmetry in the ionic strength and the amount of anionic lipid. Other factors that may be of importance in the creation of the structures, expansion forces, osmotic forces, and the bilayer-support interaction are discussed. The cap structures have the potential to be of considerable utility in examining the effect that curvature has on membrane processes.     

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.     

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 degrees 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 alpha-L beta') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 degrees C commencing at the calorimetric phase transition temperature.     

Atomic force microscopy of supported lipid bilayers

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy (AFM) has become a well-established technique for imaging SLBs at nanometer resolution. A unique feature of AFM is its ability to monitor dynamic processes, such as the interaction of bilayers with proteins and drugs. Here, we present protocols for preparing dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers supported on mica using small unilamellar vesicles and for imaging their nanoscale interaction with the antibiotic azithromycin using AFM. The entire protocol can be completed in 10 h.     

Atomic force microscopy reveals defects within mica supported lipid bilayers induced by the amyloidogenic human amylin peptide

To date, over 20 peptides or proteins have been identified that can form amyloid fibrils in the body and are thought to cause disease. The mechanism by which amyloid peptides cause the cytotoxicity observed and disease is not understood. However, one of the major hypotheses is that amyloid peptides cause membrane perturbation. Hence, we have studied the interaction between lipid bilayers and the 37 amino acid residue polypeptide amylin, which is the primary constituent of the pancreatic amyloid associated with type 2 diabetes. Using a dye release assay we confirmed that the amyloidogenic human amylin peptide causes membrane disruption; however, time-lapse atomic force microscopy revealed that this did not occur by the formation of defined pores. On the contrary, the peptide induced the formation of small defects spreading over the lipid surface. We also found that rat amylin, which has 84% identity with human amylin but cannot form amyloid fibrils, could also induce similar lesions to supported lipid bilayers. The effect, however, for rat amylin but not human amylin, was inhibited under high ionic conditions. These data provide an alternative theory to pore formation, and how amyloid peptides may cause membrane disruption and possibly cytotoxicity.     

Interleaflet coupling and domain registry in phase-separated lipid bilayers

There is clear evidence of an interleaflet coupling in model lipid/cholesterol membranes exhibiting liquid-liquid phase separation. The strength of this coupling is quantified by the mismatch free energy, γ. We calculate it using a molecular mean-field model of a phase-separated lipid/cholesterol bilayer and obtain values that increase as the concentration of saturated lipids in the coexisting phases is increased. These values lie in the range 0.01–0.03 k_BT/nm². We clarify the relationship between the interleaflet coupling and the extent of interleaflet alignment of liquid domains by analyzing a statistical mechanical model of coupled fluctuating domain interfaces. The model is solved exactly using the correspondence between statistical mechanics and quantum mechanics, yielding an expression for the characteristic size of fluctuations out of domain registry. This length scale depends only weakly on the strength of the interleaflet coupling and inevitably is only of the order of nanometers, which explains the experimental result that fluctuations out of domain registry have not been observed by optical microscopy.     

Binding of macrophages and phospholipid flip-flop in supported lipid bilayers

Subclass-specific antibody-dependent interactions (binding and triggering) between macrophages and supported lipid bilayers have been studied. Percentages of mouse macrophage binding (J774 cell line) to the lipid bilayers were dependent on mouse monoclonal IgG subclasses. The efficiencies were as follows: IgG1 = IgG2a greater than IgG2b greater than IgG3. Furthermore, macrophage triggering (spreading) was more efficient on IgG2a- or IgG1-coated lipid bilayers than on IgG2a, IgG3, or non-specific rabbit IgG. The present experiments show also that phospholipid molecules are able to flip-flop from one side of a supported planar bilayer membrane to the other with a half-life of 10 h-1 day at 25 degrees C.     

Probing ras effector interactions on nanoparticle supported lipid bilayers

Many biological processes take place in close proximity to lipid membranes. For a detailed understanding of the underlying mechanisms, tools are needed for the quantitative characterization of such biomolecular interactions. In this work, we describe the development of methods addressing the dynamics and affinities of protein complexes attached to an artificial membrane system. A semisynthetic approach provides the Ras protein with palmitoyl anchors, which allow stable membrane insertion, as a paradigm for membrane associated proteins that interact with multiple effectors. An artificial membrane system is constituted by nanoparticles covered with a lipid bilayer. Such a stable suspension allows for the characterization of the interaction between membrane-bound Ras and effector proteins using conventional fluorescence-based methods.     

Capturing the nanoscale complexity of cellular membranes in supported lipid bilayers

The lateral mobility of cell membranes plays an important role in cell signaling, governing the rate at which embedded proteins can interact with other biomolecules. The past two decades have seen a dramatic transformation in understanding of this environment, as the mechanisms and potential implications of nanoscale structure of these systems has become accessible to theoretical and experimental investigation. In particular, emerging micro- and nano-scale fabrication techniques have made possible the direct manipulation of model membranes at the scales relevant to these biological processes. This review focuses on recent advances in nanopatterning of supported lipid bilayers, capturing the impact of membrane nanostructure on molecular diffusion and providing a powerful platform for further investigation of the role of this spatial complexity on cell signaling.     

Studying membrane fusion using supported lipid bilayers on superparamagnetic beads

The fusion between two lipid membranes is a ubiquitous mechanism in cell traffic and pathogens invasion. Yet it is not well understood how two distinct bilayers overcome the energy barriers towards fusion and reorganize themselves to form a unique continuous bilayer. The magnitudes and numbers of these energy barriers are themselves an open question. To tackle these issues, we developed a new tool that allows to control the forces applied between two supported lipid bilayers (SLBs) deposited on superparamagnetic beads. By applying a magnetic field, the beads self-organize along field lines in chains of beads and compress the two membranes on the contact zone. Using the diffusion of fluorescently labelled lipids from one bilayer to the other allows us to identify fusion of the bilayers in contact. We applied increasing forces on SLBs and increased the occurrence of fusion. This experimental system allows the simultaneous study of tens of facing bilayers in a single experiment and mitigates the stochasticity of the fusion process. It is thus a powerful tool to test the various parameters involved in the membrane fusion process.     

Multivalent ligand–receptor binding on supported lipid bilayers

Fluid supported lipid bilayers provide an excellent platform for studying multivalent protein–ligand interactions because the two-dimensional fluidity of the membrane allows for lateral rearrangement of ligands in order to optimize binding. Our laboratory has combined supported lipid bilayer-coated microfluidic platforms with total internal reflection fluorescence microscopy (TIRFM) to obtain equilibrium dissociation constant (K_D) data for these systems. This high throughput, on-chip approach provides highly accurate thermodynamic information about multivalent binding events while requiring only very small sample volumes. Herein, we review some of the most salient findings from these studies. In particular, increasing ligand density on the membrane surface can provide a modest enhancement or attenuation of ligand–receptor binding depending upon whether the surface ligands interact strongly with each other. Such effects, however, lead to little more than one order of magnitude change in the apparent K_D values. On the other hand, the lipophilicity and presentation of lipid bilayer-conjugated ligands can have a much greater impact. Indeed, changing the way a particular ligand is conjugated to the membrane can alter the apparent K_D value by at least three orders of magnitude. Such a result speaks strongly to the role of ligand availability for multivalent ligand–receptor binding.     

Atomic force microscope image contrast mechanisms on supported lipid bilayers

This work presents a methodology to measure and quantitatively interpret force curves on supported lipid bilayers in water. We then use this method to correlate topographic imaging contrast in atomic force microscopy (AFM) images of phase-separated Langmuir-Blodgett bilayers with imaging load. Force curves collected on pure monolayers of both distearoylphosphatidylethanolamine (DSPE) and monogalactosylethanolamine (MGDG) and dioleoylethanolamine (DOPE) deposited at similar surface pressures onto a monolayer of DSPE show an abrupt breakthrough event at a repeatable, material-dependent force. The breakthrough force for DSPE and MGDG is sizable, whereas the breakthrough force for DOPE is too small to measure accurately. Contact-mode AFM images on 1:1 mixed monolayers of DSPE/DOPE and MGDG/DOPE have a high topographic contrast at loads between the breakthrough force of each phase, and a low topographic contrast at loads above the breakthrough force of both phases. Frictional contrast is inverted and magnified at loads above the breakthrough force of both phases. These results emphasize the important role that surface forces and mechanics can play in imaging multicomponent biomembranes with AFM.     

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.     

Direct measurement of the mechanical properties of lipid phases in supported bilayers

Biological membranes define not only the cell boundaries but any compartment within the cell. To some extent, the functionality of membranes is related to the elastic properties of the lipid bilayer and the mechanical and hydrophobic matching with functional membrane proteins. Supported lipid bilayers (SLBs) are valid biomimetic systems for the study of membrane biophysical properties. Here, we acquired high-resolution topographic and quantitative mechanics data of phase-separated SLBs using a recent atomic force microscopy (AFM) imaging mode based on force measurements. This technique allows us to quantitatively map at high resolution the mechanical differences of lipid phases at different loading forces. We have applied this approach to evaluate the contribution of the underlying hard support in the determination of the elastic properties of SLBs and to determine the adequate indentation range for obtaining reliable elastic moduli values. At ~200 pN, elastic forces dominated the force-indentation response and the sample deformation was <20% of the bilayer thickness, at which the contribution of the support was found to be negligible. The obtained Young's modulus (E) of 19.3 MPa and 28.1 MPa allowed us to estimate the area stretch modulus (k(A)) as 106 pN/nm and 199 pN/nm and the bending stiffness (k(c)) as 18 k(B)T and 57 k(B)T for the liquid and gel phases, respectively.     

The SIV tilted peptide induces cylindrical reverse micelles in supported lipid bilayers

Elucidation of the molecular mechanism leading to biomembrane fusion is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various important diseases. According to the generally admitted stalk mechanism described for membrane fusion, negatively curved lipids may play a central role during the early steps of the process. In this study, we used atomic force microscopy (AFM) to address the crucial question of whether negatively curved lipids influence the interaction of the simian immunodeficiency virus (SIV) fusion peptide with model membranes. To this end, dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers containing 0.5 mol % dioleoylphosphatidic acid (DOPA) were incubated with the SIV peptide and imaged in real time using AFM. After a short incubation time, we observed a 1.9 nm reduction in the thickness of the DPPC domains, reflecting either interdigitation or fluidization of lipids. After longer incubation times, these depressed DPPC domains evolved into elevated domains, composed of nanorod structures protruding several nanometers above the bilayer surface and attributed to cylindrical reverse micelles. Such DOPC/DPPC/DOPA bilayer modifications were never observed with nontilted peptides. Accordingly, this is the first time that AFM reveals the formation of cylindrical reverse micelles in lipid bilayers promoted by fusogenic peptides.     

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.