Stabilisation of lipid membranes by β-peptide fibres

The scission of membranes necessary for vesicle biogenesis and cytokinesis is mediated by cytoplasmic proteins, which include members of the ESCRT (endosomal sorting complex required for transport) machinery. During the formation of intralumenal vesicles that bud into multivesicular endosomes, the ESCRT-II complex initiates polymerization of ESCRT-III subunits essential for membrane fission. However, mechanisms underlying the spatial and temporal regulation of this process remain unclear. Here, we show that purified ESCRT-II binds to the ESCRT-III subunit Vps20 on chemically defined membranes in a curvature-dependent manner. Using a combination of liposome co-flotation assays, fluorescence-based liposome interaction studies, and high-resolution atomic force microscopy, we found that the interaction between ESCRT-II and Vps20 decreases the affinity of ESCRT-II for flat lipid bilayers. We additionally demonstrate that ESCRT-II and Vps20 nucleate flexible filaments of Vps32 that polymerize specifically along highly curved membranes as a single string of monomers. Strikingly, Vps32 filaments are shown to modulate membrane dynamics in vitro, a prerequisite for membrane scission events in cells. We propose that a curvature-dependent assembly pathway provides the spatial regulation of ESCRT-III to fuse juxtaposed bilayers of elevated curvature.

A sticky situation: Domain-dependent recognition of the glycosphingolipid galactosylceramide by norovirus-like particles (see picture; red/yellow) is shown using supported lipid bilayers (purple) as model membranes. Optimal ligand presentation is found to promote strong binding to GalCer. This presentation can be found at the edges of the glycosphingolipid-enriched domains (green) and binding is repressed in the absence of these domains.

Antimicrobial peptides (AMPs) and lipopeptides (LPs) represent very promising molecules to fight resistant bacterial infections due to their broad-spectrum of activity, their first target, i.e. the bacterial membrane, and the rapid bactericidal action. For both types of molecules, the action mechanism starts from the membrane of the pathogen agents, producing a disorganization of their phase structure or the formation of pores of different size altering their permeability. This mechanism of action is based on physical interactions more than on a lock-and-key recognition event and it is difficult for the pathogens to rapidly develop an effective resistance. Very small differences in the sequence of both AMPs and LPs might lead to very different effects on the target membrane. Therefore, a correct understanding of their mechanism of action is required with the aim of developing new synthetic peptides, analogues of the natural ones, with specific and more powerful bactericidal activity. Atomic force microscopy (AFM), with its high resolution and the associated force spectroscopy resource, provides a valuable technique to investigate the reorganization of lipid bilayers exposed to antimicrobial or lipopeptides. Here, we present AFM results obtained by ours and other groups on the action of AMPs and LPs on supported lipid bilayers (SLBs) of different composition. We also consider data obtained by fluorescence microscopy to compare the AFM data with another technique which can be used on different lipid bilayer model systems such as SLBs and giant unilamellar vesicles. The outcomes here presented highlight the powerful of AFM-based techniques in detecting nanoscale peptide-membrane interactions and strengthen their use as an exceptional complementary tool to in vivo investigations. Indeed, the combination of these approaches can help decipher the mechanisms of action of different antimicrobials and lipopeptides at both the micro and nanoscale levels, and to design new and more efficient antimicrobial compounds.

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

Supported Lipid Bilayers (SLBs) on Polyelectrolyte Multilayers (PEMs) have large potential as models for developing sensor devices. SLBs can be designed with receptors and channels, which benefit from the biological environment of the lipid layers, to create a sensing interface for ions and biomarkers. PEMs assembled by the Layer-by-Layer (LBL) technique and used as supports for a lipid bilayer enable an easy integration of the bilayer on almost any surface and device. For electrochemical sensors, LBL assembly enables nanoscale tunable separation of the lipid bilayer from the electrode surface, avoiding undesired effects of the electrode surface on the lipid bilayers. We study the fabrication of valinomycin-doped SLBs on PEMs as a model system for biophysical studies and for selective ion sensing. SLBs are fabricated from dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylserine (DOPS) 50:50 vesicles doped with valinomycin, as a K+-selective carrier. SLBs were deposited on electrodes coated with poly(allyl amine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS) multilayers. Lipid bilayer formation was monitored by using Quartz Crystal Microbalance with Dissipation (QCMD) technique and Atomic Force Microscopy (AFM). Electrochemical impedance spectroscopy (EIS) and potentiometric measurements were performed to assess K+ selectivity over other ions and the potential of valinomycin-doped SLBs for K+-sensing.

Topographically smooth and stable supported lipid bilayer for high-resolution AFM studies.

Understanding the properties of cell membranes is important in the fields of fundamental and applied biology. While the characterization of simplified biological membrane mimics comprising liquid phase lipids has been routinely performed due to the ease of fabrication, the characterization of more realistic membrane mimics comprising multi-phase lipids remains challenging due to more complicated fabrication requirements. Herein, we report a convenient approach to fabricate and characterize multi-phase supported lipid bilayers (SLBs). We employed the solvent-assisted lipid bilayer (SALB) formation method to fabricate mixed lipid bilayers comprising liquid phase 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and gel phase 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipids at room temperature. The fabrication procedure was performed inside a newly designed microfluidic chamber, which facilitated the subsequent characterization of the SLBs without exposure to air. The SLBs were then characterized via fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), atomic force microscopy (AFM) and AFM-based force-distance measurements. Interestingly, results from these characterization techniques revealed that regardless of the gel phase composition, the SALB formation method consistently yielded uniform SLBs at room temperature, even though the transition temperature of DPPC is considerably higher. Furthermore, the composition ratio of DOPC and DPPC in the precursor solution is well reproduced in the fabricated SLBs. We also identified from diffusivity measurements that a high ratio of gel phase lipid revitalizes lipid-lipid interactions, which led to reduced molecular fluidity and the suppression of thermal undulation within the SLBs. Taken together, our results highlight the robustness of the SALB formation method that allows the fabrication of complex lipid bilayers with a high degree of precision, which is suitable for functional studies of biological membranes.

Galactosylceramide Domain Microstructure: Impact of Cholesterol and Nucleation/Growth Conditions

Long-range ordering of DNA crossover tiles with blunt ends on lipid bilayers is investigated using atomic force microscopy. "Blunt-ended" tiles do not have single-stranded complementary ends, and thus instead of assembling via base-pairing, they can interact by π-stacking of their duplex ends. This work demonstrates that the balance of base π-stacking interactions between the ends of DNA duplexes, cholesterol-mediated DNA anchoring, and electrostatic DNA binding to supported lipid bilayers (SLBs) presents an opportunity to build dynamic materials with long-range order on a soft support. The tiles are shown to organize into novel tunable surface packing morphologies on the micrometer scale. This work focuses on three-point star (3PS) tiles that are either unmodified or modified with a cholesterol unit and investigates their interactions on supported lipid bilayers. On fluid bilayers, the cholesterol tiles form extended hexagonal arrays with few defects, while the unmodified tiles do not bind. In contrast, both modified and unmodified tiles bind to gel-phase bilayers and produce arrays of new organized morphologies. With increasing tile concentration, we observe a range of motifs, that progressively favor tile-tile packing over duplex-end π-stacking. These structures can selectively pattern domains of phase-separated lipid bilayers, and the patterning is also observed for four-arm cross-tiles. Dynamic blunt end contacts promote error correction and network reconfiguration to maximize favorable interactions with the substrate and are required for the observed tile organization. These results suggest that small blunt-ended tiles can be used as a platform to organize oligonucleotides, nanoparticles, and proteins into extensive networks at the interface with biologically relevant membrane systems or other soft surface materials for applications in cellular recognition, plasmonics, light harvesting, model systems for membrane protein assemblies, or analytical devices.

The solid-substrate-dependent structure and dynamics of molecules in a supported lipid bilayer (SLB) were directly investigated via atomic force microscopy (AFM) and single particle tracking (SPT) measurements. The appearance of either vertical or horizontal heterogeneities in the SLB was found to be strongly dependent on the underlying substrates. SLB has been widely used as a biointerface with incorporated proteins and other biological materials. Both silica and mica are popular substrates for SLB. Using single-molecule dynamics, the fluidity of the upper and lower membrane leaflets was found to depend on the substrate, undergoing coupling and decoupling on the SiO2/Si and mica substrates, respectively. The anisotropic diffusion caused by the locally destabilized structure of the SLB at atomic steps appeared on the Al2O3(0001) substrate because of the strong van der Waals interaction between the SLB and the substrate. Our finding that the well-defined surfaces of mica and sapphire result in asymmetry and anisotropy in the plasma membrane is useful for the design of new plasma-membrane-mimetic systems. The application of well-defined supporting substrates for SLBs should have similar effects as cell membrane scaffolds, which regulate the dynamic structure of the membrane.

Electrodeless QCM-D for lipid bilayer applications

Lipopolysaccharides (LPS; endotoxin) activate immunocompetent cells of the host via a transmembrane signaling process. In this study, we investigated the function of the LPS-binding protein (LBP) in this process. The cytoplasmic membrane of the cells was mimicked by lipid liposomes adsorbed on mica, and the lateral organization of LBP in these membranes and its interaction with LPS aggregates were characterized by atomic force microscopy. Using cantilever tips functionalized with anti-LBP antibodies, single LBP molecules were localized in the membrane at low concentrations. At higher concentrations, LBP formed clusters of several molecules and caused cross-linking of lipid bilayers. The addition of LPS to LBP-containing liposomes led to the formation of LPS domains in the membranes, which could be inhibited by anti-LBP antibodies. Thus, LBP mediates the fusion of lipid membranes and LPS aggregates.

Docosahexaenoic acid (DHA) is the most abundant polyunsaturated omega-3 fatty acid found in mammalian neuronal cell membranes. Although DHA is known to be important for neuronal cell survival, little is know about how DHA interacts with phospholipid bilayers. This study presents a detailed quartz crystal microbalance with dissipation monitoring (QCM-D) analysis of free DHA interactions with individual and mixed phospholipid supported lipid bilayers (SLB). DHA incorporation and subsequent changes to the SLBs viscoelastic properties were observed to be concentration-dependent, influenced by the phospholipid species, the headgroup charge, and the presence or absence of calcium ions. It was observed that 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) SLBs incorporated the greatest amount of DHA concentration, whereas the presence of phospholipids, phosphatidylserine (PS), and phosphatidylinositol (PI) in a POPC SLB significantly reduced DHA incorporation and changed the SLBs physicochemical properties. These observations are hypothesized to be due to a substitution event occurring between DHA and phospholipid species. PS domain formation in POPC/PS 8:2 SLBs was observed in the presence of calcium ions, which favored DHA incorporation to a similar level as for a POPC only SLB. The changes in SLB thickness observed with different DHA concentrations are also presented. This work contributes to an understanding of the physical changes induced in a lipid bilayer as a consequence of its exposure to different DHA concentrations (from 50 to 200 μM). The capacity of DHA to influence the physical properties of SLBs indicates the potential for dietary DHA supplementation to cause changes in cellular membranes in vivo, with subsequent physiological consequences for cell function.

Out and In: Simplifying Membrane Protein Studies by AFM

Cell membranes consist of a multitude of lipid molecules that serve as a framework for the even greater variety of membrane associated proteins [1–4]. As this highly complex (nonequilibrium) system cannot easily be understood and studied in a controlled way, a wide variety of model systems have been devised to understand the dynamics, structure, and thermodynamics in biological membranes. One such model system is a supported lipid bilayer (SLB), a two-dimensional membrane suspended on a surface. SLBs have been realized to be manageable experimentally while reproducing many of the key features of real biological membranes [5,6]. One of the main advantages of supported bilayers is the physical stability due to the solid support that enables a wide range of surface characterization techniques not available to free or unsupported membranes. As SLBs maintain some of the crucial structural and dynamic properties of biological membranes, they provide an important bridge to natural systems. In order to mimic cell membranes reliably, certain structural and dynamic features have to be reliably reproduced in the artificially constructed lipid bilayers. SLBs should display lateral mobility as in living cells, because many membrane activities involve transport, recruitment, or assembly of specific components. It is also critical for membranes to exhibit the correct thermodynamic phase, namely, a fluid lipid bilayer, to respond to environmental stress such as temperature and pressure changes [7]. There are several ways to fabricate supported lipid bilayers (SLBs) on planar substrates. One can use vesicle fusion on solid substrates [5,8–10] as well as Langmuir-Blodgett deposition [11,12]. Proteoliposome adsorption and subsequent membrane formation on a mica surface was first demonstrated by Brian and McConnell [13]. Because of its simplicity and reproducibility, this is one of the most common approaches to prepare supported membranes. A diverse range of different solid substrates has been used as support material below the bilayer [14,15]. Silicon oxide is the material of choice for vesicle fusion [16]. Polymer cushions dampen the effect of hard surfaces and therefore have been actively investigated [17–20]. However, it is not fully understood which changes the introduction of a solid support introduces into such a biomimetic system. Experimentally it is almost impossible to address such changes, as extremely highresolution data would be required.