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The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry. [ABSTRACT FROM AUTHOR]

Investigations of ligand-binding kinetics to membrane proteins are hampered by their poor stability and low expression levels, which often translates into sensitivity-related limitations impaired by low signal-to-noise ratios. Inspired by affinity capturing of water-soluble proteins, which utilizes water as the mobile phase, we demonstrate affinity capturing and local enrichment of membrane proteins by using a fluid lipid bilayer as the mobile phase. Specific membrane-protein capturing and enrichment in a microfluidic channel was accomplished by immobilizing a synthesized trivalent nitrilotriacetic acid (tris-NTA)-biotin conjugate. A polymer-supported lipid bilayer containing His6-tagged β-secretase (BACE) was subsequently laterally moved over the capture region by using a hydrodynamic flow. Specific enrichment of His6-BACE in the Ni2+-NTA-modified region of the substrate resulted in a stationary three-fold increase in surface coverage, and an accompanied increase in ligand-binding response. [ABSTRACT FROM AUTHOR]

Relatively large (~100 nm) spherical particles, e.g., virions, vesicles, or metal nanoparticles, often interact with short (<10 nm) flexible receptors immobilized in a lipid membrane or on other biologically relevant surfaces. The attachment kinetics of such particles may be limited globally by their diffusion toward a membrane or locally by diffusion around receptors. The detachment kinetics, also, can be limited by diffusion. Focusing on local diffusion limitations and using suitable approximations, we present expressions for the corresponding rate constants and identify their dependence on particle size and receptor length. We also illustrate features likely to be observed in such kinetics for particles (e.g., vesicles) with a substantial size distribution. The results obtained are generic and can be used to interpret a variety of situations. For example, we estimate upper values of virion attachment rate constants and clarify the likely effect of vesicle size distribution on previously observed non-exponential kinetics of vesicle detachment. [ABSTRACT FROM AUTHOR]

The accurate determination of the maximum turnover number and Michaelis constant for membrane enzymes remains challenging. Here, this problem has been solved by observing in parallel the hydrolysis of thousands of individual fluorescently labeled immobilized liposomes each processed by a single phospholipase A2 molecule. The release of the reaction product was tracked using total internal reflection fluorescence microscopy. A statistical analysis of the hydrolysis kinetics was shown to provide the Michaelis-Menten parameters with an accuracy better than 20 % without variation of the initial substrate concentration. The combined single-liposome and single-enzyme mode of operation made it also possible to unravel a significant nanoscale dependence of these parameters on membrane curvature. [ABSTRACT FROM AUTHOR]

Attachment of lytic peptides to the lipid membrane of virions or bacteria is often accompanied by their aggregation and pore formation, resulting eventually in membrane rupture and pathogen neutralization. The membrane rupture may occur gradually via formation of many pores or abruptly after the formation of the first pore. In academic studies, this process is observed during interaction of peptides with lipid vesicles. We present an analytical model and the corresponding Monte Carlo simulations focused on the pore formation in such situations. Specifically, we calculate the time of the first nucleation-limited pore-formation event and show the distribution of this time in the regime when the fluctuations of the number of peptides attached to a vesicle are appreciable. The results obtained are used to clarify the mechanism of the pore formation and membrane destabilization observed recently during interaction of highly active a-helical peptide with sub-100-nm lipid vesicles that mimic enveloped viruses with nanoscale membrane curvature. The model proposed and the analysis presented are generic and may be applicable to other meso- and nanosystems. [ABSTRACT FROM AUTHOR]

Abstract: In cells, an appreciable part of enzymes is associated with lipid membranes. Academic experimental studies of the function of membrane enzymes (e.g., PLA2 representing a prototype for interfacial enzymology) are often focused on the enzyme–vesicle interaction or, more specifically, on conversion of lipid forming the external leaflet of the vesicle membrane. The corresponding kinetics are complicated by many factors inherent to the interfacial physics and chemistry. The understanding of the relative role of such factors and how they should be quantitatively described is still limited. Here, we present the mean-field kinetic equations, taking the formation of rafts in the membrane and the product-induced membrane strain into account, and analyze various scenarios of lipid conversion. In particular, we scrutinize the conditions when the kinetics may exhibit a transition from a relatively long latency period to a steady-state regime with fast nearly constant reaction rate. Specifically, we discuss the likely role of the pore formation in the external lipid layer in this transition. The latter effect may be caused by the product-induced tensile strain in this layer. [Copyright &y& Elsevier]

We have investigated the pathway by which unilamellar POPC liposomes upon adsorption undergo rupture and form a supported lipid bilayer (SLB) on a SiO2 surface. Biotinylated lipids were selectively incorporated in the outer monolayer of POPC liposomes to create liposomes with asymmetric lipid compositions in the outer and inner leaflets. The specific binding of neutravidin and anti-biotin to SLBs formed by liposome fusion, prior to and after equilibrated flip-flop between the upper and lower monolayers in the SLB, were then investigated. It was concluded that the lipids in the outer monolayer of the vesicle predominantly end up on the SLB side facing the SiO2 substrate, as demonstrated by having maximum 30-40% of lipids in the liposome outer monolayer orienting towards the bulk after forming the SLB. [ABSTRACT FROM AUTHOR]

A correction to the article on the lipid membrane nanometer-scale molecular organization by ion mass spectrometry is presented.

This article summarizes our most recent contributions to the rapidly growing field of supported lipid assemblies with emphasis on current studies addressing both fundamental and applied aspects of supported lipid bilayer (SLB) and tethered lipid vesicles (TLVs) to be utilized in sensing applications. The new insights obtained from combining the quartz crystal microbalance with dissipation monitoring technique with surface plasmon resonance are described, and we also present recent studies in which nanoplasmonic sensing has been used in studies of SLBs and TLVs. To gain full control over the spatial arrangement of TLVs in both two and three dimensions, we have developed a method for site-selective and sequence-specific sorting of DNA-tagged vesicles to surfaces modified with complementary DNA. The combination of this method with nanoplasmonic sensing formats is covered as well as the possibility of using DNA-modified vesicles for the detection of unlabeled DNA targets on the single-molecule level. Finally, a new method for membrane fusion induced by hybridization of vesicle-anchored DNA is demonstrated, including new results on content mixing obtained with vesicle populations encapsulating short, complementary DNA strands. [ABSTRACT FROM AUTHOR]

Membrane enzymatic reactions can now be experimentally studied at the level of single sub-100 nm lipid vesicles. To interpret such experiments, we scrutinize theoretically various aspects of the hydrolysis of vesicles by single enzyme molecules and enzyme (e.g., PLA2) supplied with the constant rate. Using the mean-field kinetic model, we illustrate the shape of the corresponding kinetics and the dependence of the time scale of the reaction on the vesicle radius. Stochastic effects are illustrated as well. In addition, we discuss the likely mechanisms of the reaction-induced pore formation and bilayer rupture. [ABSTRACT FROM AUTHOR]

Macromolecular association commonly occurs via dynamic engagement of multiple weak bonds referred to as multivalent interactions. The distribution of the number of bonds, combined with their strong influence on the residence time, makes it very demanding to quantify this type of interaction. To address this challenge in the context of virology, we mimicked the virion association to a cell membrane by attaching lipid vesicles (100 nm diameter) to a supported lipid bilayer via multiple, identical cholesterol-based DNA linker molecules, each mimicking an individual virion-receptor link. Using total internal reflection microscopy to track single attached vesicles combined with a novel filtering approach, we show that histograms of the vesicle diffusion coefficient D exhibit a spectrum of distinct peaks, which are associated with vesicles differing in the number, n, of linking DNA tethers. These peaks are only observed if vesicles with transient changes in n are excluded from the analysis. D is found to be proportional to 1/n, in excellent agreement with the free draining model, allowing to quantify transient changes of n on the single vesicle level and to extract transition rates between individual linking states. Necessary imaging conditions to extend the analysis to multivalent interactions in general are also reported. [ABSTRACT FROM AUTHOR]

Abstract: The aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na+) or divalent (Ca2+) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC–DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC–DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA–phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine. [Copyright &y& Elsevier]

This paper presents a study of supported lipid bilayer (SLB) formation and subsequent protein binding using a sensor that combines localized surface plasmon resonance (LSPR) and quartz crystal microbalance with dissipation (QCM-D) monitoring. The LSPR activity arises from silicon oxide (SiO~) coated nanometric apertures in a thin gold film, which also serves as the active electrode of a QCM-D crystal. Both transducer principles provide signatures for the formation of a SIB upon adsorption and subsequent rupture of adsorbed lipid vesicles. However, the two techniques are sensitive over different regions of the sample: LSPR primarily inside and on the rim of the holes and QCM-D primarily on the planar areas between the holes. Although the dimension of the lipid vesicles is on the same order as the dimension of the nanoholes, it is concluded from the response of the combined system that vesicle rupture in the nanoholes and on the planar region between the holes is synchronized. Furthermore, by determining the thickness of the SIB from the QCM-D response, the characteristic decay length of the LSPR field intensity could be determined. This made it possible not only to determine the mass and refractive index of the homogeneous SIB but also to postulate a generic means to quantify the LSPR response in terms of mass-uptake also for nonhomogeneous films. This is exemplified by measuring the adsorbed lipid mass during vesicle adsorption, yielding the critical lipid vesicle coverage at which spontaneous rupture into a planar bilayer occurs. The generic applicability and versatility of the method is demonstrated from specific protein binding to a functionalized SLB. From the absolute refractive index of the protein, provided from the LSPR data alone, it was possible to determine both the effective thickness of the protein film and the molecular mass (or number) of bound protein. [ABSTRACT FROM AUTHOR]

Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO2 surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3–4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed. [Copyright &y& Elsevier]

Multivalent receptor-mediated interactions between virions and a lipid membrane can be weakened using competitive nonpathogenic ligand binding. In particular, the subsequent binding of such ligands can induce detachment of bound virions, a phenomenon of crucial relevance for the development of new antiviral drugs. Focusing on the simian virus 40 (SV40) and recombinant cholera toxin B subunit (rCTB), and using (monosialotetrahexosyl)ganglioside (GM1) as their common receptor in a supported lipid bilayer (SLB), we present the first detailed investigation of this phenomenon by employing the quartz crystal microbalance with dissipation (QCM-D) and total internal reflection fluorescence (TIRF) microscopy assisted 2D single particle tracking (SPT) techniques. Analysis of the QCM-D-measured release kinetics made it possible to determine the binding strength of a single SV40-GM1 pair. The release dynamics of SV40, monitored by SPT, revealed that a notable fraction of SV40 becomes mobile just before the release, allowing to estimate the distribution of SV40-bound GM1 receptors just prior to release. [ABSTRACT FROM AUTHOR]

Proteins are key components in a multitude of biological processes, of which the functions carried out by transmembrane (membrane-spanning) proteins are especially demanding for investigations. This is because this class of protein needs to be incorporated into a lipid bilayer representing its native environment, and in addition, many experimental conditions also require a solid support for stabilization and analytical purposes. The solid support substrate may, however, limit the protein functionality due to protein-material interactions and a lack of physical space. We have in this work tailored the pore size and pore ordering of a mesoporous silica thin film to match the native cell-membrane arrangement of the transmembrane protein human aquaporin 4 (hAQP4). Using neutron reflectivity (NR), we provide evidence of how substrate pores host the bulky water-soluble domain of hAQP4, which is shown to extend 7.2 nm into the pores of the substrate. Complementary surface analytical tools, including quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy, revealed successful protein-containing supported lipid bilayer (pSLB) formation on mesoporous silica substrates, whereas pSLB formation was hampered on nonporous silica. Additionally, electron microscopy (TEM and SEM), light scattering (DLS and stopped-flow), and small-angle X-ray scattering (SAXS) were employed to provide a comprehensive characterization of this novel hybrid organic-inorganic interface, the tailoring of which is likely to be generally applicable to improve the function and stability of a broad range of membrane proteins containing water-soluble domains. [ABSTRACT FROM AUTHOR]

The article presents a method to selectively fuse lipid vesicles by the hybridization of membrane-anchored DNA strands. The authors note that the concept will provide a tool to manipulate the composition and content of artificial and biological lipid compartments and a model for membrane fusion in cells. DNA-induced fusion was showed to be sensitive to the presence of cone shaped lipids such as cholesterol. Results of the study and its implications are presented and discussed.

Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins, we used total internal reflection fluorescence imaging to study a transmembrane protease, ß-secretase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEG-cushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms. [ABSTRACT FROM AUTHOR]

Using tethered sub-100 nm lipid vesicles that mimic envelopedviruseswith nanoscale membrane curvature, we have in this work designed atotal internal reflection fluorescence microscopy-based single vesicleassay to investigate how an antiviral amphipathic α-helical(AH) peptide interacts with lipid membranes to induce membrane curvature-dependentpore formation and membrane destabilization. Based on a combinationof statistics from single vesicle imaging, binding kinetics data,and theoretical analysis, we propose a mechanistic model that is consistentwith the experimentally observed peptide association and pore formationkinetics at medically relevant peptide concentrations (10 nM to 1μM) and unusually low peptide-to-lipid (P/L) ratio (∼1/1000).Importantly, the preference of the AH peptide to selectively rupturevirions with sub-100 nm diameters appears to be related to membranestrain-dependent pore formation rather than to previously observednanoscale membrane curvature facilitated binding of AH peptides. Comparedto other known proteins and peptides, the combination of low effectiveP/L ratio and high specificity for nm-sized membrane curvature lendsthis particular AH peptide great potential to serve as a frameworkfor developing a highly specific and potent antiviral agent for prophylacticand therapeutic applications while avoiding toxic side effects againsthost cell membranes. [ABSTRACT FROM AUTHOR]

A single-chip electrochemical method based on impedance measurements in resonance mode has been employed to study lipid monolayer and bilayer formation on hydrophobic alkanethiolate and SiO2 substrates, respectively. The processes were monitored by temporally resolving changes in interfacial capacitance and resistance, revealing information about the rate of formation, coverage, and defect density (quality) of the layers at saturation. The resonance-based impedance measurements were shown to reveal significant differences in the layer formation process of bilayers made from (i) positively charged lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), (ii) neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on SiO2, and (iii) monolayers made from POEPC on hydrophobic alkanethiolate substrates. The observed responses were represented with an equivalent circuit, suggesting that the differences primarily originate from the presence of a conductive aqueous layer between the lipid bilayers and the SiO2. In addition, by adding the ion channel gramicidin D to bilayers supported on SiO2, channel-mediated charge transport could be measured with high sensitivity (resolution around 1 pA). [ABSTRACT FROM AUTHOR]

Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hydrodynamically driven SLB can be used to induce rupture of adsorbed lipid vesicles of compositions that typically prevent spontaneous SLB formation, such as vesicles made of complex lipid compositions, containing high cholesterol content or being derived from real cell membranes. In particular, upon fusion between the moving edge of a preformed SLB and adsorbed vesicles made directly from 3T3 fibroblast cell membranes, the membrane content of the vesicles was shown to be efficiently transferred to the SLB. The molecular transfer was verified using cholera toxin B subunit (CTB) binding to monosialoganglioside receptors (GM1 and GM3), and the preserved lateral mobility was confirmed by spatial manipulation of the GM1/M3—CTB complex using a hydrodynamic flow. Two populations of CTB with markedly different drift velocity could be identified, which from dissociation kinetics data were attributed to CTB bound with different numbers of ganglioside anchors. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]