Microcavity supported lipid membranes: versatile platforms for building asymmetric lipid bilayers and for protein recognition

Microcavity supported lipid bilayers (MSLB) are contact-free membranes suspended across aqueousfilled pores that maintain the lipid bilayer in a highly fluidic state and free from frictional interactions with substrate. Such platforms offer the prospect of liposome-like fluidity with the compositional versatility and addressability of supported lipid bilayers and thus offer significant opportunity for modelling membrane asymmetry, protein-membrane interactions and aggregation at the membrane interface. Herein, we evaluate their performance by studying the effect of transmembrane lipid asymmetry on lipid diffusivity, membrane viscosity and cholera toxin- ganglioside recognition across six symmetric and asymmetric membranes including binary compositions containing both fluid and gel phase, and ternary phase separated membrane compositions. Fluorescence lifetime correlation spectroscopy (FLCS) was used to determine the lateral mobility of lipid and protein, and electrochemical impedance spectroscopy (EIS) enabled detection of protein-membrane assembly over the nanomolar range. Transmembrane leaflet asymmetry was observed to have profound impact on membrane electrochemical resistance where the resistance of a ternary symmetric phase separated bilayer was found to be at least 2.6 times higher than the asymmetric bilayer with analogous composition at the distal leaflet but where the lower leaflet comprised only 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Similarly, the diffusion coefficient for MSLBs was observed to be 2.5 fold faster for asymmetric MSLBs where the lower leaflet is DOPC alone. Our results demonstrate that interplay of lipid packing across both membrane leaflets and concentration of GM1 both affect the extent of cholera toxin aggregation and consequent diffusion of the cholera-GM1 aggregates. Given that true biomembranes are both fluidic and asymmetric, MSLBs offer the opportunity for building greater biomimicry into biophysical models and the approach described demonstrates the value of MSLBs in studying aggregation and membrane associated multivalent interactions prevalent in many carbohydrates mediated processes

Interaction of miltefosine with microcavity supported lipid membrane: biophysical insights from electrochemical impedance spectroscopy

Miltefosine an alkylphosphocholine analogue, is the only drug taken orally for the treatment of leishmaniasis-a parasitic disease caused by sandflies. Although it is believed that Miltefosine exerts its activity by acting at the lipid membrane, detailed understanding of the interaction of this drug with eukaryotic membranes is still lacking. Herein, we exploit microcavity pore suspended lipid bilayers (MSLBs) as a biomimetic platform in combination with a highly sensitive label-free electrochemical impedance spectroscopy (EIS) technique to gain biophysical insight into the interaction of Miltefosine with host cell membrane as a function of lipid membranes composition. Four membrane compositions with increasing complexity were evaluated; DOPC, DOPC:Chol (75:25), domain forming DOPC:SM:Chol (40:40:20) and mammalian plasma membrane (MPM) mimetic DOPC:DOPE:Chol:SM:DOPS (32:25:20:15:8) and used to study the interaction of Miltefosine in a concentration-dependent manner using EIS. The membrane resistance changes in response to Miltefosine were modelled by an empirical Langmuir isotherm binding model to provide estimates of binding saturation and equilibrium association constant. Miltefosine was found to have greatest impact on electrochemical properties of the simpler membrane systems; DOPC and DOPC:Chol, where these membranes were found to be more susceptible to membrane thinning, attributed to strong permeation/penetration of the drug whilst, compositions that included both Chol and SM, expected to contain large liquid-ordered domains exhibited weaker changes to membrane resistance but strongest drug association. In contrast, at the MPM membrane, Miltefosine exerts weakest association, which is tentatively attributed to electrostatic effects from the anionic DOPS but some membrane thinning is observed reflected in change in resistance and capacitance values attributed to some weak permeation

Microcavity supported lipid bilayers; evaluation of drug- lipid membrane Interactions by electrochemical impedance and fluorescence correlation spectroscopy

Many drugs have intracellular or membrane-associated targets thus understanding their interaction with the cell membrane is of value in drug development. Cell-free tools used to predict membrane interactions should replicate the molecular organization of the membrane. Microcavity array supported lipid bilayer (MSLB) platform are versatile biophysical models of the cell membrane that combine liposome-like membrane fluidity with stability and addressability. We used an MSLB herein to interrogate drugmembrane interactions across seven drugs from different classes, including non-steroidal antiinflammatories; Ibuprofen (Ibu) and Diclofenac (Dic), antibiotics; Rifampicin (Rif), Levofloxacin (Levo) and Pefloxacin (Pef), and bisphosphonates; Alendronate (Ale) and Clodronate (Clo). Fluorescence lifetime correlation spectroscopy (FLCS) and electrochemical impedance spectroscopy (EIS) were used to evaluate the impact of drug on DOPC and binary bilayers over physiologically relevant drug concentrations. Whereas FLCS data revealed Ibu, Levo, Pef, Ale and Clo had no impact on lipid lateral mobility, EIS which is more sensitive to membrane structural change, indicated modest but significant decreases to membrane resistivity consistent with adsorption but weak penetration of drugs at the membrane. Ale and Clo, evaluated at pH 5.25, did not impact the impedance of the membrane except at concentrations exceeding 4mM. Conversely, Dic and Rif dramatically altered bilayer fluidity, suggesting their translocation through the bilayer and, EIS data, showed resistivity of the membrane decreased substantially with increasing drug concentration. Capacitance changes to the bilayer in most cases were insignificant. Using a Langmuir-Freundlich model to fit the EIS data, we propose Rsat as an empirical value that reflects permeation. Overall, the data indicate that Ibu, Levo, and Pef, adsorb at the interface of the lipid membrane but Dic and Rif interact strongly, permeating the membrane core modifying the water/ion permeability of the bilayer structure. These observations are discussed in the context of previously reported data on drug permeability and Log P

Interaction of miltefosine with microcavity supported lipid membrane: biophysical insights from electrochemical impedance spectroscopy

Miltefosine an alkylphosphocholine analogue, is the only drug taken orally for the treatment of leishmaniasis-a parasitic disease caused by sandflies. Although it is believed that Miltefosine exerts its activity by acting at the lipid membrane, detailed understanding of the interaction of this drug with eukaryotic membranes is still lacking. Herein, we exploit microcavity pore suspended lipid bilayers (MSLBs) as a biomimetic platform in combination with a highly sensitive label-free electrochemical impedance spectroscopy (EIS) technique to gain biophysical insight into the interaction of Miltefosine with host cell membrane as a function of lipid membranes composition. Four membrane compositions with increasing complexity were evaluated; DOPC, DOPC:Chol (75:25), domain forming DOPC:SM:Chol (40:40:20) and mammalian plasma membrane (MPM) mimetic DOPC:DOPE:Chol:SM:DOPS (32:25:20:15:8) and used to study the interaction of Miltefosine in a concentration-dependent manner using EIS. The membrane resistance changes in response to Miltefosine were modelled by an empirical Langmuir isotherm binding model to provide estimates of binding saturation and equilibrium association constant. Miltefosine was found to have greatest impact on electrochemical properties of the simpler membrane systems; DOPC and DOPC:Chol, where these membranes were found to be more susceptible to membrane thinning, attributed to strong permeation/penetration of the drug whilst, compositions that included both Chol and SM, expected to contain large liquid-ordered domains exhibited weaker changes to membrane resistance but strongest drug association. In contrast, at the MPM membrane, Miltefosine exerts weakest association, which is tentatively attributed to electrostatic effects from the anionic DOPS but some membrane thinning is observed reflected in change in resistance and capacitance values attributed to some weak permeation

Pathways for creation and annihilation of nanoscale biomembrane domains reveal alpha and beta-toxin nanopore formation processes

Raft-like functional domains with putative sizes of 20-200 nm and which are evolving dynamically are believed to be the most crucial regions in cellular membranes which determine cell signaling and various functions of cells. While the actual sizes of these domains are believed to vary from cell to cell no direct determination of their sizes and their evolution when cells interact with external agents like toxins and relevant biomolecules exists. Here, we report the first direct determination of the size of these nanoscale regions in model raft-forming biomembranes using the method of super-resolution stimulated emission depletion nanoscopy coupled with fluorescence correlation spectroscopy (STED-FCS). We also show that the various pathways for creation and destruction of such nanoscale membrane regions due to interaction with prototypical and nanopore-forming toxins, can reveal the nature of the respective pore formation processes. The methodology, in turn, establishes a new nano-biotechnological protocol which could be very useful in preventing their cytotoxic effects in particular but also enable microscopic understanding of biomolecule-cell membrane interactions in general

Preferential binding and re-organization of nanoscale domains on model lipid membranes by pore-forming toxins: insight from STED-FCS

Potential nanodomains in cellular membranes are widely believed to be targeted by proteins and other biomolecules to enable execution of critical cellular functions. However, characterization of these nanodomains remains elusive, primarily due to the diffraction limit of a conventional optical microscope. Herein using super-resolution STED microscopy coupled with fluorescence correlation spectroscopy (STED-FCS), we provide experimental evidence of lipid nanodomains present in model membranes comprised of phosphocholine-cholesterol binary mixture, and its reorganization induced by pore-forming toxins (PFTs). In this study, we used two different types of PFTs, namely alpha-PFT (cytolysis A) and beta-PFT (listeriolysin O), that preferentially associate as well as reorganize cholesterol-containing saturated and unsaturated phosphocholine membranes. The emergence of nanodomains due to the lipid-lipid and lipid-PFT interactions was quantified at a length scales of similar to 50-150nm using FCS diffusion law enabled by variation of spot sizes in the super-resolution STED microscopy. Our results shed light on the usefulness of super-resolution microscopy to quantify the underlying nanoscale domains in model membranes with implications for a wide variety of membrane-mediated cellular events observed in real cell membranes

Complex dynamics at the nanoscale in simple biomembranes

Nature is known to engineer complex compositional and dynamical platforms in biological membranes. Understanding this complex landscape requires techniques to simultaneously detect membrane reorganization and dynamics at the nanoscale. Using super-resolution stimulated emission depletion (STED) microscopy coupled with fluorescence correlation spectroscopy (FCS), we reveal direct experimental evidence of dynamic heterogeneity at the nanoscale in binary phospholipid-cholesterol bilayers. Domain formation on the length scale of similar to 200-600 nm due to local cholesterol compositional heterogeneity is found to be more prominent at high cholesterol content giving rise to distinct intra-domain lipid dynamics. STED-FCS reveals unique dynamical crossover phenomena at length scales of similar to 100-150 nm within each of these macroscopic regions. The extent of dynamic heterogeneity due to intra-domain hindered lipid diffusion as reflected from the crossover length scale, is driven by cholesterol packing and organization, uniquely influenced by phospholipid type. These results on simple binary model bilayer systems provide novel insights into pathways leading to the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events

Unraveling complex nanoscale lipid dynamics in simple model biomembranes: Insights from fluorescence correlation spectroscopy in super-resolution stimulated emission depletion mode

Dynamic heterogeneity (DH) at nanoscale due to lipid-lipid and/or lipid-protein interactions in cell membranes plays a crucial role in determining a broad range of important cell functions. In cell membranes, the dimensions of these nanodomains have been postulated to be in the order of 10's of nm and transient in nature. While the structural features of membranes have been studied in detail, little is known about their dynamical characteristics due to paucity of techniques which can probe nanoscale phenomena with simultaneous high temporal resolution. A combination of super-resolution stimulated emission depletion (STED) and fluorescence correlation spectroscopy (FCS) technique can overcome this limitation and provide information about the nanoscale dynamic heterogeneity in cell membranes. Using STED-FCS and FCS diffusion law, we provide an understanding of how nanoscale dynamically organizing lipid platforms can emerge in minimal system of model biomembranes. To illustrate the utility of the technique we have chosen cholesterol containing supported lipid bilayers and demonstrated the role of cholesterol concentration and/or added pore-forming protein, Listeriolysin O (LLO) in determining onset of lipid DH. In addition we have also looked at multi-component lipid bilayers with and without cholesterol to infer about the role of phospholipid and cholesterol composition on lipid dynamics. These results on simple biomimetic systems provide insights into fundamental pathways for the emergence of complex nanodomain substructures with implications for a wide variety of membrane mediated cellular events and depict the significant contribution that STED-FCS can make in resolving several outstanding issues in membrane biology. (C) 2017 Elsevier Inc. All rights reserved

Establishing the Ellipsoidal Geometry of a Benzoic Acid-Based Amphiphile via Dimer Switching: Insights from Intramolecular Rotation and Facial H‑Bond Torsion

Soft molecular ellipsoids conceived from 3,4-di­(dodecyloxy)­benzoic acid (DDBA) amphiphile draw attention to monomer structure design, intramolecular −COOH headgroup twist (ϕ°) and cyclic–acyclic dimer switching through facial H-bond torsion (ψ°). Generically, precipitation in hydrogen bonded systems has been the prime phenomenon once the critical aggregation concentrations were reached in the bulk solution. DDBA was no exception to this generalization. It formed precipitates in chloroform and methanol with no specific geometry but with cyclic dimer motifs in them. On the contrary, surface pressure modulated interfacial aggregation with ellipsoidal geometry followed acyclic dimerization (catemer motif) with various levels of headgroup torsion, established through real-time polarization modulated infrared reflection–absorption spectroscopy (IRRAS) and density functional theory (DFT) calculations, that estimated the energy costs for these unexplored pathways. The reaction coordinates ϕ° and ψ° in consonance with 2D surface pressure modulation thus directed the shape anisotropy during the dynamic self-assembly of DDBA. Changes in subphase pH and metal ionic environment had a derogatory effect on the ellipsoid formation, the structural requirement for which strictly followed a stringent need for twin alkyl chains in an asymmetric unit cell, as 4-dodecyloxybenzoic acid (MABA) with a single alkyl chain formed exclusively spherical assemblies with no dimer modulation. The investigation thus reports unexplored energy pathways toward ellipsoidal geometry of the amphiphile in the course of its interfacial aggregation

Nanoscale dynamics of phospholipids reveals an optimal assembly mechanism of pore-forming proteins in bilayer membranes

Cell membranes are believed to be highly complex dynamical systems having compositional heterogeneity involving several types of lipids and proteins as the major constituents. This dynamical and compositional heterogeneity is suggested to be critical to the maintenance of active functionality and response to chemical, mechanical, electrical and thermal stresses. However, delineating the various factors responsible for the spatio-temporal response of actual cell membranes to stresses can be quite challenging. In this work we show how biomimetic phospholipid bilayer membranes with variable lipid fluidity determine the optimal assembly mechanism of the pore-forming protein, listeriolysin O (LLO), belonging to the class of cholesterol dependent cytolysins (CDCs). By combining atomic force microscopy (AFM) and super-resolution stimulated emission depletion (STED) microscopy imaging on model membranes, we show that pores formed by LLO in supported lipid bilayers can have variable conformation and morphology depending on the fluidity of the bilayer. At a fixed cholesterol concentration, pores formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes were larger, flexible and more prone to coalescence when compared with the smaller and more compact pores formed in the lower fluidity 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. In contrast, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes did not show any evidence of pore formation. Fluorescence correlation spectroscopy (FCS) in STED mode revealed the appearance of a length scale, xi, below which lipid dynamics, under the influence of LLO protein binding and assembly, becomes anomalous. Interestingly, the magnitude of xi is found to correlate with both lipid fluidity and pore dimensions (and flexibility) in DOPC and POPC bilayers. However this length scale dependent crossover, signalling the onset of anomalous diffusion, was not observed in DMPC bilayers. Our study highlights the subtle interplay of lipid membrane mediated protein assembly and lipid fluidity in determining proteolipidic complexes formed in biomembranes and the significant insight that STED microscopy provides in unraveling critical aspects of nanoscale membrane biophysics