Multicomponent Lipid Bilayers Research Articles

Coupling the role of lipids to the conformational dynamics of the ABC transporter P-glycoprotein

The ATP-binding cassette transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug-resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, “straight” and “kinked,” respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of two human P-gp inward-open conformers, straight and kinked, employing all-atom molecular dynamics (MD) simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter into the cavity of the straight P-gp conformer solved in detergent. However, access to the cavity of the kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp conformers and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, the findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-gp, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane helix (TM10) of the straight conformer underwent a spontaneous transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between the two P-gp conformers.

Membrane lateral organization from potential energy disconnectivity graph

Understanding the thermodynamic and kinetic properties of biomolecules requires elucidation of their complex energy landscape. A disconnectivity graph analysis of the energy landscape provides a framework for mapping the multi-dimensional landscape onto a two-dimensional representation while preserving the key features of the energy landscape. Several studies show that the structure or shape of the disconnectity graph is directly associated with the function of protein and nucleic acid molecules. In this review, we discuss how disconnectivity analysis of the potential energy surface can be extended to lipid molecules to glean important information about membrane organization. The shape of the disconnectivity graphs can be used to predict the lateral organization of multi-component lipid bilayer. We hope that this review encourages the use of disconnectivity graphs routinely by membrane biophysicists to predict the lateral organization of lipids.

Extension of the iSoLF implicit-solvent coarse-grained model for multicomponent lipid bilayers.

iSoLF is a coarse-grained (CG) model for lipid molecules with the implicit-solvent approximation used in molecular dynamics (MD) simulations of biological membranes. Using the original iSoLF (iSoLFv1), MD simulations of lipid bilayers consisting of either POPC or DPPC and these bilayers, including membrane proteins, can be performed. Here, we improve the original model, explicitly treating the electrostatic interactions between different lipid molecules and adding CG particle types. As a result, the available lipid types increase to 30. To parameterize the potential functions of the new model, we performed all-atom MD simulations of each lipid at three different temperatures using the CHARMM36 force field and the modified TIP3P model. Then, we parameterized both the bonded and non-bonded interactions to fit the area per lipid and the membrane thickness of each lipid bilayer by using the multistate Boltzmann Inversion method. The final model reproduces the area per lipid and the membrane thickness of each lipid bilayer at the three temperatures. We also examined the applicability of the new model, iSoLFv2, to simulate the phase behaviors of mixtures of DOPC and DPPC at different concentrations. The simulation results with iSoLFv2 are consistent with those using Dry Martini and Martini 3, although iSoLFv2 requires much fewer computations. iSoLFv2 has been implemented in the GENESIS MD software and is publicly available.

Interactions of Bacterial Quorum Sensing Signals with Model Lipid Membranes: Influence of Membrane Composition on Membrane Remodeling.

We report the influence of membrane composition on the multiscale remodeling of multicomponent lipid bilayers initiated by contact with the amphiphilic bacterial quorum sensing signal N-(3-oxo)-dodecanoyl-l-homoserine lactone (3-oxo-C12-AHL) and its anionic headgroup hydrolysis product, 3-oxo-C12-HS. We used fluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) to characterize membrane reformation that occurs when these amphiphiles are placed in contact with supported lipid bilayers (SLBs) composed of (i) 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) containing varying amounts of cholesterol or (ii) mixtures of DOPC and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, a conical zwitterionic lipid) or 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS, a model anionic lipid). In general, we observe these mixed-lipid membranes to undergo remodeling events, including the formation and subsequent collapse of long tubules and the formation of hemispherical caps, upon introduction to biologically relevant concentrations of 3-oxo-C12-AHL and 3-oxo-C12-HS in ways that differ substantially from those observed in single-component DOPC membranes. These differences in bilayer reformation and their associated dynamics can be understood in terms of the influence of membrane composition on the time scales of molecular flip-flop, lipid packing defects, and lipid phase segregation in these materials. The lipid components investigated here are representative of classes of lipids that comprise both naturally occurring cell membranes and many useful synthetic soft materials. These studies thus represent a first step toward understanding the ways in which membrane composition can impact interactions with this important class of bacterial signaling molecules.

Conformations of Three Types of Ultra-Long-Chain Fatty Acids in Multicomponent Lipid Bilayers.

Ultra-long-chain fatty acids (ULCFAs) are biosynthesized in certain types of tissues, but their biological roles remain unknown. Here, we report how the conformation of ULCFAs depends on the length and unsaturated-bond ratio of the ultra-long chains and the composition of the host bilayer membrane using molecular dynamics simulations. The ultra-long chain of ULCFAs flips between the two leaflets and fluctuates among three conformations: elongated, L-shaped, and turned. Furthermore, we found that the saturated ultra-long chain exhibited an elongated conformation more frequently than the unsaturated chain. In addition, the truncation of the ultra-long chain at C26 had little effect on the remaining ULCFAs. ULCFAs respond to lipid-density differences in the two leaflets, and the ratio of the elongated and turned conformations changed to reduce this difference. However, in cholesterol-containing membranes, ULCFAs exhibit no density difference after the flip-flop of cholesterol removes the difference.

Polyvinyl Chloride Nanoparticles Affect Cell Membrane Integrity by Disturbing the Properties of the Multicomponent Lipid Bilayer in Arabidopsis thaliana.

The ubiquitous presence of nanoplastics (NPs) in natural ecosystems is a serious concern, as NPs are believed to threaten every life form on Earth. Micro- and nanoplastics enter living systems through multiple channels. Cell membranes function as the first barrier of entry to NPs, thus playing an important biological role. However, in-depth studies on the interactions of NPs with cell membranes have not been performed, and effective theoretical models of the underlying molecular details and physicochemical behaviors are lacking. In the present study, we investigated the uptake of polyvinyl chloride (PVC) nanoparticles by Arabidopsis thaliana root cells, which leads to cell membrane leakage and damage to membrane integrity. We performed all-atom molecular dynamics simulations to determine the effects of PVC NPs on the properties of the multicomponent lipid bilayer. These simulations revealed that PVCs easily permeate into model lipid membranes, resulting in significant changes to the membrane, including reduced density and changes in fluidity and membrane thickness. Our exploration of the interaction mechanisms between NPs and the cell membrane provided valuable insights into the effects of NPs on membrane structure and integrity.

Brillouin Spectroscopy of Binary Phospholipid-Cholesterol Bilayers.

Multicomponent lipid bilayers are used as models for searching the origin of spatial heterogeneities in biomembranes called lipid rafts, implying the coexistence of domains of different phases and compositions within the lipid bilayer. The spatial organization of multicomponent lipid bilayers on a scale of a hundred nanometers remains unknown. Brillouin spectroscopy providing information about the acoustic phonons with the wavelength of several hundred nanometers has an unexplored potential for this problem. Here, we applied Brillouin spectroscopy for three binary bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol. The Brillouin experiment for the oriented planar multibilayers was realized for two scattering geometries involving phonons for the lateral and normal directions of the propagation. The DPPC-DOPC mixtures known for the coexistence of the solid-ordered and liquid-disordered phases had bimodal Brillouin peaks, revealing the phase domains with sizes more than a hundred nanometers. Analysis of the Brillouin data for the binary mixtures concluded that the lateral phonons are preferable for testing the lateral homogeneity of the bilayers, while the phonons spreading across the bilayers are sensitive to the layered packing at the mesoscopic scale.

Dynamic response on a nanometer scale of binary phospholipid-cholesterol vesicles: Low-frequency Raman scattering insight.

Low-frequency Raman spectroscopy was used to study the dynamic response on a nanometer scale of aqueous suspensions of two-component lipid vesicles. Binary mixtures of saturated phospholipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) and cholesterol are interesting for possible coexistence of solidlike and liquid-ordered phases, while the phase coexistence was not reported for unsaturated phospholipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC) and cholesterol mixtures. The DOPC-DPPC mixtures represent the well-documented case of coexisting domains of solidlike and liquid-disordered phases. These three series of lipid mixtures are studied here. A broad peak with the maximum in the range of 30-50cm^{-1} and a narrow peak near 10cm^{-1} are observed in the Raman susceptibility of the binary mixtures and attributed to the acousticlike vibrational density of states and layer modes, respectively. Parameters of the broad and narrow peaks are sensitive to lateral and conformational hydrocarbon chain ordering. It was also demonstrated that the low-frequency Raman susceptibility of multicomponent lipid bilayers allows one to determine the phase state of lipid bilayers and distinguish the homogeneous distribution of molecular complexes from coexisting domains with sizes above several nanometers. Thus, the low-frequency Raman spectroscopy provides unique information in studying phase coexistence in lipid bilayers.

Nonconverged Constraints Cause Artificial Temperature Gradients in Lipid Bilayer Simulations.

Molecular dynamics (MD) simulations have become an indispensable tool to investigate phase separation in model membrane systems. In particular, simulations based on coarse-grained (CG) models have found widespread use due to their increased computational efficiency, allowing for simulations of multicomponent lipid bilayers undergoing phase separation into liquid-ordered and liquid-disordered domains. Here, we show that a significant temperature difference between molecule types can artificially arise in CG MD membrane simulations with the standard Martini simulation parameters in GROMACS. In particular, the linear constraint solver (LINCS) algorithm does not converge with its default settings, resulting in serious temperature differences between molecules in a time step-dependent manner. We demonstrate that the underlying reason for this behavior is the presence of highly constrained moieties, such as cholesterol. Their presence can critically impact numerous structural and dynamic membrane properties obtained from such simulations. Furthermore, any preference of these molecules toward a certain membrane phase can lead to spatial temperature gradients, which can amplify the degree of phase separation or even induce it in compositions that would otherwise mix well. We systematically investigated the effect of the integration time step and LINCS settings on membrane properties. Our data show that for cholesterol-containing membranes, a time step of 20 fs should be combined with at least lincs_iter = 2 and lincs_order = 12, while using a time step of 30 fs requires at least lincs_iter = 3 and lincs_order = 12 to bring the temperature differences to a level where they do not perturb central membrane properties. Moreover, we show that in cases where stricter LINCS settings are computationally too demanding, coupling the lipids in multiple groups to the temperature bath offers a practical workaround to the problem, although the validity of this approach should be further verified. Finally, we show that similar temperature gradients can also emerge in atomistic simulations using the CHARMM force field in combination with settings that allow for a 5 fs integration step.

Effect of alcohol on the phase separation in model membranes

The discovery of coexisting liquid-ordered and liquid-disordered phases in multicomponent lipid bilayers has received widespread attention due to its potential relevance for biological systems. One of the many open questions is how the presence of additional components affects the nature of the coexisting phases. Of particular interest is the addition of alcohols because their anesthetic properties may arise from modulating bilayer behavior. We use coarse-grained Molecular Dynamics simulations to gain insight into the partitioning preferences of linear n-alcohols into ordered and disordered bilayers alongside their effects on local membrane structure. We find that alcohols cause only small changes to membrane composition alongside a lack of significant effects on membrane thickness and lipid tail order. Cholesterol and n-alcohol trans-bilayer motion is measured and found to be near or within the range of previous atomistic results. The cholesterol flip-flop rates increase with both n-alcohol length and concentration for octanol, dodecanol, and hexadecanol, indicating a decrease in lipid order. Umbrella sampling simulations of removing cholesterol from tertiary membranes find no significant difference with or without n-alcohols at various concentrations. Simulations of a phase-separated bilayer show that octanol preferentially partitions into the liquid-disordered phase in a ratio of approximately 3:1 over the liquid-ordered phase. Furthermore, partition coefficients of alcohol in single-phase membranes show a preference for longer alcohols (dodecanol and hexadecanol) to partition preferentially into the liquid-ordered phase, while decreasing the length of the alcohol reverses this trend. Our work tests experimental results while also investigating the ability for coarse-grained MARTINI simulations to capture minute differences in model membrane spatial arrangements on the nanoscale level.

Activation Microswitches in Adenosine Receptor A2A Function as Rheostats in the Cell Membrane.

Although multiple components of the cell membrane modulate the stability and activation of G protein-coupled receptors (GPCRs), insights into the dynamics of GPCR structures come from biophysical studies conducted in detergents. This is because of the challenges of studying activation in a multicomponent lipid bilayer. To understand the role of cellular membrane lipids and cations in GPCR activation, we performed multiscale molecular dynamics simulations (56 μs) on three different conformational states of adenosine receptor A2AR, in both the cell membrane-like lipid bilayer and in detergent micelles. Molecular dynamics (MD) simulations show that the phosphatidylinositol bisphosphate (PIP2) interacts with the basic residues in the intracellular regions of A2AR, thereby reducing the flexibility of the receptor in the inactive state and limiting the transition to the active-intermediate state. In the G protein-coupled fully active state, PIP2 stabilizes the GPCR:G protein complex. Such stiffening effects are absent in non-ionic detergent micelles, and therefore, more transitions have been observed in detergents. The inter-residue distances that change significantly upon GPCR activation are known as activation microswitches. The activation microswitches show different levels of activation in the cell membrane, in the pure POPC bilayer, and in detergents. Thus, the temporal heat map of different activation microswitches calculated from the MD simulations suggests a rheostat model of GPCR activation microswitches rather than the binary switch model. These simulation results connect the chemistry of cell membrane lipids to receptor activity, which is useful for the design of detergents mimicking the cell membrane.

Modulated and spiral surface patterns on deformable lipid vesicles.

We investigate the behavior of two-dimensional systems that exhibit a transition between homogeneous and spatially inhomogeneous phases, which have spherical topology, and whose mechanical properties depend on the local value of the order parameter. One example of such a system is multicomponent lipid bilayer vesicles, which serve as a model to study cellular membranes. Under certain conditions, such bilayers separate into coexisting liquid-ordered and liquid-disordered regions. When arranged into the shape of small vesicles, this phase coexistence can result in spatial patterns that are more complex than the basic two-domain configuration encountered in typical bulk systems. The difference in bending rigidity between the liquid-ordered and liquid-disordered regions couples the shape of the vesicle to the local composition. We show that this interplay gives rise to a rich phase diagram that includes homogeneous, separated, and axisymmetric modulated phases that are divided by regions of spiral patterns in the surface morphology.

Geometric pinning and antimixing in scaffolded lipid vesicles

Previous studies on the phase behaviour of multicomponent lipid bilayers found an intricate interplay between membrane geometry and its composition, but a fundamental understanding of curvature-induced effects remains elusive. Thanks to a combination of experiments on lipid vesicles supported by colloidal scaffolds and theoretical work, we demonstrate that the local geometry and global chemical composition of the bilayer determine both the spatial arrangement and the amount of mixing of the lipids. In the mixed phase, a strong geometrical anisotropy can give rise to an antimixed state, where the lipids are mixed, but their relative concentration varies across the membrane. After phase separation, the bilayer organizes in multiple lipid domains, whose location is pinned in specific regions, depending on the substrate curvature and the bending rigidity of the lipid domains. Our results provide critical insights into the phase separation of cellular membranes and, more generally, two-dimensional fluids on curved substrates.

C24:0 and C24:1 sphingolipids in cholesterol-containing, five- and six-component lipid membranes

The biophysical properties of sphingolipids containing lignoceric (C24:0) or nervonic (C24:1) fatty acyl residues have been studied in multicomponent lipid bilayers containing cholesterol (Chol), by means of confocal microscopy, differential scanning calorimetry and atomic force microscopy. Lipid membranes composed of dioleoyl phosphatidylcholine and cholesterol were prepared, with the addition of different combinations of ceramides (C24:0 and/or C24:1) and sphingomyelins (C24:0 and/or C24:1). Results point to C24:0 sphingolipids, namely lignoceroyl sphingomyelin (lSM) and lignoceroyl ceramide (lCer), having higher membrane rigidifying properties than their C24:1 homologues (nervonoyl SM, nSM, or nervonoyl Cer, nCer), although with a similar strong capacity to induce segregated gel phases. In the case of the lSM-lCer multicomponent system, the segregated phases have a peculiar fibrillar or fern-like morphology. Moreover, the combination of C24:0 and C24:1 sphingolipids generates interesting events, such as a generalized bilayer dynamism/instability of supported planar bilayers. In some cases, these sphingolipids give rise to exothermic curves in thermograms. These peculiar features were not present in previous studies of C24:1 combined with C16:0 sphingolipids. Conclusions of our study point to nSM as a key factor governing the relative distribution of ceramides when both lCer and nCer are present. The data indicate that lCer could be easier to accommodate in multicomponent bilayers than its C16:0 counterpart. These results are relevant for events of membrane platform formation, in the context of sphingolipid-based signaling cascades.

Bilayer‐mediated assembly of cationic nanoparticles adsorbed to lipid bilayers: Insights from molecular simulations

AbstractUnderstanding interactions between functionalized gold nanoparticles (NPs) and lipid bilayers is essential for biomedical applications. Experiments have shown that NPs that are stable in solution can assemble into clusters when adsorbed to a lipid bilayer, suggesting that bilayer‐mediated interactions facilitate assembly. In this work, we use coarse‐grained molecular dynamics simulations to study bilayer‐mediated interactions between NPs adsorbed to single‐ and multicomponent lipid bilayers. We perform unbiased simulations and umbrella sampling calculations using an implicit solvent force field to determine the thermodynamic contributions to assembly. We show that bilayer‐mediated interactions drive the assembly of NPs into linear aggregates on liquid‐disordered bilayers, which we attribute to a reduction in bilayer curvature. Similar bilayer‐mediated interactions induce the alignment of NP clusters with phase boundaries in phase‐separated bilayers. Together, these simulation results provide new physical insight into the balance of forces that dictate the assembly of charged NPs at multicomponent lipid bilayer interfaces.

Impact of Dyslipidemic Levels of Oxidized Cholesterol on Endothelial Membranes

Dyslipidemia, or abnormally high levels of blood cholesterol in the form of low-density lipoprotein (LDL), increases the chance of developing cardiovascular diseases, potentially leading to heart disease, diabetes, or stroke. Blood levels of oxidative modifications of low-density lipoprotein (oxLDL - a major proinflammatory and proatherogenic factor) are also increased under dyslipidemic conditions. In this study, we first performed mass spectrometric analyses of the sterol composition of human aortic endothelial cells (HAECs) exposed to the pathological levels of oxLDL observed in hypercholesterolemia in vivo. We found that exposure of HAECs to oxLDL does not lead to an increase in cellular cholesterol, but results instead in significant incorporation of oxysterols, particularly, 7-ketocholesterol. In this study, we computationally investigate the impact of the physiological increases of 7-ketocholesterol on the physical properties of endothelial membranes using coarse-grained molecular dynamics simulations to compare dyslipidemic lipid compositions to normal physiological profiles. A topology of 7-ketocholesterol based on the Martini coarse-graining scheme was created, validated, and subsequently incorporated into multi-component lipid bilayers representing endothelial membranes at both physiological and dyslipidemic concentrations, determined by mass spectrometric analyses. Molecular simulations show differential membrane perturbation effects of the various concentrations of oxidized sterol present, including increased fluidization and decreased packing of the membrane lipids accompanying increased 7-ketocholesterol levels, effects contrary to those expected to be caused by non-oxidized cholesterol. Differences in sterol diffusion rates, water, and lipid distribution within the model membranes were also observed. These changes in membrane structure and dynamics in response to varying levels of 7-ketocholesterol would suggest that, in addition to altering lipid composition, dyslipidemic conditions involving this oxidized sterol, along with several other bioactive molecules, disrupt the biomechanical integrity of the endothelial membrane.

Permeation of nanoparticles across the intestinal lipid membrane: dependence on shape and surface chemistry studied through molecular simulations.

Nanoparticles are being explored for topical and oral drug delivery applications as they can cross various biological barriers, for example, the intestinal epithelium. The ability of nanoparticles to cross barriers depends on their morphological and surface properties such as size, surface chemistry and shape, among others. The effect of nanoparticle size on their membrane permeability has been well studied both experimentally and theoretically. However, less attention has been given to understand the role of nanoparticle shape in their translocation across biological barrier membranes. Here, we report on the influence of the nanoparticle's shape, surface chemistry and concentration on their permeation across a human intestinal apical cell membrane model. A representative multicomponent lipid bilayer model of the human intestinal apical membrane was built. The free energy of permeation of nanoparticles across the model lipid bilayer was calculated using multiple umbrella sampling simulations. The interaction of these nanoparticles with the model lipid bilayer was captured using extensive microsecond unrestrained molecular dynamics simulations. We observed that: (a) irrespective of the surface chemistry, the efficacy of nanoparticle penetration across the lipid layer was in the order of rod > disc > sphere; (b) irrespective of the shape, apolar and nonpolar nanoparticles were found to locate in the interior of the lipid bilayer, whereas charged and polar nanoparticles were either adsorbed on the lipid headgroups or remained in the water layer; (c) apolar and nonpolar disc shaped nanoparticles had higher efficacy in permeation across the lipid bilayer as compared to disc and sphere shaped nanoparticles; and (d) at a higher concentration of nanoparticles, sphere and disc shaped nanoparticles exhibited more agglomeration as compared to rod shaped nanoparticles. Based on these outcomes, a few nanoparticles were designed which penetrated readily into the lipid layer and these nanoparticles were also able to co-deliver a therapeutic protein inside the lipid layer. The apical model lipid membrane and protocols used in this study can thus be utilized for the in silico design of nanoparticles for the oral delivery of therapeutics.

Exploring the Structure and Stability of Cholesterol Dimer Formation in Multicomponent Lipid Bilayers

For 40 years, the existence and possible functional importance of cholesterol dimer formation has been discussed. Due to challenges associated with structural studies of membrane lipids, there has as yet been no direct experimental verification of the existence and relevance of the cholesterol dimer. Building on recent advances in lipid force fields for molecular simulation, in this work the structure and stability of the cholesterol dimer is characterized in POPC bilayers in absence and presence of sphingomyelin. The cholesterol dimer structural ensemble is found to consist of sub-states that reflect, but also differ from, previously proposed dimer structures. While face-to-face dimer structures predominate, no evidence is found for the existence of tail-to-tail dimers in POPC lipid bilayers. Near stoichiometric complex formation of cholesterol with sphingomyelin is found to effect cholesterol dimer structure without impacting population. Comparison with NMR-derived order parameters provide validation for the simulation model employed and conclusions drawn related to the structure and stability of cholesterol dimers in multicomponent lipid bilayers. © 2016 Wiley Periodicals, Inc.

Nanophotonic Tools to Resolve Nanoscale Dynamics on Biological Membranes

The plasma membrane of living cells is dynamically compartmentalized at multiple spatial scales ranging from the nano- to the mesoscale. This compartmentalization is crucial for a large number of cellular functions as it allows different receptors and proteins to efficiently interact, in space and time, with their partner ligands and to trigger cell signaling events. At the nanoscale, cell membranes organize into dynamic nano-assemblies known as lipid rafts, enriched by cholesterol, sphingolipids, and certain types of proteins. However, despite many years of research, the field of lipid rafts has remained elusive given their small size (nanometer scale) and their transient character. Visualizing lipid rafts in living cells thus requires simultaneous nanometer spatial precision and microsecond temporal resolution, which is beyond the reach of diffraction-limited optical microscopy. Optical antennas based on metallic nanostructures efficiently enhance and confine light into nanometer dimensions, breaching the diffraction limit. In here, I will discuss recent progress combining optical antennas with fluorescence correlation spectroscopy (FCS) to enquire on the nanoscale dynamics of multicomponent lipid bilayers and living cells. Our results reveal the coexistence of cholesterol-enriched fluctuating nanoscopic domains on mimetic and living cell membranes, in the microsecond scale and with characteristic sizes below 10nm. These nanoscale assemblies might represent lipid raft precursors that in the absence of proteins and/or other molecular stabilizing factors, are poised to be highly transient.

Direct manipulation of liquid ordered lipid membrane domains using optical traps

Multicomponent lipid bilayers can give rise to coexisting liquid domains that are thought to influence a host of cellular activities. There currently exists no method to directly manipulate such domains, hampering our understanding of their significance. Here we report a system that allows individual liquid ordered domains that exist in a liquid disordered matrix to be directly manipulated using optical tweezers. This allows us to drag domains across the membrane surface of giant vesicles that are adhered to a glass surface, enabling domain location to be defined with spatiotemporal control. We can also use the laser to select individual vesicles in a population to undergo mixing/demixing by locally heating the membrane through the miscibility transition, demonstrating a further layer of control. This technology has potential as a tool to shed light on domain biophysics, on their role in biology, and in sculpting membrane assemblies with user-defined membrane patterning.