Fluid Supported Lipid Bilayers Research Articles

A Membrane-Associated Light-Harvesting Model is Enabled by Functionalized Assemblies of Gene-Doubled TMV Proteins.

Photosynthetic light harvesting requires efficient energy transfer within dynamic networks of light-harvesting complexes embedded within phospholipid membranes. Artificial light-harvesting models are valuable tools for understanding the structural features underpinning energy absorption and transfer within chromophore arrays. Here, a method for attaching a protein-based light-harvesting model to a planar,fluidsupported lipid bilayer (SLB) is developed. The protein model consists of the tobacco mosaic viral capsid proteins that are gene-doubled to create a tandem dimer (dTMV). Assemblies of dTMV break the facial symmetry of the double disk to allow for differentiation between the disk faces. A single reactive lysine residue is incorporated into the dTMV assemblies for the site-selective attachment of chromophores for light absorption. On the opposing dTMV face, a cysteine residue is incorporated for the bioconjugation of a peptide containing a polyhistidine tag for association with SLBs. The dual-modified dTMV complexes show significant association with SLBs and exhibit mobility on the bilayer. The techniques used herein offer a new method for protein-surface attachment and provide a platform for evaluating excited state energy transfer events in a dynamic, fully synthetic artificial light-harvesting system.

Reconstitution of Membrane-tethered Minimal Actin Cortices on Supported Lipid Bilayers.

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

Endothelial cell spreading on lipid bilayers with combined integrin and cadherin binding ligands

Endothelial cells play a central role in the vascular system, where their function is tightly regulated by both cell-extracellular matrix (e.g., via integrins) and cell–cell interactions (e.g., via cadherins). In this study, we incorporated cholesterol-modified integrin and N-cadherin peptide binding ligands in fluid supported lipid bilayers. Human umbilical vein endothelial cell adhesion, spreading and vinculin localization in these cells were dependent on ligand density. One composition led to observe a higher extent of cell spreading, where cells exhibited extensive lamellipodia formation and a qualitatively more distinct N-cadherin localization at the cell periphery, which is indicative of N-cadherin clustering and a mimic of cell–cell contact formation. The results can be used to reconstitute the endothelial-pericyte interface on biomedical devices and materials.

A Multimodal Platform for Simultaneous T-Cell Imaging, Defined Activation, and Mechanobiological Characterization.

T-cell antigen recognition is accompanied by extensive morphological rearrangements of the contact zone between the T-cell and the antigen-presenting cell (APC). This process involves binding of the T-cell receptor (TCR) complex to antigenic peptides presented via MHC on the APC surface, the interaction of costimulatory and adhesion proteins, remodeling of the actin cytoskeleton, and the initiation of downstream signaling processes such as the release of intracellular calcium. However, multiparametric time-resolved analysis of these processes is hampered by the difficulty in recording the different readout modalities at high quality in parallel. In this study, we present a platform for simultaneous quantification of TCR distribution via total internal reflection fluorescence microscopy, of intracellular calcium levels, and of T-cell-exerted forces via atomic force microscopy (AFM). In our method, AFM cantilevers were used to bring single T-cells into contact with the activating surface. We designed the platform specifically to enable the study of T-cell triggering via functionalized fluid-supported lipid bilayers, which represent a widely accepted model system to stimulate T-cells in an antigen-specific manner. In this paper, we showcase the possibilities of this platform using primary transgenic T-cells triggered specifically via their cognate antigen presented by MHCII.

Surface-Enhanced Raman Spectroscopy of Fluid-Supported Lipid Bilayers.

Supported lipid bilayers are essential model systems for studying biological membranes and for membrane-based sensor development. Surface-enhanced Raman spectroscopy (SERS) stands to add considerably to our understanding of the dynamics and interactions of these systems through direct chemical information. Despite this potential, SERS of lipid bilayers is not routinely achieved. Here, we carried out the first measurements of a solid-supported lipid bilayer on a SERS-active substrate and characterized the bilayer using SERS, atomic force microscopy, surface plasmon resonance spectroscopy, ellipsometry, and fluorescence recovery after photobleaching (FRAP). The creation of a fluid, SERS-active supported lipid bilayer was accomplished through use of a novel silica-coated silver film-over-nanosphere substrate. These substrates offer a powerful new platform to couple common surface techniques that are challenging on the nanoscale, for example, ellipsometry and FRAP, with SERS for studying biological membranes and their dynamics.

Sensing Ability and Formation Criterion of Fluid Supported Lipid Bilayer Coated Graphene Field-Effect Transistors.

Supported lipid bilayers (SLBs) have been widely used to provide native environments for membrane protein studies. In this study, we utilized graphene field-effect transistors (GFETs) coated with a fluid SLB to perform label-free detection of membrane-associated ligand-receptor interactions in their native lipid bilayer environment. It is known that the analyte-binding event needs to occur within the Debye length for it to be significantly sensed by an FET sensor. However, the thickness of a lipid bilayer is around 4-5-nm-thick, which is larger than the Debye length of a solution with physiologically relevant ionic strength. There is thus a question of whether an FET sensor can detect the binding event above the bilayer. In this study, we show how the existence of an SLB can influence the effective detection distance and the formation criterion of a fluid and continuous SLB on a graphene surface. We discovered that the water intercalation between the graphene and the underlying silica substrate hinders the SLB formation but is required for the stable electrical recording by a GFET. To verify the existence of a fluid SLB on graphene, which was previously complicated by the graphene fluorescence quenching effect, we developed a modified fluorescence recovery after photobleaching method. In addition, our results showed that SLB coated GFETs can quantitatively detect ligand binding onto the receptors embedded in the SLBs. The comparison of our experimental data with a theoretical model shows that the contribution of the SLB acyl chain hydrophobic region to the screening effect can be negligible and, therefore, that the effective detection region can extend beyond the SLB.

T Cells on Engineered Substrates: The Impact of TCR Clustering Is Enhanced by LFA-1 Engagement.

We created APC-mimetic synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit antibodies directed against the TCR-complex in the form of a patterned array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. We show that for T cell adhesion mediated by T cell receptor (TCR) alone, in the patterned, but not in the corresponding homogeneous controls, the TCR, ZAP-70 and actin are present in the form of clusters or patches that co-localize with the ligand-dots. However, global cell scale parameters like cell area and actin distribution are only weakly impacted by ligand clustering. In presence of ICAM-1 - the ligand of the T cell integrin LFA-1 - on the SLB, the TCR is still clustered due to the patterning of its ligands, but now global parameters are also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, whereas the cell area increases significantly. These observations taken together point to a possible pivotal role for LFA-1 in amplifying the effect of TCR-clustering. No such effect is evident for co-engagement of CD28, affected via its ligand B7.2. Unlike on ICAM-1, on B7.2 cell spreading and actin organization are similar for homogeneous and patterned substrates. However, TCR and ZAP-70 clusters are still formed in the patterned case. These results indicate complementary role for LFA-1 and CD28 in the regulation and putative coupling of TCR micro-clusters to actin. The engineered substrates presented here clearly have the potential to act as platform for fundamental research in immune cell biology, as well as translational analyses in immunotherapy, for example to screen molecules for their role in T cell adhesion/activation.

Nanometer-scale molecular organization in lipid membranes studied by time-of-flight secondary ion mass spectrometry.

The organization of lipid membranes plays an important role in a wide range of biological processes at different length scales. Herein, the authors present a procedure based on time-of-flight secondary ion mass spectrometry (ToF-SIMS) to characterize the nanometer-scale ordering of lipids in lipid membrane structures on surfaces. While ToF-SIMS is a powerful tool for label-free analysis of lipid-containing samples, its limited spatial resolution prevents in-depth knowledge of how lipid properties affect the molecular assembly of the membrane. The authors overcome this limitation by measuring the formation of lipid dimers, originating in the same nanometer-sized primary ion impact areas. The lipid dimers reflect the local lipid environment and thus allow us to characterize the membrane miscibility on the nanometer level. Using this technique, the authors show that the chemical properties of the constituting lipids are critical for the structure and organization of the membrane on both the nanometer and micrometer length scales. Our results show that even at lipid surface compositions favoring two-phase systems, lipids are still extracted from solid, gel phase, domains into the surrounding fluid supported lipid bilayer surrounding the gel phase domains. The technique offers a means to obtain detailed knowledge of the chemical composition and organization of lipid membranes with potential application in systems where labeling is not possible, such as cell-derived supported lipid bilayers.

Supported Lipid Bilayers with Phosphatidylethanolamine as the Major Component.

Phosphatidylethanolamine (PE) is notoriously difficult to incorporate into model membrane systems, such as fluid supported lipid bilayers (SLBs), at high concentrations because of its intrinsic negative curvature. Using fluorescence-based techniques, we demonstrate that having fewer sites of unsaturation in the lipid tails leads to high-quality SLBs because these lipids help to minimize the curvature. Moreover, shorter saturated chains can help maintain the membranes in the fluid phase. Using these two guidelines, we find that up to 70 mol % PE can be incorporated into SLBs at room temperature and up to 90 mol % PE can be incorporated at 37 °C. Curiously, conditions under which three-dimensional tubules project outward from the planar surface as well as conditions under which domain formation occurs can be found. We have employed these model membrane systems to explore the ability of Ni2+ to bind to PE. It was found that this transition metal ion binds 1000-fold tighter to PE than to phosphatidylcholine lipids. In the future, this platform could be exploited to monitor the binding of other transition metal ions or the binding of antimicrobial peptides. It could also be employed to explore the physical properties of PE-containing membranes, such as phase domain behavior and intermolecular hydrogen bonding.

Mobile lipid bilayers on gold surfaces through structure-induced lipid vesicle rupture.

Forming fluid supported lipid bilayers (SLBs) on a gold surface can enable various lipid-membrane-associated biomolecular interactions to be investigated by several surface sensing techniques, such as surface plasmon resonance and scanning tunneling microscopy. However, forming fluid SLBs on a gold surface through lipid vesicle deposition continues to pose a challenge. In this study, we constructed nanograting structures on a gold surface to induce lipid vesicle rupture for forming a mobile layer of SLBs. Observations based on fluorescence recovery after photobleaching showed that SLBs on the prepared grating supports had some fluidity, while SLBs on the planar support had no fluidity. The anisotropic fluorescence intensity recovery shape changes observed in the SLBs on the grating support suggested that a second layer of SLBs partially formed on top of the first layer in contact with the gold surface and extended along the grating structure. Comparisons of the relative amounts of second bilayer and the fluorescence recovery fractions on supports with various grating edge densities suggested that the second layer formed at the edge regions and that the coverage ratio was directly proportional to the grating edge density. All of these results showed that the grating edges could serve as vesicle-rupture-inducing sites for the formation of a mobile second SLB on a gold surface. The formation of the second layer of SLBs at the edge regions but not in the flat regions enabled us to determine the second layer locations and provided us with an opportunity to pattern mobile lipid bilayers on gold surfaces by controlling the edge locations.

Creating Fluid Supported Lipid Bilayers with High Amounts of Phosphatidylethanolamine

Phosphatidylethanolamine (PE) comprises 20-50% of overall phospholipid content in human cell membranes and constitutes 70-80% of the membranes in gram-negative bacteria. Its presence is specifically required for the proper folding of numerous membrane proteins, the function of active transport systems, cell division, fusion, blood coagulation, and may play a role in neurodegenerative diseases. Unfortunately, it is hard to work with this lipid in model systems like supported lipid bilayers and there is correspondingly less information known about its basic physical properties in bilayers. Specifically, the role of PE in lipid raft formation, vesicle fusion, cholesterol interactions, ion binding, and lipid flip-flop needs to be elucidated to understand its part in cell membrane function and disease pathways. To this end, my research has two goals. First, I have developed supported lipid bilayer systems that can operate with high mole percentages of PE. Second, I have begun to exploit such systems to explore the properties and functions of PE in membranes.

A versatile approach to the generation of fluid supported lipid bilayers and its applications.

Establishing supported lipid bilayers with biologically relevant composition, including transmembrane proteins and various classes of lipids, presents a significant challenge. We describe a generic and facile approach to the production of fluid polymer-supported lipid bilayers that allows for the incorporation of a wide variety of lipids and transmembrane proteins. The method is based on the formation of a polymer brush displaying lipid groups, followed by spin-coating of membrane lipids. Subsequentially, transmembrane proteins are incorporated by the fusion of proteoliposomes with the bilayer. Several applications, including the incorporation and single-molecule tracking of transmembrane proteins in a bilayer and the visualization of the fusion of individual, membrane-enveloped viruses with a supported membrane, are demonstrated. Our results suggest that the membrane properties are consistent with those found in physiologically relevant conditions and underscore the wide applicability of our approach to synthetic biology, lab-on-a-chip applications, biophysical and pharmaceutical studies.

The Nanomechanical Properties of Lipid Membranes are Significantly Influenced by the Presence of Ethanol

Ethanol has a profound impact on biological systems and is moreover used in various medical and nonmedical applications. Its interaction with the lipid part of biological membranes has been the subject of intensive studies, but surprisingly, to our knowledge, no study has examined the influence of ethanol on lipid bilayer nanomechanics. We performed atomic force microscopy-based measurements to assess the influence of ethanol on the nanomechanical properties of fluid supported lipid bilayers. Ethanol significantly reduces membrane stability, bilayer thickness, Young’s modulus, area stretch modulus, and bending stiffness. Altogether, our data suggest that ethanol addition to supported lipid bilayers supports both the hydrophobic and the hydrophilic permeation pathways by a decrease of bilayer thickness and reduced stability, respectively.

Continuous Lipid Bilayers Derived from Cell Membranes for Spatial Molecular Manipulation

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 (G(M1) and G(M3)), and the preserved lateral mobility was confirmed by spatial manipulation of the G(M1/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.

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability, and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma (HCC) exhibit a 10,000-fold greater affinity for HCC than for hepatocytes, endothelial cells, and immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, siRNA, and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer allow a single protocell loaded with a drug cocktail to kill a drug-resistant HCC cell, representing a 106-fold improvement over comparable liposomes.

Thermal Stress of Supported Lipid Bilayer Induces Formation and Collapse of Uniform Radius Tubules

Lipid bilayer morphologies and the transitions between them are important to many cellular processes. Supported lipid bilayer (SLB) provides a model system in which to quantitatively investigate transitions from planar to tubular and tubular to spherical morphologies. Following a small increase in temperature (∼5-10°C) flexible filaments extrude from a fluid SLB. Individual filaments can reach hundreds of microns in length before spontaneously collapsing into discs. We demonstrate that the filaments are tubular by decreasing the external buffer concentration, which causes them to swell, first into resolvable tubules with capped ends and then into giant vesicles. At high ionic strength, the sub-resolution tubules are adsorbed to the SLB, enabling the measurement of their radius to within ±5 nm using conventional fluorescence microscopy. The radius depends on the lipid tail composition and varies <10% along the tubule length. Under tension, tubules are even more uniform, having no measurable variation in radius.

Detection of Rare Interaction Events Via Combined Photobleaching and Single Molecule Microscopy

Gathering information on the interplay between different membrane components and the characteristics of protein interactions in live cells requires the design of sensitive methods that allow for the simultaneous study of different probe molecules. Especially the aim to single out rare interaction events under a vast excess of non-interacting molecules remains challenging. The two-color single molecule imaging technique presented here is the advancement of a recently presented approach to virtually dilute fluorescently labeled membrane constituents by photobleaching [1]. Using this technique, single molecule microscopy can be performed at almost arbitrarily high surface densities of fluorescent probe molecules. The method yields information on the fraction of colocalized particles and their position within 40nm accuracy. Supplemental data on the mobility and stoichiometry of the labeled molecule species can also be deduced. The sensitivity of our two-color single molecule imaging technique is significantly increased by tracking colocalized spots over consecutive images. We present a detailed statistical description of false positives and false negatives and quantify the sensitivity of our method. Proof of principle experiments were performed by measuring the interaction between Alexa647-labeled Cholera Toxin B (CTX-B-Alexa647) and Bodipy-labeled GM1 (Bodipy-GM1) diffusing in a fluid supported lipid bilayer. We directly observed single Cholera Toxin molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. We demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified by tracking colocalized spots. We also present preliminary results on the application to live cells investigating the interaction between Bodipy-GM1 and Ab-Alexa647 labeled CD59. [1] Moertelmair et al., Appl. Phys Lett. 87, 263903, 2005.

Distribution and Dynamics of RBL IgE Receptors (FcɛRI) Quantitatively Observed on Planar Ligand-Presenting Surfaces

There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. When T cells, B cells, or mast cells bind to monovalent ligands on fluid supported lipid bilayers, receptor clustering, signaling, and receptor redistribution into immunological synapses follow. We have quantitatively studied the kinetics of redistribution of IgE receptors (FcɛRI) on RBL-2H3 mast cell surfaces. To separate the kinetics of receptor redistribution from cell spreading, the initial cell-substrate contact time was precisely defined (±50 ms) by micropipette cell manipulation. Using total internal reflection fluorescence, the distribution and dynamics of receptor clusters were imaged. We find strong quantitative evidence that initial receptor aggregation occurs at cellular protrusions, which are visible bright spots/regions on cells contacting ligand-free surfaces. The initial size of these regions is independent of the substrate and the presence or absence of ligand, and they were found to be randomly distributed over the interfacial contact area. Using a finite-element diffusion model, we found that the initial rate of accumulation of receptors at the protrusions is consistent with diffusion-limited trapping with D ∼10−1 μm2/s. At longer timescales, individual clusters on ligand-bearing membranes were observed to move with both a diffusive and a directed component of motion; clusters eventually coalesced near the center of the contact region (∼ 1 minute). The dynamics of the early cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.Sandia is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DE-ACO4-94AL85000.

Two-color single molecule tracking combined with photobleaching for the detection of rare molecular interactions in fluid biomembranes

Experimentalists are increasingly confronted with the demand to single out rare interaction events under a vast excess of non-interacting molecules. We recently presented an approach how to virtually dilute fluorescently labeled membrane constituents by combining photobleaching and single molecule microscopy, termed “Thinning Out Clusters while Conserving the Stoichiometry of Labeling” (TOCCSL; Moertelmaier et al., Appl. Phys. Lett., 2005, 87, 263903). Using this approach, single molecule microscopy can be performed even at arbitrarily high surface densities of fluorescent probe molecules. Here, we extended this method for two color microscopy. We provide a detailed statistical description of false positives and false negatives. In particular, we quantified the increase in sensitivity by tracking the colocalized objects over successive images. Proof of principle experiments were performed by measuring the interaction between Alexa647-labeled Cholera Toxin B (CTX-B-Alexa647) and Bodipy-labeled GM1 (Bodipy-GM1) diffusing in a fluid supported lipid bilayer. We directly observed single Cholera Toxin B molecules bound to Bodipy-GM1 and quantified their occupancy via brightness analysis. Each colocalized spot could be further analyzed with respect to its diffusion constant, yielding a clear anticorrelation between occupancy and mobility. We finally demonstrate that extremely low interaction probabilities of only 2.5% can be unambiguously identified.