Pore-spanning Lipid Bilayers Research Articles

High-Throughput Monitoring of Single Vesicle Fusion Using Freestanding Membranes and Automated Analysis.

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO2 holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

Continuous Pore-Spanning Lipid Bilayers on Silicon Oxide-Coated Porous Substrates.

A number of techniques has been developed and analyzed in recent years to generate pore-spanning membranes (PSMs). While quite a number of methods rely on nanoporous substrates, only a few use micrometer-sized pores to be able to individually resolve suspending membranes by means of fluorescence microscopy. To be able to produce PSMs on pores that are micrometer in size, an orthogonal functionalization strategy resulting in a hydrophilic surface is highly desirable. Here, we report on a method to prepare PSMs based on the evaporation of a thin layer of silicon monoxide on top of the porous substrate. PM-IRRAS experiments demonstrate that the final surface is composed of SiOx with 1 < x < 2. The hydrophilic surface turned out to be well suited to spread giant unilamellar vesicles forming PSMs. As the method does not rely on a gold coating as frequently used for orthogonal functionalization, fluorescence micrographs provide information not only from the freestanding membrane areas but also from the supported ones. The observation of the entire PSM area enabled us to observe phase-separation in these membranes on the freestanding and supported parts as well as protein binding and possible lipid reorganization of the membranes induced by binding of the protein Shiga toxin.

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed. The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates. High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results. The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

Self-Organization of Actomyosin Networks Attached to Artificial Membranes

The shape and mechanical stability of cells are highly dependent on their cytoskeleton. Actin is one of the main proteins which contributes to this biological network. The mechanical movements of the cell rely on the interaction of F-actin with several other proteins. One of these proteins is myosin II, a molecular motor protein. By hydrolysing ATP myosin is able to walk along F-actin or to induce tension on these filaments.In our model system F-actin networks are attached to pore spanning lipid bilayers (PSLBs) via electrostatical interactions or the linker protein ezrin which mimics the biological situation. Ezrin has a PIP2 binding site located at the N-terminus and a F-actin binding site at the C-terminus and is responsible for the linkage of F-actin to PIP2 present in the PSLB. Several actin binding proteins and cross-linkers are introduced during the polymerization of actin filaments.Besides the visual self-organization the mechanical properties of different F-actin networks are examined. Atomic force microscopy is used to determine the lateral membrane tension of the PSLB dependent on different actomyosin networks. The viscoelastic properties of the network will be recorded by passive microrheology using the mean square displacement of a polymer beads Brownian movement.

Permeabilization Assay for Antimicrobial Peptides Based on Pore-Spanning Lipid Membranes on Nanoporous Alumina

Screening tools to study antimicrobial peptides (AMPs) with the aim to optimize therapeutic delivery vectors require automated and parallelized sampling based on chip technology. Here, we present the development of a chip-based assay that allows for the investigation of the action of AMPs on planar lipid membranes in a time-resolved manner by fluorescence readout. Anodic aluminum oxide (AAO) composed of cylindrical pores with a diameter of 70 nm and a thickness of up to 10 μm was used as a support to generate pore-spanning lipid bilayers from giant unilamellar vesicle spreading, which resulted in large continuous membrane patches sealing the pores. Because AAO is optically transparent, fluid single lipid bilayers and the underlying pore cavities can be readily observed by three-dimensional confocal laser scanning microscopy (CLSM). To assay the membrane permeabilizing activity of the AMPs, the translocation of the water-soluble dyes into the AAO cavities and the fluorescence of the sulforhodamine 101 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanol-l-amine triethylammonium salt (Texas Red DHPE)-labeled lipid membrane were observed by CLSM in a time-resolved manner as a function of the AMP concentration. The effect of two different AMPs, magainin-2 and melittin, was investigated, showing that the concentrations required for membrane permeabilization and the kinetics of the dye entrance differ significantly. Our results are discussed in light of the proposed permeabilization models of the two AMPs. The presented data demonstrate the potential of this setup for the development of an on-chip screening platform for AMPs.

Mechanics of F-Actin-Membrane Composites Investigated by Atomic Force Microscopy

The cortical cytoskeleton attached to the plasma membrane is an essential mechanical component within the cell. It regulates fundamental processes including cell division, motility and shape. In order to investigate how the individual components of the cortical cytoskeleton contribute to the mechanical properties of the plasma membrane, a model system was established. Pore spanning lipid bilayers (PSLBs) containing a specific receptor lipid were prepared on porous silicon nitride substrates. Subsequently, F-actin was bound to the PSLBs either directly via electrostatic interaction or via the linker protein ezrin.Fluorescence microscopy was utilized to visualize the prepared PSLBs and the F-actin binding. Indentation experiments were performed by atomic force microscopy (AFM) to elucidate the mechanical properties of the PSLBs. The apparent spring constants of the PSLBs were extracted from the measured force-indentation curves. In our system, the determined apparent spring constants directly correlate with the lateral tension of the PSLBs. We analyzed the lateral tension as a function of F-actin coverage.Figure 1: Experimental setup. PSLBs are prepared on hydrophilic functionalized porous silicon nitride substrates and prepolymerized F-actin is bound to them. The mechanical properties of the composite are determined by force indentation measurements.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Smart polymer brush nanostructures guide the self-assembly of pore-spanning lipid bilayers with integrated membrane proteins

Nanopores in arrays on silicon chips are functionalized with pH-responsive poly(methacrylic acid) (PMAA) brushes and used as supports for pore-spanning lipid bilayers with integrated membrane proteins. Robust platforms are created by the covalent grafting of polymer brushes using surface-initiated atom transfer radical polymerization (ATRP), resulting in sensor chips that can be successfully reused over several assays. His-tagged proteins are selectively and reversibly bound to the nitrilotriacetic acid (NTA) functionalization of the PMAA brush, and consequently lipid bilayer membranes are formed. The enhanced membrane resistance as determined by electrochemical impedance spectroscopy and free diffusion of dyed lipids observed as fluorescence recovery after photobleaching confirmed the presence of lipid bilayers. Immobilization of the His-tagged membrane proteins on the NTA-modified PMAA brush near the pore edges is characterized by fluorescence microscopy. This system allows us to adjust the protein density in free-standing bilayers, which are stabilized by the polymer brush underneath. The potential application of the integrated platform for ion channel protein assays is demonstrated.

Separating Attoliter-Sized Compartments Using Fluid Pore-Spanning Lipid Bilayers

Anodic aluminum oxide (AAO) is a porous material having aligned cylindrical compartments with 55-60 nm diameter pores, and being several micrometers deep. A protocol was developed to generate pore-spanning fluid lipid bilayers separating the attoliter-sized compartments of the nanoporous material from the bulk solution, while preserving the optical transparency of the AAO. The AAO was selectively functionalized by silane chemistry to spread giant unilamellar vesicles (GUVs) resulting in large continuous membrane patches covering the pores. Formation of fluid single lipid bilayers through GUV rupture could be readily observed by fluorescence microscopy and further supported by conservation of membrane surface area, before and after GUV rupture. Fluorescence recovery after photobleaching gave low immobile fractions (5-15%) and lipid diffusion coefficients similar to those found for bilayers on silica. The entrapment of molecules within the porous underlying cylindrical compartments, as well as the exclusion of macromolecules from the nanopores, demonstrate the barrier function of the pore-spanning membranes and could be investigated in three-dimensions using confocal laser scanning fluorescence imaging.

Pore Spanning Lipid Bilayers on Mesoporous Silica Having Varying Pore Size

Synthetic lipid bilayers have similar properties as cell membranes and have been shown to be of great use in the development of novel biomimicry devices. In this study, lipid bilayer formation on mesoporous silica of varying pore size, 2, 4, and 6 nm, has been investigated using quartz crystal microbalance with dissipation monitoring (QCM-D), fluorescent recovery after photo bleaching (FRAP), and atomic force microscopy (AFM). The results show that pore-spanning lipid bilayers were successfully formed regardless of pore size. However, the mechanism of the bilayer formation was dependent on the pore size, and lower surface coverages of adsorbed lipid vesicles were required on the surface having the smallest pores. A similar trend was observed for the lateral diffusion coefficient (D) of fluorescently labeled lipid molecules in the membrane, which was lowest on the surface having the smallest pores and increased with the pore size. All of the pore size dependent observations are suggested to be due to the hydrophilicity of the surface, which decreases with increased pore size.

Preparation of Solvent-Free, Pore-Spanning Lipid Bilayers: Modeling the Low Tension of Plasma Membranes

Plasma membrane tension, produced by the underlying cytoskeleton, governs many dynamic processes such as fusion, blebbing, exo- and endocytosis, cell migration, and adhesion. Here, a new protocol is introduced to model this intricate and often overlooked aspect of the plasma membrane. Lipid bilayers spanning pores of 600 nm radius were prepared by adsorption and spreading of giant unilamellar vesicles (GUVs) on moderately hydrophilic porous substrates prepared by gold-coating and subsequent self-assembly of a mercaptoethanol monolayer. Rupture of GUVs formed tens of micrometer sized pore-spanning membrane patches displaying low tension of σ ≤ 3.5 mN m(-1) and lateral diffusion constants of about 8 μm(2) s(-1). Site-specific force indentation experiments were performed to determine membrane tension as a function of lipid composition: for pure DOPC bilayers, a tension of 1.018 ± 0.014 mN m(-1) was measured, which was increased by the addition of cholesterol to 3.50 ± 0.15 mN m(-1). Compared to DOPC, POPC bilayers displayed a larger tension of 2.00 ± 0.09 mN m(-1). Addition and subsequent partitioning of 2-propanol was shown to significantly reduce the membrane tension as a function of its concentration.

A membrane fusion assay based on pore-spanning lipid bilayers

Pore-spanning planar membranes on highly ordered porous silicon substrates were shown to be well suited to monitor the calcium ion mediated fusion of large unilamellar vesicles by means of confocal laser scanning fluorescence microscopy and scanning ion conductance microscopy in real-time.

Mechanical Properties of Pore-Spanning Lipid Bilayers Probed by Atomic Force Microscopy

We measure the elastic response of a free-standing lipid membrane to a local indentation by using an atomic force microscope. Starting point is a planar gold-coated alumina substrate with a chemisorbed 3-mercaptopropionic acid monolayer displaying circular pores of very well defined and tunable size, over which bilayers composed of N, N,-dimethyl- N, N,-dioctadecylammonium bromide or 1,2-dioleoyl-3-trimethylammonium-propane chloride were spread. Centrally indenting these “nanodrums” with an atomic force microscope tip yields force-indentation curves, which we quantitatively analyze by solving the corresponding shape equations of continuum curvature elasticity. Since the measured response depends in a known way on the system geometry (pore size, tip radius) and on material parameters (bending modulus, lateral tension), this opens the possibility to monitor local elastic properties of lipid membranes in a well-controlled setting.

Pore-Spanning Lipid Bilayers Visualized by Scanning Force Microscopy

Pore-Spanning Lipid Bilayers Visualized by Scanning Force Microscopy