This material is based upon work supported by the National Science Foundation under Grant No. 1942418 awarded to A.S., and a National Science Foundation Graduate Research Fellowship awarded to C.M.B.H., under Grant No. 1644760. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. The authors thank Dr. Noel Vera-Gonz&#225;lez for lipid vesicle characterization data acquisition. The authors thank Professor Robert Hurt (Brown University) for the use of his Zetasizer. The authors thank the Brown University Mass Spectrometry Facility, in particular, Dr. Tun-Li Shen for assistance with quantifying lipid composition.

The authors declare that they have no conflict of interest or competing financial interests.

This protocol allows for the formation of lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. Here, critical steps are presented to form each of these structures. When forming lipid vesicles, it is important to extrude above the transition temperature of the lipid39. When below the transition temperature, the lipid is physically present in its ordered gel phase39. In this ordered phase the hydrocarbon lipid tails are fully extended allowing for close pac...

The cell membrane, composed primarily of phospholipids, cholesterol, and proteins, is a crucial component of all living cells1. With organization driven by lipid amphiphilicity, the cell membrane functions as a protective barrier and regulates how the cell interacts with its surrounding environment2. Several cellular processes are dependent on the lipid and protein composition of the membrane1,2. For example, cell membrane interactions are important for effective drug delivery3. Pharmaceuticals, biologics, nanomaterials, biological toxins, and environmental toxicants can impact the integrity of a cell membrane, thereby affecting cellular function4. The construction of in vitro cell mimicking membrane models based on the lipid composition of cell membranes has the potential to provide facile tools to greatly enhance the study of the potential impact of these materials on cells.
Model lipid bilayers include lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. Supported lipid bilayers are a model of the phospholipid cell membrane commonly used in biotechnology applications where lipid vesicles are ruptured on a supported substrate material5,6,7,8,9. One common technique used to monitor bilayer formation is quartz crystal microbalance with dissipation monitoring (QCM-D), which examines the adsorption of vesicles in comparison to the bulk liquid properties in situ8,10,11,12,13,14. Previously, QCM-D has been used to demonstrate that under flow conditions, once a critical vesicle coverage of phosphatidylcholine (PC) lipid vesicles is achieved on the surface, they spontaneously rupture into rigid lipid bilayers15. Previous work has also investigated supported lipid bilayer formation with varying lipid compositions16, incorporation of lipid proteins17,18,19, and utilizing polymer cushions20, yielding supported lipid bilayers capable of mimicking various aspects of cell membrane function.
Lipid bilayers have been used to mimic various biological barriers from sub-cellular to organ levels including mitochondrion, red blood cell, and liver cell membranes by altering the phospholipid, cholesterol, and glycolipid components21. These more complex multi-lipid vesicles may require additional methods to achieve vesicle rupture, depending on the lipid composition. For example, previous studies have utilized an &#945;-helical (AH) peptide derived from the hepatitis C virus&#39;s nonstructural protein 5A to induce bilayer formation by destabilizing the adsorbed lipid vesicles22,23. Using this AH peptide, supported lipid bilayers mimicking placental cells have previously been formed24. The great potential of supported lipid bilayers for biomedical applications has been demonstrated with investigations spanning molecular and nanoparticle transport25, 26, environmental toxicant interactions27, protein assembly and function17,18,19, peptide arrangement and insertion28, 29, drug screening30, and microfluidic platforms31.
Suspended lipid bilayers have been used for pharmaceutical screening studies via a parallel artificial membrane permeability assay (PAMPA) where a lipid bilayer is suspended across a porous hydrophobic insert32,33,34,35. PAMPA lipid models have been developed for different biological interfaces including the blood-brain, buccal, intestinal, and transdermal interfaces36. By combining both the supported lipid bilayer and PAMPA techniques, adsorption, permeability, and embedment of compounds within lipid components of a desired tissue or cell type can be thoroughly studied.
This protocol describes the fabrication and application of in vitro cell membrane lipid bilayer models to investigate several molecular interactions. Preparation of both uni-lipid and multi-lipid supported and suspended lipid bilayers is detailed. To form a supported lipid bilayer, lipid vesicles are first developed using thin-film hydration and extrusion methods followed by physicochemical characterization. Formation of a supported lipid bilayer using QCM-D monitoring and fabrication of suspended lipid membranes for use in PAMPA is discussed. Finally, multi-lipid vesicles for the development of more complex cell mimicking membranes are examined. Using both types of fabricated lipid membranes, this protocol demonstrates how this tool can be used to study molecular interactions. Overall, this technique constructs cell mimicking lipid bilayers with high reproducibility and versatility.

<table><tbody><tr><td>1-palmitoyl-2-oleoyl-glycero-3-phosphocholine&amp;nbsp; (POPC, 16:0-18:1 PC)</td><td>Avanti Polar Lipids</td><td>850457</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS, 16:0-18:1 PS)</td><td>Avanti Polar Lipids</td><td>840034</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0-18:1 PE)</td><td>Avanti Polar Lipids</td><td>850757</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-2-phospho-L-serine (DOPS, 18:1 PS)</td><td>Avanti Polar Lipids</td><td>840035</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 18:1 (&amp;Delta;9-Cis) PC)</td><td>Avanti Polar Lipids</td><td>850375</td><td></td></tr><tr><td>1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, 18:1 (&amp;Delta;9-Cis) PE)</td><td>Avanti Polar Lipids</td><td>850725</td><td></td></tr><tr><td>1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (18:0 EPC (Cl Salt))</td><td>Avanti Polar Lipids</td><td>890703</td><td></td></tr><tr><td>3 mL Luer-Loc syringes</td><td>BD</td><td>309657</td><td></td></tr><tr><td>40 mL sample vial, amber with polytetrafluoroethylene (PTFE)/rubber liner</td><td>Duran Wheaton Kimble</td><td>W224605</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td></td></tr><tr><td>Alconox</td><td>Fisher Scientific</td><td>50-821-781</td><td></td></tr><tr><td>Ammonium formate</td><td>Millipore Sigma</td><td>LSAC70221</td><td></td></tr><tr><td>C18, 3.5 um x 50 mm column, SunFire</td><td>Waters</td><td>&amp;nbsp;186002551</td><td></td></tr><tr><td>Chloroform</td><td>Millipore Sigma</td><td>LSAC288306</td><td></td></tr><tr><td>Cuvette UV Micro LCH 8.5 mm, 50 um, RPK</td><td>Sarstedt</td><td>67.758.001</td><td></td></tr><tr><td>Di(2-ethylhexyl) phthalate (DEHP)</td><td>Millipore Sigma</td><td>36735</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Millipore Sigma</td><td>LSAC472301</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco</td><td>111000200</td><td></td></tr><tr><td>Filter supports, 10 mm</td><td>Avanti Polar Lipids</td><td>610014</td><td>Size for mini extruder</td></tr><tr><td>Folded capillary zeta cell</td><td>Malvern Panalytical</td><td>DTS1070</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>190764-4L</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly Clark</td><td>34256</td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol (soy) (Soy PI)</td><td>Avanti Polar Lipids</td><td>840044</td><td></td></tr><tr><td>L-&amp;alpha;-phosphitidylcholine (Egg, Chicken)</td><td>Avanti Polar Lipids</td><td>840051</td><td></td></tr><tr><td>LiposoFast &amp;reg; LF-50</td><td>Avestin, Inc.</td><td></td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>179337 - 4L</td><td></td></tr><tr><td>Mini-extruder set with holder/heating block</td><td>Avanti Polar Lipids</td><td>610000</td><td></td></tr><tr><td>MultiScreen-IP Filter Plate, 0.45&amp;nbsp;&amp;micro;m, clear, sterile</td><td>Millipore Sigma</td><td>MAIPS4510</td><td>for PAMPA studies</td></tr><tr><td>Nitrogen gas, ultrapure</td><td>TechAir</td><td>NI T5.0</td><td></td></tr><tr><td>Nuclepore hydrophilic membranes, polycarbonate, 19 mm, 0.1 um</td><td>Whatman</td><td>800309</td><td>Size for mini extruder</td></tr><tr><td>Nuclepore hydrophilic membranes, polycarbonate, 25 mm, 0.1 um</td><td>Whatman</td><td>110605</td><td>Size for large extruder</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM999</td><td></td></tr><tr><td>Phosphate buffer saline (PBS), 10x</td><td>Genesee Scienfitic</td><td>25-507X</td><td>Dilute to 1x</td></tr><tr><td>Qsoft 401 software</td><td>Biolin Scientific</td><td></td><td></td></tr><tr><td>Quartz Crystal Microbalance with Dissipation Q-Sense Analyzer</td><td>Biolin Scientific</td><td></td><td></td></tr><tr><td>Scintillation vials, borosilicate glass vials, 20 mL</td><td>Duran Wheaton Kimble</td><td>986561</td><td></td></tr><tr><td>Silicon Dioxide, thin QSensors</td><td>Biolin Scientific</td><td>QSX 303</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Millipore Sigma</td><td>LSACS5886</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Fisher Scientific</td><td>BP166-100</td><td></td></tr><tr><td>Solvent Safe pipette tips</td><td>Sigma-Aldrich</td><td>S8064</td><td></td></tr><tr><td>Sphingomyelin (Egg, Chicken)</td><td>Avanti Polar Lipids</td><td>860061</td><td></td></tr><tr><td>Trizma base</td><td>Millipore Sigma</td><td>LSACT1503</td><td></td></tr><tr><td>Trypsin-ethylenediaminetretaacetic acid</td><td>Caisson Labs</td><td>TRL01-6X100ML</td><td></td></tr><tr><td>Whatman drain disc, 25 mm</td><td>Whatman</td><td>230600</td><td>Size for large extruder</td></tr><tr><td>Zetasizer ZS90</td><td>Malvern Panalytical</td><td></td><td></td></tr><tr><td>Zetasizer 7.01 software</td><td>Malvern Panalytical</td><td></td><td></td></tr></tbody></table>

assembly of cell mimicking supported and suspended lipid bilayer models for the study of molecular interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol details methods for forming supported and suspended lipid bilayers (Figure 1). The first step to forming a supported lipid bilayer is to develop lipid vesicles. The mini extruder allows for small volumes of lipid vesicles to be prepared (1 mL or less), while the large extruder allows for 5-50 mL of lipid vesicles to be prepared in one batch. Size distributions of uni-lipid vesicles formed by either the mini or large extruder are shown in Figure 2A....

Watch this Scientific Journal Video about Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions at JoVE.com

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

assembly-cell-mimicking-supported-suspended-lipid-bilayer-models-for

Research

JoVE Journal

Bioengineering

3.4K Views.  Worcester Polytechnic Institute. De constructie van lipide dubbellaagse membranen beschreven in dit protocol kan worden gebruikt om cruciale informatie te verkrijgen over membraaninteracties met farmaceutische agentia, milieutoxische stoffen en zelfs biologische verbindingen. Deze technieken kunnen worden gebruikt om membranen van verschillende vloeistofcomplexiteit te fabriceren en om de permeabiliteit, absorptie en inbedding van verbindingen in membranen te beoordelen. Voeg om te beginnen het juiste volume lipidenbouillono...

Video: Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

We provide a protocol for in vitro self-organization assays of MinD and MinE on a supported lipid bilayer in an open chamber. Additionally, we describe how to enclose the assay in lipid-clad PDMS microcompartments to mimic in vivo conditions by reaction confinement.

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Biochemistry

Formation of Biomembrane Microarrays with a Squeegee-based Assembly Method

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are heterogeneous mixtures of many types of lipids, contain membrane-bound proteins and are decorated with carbohydrates. In some experiments, it is desirable to decouple the biophysical or biochemical properties of the lipid bilayer from those of the natural membrane. Such cases call for the use of model systems such as giant vesicles, liposomes or supported lipid bilayers (SLBs). Arrays of SLBs are particularly attractive for sensing applications and mimicking cell-cell interactions. Here we describe a new method for forming SLB arrays. Submicron-diameter SiO2 beads are first coated with lipid bilayers to form spherical SLBs (SSLBs). The beads are then deposited into an array of micro-fabricated submicron-diameter microwells. The preparation technique uses a "squeegee" to clean the substrate surface, while leaving behind SSLBs that have settled into microwells. This method requires no chemical modification of the microwell substrate, nor any particular targeting ligands on the SSLB. Microwells are occupied by single beads because the well diameter is tuned to be just larger than the bead diameter. Typically, more 75% of the wells are occupied, while the rest remain empty. In buffer SSLB arrays display long-term stability of greater than one week. Multiple types of SSLBs can be placed in a single array by serial deposition, and the arrays can be used for sensing, which we demonstrate by characterizing the interaction of cholera toxin with ganglioside GM1. We also show that phospholipid vesicles without the bead supports and biomembranes from cellular sources can be arrayed with the same method and cell-specific membrane lipids can be identified.

Supported lipid bilayers and natural membrane particles are convenient systems that can approximate the properties of cell membranes and be incorporated in a variety of analytical strategies. Here we demonstrate a method for preparing microarrays composed of supported lipid bilayer-coated SiO2 beads, phospholipid vesicles or natural membrane particles.

Lipid bilayer membranes form the plasma membranes of cells and define the boundaries of subcellular organelles. In nature, these membranes are ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

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.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

Biology

Simulating and Analyzing Lipid Bilayers Using Molecular Dynamics

Realistic Membrane Modeling Using Complex Lipid Mixtures in Simulation Studies

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different mechanical and structural properties in the membrane that directly impact the molecules and processes that occur at this interface. Lipid composition is dynamic and can serve to modulate cell signaling processes. Computational approaches are increasingly used to predict interactions between biomolecules and provide molecular insights to experimental observables. Molecular dynamics (MD) is a technique based on statistical mechanics that predicts the movement of atoms based on the forces that act on them. MD simulations can be used to characterize the interaction of biomolecules. Here, we briefly introduce the technique, outline practical steps for beginners who are interested in simulating lipid bilayers, demonstrate the protocol with beginner-friendly software, and discuss alternatives, challenges, and important considerations of the process. Particularly, we emphasize the relevance of using complex lipid mixtures to model a cell membrane of interest to capture the appropriate hydrophobic and mechanical environments in simulation. We also discuss some examples where membrane composition and properties modulate the interactions of bilayers with other biomolecules.

Author Spotlight: Advancing Cell Membrane Biophysics - Exploring Interactions and Challenges Through Experimental and Computational Approaches 

Membrane lipid diversity in structure and composition is an important contributor to cellular processes and can be a marker of disease. Molecular dynamics simulations allow us to study membranes and their interactions with biomolecules at atomistic resolution. Here, we provide a protocol to build, run, and analyze complex membrane systems.

Lipids are structural building blocks of cell membranes; lipid species vary across cell organelles and across organisms. This variety results in different ...

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

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