The authors wish to acknowledge support from the National Research Foundation (NRF -NRFF2011-01 and NRF2015NRF-POC0001-19), the National Medical Research Council (NMRC/CBRG/0005/2012), and Nanyang Technological University to N.J.C.

1. Formation of Supported Lipid Bilayer on a Hydrophilic Solid Support
<ol>
	<li>Prepare lipid stock solutions of 10 mg/ml 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1 mg/ml 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) by dissolving the respective lipid powders (appropriately weighed beforehand with an analytical mass balance) in isopropanol solution.</li>
	<li>Dilute and mix the stock solutions in isopropanol in order to prepare the desired lipid mixture at the final concentration. For fluorescence microscopy and FRAP experiments,0.5 wt% Rh-PE should be included in the lipid mixture.</li>
	<li>Inject the lipid mixture in isopropanol into the microfluidic channel until it is filled.</li>
	<li>Incubate the lipid mixture on the glass surface for about 10 min.</li>
	<li>Gradually replace the lipid solution with water or buffer solution using a peristaltic pump at a very low flow rate (10-50 &#181;l/min). Alternatively, replace the lipid mixture by repeated pipetting.</li>
	<li>Rinse the channel thoroughly with excess buffer in order to remove the residual isopropanol.</li>
</ol>
2. Formation of Cholesterol-enriched Supported Membranes
Note: The solid surface (SiO2) supports vesicle fusion, but the membrane composition (high cholesterol) inhibits vesicle fusion because cholesterol-enriched vesicles have high bending rigidity30.
<ol>
	<li>Prepare a stock solution containing 10 mg/ml DOPC lipid, 10 mg/ml cholesterol, and 1 mg/ml Rh-PE by first dissolving the respective powders in isopropanol.</li>
	<li>Repeat steps 1.2-1.6 using the stock solutions prepared in step 2.1.</li>
</ol>
3. Membrane Fluidity Assay
<ol>
	<li>Immerse the glass slide in sodium dodecyl sulfate (SDS) solution (1%) for 10 min.</li>
	<li>Wash the slides thoroughly with deionized water and rinse with ethanol.</li>
	<li>Blow-dry slides using a gentle stream of nitrogen.</li>
	<li>Expose the slides to oxygen plasma for 30 sec at maximum radiofrequency power in the oxygen plasma chamber.</li>
	<li>Remove the protective film coating of the bottomless commercial microfluidic chamber (Figure 1A) using a tweezer and attach the glass slide onto the sticky side of the chamber (Figure 1B).</li>
	<li>Assemble the connectors and tubing into the inlet and outlet positions of the chamber and place the microfluidic channel on the microscope stage (Figure 1C).</li>
	<li>Form a fluorescently-labeled supported lipid bilayer in the microfluidic channel [repeat step 1 using the desired lipid composition (e.g., 0.5 mg/ml DOPC) including 0.5 wt% Rh-PE in organic solvent].</li>
	<li>Locate the bilayer plane with a 60X oil immersion objective (NA 1.49) in order to capture images.</li>
	<li>Take two pre-bleach images, then photo-bleach a 30 &#181;m wide circular spot with a 532 nm, 100 mW laser beam. Before photobleaching, warm up the laser until its intensity has become stable. Immediately after photobleaching, capture a series of images every 1 sec for 2 min in order to follow the recovery in fluorescence intensity at the bleached spot.</li>
</ol>
4. Cholesterol Quantification Assay
<ol>
	<li>Expose the silicon dioxide-coated quartz crystal sensor chip to oxygen plasma for 30 sec at maximum radiofrequency power in the oxygen plasma chamber. Remove the chip from the chamber and immediately mount the chip in the measurement chamber.</li>
	<li>Before experiment, first acquire the frequency and dissipation of the sensor chip in air in order to ensure proper mounting.
	<ol>
		<li>For the commercial Q-Sense E1 or E4 QCM-D instrument, run the Q-Soft software program and click &#8220;Acquisition&#8221; and select &#8220;Setup Measurement&#8221;.</li>
		<li>In the new window which appears, check the 3, 5, 7, 9, 11 and 13 overtones and then click Find and Run in order to check the resonance spectra. Set the temperature at 24 &#176;C. If one or more resonance frequencies do not agree with the expected values, check the chip mounting and re-perform this step until an agreement is reached.</li>
	</ol>
	</li>
	<li>Start the peristaltic pump and flow buffer solution (10 mM Tris, 150 mM NaCl, pH 7.5) into the measurement chamber at a flow rate of 100 &#181;l/min.</li>
	<li>In the software program, click &#8220;Acquisition&#8221; and select &#8220;Restart Measurement&#8221; in order to record the resonance frequency and energy dissipation signals. Repeat this step until a stable baseline is obtained for the frequency and dissipation shifts. Note: For steps hereafter, there will be additional frequency and dissipation shifts which can be recorded as a function of time.</li>
	<li>Inject isopropanol (without lipid) for 10 min.</li>
	<li>Inject the mixture of DOPC lipid/cholesterol at the desired molar ratio with a total lipid concentration of 0.5 mg/ml in isopropanol for 10 min.</li>
	<li>Inject buffer for 20 min at a flow rate of 100 &#181;l/min.</li>
	<li>Inject a solution of 1 mM methyl-&#946;-cyclodextrin (M&#946;CD) prepared in buffer (flow rate 100 &#181;l/min) until the frequency signal reaches a stable value.</li>
	<li>Measure the relative positive frequency shifts that is caused by the M&#946;CD treatment step and calculate the mass of cholesterol and DOPC lipids by converting the &#916;fcho and &#916;fDOPC values to mass values by using the Sauerbrey equation [&#916;m = -(C/n)&#215;&#916;f, where C=17.7 ng/cm2, n:overtone].</li>
	<li>Calculate the mole fraction of cholesterol in the SLB, taking into account the molecular weights of DOPC lipid (786.1 g/mol) and cholesterol (386.6 g/mol).</li>
</ol>

The authors have no competing financial interests to disclose.

In this work, a solvent-exchange protocol is presented in which lipids in alcohol (isopropanol, ethanol, or n-propanol) are incubated with a solid support and then the alcohol is replaced gradually with an aqueous buffer solution in order to drive a series of phase transitions eventually producing lamellar-phase lipid bilayers24. It is shown that the method enables fabrication of supported lipid bilayers on surfaces such as gold, which is intractable to the vesicle fusion method.
An optimal lipid concentration range (0.1 - 0.5 mg/ml) has been determined for complete bilayer formation in standard experimental formats tested thus far. At lipid concentrations below 0.1 mg/ml, discrete, microscopic patches of bilayers formed. On the other hand, at concentrations above 0.1 mg/ml and lower than 0.5 mg/ml, a complete and uniform bilayer is formed. At lipid concentrations above this range, a fluid bilayer was still formed as verified by FRAP analysis, however, fluorescence microscopy reveals the presence of additional lipid structures on top of the bilayer. Strikingly, the morphology of these additional lipid structures, as determined by QCM-D analysis, depended on the alcohol which was used during the incubation step. In the case of ethanol, the relatively high &#916;f and &#916;D shifts resemble the QCM-D signature obtained for an adsorbed vesicle layer. When isopropanol or n-propanol was instead used, the &#916;f was slightly higher than the value expected for a bilayer (final &#916;f between -30 to -40 Hz), while the &#916;D was appreciably higher. Such QCM-D responses would be expected for extended lipid structures (e.g., worm-like micelles) protruding outward from the membrane surface (as visible by fluorescence microscopy in some cases).
The rate of solvent exchange is another important parameter which can be critical, especially when lower lipid concentrations (e.g., 0.1 mg/ml) are used. Rapid solvent exchange at low lipid concentration can lead to the formation of incomplete bilayers. In the standard measurement chamber used for QCM-D measurements in this paper (Q-Sense E4 measurement chamber), flow rates of around 100 &#181;l/min, were suitable for highly reproducible complete bilayer formation. For flow cells with other geometries and volume, the optimal flow rate may vary and must be empirically determined based on the steps suggested herein.
In addition to form supported lipid bilayers on surfaces which are intractable to vesicle fusion, the SALB can be employed to circumvent the need for lipid vesicles which can rupture, thereby opening the door to the fabrication of supported membranes with complex compositions. As an illustrative example composition, lipid mixtures with a high fraction of cholesterol were examined. Cholesterol is an important component of mammalian cell membranes, and its fraction can approach 45&#8722;50 mol% of the membrane lipid composition (e.g., in erythrocytes). Thus, even a simple model of a lipid bilayer representing a human cell membrane should include cholesterol.
While vesicle fusion could be used to fabricate fluidic lipid bilayers containing only 10-15% cholesterol, the SALB method enables formation of fluidic lipid bilayers containing high fractions of cholesterol (up to 57 mol%, as quantified by QCM-D measurements)36. However, when the level of cholesterol was further elevated (up to 63 mol%), stripe-shape domains were observed37. The co-existing domains were liquid, reminiscent of those observed in the &#946; region in the phase diagram of the cholesterol/phospholipids monolayer at the air-water interface.
Overall, the SALB method is shown to be a simple and efficient approach to form supported lipid bilayers, especially in cases beyond the scope of the conventional vesicle fusion method. Thus far, the QCM-D technique and fluorescence microscopy were mainly used to characterize the supported lipid bilayers formed by the SALB method. Looking forward, a broad range of surface-sensitive analytical measurements techniques, including surface plasmon resonance (SPR)38, atomic force microscopy (AFM)39,40, Fourier-transform infrared spectroscopy41, X-ray42 and neutron reflectivity43, can be used to further characterize and study simple and complex bilayer configurations prepare by the SALB method. These emerging capabilities open the door to a greater number of scientists who can explore artificial cell membranes by taking advantage of a simple and robust experimental protocol.

The solid-supported lipid bilayer1 (SLB) is a versatile platform which preserves the basic characteristics of biomembranes such as the bilayer thickness, two-dimensional lipid diffusivity, and the ability to host membrane-associated biomolecules. Due to the complexity of natural cell membranes, this simple platform has been shown to function as an efficient platform for in vitro studies of membrane-related processes such as raft formation2, protein binding3, virus and virus-like particle binding4,5, and cell signaling6. Formed in close proximity to a solid support, the SLB platform is compatible with a range of surface-sensitive measurements techniques such as total internal reflection microscopy (TIRF), quartz crystal microbalance-dissipation (QCM-D), and impedance spectroscopy.
Several methods have been developed to produce different types of SLBs, including air bubble collapse7 and dip-pen nanolithography8 for submicron-sized lipid spots, spin-coating9 for bilayer stacks and Langmuir&#8722;Blodgett (LB)10 and vesicle fusion (VF)11 for full-spanning, single lipid bilayer coatings. The VF method consists of the adsorption of small unilamellar vesicles to a solid support and subsequent spontaneous rupture and fusion to form a continuous lipid bilayer. However, under physiological conditions, spontaneous vesicle rupture is mainly limited to silicon-based materials such as silicon dioxide, glass, and mica. In addition, vesicle rupture does not occur spontaneously for vesicles of complex lipid compositions such as those containing high fractions of cholesterol or negatively charged lipids. Depending on the system, vesicle rupture may be induced by further tailoring the experimental conditions such as temperature12, solution pH13, and salinity14, osmotic shock15 or pressure16, or addition of divalent ions such as Ca2+ 17 . Alternatively, the membrane-active AH peptide can be introduced in order to destabilize a layer of adsorbed vesicles, leading to vesicle rupture and bilayer formation on a range of surfaces18-22.
Moreover, successful bilayer formation requires preparation of a well-controlled population of small unilamellar vesicles that can be time consuming and difficult to achieve for certain membrane compositions. Therefore, despite its high efficiency in optimal cases (e.g., after extensive freeze-thaw pretreatment of vesicles23), the general application of vesicle fusion is limited by the scope of suitable substrates and membrane compositions .
The solvent-assisted lipid bilayer (SALB) method24-28 is an alternative fabrication technique which does not require lipid vesicles. The method is based on the deposition of lipid molecules onto a solid surface in the presence of a water miscible organic solvent followed by gradual exchange of this solvent with an aqueous buffer solution in order to trigger SLB formation. During the solvent-exchange step, the ternary mixture of lipids, organic solvent, and water undergoes a series phase transitions with increasing water fraction, which leads to the formation of lamellar phase structures in the bulk solution and an SLB on the solid substrate. Importantly, this self-assembly route bypasses the need for vesicle rupture, which is usually the limiting step for the transformation of adsorbed vesicles into an SLB. The protocol is applicable to a wide variety of surfaces including silicon dioxide, aluminum oxide, chrome, indium tin oxide, and gold. In this paper and in the accompanying video, a comparison of lipid deposition by the SALB and vesicle fusion methods is presented. In particular, the influence of experimental parameters, including lipid concentration, flow rate, and the choice of water miscible organic solvent, on the quality of the bilayer formed by the SALB method are discussed. Analytical characterization of the fabricated SLBs is performed by the QCM-D, fluorescence microscopy, and fluorescence recovery after photobleaching (FRAP) techniques. QCM-D monitoring is a surface-sensitive mass measurement technique which, since the pioneering work conducted by Keller and Kasemo29, has been extensively used to quantitatively investigate bilayer formation. Fluorescence microscopy permits inspection of membrane homogeneity as well as the visualization of membrane domains. The FRAP technique is a standard tool to determine the lateral mobility of lipid molecules in an SLB, which is an essential property of fluidic membranes.
The first part of this study involves QCM-D analysis of the SALB and vesicle fusion methods applied to attempt bilayer formation on silicon dioxide and gold. In the second part, the preparation and characterization of supported membranes containing a range of cholesterol concentrations with the SALB method are demonstrated and the results are compared with those obtained by the vesicle fusion method.

<table><tbody><tr><td>QCM-D silicon dioxide-coated substrates</td><td>QSense AB,&amp;nbsp; Sweden</td><td></td><td></td></tr><tr><td>QCM-D gold-coated substrates</td><td>QSense AB,&amp;nbsp; Sweden</td><td></td><td></td></tr><tr><td>Q-Sense E4 module</td><td>QSense AB,&amp;nbsp; Sweden</td><td></td><td></td></tr><tr><td>Plasma Cleaner, PDC-32G</td><td>Harrick Plasma, Ithaca, NY</td><td>PDC-001 (115V)&amp;nbsp;</td><td></td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DOPC)</td><td>Avanti Polar Lipids</td><td>850375P</td><td></td></tr><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphoethanolamine-&lt;em&gt;N&lt;/em&gt;-(lissamine rhodamine B sulfonyl) (ammonium salt) (Rh-PE)</td><td>Avanti Polar Lipids</td><td>810150P</td><td></td></tr><tr><td>cholesterol</td><td>Avanti Polar Lipids</td><td>700000P</td><td></td></tr><tr><td>Methyl-&amp;beta;-cyclodextrin</td><td>Sigma&amp;nbsp;</td><td>C4555</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma&amp;nbsp;</td><td>673773</td><td></td></tr><tr><td>Ethanol</td><td>Sigma&amp;nbsp;</td><td>459844</td><td></td></tr><tr><td>n-propanol</td><td>Sigma&amp;nbsp;</td><td>279544</td><td></td></tr><tr><td>Sticky-Slide I 0.1 Luer</td><td>IBIDI</td><td>81128</td><td></td></tr><tr><td>Male elbow 1/8&amp;rdquo;</td><td>Cole-Parmer</td><td>30505-70</td><td></td></tr><tr><td>Silicon tubing 1.6&amp;#160;mm ID</td><td>IBIDI</td><td>10842</td><td></td></tr><tr><td>Glass coverslip No. 1.5H, 25 mm x 75 mm</td><td>IBIDI</td><td>10812</td><td></td></tr><tr><td>Reglo Digital M2-2/12 Peristaltic Pump</td><td>Ismatec</td><td></td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma&amp;nbsp;</td><td>71725</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method at JoVE.com

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.

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 ...

biomembrane-fabrication-solvent-assisted-lipid-bilayer-salb

Research

JoVE Journal

Bioengineering

14.8K Views.  Nanyang Technological University. 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.

Video: Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

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 ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

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 ...

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 ...

Biochemistry

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

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 ...

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 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 ...

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 &#181;m in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...