The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).

1. Scaffold Liposome Preparation
NOTE: Ideally, initial experiments should use a relatively simple and featureless scaffold (comprised of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine or PC) and DGS-NTA(Ni2+) (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl](nickel salt)). Building off of these experiments, lipid charge, flexibility, and curvature can be introduced as individual factors with the potential to alter membrane-proximal interactions. These changes can be achieved by adding defined amounts of specific lipid constituents, including phosphatidylserine or cardiolipin (CL), phosphatidylethanolamine (DOPE or PE), or galactosyl(&#946;) ceramide.
<ol>
	<li>Combine lipids dissolved in chloroform in a clean glass test tube. Evaporate the solvent with dry nitrogen gas while rotating the tube to form a thin lipid film. Remove residual solvent with a centrifugal evaporator for 1 h at 37 &#176;C. 
	NOTE: Various liposome formulations are used in the protocols described below: scaffold liposomes (3.3 mol% DGS-NTA(Ni2+) / 96.7 mol% DOPC), scaffold liposomes with cardiolipin (3.3 mol% DGS-NTA(Ni2+) / 10 mol% cardiolipin / 86.7 mol% DOPC), flexible scaffold liposomes with cardiolipin (3.3 mol% DGS-NTA(Ni2+) / 10 mol% cardiolipin / 35 mol% DOPE / 51.7 mol% DOPC), and enriched scaffold liposomes (10 mol% DGS-NTA(Ni2+) / 15 mol% cardiolipin / 35 mol% DOPE / 40 mol% DOPC).</li>
	<li>Add Buffer A (25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mM KCl, pH adjusted to 7.5 with KOH) preheated to 37 &#176;C such that the final lipid concentration is 1 - 2 mM. Incubate 30 min at 37 &#176;C with occasional vortexing to fully resuspend the lipid mixture (Figure 1a).</li>
	<li>Transfer to a plastic test tube, place the tube in liquid nitrogen until completely frozen (roughly 30 s), and place in a 37 &#176;C water bath until fully thawed (roughly 1 - 2 min). Repeat for a total of 4 freeze-thaw cycles (Figure 1b).</li>
	<li>Prepare a lipid extruder by soaking 4 filter supports and a polycarbonate filter in buffer and assembling the extruder according to manufacturer instructions. Extrude the lipid solution through the filter 21 times. Use gentle, constant pressure to ensure a homogenous size distribution (Figure 1c). 
	NOTE: For all experiments described in this protocol, a 1.0 &#181;m polycarbonate filter was used for extrusion. Drp1 interaction with anionic lipids can be observed with a variety of liposome diameters ranging from 50 nm to 400 nm12 or larger13. Hence, the filter size of 1 &#181;m was chosen to be ideal for both GTPase activity and for electron microscopy. If other liposome diameters are desired, preparation of giant unilamellar vesicles14,15 (GUVs) or small unilamellar vesicles16 (SUVs) can be used. Dynamic light scattering can be used to assess liposome size heterogeneity13.</li>
	<li>Store extruded liposomes at 4 &#176;C and discard after 3 - 5 d.</li>
</ol>
2. Use of Scaffold Liposomes for Protein Binding Analysis
<ol>
	<li>Sample Preparation
	<ol>
		<li>Incubate His-tagged Mff&#916;TM (5 &#181;M final) with scaffold liposomes (40 mol% PC / 35 mol% PE / 15 mol% CL / 10 mol% DGS-NTA(Ni2+); 50 &#181;M final) for at least 15 min at RT in Buffer A + BME (25 mM HEPES, 150 mM KCl, 10 mM &#946;-mercaptoethanol (BME), pH adjusted to 7.5 with KOH). For an Mff-free control, incubate liposomes with a his-tagged control protein (such as GFP) to bind and shield exposed NTA(Ni2+). 
		NOTE: Mff&#916;TM was expressed and purified as described in a previous study2. GFP was purified in a similar manner, but the ion-exchange step was omitted. BME was required for these experiments because Drp1 is sensitive to oxidation, which can alter its activity and assembly properties.</li>
		<li>Add Drp1 (2 &#181;M final) and incubate for 1 h at RT. 
		NOTE: Drp1 was expressed and purified as described in a previous study2. After incubation with Drp1, the effect of nucleotide binding on membrane deformation can be investigated by incubating one additional hour with 2 mM MgCl2 and either 1 mM GTP, 1 mM GMP-PCP, or Buffer A + BME.</li>
	</ol>
	</li>
	<li>Negative Stain Transmission Electron Microscopy (EM) Analysis
	<ol>
		<li>Transfer 5 &#181;L of sample to a sheet of laboratory film, and lay a carbon-coated Cu/Rh grid on the sample. Incubate the grid 1 min on the sample, blot away excess liquid on filter paper, and transfer to a drop of 2% uranyl acetate. Incubate 1 min, blot excess stain on filter paper, and transfer to a grid box. Store under vacuum O/N to ensure full desiccation.</li>
		<li>Image samples using a transmission electron microscope at 18,500 - 30,000X&#160;magnification to observe ultrastructural changes in protein and liposome morphologies17.&#160; 
		Note: Ultrastructural changes can be quantified using image analysis software, such as ImageJ13 (http://imagej.nih.gov/ij/). Protein decoration can be measured when compared with naked lipid templates. Additionally, the diameters of tubular segments can be measured from the outermost portion of the assemblies13. A more detailed analysis can be performed using cryo-electron microscopy17. This method can be used to image native protein-lipid assemblies in solvent without the use of heavy metal stains that coat the sample. In this way, detailed structural features not apparent in negative stain, including changes in the underlying lipid morphology, can be examined and quantified.</li>
	</ol>
	</li>
</ol>
3. Use of Scaffold Liposomes for Enzymatic Assay
Note: A colorimetric GTPase assay18 was used to measure phosphate liberation via GTP hydrolysis. Alternative GTPase assays are available19 and can be implemented as needed.
<ol>
	<li>Incubate His-tagged Mff&#916;TM (Mff), Fis1&#916;TM(Fis1), or GFP (5 &#181;M final for all) with scaffold liposomes (150 &#181;M final) for 15 min at RT in Buffer A + BME (volume = 30 &#181;L). Add Drp1 (500 nM final) and incubate an additional 15 min at RT (volume = 80 &#181;L). 
	NOTE: Fis1 was purified in a similar manner to Mff2, but the ion-exchange chromatography step was omitted. The purpose of His-tagged GFP is to shield the NTA(Ni2+) headgroups and prevent nonspecific charge interactions with other proteins. If no effect is observed in the absence of GFP, then this control may not be required. Alternative blocking proteins (of comparable size to the protein of interest) can be used as well, but GFP allows for direct visualization of the interactions with scaffold liposomes.</li>
	<li>Transfer tubes to a thermocycler set to 37 &#176;C, and initiate reactions by addition of GTP and MgCl2 (1 mM and 2 mM final, respectively; volume = 120 &#181;L).</li>
	<li>At desired time points (i.e. T = 5, 10, 20, 40, 60 min), transfer 20 &#181;L of reaction to wells of a microtiter plate containing 5 &#181;L of 0.5 M EDTA to chelate Mg2+ and stop the reaction.</li>
	<li>Prepare a set of phosphate standards by diluting KH2PO4 in Buffer A + BME to calibrate results. A useful set of standards is 100, 80, 60, 40, 20, 10, 5, and 0 &#181;M. Add 20 &#181;L of each to wells containing 5 &#181;L of 0.5 M EDTA.</li>
	<li>Add 150 &#181;L of Malachite green reagent (1 mM malachite green carbinol, 10 mM ammonium molybdate tetrahydrate, and 1 N HCl) to each well, and read OD650 5 min after addition. 
	NOTE: GTP is acid labile, and will hydrolyze in the presence of malachite green reagent. Ensure that the time between adding malachite reagent and reading is constant to ensure reproducible results.</li>
	<li>Generate a standard curve by plotting OD650 of the standards as a function of phosphate concentration. Use linear regression to determine the relationship between OD650 and phosphate concentration in a sample.</li>
	<li>Using the linear regression, convert the OD650 of the protein reaction samples to &#181;M phosphate. Determine the rate of phosphate generation for each reaction mixture by plotting phosphate concentration as a function of time, and convert to kcat by dividing the rate by the Drp1 concentration (0.5 &#181;M). 
	NOTE: Only the initial linear rate should be used to determine the rate of phosphate generation, and a minimum of 3 data points must be used. If the rate of reaction is sufficiently rapid that the first three data points are not linear (i.e. the r2 of the linear fit is less than 0.9) a significantly shorter time course with at least 3 time points should be performed.</li>
</ol>

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The authors have nothing to disclose.

This protocol offers a method for investigating protein-protein interactions involving integral membrane proteins. By utilizing a modular liposome scaffold, investigators are capable of assessing the activity of one or more proteins in a lipid-proximal environment. Previous studies have demonstrated a similar method for receptor enzymes of the plasma membrane24-26. We have expanded this method to incorporate lipid cofactors and explore interactions between proteins that make up the mechan...

Studying membrane-proximal protein-protein interactions is a challenging endeavor due to difficulty in recapitulating the native environment of the integral membrane proteins involved1. This is due to the necessity of detergent solubilization and the inconsistent orientation of proteins in proteoliposomes. In order to avoid these issues, we have employed a strategy whereby soluble domains of integral membrane proteins are expressed as His-tag fusion proteins, and these soluble fragments are anchored to scaffold liposomes via interactions with NTA(Ni2+) headgroups at the lipid surface. Using these scaffolds, lipid-proximal protein interactions can be investigated over a range of lipid and protein compositions.
We have effectively applied this method to investigate the critical protein-protein interactions that govern assembly of the mitochondrial fission complex and examine lipid interactions that modulate this process2. During mitochondrial fission, a conserved membrane remodeling protein, called Dynamin-related protein 1 (Drp1)3, is recruited to the surface of the Outer Mitochondrial Membrane (OMM) in response to cellular signals that regulate energy homeostasis, apoptotic signaling, and several other integral mitochondrial processes. This large, cytosolic GTPase is recruited to the surface of mitochondria through interactions with integral OMM proteins4-8. The role of one such protein, Mitochondrial Fission Factor (Mff), has been difficult to elucidate due to an apparent weak interaction with Drp1 in vitro. Nevertheless, genetic studies have clearly demonstrated that Mff is essential for successful mitochondrial fission7,8. The method described in this manuscript was able to overcome previous shortcomings by introducing simultaneous lipid interactions that promote Drp1-Mff interactions. Overall, this novel assay revealed fundamental interactions guiding assembly of the mitochondrial fission complex and provided a new stage for ongoing structural and functional studies of this essential molecular machine.
To date, examination of interactions between Drp1 and Mff have been complicated by the inherent flexibility of Mff9, the heterogeneity of Drp1 polymers2,10, and the difficulty in purifying and reconstituting full-length Mff with an intact transmembrane domain11. We addressed these challenges by using NTA(Ni2+) scaffold liposomes to reconstitute His-tagged Mff lacking its transmembrane domain(Mff&#916;TM-His6). This strategy was advantageous because Mff&#916;TM was extremely soluble when over-expressed in E. coli, and this isolated protein was easily reconstituted on scaffold liposomes. When tethered to these lipid templates, Mff assumed an identical, outward facing orientation on the surface of the membrane. In addition to these advantages, mitochondrial lipids, such as cardiolipin, were added to stabilize Mff folding and association with the membrane11. Cardiolipin also interacts with the variable domain of Drp12,12 which may stabilize this disordered region and facilitate assembly of the fission machinery.
This robust method is widely applicable for future studies that seek to evaluate membrane-proximal protein interactions. Through the use of additional tethering/affinity interactions, the sophistication of these membrane reconstitution studies can be enhanced to mimic additional complexity found at the surface of membranes within cells. At the same time, lipid compositions can be modified to more accurately mimic the native environments of these macromolecular complexes. In summary, this method provides a means to examine the relative contributions of proteins and lipids in shaping membrane morphologies to during critical cellular processes.

<table><tbody><tr><td>Phosphatidylcholine (DOPC)</td><td>Avanti Polar Lipids</td><td>850375</td><td></td></tr><tr><td>Phosphatidylethanolamine (DOPE)</td><td>Avanti Polar Lipids</td><td>850725</td><td></td></tr><tr><td>DGS-NTA(Ni&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Avanti Polar Lipids</td><td>790404</td><td></td></tr><tr><td>Bovine Heart Cardiolipin (CL)</td><td>Avanti Polar Lipids</td><td>840012</td><td></td></tr><tr><td>Chloroform</td><td>Acros Organics</td><td>268320010</td><td></td></tr><tr><td>Liposome Extruder</td><td>Avanti Polar Lipids</td><td>610023</td><td></td></tr><tr><td>Cu/Rh Negative Stain Grids</td><td>Ted Pella</td><td>79712</td><td></td></tr><tr><td>Microfuge Tube</td><td>Beckman</td><td>357448</td><td></td></tr><tr><td>GTP</td><td>Jena Biosciences</td><td>NU-1012</td><td></td></tr><tr><td>GMP-PCP</td><td>Sigma Aldrich</td><td>M3509</td><td></td></tr><tr><td>Microtiter Plate strips</td><td>Thermo Scientific</td><td>469949</td><td></td></tr><tr><td>EDTA</td><td>Acros Organics</td><td>40993-0010</td><td></td></tr><tr><td>Instant Blue Coomassie Dye</td><td>Expedeon</td><td>ISB1L</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310</td><td></td></tr><tr><td>BME</td><td>Sigma Aldrich</td><td>M6250</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P330</td><td></td></tr><tr><td>KOH</td><td>Fisher Scientific</td><td>P250</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Acros Organics</td><td>223211000</td><td></td></tr><tr><td>4 - 20% SDS-PAGE Gel</td><td>Bio Rad</td><td>456-1096</td><td></td></tr><tr><td>4x Laemmli Loading Dye</td><td>Bio Rad</td><td>161-0747</td><td></td></tr><tr><td>HCL</td><td>Fisher Scientific</td><td>A144S</td><td></td></tr><tr><td>Malachite Green Carbinol</td><td>Sigma Aldrich</td><td>229105</td><td></td></tr><tr><td>Ammonium Molybdate Tetrahydrate</td><td>Sigma Aldrich</td><td>A7302</td><td></td></tr><tr><td>Laboratory Film</td><td>Parafilm</td><td>PM-996</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Polysciences</td><td>21447</td><td></td></tr><tr><td>Tecnai T12 100 keV Microscope</td><td>FEI</td><td></td><td></td></tr><tr><td>Optima MAX</td><td>Beckman</td><td></td><td></td></tr><tr><td>TLA-55 Rotor</td><td>Beckman</td><td></td><td></td></tr><tr><td>Refrigerated CentriVap Concentrator</td><td>Labconico</td><td></td><td></td></tr><tr><td>Mastercycler Pro Thermocycler</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>VersaMax Microplate reader</td><td>Molecular Devices</td><td></td><td></td></tr></tbody></table>

using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

While the interaction between Drp1 and Mff has been demonstrated to be important for mitochondrial fission, this interaction has been difficult to recapitulate in vitro. Our goal was to better emulate the cellular environment wherein Drp1 and Mff interact. To this end, liposomes containing limiting concentrations of NTA(Ni2+) headgroups were prepared by rehydrating a lipid film as described above. The lipid solution initially consists of unilamellar and multilamellar v...

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Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

using-scaffold-liposomes-to-reconstitute-lipid-proximal-protein

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.8K Views.  Case Western Reserve University School of Medicine. This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment. 

Video: Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

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 ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

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 ...

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Neuroscience

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 ...

Bioengineering

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

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 ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Summary

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Chapters in this video

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

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

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

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

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

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