Name: detergent-assisted reconstitution of recombinant drosophila atlastin into liposomes for lipid-mixing assays
Uploaded: 2019-07-03T13:00:00.000+00:00
Duration: 8 min 43 s
Description: Watch this Scientific Journal Video about Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays at JoVE.com

We thank Dr. Michael Stern and his lab for their insights and feedback on atlastin related projects. This work was supported by the National Institute of General Medical Sciences [R01GM101377] and the National Institute of Neurological Disorders and Stroke [R01NS102676].

1. Purification of GST-DAtl-His8
<ol>
	<li>Protein expression and lysate preparation
	<ol>
		<li>Transform BL21 (DE3) E. coli with the GST-DAtl-His8 construct in pGEX4-T32 and select on an ampicillin plate.</li>
		<li>Select a single transformant and inoculate 5 mL of LB + ampicillin (5 &#181;L of 100 mg/mL ampicillin) in a 14 mL culture tube and incubate at 25 &#176;C with shaking at 200 rpm for 6-8 h. 
		NOTE: Due to leaky expression, it is not recommended to incubate at higher temperatures. Growth at 25 &#176;C reduces aggregation of protein during this growth period.</li>
		<li>Inoculate 200 mL of LB + ampicillin with 1 mL of the 5 mL culture and incubate overnight (~15&#8211;18 h) at 25 &#176;C.</li>
		<li>The next morning, harvest the cells by centrifugation (2,000 x g for 10 min) and resuspend in 5 mL of LB.</li>
		<li>Inoculate 4 L of LB + ampicillin and measure OD600 (0.05&#8211;0.15). Incubate at 25 &#176;C with shaking. 
		NOTE: Reserve some media to serve as a blank for the OD600 measurements before adding bacteria.</li>
		<li>Measure OD600 every hour until it reaches an OD between 0.4&#8211;0.5. At this point, reduce the temperature to 16 &#176;C.</li>
		<li>Induce with 0.2 mM IPTG (800 &#181;L of 1 M stock), 10 min after the incubator reaches 16 &#176;C. Incubate overnight (~15&#8211;18 h) at 16 &#176;C. 
		NOTE: The lower temperature improves the yield of functional protein by reducing aggregation of atlastin.</li>
		<li>The next morning, harvest the cells by centrifuging at 7,500 x g at 4 &#176;C.</li>
		<li>Resuspend the cells in 200 mL of A200 (25 mM HEPES (pH 7.4) and 200 mM KCl).</li>
		<li>Centrifuge the cells at 11,000 x g for 5 min at 4 &#176;C.</li>
		<li>Resuspend the pellet in 40 mL of breaking buffer (A200 plus 10% glycerol, 2 mM 2-mercaptoethanol, 4% Triton X-100 (TX100), 40mM imidazole, and one EDTA-free protease inhibitor cocktail tablet). 
		NOTE: Add TX-100 after resuspending to avoid generating bubbles and foam.</li>
		<li>Pass through an 18 G needle and run the cells through a cell disrupter three times at ~10,000 psi.</li>
		<li>Centrifuge the extract at 125,000 x g for 1 h. 
		NOTE: Optionally dissolve the pellet 1:2 (w:v) in 8 M Urea and nutate at room temperature overnight. Urea as a denaturant will dissolve slowly any insoluble pelleted protein for SDS-PAGE analysis. Save 1 &#956;L for SDS-PAGE and Coomassie stain analysis.</li>
		<li>Filter the extract through a 0.45 &#181;m cellulose nitrate sterile membrane filter to remove bacteria and large bacterial debris. 
		NOTE: Optionally, save 1 &#956;L for SDS PAGE and Coomassie stain analysis.</li>
	</ol>
	</li>
	<li>Protein purification by affinity chromatography
	<ol>
		<li>Load the filtered lysate onto an immobilized metal affinity chromatography (IMAC) resin column charged with Ni2+, pre-equilibrated with low imidazole buffer (A100 plus 10% glycerol, 2 mM 2-mercaptoethanol, 1% TX-100, 40 mM imidazole) at a rate of 1 mL/min at 4 &#176;C.</li>
		<li>Wash the column with 25 mL of A100 plus 10% glycerol, 2 mM 2-mercaptoethanol, 0.1% Anapoe X-100, 40 mM imidazole with a rate of 1 mL/min at 4&#176;C. Elute the protein with a 30 mL linear gradient of imidazole from 40 mM to 500 mM, and a final 5 mL wash at 500 mM.</li>
		<li>Pool together peak fractions and incubate for 1 h at 4 &#176;C with swollen GSH-Agarose beads, previously swollen in water and equilibrated in A100 with 10% glycerol, 2 mM 2-mercaptoethanol, 0.1% Anapoe X-100, and 1 mM EDTA. 
		NOTE: GSH-Agarose beads can be swollen in 50 mL of water the day before and incubated overnight at 4 &#176;C, or for 1 h at RT. To remove water and equilibration buffer, centrifuge in a swinging bucket rotor at 500 x g for 1 min without brake. Aspirate the supernatant with a 26 G needle.</li>
		<li>Pellet GSH-Agarose beads by centrifuging in a swinging bucket rotor at 500 x g without brake and remove the lysate supernatant by aspiration with a 26 G needle.</li>
		<li>Transfer the beads to a 10 mL polyprep column and wash five times with 5 mL of equilibration buffer (A100 with 10% glycerol, 2 mM 2-mercaptoethanol, 0.1% Anapoe X-100, and 1 mM EDTA) by centrifuging at 500 x g.</li>
		<li>Elute the protein with 1&#8211;1.5 mL of equilibration buffer supplemented with 10 mM reduced glutathione. Aliquot eluted protein and flash freeze in liquid nitrogen. Protein can be stored at -80 &#176;C indefinitely. 
		NOTE: Adjust pH of elution buffer to 7.4.</li>
		<li>Quantify protein concentration by amido black protein assay9 and assess purity by SDS-PAGE and Coomassie staining.</li>
	</ol>
	</li>
</ol>
2. Reconstitution of Recombinant Atlastin into Liposomes
<ol>
	<li>Liposome production by extrusion method10
	<ol>
		<li>Make lipid mix stocks in chloroform (10 mM total lipid). The necessary lipids are 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-DPPE), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-DPPE). Acceptor liposomes consist of POPC: DOPS (85:15 molar ratio) and donor liposomes of POPC:DOPS:Rh-PE:NBD-PE (83:15:1.5:1.5 molar ratio). 
		NOTE: While POPC:DOPS lipid mixes have been traditionally used for in vitro lipid mixing assays, alternative lipid compositions may be adapted for different experimental purposes. POPC:DOPS liposomes are very stable and harder to fuse, therefore is a very stringent system for fusion.</li>
		<li>Add 1 &#956;Ci/mL of L-&#945;-dipalmitoyl-phosphatidylcholine (choline methyl-3H) to lipid mixes in order to determine lipid concentrations at later steps by liquid scintillation counting. Reserve at least 8 &#956;L of this stock.</li>
		<li>Transfer desired amount of lipid mixes to flint glass tubes.</li>
		<li>Dry the lipid mixes under a gentle stream of N2 gas for ~10 min until no more chloroform is visible.</li>
		<li>Dry the lipid film further in a desiccator by vacuuming for 30&#160;min.</li>
		<li>Add enough aqueous A100 with 10% glycerol, 2 mM 2-mercaptoethanol, and 1 mM EDTA to the lipid film and bring back the concentration to 10 mM. Resuspend the lipid film by vortexing lightly for 15 min at room temperature. A vortexer that can accommodate flint glass tubes is recommended.</li>
		<li>Freeze-thaw the hydrated lipids in liquid nitrogen ten times. After freezing in liquid nitrogen, thaw the liposomes by letting the solution sit at room temperature for 30 s, then transfer to water for faster thawing. This freeze-thaw cycling will minimize multilamellar vesicles. 
		CAUTION: Tubes may crack if they contain volumes larger than 0.5 mL and if they are transferred directly form liquid nitrogen to water.</li>
		<li>Pass the lipid through polycarbonate filters with 100 nm pore size 19 times using mini-extruder.</li>
		<li>Determining total lipid concentration of liposomes by scintillation counting 
		NOTE: Some of the lipid may be left behind in glass tubes and in mini-extruder.
		<ol>
			<li>Add 4 &#956;L of lipid stock and liposomes in 3 mL of scintillation cocktail.</li>
			<li>Measure average counts per minute (CPMA) for stock and liposomes. And calculate liposomes concentration with the following formula: <img alt="Equation 1" src="/files/ftp_upload/59867/59867equ01.jpg" /></li>
		</ol>
		</li>
		<li>Store the liposomes at 4 &#176;C for up to a week. Longer storage is not recommended as liposomes might aggregate with time, decreasing reconstitution efficiencies.</li>
	</ol>
	</li>
	<li>Reconstitution by detergent assisted incorporation into preformed liposomes
	<ol>
		<li>Calculate the amount of buffer, protein, liposomes and extra detergent to be mixed together:
		<ol>
			<li>Determine the desired total volume. This volume is made up of buffer A100 with 10% glycerol, 2 mM 2-mercaptoethanol, and 1 mM EDTA. Volumes less than 250 &#956;L do not mix well in 0.5 mL microcentrifuge tubes.</li>
			<li>Calculate the volume of liposomes needed to give a final lipid concentration of about 1 mM. Subtract the volume from the buffer volume.</li>
			<li>Calculate the volume of protein needed to give the desired protein to lipid molar ratio (usually 1:400). Decrease the buffer volume accordingly.</li>
			<li>Determine how much extra detergent needs to be added to saturate the liposomes aiming for an effective detergent to lipid ratio (Reff) between 0.64&#8211;0.8. Remember to consider the detergent that is added with the protein. The (Reff) is determined by the equation <img alt="Equation 2" src="/files/ftp_upload/59867/59867equ02.jpg" /> Dtotal is the total detergent concentration and Dwater is the monomeric detergent concentration (0.18&#160;mM for TX-100 and Anapoe X-100 in the presence of detergent)4,11.</li>
		</ol>
		</li>
		<li>Mix the solutions together in a 0.5 mL tube in the following order: buffer, detergent, protein, and liposomes. Add the liposomes rapidly and vortex immediately for 5 s to homogenize the mixture.</li>
		<li>Incubate the reaction in a nutator for 1 h at 4 &#176;C.</li>
		<li>Make 0.2 g/mL nonpolar polystyrene adsorbent bead &#8220;slurry&#8221; in water. 
		NOTE: Make the polystyrene adsorbent bead &#8220;slurry&#8221; during previous the 1 h incubation in step 2.2.3.</li>
		<li>Weigh out 0.2 g of polystyrene adsorbent beads and transfer to a microcentrifuge tube.</li>
		<li>Degas the beads by adding 1 mL of methanol to the tube and mix for 1 min. Degassed beads will sink.</li>
		<li>Aspirate the methanol and add water to the beads. Let the beads mix with water for 5 min, then aspirate the water. Repeat four times, then bring the polystyrene adsorbent bead &#8220;slurry&#8221; to 1 mL volume with water and a final concentration of 0.2 g/mL. 
		NOTE: Beads should still settle to the bottom of the tube. If they do not, degas again.&#160;Use a 21 G or higher needle to aspirate methanol and water from beads.&#160;</li>
		<li>Calculate the amount of polystyrene adsorbent beads needed to absorb all the detergent in each sample. 1 g of polystyrene adsorbent beads absorbs 70 mg of TX-100. To calculate the volume of polystyrene adsorbent bead slurry needed for each reaction, divide the total detergent in the reaction (step 2.2.1.4) by 70 mg, and then by the concentration of the bead slurry (0.2 g/mL (step 2.2.7).</li>
		<li>Transfer calculated amount of polystyrene adsorbent bead slurry to a 0.5 mL tube and aspirate the water. 
		NOTE: Cut the end of a 20&#8211;200 &#181;L tip to transfer the beads, vortex the tube just prior to pipetting to resuspend settled beads.</li>
		<li>Add the samples to the 0.5 mL tube containing polystyrene adsorbent beads and incubate the sample nutating for 1 h at 4 &#176;C.</li>
		<li>Repeat twice leaving old beads behind and transferring the sample to fresh beads.</li>
		<li>Add the sample to a fourth tube with fresh beads and incubate overnight (~15&#8211;18 h) at 4 &#176;C.</li>
	</ol>
	</li>
	<li>In the morning, remove the sample from polystyrene adsorbent beads and pellet insoluble protein aggregates by centrifuging for 10&#160;min at 16,000 x g at 4 &#176;C.</li>
	<li>Recover the supernatant and determine the final lipid concentration by liquid scintillation counting (see step 2.1.9.2). Optionally, protein concentration may be determined by amido black protein assay9. 
	NOTE: For atlastin proteoliposomes, enzymatic assays should be performed with fresh liposomes. Long storage at 4 &#176;C or freezing leads to significant loss in atlastin activity.</li>
</ol>
3. Lipid Mixing Assays
<ol>
	<li>Bring the donor and acceptor proteoliposomes to a concentration of 0.15 mM each in A100 with 10% glycerol, 2 mM &#946;-mercaptoethanol, 1 mM EDTA and 5 mM MgCl2. Each reaction (50 &#956;L) should be added to a well in a flat white 96 well plate suitable for fluorescence readings. Prepare at least 4 reactions including a triplicate, and a no-GTP negative control.</li>
	<li>Place the plate in a preheated plate reader at 37 &#176;C. Measure NBD fluorescence (excitation 460 nm and emission 535 nm) for 5 min every min.</li>
	<li>Induce fusion by adding 5 mM GTP (5 &#956;L of 50 mM GTP).</li>
	<li>Measure NBD fluorescence every minute for 1 h.</li>
	<li>Add 5 &#956;L of 2.5% w/v n-Dodecyl &#946;-D-maltoside to dissolve the proteoliposomes and measure the maximum NBD fluorescence. Read NBD fluorescence for 15 min every min.</li>
</ol>
4. Liposome Floatation on Iohexol Discontinuous Gradient12
<ol>
	<li>(optional) Analyze the efficiency of reconstitution by floatation assays13.</li>
	<li>Prepare an 80% and a 30% w/v iohexol in A100 with 10% glycerol, 2 mM 2-mercaptoethanol, and 1 mM EDTA. Iohexol does not dissolve readily, so this must be prepared a day before by nutating overnight at 4 &#176;C.</li>
	<li>Thoroughly mix 150 &#956;L of 80% iohexol stock with 150 &#956;L of proteoliposomes sample to bring it to a 40% iohexol. Add this to a 5 x 41 mm2 Ultra-clear tube avoiding bubbles.</li>
	<li>Layer 250 &#956;L of 30% iohexol stock slowly on top of the sample to make a middle layer. Avoid any bubbles and disturbing the bottom layer. On top of the middle layer, slowly add 50 &#956;L of A100 with 2 mM 2-mercaptoethanol and 1 mM EDTA.</li>
	<li>Centrifuge the gradient in a swinging bucket rotor at 220,000 x g for 4 h at 4 &#176;C with slow acceleration and no break.</li>
	<li>Harvest the layers of the gradient and analyze by SDS-PAGE and Coomassie stain, quantification may be done by densitometry. Reconstituted protein should float to the top layer, while protein and lipid aggregates will sediment at the bottom or in the middle layer.</li>
</ol>
5. Analysis of Orientation of Reconstituted Protein by Thrombin Proteolysis
NOTE: The atlastin construct reported here has a thrombin cut site between the end of the N-terminal GST tag and the beginning of atlastin. Atlastin in the correct orientation will have this cut site accessible to the protease, while protein in the wrong orientation will be protected by the lipid bilayer.
<ol>
	<li>To assay atlastin proteoliposome orientation, reserve at least 8 &#956;L of fresh reconstituted proteoliposomes.</li>
	<li>Add 8 &#956;L of proteoliposomes and 1 &#956;L of 1 U/&#956;L thrombin. Incubate at 37 &#176;C for 1 h.</li>
	<li>Quench the protease with 1 &#956;L of 5 mg/mL of EDTA-free protease inhibitor cocktail and incubate for 30 min at 37 &#176;C.</li>
	<li>Analyze the sample by SDS-PAGE and Coomassie stain. Proportion of cleaved and uncleaved protein may be determined by densitometry.</li>
</ol>

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The authors have nothing to disclose.

The methods here delineate an efficient method for purifying, reconstituting, and measuring fusion activity of recombinant atlastin. To ensure high yields of functional atlastin some critical steps must be considered. Expression of atlastin must be done at low temperatures (16 &#176;C) to avoid aggregation and one should aim for a final concentration between 0.4&#8211;1.5 mg/mL. Very dilute protein will not be reconstituted optimally at a 1:400 protein to lipid ratio. Reconstitution efficiency can be optionally analyzed ...

Membrane fusion is a critical process in many biological reactions. Under biological conditions, membrane fusion is not spontaneous and requires specialized fusion proteins to catalyze such reactions1. ER homotypic membrane fusion is mediated in animals by the dynamin related GTPase atlastin2. Atlastin&#8217;s role in homotypic fusion is fundamental for three-way junctions in peripheral ER, which constitutes a large interconnected network of tubules that extend throughout the cell. Atlastins have a conserved domain morphology consisting of a large GTPase, a three helix bundle middle domain, a hydrophobic membrane anchor, and a short cytoplasmic C-terminal tail3. In vitro studies with recombinant Drosophila atlastin have shown that when reconstituted to liposomes, it maintains its fusogenic properties. Other atlastins, including human homologs have not been able to recapitulate fusion in vitro. We describe here a methodology for purifying a GST and poly-histidine tagged recombinant Drosophila atlastin, reconstituting it to liposomes, and assaying fusion.
Studying membrane fusion in vitro presents a challenge as fusogenic proteins usually have a membrane anchor. In order to study them, it is necessary to reconstitute them into model lipid bilayers. Large unilamellar vesicles (LUV) are a useful tool to study lipid protein interactions. We present here a system to make LUVs of different lipid compositions for protein reconstitution and fusion assays. Reconstitution of integral proteins into LUVs can be achieved by a variety of methods including, organic solvent-mediated reconstitution, mechanical mechanisms, or detergent assisted reconstitution4. We present here a method for reconstituting Drosophila atlastin into preformed liposomes by detergent removal. Advantages of this reconstitution method include high reconstitution yields and proper orientation of atlastin in the lipid bilayer. Additionally, through this method, the protein is not dried or exposed to organic solvents thereby maintaining structure and function. Among its disadvantages, the presence of detergents may not be ideal for all proteins and the final proteoliposomes may have some incorporated detergent in the lipid bilayer. Further dialysis may be used to eliminate more of the detergent. However, dialysis may take a long time and can therefore lead to loss of protein activity.
Assessing atlastin&#8217;s fusion activity can be determined by lipid mixing assays as previously described2. Here, we delineate a method for measuring atlastin mediated fusion through N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)/Lissamine rhodamine-B sulfonyl (rhodamine) labeled lipids. This assay requires fusion of donor (labeled) proteoliposomes and acceptor (unlabeled) proteoliposomes. A FRET release can be measured during the reaction as the dilution&#160;of a donor&#8211;acceptor pair from &#8220;labeled&#8221; liposomes to &#8220;unlabeled&#8221; liposomes as a result of lipid mixing during membrane fusion (Figure 1)5. While this assay serves as a proxy for membrane fusion, it is limited in distinguishing between membrane fusion and hemifusion, a state where only the outer leaflets mix. To address this issue, an alternative is outer leaflet quenching of NBD by dithionite. Following the same methodology as NBD/rhodamine lipid mixing assays, upon quenching the outer leaflet any NBD FRET release by fusion will be due to inner leaflet mixing8.
Alternative fusion assays by inner aqueous content mixing address full fusion only5. Examples of this are terbium (Tb)/dipicolinic acid (DPA) assays and aminonaphthalene trisulfonic acid (ANTS)/p-xylene bis(pyridinium) bromide (DPX) assays. In Tb/DPA assays, a pool of liposomes with encapsulated Tb are mixed and fused with liposomes with encapsulated DPA; upon fusion, fluorescence is increased via internal energy transfer from DPA to Tb within the [Tb(DPA)3]3- chelation complex6. In contrast, for ANTS/DPX assays, ANTS fluorescence is quenched by DPX7. While these systems address inner content mixing, more in-depth preparation of liposomes is required for removal of non-encapsulated reagents, as well as unintended interaction of the fluorophores.

<table><tbody><tr><td>10 mL poly-prep chromatography columns</td><td>Biored</td><td>731-1550</td><td></td></tr><tr><td>10 mm x 75 mm Flint glass tubes</td><td>VWR</td><td>608225-402</td><td></td></tr><tr><td>47 mm diameter, 0.45 &amp;mu;m pore whatman sterile membrane filters</td><td>Whatman</td><td>7141 104</td><td></td></tr><tr><td>96 well white plate</td><td>NUNC</td><td>437796</td><td></td></tr><tr><td>Anapoe X-100</td><td>Anatrace</td><td>9002-931-1</td><td></td></tr><tr><td>Cell disrupter</td><td>Avestin</td><td>Avestin Emulsiflex C3</td><td></td></tr><tr><td>DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt))</td><td>Avanti</td><td>840035P-10mg</td><td>DOPS</td></tr><tr><td>EDTA</td><td>Research organics inc.</td><td>6381-92-6</td><td>Ethylenediaminetetraacetic acid</td></tr><tr><td>EDTA-free protease inhibitor cocktail</td><td>Roche</td><td>11873580001</td><td>Complete protease inhibitor</td></tr><tr><td>Extruder</td><td>Sigma Aldrich</td><td>Z373400&amp;nbsp;</td><td>Liposofast Basic Extruder</td></tr><tr><td>GE Akta Prime liquid chromatography system</td><td>GE Pharmacia</td><td>8149-30-0006</td><td></td></tr><tr><td>Glutathione agarose beads</td><td>Sigma aldrich</td><td>G4510-50ml</td><td></td></tr><tr><td>Glycerol</td><td>EMD</td><td>GX0185-5</td><td></td></tr><tr><td>GTP</td><td>Sigma Aldrich</td><td>36051-31-7</td><td>Guanosine 5&#039; triphosphate sodium salt hydrate</td></tr><tr><td>HEPES, acid free</td><td>Omnipur</td><td>5330</td><td></td></tr><tr><td>Imidazole</td><td>fluka</td><td>5670</td><td></td></tr><tr><td>Immobilized metal affinity chromatography (IMAC) resin column</td><td>GE Healthcare</td><td>17040801</td><td>1 mL HiTrap Chelating HP immobilized metal affinity chromatography columns</td></tr><tr><td>Iohexol</td><td>Accurate chemical and scientific corporation</td><td>AN 7050 BLK</td><td>Accudenz/Nycodenz</td></tr><tr><td>IPTG</td><td>Research products international corp.</td><td>I56000-100.0</td><td>IPTG, dioxane free</td></tr><tr><td>L-Glutathione reduced</td><td>Sigma-Aldrich</td><td>G4251-5g</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>7791-18-6</td><td></td></tr><tr><td>Methanol</td><td>Omnisolv</td><td>MX0488-1</td><td></td></tr><tr><td>NBD-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt))</td><td>Avanti</td><td>236495</td><td>NBD-DPPE</td></tr><tr><td>n-Dodecyl &amp;beta;-D-maltoside</td><td>Chem-Impex International</td><td>21950</td><td></td></tr><tr><td>Nonpolar polystyrene adsorbent beads</td><td>BioRad</td><td>152-3920</td><td>SM2 Biobeads</td></tr><tr><td>Nuclepore track-etch polycarbonate 19 mm 0.1 &amp;mu;m pore membrane</td><td>Whatman</td><td>800309</td><td></td></tr><tr><td>Optima LE80K Ultra centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Phosphatidylcholine, L-&amp;alpha;-dipalmitoyl [choline methyl-3H]</td><td>ARC</td><td>ART0284</td><td>Titriated lipids</td></tr><tr><td>Plate reader</td><td>TECAN</td><td>TECAN infinite M200 plate reader</td><td></td></tr><tr><td>POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)</td><td>Avanti</td><td>850457C-25mg</td><td>POPC</td></tr><tr><td>Potassium chloride</td><td>MP</td><td>151944</td><td></td></tr><tr><td>Rh-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt))</td><td>Avanti</td><td>236495</td><td>Rh-DPPE</td></tr><tr><td>Scintillation Cocktail</td><td>National Diagnostics</td><td>LS-272</td><td>Ecoscint XR Scintillation solution for aqueous or non-aqueous samples</td></tr><tr><td>Scintillation vials</td><td>Beckman</td><td>592928</td><td>Fast turn cap Mini Poly-Q Vial</td></tr><tr><td>Thrombin</td><td>Sigma</td><td>T1063-1kU</td><td>Thrombin from human plasma</td></tr><tr><td>Triton X-100</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Ultra-clear centrifuge tubes 5 mm x 41 mm</td><td>Beckman</td><td>344090</td><td></td></tr><tr><td>Vortex 9 to 13 mm Tube Holder</td><td>VWR</td><td>58816-138</td><td>Insert for vortexing flint glass tubes</td></tr><tr><td>Vortex Insert Retainer</td><td>VWR</td><td>58816-132</td><td>Retainer needed for vortex tube holder</td></tr><tr><td>Vortexer</td><td>VWR</td><td>2.235074</td><td>Vortex Genie 2 model G560</td></tr><tr><td>&amp;beta;-mercaptoethanol molecular biology grade</td><td>Calbiochem</td><td>444203</td><td></td></tr></tbody></table>

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.

The efficiency of atlastin reconstitution is presented in Figure 2. Reconstituted proteoliposomes were floated in an iohexol discontinuous gradient. Unincorporated protein was sedimented in the bottom layer (B) or in the middle layer (M). Reconstituted protein would float to the top layer (T). Samples of the gradient were harvested and analyzed by SDS-PAGE and Coomassie staining. The quantification of the gel by densitometry shows a very high efficiency of reconstitution with negligible lose...

Watch this Scientific Journal Video about Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays at JoVE.com

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.

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 ...

detergent-assisted-reconstitution-recombinant-drosophila-atlastin

Research

JoVE Journal

Biochemistry

7.7K Views.  Rice University. 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.

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

A Fluorescence-based Assay of Phospholipid Scramblase Activity

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and biochemically verified was opsin, the apoprotein of the photoreceptor rhodopsin. Rhodopsin is a G protein-coupled receptor localized in rod photoreceptor disc membranes of the retina where it is responsible for the perception of light. Rhodopsin&#39;s scramblase activity does not depend on its ligand 11-cis-retinal, i.e., the apoprotein opsin is also active as a scramblase. Although constitutive and regulated phospholipid scrambling play an important role in cell physiology, only a few phospholipid scramblases have been identified so far besides opsin. Here we describe a fluorescence-based assay of opsin&#39;s scramblase activity. Opsin is reconstituted into large unilamellar liposomes composed of phosphatidylcholine, phosphatidylglycerol and a trace quantity of fluorescent NBD-labeled PC (1-palmitoyl-2-{6-[7-nitro-2-1,3-benzoxadiazole-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine). Scramblase activity is determined by measuring the extent to which NBD-PC molecules located in the inner leaflet of the vesicle are able to access the outer leaflet where their fluorescence is chemically eliminated by a reducing agent that cannot cross the membrane. The methods we describe have general applicability and can be used to identify and characterize scramblase activities of other membrane proteins.

We describe a fluorescence-based assay to measure phospholipid scrambling in large unilamellar liposomes reconstituted with opsin.

Scramblases translocate phospholipids across the membrane bilayer bidirectionally in an ATP-independent manner. The first scramblase to be identified and ...

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.

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.

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

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 ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity results in the hydrolysis of the membrane lipid phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] leading ultimately to the activation of transient receptor potential (TRP) and TRP like (TRPL) channels. The activity of PLC&#946; also leads subsequently to the generation of many lipid species several of which have been proposed to play a role in TRP and TRPL activation. In addition, several classes of lipids have been proposed to play key roles in organizing the cell biology of photoreceptors to optimize signaling reactions for optimal sensory transduction. Historically, these discoveries have been driven by the ability to isolate Drosophila mutants for enzymes that control the levels of specific lipids and perform analysis of photoreceptor physiology in these mutants. More recently, powerful mass spectrometry methods for isolation and quantitative analysis of lipids with high sensitivity and specificity have been developed. These are particularly suited for use in Drosophila where lipid analysis is now possible from photoreceptors without the need for radionuclide labeling. In this article, the conceptual and practical considerations in the use of lipid mass spectrometry for the robust, sensitive, and accurate quantitative assessment of various signaling lipids in Drosophila photoreceptors are covered. Along with existing methods in molecular genetics and physiological analysis such lipid is likely to enhance the power of photoreceptors as a model system for discoveries in biology.

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

The manuscript presents versatile, robust, and sensitive mass spectrometry protocols to identify and quantify several classes of lipids from Drosophila photoreceptors.

The activation of phospholipase C&#946; (PLC&#946;) is an essential step during sensory transduction in Drosophila photoreceptors. PLC&#946; activity ...

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

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 ...

Bioengineering

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

Biology

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to assemble. Therefore, to identify these functional protein complexes, it is important to stabilize them in vivo before cell lysis and subsequent purification. Here we describe a method used to isolate large bona fide protein complexes from Drosophila embryos. This method is based on embryo permeabilization and stabilization of the complexes inside the embryos by in vivo crosslinking using a low concentration of formaldehyde, which can easily cross the cell membrane. Subsequently, the protein complex of interest is immunopurified followed by gel purification and analyzed by mass spectrometry. We illustrate this method using purification of a Tudor protein complex, which is essential for germline development. Tudor is a large protein, which contains multiple Tudor domains - small modules that interact with methylated arginines or lysines of target proteins. This method can be adapted for isolation of native protein complexes from different organisms and tissues.

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Multi-component protein complexes play crucial roles during cellular function and development. Here we describe a method used to isolate native protein complexes from Drosophila embryos after in vivo crosslinking followed by purification of the crosslinked complexes for subsequent structure-function analysis.

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

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

Summary

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Chapters in this video

A Fluorescence-based Assay of Phospholipid Scramblase Activity

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.

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

Analysis of Lipid Signaling in Drosophila Photoreceptors using Mass Spectrometry

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

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos