In this particle, we introduce an in vitro reconstitution platform that mimics the lipid environment of mitochondria inner membrane. This platform can be used to investigate molecular mechanisms of mitochondria inner membrane fusion. The main advantage of this technique is that it allows quantitative investigation of integral membrane proteins and membrane-associated proteins in a near native environment.
To begin, mix solutions A and B according to manuscript directions, then generate the lipid mixture by adding the calculated volume of storage solution into amber vials with a glass syringe. Match the final volume by adding extra chloroform into the vials. Bake microscope coverglass slides at 520 degrees Celsius for 30 minutes.
After baking, cool them to room temperature. Add approximately 10 grams of sodium hydroxide to 500 milliliters of methanol while stirring. Stir for two hours, continuing to add sodium hydroxide to the solution until precipitate start to show.
Clean the glass slides in 10%sodium dodecyl sulfate solution, methanol saturated with sodium hydroxide, and 50 millimolar hydrochloric acid, sequentially bath sonicating the slides under each condition for 30 minutes. Clean the glass slides in ultrapure water for 10 minutes between each condition. Store the cleaned coverglass sealed in hydrochloric acid solution for up to two weeks to ensure good bi-layer quality.
Clean the polytetrafluoroethylene trough of the Langmuir-Blodgett dipping system using chloroform and ultrapure water until no wetting is observed. Spray chloroform on the trough surface and wipe it thoroughly three times with cellulose wipes, then rinse it three times with ultrapure water and remove the water via suctioning. When finished, cover the surface of the trough with clean ultrapure water.
Take two pieces of surface treated coverglass from the cleaning solution and rinse them with ultrapure water for approximately 30 seconds. Place the coverglass in a back-to-back manner using the substrate clamp to hold the glass slides. Immerse the glass slide underneath the water surface by manually clicking dipper down on the Langmuir control system.
Zero the film balance and carefully spread solution B drop by drop at the air water interface. Make sure lipids are only spreading at the air water interface with no chloroform and lipid droplets sinking to the bottom of the polytetrafluoroethylene surface, which will create a lipid channel and prevent the monolayer formation. Stop adding lipids when the film balance readout is around 15 to 20 millinewton per meter.
Wait for 10 to 15 minutes, then initiate the barrier controller to alter the surface area by clicking start experiment. Wait until the film balance readout increases to 37 millinewton per meter and keep the pressure for approximately 20 to 30 minutes. Raise the coverglass at the speed of 22 millimeters per minute while maintaining the surface tension at 37 millinewton per meter.
A lipid monolayer with polymer tethering will be transferred from the air water interface to the surface of coverglass through the Blodgett dipping process, forming the bottom leaflet of the lipid bi-layer. Clean the air water interface by suction and rinse the trough with ultrapure water. After cleaning a one-welled glass slide with chloroform, ethanol, and ultrapure water, set it on the trough underneath the water layer.
Make sure that the well is facing up toward the air water interface and pour fresh ultrapure water until the glass slide is fully covered, then immerse the glass slide under the water surface as previously described. Hold the coverglass with the lipid monolayer using a silicon suction cup and gently push the lipid monolayer to the air water interface. Hold the cover glass for two to three seconds at the interface, then push it against the slide.
Take the slide out with the coverglass. Take the coverglass and the bi-layer to an epifluorescence microscope and image the lipid bi-layer according to text manuscript directions. Prepare a crystallization dish containing ultrapure water and place a clean microscope image ring underneath the dish.
Immerse this slide and coverglass that contain the lipid bi-layer underneath the water. Gently separate the slide and coverglass, holding the coverglass slide from the bottom and transfer the coverglass into the image ring. Replace the ultrapure water in the image ring with bis-tris sodium chloride buffer, making sure that the lipid bi-layer is not exposed to any air bubbles.
Add 1.1 nanomolar n-octyl-beta-glucopyranoside to the lipid bi-layer, then immediately add the mixture of 1.2 nanomolar DDM and 1.3 picomoles of purified L-OPA1 into the image ring. Incubate the sample on a bench-top shaker at low speed for two hours. Distribute 30 milligrams SM-2 resin beads into three milliliters of bis-tris buffer and shake.
Use a plastic pipette to add 5 to 10 microliters of the SM-2 resin beads to the image ring and incubate it for 10 minutes, then rinse the resin beads. The final volume of the buffer in the image ring should be 1.5 milliliters. Prepare one milligram of lipid mixture A in chloroform solution, then evaporate chloroform under nitrogen flow for 20 minutes.
Keep the mixture under vacuum overnight to form a lipid film. Prepare 50 millimolar calcium containing buffer by dissolving 15.56 grams of calcein in 50 milliliters of 1.5 molar sodium hydroxide solution. Stir the mixture at room temperature until calcein is completely dissolved.
Add 12.5 millimolar bis-tris and ultrapure water for a final volume of 500 milliliters and adjust the pH to 7.5. Dispense lipid film in calcein containing buffer, then fully hydrate the lipid by heating the suspension at 65 degrees Celsius for 20 minutes. Form 200 nanometer liposomes via extrusion with a polycarbonate membrane.
Add two micrograms of L-OPA1 in 0.5 micromolar DDM, 2.2 milligrams liposome and incubate the solution at four degrees Celsius for 1.5 hours. Remove the surfactant by dialysis with a 3.5 kilodalton dialysis cassette against 250 milliliters of 25 millimolar bis-tris, 150 millimolar sodium chloride, and 50 millimolar calcium buffer at four degrees Celsius overnight, changing the buffer twice. Remove extra calcein using a PD10 desalting column, then proceed with imaging and data analysis as described in the text manuscript.
Epifluorescence microscopy images of a lipid bi-layer and its lipid fluidity are shown here. The lipid distribution is shown before and after photo bleaching and the homogeneity is shown before and after reconstitution. L-OPA1 reconstituted and lipid bi-layer was validated by fluorescence correlation spectroscopy or FCS.
The FCS curves indicated that 75%of L-OPA1 was reconstituted into the lipid bi-layer suggesting that L-OPA1 freely diffuses in the polymer tethered lipid bi-layer with the potential to self-assemble into functional complexes. Fluorescence step bleaching indicated that an average of two to three copies of L-OPA1 were reconstituted in a given liposome. The size distribution of L-OPA1 reconstituted proteoliposomes was tested after reconstitution using DLS and verified with FCS.
Membrane tethering was monitored by observing the signal of Texas Red on the surface of the lipid bi-layer using TIRF microscopy. Membrane lipid de-mixing or hemifusion was monitored through Texas Red as the liposome marker diffused into the lipid bi-layer. Calcein de-quenching helped distinguish full fusion pore formation from only lipid de-mixing, allowing comparison between conditions where particles stall at hemifusion and particles that proceed to full fusion.
Membrane tethering was indicated by a stable lipid signal from liposomes and effusion signal featured no de-quenching in the calcein signal, but a rapid decay of the Texas Red signal indicated diffusion of the dye into the lipid bi-layer. Full fusion featured both lipid decay and content release. When attempting this particle, remember to thoroughly clean both the coverglass and Langmuir trough to ensure bi-layers of high quality are fabricated.
Good air quality is also critical to prevent defects in the bi-layer. This technique paves the way for us to explore new questions in mitochondria membrane dynamics and organization. Exciting future experiment includes exploring the influence of bi-layer assymetry, membrane potential, and disease-related mutants in mitochondria inner membrane dynamics.
