The overall goal of this procedure is to visualize HIV-1 Gag binding on giant unilamellar vesicle membranes. This method can help answer key questions in the Bio Assembly field. Such as the roles created by lipids with different osteo chains in membrane binding or virostructural proteins, such as vitro viral Gag proteins.
The main advantage of this technique is that the membrane binding of Gag can be detected visually on giant unilamellar vesicle membranes. directly after mixing result potentially deleterious, lengthy Post Mixing steps. Preparation of HIV-1 Gag is covered in the text protocol, and must be done one day in advance of preparing the GUVs.
Take out lipid stocks from the freezer and equilibrate to room temperature. Meanwhile, set the temperature of the heat block at least five degrees celsius higher than the highest melting temperature of the lipids in use. In this demonstration, the temperature is set to 65 degrees celsius, the highest temperature we have used.
The lipids must be dissolved in inorganic solvents, stored in glass vials with Teflon-lined screw caps, and wrapped in Parafilm. Once thawed, make sure the suspensions are cleared. Calculate the required lipid volumes according to the desired moldar/lipid ratios.
Then, aliquot the calculated volumes into a clean, screw-capped, glass vial using a chloroform-resistant device, such as a Hamilton-type glass syringe. Adjust the total volume with an appropriate organic solvent, such as chloroform. Next, prepare two new ITO coated slides for each electroformation chamber.
Verify that the conductive side of the slide is facing up by checking the resistance using a multimeter, which should be about 200 ohms. Then, gently clean their surfaces using 70%ethanol and lint free wipes. Next, clean the outer surface of the syringe needle by rinsing it with chloroform, and place the syringe on the heat block.
Also place the cleaned ITO-coated slides on the heat block, conductive side up. In a few minutes, the slides will reach temperature. Then, add 40 to 50 microliters of the lipid mixture to one of the slides.
The lipid should be spread out quickly, but as uniformly as possible. Next, add another half volume of the lipid mixture to another glass slide, and immediately spread it out to make a uniform lipid film. Be quick and gentle to avoid damaging the thin ITO coating.
If the opacity of the film appears excessively nonuniform, redo the procedure using a new slide. Next, make a second pair of slides for a different lipid mixture as the first. A uniform layer of lipids is needed for optimal GUV yields, and compositional uniformity of GUVs.
Store the slides in a vacuum dessication chamber for 60 to 90 minutes to remove trace levels of organic solvent. For the next step, set an incubator to the temperature of the heat block used in the previous steps, and warm a stock of 300 millimolar sucrose solution to that temperature. Also, prepare a PDMS gasket by gently wiping it with 70%ethanol, and letting it dry completely.
Once the slides have dried, place them on a clean surface and gently but firmly press the gasket onto one slide so that the gasket forms a watertight seal. Confirm the absence of gas between the PDMS gasket and the slide. Then, completely fill the gasket with the prewarmed sucrose solution.
Do not leave any bubbles. Now, press the other coated slide on top of the gasket aligned with the other slide. Ensure that there are no bubbles, and fasten the chamber using binder clips.
Next, remove the excess sucrose solution by aspiration, and clean the slides using lint free wipes. To proceed, fasten the slide assembly to a copper bar such that the conductive side of each slide is in uniform contact with the bar. Then, connect the copper bar to the function generator output, and place the assembly inside the incubator with the function generator remaining outside.
Once at temperature, program the function generator to apply a 10 Hz sine wave and a potential difference of one volt for 90 minutes. Make certain that the voltage is one volt using a multimeter. After 90 minutes, slowly decrease the frequency to two Hz and continue the electroformation for another 10 minutes.
During this time, harvest the HIV-1 Gag from the in vitro translation reaction set up the day before. Remove unwanted aggregates by spinning down the lysates and carefully transfer the supernatant into another tube. After 10 minutes at two Hz, switch off the function generator, turn off the incubator, and leave the incubator door open until the assembly returns to room temperature.
Once cooled, carefully bring the slide assembly to the bench and gently disassemble the chamber by removing the top slide with forceps. Transfer the GUVs with a clipped 1000 microliter pipette tip to a new tube. Handle this tube gently and use the GUVs within 90 minutes.
Next, prepare an imaging chamber. Attach a PDMS sheet containing a small hole onto a clean cover slip. Press it down gently and firmly for a tight seal.
Then, mix five microliters of the in vitro translation reaction with five microliters of GUVs. After a few minutes, transfer the mixture to the imaging chamber and proceed with imaging. To prevent evaporation, the imaging chamber can be covered with a cover slip.
Using the described protocol, GUVs were prepared composed of POPC, POPS, and cholesterol in a 4.7 to 2.3 to 3 molar ratio. Florescence measurement of Gag YFP signals on a GUV shows that they are not detectable above the background at the GUV surface. However, Gag-YFP protein bound efficiently to the GUVs when brain-PIP2 was included into the lipid mixture.
Gag binding is evident by the increase in florescence intensity on the membrane surface, which, when scanned, appears as a sharp peak. Once mastered, provided that the protein is available in a florescently labeled form, this technique can be done in four to five powers. While attempting this procedure, it's important to remember to be gentle when handling the GUVs, and always use fresh lipids when possible.
This technique has allowed researchers in the field of retrovirus assembly to examine membrane interactions of retroviral Gag proteins in vitro without lengthy Post Mixing procedures, which may alter the latent state of the interactions.
