Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

In recent years, encapsulation within cell-sized lipid bilayer vesicles has proven to be a useful method for in vitro reconstitution experiments. The method we present will greatly help with cytoskeleton reconstitution in synthetic cell research. With this method, we can generate heterogeneously sized giant unilamellar vesicles with a high yield.

The vesicle generation time is significantly decreased compared to the conventional cDICE technique. We can encapsulate cell-free transcription-translation systems isolated from complex, simultaneously occurring cellular processes to examine molecular pathways. This method can be utilized to assemble minimal protocells towards creating functional synthetic cells.

Start preparing an oil-lipid mixture by transferring dioleoyl-phosphocholine, cholesterol, and rhodamine PE to a 15-milliliter glass vial. Then, add 0.5 milliliters of chloroform in the vial. Pipette 7.2 milliliters of silicone oil and 1.8 milliliters of mineral oil in a separate 15-milliliter tube and mix the oils by vortexing at a speed of 3, 200 rotations per minute for 10 seconds.

Then, add the oil mixture to the vial containing the lipid and chloroform mixture and vortex the mixture at the speed of 3, 200 rotations per minute for 10 to 15 seconds until the resulting lipids-in-oil mix is slightly cloudy, as the lipids are not fully dissolved in the oil. Next, sonicate the lipid in oil dispersion in a bath sonicator with ultrasonic power of 80 watts and an operating frequency of 40 kilohertz for 30 minutes at room temperature. If not used immediately, store the lipid-oil mixture at four degrees Celsius for 24 hours.

To generate the vesicles, use the assembly with a 3D-printed shaft made from black resin mounted on the benchtop stir plate at the speed of 1, 200 rotations per minute. Then, mount the 3D-printed continuous droplet interface crossing encapsulation, or cDICE chamber, made from the clear resin on the black resin shaft. Prepare 1-to 10-micromolar actin solution in globular actin buffer, or G buffer, including 10%ATO 488 actin.

To begin the actin polymerization, add filamentous actin polymerization buffer, or F buffer, in actin solution on ice and then keep the solutions on ice to slow down actin polymerization before adding a crosslinker. To prepare the actin binding proteins, or ABPs, from 1.75 milligrams per milliliter of fascin stock, aliquot 1.57 microliters of fascin in a separate microtube. Next, add 7.5%of density gradient medium into the actin solution to create a density gradient between the outer and the inner aqueous phase and facilitate giant unilamellar vesicles or GUVs, sedimentation.

Then, dispense 700 microliters of 200-millimolar glucose as the outer solution into the chamber. Add enough lipid-oil mixture into the chamber until 60%to 80%of the chamber is filled. An interface will be formed between the lipid-oil mixture and the outer solution.

Prepare an actin-ABP mixture by transferring ABPs into the actin solution. Then, use a regular 100-to 1, 000-microliter pipette to transfer the 700 microliters of the lipid-oil mixture into the actin-ABP mixture. Pipette up and down eight times to generate cell-sized lipid monolayer emulsion droplets with a diameter of 7 to 100 microns.

Use the same 100-to 1, 000-microliter pipette to dispense the entire emulsion into the rotating chamber. The droplets will acquire a second leaflet of lipids by crossing the lipid monolayer at the oil-outer solution interface, thereby forming GUVs. When done, remove the chamber from the stir plate and discard most of the lipid-oil mixture by tilting the chamber in the waste container.

By holding the chamber with its lid facing inside, open the chamber lid and slightly tilt the chamber inwards. The interface between the outer solution containing GUVs and the lipid-oil mixture will be visible from the chamber opening where the lid is located. Use a pipette to collect enough outer solution containing GUVs and dispense 50 to 300 microliters of the outer solution into a 96-well plate to obtain an appropriate density of GUVs.

For imaging the GUVs, set up the 96-well plate on the stage of an inverted microscope equipped with an oil immersion 60X objective lens. Open an image sequence of interest in an image processing software, ImageJ Fiji, and identify the image with the highest intensity. Hold Control Shift C to open the brightness and contrast window and click on the reset tab.

In the ImageJ Fiji menu, go to the Analyze and Set Scale tabs to enter the known physical distance and unit for each image pixel. Once done, go to the Image, Stacks, and then 3D Project buttons to reconstruct a 3D image from the Z stack. Set the Projection method as Brightness Point, Slice spacing as 0.5 micrometers, and then check mark the Interpolate box.

Use the default options for the rest of the settings and capture images. The representative image shows a confocal image of rhodamine PE-labeled GUVs with encapsulated fascin-actin bundles. Transferring the actin binding proteins into the actin solution, then adding lipid-oil mixture and generation of lipid monolayer droplets should take place in a few seconds to avoid the formation of actin networks before the encapsulation.

We have used this modified cDICE technique to examine the roles of different actin net cross-linkers in organizing actin networks. We expect this method will help other researchers to explore the regulation of other cytoskeleton proteins.