Bioengineering
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In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles
Chapters
Summary August 25th, 2022
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.
Transcript
This measure builds a minimum model of the cell devoid of complex biochemical regulations. The technique used can generate high yield of liposomes and have high encapsulation efficiencies for cytoskeleton proteins. This measure can be applied to encapsulation of variety of proteins and large objects such as microparticles and self preparing micro swimmers.
An individual is struggling increasing the liposome yield for the first several attempts. It is recommended to regularly discussion section of the manuscript for more details to increase the yield. To begin, prepare the aqueous inner non polymerization buffer in a total volume of five milliliter by mixing 0.1 millimolar CACL two, 10 millimolar HEPES, one millimolar DTT, 0.5 millimolar DAPCO, 320 millimolar sucrose, and 0.2 millimolar ATP.
Prepare the protein mix by adding proteins to the inner non polymerization buffer at four degrees Celsius. To form an actin layer, add 100 nanomolar gellcalin, four micromolar coughalin and 2.2 micromolar VCA HEZ to the protein mix. As a control experiment, replace protein mix with 100 micrograms per milliliter fluorescent dye.
Prepare the aqueous inner polymerization buffer in a total volume of five milliliters by mixing 100 millimolar potassium chloride. Four millimolar magnesium chloride, 10 millimolar HEPES, one millimolar DTT, 0.5 millimolar DABCO, 10 millimolar ATP and 80 millimolar sucrose. Prepare the final buffer by mixing inner non polymerization buffer and inner polymerization buffer at a volume ratio of one to one, to yield the inner aqueous solution in a total volume of 30 microliters that will be encapsulated within the liposome.
Prepare the aqueous outside buffer in a total volume of 150 microliters by mixing 10 millimolar HEPES, 50 millimolar potassium chloride, two millimolar magnesium chloride, 0.2 millimolar calcium chloride, two millimolar ATP, one millimolar DTT, 0.5 millimolar DAPCO, 212 millimolar glucose and 0.1 milligrams per milliliter beta casing. To prepare the lipid oil mixture, begin by adding 100 microliters of 25 milligrams per milliliters EGGPC into a glass vial. Evaporate chloroform with argon gas leaving a dry solid lipid film at the bottom of the vial.
To form an actin layer, add nickel lipid at a 10 to one ratio of EGGPC to nickel lipid and mix, before the evaporation of chloroform. Add two milliliters of mineral oil. Evaporate the chloroform and sonicate the lipid oil mixture at room temperature in a bath for one hour, to resuspend the lipids.
Prepare a final buffer in oil emulsion for preparing monolayer lipid droplets containing the protein of interest. Start by taking 100 microliters of the lipid oil mixture in a plastic tube and add 10 microliters of final buffer to the lipid oil mixture. Ensure that final buffer is in one droplet.
Using a glass syringe draw a small amount of lipid oil mixture first and then the final buffer droplet by placing the tip of the syringe at the periphery of the droplet to break it up into tiny droplets. Gently aspirate up and down multiple times until a cloudy emulsion is formed. Put 30 microliters of outside buffer in a separate plastic tube.
Place 30 microliters of the lipid oil mixture on top of outside buffer and let it sit for approximately 10 minutes to develop a lipid monolayer at the interface. To prepare liposomes, carefully add 50 microliters of the final buffer oil emulsion, to the top oil phase of the tube. Centrifuge the plastic tube at 100 times G for 15 minutes at four degrees Celsius.
Vary time and centrifugation speed, to optimize for liposome formation. Carefully remove the oil phase by pipette aspirate extra volume if needed. Ensure not to put the pipette tip at the side of the tube to avoid creating a meniscus of oil on top of the liposome phase.
With a new pipette, cut the tip of the pipette. Slowly stick the pipette into the remaining bottom phase and aspirate the aqueous volume to collect liposomes. Pour 100 microliters of outside buffer into the well of an incubation chamber.
Gently deposit the collected liposomes into the outside buffer and then place another cover slip on top of the chamber. Observe liposomes with a confocal microscope using a 63 x oil immersion objective. Use 488, 647 and 561 nanometers laser lines.
To observe fluorescently labeled lipid, encapsulated fluorescent dye and encapsulated fluorescently labeled actin respectively. Capture the frames of interest and save the images in TIF format. Process and analyze images in image J.Start by opening the TIF file, using image J software.
Go to image, then click adjust. Followed by brightness contrast, adjust the brightness and contrast of the image to the desired scale by adjusting the minimum and maximum settings and the brightness and contrast sliders. Click on the rectangle selection tool in the toolbar and select the region of interest.
Go to image, then click crop to crop the ROI. Go to analyze, then click set scale. In the pop up window, enter 1.0 in the pixel aspect ratio field and set micrometer as the unit of length.
In the distance in pixels field, enter the image width in pixels. In the known distance field, enter the real image width in microns. To measure the size of the liposome, using the oval selection tool in the toolbar, draw a circle along the edge of the liposome.
Go to analyze, then click measure, to measure the area of the circle, from which the diameter of the liposome can be calculated. Successful liposome generation is verified through the visualization of the thin circular ring which is the green fluorescent lipid bilayer. Under a 488 nanometer laser using confocal microscopy to confirm encapsulation of the fluorescent dye.
The internal environment of the liposomes should be uniformly fluorescent under a 647 nanometer laser. The formation of liposomes could be visualized by the thin green circular rings. The reconstituted affect in networks inside liposomes are heterogeneous, manifesting as branched network structures of actin filaments.
The branch architecture was triggered by the introduction of the ARP two three complex, which simultaneously controls the nucleation and branching of actin filaments, along with VCA HEZ. A thin, but densely branched actin layer is created at the inner leaflet of the bilayer of liposomes which can be visualized as a fluorescent shall. The lipid audio final buffer mixture must be generally pumped back and forth through a glass syringe, to avoid introducing air bubbles.
The mixture must be whitish after process. This technique paved the way for building a minimal model of the cell, devoid of complex biochemical regulations. With this cell mimicking system researchers can explore the mechanical and dynamic properties of cytoskeleton proteins, nucleator and the molecular motor.
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