We acknowledge funding ARO MURI W911NF-14-1-0403 to M.P.M., the National Institutes of Health (NIH) R01 1R01GM126256 to M.P.M., the National Institutes of Health (NIH) U54 CA209992, NIH RO1 GM126256, NIH U54 CA209992, University of Michigan / Genentech, SUBK00016255 and Human Frontiers Science Program (HFSP) grant number RGY0073/2018 to M.P.M. Any opinion, findings, conclusions, or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the ARO, NIH, or HFSP. S.C. acknowledges fruitful discussions with V. Yadav, C. Muresan, and S. Amiri.

1. Preparation of buffers and protein solutions
<ol>
	<li>Prepare the aqueous Inner Non-Polymerization (INP) Buffer in a total volume of 5 mL by mixing 0.1 mM CaCl2, 10 mM HEPES (pH 7.5), 1 mM DTT, 0.5 mM Dabco, 320 mM sucrose, and 0.2 mM ATP.</li>
	<li>Prepare the Protein Mix (PM) by adding proteins to the INP buffer at 4 &#176;C with the following concentrations: 11.2 &#181;M non-fluorescent G-actin, 2.8 &#181;M fluorescently labeled actin, and 0.24 &#181;M Arp2/3 (Table of Materials). To form an F-actin layer, add 100 nM gelsolin, 4 &#181;M cofilin, and 2.2 &#181;M VCA-His to the PM. As a control experiment, replace PM by 100 &#181;g/mL fluorescent dye (Table of Materials).</li>
	<li>Prepare the aqueous Inner Polymerization (IP) Buffer (aqueous) in a total volume of 5 mL by mixing 100 mM KCl, 4 mM MgCl2, 10 mM HEPES (pH 7.5), 1 mM DTT, 0.5 mM Dabco, 10 mM ATP, and 80 mM sucrose.</li>
	<li>Prepare the Final Buffer (FB) by mixing INP buffer (containing PM) and IP buffer at a volume ratio of 1:1 to yield the inner aqueous solution in a total volume of 30 &#181;L that will be encapsulated within the liposome.</li>
	<li>Prepare the aqueous Outside Buffer (OB) in a total volume of 150 &#181;L by mixing 10 mM HEPES (pH 7.5), 50 mM KCl, 2mM MgCl2, 0.2 mM CaCl2, 2mM ATP, 1 mM DTT, 0.5 mM Dabco, 212 mM glucose, and 0.1 mg/mL &#946;-casein. 
	NOTE: The osmolarity of the OB can be adjusted with glucose to ensure the osmotic pressure of the OB is slightly larger than that of the FB (20-60 mOsm). The density of the FB should be slightly higher than the OB.</li>
</ol>
2. Preparation of liposomes based on the inverted emulsion techniques
<ol>
	<li>Prepare the lipid-oil mixture
	<ol>
		<li>Add 100 &#181;L of 25 mg/mL L-&#945;-phosphatidylcholine (non-fluorescent Egg PC, also called EPC, including 1% DHPE) into a glass vial. Evaporate chloroform with argon gas, leaving a dry solid lipid film (2.5 mg) at the bottom of the vial.
		<ol>
			<li>To form an F-actin layer, add 1,2-dioleoyl-sn-glycero-3-{[n(5-amino-1-carboxypentyl) iminodiacetic acid] succinyl} nickel salt (DOGS-NTA-Ni) at a 10:1 ratio of EPC to DOGS-NTA-Ni and mix before the evaporation of chloroform.</li>
		</ol>
		</li>
		<li>Add 2 mL of mineral oil and sonicate the lipid-oil mixture at room temperature in a bath for 1 h to re-suspend the lipids. 
		NOTE: The lipid-oil mixture after sonication can be kept for 1 week at 4 &#176;C. Re-sonication is suggested before use.</li>
	</ol>
	</li>
	<li>Prepare a Final Buffer in oil (FB/oil) emulsion for preparing monolayer lipid droplets containing the protein of interest.
	<ol>
		<li>To 100 &#181;L of lipid-oil mixture taken in a plastic tube, add 10 &#181;L of FB. Ensure that FB is in one droplet.</li>
		<li>Using a glass syringe, draw the lipid-oil-FB mixture and gently aspirate up and down multiple times to emulsify it; draw a small amount of lipid-oil mixture first, and then the FB droplet by placing the tip of the syringe at the periphery of the droplet to break it up into tiny droplets. Repeat the up and down aspiration until a whitish and cloudy emulsion is formed.</li>
	</ol>
	</li>
	<li>Put 30 &#181;L of OB in a separate plastic tube. Place 30 &#181;L of the lipid-oil mixture on top of OB and let it sit for ~10 mins to develop a lipid monolayer at the interface. 
	NOTE: If the lipids are charged or the protein is incorporated, the duration of this step should be extended38.</li>
	<li>Prepare the liposomes
	<ol>
		<li>Carefully add 50 &#181;L of the FB/oil emulsion (step 2.2) to the top oil phase of the tube from step 2.3.</li>
		<li>Centrifuge the plastic tube at 100 x g for 15 mins at 4 &#176;C. Vary time and centrifugation speed to optimize for liposome formation. 
		NOTE: After centrifugation, the top oil phase should be clear, and the bottom OB (containing liposomes) should be slightly cloudy.</li>
		<li>Carefully remove the oil phase away 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.</li>
		<li>With a new pipette, slowly stick the pipette tip into the remaining bottom phase and aspirate the aqueous volume to collect liposomes. 
		NOTE: It is better to lose volume than to incorporate the oil. The inclusion of oil will cause liposomes to rupture, and the internal components will be released into the external buffer. Cut the tip of a pipette to reduce shear.</li>
	</ol>
	</li>
</ol>
3. Microscopy observation
<ol>
	<li>Pour 100 &#181;L of OB into the well of an incubation chamber (4-well plate containing 12 mm round coverslips). Gently deposit the collected liposomes (step 2.4.4) into OB, and then place another coverslip on top of the chamber. 
	NOTE: The OB contains the &#946;-casein to passivate the glass surface and minimize sticking between the liposomes20.</li>
	<li>Observe liposomes with a confocal microscope using a 63x oil-immersion objective. Use 488 nm, 647 nm, and 561 nm 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 TIFF format.</li>
	<li>Process and analyze images in ImageJ/Fiji39. For new users of ImageJ/Fiji, an online tutorial is available (see Table of Materials).
	<ol>
		<li>Open the TIFF file using ImageJ/Fiji software.</li>
		<li>Go to Image &#62; Adjust &#62; 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.</li>
		<li>Click on the Rectangle selection tool in the toolbar and select the region of interest (ROI). Go to Image &#62; Crop to crop the ROI.</li>
		<li>Go to Analyze &#62; Set Scale. In the pop-up window, enter 1.0&#160;in the Pixel Aspect Ratio field and set &#956;m 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.</li>
		<li>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 &#62; Measure to measure the area of the circle, from which the diameter of the liposome can be calculated.</li>
	</ol>
	</li>
</ol>

The authors declare no conflicts of interest.

Several key steps determine the success of a high yield of liposomes during the preparation process. To completely dissolve the lipid film in the oil, the sample must be sonicated until the lipid film at the bottom of the glass vial disappears completely. After the sonication, the lipid-oil mixture must be stored overnight at room temperature under dark conditions for the lipid molecules to disperse further29. The mixture can be stored at 4 &#176;C for up to a week. When preparing an FB/oil emulsion with monolayer lipid droplets containing protein of interest, the lipid-oil-FB mixture must be gently pumped back and forth through a glass syringe to avoid introducing air bubbles. During this process, one big droplet is sheared off at the tip of the glass syringe into many smaller droplets (~5-100 &#181;m in diameter) after several repetitions. The mixture must be whitish after the process30. To check the quality of phospholipids, and whether a lipid monolayer had successfully formed in the peripheral of the FB containing droplet, monolayer lipid droplets incorporating fluorescent dye were examined in the emulsion, as shown in Figure 2A. To ensure the passage of the monolayer lipid droplets through the OB/oil interface during centrifugation, and allow the collected liposomes in OB to settle on the glass surface, the density of FB should be slightly higher than the OB. Therefore, sucrose and glucose were chosen as the main component of the inner (FB) and outer (OB) aqueous solutions, respectively. In the previous work, dextran was used to adjust the density of the inner aqueous solution20, which is not included in this protocol. Moreover, the osmolarity of the OB was adjusted with glucose such that the osmotic pressure of the OB is slightly larger than that of FB (20-60 mOsm) as reported in the previous work20,22. Large osmolarity difference led to the shrinkage (when the osmolarity of the OB is greater than that of FB) or rupture (when the osmolarity of the FB is greater than that of OB) of the liposomes. An osmometer was used to check the osmolarities of the buffers. In the protocol, &#946;-casein was added in the OB to passivate both plastic tube surfaces and glass surfaces of the imaging chamber and minimize sticking between the liposomes20,49. Mineral oil was used as the oil solution (i.e., the lipid solvent). Different lipid solvents have been reported elsewhere, including dodecane50, liquid paraffin30, squalane51, etc. Accordingly, different centrifugation speeds and durations are used for various lipid solvents because of their viscosity differences and the influence they bring on the liposome surface tension. The duration of step 2.3 needs to be extended for charged lipids because, in the monolayer, these charged molecules repel each other, thus requiring more time to diffuse and organize well at the interface between the lipid/oil mixture and OB38. For lipids incorporated with proteins of interest, an extension of this step is also recommended to ensure better monolayer formation52. For the liposome yield of liposomes encapsulating polymerized F-actin network, the number of liposomes per field of view (100 &#181;m x 100 &#181;m) is around 5-10 with an average diameter of 20 &#181;m. A zoomed-out image of a typical microscopy sample is shown in Figure 3B. More zoomed-out confocal images of the liposomes produced through the inverted emulsion method can be found in a recent study52&#160;where various parameters (density difference, centrifugation speed/time, lipid concentration, pH, temperature, etc.) have been optimized for high-yield production of liposomes.
For the reconstitution of the F-actin network, it is essential to choose an appropriate concentration of the adenosine triphosphate (ATP), dithiothreitol (DTT), the associated salts, and the pH condition. An intermediate checkpoint of the reconstituted F-actin network can be done by observing monolayer lipid droplets with encapsulated actin inside. Different forms of encapsulated actin inside the monolayer lipid droplet are shown in Figure 2B. Actin polymerization occurs at &#62;20 mM K+ salt and in &#62;0.2 mM Mg2+ salt with the pH stabilized between 6.5 and 8.5, as reported previously53,54,55. Before the imaging of liposomes, all solutions were kept on ice to prevent early actin polymerization. The samples were imaged at room temperature to trigger actin polymerization. The architecture of the reconstituted F-actin network is predominantly regulated by the activity of the Arp2/3 complex. The Arp 2/3 complex nucleates and branches actin filaments from the side of an existing mother filament to generate dendritic architectures56,57. The Nickel-lipid (DOGS-NTA-Ni) and the VCA-His are critical for the formation of the F-actin layer. VCA-His serves as a nucleation-promoting factor, which is linked to Nickel-lipids at the membrane22,58. A branched F-actin network is then nucleated from VCA-His facilitating the Arp2/3 complex, and as a result, a dense F-actin shell is formed at the inner leaflet of the lipid membrane. The concentration of VCA-His within the liposome determines the thickness of the F-actin layer. The addition of the actin regulatory proteins (cofilin and gelsolin) also promotes the formation of the F-actin layer. The cofilin severs actin filaments to increase the number of filament ends, while the gelsolin binds to the barbed ends of actin filaments to limit their growth. Thereby, in the presence of cofilin and gelsolin, a larger number of filaments can be nucleated by Arp2/3, and then recruited by VCA-His to form the F-actin layer.
Similar papers based on the emulsion transfer method for protein encapsulation have been published in recent years28,30,34. One major difference between the protocol here and the protocols in these papers is the choice of the primary lipid for liposomes. In this work, we use the L-&#945;-phosphatidylcholine (EPC, a mixture of different phosphatidylcholine species containing approximately 60% POPC59) as the main component of the liposome membrane. To form an F-actin layer, a mixture of the EPC and the Nickel-lipid (DOGS-NTA-Ni) was used at a ratio of 10:1. In a previous work, liposomes were spread on polyhistidine coated glass, and&#160;EPC, cholesterol, and Nickel-lipid were mixed at a ratio of 53:37:1020. In comparison, Natsume et al. and Bashirzadeh et al. chose the dioleoyl-phosphocholine (DOPC, a single type of phospholipid) to encapsulate microspheres28&#160;and fascin-actin bundles34, respectively, while Fujii et al. recommended 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, a pure synthetic phospholipid) as the primary lipid to establish a liposomes-based membrane protein synthesis technology30. The choice of the primary lipids affects the physical properties of liposomes. For example, as the degree of unsaturation of the acyl chain increases (i.e., POPC&#60; EPC&#60; DOPC60), the permeability properties of various molecules through the bilayer decrease61. The choice also depends on the lipids of interest being mixed. For instance, the addition of the cholesterol makes EPC liposomes more stable, while it destabilizes the bilayer structure of DOPC/DOPE mixture61.
The similarities between this work and a recent publication from Bashirzadeh et al.34&#160;fall on the same topic of actin encapsulation. Meanwhile, there are clear distinctions. Bashirzadeh et al.&#160;reported a modified cDICE approach utilizing a 3D-printed rotating chamber. Here, we adopt the simple, traditional inverted emulsion transfer method using a syringe and a centrifuge machine. Both of these two methods generate a high yield of liposomes and have high encapsulation efficiencies for large biomolecules24. Besides the difference in the lipid components as mentioned above, the architectures of the encapsulated actin network alter as well. Bashirzadeh et al. encapsulated fascin-actin bundles within the liposome, while in this work, we encapsulated a branched network triggered by the Arp2/3 complex. In addition, the formation of an F-actin layer to create a cortex-biomimicking system in the presence VCA-His has been elaborated here.
The method reported here has two limitations. One is that the size of the liposomes that incorporate in vitro reconstituted actin networks cannot be precisely manipulated (varying from 5 to 50 &#181;m in diameter), and the yield is low compared to the electroformation method. The other limitation of a water-oil-based method is that trace amounts of oil are trapped between the bilayer leaflets24. Although it does not significantly affect the membrane thickness or the biocompatibility of these bio-mimicking liposomes24,35,62,63, the membrane dynamics of the oil-inserted liposomes may be altered62. In this method, the actin nucleator Arp2/3 is incorporated. Future studies may include other cytoskeleton proteins64,65,66,67, nucleators, such as formin68, or molecular motors such as myosin II69.

The actin cytoskeleton plays a fundamental role in constructing the intracellular architecture of the cell by coordinating molecular-level contractility and force generation1,2,3. As a result, it mediates numerous essential cellular activities, including cell deformation4,5, division6, migration7,8, and adhesion9. The in vitro reconstitution of actin networks has gained tremendous attention in recent years10,11,12,13,14,15,16,17. The goal of reconstitution is to build a minimal model of the cell devoid of the complex biochemical regulation that exists within live cells. This offers a controllable environment to probe specific intracellular activities and facilitates the identification and analysis of different components of the actin cytoskeleton18,19. Further, the encapsulation of in vitro actin networks inside phospholipid giant unilamellar vesicles (GUVs, liposomes) provides a confined but deformable space with a semi-permeable boundary. It mimics the physiological and mechanical microenvironment of the actin machinery within the cell9,20,21,22.
Among various methods to prepare liposomes, the lipid film hydration method (also known as the swelling method) is one of the earliest techniques23. The dry lipid film hydrates with the addition of buffers, forming membranous bubbles that eventually become vesicles24. To produce larger vesicles with a higher yield, an improved method advancing from the film hydration method, known as the electroformation method25, applies an AC electric field to efficiently promote the hydration process26. The major limitations of these hydration-based methods for actin encapsulation are that it has low encapsulation efficiency of highly concentrated proteins, and it is only compatible with specific lipid compositions24. The inverted emulsion technique, in comparison, has fewer limitations for lipid components and protein concentrations20,27,28,29. In this method, a mixture of proteins for encapsulation is added to the inner aqueous buffer, which is later emulsified in a lipid-containing mineral oil solution, forming lipid-monolayer droplets. The monolayer lipid droplets then cross through another lipid/oil-water interface through centrifugation to form bilayer lipid vesicles (liposomes). This technique has proven to be one of the most successful strategies for actin encapsulation24,30. Separately, there are some microfluidic device methods, including pulsed jetting31,32, transient membrane ejection33, and the cDICE method34. The similarities between the inverted emulsion method and the microfluidic method are the lipid solvent (oil) that is utilized and the introduction of lipid/oil-water interface for the formation of the outer leaflet of liposomes. By contrast, the generation of liposomes by the microfluidic method requires a set-up of microfluidic devices and is accompanied by oil trapped between the two leaflets of the bilayer, which requires an extra step for oil removal35.
In this manuscript, we used the inverted emulsion technique to prepare liposomes encapsulating a polymerized F-actin network as used previously22. The protein mixture for encapsulation was first placed in a buffer with nonpolymerizing conditions to maintain actin in its globular (G) form. The whole process was carried out at 4 &#176;C to prevent early actin polymerization, which was later triggered by allowing the sample to warm to room temperature. Once at room temperature, the actin polymerizes into its filamentous (F) form. A variety of actin-binding proteins can be added to the inner aqueous buffer solution to study protein functionalities and properties, thus, further providing insights into its interaction with the actin network and membrane surface. This method can also be applied to the encapsulation of various proteins of interest36&#160;and large objects (microparticles, self-propelled microswimmers, etc.) close to the size of the final liposomes28,37.

<table><tbody><tr><td>1,2-dioleoyl-sn-glycero-3-{[n(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DOGS-NTA-Ni)&amp;nbsp;</td><td>Avanti Polar Lipids Inc.</td><td>231615773</td><td>Nickel Lipid</td></tr><tr><td>1,4-Diazabicyclo[2.2.2]octane</td><td>Sigma</td><td>D27802-25G</td><td>DABCO</td></tr><tr><td>Actin protein (&gt;99% pure): rabbit skeletal muscle</td><td>Cytoskeleton, Inc</td><td>AKL99-D</td><td>non-fluorescent G-actin</td></tr><tr><td>Actin protein (rhodamine): rabbit skeletal muscle</td><td>Cytoskeleton, Inc</td><td>AR05</td><td>fluorescently labeled actin</td></tr><tr><td>Adenosine 5&amp;prime;-triphosphate disodium salt hydrate</td><td>Sigma</td><td>A2383-10G</td><td>ATP</td></tr><tr><td>Alexa Fluor 647 dye</td><td>ThermoFisher</td><td></td><td>fluorescent dye</td></tr><tr><td>Andor iQ3</td><td>Andor Technologies</td><td></td><td>control and acquisition software for confocal microscope</td></tr><tr><td>Arp2/3 Protein Complex: Porcine Brain</td><td>Cytoskeleton, Inc</td><td>RP01P-A</td><td>Arp 2/3</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>10035048</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Chamlide Chambers (4-well for 12 mm round coverslip)</td><td>Quorum Technologies</td><td></td><td>incubation chamber</td></tr><tr><td>Cofilin protein: human recombinant</td><td>Cytoskeleton, Inc</td><td>CF01-C</td><td>cofilin</td></tr><tr><td>Confocal Microscope (63&amp;times; oil-immersion objective)</td><td>Andor Technologies</td><td>LEICA DMi8</td><td></td></tr><tr><td>D-(+)-GLUCOSE BIOXTRA</td><td>Sigma</td><td>G7528</td><td>glucose</td></tr><tr><td>Dithiothreitol</td><td>DOT Scientific&amp;nbsp;</td><td>DSD11000-10</td><td>DTT</td></tr><tr><td>Gelsolin Protein: Homo Sapiens Recombinant</td><td>Cytoskeleton, Inc</td><td>HPG6</td><td>gelsolin</td></tr><tr><td>Hamilton 1750 Gastight Syringe, 500 &amp;micro;L, cemented needle, 22 G, 2&quot; conical tip</td><td>Cole-Parmer</td><td>UX-07940-53</td><td>glass syringe</td></tr><tr><td>HEPES</td><td>AmericanBio</td><td>7365-45-9</td><td></td></tr><tr><td>ImageJ/Fiji</td><td></td><td></td><td>https://imagej.net/tutorials/</td></tr><tr><td>L-alpha-Phosphatidylcholine</td><td>Avanti Polar Lipids Inc.</td><td>97281442</td><td>EPC</td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>7786303</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Mineral oil, BioReagent, for molecular biology, light oil</td><td>Sigma</td><td>8042475</td><td>mineral oil</td></tr><tr><td>N-WASP fragment WWA (aa400&amp;ndash;501, VCA-His)</td><td></td><td></td><td>VCA-His is purified using lab protocol. The protocol can be provided upon reasonable requests</td></tr><tr><td>Oregon Green 488 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (Oregon Green 488 DHPE)</td><td>Thermo Fisher</td><td>O12650</td><td>DHPE</td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>7447407</td><td>KCl</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>57-50-1</td><td>sucrose</td></tr><tr><td>&amp;beta;-Casein from bovine milk</td><td>Sigma</td><td>C6905-250MG</td><td></td></tr></tbody></table>

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.

The preparation of liposomes based on the inverted emulsion technique is illustrated graphically and schematically in Figure 1.
First, empty (bare) liposomes (~5-50 &#181;m in diameter) that were composed of phospholipid (EPC) and fluorescent lipid (DHPE) were prepared. A bright, far-red fluorescent dye was encapsulated within bare liposomes as a control experiment. Whether a lipid monolayer has successfully formed in the peripheral of the droplet could be determined by observing monolayer lipid droplets incorporating fluorescent dye in the emulsion, as shown in Figure 2A. Successful liposome generation could be verified through the visualization of the thin circular ring, which is the green-fluorescent lipid bilayer (conjugation of lipids with DHPE), under 488 nm laser using confocal microscopy (Figure 2A, right-most panel). To confirm encapsulation of the fluorescent dye, the internal environment of the liposomes should be uniformly fluorescent under a 647 nm laser (Figure 2A; overlay image) due to the far-red fluorescent dye (Table of Materials).
Different forms of encapsulated actin inside the monolayer lipid droplet are shown in Figure 2B. Images from left to right show globular actin (G-actin), filamentous actin (F-actin), actin forming a thin F-actin layer with the addition of VCA-His to the final buffer and Nickel-lipid to the membrane, and actin forming a thin F-actin layer with the addition of VCA-His, cofilin, and gelsolin to the final buffer and Nickel-lipid to the membrane.
Next, purified G-actin and associated F-actin binding proteins were encapsulated within the liposome. Composed of the same membrane components as above (EPC mixed with DHPE), liposomes here were ~5-50 &#181;m in diameter. The formation of liposomes could be visualized by the thin green circular rings shown in Figure 3A,B. Actin polymerization was triggered by allowing the sample to warm to room temperature. As shown in Figure 3A, the reconstituted F-actin networks (with 20% fluorescently labeled actin) inside liposomes are heterogeneous, manifesting as branched network structures of actin filaments. The branched architecture was triggered by the introduction of the Arp2/3 complex, which simultaneously controls the nucleation and branching of actin filaments, along with VCA-His40,41,42,43,44,45.
Finally, an F-actin cortex-biomimicking system was created. A thin, but densely branched F-actin layer was created at the inner leaflet of the bilayer of liposomes22, which could be visualized as a fluorescent shell (Figure 3C). In this case, additionally, VCA-His, cofilin, and gelsolin were encapsulated into liposomes. Nickel-lipids were required in the component of the lipid membrane. VCA-His, a WASP fragment, carries a histidine tag in interaction with the Nickel-lipids of the lipid membrane. In the meantime, it recruits the Arp2/3 complex20,46,47,48. As a result, actin nucleated by the Arp2/3 complex generates an F-actin layer coated along the inner layer of the membrane in the presence of cofilin and gelsolin.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64026/64026fig01.jpg" /> 
Figure 1: Preparation of liposomes based on the inverted emulsion technique. (A) Liposome preparation consists of two steps. Step one: A droplet of 10 &#181;L of Final Buffer (FB) carrying proteins of interest was added to 100 &#181;L of the phospholipid-oil mixture at a ratio of 1:10 and suspended by gently aspirating up and down with a glass syringe. Solutions containing monolayer lipid droplets became whitish after the back-and-forth aspiration. Step two: In a separate plastic tube, a few microliters of the lipid-oil mixture were placed on top of the same amount of OB and allowed to sit for ~10 min to develop a lipid monolayer at OB/oil interface. The emulsion from step one was poured on top of the oil phase from step two and was centrifuged (100 x g; 15 mins) at 4 &#176;C. After centrifugation, the top oil solution should be clear, and the bottom OB solution (containing liposomes) should be slightly cloudy. (B) Schematics of the liposome preparation from an inverted emulsion. The whitish emulsion on the top contained monolayer lipid droplets that incorporated a branched actin network inside. During centrifugation, monolayer lipid droplets passed through the OB/oil interface to form an outer leaflet such that liposomes were created and accumulated at the bottom of the plastic tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64026/64026fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64026/64026fig02.jpg" /> 
Figure 2: Microscopic images of the monolayer lipid droplet. (A) The monolayer lipid droplet incorporating fluorescent dye in the emulsion. Images from left to right show a monolayer lipid droplet under the DIC channel, fluorescent 640 nm channel, overlay of the DIC and 640 nm channel, and fluorescent 488 nm channel, respectively. (B) Different forms of encapsulated actin inside the monolayer lipid droplet under a fluorescent 561 nm channel. Images from left to right show globular actin (G-actin), filamentous actin (F-actin), actin forming a thin F-actin layer with the addition of VCA-His to the Final Buffer and Nickel-lipid to the membrane, and actin forming a thin F-actin layer with the addition of VCA-His, cofilin, and gelsolin to the Final Buffer and Nickel-lipid to the membrane. Scale bars are 10 &#181;m at 63x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64026/64026fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64026/64026fig03.jpg" /> 
Figure 3: Microscopic images of the encapsulation of actin networks inside liposomes. (A) A liposome encapsulating polymerized branched (Arp 2/3 nucleated) F-actin network. Left to right: fluorescent 488 nm channel (left), fluorescent 561 nm channel (center), overlay (right). (B) A representative result of suspensions of liposomes encapsulating polymerized branched (Arp 2/3 nucleated) F-actin networks. Left to right: fluorescent 488 nm channel (left), fluorescent 561 nm channel (center), and overlay (right). (C) The appearance of the formation of a thin but dense F-actin layer coated along the inner layer of the lipid membrane of a liposome. Top to bottom: fluorescent 488 nm channel (top), fluorescent 561 nm channel (center), overlay (bottom). Scale bars are 10 &#181;m at 63x magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64026/64026fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles at JoVE.com

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Bioengineering

3.3K Views.  Yale University. 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.

Video: In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Biochemistry

In Vitro Polymerization of F-actin on Early Endosomes

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto the endosomal membrane. We have established a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to genetic and biochemical manipulations. Endosomal fractions are prepared by floatation in sucrose gradients from cells expressing the early endosomal protein GFP-RAB5. Cytosolic fractions are prepared from separate batches of cells. Both endosomal and cytosolic fractions can be stored frozen in liquid nitrogen, if needed. In the assay, the endosomal and cytosolic fractions are mixed, and the mixture is incubated at 37 &#176;C under appropriate conditions (e.g., ionic strength, reducing environment). At the desired time, the reaction mixture is fixed, and the F-actin is revealed with phalloidin. Actin nucleation and polymerization are then analyzed by fluorescence microscopy. Here, we report that this assay can be used to investigate the role of factors that are involved either in actin nucleation on the membrane, or in the subsequent elongation, branching, or crosslinking of F-actin filaments.

In Vitro Polymerization of F-actin on Early Endosomes

Early endosome functions depend on F-actin polymerization. Here, we describe a microscopy-based in vitro assay that reconstitutes the nucleation and polymerization of F-actin on early endosomal membranes in test tubes, thus rendering this complex series of reactions amenable to biochemical and genetic manipulations.

Many early endosome functions, particularly cargo protein sorting and membrane deformation, depend on patches of short F-actin filaments nucleated onto ...

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Chemistry

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

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, ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

Immunology and Infection

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular ...

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Biology

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

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Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

In Vitro Polymerization of F-actin on Early Endosomes

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles