APL acknowledges support by a Humboldt Research Fellowship for Experienced Researchers and from the National Science Foundation (1939310 and 1817909) and National Institutes of Health (R01 EB030031).

1. Preparation of oil-lipid-mixture
NOTE: The step needs to be performed in a fume hood following all the safety guidelines for handling chloroform.
<ol>
	<li>Take 0.5 mL of chloroform in a 15 mL glass vial. Add 88 &#181;L of 25 mg/mL dioleoyl-phosphocholine (DOPC), 9.3 &#181;L of 50 mg/mL cholesterol, and 5 &#181;L of 1 mg/mL dioleoyl-phosphoethanolamine-lissamine rhodamine B (rhodamine PE) (see Table of Materials) into the 15 ml glass vial. 
	NOTE: Th...

<ol>
	<li>Groaz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+spatiotemporal+organization+and+dynamics+in+synthetic+cells.">Engineering spatiotemporal organization and dynamics in synthetic cells.</a> Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 13 (3), 1685 (2021).</li><li>Diltemiz, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+artificial+cells+as+drug+carriers.">Use of artificial cells as drug carriers.</a> Materials Chemistry Frontiers. 5, 6672-6692 (2021).</li><li>Liu, A. P., Fletcher, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+under+construction:+In+vitro+reconstitution+of+cellular+function.">Biology under construction: In vitro reconstitution of cellular function.</a> Nature Reviews Molecular Cell Biology. 10 (9), 644-650 (2009).</li><li>Ganzinger, K. A., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+from+less+-+bottom-up+reconstitution+of+cell+biology.">More from less - bottom-up reconstitution of cell biology.</a> Journal of Cell Science. 132 (4), 227488 (2019).</li><li>Bashirzadeh, Y., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+the+cytoskeleton:+Towards+mimicking+the+mechanics+of+a+cell.">Encapsulation of the cytoskeleton: Towards mimicking the mechanics of a cell.</a> Soft Matter. 15 (42), 8425-8436 (2019).</li><li>Blanchoin, L., Boujemaa-Paterski, R., Sykes, C., Plastino, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics,+architecture,+and+mechanics+in+cell+motility.">Actin dynamics, architecture, and mechanics in cell motility.</a> Physiol reviews. 94 (1), 235-263 (2014).</li><li>Angelova, M. I., Dimitrov, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome+electroformation.">Liposome electroformation.</a> Faraday Discussions of the Chemical Society. 81, 303-311 (1986).</li><li>Tsumoto, K., Matsuo, H., Tomita, M., Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+formation+of+giant+liposomes+through+the+gentle+hydration+of+phosphatidylcholine+films+doped+with+sugar.">Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar.</a> Colloids and Surfaces B: Biointerfaces. 68 (1), 98-105 (2009).</li><li>Bailey, A. L., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+fusion+with+cationic+liposomes:+Effects+of+target+membrane+lipid+composition.">Membrane fusion with cationic liposomes: Effects of target membrane lipid composition.</a> Biochemistry. 36 (7), 1628-1634 (1997).</li><li>Haluska, C. K., Riske, K. A., Marchi-Artzner, V., Lehn, J. M., Lipowsky, R., Dimova, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+scales+of+membrane+fusion+revealed+by+direct+imaging+of+vesicle+fusion+with+high+temporal+resolution.">Time scales of membrane fusion revealed by direct imaging of vesicle fusion with high temporal resolution.</a> Proceedings of the National Academy of Sciences. 103 (43), 15841-15846 (2006).</li><li>Nishimura, K., Suzuki, H., Toyota, T., Yomo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+control+of+giant+unilamellar+vesicles+prepared+from+inverted+emulsion+droplets.">Size control of giant unilamellar vesicles prepared from inverted emulsion droplets.</a> Journal of Colloid and Interface Science. 376 (1), 119-125 (2012).</li><li>Pautot, S., Frisken, B. J., Weitz, D. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+age+of+cell-free+biology.">The new age of cell-free biology.</a> Annual Review of Biomedical Engineering. 22, 51-77 (2020).</li><li>Ho, K. K. Y., Murray, V. L., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+artificial+cells+by+combining+HeLa-based+cell-free+expression+and+ultrathin+double+emulsion+template.">Engineering artificial cells by combining HeLa-based cell-free expression and ultrathin double emulsion template.</a> Methods in Cell Biology. , 303-318 (2015).</li><li>Akashi, K., Miyata, H., Itoh, H., Kinosita, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+giant+liposomes+in+physiological+conditions+and+their+characterization+under+an+optical+microscope.">Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope.</a> Biophysical Journal. 71 (6), 3242-3250 (1996).</li><li>M&#233;l&#233;ard, P., Bagatolli, L. A., Pott, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+unilamellar+vesicle+electroformation:+From+lipid+mixtures+to+native+membranes+under+physiological+conditions.">Giant unilamellar vesicle electroformation: From lipid mixtures to native membranes under physiological conditions.</a> Methods in Enzymology. 465, 161-176 (2009).</li><li>Walde, P., Cosentino, K., Engel, H., Stano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+vesicles:+Preparations+and+applications.">Giant vesicles: Preparations and applications.</a> ChemBioChem. 11 (7), 848-865 (2010).</li><li>P&#233;cheur, E. I., Martin, I., Ruysschaert, J. M., Bienven&#252;e, A., Hoekstra, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+fusion+induced+by+11-mer+anionic+and+cationic+peptides:+A+structure&#8722;+function+study.">Membrane fusion induced by 11-mer anionic and cationic peptides: A structure&#8722; function study.</a> Biochemistry. 37 (8), 2361-2371 (1998).</li><li>Morigaki, K., Walde, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Giant+vesicle+formation+from+oleic+acid/sodium+oleate+on+glass+surfaces+induced+by+adsorbed+hydrocarbon+molecules.">Giant vesicle formation from oleic acid/sodium oleate on glass surfaces induced by adsorbed hydrocarbon molecules.</a> Langmuir. 18 (26), 10509-10511 (2002).</li><li>Majumder, S., Wubshet, N., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+complex+solutions+using+droplet+microfluidics+towards+the+synthesis+of+artificial+cells.">Encapsulation of complex solutions using droplet microfluidics towards the synthesis of artificial cells.</a> Journal of Micromechanics and Microengineering. 29 (8), 83001 (2019).</li><li>Abkarian, M., Loiseau, E., Massiera, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+droplet+interface+crossing+encapsulation+(cDICE)+for+high+throughput+monodisperse+vesicle+design.">Continuous droplet interface crossing encapsulation (cDICE) for high throughput monodisperse vesicle design.</a> Soft Matter. 7 (10), 4610-4614 (2011).</li><li>Van de Cauter, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cDICE+for+efficient+reconstitution+of+biological+systems+in+giant+unilamellar+vesicles.">Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles.</a> ACS Synthetic Biology. 10 (7), 1690-1702 (2021).</li><li>Claudet, C., In, M., Massiera, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+to+disperse+lipids+as+aggregates+in+oil+for+bilayers+production.">Method to disperse lipids as aggregates in oil for bilayers production.</a> The European Physical Journal E. 39 (1), 1-6 (2016).</li><li>Bashirzadeh, Y., Wubshet, N. H., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confinement+Geometry+tunes+fascin-actin+bundle+structures+and+consequently+the+shape+of+a+lipid+bilayer+vesicle.">Confinement Geometry tunes fascin-actin bundle structures and consequently the shape of a lipid bilayer vesicle.</a> Frontiers in Molecular Biosciences. 7, 610277 (2020).</li><li>Bashirzadeh, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+crosslinker+competition+and+sorting+drive+emergent+GUV+size-dependent+actin+network+architecture.">Actin crosslinker competition and sorting drive emergent GUV size-dependent actin network architecture.</a> Communications Biology. 4, 1136 (2021).</li><li>Bashirzadeh, Y., Moghimianavval, H., Liu, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+actomyosin+patterns+drive+cell-like+membrane+shape+changes.">Encapsulated actomyosin patterns drive cell-like membrane shape changes.</a> bioRxiv. , (2021).</li><li>Litschel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+contractile+actomyosin+rings+in+vesicles.">Reconstitution of contractile actomyosin rings in vesicles.</a> Nature Communications. 12 (1), 1-10 (2021).</li><li>Vitale, S. A., Katz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+droplet+dispersions+formed+by+homogeneous+liquid&#8722;+liquid+nucleation:&amp;#34;The+Ouzo+effect.&amp;#34.">Liquid droplet dispersions formed by homogeneous liquid&#8722; liquid nucleation:&amp;#34;The Ouzo effect.&amp;#34.</a> Langmuir. 19 (10), 4105-4110 (2003).</li><li>Pautot, S., Frisken, B. J., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+asymmetric+vesicles.">Engineering asymmetric vesicles.</a> Proceedings of the National Academy of Sciences. 100 (19), 10718-10721 (2003).</li><li>Deshpande, S., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+microfluidic+production+of+cell-sized+liposomes.">On-chip microfluidic production of cell-sized liposomes.</a> Nature Protocols. 13 (5), 856-874 (2018).</li><li>Deshpande, S., Caspi, Y., Meijering, A. E. C., Dekker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Octanol-assisted+liposome+assembly+on+chip.">Octanol-assisted liposome assembly on chip.</a> Nature Communications. 7, 10447 (2016).</li><li>Wubshet, N. 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Different methods of generating GUVs have been explored for the creation of synthetic cells&#160;However, the complexity of the procedures, extended time to attain encapsulation, restriction of lipid types and molecular composition of the encapsulant, need for non-physiological chemicals to facilitate encapsulation, low GUV yield, and inconsistencies in encapsulation efficiency have continued to challenge researchers in this field. Considering the wide range of potential studies that can be embarked in bottom-up syntheti...

Lipid bilayer compartments are used as model synthetic cells for studying enclosed organic reactions and membrane-based processes or as carrier modules in drug delivery applications1,2. Bottom-up biology with purified components requires minimal experimental systems to explore properties and interactions between biomolecules, such as proteins and lipids3,4. However, with the advancement of the field, there is an increased need for more complex experimental systems that better imitate the conditions in biological cells. Encapsulation in GUVs is a practical approach that can offer some of these cell-like properties by providing a deformable and selectively permeable lipid bilayer and a confined reaction space. In particular, in vitro reconstitution of cytoskeletal systems, as models of synthetic cells, can benefit from encapsulation in membrane compartments5. Many cytoskeletal proteins bind and interact with the cell membrane. As most cytoskeletal assemblies form structures that span the entirety of the cell, their shape is naturally determined by cell-sized confinement6.
Different methods are used to generate GUVs, such as the swelling7,8, small vesicle fusion9,10, emulsion transfer11,12, pulsed jetting13, and other microfluidic approaches14,15. Although these methods are still utilized, each has its limitations. Thus, a robust and straightforward approach with a high yield of GUV encapsulation is highly desirable. Although techniques such as spontaneous swelling and electroswelling are widely adopted for the formation of GUVs, these methods are primarily compatible with specific lipid compositions16, low salt concentration buffers17, smaller encapsulant molecular size18, and require a high volume of the encapsulant. Fusing multiple small vesicles into a GUV is inherently energetically unfavorable, thus requiring specificity in charged lipid compositions9 and/or external fusion-inducing agents, such as peptides19&#160;or other chemicals. Emulsion transfer and microfluidic methods, on the other hand, may require droplet stabilization through surfactant and solvent removal after bilayer formation, respectively18,20. The complexity of experimental setup and device in microfluidic techniques such as pulsed jetting impose an additional challenge21. cDICE is an emulsion-based method derived from similar principles governing emulsion transfer22,23. An aqueous solution (outer solution) and a lipid-oil mixture are stratified by centrifugal forces in a rotating cylindrical chamber (cDICE chamber) forming a lipid saturated interface. Shuttling lipid monolayered aqueous droplets into the rotating cDICE chamber results in zipping of a bilayer as droplets cross the lipid-saturated interface into the outer aqueous solution22,24. The cDICE approach is a robust technique for GUV encapsulation. With the presented modified method, not only the high vesicle yield typical for cDICE with a significantly shorter encapsulation time (a few seconds) is achieved but GUV generation time that allow for the observation of time-dependent processes (e.g., actin cytoskeletal network formation) is significantly reduced. The protocol takes about 15-20 min from the start to GUV collection and imaging. Here, GUV generation is described using the modified cDICE method for encapsulating actin and actin-binding proteins (ABPs). However, the presented technique is applicable for encapsulating a wide range of biological reactions and membrane interactions, from the assembly of biopolymers to cell-free protein expression to membrane fusion-based cargo transfer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18:1 Liss Rhod PE lipid in chloroform</td>
			<td>Avanti Polar Lipids</td>
			<td>810150C</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Optical Btm Pit PolymerBase</td>
			<td>ThermoFisher Scientific</td>
			<td>165305</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin from rabbit skeletal muscle</td>
			<td>Cytoskeleton</td>
			<td>AKL99-A</td>
			<td></td>
		</tr>
		<tr>
			<td>ATTO 488-actin from rabbit skeletal muscle</td>
			<td>Hypermol</td>
			<td>8153-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Axygen microtubes (200 &#181;L)</td>
			<td>Fisher Scientific</td>
			<td>14-222-262</td>
			<td>for handling ABPs</td>
		</tr>
		<tr>
			<td>Black resin</td>
			<td>Formlabs</td>
			<td>RS-F2-GPBK-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol (powder)</td>
			<td>Avanti Polar Lipids</td>
			<td>700100P</td>
			<td></td>
		</tr>
		<tr>
			<td>Choloroform</td>
			<td>Sigma Aldrich</td>
			<td>67-66-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear resin</td>
			<td>Formlabs</td>
			<td>RS-F2-GPCL-04</td>
			<td></td>
		</tr>
		<tr>
			<td>CSU-X1 Confocal Scanner Unit</td>
			<td>YOKOGAWA</td>
			<td>CSU-X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Density gradient medium (Optiprep)</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC lipid in chloroform</td>
			<td>Avanti Polar Lipids</td>
			<td>850375C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fascin</td>
			<td>homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>F-buffer</td>
			<td>homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand microtubes (1.5 mL)</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>FS02 Sonicator</td>
			<td>Fischer Scientific</td>
			<td>FS20</td>
			<td></td>
		</tr>
		<tr>
			<td>G-buffer</td>
			<td>homemade</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>158968</td>
			<td></td>
		</tr>
		<tr>
			<td>iXon X3 camera</td>
			<td>Andor</td>
			<td>DU-897E-CS0</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Acros Organics</td>
			<td>8042-47-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX81 Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX21</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus PlanApo N 60x Oil Microscope Objective</td>
			<td>Olumpus</td>
			<td>1-U2B933</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone oil</td>
			<td>Sigma-Aldrich</td>
			<td>317667</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To demonstrate the successful generation of cytoskeletal GUVs using the current protocol, fascin-actin bundle structures in GUVs were reconstituted. Fascin is a short crosslinker of actin filaments which forms stiff parallel-aligned actin bundles and is purified from E. coli as Glutathione-S-Transferase (GST) fusion protein26. 5 &#181;M of actin was first reconstituted, including 0.53 &#181;M of ATTO488 actin in the actin polymerization buffer and 7.5% of the density gradient medium. Upon...

Watch this Scientific Journal Video about Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles at JoVE.com

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

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 ...

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.

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Research

JoVE Journal

Bioengineering

4.0K Views.  University of Michigan, Ann Arbor. 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 transcriptio...