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 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.
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 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.
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.
2. Vesicle generation
3. Imaging and 3D image reconstruction
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 µM of actin was first reconstituted, including 0.53 µM of ATTO488 actin in the actin polymerization buffer and 7.5% of the density gradient medium. Upon adding fascin at a concentration of 2.5 µM and encapsulating the fascin-actin mixture, actin bundle structures were formed in GUVs. Z-stack confocal image sequences of the encapsulated actin bundle structures in the rhodamine PE-labeled GUVs were captured 1 h post-encapsulation (Figure 2A). Using this protocol, inherent competition and sorting of the encapsulated actin crosslinkers, α-actinin, and fascin, which, together, form different actin bundle patterns in a GUV size-dependent manner, was previously demonstrated26.
Like the modified inverted emulsion approach presented here, the traditional cDICE process generates cytoskeletal GUVs with high yield yet requires a syringe pump and tubing setup for controlled injection of protein solution into the rotating chamber at low flow rates in the order of nanoliters per second22,28. In this approach, the emulsion is directly generated in the rotating cDICE chamber; a thin capillary is inserted in the oil phase. The protein solution is injected through a syringe pump. Droplets form and are sheared off at the capillary tip before they travel towards the aqueous outer phase, where they turn into GUVs, similar to the method described above. Figure 2B shows vesicles that encapsulate a reaction mixture using this approach. The reaction mix contains 6 µM of actin which is bundled by 0.9 µM of fascin. Here, the two methods and their results are not being compared but note that they both generate a high yield of GUVs.
Figure 1: Experimental setup for generating GUVs. (A) Top view and side section view of the cDICE chamber. (B) Photos of the setup for the spinning chamber. (C-E) Schematic illustrations of stepwise procedures for generation of GUVs. Please click here to view a larger version of this figure.
Figure 2: Encapsulation of actin bundle structures. (A) The images show representative fluorescence confocal slices of GUVs (left) and maximum projections of a confocal z-stack of actin and lipid channels (right). Fascin, 2.5 µM; actin, 5 µM (including 10% ATTO 488 actin). Scale bar = 10 µm. (B) Encapsulation of actin bundle structures using conventional cDICE. The image shows a representative maximum projection of confocal fluorescence images of encapsulated actin bundles formed in the presence of fascin. Fascin, 0.9 µM; Actin, 6 µM. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Supplementary File 1: 3D printed shaft design. Please click here to download this File.
Supplementary File 2: Design for the 3D-printed cDICE chamber. Please click here to download this File.
Different methods of generating GUVs have been explored for the creation of synthetic cells 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 synthetic biology, a seamless high throughput GUV encapsulation approach that is compatible with different lipid compositions and can encapsulate any molecules regardless of size may spur new opportunities to study complex biomimicking synthetic systems. The cDICE method has eliminated most challenges and limitations inherent to prior GUV generation methods.
The approach and governing principles to generate GUVs using the cDICE method predates the platform and have been implemented in earlier techniques such as the inverted emulsion transfer12. However, the inverted emulsion transfer method has limitations such as low vesicle yield and heterogeneity of vesicles. For the cDICE method presented here, lipids are dispersed in oil in the form of aggregates of tens of nanometers (size of lipid aggregate is dependent upon the overall concentration of lipids)24. Dispersion of lipids is in two miscible oils, where one (mineral oil) can dissolve lipids and a second oil (silicon oil) that is not miscible with lipids. This creates lipid aggregate coacervates by way of solvent-shifting29. This particular dispersion approach facilitates instant monolayer saturation of aqueous droplets and faster renewal of lipids at the oil-aqueous interface as aqueous droplets continuously cross the lipid-saturated oil-aqueous interface. This also subsequently improves bilayer zipping to form GUVs and increases GUV throughput. The centrifugal forces generated by the rotating chamber are optimal for shuttling polydispersed droplets across the lipid-saturated interface. The original version of the cDICE method utilizes a microcapillary nozzle to inject the inner solution into the oil-lipid mixture. In this approach, shearing forces created by the rotating oil-lipid mixture generate aqueous droplets, eventually transforming into GUVs as described. However, with the intent to reduce the time taken to prepare injection platform, especially critical for fast reactions, such as actin network assembly and potential clogging of the microcapillary, aqueous droplets with lipid monolayers are now generated by adding the oil-lipid mixture directly to the inner solution and pipetting up and down. This approach eliminates the time lag in GUV encapsulation for a fast reaction experiment.
Amongst the challenges caused by earlier GUV generation methods is the restriction of lipid types (charge of lipid and phase of lipid) depending on the technique of GUV generation. Multiple lipid types, including DOPC, dioleoyl-glycero-hosphoserine (DOPS), dioleoyl-glycero-succinate (DGS), dimyristoyl-glycero-phosphocholine (DMPC), and combinations of different lipids and cholesterol at varying concentrations were tested. For all conditions, the cDICE method is shown to form GUVs with a high encapsulation efficiency at a consistently high GUV yield. Furthermore, the cDICE method has also been shown to effectively encapsulate different cellular components, including cytoskeletal proteins, cell-free expression reactions, crowding agents, dyes, and other cellular molecules of different sizes without a loss of encapsulation efficiency or decreased throughput. Furthermore, like normal inverted emulsion transfer methods30, the modified cDICE can potentially permit the generation of asymmetric GUVs for future work. Different lipid compositions can be used for the inner leaflet and the outer leaflet since monolayer droplets are formed separately (inside a microtube by pipetting up and down) before zipping the bilayer inside the cDICE chamber. Sedimentation of lipids is observed when the lipid-oil mixture is kept for long; however, one can simply vortex the lipid-oil mixture before encapsulation to re-disperse lipid aggregates. It is important to note that encapsulation quality can become compromised when the lipid-oil mixtures are kept longer, as indicated by more significant than usual lipid aggregates in the lipid-oil mix. Although not tested, these aggregates could potentially result in imperfection in bilayer zipping, and the aggregates may also end up being encapsulated with inner solution compromising the desired chemical environment.
The limitation of the presented modified approach to form aqueous droplets is in generating uniformity in the size of droplets. Although this can be improved by using microcapillary injection of inner solution at different flow rates to regulate GUV sizes, it is less desirable to monitor fast reactions like actin assembly in encapsulated GUVs. By making droplets by pipetting up and down that results in different GUV sizes, one can analyze populations of similar size vesicles. Concerns about possible oil retention in bilayers impede the adoption of most GUV generation techniques, including emulsion-based GUV generation techniques such as cDICE21. However, the amount of oil remaining in the membrane may be reduced by using organic agents such as 1-octanol, which can be removed after generating the vesicles31,32. Future modifications to the method, possibly by changing solvent composition, need to be investigated.
There are many areas in bottom-up synthetic biology that are yet to be investigated and perhaps require cell-mimicking confinements of GUVs. Such experimental endeavors necessitate GUV generation platforms like cDICE to generate GUVs robustly while efficiently encapsulating various molecules of interest. Many cellular processes occur faster than the time it takes to encapsulate molecules using prior GUV generation techniques. As described here, actin solutions are encapsulated quickly enough to observe vesicle deformation resulting from actin network assembly. Such synthetic cells with reconstituted actin cytoskeleton have revealed features of actin network organization in the presence of different crosslinkers5,25,33 and membrane remodeling25,27,28. They will inspire future work to create more sophisticated synthetic cells.
The authors have nothing to disclose.
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).
18:1 Liss Rhod PE lipid in chloroform | Avanti Polar Lipids | 810150C | |
96 Well Optical Btm Pit PolymerBase | ThermoFisher Scientific | 165305 | |
Actin from rabbit skeletal muscle | Cytoskeleton | AKL99-A | |
ATTO 488-actin from rabbit skeletal muscle | Hypermol | 8153-01 | |
Axygen microtubes (200 µL) | Fisher Scientific | 14-222-262 | for handling ABPs |
Black resin | Formlabs | RS-F2-GPBK-04 | |
Cholesterol (powder) | Avanti Polar Lipids | 700100P | |
Choloroform | Sigma Aldrich | 67-66-3 | |
Clear resin | Formlabs | RS-F2-GPCL-04 | |
CSU-X1 Confocal Scanner Unit | YOKOGAWA | CSU-X1 | |
Density gradient medium (Optiprep) | Sigma-Aldrich | D1556 | |
DOPC lipid in chloroform | Avanti Polar Lipids | 850375C | |
Fascin | homemade | N/A | |
F-buffer | homemade | N/A | |
Fisherbrand microtubes (1.5 mL) | Fisher Scientific | 05-408-129 | |
FS02 Sonicator | Fischer Scientific | FS20 | |
G-buffer | homemade | N/A | |
Glucose | Sigma-Aldrich | 158968 | |
iXon X3 camera | Andor | DU-897E-CS0 | |
Mineral oil | Acros Organics | 8042-47-5 | |
Olympus IX81 Inverted Microscope | Olympus | IX21 | |
Olympus PlanApo N 60x Oil Microscope Objective | Olumpus | 1-U2B933 | |
Silicone oil | Sigma-Aldrich | 317667 |