This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.
Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.
Synthetic model membranes are powerful tools in the investigation of the fundamental properties and functions of biomembranes. Giant unilamellar vesicles (GUVs) are one of the most prominent platforms to study a variety of plasma membrane properties and can be engineered to mimic different physiological conditions1,2,3,4,5,6,7,8. It is well established that the plasma membrane and its organization play a key role in a multitude of cellular processes, such as signal transduction, adhesion, endocytosis, and transport9,10,11,12,13,14,15.
GUVs have been fabricated using various methods, including gentle hydration16, hydrogel swelling17, electroformation18, microfluidic techniques19,20,21,22, jetting23, and solvent exchange24,25,26. Due to challenges in handling integral membrane proteins (IMPs), in vitro platforms to study them have been limited. GUVs present a simplified platform for studying IMPs in an environment that mimics their native environment. Although there have been several approaches for protein reconstitution in GUVs, challenges arise from incorporating proteins with the correct orientation and maintaining protein functionality27.
Most successful protein-reconstitution in GUVs requires the detergent exchange method; which involves solubilizing the proteins from their native environment by detergents, followed by protein purification, and then replacing the detergent molecules with lipids through various methods28. While detergents serve to stabilize the tertiary structure of IMPs during purification, detergent micelles are a relatively unnatural environment for these proteins, which are better stabilized, particularly for functional studies, in lipid bilayers28,29,30. Moreover, incorporating functional transmembrane proteins into the lipid bilayer using traditional GUV fabrication techniques has been difficult due to the size, the delicacy of these proteins, and the additional detergent exchange steps that would be needed27,31,32,33. The use of organic solvent to remove detergents causes protein aggregation and denaturing34. An improved detergent-mediated method has been promising, however, caution is needed for the detergent removal step and optimization might be needed for specific proteins31,35. Additionally, methods that utilize electroformation could restrict the choice of protein and may not be suitable for all lipid compositions especially charged lipids31,36,37. Another technique that has been used is peptide-induced fusion of large unilamellar vesicles (LUVs) containing the desired protein with GUVs, though it was found to be laborious and can lead to the insertion of foreign molecules-the fusogenic peptides33,38,39. Giant plasma membrane vesicles (GPMVs), which are derived from living cells, can be used to overcome some of these issues, however they allow minimal control of the resultant lipid and protein composition14,40,41. Therefore, the integration of IMPs in the bilipid layer of GUVs using our modified agarose swelling method presents a reliable method to further examine these proteins in the membrane environment42,43,44,45.
Cellular signaling and communication involves a family of proteins known as G protein-coupled receptors (GPCRs); GPCRs are among the largest family of proteins and are associated with modulating mood, appetite, blood pressure, cardiovascular function, respiration, and sleep among many other physiological functions46. In this study, we used human serotonin 1A receptor (5-HT1AR) which is a prototypical member of the GPCR family. 5-HT1AR can be found in the central nervous system (CNS) and blood vessels; it influences numerous functions such as cardiovascular, gastrointestinal, endocrine functions, as well as participating in the regulation of mood47. A large barrier to GPCR research arises from their complex amphiphilic structure, and GUVs present a promising platform for the investigation of various properties of interest, ranging from protein functionality, lipid-protein interactions, and protein-protein interactions. Various approaches have been utilized to study lipid-protein interactions such as surface plasmon resonance (SPR)48,49, nuclear magnetic resonance spectroscopy (NMR)50,51, protein lipid overlay (PLO) assay51,52,53,54, native mass spectrometry55, isothermal titration calorimetry (ITC)56,57, and liposome sedimentation assay58,59. Our lab has used the simplified GUV approach to investigate the effect of lipid-protein interactions on protein functionality by incapsulating BODIPY-GTPγS, which binds with the Giα subunit in the active state of the receptor. Their binding unquenches the fluorophore producing a fluorescence signal that could be detected over time45. Moreover, various studies investigated Lipid-protein interactions and the role of proteins in sensing or stabilizing membrane curvature60,61, and utilizing a feasible GUV approach could be a key advantage.
This protocol demonstrates a straightforward method to incorporate GPCRs into the membrane of GUVs using a modified agarose hydrogel system17,42. Furthermore, based on our previous work, our method could be suitable for IMPs that can bear short-term exposure to 30-40 °C. Briefly, we spread a thin film of agarose combined with membrane fragments containing the GPCR of interest. Following gelation of this layer, we deposit a lipid solution atop the agarose and allow the solvent to evaporate. Rehydration of the system was then performed with an aqueous buffer, resulting in the formation of GUVs with protein incorporated in the lipid bilayer.
1. Protein labeling
2. GUVs with membrane-incorporated 5-HT 1A
Figure 1: Illustration of the detailed protocol steps. Created with BioRender.com Please click here to view a larger version of this figure.
The concentration of protein was measured, and the degree of labeling was calculated as the molar ratio between the dye and the protein to be 1:1. By examining the GUVs using confocal microscopy, we were able to confirm successful formation and protein integration of the vesicles. The lipids were labeled with 0.4 mol% ATTO 488-DPPE, and the protein was covalently labeled via rhodamine NHS-ester modification of primary amines. Figure 2a and Figure 2b show a protein-incorporated vesicle in the ATTO 488 and rhodamine channels, respectively. All micrographs have been dark current and flatfield corrected. Figure 2c and Figure 2d show a negative control GUV with no protein incorporated. Figure 3a and Figure 3b show a protein incorporated GUV with line intensity profiles given by the dashed white line of the same vesicle in both channels. The line intensity profile shows a two-dimensional plot of the intensities of the pixels along the white drawn line within the image. The x-axis is the distance along the line and the y-axis is the pixel intensity. ImageJ software was used to plot the profile intensity of the indicated line.
Figure 2: Micrographs comparing protein incorporated GUVs and GUVs without protein (control). Micrographs (a) and (b) show protein incorporated GUV fluorescence with the respective ATTO 488 and rhodamine channels, respectively. Micrographs (c) and (d) show a protein omitted GUV when excited with ATTO 488 and rhodamine channels, respectively. Please click here to view a larger version of this figure.
Figure 3: Top row shows micrographs of protein incorporated GUVs in ATTO 488 (a) and rhodamine (b) channels. Line intensity profiles for the indicated white-dashed lines are below. The analysis was performed using ImageJ software. Please click here to view a larger version of this figure.
We have identified two steps that are critical to the success of the overall protocol: plasma treatment and lipid deposition. Plasma cleaning of the coverslips is essential in ensuring that there is adequate coverage and adhesion of the agarose hydrogel to the glass coverslip. Plasma cleaning accomplishes two things: first, it removes traces of organic matter from the glass surface; second, it activates the coverslip surface, allowing for an increase in wettability as the glass surface hydrophilicity increases62,63. Touching the coverslip surface post-plasma cleaning will inactivate and contaminate the ultraclean surface and is strongly advised against. Our recommendation is to only touch the very edges and undersides of the coverslip when handling the coverslips for the agarose slip casting step. The second critical step is the deposition of lipids onto the dry hydrogel surface. This method uses a dropwise lipid deposition, which requires a gas chromatography (GC) needle and an air stream to deposit a few microliters of lipid solution at a time, allowing for precise control of the amount of lipid added and the placement of the lipid film on the hydrogel surface. The drawback of this method is that if not done carefully, it can result in a few select areas with a thicker lipid film, resulting in reduced GUV yields. Thus, it is critical to ensure that there is as uniformly thin of a lipid layer as possible on the surface of the agarose.
One of the most significant benefits of this protocol is the flexibility of the platform itself; this method lends itself very well to changes in protein and lipid composition, as well as encapsulation and buffer modifications. This protocol can, in principle, include any transmembrane protein, as we have been able to successfully incorporate a number of different transmembrane proteins, ranging from the adenosine receptor (A2AR) to plant aquaporins without sacrificing functionality42,45,64. Traditionally, proteins have been incorporated into GUVs following solubilization by detergents or incorporation into proteo-liposomes or small unilamellar vesicles that can be subsequently integrated into a preformed GUV65. The advantage of our modified hydrogel swelling method is that it removes the dependency of detergents or intermediate vesicles and provides an intermediate hydrated scaffold. The benefits of this are twofold: we can stably incorporate functional GPCRs into the membrane in a more physiologically relevant buffer without relying on detergent exchange methods that require more preparation and care regarding the concentration of the said detergents, and that the process by which GUVs bud off the surface of the hydrogel allows for the correct orientation of the proteins in the bilayer66. We have shown that the GUV budding process involves the coalescence of many smaller nanometer-scale vesicles into larger, micron-scale vesicles, which encourages correct protein orientation from the beginning. We have shown this to be the case in our previous work; in short, we covalently labeled an antibody targeting a specific cytosolic loop of the Adenosine receptor and incubated the labeled antibody with the protein, and then incorporated the labeled protein into lipid-dye-labeled GUVs using the modified hydrogel swelling method. We then exposed the protein-incorporated vesicles to a charged quencher, which is unable to cross the bilayer. We subsequently see a 50% reduction in fluorescence of the lipid dye, but the fluorescence of the labeled protein remains unaffected by the quencher, demonstrating proper orientation44.
Previous work out of our lab has investigated the role in which lipid headgroup charge, zwitterionic and net-ionic charged lipids, as well as buffer and hydrogel properties such as pH, ionic strength, osmolarity, and hydrogel concentration have on the dynamics of GUV formation67. In short, lipid charge does not largely affect GUV formation, while buffer properties such as increases in sucrose concentration (e.g., 500 mM Sucrose in 185 mM ionic strength PBS buffer) negatively affect GUV formation, resulting in irregularly shaped vesicles that most likely will not readily lend themselves to harvesting. Acidic solutions (pH = 3) increase the rate of formation, while a more basic solution (pH = 8) suppresses the rate of GUV formation. GUVs still form at both the acidic and basic buffers, with only marginal differences in vesicle size. Low agarose concentrations (~0.1-1 w/v%) also negatively affect GUV formation due to a lack of homogenous surface coverage and a decrease in hydrogel swelling, a necessary force in the coalescence and budding of GUVs off the hydrogel surface. Thus, we have determined that a 2 w/v% final agarose concentration with a sucrose/glucose solution of 100-200 mM, combined with a buffer ionic strength of 185 mM PBS at pH 7.4 achieves a good balance of agarose swelling, GUV formation rate, and subsequent vesicle size. For vesicles that contain protein, increasing the initial agarose concentration to 3 w/v% allows for a final agarose concentration of 2 w/v% after the addition of the protein solution. In addition to formation dynamics, the sucrose/glucose buffer system also facilitates the sedimentation and subsequent collection of formed GUVs, as well as visualization under phase contrast microscopy65,68.
There are some points of caution regarding this protocol, specifically regarding the agarose and the selection of vesicles. For instance, while we use an ultralow melting temperature agarose, the agarose-water suspension needs to reach at least 60 °C to become molten, and the agarose-protein mixture is incubated at 45 °C. In our experience, this temperature does not eliminate the activity of 5-HT1AR, but caution is warranted for other proteins. In general, the agarose we use begins to gel at 20 °C and thus the swelling reaction can take place at temperatures above 20 °C, but this process cannot function below that temperature. It should also be noted that the closer the temperature gets to 20 °C, the less efficient the swelling step becomes, leading to subsequent decreases in GUV yields. The agarose can also present an issue during the settling and visualization steps, as it can persist at the bottom of the settling/collection tube as debris. Thus, caution is required for the temperature required to maintain the molten agarose and ensuring that the said temperature will not denature the protein of interest as well as aspirating the settling solution to avoid any excess suspended agarose from being included in the final sample. This method in its current state also results in a heterogeneous GUV population size, with some vesicles displaying multilamellarity and other flawed vesicle phenomena such as vesicles within vesicles. This is typical of common GUV formation methods and requires vigilance and discretion when selecting vesicles for microscopy and analysis. GUVs that display unusually high levels of fluorescence are also not recommended for analysis, as agarose can be found on the interior of some of these vesicles. Unpublished work out of our lab has been able to run micropipette aspiration experiments using vesicles made using this technique, illustrating that the agarose method produces vesicles without mechanics-altering agarose in the lumen.
Limitations aside, this protocol presents a robust and straightforward method for generating protein incorporated GUVs. It can generate high yields of GUVs in physiologically relevant conditions that incorporate properly oriented transmembrane proteins into the bilayer without compromising their functionality. This is a departure from other methods of vesicle formation, which involve electric currents or gentle hydration, that would significantly damage the structure of the protein and render it nonfunctional or require further detergent solubilization and removal steps. Given that GPCRs represent upwards of a third of all pharmaceutical targets, there is significant interest in being able to study this family of proteins in a highly tunable, high-throughput, biomimetic platform. More specifically, the applications of this work range from the study of protein-lipid interactions, how the lipid microenvironment influences protein functionality and localization, and other basic biophysical questions that can inform pharmaceutical drug development and discovery. An example of this can be found in the work completed within our lab, which has been able to discern variances in receptor functionality as a result of lipid oxidation.
The authors have nothing to disclose.
We thank Matthew Blosser for valuable discussion and advice. This work was supported by the Office of Naval Research (N00014-16-1-2382) and the National Science Foundation (PHY-1915017).
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) | Avanti Polar Lipids, Alabaster, AL | 850375C-25mg | |
TI-Eclipse inverted microscope | Nikon, Melville, NY | Eclipse Ti | |
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) | Avanti Polar Lipids, Alabaster, AL | 850355C-25mg | |
13/16″ ID, 1″ OD silicon O-rings | Sterling Seal & Supply, Neptune, IN | 5-003-8770 | |
16-bit Cascade II 512 electron-multiplied charge coupled device camera | Photometrics, Huntington Beach, CA | Cascade II 512 | |
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) | Avanti Polar Lipids, Alabaster, AL | 850457C-25mg | |
50 mW solid-state lasers at 488 nm and emission filter centered at 525 nm, and 561 nm with emission filter centered at 595 nm | Coherent, Santa Clara, CA | 488/561-50-LS | |
5-HT1AR membrane fragments | Perkin Elmer, Waltham, MA | RBHS1AM400UA | |
ATTO-488-1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) | ATTO-TEC, Siegen, Germany | AD 488-155 | |
Bench top plasma cleaner | Harrick Plasma, Ithaca, NY | PDC-32G | |
bovine serum albumin (BSA) | Sigma Aldrich, St. Louis, MO | A9418 | |
chloroform (CHCl3) | Millipore Sigma, Burlington, MA | CX1055 | |
Cholesterol (Chol) | Sigma Aldrich, St. Louis, MO | C8667-5G | |
Corning 96-well Flat Clear Bottom | Corning, Corning, NY | 3904 | |
Elmasonic E-Series E15H Ultrasonic | Elma, Singen, Germany | [no longer sold on main website] | |
glucose | Sigma Aldrich, St. Louis, MO | G7528 | |
methanol (MeOH) | Millipore Sigma, Burlington, MA | MX0485 | |
NanoDrop ND-1000 | Thermo Fisher Scientific, Waltham, MA | ND-1000 | |
NHS-Rhodamine | Thermo Fisher Scientific, Waltham, MA | 46406 | |
phosphate buffered saline (PBS) (10x PBS) | Corning, Corning, NY | 21-040 | |
spinning-disc CSUX confocal head | Yokogawa,Tokyo, Japan | CSU-X1 | |
standard 25 mm no. 1 glass coverslips | ChemGlass, Vineland, NJ | CLS-1760 | |
sucrose | Sigma Aldrich, St. Louis, MO | S7903 | |
Sykes-Moore chambers | Bellco, Vineland, NJ | 1943-11111 | |
Ultra-low melting temperature agarose | Sigma Aldrich, St. Louis, MO | A5030 | |
VWR Analog Heatblock | VWR International, Radnor, PA | [no longer sold on main website] | |
VWR Tube Rotator | VWR International, Radnor, PA | 10136-084 | |
Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL | Thermo Fisher Scientific, Waltham, MA | 89882 |