Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Summary

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-HT_1AR), one of the classes of pharmacologically important G protein-coupled receptors.

Abstract

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-HT_1AR) 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-HT_1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT_1AR 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.

Introduction

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 conditions^{1,2,3,4,5,6,7,8}. 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 transport^{9,10,11,12,13,14,15}.

GUVs have been fabricated using various methods, including gentle hydration¹⁶, hydrogel swelling¹⁷, electroformation¹⁸, microfluidic techniques^19,20,21,22, jetting²³, and solvent exchange^24,25,26. 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 bilayers^28,29,30. 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 needed^27,31,32,33. 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 proteins^31,35. Additionally, methods that utilize electroformation could restrict the choice of protein and may not be suitable for all lipid compositions especially charged lipids^31,36,37. 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 peptides^33,38,39. 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 composition^14,40,41. 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 environment^42,43,44,45.

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-HT_1AR) which is a prototypical member of the GPCR family. 5-HT_1AR 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) assay^51,52,53,54, native mass spectrometry⁵⁵, isothermal titration calorimetry (ITC)^56,57, and liposome sedimentation assay^58,59. 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 G_iα 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 curvature^60,61, 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 system^17,42. 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.

Protocol

1. Protein labeling

1. Allow NHS-Rhodamine, 5-HT_1A membrane fragments, and one 7 K MWCO spin desalting column to equilibrate at room temperature.
2. Dissolve 1 mg of NHS-rhodamine in 100 µL of dimethyl sulfoxide (DMSO).
3. Add 5 µL of 1 M sodium bicarbonate solution to increase the pH of 5-HT_1AR solution to pH 8.
4. Add 3.66 µL of the NHS-rhodamine solution to 50 µL of the 5-HT_1AR solution and pipette gently up and down in a microcentrifuge tube.
   NOTE: Ensure to have at least 10x molar excess of NHS-rhodamine.
5. Keep the mixture protected from light and put on rotator at room temperature for 1 h.
6. Wash a 7 k MWCO spin column with 200 µL of 1x phosphate buffer saline (1x PBS) three times for 1.5 min at 1.5 RCF for each wash.
7. Add the labeled protein to one column and balance the amount in another microcentrifuge tube.
8. Spin down the labeled protein once for 5 min at 1.5 RCF.
9. Take a UV-vis spectrum using a nanodrop spectrophotometer at 280 nm and 554 nm and calculate the labeling efficiency following the manufacturer's manual.
10. Store the labeled protein covered at 5 °C until further use. The solution is stable for approximately a week after labeling.

2. GUVs with membrane-incorporated 5-HT _1A

1. Preparation of materials and reagents
   1. Allow the protein, lipids and BSA (Bovine serum albumin) to equilibrate to room temperature.
   2. During this time, clean the coverslips by placing them in methanol and sonicating for 30 min at 40 °C. Ensure that the methanol completely covers the coverslips and the water level in the water bath is above the level of the methanol in the container.
      NOTE: Methanol is toxic and should be handled in appropriate chemical hood.
   3. Dry off the excess methanol on the coverslips with a gentle stream of air. Place the coverslip rack covered in a 40 °C oven for 15 min to ensure that the excess coverslips dry off.
   4. Begin the plasma cleaning process. First, place the coverslips into the plasma cleaner and close off the air intake valve to evacuate all the air inside the chamber.
   5. Once the chamber is under vacuum, clean the coverslips for 5 min using high RF power setting and a near complete vacuum, with only a slight air intake into the vacuum chamber. To ensure the proper level of plasma, adjust the opening of the vacuum chamber such that the resultant color of the plasma is a steady, bright pink.
      NOTE: It is crucial when using air that the plasma remains a bright pink color for the duration of the plasma treatment step, as a darker purple color indicates that there is an improper amount of air in the chamber and will result in a suboptimal plasma treatment.
   6. Once the 5 min have passed, shut off the RF power and release the vacuum.
      NOTE: Upon removal from the plasma chamber, please ensure that the coverslips remain covered.
2. Hydrogel preparation
   1. Combine 6 mg of ultra-low melting temperature agarose with 300 µL of ultrapure water (i.e., 2% (w/v) agarose).
      NOTE: 2% agarose will be used to make protein-free GUVs. Agarose solution can be kept at 45 °C for two days.
   2. Combine 9 mg of ultra-low temperature agarose with 300 µL of ultrapure water for 3 w/v% agarose by as prepared in step 3.1. 3% agarose will be used to make protein incorporated GUVs.
   3. Vortex the solution briefly before placing them on the 90 °C heat block for 10 min. Then, vortex the tube again before transferring it to a 45 °C heat block to keep it in the molten form until further use.
3. Agarose and protein mixing
   1. Mix 21 µL of 3% agarose with the 7 µL of 5-HT_1AR membrane fragments. Pipette up and down slowly many times to ensure adequate mixing. Then, incubate at 45 °C for 1 min.
4. Hydrogel and lipid deposition
   1. For protein-free GUVs: Make a thin film on freshly plasma-cleaned coverslips using 20 µL of 2% agarose. Quickly drop another coverslip on top of the agarose droplet and gently slide the coverslips apart to make a thin film on both coverslips.
      NOTE: This step is tricky in that the sliding of the droplet must occur while the agarose is still in the molten form.
   2. For protein-incorporated GUVs: Pipette the protein/agarose mixture up and down one more time, and then deposit 20 µL of the 2% agarose on a plasma-cleaned coverslip. Follow the slip-casting directions as described above.
   3. Allow the agarose to gel protected from light for 30 min at room temperature.
   4. Deposit the lipids dropwise on top of the agarose layer. Use a total of 10 µL of 2 mg/mL of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) with 0.4 Mol% 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) labeled with ATTO 488 (ATTO-488-DPPE) (or lipid mixture of interest) in chloroform on top of the agarose film. Deposit the droplets using a gas chromatography needle and spread one droplet at a time around via a gentle air stream.
      NOTE: Caution is needed with this step to make a relatively uniform layer of lipids on top of the hydrogel. Also, chloroform is toxic and should be handled in appropriate chemical hood.
   5. Assemble the Sykes-Moore (S-M) chambers by placing an O-ring on top of the coverslip, and then placing the top component of the chamber on top of the O-ring. Use the key provided by the manufacturer to assemble the chamber by screwing the chamber components together to seal the chamber and prevent any leakage.
      NOTE: The top of the chamber should be tightened on the O-ring but caution is needed to ensure the coverslip stays intact as the coverslip can crack if the O-ring doesn't sit properly in the chamber. Also, ensure that the chamber is sealed tight enough such that the chamber does not leak when the swelling solution is added. Failure to tighten the chamber enough will result in leaks and loss of sample.
5. Swelling and harvesting of vesicles
   1. Hydrate the entire system by gently pipetting 450 µL of 200 mM sucrose in 1x PBS and gently tapping the chambers to ensure adequate buffer coverage of the hydrogel-lipid layers.
      NOTE: The sucrose solution can be replaced with a rehydration buffer containing biological probes of interest.
   2. Place the chambers at 45 °C and cover the top part of the chamber with a coverslip to prevent evaporation. Allow the sample to swell, protected from debris and light for 1 h.
   3. Add 100 µL of 1 mg/mL BSA in ultrapure water into each well of a 96-well plate intended to be used. Incubate at room temperature for 1 h.
   4. Wash three times with ultra-pure water and once with 200 mM sucrose in 1x PBS.
   5. Finally, add 200 mM of glucose in 1x PBS until the addition of the GUV sample solution.
      NOTE: BSA was used to block GUV adsorption.
   6. After allowing the hydrogel to swell, gently shake and tap the chamber to dislodge any GUVs that may remain attached to the hydrogel surface. Then, carefully pipette up the GUV-sucrose solution.
      NOTE: As an optional step to ensure all vesicles are detached from the surface, gently pipette some of the sucrose suspension back onto the hydrogel surface.
   7. Move the suspension into a previously prepared microcentrifuge tube containing 700 µL of 200 mM glucose in 1x PBS.
      NOTE: The density gradient will lead to settling of the vesicles to the bottom of the centrifuge tube.
   8. Allow the vesicles to settle for another hour to ensure that the vesicles can sink to the bottom of the microcentrifuge tube, allowing for optimal collection.
   9. After the settling of GUVs in glucose, transfer 300 µL from the bottom of the centrifuge tube (the settled vesicles) into the previously prepared and BSA-treated 96-well plate to examine the vesicles under the confocal microscope.
      NOTE: Be sure to avoid the very bottom of the microcentrifuge tube to minimize the amount of debris collected in the final sample.
6. Check the samples under the microscope.
   1. Shine a 488 nm laser on the sample (that allows us to visualize the membrane, as the bilayer has been labeled with ATTO-488-DPPE).
   2. Shine a 561 nm laser on the sample (that allows us to visualize the protein, since it has been labeled with NHS-Rhodamine).
      ​NOTE: Caution is needed while imaging the sample as photooxidation can destabilize the vesicles. Vesicles were observed on the same day.

Figure 1: Illustration of the detailed protocol steps. Created with BioRender.com Please click here to view a larger version of this figure.

Results

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 prote...

Discussion

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 hydrophili...

Materials

Name	Company	Catalog Number	Comments
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