Lipid Exchange Assay in Living Cells

Summary

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

Abstract

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane's propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

Introduction

The plasma membrane is composed of a lipid bilayer enriched with various membrane proteins, including transmembrane receptors and ion channels. Lipid domains within the membrane have been elucidated through detergent-soluble and insoluble regions identified in detergent-resistant membrane (DRM) fractionation experiments¹. The insoluble fractions were characterized by being enriched in cholesterol, tightly packed sphingomyelins and saturated phospholipids, exhibiting higher melting points, in contrast to the soluble fractions that predominantly consist of lower melting temperature and loosely packed unsaturated phospholipids. The tightly packed regions are referred to as liquid-ordered (Lo) lipid domains, or lipid rafts, while the more loosely organized liquid-disordered (Ld) lipid domains are the non-raft regions of the plasma membrane^2,3. Lipid raft regions are known to facilitate signaling processes, with evidence indicating that the active insulin receptor associates with these rafts^4,5. However, due to the dynamic nature of the cell membrane and the generally small size of domains, directly visualizing the presence of rafts in live cells presents significant challenges. In this context, we present a method to investigate the impact of lipid rafts on the insulin receptor through lipid exchange techniques.

Cyclodextrins (CDs) are formed by linked glucose monomers that create a ring-like structure with a central cavity. The size of this cavity is determined by the number of glucose units: six units form alpha-cyclodextrin (α-CD), while seven units create beta-cyclodextrins (β-CDs). CDs are highly water-soluble molecules capable of encapsulating lipids within their cavity, thus facilitating their transport to the cell membrane⁶. Beta-cyclodextrins have been extensively used to add and remove lipids from membranes⁷; however, its larger cavity lacks specificity for cholesterol or phospholipids⁸. In contrast, alpha-cyclodextrins, with their smaller cavity, exhibit greater selectivity in binding lipid molecules over sterols. Specifically, methyl-α-cyclodextrin (methyl-α-CDs) does not interact with sterols and has been effectively used to exchange phospholipids and sphingomyelins without altering the cholesterol composition of the cell membrane^8,9.

In this manuscript, we provide a detailed protocol for using methyl-α-CDs (MαCD) to exchange lipids in the outer leaflet of the cell membrane with exogenous lipids that have properties either promoting or disrupting lipid raft formation. This exchange is used to investigate the impact of lipid rafts on insulin receptor activity. The demonstration will focus on the introduction of a phospholipid and sphingomyelin affecting the formation of liquid-ordered (Lo) domains in the plasma membrane of Chinese Hamster Ovary (CHO) cell lines stably overexpressing the insulin receptor (IR)¹⁰. The extent of lipid exchange in the CHO IR cells will be assessed through high-performance thin-layer chromatography (HP-TLC), while changes in insulin receptor activity will be quantified by western blot analysis following insulin stimulation post-lipid exchange.

Protocol

1. Preparation of Methyl-α-CD solution

1. Add 20 mL of phosphate-buffered saline (PBS) to ~10 g of MαCD powder in a glass bottle. Incubate in a warm water bath (45 °C) to dissolve, stirring occasionally until well dissolved.
   NOTE: The solution may still be cloudy due to insoluble CD species.
2. Pass solution through a 0.22 µm syringe filter. The solution will become clear.
3. Use a refractometer to determine the exact concentration of MαCD.
   1. Place 10 µL of sample on the sample area of a refractometer, illuminate it using a white incandescent bulb, and record the solution's refractive index.
   2. Calculate the MαCD concentration using the equation
      RI = (1.49 x 10 x C) + 1.33
      where RI = Refractive index, C = concentration of MαCD (mM). The equation was obtained through gravimetric analysis, or measuring the refractive index of a known weight of MαCD dissolved in a known volume.
4. Close the glass bottle with a lid and wrap the lid in a transparent film to prevent evaporation. Store at 4 °C.

2. Preparation of multilamellar vesicles (MLVs)

1. Maintain stock solutions of desired exogenous lipids dissolved in chloroform at concentrations of 20 mM to 50 mM and store at -20 °C or below to minimize solvent evaporation.
   NOTE: All lipids in Table 1 can be fully dissolved in chloroform stocks. However, it is possible that alternative lipids may require 1:1 chloroform: methanol to fully dissolve.
2. Aliquot lipid stock into borosilicate tubes using a positive-displacement pipette with a glass tip. Alternatively, a glass microsyringe can be used.
3. Dry lipid aliquot on a heating block at a low setting of about 50 °C under a stream of N₂ gas until all apparent chloroform evaporates.
4. Remove the remaining solvent from the dried lipid by placing the tube in a vacuum chamber and exposing it to a high vacuum (under 200 mTorr) for 1 h.
5. Add serum-free Ham's F-12 media to dry lipid film to reach a final concentration of 20 mM. Cover with a lid or Teflon tape and heat in a 70 °C water bath for 5 min.
6. Vortex to suspend lipids and form MLVs. The media should now appear cloudy.
   NOTE: Some saturated lipids may not be fully suspended by vortexing alone and may need to be suspended by pipetting up and down until the film is no longer visible.
   1. Transfer the entire volume to a microcentrifuge tube. MLVs can be stored for up to 3 days at 4 °C.

3. Preparation of lipid exchange media

1. Add MαCD stock to a final concentration of 40 mM, along with prepared MLVs to the final lipid concentration found in Table 1.
   NOTE: If using MLVs stored at 4 °C, they will be settled at the bottom of the tube. Warm up to room temperature and ensure they are resuspended by flicking or agitating the tube.
   1. Load lipids onto MαCD by incubating for 30 min in a 37 °C or 55 °C water bath according to Table 1. The temperature chosen should be above the gel-to-liquid phase melting point of the chosen lipid. The media should go from cloudy to clear.
   2. Let lipid exchange media cool to room temperature for 30-60 min.

4. Lipid exchange treatment of cells

1. Grow CHO IR cells at 37 °C and 5 % CO₂ in Dulbecco's modified Eagle's medium (DMEM, 4.5 g/L glucose) supplemented with 10% fetal bovine serum (FBS), 300 µg/mL L-glutamine, 100 µg/mL non-essential amino acids, 50 µg/mL G418, 2 µM methotrexate, and 1x antibiotic-antimycotic.
   1. Seed 1.5 x 10⁶ cells in 60 mm plates and grow until they are 80%-90% confluent.
   2. Wash cells 3x by adding 1 mL of PBS and then aspirating. Starve cells overnight in 2 mL of serum-free Ham's F12 media.
2. Wash cells 3x with 1 mL of PBS. Add 1 mL of prepared exchange media or serum-free media as a control to the cells and incubate for 1 h at room temperature (25 °C -27 °C). Swirl cells every 15 min to ensure even exposure to exchange media.
   1. Wash cells 3x with 1 mL of PBS. Each plate can be subsequently processed for lipid extraction in step 5, or for IR autophosphorylation in step 7.

5. Lipid extraction on cell culture plate

1. Completely remove PBS and set the plate at a 45° angle for 10 min or until fully dry, removing any buffer that may collect along the bottom.
2. Add 1 mL of 3:2 (v:v) hexane: isopropanol solution to the dried cells and incubate for 10 min on a shaker at room temperature.
3. Transfer the solution to a borosilicate tube. Cover with Teflon tape and store at -20 °C.
4. Dissolve the remaining cell debris by adding 500 µL of 1N NaOH and shaking for 10 min at room temperature.
   1. Use this solution in a protein quantification assay such as the Bradford Assay to determine the protein concentration of each sample. These concentration values can be used to normalize loading volumes of the lipid extracts on HP-TLC plates for equal lipid loading across all samples.

6. Checking exchange efficiency with HP-TLC

1. Make 100 mL of 65:25:5 (v:v:v) chloroform:methanol:30% (v/v) ammonium hydroxide and pour it into a glass TLC tank. Cover tightly and allow vapor to equilibrate for at least 1 h.
2. Dry lipid extract sample on a heating block at low setting under a stream of N₂ gas until all apparent organic solvent evaporates.
3. Dissolve the lipid film in 50 µL of 1:1 (v:v) chloroform: methanol.
4. Use a 10 µL Hamilton syringe to load 1-10 µL of sample in 1 cm bands on a silica HP-TLC plate. Load a maximum of 10 bands on a 20 cm plate. Use normalized values to determine volumes needed to load equal amounts of lipids across all samples.
   NOTE: Before loading each sample, the plate should be activated by placing it on a hot plate at a medium-high setting.
5. Place the plate upright in the TLC tank and allow the solvent front to travel 8 cm to ensure the separation of phospholipid species.
6. Let the plate dry for 10 min. Spray with an aqueous solution of 3% (w/v) cupric acetate and 8% (v/v) phosphoric acid.
   1. Let the plate dry for 30 min at room temperature or with a heat gun. The plate should turn from translucent blue to opaque white.
7. Char plate in a 180 °C-200 °C oven for 5-10 min or until the black lipid bands become detectable.

7. Checking receptor activation with autophosphorylation assay and western blot

1. Incubate cells with 500 µL of 100 nM Insulin in serum-free media at room temperature for 5 min.
2. Wash cells with ice-cold PBS and put cells on ice to halt stimulation. Promptly add 1 mL of ice-cold PBS to the cells and harvest with a cell scraper. Pellet cells at 3000 x g for 5 min at 4 °C.
3. Add 100-200 µL of complete RIPA lysis buffer (50 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 1% (w/v) sodium deoxycholate, 1 mM activated sodium orthovanadate, 10 µg/mL aprotinin, 10 µg/mL leupeptin) to cell pellet on ice. Pipette up and down 30x-40x to lyse.
4. Incubate lysate on ice for 10 min. Clear lysate of cell debris by spinning at 16,000 x g for 10 min at 4 °C and collecting the supernatant. Reserve about 20 µL of lysate to determine protein concentration with Bradford assay.
5. Combine lysate with 5x Laemmli buffer (350 mM Tris HCl pH 6.8, 30% v/v glycerol, 10% w/v SDS, 25% v/v β-mercaptoethanol, 0.002 g of bromophenol blue) and boil at 95 °C for 5 min. The volume of buffer added should be sufficient to achieve a final concentration of 1x Laemmli buffer.
6. Run lysates through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
   1. Load equal mass of protein for all samples along with molecular weight marker onto an SDS-PAGE gel. Run at 100 V-150 V in running buffer (2.5 mM Tris pH 8, 19.2 mM glycine, 0.01% (w/v) SDS) until well resolved in the 100-250 kDa range.
7. Transfer onto a polyvinylidene fluoride (PVDF) membrane in transfer buffer (2.5 mM Tris pH 8, 19.2 mM glycine) for 1 h at 100V and 4 °C.
8. Block PVDF membrane for 30 min at room temperature in 5% BSA solution in TBST (50 mM Tris pH 8, 150 mM NaCl, 0.1% (v/v) Tween 20).
9. Incubate with pYpY IR antibody or IR-β antibody at a 1:1000 concentration in 5% BSA solution in TBST for 1 h at room temperature or overnight at 4 °C.
   NOTE: The pYpY IR antibody specifically recognizes phosphorylated tyrosines 1162 and 1163 and serves to indicate autophosphorylation of the receptor. The IR-β antibody recognizes the beta subunit of the receptor indiscriminately regardless of phosphorylation state and serves to indicate global levels of the receptor.
10. Wash 3x with TBST at room temperature. Incubate with α Rabbit-HRP antibody at a 1:3000 concentration in 1% BSA solution in TBST for 30 min at room temperature.
11. Wash 3x with TBST at room temperature. Incubate with enhanced chemiluminescent (ECL) substrate for 1 min and image the film.

Results

To demonstrate the observable change in cellular lipid composition after the exchange, we performed HP-TLC on CHO IR cells following brain SM (bSM) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) exchange (Figure 1). In cases where sphingomyelins like bSM are being used for exchange, an increase in the SM band intensity is apparent, along with a decrease in PC band intensity relative to the untreated control. Conversely, when exchanging phosphatidylcholines like DOPC, the PC band becomes...

Discussion

Since the conceptualization of the existence of lipid rafts in the cell membrane there have been numerous attempts to visualize them in cells and study lipid and receptor association. Experiments involving microscopy¹¹ in cells used fluorescently tagged biomarkers, usually, proteins and lipids known to associate with rafts, to visually study the localization of ordered lipid domains in the cell¹². However, the cell membrane is full of folds^13,

Materials

Name	Company	Catalog Number	Comments
1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)	Avanti Polar Lipids	850335
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)	Avanti Polar Lipids	850345
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)	Avanti Polar Lipids	850375
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)	Avanti Polar Lipids	850355
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)	Avanti Polar Lipids	850365
1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC)	Avanti Polar Lipids	850457
Anti-insulin receptor β antibody	Cell Signaling Technology	CST3025
Anti-pYpY1162/1163 Insulin receptor antibody	R&D Systems Inc.	AF2507
Anti-rabbit IgG, HRP-linked Antibody	Cell Signaling Technology	7074
Borosilicate glass test tubes (12 x 75 mm)	Thermo Fisher Scientific	14-961-26
Brain sphingomyelin (bSM)	Avanti Polar Lipids	860062
Egg sphingomyelin (eSM)	Avanti Polar Lipids	860061
Fetal bovine serum (FBS)	Corning	35-016-CV
G418 disulfate salt	Sigma Aldrich	A1720
Gibco Antibiotic-antimycotic solution (100x)	Thermo Fisher Scientific	15240062
Gibco Dulbecco’s modified eagle medium (DMEM, 4.5 g/L glucose, L-glutamine, sodium pyruvate)	Thermo Fisher Scientific	11965092
Gibco ham’s F12 media	Thermo Fisher Scientific	11765054
Gibco L-glutamine	Thermo Fisher Scientific	25030032
Gibco MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher Scientific	11140050
Gibco phosphate buffered saline (PBS) without calcium and magnesium (0.144 g/L KH2PO4, 9 g/L NaCl, 0.795 g/L Na2- HPO4 (anhydrous))	Thermo Fisher Scientific	10010023
Gibco Trypsin-EDTA (0.05%), phenol red	Thermo Fisher Scientific	25300054
High performance thin layer chromatography (HP-TLC)	Merck	HP-TLC Silica Gel 60 plates
Immobilon-P PVDF Membrane	Millipore	IPVH00010
Methotrexate	Sigma Aldrich	454126
Methyl-α-cyclodextrin (MαCD)	AraChem	CDexA076/BR
Pierce ECL Western Blotting Substrate	Thermo Fisher Scientific	32106
Sodium orthovanadate, Activated	Sigma Aldrich	5.08605