Characterization of Lipid Order and Domain Formation in Model Membranes Using Fluorescence Microscopy and Spectroscopy

Abstract

Fluorescence-based techniques have been an integral factor in the study of cellular and model membranes. Fluorescence studies carried out on model membranes have provided valuable structural information and have helped reveal mechanistic detail regarding the formation and properties of ordered lipid domains, commonly known as lipid rafts. This chapter focuses on four techniques, based on fluorescence spectroscopy or microscopy, which are commonly used to analyze lipid rafts. The techniques described in this chapter may be used in a variety of ways and in combination with other techniques to provide valuable information regarding lipid order and domain formation, especially in model membranes.

1. Introduction

The plasma membrane has long been of interest to molecular biologists, biochemists, and biophysicists as it represents the first point of contact between the cell and foreign substances. The membrane plays a crucial role in a variety of important biological functions, such as cell adhesion, cell signaling, and the transport of molecules in and out of the cell [1, 2]. The membrane’s functions depend on the structure of its lipid bilayer. Lipid bilayers are not static constructs, rather they display remarkably complex dynamics. The membrane may exhibit various degrees of phase segregation depending on its composition and environment. Two phases are typically observed in cell membranes: the liquid disordered phase (L_d), which is a fluid phase characteristic of loosely packed lipids and high lateral mobility of lipids and membrane proteins, and the liquid ordered phase (L_O), which has a high degree of lipid packing order and more restricted lateral fluidity [3]. Of growing interest in membrane research is the influence that microdomains of densely packed saturated sphingolipids, cholesterol, and associated proteins have on membrane function and interactions. These microdomains, commonly referred to as lipid rafts, are hypothesized to play special roles in cell signaling, protein trafficking, and many other biological functions [4]. In this chapter, we outline some of the fluorescent techniques commonly used for the study of membrane rafts.

One of the more common fluorescent-based techniques used in the study of lipid rafts is the measurement of steady-state fluorescence anisotropy. Such data is often analyzed to provide information on lipid order and packing in the lipid bilayer. In this technique, a fluorescent probe must first be incorporated into a suspension of vesicles. When excited by a polarized light source, the probe emits light and intensity is measured along each of the axes of polarization. The ratio of polarized emitted light intensity to total emitted light intensity provides the anisotropy, a value between zero and one [5]. When polarized light strikes a homogenous suspension of vesicles with incorporated fluorophore, those fluorophores which have dipole moments randomly oriented along the axis of polarization will be preferentially excited [6]. Thus, the light emitted by the fluorophores is not randomly oriented, rather the emitted light is also polarized (Fig. 1a). When fluorophores are free to rotationally diffuse in suspension, and if this diffusion occurs at a timescale comparable to the absorption and reemission of light, the reorientation of fluorophores can diminish the polarizing effect (Fig. 1b) [6]. A fluorophore’s ability to rotationally reorient itself depends on its size and shape and on the viscosity of the suspension [6]. Thus, a lipid molecule that is rigidly packed in a membrane (highly saturated lipids tend to exhibit this behavior) will experience less reorientation following excitation and the emitted light will retain polarization, resulting in a high anisotropy value. Loosely packed lipids, such as highly unsaturated lipids, tend to experience greater reorientation and thus polarization of emitted light will be diminished, resulting in a low anisotropy value.

Fig. 1.

Fluorescence anisotropy values depend on the property of emitted light upon fluorophore excitation by polarized light source. (a) High anisotropy: fluorophore experiences little spatial reorientation in an ordered membrane. (b) Low anisotropy: fluorophore experiences spatial reorientation at a timescale comprable to absorption and emission in a disordered membrane

Another widely used technique in lipid raft research is the measurement of Förster Resonance Energy Transfer (FRET) between two fluorophores. FRET is used to provide information on lipid phase segregation. FRET describes the nonradiative transfer of energy from one chromophore to another. The molecule which energy is transferred from is referred to as the “donor” and the molecule to which energy is transferred is the “acceptor”; the two constitute a FRET pair. FRET requires that the emission spectrum of the donor molecule overlap with the excitation spectrum of the acceptor molecule. The efficiency of the energy transfer process between the pair is dependent on the inverse sixth power of the distance between the FRET pair, making FRET highly sensitive to measurements of distances in the 1–10 nm range [7]. FRET has been adapted to lipid order studies by conjugating small fluorescent probes on to the head groups of various lipids. One member of a FRET pair may be attached to a lipid known or thought to exhibit ordered packing behavior (such as a saturated lipid) and the other member may be attached to a lipid that exhibits less ordered packing behavior (such as an unsaturated lipid). FRET efficiency can be measured as temperature and membrane composition is varied [8]. Changes in FRET efficiency are related to changes in FRET pair proximity. A high FRET efficiency indicates little to no segregation of donor and acceptor probe in space, a low efficiency indicates that the donor and acceptor have segregated into distinct domains. The concept of FRET is displayed schematically in Fig. 2.

Fig. 2.

Use of FRET technique to determine model membrane order. Low FRET indicates segregation of donor and acceptor probes into distinct domains. High FRET indicates a homogeneous bilayer with donor and acceptor lipids randomly dispersed throughout the membrane

Lipid rafts in cell membranes are typically too small (100 nm or less) to be observed via conventional microscopy. Researchers have developed methods to create giant unilamellar vesicles (GUVs) that exhibit micron sized domains; these methods include the gentle hydration method, gel assisted swelling, and the droplet transfer method. A more commonly used process for creating GUVs is the electroformation technique. Using this technique, an electric field is applied to an aqueous solution of lipids, causing them to swell into vesicles of a large size [9]. GUVs may be incorporated with fluorescently labeled lipid probes and imaged under fluorescent microscope. Confocal microscopy with z-stacking can be used to provide three-dimensional images of GUVs. The clustering of the probe into distinct regions of the vesicle provides visual evidence of domain formation. GUV imaging is more qualitative in nature than quantitative and is thus often performed alongside other techniques (such as those already mentioned) to reinforce their discoveries.

The leakage assay is another fluorescence-based technique commonly used in membrane studies [10]. While the assay does not provide the order of a membrane or detail the phases present in the bilayer, it is used to examine membrane stability of raft-forming membranes and the interactions between membranes and particles. This assay is used to determine when a model membrane has been disrupted, causing its contents to leak out from the interior of the membrane. The assay involves incorporating a self-quenching fluorescent dye into the membrane during the vesicle assembly step. When present inside a vesicle at concentrations above the self-quenching concentration, the fluorescent dye is quenched. However, when the membrane is disrupted, dye leaks from the membrane and is diluted, resulting in a spike in fluorescence (Fig. 3). This assay can be used to study model membrane integrity over time, across a range of temperatures, or under various conditions (such as exposure to foreign materials). A per cent leakage can be quantified by treating a vesicle solution with strong detergent, completely disrupting all membranes. The fluorescence measured after such treatment coincides with 100% leakage, or total disruption of the vesicle.

Fig. 3.

Upon disruption of model membrane, the self-quenching dye is released from the vesicle interior and fluoresces

2. Materials

2.1. Fluorescence Anisotropy

2.1.1. Reagents

1. Dry powder lipids, selected based on desired vesicle composition.

2. HPLC grade organic solvent, such as chloroform.

3. Phosphate buffered saline solution (PBS: 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) or other desired buffer solution.

4. Lipophilic fluorescent probe, such as 1,6-diphenyl-1,3,5-hexatriene (DPH).

5. Acetone.

2.1.2. Equipment

1. Micro dispenser compatible with strong organic solvent.

2. Desiccator, SpeedVac™, or similar solvent remover.

3. Heated water bath.

4. Membrane extruding device.

5. High sensitivity spectrofluorometer.

2.2. FRET

2.2.1. Reagents

1. Dry powder lipids.

2. Additional lipid probes with conjugated FRET pair (e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl), abbreviated: NBD-DPPE; and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, abbreviated Rho-DOPE; see Note 1).

3. HPLC grade organic solvent.

4. PBS or other desired buffer.

2.2.2. Equipment

1. Micro dispenser compatible with strong organic solvent.

2. Desiccator, SpeedVac™, or similar solvent remover.

3. Heated water bath.

4. Membrane extruding device.

5. High sensitivity spectrofluorometer.

2.3. GUV Imaging

2.3.1. Reagents

1. Dry powder lipids.

2. Fluorescently tagged lipid marker (such as Rho-DOPE).

3. HPLC grade organic solvent.

4. Purified water (18 MΩ cm).

2.3.2. Equipment

1. Chamber in which to conduct electro-formation method (see Note 6).

2. Indium tin oxide coated coverslips.

3. Desiccator.

4. Grease for sealing.

5. Hot plate.

6. Copper tape.

7. Electric field generator with alligator clip electrodes.

8. Confocal microscope.

2.4. Leakage Assay

2.4.1. Reagents

1. Dry powder lipids.

2. Self-quenching fluorescent dye, such as 6-carboxyfluorescein (CF).

3. HPLC grade organic solvent.

4. PBS or other desired buffer.

5. Strong detergent, such as Triton-X.

2.4.2. Equipment

1. Micro dispenser compatible with strong organic solvent.

2. Desiccator, SpeedVac™, or similar solvent remover.

3. Heated water bath.

4. Membrane extruding device.

5. Desalting columns.

6. High sensitivity spectrofluorometer.

3. Methods

3.1. Fluorescence Anisotropy

1. Prepare lipid stock solutions by dissolving dry powder lipids in organic solvent.

2. Mix lipids by pipetting aliquots of lipid stock solutions into glass tube at desired concentrations.

3. Remove organic solvent from lipid mixture such that only a thin film of lipids remains at bottom of glass tube (drying under nitrogen and placing under vacuum for several hours is sufficient; alternatively, use a SpeedVac™).

4. Heat lipids and PBS in water bath for 5–15 min to facilitate next step; select a temperature for the water bath above the melting temperature of individual lipid components.

5. Rehydrate lipids with appropriate volume of PBS (or similar buffer) to achieve desired concentration of resuspended lipids.

6. Vortex mix rehydrated lipid suspension until opaque white lipid film is completely removed from bottom of glass tube; this step assembles multilamellar vesicles (MLVs).

7. If large unilamellar vesicles (LUVs) are desired, expose MLV suspension to several cycles of freeze/thaw by placing in acetone chilled by dry ice and removing to heated water bath (see Note 1).

8. If vesicles of uniform size are desired, pass vesicle suspension through membrane extruder.

9. Dissolve lipophilic fluorescent probe in acetone and add to MLV or LUV suspension to incorporate probe, required concentration of probe may vary based on chemical identity, for DPH, 0.2 mol% is sufficient (see Note 2).

10. Conduct anisotropy measurements of fluorescent probe at appropriate excitation and emission wavelengths using a spectrofluorometer (measurements can be made over a range of temperatures to observe possible melting temperature and phase transition of lipids in system).

3.2. FRET

1. Prepare lipid stock solutions by dissolving dry powder lipids in organic solvent.

2. Mix lipids in glass tubes, creating four samples (see Note 3):

   Sample 1: Vesicles that contain both donor and acceptor lipid probes, referred to as F sample.

   Sample 2: Vesicles with acceptor lipid probe, to serve as blank for F sample.

   Sample 3: Vesicles with donor lipid probe, referred to as F₀ sample.

   Sample 4: Vesicles without lipid probe, to serve as blank for F₀ sample (see Note 4).

3. Remove organic solvent from lipid mixture such that only a thin film of lipids remains at bottom of glass tube.

4. Heat lipids and PBS in water bath for 5–15 min to facilitate next step; select a temperature for the water bath above the melting temperature of individual lipid components.

5. Rehydrate lipids with appropriate volume of PBS (or similar buffer) to achieve desired concentration of resuspended lipids.

6. Vortex mix rehydrated lipid suspension until opaque lipid film is completely removed from bottom of glass tube; this step assembles multilamellar vesicles (MLVs).

7. If vesicles of uniform size are desired, pass vesicle suspension through membrane extruder (see Note 5).

8. Conduct fluorescence measurements of samples using appropriate excitation and emission wavelengths corresponding to donor probe spectrum.

9. After subtracting blank fluorescence from F and F₀ vesicles, calculate a value for F/F₀, FRET efficiency as a percent is equivalent to (1 – F/F₀) × 100.

3.3. GUV Imaging

1. Prepare lipid stock solutions by dissolving dry powder lipids in organic solvent.

2. Mix lipids by pipetting aliquots of lipid stock solutions into glass tube at desired concentrations. For the purposes of imaging GUVs under microscope, it is best to keep total lipid concentration close to 1 mg/mL.

3. Add probe to lipid mixture at effective concentration (approx. 0.2 mol% for Rho-DOPE).

4. Streak a small volume (1–1.5 μL) of lipid mixture across indium tin oxide coated coverslip, taking special care to apply lipids near the center of the slip.

5. Place coverslip in desiccator to remove organic solvent (this solvent removal method is preferred over others that might splash lipids from the coverslip, such as drying with nitrogen).

6. Add one strip of conductive copper tape to a corner of each coverslip (lipid coverslip and a clean coverslip) on the conductive side.

7. Place dried coverslip and clean coverslip on each side of central channel piece, such that the coverslips “sandwich” the central channel and that the lipids face in toward the clean slip through the hole in the plate. Copper tape should be accessible to electrodes. Coverslips and channel piece should be sealed with grease and placed in the chamber frame.

8. Inject purified water into the channel and place entire chamber on hotplate at 70 °C.

9. Connect electrodes to copper tape and set generator to 0.7 V and 10 Hz.

10. After 2 h, turn off hotplate and let generator run for another 2 h.

11. Perform confocal microscopy on sample, being careful not to disturb chamber during transfer to microscope (see Note 6 for chamber assembly).

3.4. Leakage Assay

1. Prepare lipid stock solutions by dissolving dry powder lipids in organic solvent.

2. Mix lipids by pipetting aliquots of lipid stock solutions into glass tube at desired concentrations.

3. Remove organic solvent from lipid mixture such that only a thin film of lipids remains at bottom of glass tube.

4. Prepare rehydration solution by dissolving self-quenching fluorescent dye in PBS or similar buffer solution such that concentration of dye is above the self-quenching concentration; depending on the dye used, pH adjustment may be necessary to completely dissolve dye.

5. Heat dried lipids and rehydration solution in water bath at high temperature (above T_m of individual lipid components).

6. Rehydrate dry lipids with appropriate volume of previously prepared solution containing self-quenching dye to achieve desired lipid concentration.

7. Vortex-mix the solution to remove lipids from bottom of tube and create a suspension of MLVs; MLVs will be incorporated with self-quenching dye.

8. If large unilamellar vesicles (LUVs) are desired, expose MLV suspension to several cycles of freeze and thaw (this step will produce LUVs from MLVs, reduce vesicle size, and help to distribute dye to vesicles).

9. If vesicles of uniform size are desired, pass vesicle suspension through membrane extruder.

10. Remove unincorporated dye from solution by passing solution through desalting column.

11. Conduct fluorescence measurements at appropriate wavelengths; if measuring leakage as a function of time after exposure to foreign particles, be sure to also measure fluorescence of a blank sample without added particles.

12. After acquiring fluorescence data over desired time period, add strong detergent to samples to totally disrupt vesicles.

13. Calculate a % leakage for all time points by subtracting the blank value at time zero and taking the ratio of fluorescence before addition of detergent to that of fluorescence after addition of detergent.

14. A baseline leakage can be calculated by performing the same calculation using data from the blank sample.

Fig. 4.

(Left) Chamber for the synthesis of GUVs: (1) Top plate, (2) central plate with tubes attached for water injection, (3) bottom plate, screws for assembly, and water tube clamp, (4) conductive coverslip with dried lipids and copper tape on conductive side (to be placed between top plate and central plate, conductive side down), (5) clean conductive coverslip (placed between bottom plate and central plate, conductive side up, such that the conductive sides of both slips are facing each other, both slips sealed with grease), (6) syringe for water injection. (Right) Assembled chamber (not pictured, hot plate on which chamber is placed)