Detergent-Resistant Membranes and the Use of Cholesterol Depletion

Detergent-Resistant Membranes and the Use of Cholesterol Depletion
I. INTRODUCTION
Lipid rafts have changed our view of membrane organization. Rafts are small platforms in cell membranes, composed of a specialized set of lipids (Ikonen and Simons, 1997). In the exoplasmic leaflet, rafts consist mainly of sphingolipids and cholesterol, which are connected to saturated glycerophospholipids and cholesterol in the cytoplasmic leaflet. Rafts are fluid assemblies, but they are more tightly packed and hence more ordered than the surrounding membrane. This difference is due to the more saturated hydrocarbon chains of sphingolipids and raft-associated glycerophospholipids as compared to the mostly unsaturated glycerophospholipids outside rafts. Cholesterol is thought to fill the spaces between the hydrocarbon chains of the sphingolipids, thus stabilizing rafts.

The raft concept transforms the classical membrane model (Singer and Nicolson, 1972) into a more complex system, in which proteins are embedded in a two-dimensional liquid that is itself a mosaic of lipid microdomains. Membrane proteins partition into raft and nonraft membranes according to their preferred lipid environment. Proteins with a high affinity for ordered membrane domains will mostly reside in rafts. Typical raft-associated proteins include glycosylphosphatidylinositol (GPI)-anchored proteins, doubly acylated proteins such as tyrosine kinases of the src family, and certain integral membrane proteins, especially palmitoylated ones such as caveolin and influenza virus hemagglutinin. Proteins with an intermediate affinity will move in and out of rafts, whereas proteins that do not pack well into ordered domains will be excluded from rafts.

Important for understanding the physiological functions of rafts, e.g., in signal transduction, is that their composition is dynamic and can be regulated (Simons and Toomre, 2000). Some receptors, such as the T- and the B-cell receptor (Janes et al., 2000; Pierce, 2002), have a weak raft affinity in the unligated state. After ligand binding, they undergo a conformational change and/or become oligomerized, which increases their raft affinity and allows them to interact with proteins constitutively present in rafts. Other signalling molecules, such as the endothelial nitric oxide synthase, α subunits of heterotrimeric G proteins, and possibly ras, can undergo signal-induced depalmitoylation, leading to loss of raft association (Bijlmakers and Marsh, 2003; Hancock, 2003). Thus, rafts can serve to segregate signalling molecules at steady state and promote their interaction upon stimulation.

Isolation of detergent-resistant membranes (DRMs) is a simple and useful biochemical method for analyzing the possible raft association of proteins and lipids. Especially when used to monitor changes in DRM composition during processes such as exocytosis (Brown and Rose, 1992) immune cell activation and viral infection (Manes et al., 2000), this technique can provide important clues about the mechanisms underlying protein sorting, signal transduation and pathogen entry into host cells (Ikonen, 2001; Simons and Toomre, 2000; van der Goot and Harder, 2001). Rafts largely resist solubilization with mild detergents due to their tight lipid packing, whereas less ordered membrane domains are disrupted. Raft proteins do not become solubilized, but remain associated with lipids. As a result, they have a lower apparent density than detergent-soluble membrane proteins and can be isolated by equilibrium density centrifugation (often referred to as "flotation"). Detergent lysates are adjusted to high density, placed at the bottom of a density gradient, and centrifuged. Contrary to fully solubilized material, DRMs and DRM-associated proteins float up the gradient, allowing their recovery from low-density fractions. As the integrity of lipid rafts depends on cholesterol, its removal renders raftassociated proteins detergent soluble. Detergent solubility of an otherwise detergent-insoluble protein after cholesterol depletion with methyl-β-cyclodextrin (cyclodextrin) can therefore serve as an additional criterion for raft association.

However, the composition of DRMs may only imperfectly reflect the association of membrane components with lipid rafts in cell membranes (Shogomori and Brown, 2003). DRMs obtained with different detergents differ considerably in their protein and lipid content (Schuck et al., 2003). Different detergents clearly reflect the organization of membranes in different ways, with Triton X-100 and CHAPS seemingly being the most informative ones. In addition, the interpretation of experimental results obtained with cyclodextrin is complicated by the fact that severe cholesterol depletion may have pleiotropic effects on membrane functions. Cyclodextrin might be more effective on cell homogenate than on living cells, but the reasons for this are not completely clear (Schuck et al., 2003).

Finally, neither detergent insolubility nor loss of detergent insolubility after cyclodextrin treatment is a strict criterion. If a protein is detergent soluble, it may still have a weak affinity for rafts in native membranes, and persistent detergent insolubility after cholesterol depletion can be caused by the remaining cholesterol. Therefore, neither criterion allows excluding raft association.

II. MATERIALS AND INSTRUMENTATION
Standard chemicals are of the highest purity available. Minimal essential medium with Earle's salts (MEM, Cat. No. 21090-022), glutamine (Cat. No. 25030- 024), and penicillin-streptomycin (Cat. No. 15140-122) are from Invitrogen. Fetal calf serum (FCS, Cat. No. A15-042) is from PAA Laboratories. One hundred-millimeter (Cat. No. 150350) and 35-mm (Cat. No. 153066) plastic tissue dishes are from Nunc. Chymostatin (Cat. No. C7268), leupeptin (Cat. No. L2884), antipain (Cat. No. A6191), pepstatin (Cat. No. P5318), DL-mevalonic acid lactone (Cat. No. M4667), and methyl-β-cyclodextrin (Cat. No. C4555) are from Sigma. Lovastatin (Cat. No. 438185) is from Merck Biosciences. Sixty percent (w/v) iodixanol (Optiprep, Cat. No. 1030061) is from Progen Biotechnik, sucrose (Cat. No. 21938) is from USB, and 10% (w/v) Triton X-100 (Surfact-Amps X- 100, Cat. No. 28314) is from Perbio.

Ultraclear centrifuge tubes for an SW40 rotor (14 × 95mm, Cat. No. 344060) and for a TLS55 rotor (11 × 34 mm, Cat. No. 347356), as well as SW 40 Ti and TLS55 rotors, are from Beckman. Ultracentrifugations are carried out using an Optima XL-100K or an Optima MAX ultracentrifuge from Beckman.

In addition, the following equipment is required: 1.5-ml microfuge tubes, cell scrapers, 15-ml plastic tubes, 25-gauge needles, and 1-ml plastic syringes.

III. PROCEDURES
A. Preparation of DRMs by Flotation on a Sucrose Step Gradient
Solutions

1. Phosphate-buffered saline (PBS): 155mM NaCl, 1.5 mM KH₂PO₄, 2.7 mM Na₂HPO₄, pH 7.2. To make 1 litre, dissolve 0.21 g KH₂PO₄, 0.73g Na₂HPO₄·7H₂O, and 9 g NaCl in double-distilled water and make up to 1 litre.
2. 1 M EDTA: To make 1 litre, dissolve 372.2 g EDTA in double-distilled water by adjusting the pH to 8 with NaOH and make up to 1 litre.
3. 5× TNE: 750 mM NaCl, 10 mM EDTA, 250 mM Tris-HCl, pH 7.4. To make 1 litre, dissolve 43.8 g NaCl and 30.3g Tris in double-distilled water, add 10ml 1 M EDTA, adjust pH to 7.4 with HCl, and make up to 1 litre.
4. 1000× CLAP: To make 1 ml, dissolve 25 mg each of chymostatin, leupeptin, antipain, and pepstatin in 1 ml dimethyl sulfoxide and store at -20°C in small aliquots.
5. TNE and TNE with protease inhibitors (TNE+): To make TNE, dilute 5× TNE 1:5 in double-distilled water. To make TNE+, dilute 1000× CLAP 1:1000 in TNE just before use. TNE+ has to be prepared freshly each time.
6. 56, 35, and 5% (w/w) sucrose: To make 100 ml, dissolve 70.75, 40.29, or 5.1 g sucrose in 20ml 5× TNE and bring to 100ml with double-distilled water. Check refractive indices of the resulting solutions, which should be 1.266, 1.154, and 1.021, respectively. Store at 4°C.
7. 2% (w/v) Triton X-100: To make 10ml, dissolve 0.2 g Triton X-100 in 2ml 5× TNE and bring to 10ml with double-distilled water. For short-term storage, keep at 4°C and protect from light to prevent autoxidation. For long-term storage, store aliquots at -20°C.

Steps

1. Grow MDCK strain II cells in MEM with 5% FCS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin on a 10-cm plastic tissue dish until confluent.
2. All subsequent steps are performed at 4°C unless stated otherwise. Remove culture medium and wash the cells once with PBS and once with TNE.
3. Collect the cells in 1 ml TNE with a cell scraper. Transfer suspension into a 1.5-ml microfuge tube and centrifuge for 5 min at 350×g.
4. Resuspend the cell pellet in 550µl TNE+. Homogenize by 20 passages through a 25-gauge needle fitted on a 1-ml syringe. This treatment should break >90% of the cells without damaging nuclei (check microscopically).
5. Take 500gl cell homogenate (about 1 mg total protein) and transfer into a new microfuge tube. Add 500µl 2% Triton X-100. From now on, strictly avoid warming of the sample. Mix well by inverting the tube and place on ice for 30min.
6. Transfer the sample into a 15-ml tube and bring to 40% (w/w) sucrose by adding 2ml 56% sucrose. Mix well by inverting the tube.
7. Place the sample at the bottom of an SW40 centrifuge tube. Sequentially overlay with 8.5 ml 35% sucrose and 0.5 ml 5% sucrose.
8. Centrifuge for 18 h at 39,000rpm (271,000 ×g) using an SW40 rotor. DRMs float to the top of the gradient during centrifugation. Carefully collect 2.5ml from the top of the gradient with a smooth pipette.
9. To further concentrate DRMs, dilute 1:4 by adding 7.5ml TNE and transfer into a new SW40 centrifuge tube. Following centrifugation for 2h at 24,000 rpm (100,000 ×g) with an SW40 rotor, DRMs can be recovered from the pellet.

B. Analysis of DRMs by Flotation on a Linear Sucrose Gradient
Solutions

1. Phosphate-buffered saline (PBS): 155mM NaCl, 1.5 mM KH₂PO₄, 2.7 mM Na₂HPO₄, pH 7.2. To make 1 litre, dissolve 0.21 g KH₂PO₄, 0.73g Na₂HPO₄·7H₂O, and 9 g NaCl in double-distilled water and make up to 1 litre.
2. 1 M EDTA: To make 1 litre, dissolve 372.2 g EDTA in double-distilled water by adjusting the pH to 8 with NaOH and make up to 1 litre.
3. 5× TNE: 750 mM NaCl, 10 mM EDTA, 250 mM Tris-HCl, pH 7.4. To make 1 litre, dissolve 43.8 g NaCl and 30.3g Tris in double-distilled water, add 10ml 1 M EDTA, adjust pH to 7.4 with HCl, and make up to 1 litre.
4. 1000× CLAP: To make 1 ml, dissolve 25 mg each of chymostatin, leupeptin, antipain, and pepstatin in 1 ml dimethyl sulfoxide and store at -20°C in small aliquots.
5. TNE and TNE with protease inhibitors (TNE+): To make TNE, dilute 5× TNE 1:5 in double-distilled water. To make TNE+, dilute 1000× CLAP 1:1000 in TNE just before use. TNE+ has to be prepared freshly each time.
6. 56, 35, and 5% (w/w) sucrose: To make 100 ml, dissolve 70.75, 40.29, or 5.1 g sucrose in 20ml 5× TNE and bring to 100ml with double-distilled water. Check refractive indices of the resulting solutions, which should be 1.266, 1.154, and 1.021, respectively. Store at 4°C.
7. 2% (w/v) Triton X-100: To make 10ml, dissolve 0.2 g Triton X-100 in 2ml 5× TNE and bring to 10ml with double-distilled water. For short-term storage, keep at 4°C and protect from light to prevent autoxidation. For long-term storage, store aliquots at -20°C.

Steps

1. Grow MDCK strain II cells in MEM with 5% FCS, 2 mM glutamine, and 100 U/ml penicillin and streptomycin on a 10-cm plastic tissue dish until confluent.
2. All subsequent steps are performed at 4°C unless stated otherwise. Remove culture medium and wash the cells once with PBS and once with TNE.
3. Collect the cells in 1 ml TNE with a cell scraper. Transfer suspension into a 1.5-ml microfuge tube and centrifuge for 5 min at 350×g.
4. Resuspend the cell pellet in 550µl TNE+. Homogenize by 20 passages through a 25-gauge needle fitted on a 1-ml syringe. This treatment should break >90% of the cells without damaging nuclei (check microscopically).
5. Take 500gl cell homogenate (about 1 mg total protein) and transfer into a new microfuge tube. Add 500µl 2% Triton X-100. From now on, strictly avoid warming of the sample. Mix well by inverting the tube and place on ice for 30min.
6. Transfer the sample into a 15-ml tube and bring to 40% (w/w) sucrose by adding 2ml 56% sucrose. Mix well by inverting the tube.
7. Using a gradient maker, prepare a linear 5-35% sucrose gradient in an SW40 centrifuge tube with 4.5 ml each of 35 and 5% sucrose. Cool to 4°C.
8. Place the sample under the gradient using a Pasteur pipette, disturbing the gradient as little as possible (alternatively, first transfer the sample into an SW40 centrifuge tube and then overlay with the gradient).
9. Centrifuge for 18h at 39,000 rpm (271,000 ×g) using an SW40 rotor. Collect twelve 1-ml fractions from the top of the gradient and resuspend the pellet in 1 ml TNE. Fractions 9-12 contain the soluble material; DRMs are found in the lighter fractions.

C. Analysis of DRMs by Flotation on an Optiprep Step Gradient
Solutions
PBS, 5× TNE, TNE, and TNE+; see Section III,A.

1. 10% (w/v) Triton X-100: To make 10 ml, dissolve 1 g of Triton X-100 in 2 ml of 5× TNE and make up to 10ml with double-distilled water. Alternatively, use Surfact-Amps X-100.
2. 30% (w/v) iodixanol: To make 5ml, mix 2.5ml Optiprep, 1 ml 5× TNE, and 1.5ml double-distilled water.

Steps

1. Grow MDCK strain II cells to confluence on a 3.5-cm plastic tissue dish.
2. At 4°C, remove culture medium and wash the cells once each with PBS and TNE.
3. Collect and spin down the cells as in Section III,A.
4. Resuspend the cell pellet in 200µl TNE+. Homogenize as in Section III,A.
5. Take 180 µl cell homogenate and transfer into a new microfuge tube. Add 20µl 10% Triton X-100 solution, mix, and place on ice for 30min.
6. Add 400µl Optiprep to bring the sample to 40% (w/v) iodixanol and mix well by inverting the tube.
7. Place the sample at the bottom of a TLS55 centrifuge tube. Sequentially overlay with 1.2ml 30% iodixanol and 0.2ml TNE.
8. Centrifuge for 2h at 55,000rpm (259,000×g) with a TLS55 rotor. Collect two fractions of 1 ml each. The top fraction contains the DRMs.

D. Cholesterol Depletion of Live Cells
Solutions
PBS; see Section III,A.

1. 20mM lovastatin: To make 2.5 ml, dissolve 20mg lovastatin in 0.5ml 100% ethanol and 0.75ml 0.1M NaOH. Incubate at 50°C for 2 h, cool briefly on ice, and adjust pH to 7.2 with HCl. Bring to 2.5 ml with doubledistilled water and store aliquots at -20°C.
2. 500mM mevalonate: To make 5ml, dissolve 325 mg in 5 ml PBS. Store aliquots at -20°C.
3. 100mM methyl-β-cyclodextrin in MEM: To make 1 ml, dissolve 150mg methyl-β-cyclodextrin in MEM just before use and make up to 1 ml with MEM.

Steps

1. Grow MDCK strain II cells on a plastic tissue dish or on filter supports. For the last 48 h before the experiment, culture the cells in normal medium supplemented with 4µM lovastatin (dilute stock solution 1:5000) and 0.25 mM mevalonate (dilute stock solution 1:2000).
2. Wash the cells twice with PBS and add MEM containing 4µM lovastatin, 0.25mM mevalonate, and 10mM methyl-β-cyclodextrin. In the case of filtergrown cells, apply cyclodextrin to both the apical and the basolateral side.
3. Incubate the cells at 37°C for 30min.

E. Cholesterol Depletion of Cell Homogenate
Solutions
PBS, TNE, TNE+, and 10% (w/v) Triton X-100; see Sections III,A and III,C.

1. 100mM methyl-β-cyclodextrin in TNE: To make 100µl, dissolve 15mg methyl-β-cyclodextrin in TNE just before use and make up to 100 µl with TNE.

Steps

1. Grow MDCK strain II cells to confluence on a 3.5-cm plastic tissue dish.
2. At 4°C, remove culture medium and wash the cells once each with PBS and TNE.
3. Collect and spin down the cells as in Section III,A.
4. Resuspend the cell pellet in 200µl TNE+. Homogenize as in Section III,A.
5. Take 180µl cell homogenate and transfer into a new microfuge tube. Add 20µl 100mM methyl-β- cyclodextrin, mix, and incubate at 37°C for 30min.
6. For subsequent DRM analysis, cool to 4°C, add 20 µl 10% Triton X-100, and incubate on ice for 30 min.
7. Add 440µl Optiprep to bring to 40% (w/v) iodixanol. Mix and take 600 µl and continue as in Section III,C, step 7.

IV. COMMENTS
Separating DRMs from detergent-soluble material by flotation using equilibrium density centrifugation is usually preferable to pelleting. Pelleting of DRMs from detergent extracts leads to contamination with other sedimentable material, e.g., cytoskeletal components. In addition, a protein might be pelleted because of its association with the cytoskeleton rather than DRMs.

Which flotation gradient to use for DRM analysis depends on the purpose of the experiment. Continuous gradients give more information about the flotation behaviour of proteins and lipids, but to monitor changes in DRM composition after, e.g., cyclodextrin treatment, simple step gradients as in Section III,C are more convenient. Various density gradient media can be used, and the choice between sucrose and iodixanol is largely a question of personal preference. However, sucrose might be superior if lipids are to be analyzed by a very sensitive method such as mass spectrometry, as iodixanol seems to have a weak tendency to follow lipids during extraction from DRMs.

How to apply cyclodextrin strongly depends on the cell type used. Hippocampal neurons cannot be exposed to 5 mM cyclodextrin for longer than 20min (Simons et al., 1998), whereas MDCK cells remain intact even when treated with 20mM for 1 h. Hence, the conditions for cyclodextrin treatment have to be established in each case. In particular, it needs to be ensured that cholesterol is the only lipid extracted. Treating live cells will preferentially extract cholesterol from the plasma membrane and allows intracellular cholesterol transport to counteract the effects of cyclodextrin, whereas extracting cholesterol from cell homogenate will affect all cell membranes.

V. PITFALLS
Membrane solubilisation by detergent depends on the molar ratio of detergent to lipid. Therefore, this parameter, rather than detergent concentration alone, always needs to be taken into account. In the case of Triton X-100, a mass ratio of detergent to protein (taken as a measure for the molar detergent-to-lipid ratio) of greater than 5:1 seems to be necessary for maximum solubilization (Ostermeyer et al., 1999).

DRM-associated proteins are separated from soluble proteins by flotation gradients because of their low density; they float due to their detergent-resistant association with lipids. In contrast, the separation of DRMs from detergent micelles, which contain solubilized membrane lipids, is based largely on differences in size. Given enough time, detergent micelles will also float as a result of their low density. However, DRMs are larger than detergent micelles and therefore float up faster [the sedimentation or flotation velocity s is given by s = V(ρ - ρ_m)/f, where V is particle volume, p is particle density, Pm is density of the solvent, and f is friction coefficient. For a spherical particle, V is proportional to r³, while f is proportional to r, so that s is proportional to r². Refer to biophysical textbooks for a more comprehensive treatment]. To avoid contamination of DRMs with detergent molecules and solubilized lipids, it is crucial to ensure that detergent micelles are well separated from DRMs under the centrifugation conditions used. The distribution of Triton in the gradient can be measured by its absorption at 280nm. For other detergents, their distribution can be determined semiquantitatively by thin-layer chromatography.

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