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Detergent and Detergent-Free Methods to Define
Lipid Rafts and Caveolae
Rennolds S. Ostrom and Xiaoqiu Liu
Summary
Lipid rafts and their related membrane vesicular structures, caveolae, are cholesterol- and sphingolipid-rich
microdomains of the plasma membrane that have attracted considerable interest because of their ability to concentrate numerous signaling proteins. Efforts to define the proteins that reside in lipid rafts and caveolae as well
as investigations into the functional role of these microdomains in signaling, endocytosis, and other cellular
processes have led to the hypothesis that they compartmentalize or prearrange molecules involved in regulating
these pathways. This chapter describes biochemical approaches for defining lipid rafts and caveolae. Included are
detergent- and nondetergent-based fractionations on sucrose-density gradients that isolate buoyant lipid rafts and
caveolae as well as caveolin antibody-based immunoisolation of detergent-insoluble membranes that selectively
isolates caveolae and not lipid rafts. Also, a general method to disrupt lipid rafts and caveolae using β-cyclodextrin that is useful for probing the role of these microdomains in cellular processes is described. The advantages
and disadvantages of the respective approaches are discussed. Taken together, these methods are useful for defining the role of lipid rafts and caveolae in cell signaling.
Key Words: β-cyclodextrin; caveolae; density gradient centrifugation; immunoisolation; lipid rafts; membrane microdomains.
1. Introduction
Lipid rafts are plasma membrane microdomains formed through the association of sphingolipid and cholesterol that have rapidly become recognized as important to many types of cellular signal transduction. Lipid rafts along with caveolae, which are thought to form from lipid
rafts because of their similar lipid composition, appear to be signaling “hot spots” because of
their ability to attract and retain numerous and diverse signaling molecules (1). Thus, caveolae and lipid rafts concentrate, and perhaps, promote the formation of signaling complexes that
are essential for rapid and specific signal transduction (2,3). In this chapter, caveolae and lipid
rafts will be introduced, their similarities and differences with respect to how they can be studied will be discussed, and several detailed methodologies for defining the proteins associated
with lipid raft and caveolar structures will be presented.
Caveolae were originally identified in endothelial cells as 50–100-nm flask-like invaginations of the plasma membrane (4). Caveolae were later shown to be involved in the transcellular movement of molecules (potocytosis and endocytosis) (5). Endocytosis by caveolae
represents a parallel but distinct pathway from clathrin-coated pits for the removal and destruction or recycling of plasma membrane receptors (5,6). Caveolae are similar to lipid rafts in that
they are both enriched in sphingolipid and cholesterol, but caveolae also express a coat of
From: Methods in Molecular Biology, vol. 400: Methods in Membrane Lipids
Edited by: A. M. Dopico © Humana Press, Inc., Totowa, NJ
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caveolin proteins on the inner leaflet of the membrane bilayer (7). All mammalian cells appear
to contain plasma membrane-lipid rafts because of the ubiquitous nature of sphingolipid and
cholesterol, but only cells expressing one of the three isoforms of caveolin appear to contain
caveolae (8). Of the three isoforms of caveolin, caveolin-1, -2, and -3 only caveolin-1 (the predominant isoform) and caveolin-3 (the striated muscle-specific isoform) are capable of inducing caveolar biogenesis (9,10). Caveolin-2, when expressed alone, cannot induce caveolar
formation, but this isoform is found in hetero-oligomers with caveolin-1 and caveolin-3
(11–15). Whereas it is unclear whether different caveolin isoform compositions create functionally distinct caveolae (16), it is clear that lipid rafts and caveolae differ in a variety of ways
(17–19). Approaches to differentiate between lipid rafts and caveolae as well as to manipulate
caveolin expression in cells, will likely lead to much more information regarding the differences
between these structures in the near future.
Despite recent advances in microscopic approaches (including atomic force microscopy to
visualize lipid rafts [20]) lipid rafts and caveolae are most readily defined using biochemical
approaches. Lipid rafts and caveolae can be extracted from other cellular material in cell
homogenates based on their relative insolubility in particular detergent or nondetergent conditions and their high buoyancy when centrifuged on a density gradient. These approaches, a
few of which are described in this chapter, rely on properties common to both caveolae and
lipid rafts, thus cannot distinguish between these domains. Caveolar domains can be specifically isolated using immunological approaches to trap caveolin proteins from plasma membrane preparations (21,22). One method that is applicable for specifically isolating caveolae
and not lipid rafts from numerous types of cells and tissues is described in this chapter.
The function of lipid rafts and caveolae in signaling or other cellular processes can also be
inferred from studies in which the microdomains are disrupted. β-cyclodextrin, a chemical
that does not enter cells but can bind cholesterol and remove it from the plasma membrane, disrupts lipid rafts and caveolae (23,24). Filipin, a polyene antibiotic and sterol-binding agent, also
disrupts lipid rafts and caveolae (25,26). This chapter describes a method using β-cyclodextrin
to disrupt lipid rafts and caveolae that can be applied in many experimental paradigms. For
more detailed information regarding the manipulation of cellular cholesterol using cyclodextrins, refer to Christian et al. (27).
Microscopic as well as other types of nonbiochemical studies are often important for
corroborating results from biochemical studies of lipid rafts and caveolae (see Note 1).
Microscopic approaches for defining caveolae are limited to electron microscopic studies,
because the resolution of light (including fluorescent microscopy) precludes the detection of
50–100 nm structures. However, laser confocal microscopy using deconvolution can be used
to define the colocalization of two proteins, and when combined with detection of caveolins,
can infer caveolar localization (28). Microscopy is also limited by the suitability of antibodies to detect native proteins of interest in fixed cells and tissues. One can utilize fluorescent
or epitope tags to circumvent the antibody problem and take advantage of other powerful
technologies such as fluorescence resonance energy transfer and bioluminescence energy
transfer that define the interaction of components in living cells (29). However, the usefulness of tagged proteins is limited to the examination of exogenously expressed proteins,
which can localize differently than their native counterparts (30). Thus, a combination of
biochemical methods, several of which are described herein, and microscopic approaches
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(described in ref. 28) should be used to provide the most complete definition of lipid rafts
and caveolae.
2. Materials
1. Phosphate-buffered saline (PBS) 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4.
2. 500 mM Na2CO3 (should be ~pH 11.0, but do not adjust).
3. MES buffered saline (MBS): 25 mM 2-(N-Morpholino) ethanesulfonic acid 4-Morpholineethane
sulfonic acid (MES), 150 mM NaCl, pH 6.0.
4. MBS/Na2CO3: MBS, 250 mM Na2CO3.
5. 90% Sucrose/MBS: dissolve 45 g sucrose with MBS until volume equals 50 mL. Heat in a
microwave oven (in 10-s intervals) to dissolve/melt.
6. 35% Sucrose in MBS/Na2CO3: 5.83 mL 90% sucrose/MBS and 9.17 mL MBS/Na2CO3.
7. 5% Sucrose in MBS/Na2CO3: 0.83 mL 90% sucrose/MBS and 14.17 mL MBS/Na2CO3.
8. Triton X-100 Sigma-Aldrich, St. Louis, MO. Catalog # ×100 buffer: MBS, 1% Triton X-100, protease inhibitor mix (Sigma P-8340) (diluted 1:100).
9. 35% Sucrose in MBS/Triton X-100: 5.83 mL 90% sucrose/MBS and 9.17 mL Triton X-100
buffer.
10. 5% Sucrose in MBS/Na2CO3: 0.83 mL 90% sucrose/MBS and 14.17 mL Triton X-100 buffer.
11. Membrane buffer: 0.25 M sucrose, 1 mM ethylenediaminetetraacetic acid, 20 mM Tricine, pH 7.8.
12. 30% Percoll Sigma-Aldrich, St. Louis, MO. Catalog # 7737: 3 mL Percoll stock solution diluted
in 9 mL PBS 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4.
13. Modified lysis buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM ethylene glycolbis
(2-aminoethylether)-N, N, N, N-tetraacetic acid (EGTA), 10 mM MgCl2, 0.5% Triton X-100,
and protease inhibitor mix (Sigma P-8340, diluted 1:100). Sigma-Aldrich, St. Louis, MO.
Catalog # P-8340.
14. Protein A-agarose and protein G-agarose.
15. Immunoprecipitation (IP) wash buffer 1: 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.2% Triton
X-100.
16. IP wash buffer 2: 10 mM Tris-HCl, pH 7.5, 0.2% Triton X-100.
17. Methyl-β-cyclodextrin (MBCD) media: serum- and NaHCO3-free Dubelco’s Modified
Eagle’s Medium (DMEM), 20 mM HEPES, 2% (2-hydroxypropyl)-β-cyclodextrin, pH 7.4.
Solution may require sonication to fully solubilize.
18. MBCD vehicle media: serum- and NaHCO3-free DMEM, 20 mM HEPES, pH 7.4.
19. MBCD-cholesterol media: serum- and NaHCO3-free DMEM, 20 mM HEPES, β-cyclodextrin
(βCD)–cholesterol complexes (10 µg/mL cholesterol: β-cyclodextrin in 1:6 molar ratio, such as
Sigma cat no. C4951), pH 7.4. Solution may require sonication to fully solubilize.
3. Methods
3.1. Isolation of Lipid Rafts and Caveolae by Sucrose-Density Centrifugation
The unique lipid composition (i.e., enrichment in sphingolipid and cholesterol) of lipid
rafts and caveolae makes them resistant to solubilization in detergents and certain other
conditions. Once other cellular material is dissolved, lipid rafts and caveolae can be separated from the rest of the cellular contents using sucrose-density centrifugation. The
method for adherent cells in tissue culture is described, but it can be readily adapted for
cells in suspension or for tissue samples. The authors have found that two 150-mm plates
of cells are adequate for one preparation, but studies to optimize the amount of starting
material are recommended.
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3.1.1. Nondetergent Isolation of Lipid Rafts and Caveolae
1. Check that cells are at least 70% confluent. Aspirate medium and wash three times with ice-cold
PBS. On the last wash, be sure to remove all PBS by tilting the plate at a steep angle for 30 s
then aspirating all liquid. This step will ensure the lysis buffer is not overly diluted.
2. Apply 1 mL of 500 mM Na2CO3 to each 150-mm plate and make sure it covers the entire
monolayer. Scrape cells from the plate with a cell scraper in a circular motion from top to
bottom, making sure to retain as much cellular material as possible in a pool at the bottom of
the tilted plate.
3. Transfer the cells and all liquid from two 150-mm plates (2 mL total) to a prechilled Dounce
(glass–glass) homogenizer Wheaton Science Products, Millville, NJ: homogenize the cells with
20 strokes (one stroke is all the way down then all the way up) on ice.
4. Transfer the homogenate to a prechilled 50-mL conical tube and homogenize with a polytron
three times for 10 s with intervals of 10–15 s. Rinse the polytron blade with 0.5 mL of 500 mM
Na2CO3 into the sample to recover all possible material.
5. Homogenize the sample using an ultrasonic cell disruptor equipped with a stainless steel probe
using high power three times for 20 s each with a full 60 s rest between each homogenization.
Ultrasonic disruptors can vary from model to model in their power output. Thus, the power setting may need to be optimized (see Note 2).
6. Proceed to Subheading 3.1.4.
3.1.2. Detergent Isolation of Lipid Rafts and Caveolae
1. Check that cells are at least 70% confluent. Aspirate medium and wash three times with ice-cold
PBS. On the last wash, be sure to remove all PBS by tilting the plate at a steep angle for 30 s
and then aspirating all liquid. This step will ensure the lysis buffer is not overly diluted.
2. Apply 1 mL of 1% Triton-X 100 buffer to each 150-mm plate so that it covers the entire monolayer (see Note 3). Scrape cells from the plate with a cell scraper in a circular motion from top
to bottom, making sure to retain as much cellular material as possible in a pool at the bottom of
the tilted plate.
3. Transfer the cells from two plates (2 mL total) to a prechilled Dounce (glass–glass) homogenizer
and incubate on ice for 20 min. Homogenize the cells with 20 strokes (one stroke is all the way
down then all the way up) on ice.
4. Proceed to Subheading 3.1.4.
3.1.3. Variation: Isolation of Lipid Rafts and Caveolae From Plasma Membranes
1. Check that cells are at least 70% confluent. Aspirate medium and wash three times with ice-cold
PBS. On the last wash, be sure to remove all PBS by tilting the plate at a steep angle for 30 s
and then aspirating all liquid. This step will ensure the lysis buffer is not overly diluted.
2. Apply 1 mL of membrane buffer to each 150-mm plate so that it covers the entire monolayer.
Scrape cells from the plate with a cell scraper in a circular motion from top to bottom, making
sure to retain as much cellular material as possible in a pool at the bottom of the tilted plate.
3. Collect the cells from two plates (2 mL total) and homogenize cells with 20 strokes (one stroke
is all the way down then all the way up) in a Dounce (glass–glass) or Teflon-glass homogenizer
Wheaton Science Products, Millville, NJ. on ice then centrifuge at 300g for 5 min and collect
the supernatant.
4. Layer the supernatant on top of 30% Percoll and centrifuge at 64,000g (19,000 rpm on a SW41
ultracentrifuge rotor [Beckman Coulter, Fullerton, CA]) for 30 min.
5. Collect the opaque band near the top of the Percoll layer as the plasma membrane fraction.
6. Adjust the plasma membrane fraction to a final concentration of 500 mM Na2CO3 by adding an
equal volume of 1 M Na2CO3 and sonicate three times for 20 s with full 60 s rests between intervals (see Note 2).
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Fig. 1. Schematic diagram illustrating the isolation of buoyant lipid-raft fractions. The primary
biochemical approach for defining lipid rafts involves the use of sucrose-density centrifugation.
Following homogenization of cells using either detergent- or nondetergent- based approaches (see
Subheading 3.1.), the sample is adjusted to 45% sucrose and bottom-loaded in an ultracentrifuge
tube. A discontinuous sucrose gradient consisting of 35% sucrose and 5% sucrose is constructed on top
of the sample and the entire gradient is centrifuged at approx 240,000g (maximum relative centrifugal
force rcf) for 16–20 h. Buoyant lipid rafts (as well as caveolae, see Subheading 1.) “float” in the gradient, whereas the bulk of the “heavy” cellular material remains in the 45% sucrose layer. The gradient is typically collected in 0.5 mL fractions starting at the top and analyzed by immunoblot.
3.1.4. Sucrose-Density Centrifugation to Fractionate Cell Homogenates
Cells or tissues should be prepared and homogenized using one of the aforementioned
approaches (see Subheadings 3.1.1., 3.1.2., or 3.1.3., see also Note 4). Once the homogenate
is prepared, the bouyant lipid-raft fraction can be isolated by floatation on a sucrose-density
gradient. The method described here utilizes a discontinuous sucrose gradient (Fig. 1) but
continuous gradients can also be used (see Note 5).
1. Mix 1 mL of homogenized sample (leaving any foam behind) with 1 mL of 90% sucrose/MBS
in a 5-mL Beckman ultraclear ultracentrifuge tube. Save any remaining sample as whole cell
lysate. Leftover lysates can also be frozen at −80°C and fractionated by sucrose density centrifugation at a later date.
2. Carefully layer 2 mL of either 35% sucrose in MBS/Na2CO3 (if sample was homogenized by
nondetergent method, see Subheadings 3.1.1. or 3.1.3.) or 35% sucrose in MBS/Triton X-100
buffer (if sample was homogenized by detergent method; see Subheading 3.1.2.) on top of the
sample/90% sucrose/MBS layer. A visible interface should exist between the two density layers.
3. Carefully layer 1 mL of either 5% sucrose in MBS/Na2CO3 (if sample was homogenized by nondetergent method, see Subheadings 3.1.1. or 3.1.3.) or 5% sucrose in MBS/Triton X-100 buffer (if
sample was homogenized by detergent method, see Subheading 3.1.2.) on top of the 35% sucrose
layer. A second interface should be visible between the 35% and the 5% sucrose layers, and the ultracentrifuge tube should be nearly full. Although the gradient is not highly sensitive, all movement of
the gradient should be made carefully and deliberately in order to not disrupt the gradient interfaces.
4. Centrifuge for 16–20 h at 46,000 rpm at 4°C in a SW55Ti rotor (Beckman), equivalent to a maximum force (bottom of the tube) of approx 260,000g and an average force (middle of the tube)
of approx 200,000g (see Note 6).
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5. At the completion of the centrifugation, carefully remove the ultracentrifuge tube from the
bucket. A faint light-scattering band, which consists of the buoyant lipid raft/caveolar material,
is often visible at the 35% sucrose –5% sucrose interface.
6. Collect samples from the gradient from the top down in 0.5 mL volumes, putting each fraction
in a labeled tube and yielding 10 fractions. One should be careful to keep the pipet at the top of
the liquid in order to draw each fraction appropriately. If cellular material is visible at the upper
gradient interface, care should be taken to collect this material in fractions two and three.
7. Fractions can then be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and immunoblotting (see Note 7).
3.2. Immunoisolation of Caveolae
This method takes advantage of the reduced solubility of lipid rafts and caveolae to detergent
in order to isolate these domains from the rest of the cellular material (as in Subheading 3.1.2.)
and then specifically “traps” caveolae (and not lipid rafts) by using an antibody to immunoprecipitate caveolin. This method will also pull down the caveolar lipids, cholesterol, and
associated proteins. One needs to first define the caveolin isoform expression in a given cell
or tissue type in order to select an appropriate caveolin antibody. For cells expressing multiple isoforms, an antibody to the most predominant or readily detectible caveolin can be used
because coexpressed isoforms form hetero-oligomers (31). Particular advantages of this
method are that one can maintain enzyme and receptor-binding activity in the isolates, facilitating the assessment protein function. The authors as well as others have used this approach
to assay adenylyl cyclase activity regulated by G protein-coupled receptors in caveolar
domains isolated from cardiac myocytes (22,28).
1. Check that cells are at least 70% confluent. Aspirate medium and wash three times with ice-cold
PBS. On the last wash, be sure to remove all PBS by tilting plate at a steep angle for 30 s and
then aspirating all liquid. This step will ensure the lysis buffer is not overly diluted.
2. Add 2 mL of modified lysis buffer to each 15-cm plate. Homogenize cells with 20 strokes (one
stroke is all the way down then all the way up) in a Dounce (glass–glass) homogenizer.
3. Transfer to a 1.5-mL microtube and add 50 µL of either protein G- or protein A-agarose suspension (see Note 8) to preclear any native antibodies. Incubate at 4°C on a rocking platform for 1 h.
4. Centrifuge in a microcentrifuge at maximum speed (12,000–14,000 rpm) for 30 s to pellet the
agarose and then transfer the supernatant to a new tube. The pellet can be discarded.
5. Add primary antibody (1–3 µL, depending on the antibody concentration and affinity) and continually mix (preferably by rocking) at 4°C for 1 h to allow antibody binding to epitope.
6. Add 50 µL protein A- or protein G-agarose to tube and continually mix (preferably by rocking)
at 4°C for 1 h to allow binding to the antibody–epitope complexes.
7. Centrifuge in a microcentrifuge at maximum speed (12,000–14,000 rpm) for 30 s to pellet agarose.
Supernatant should be saved as the IP supernatant. IP supernatant should be assessed for the
amount of epitope not trapped in the immunoprecipitates and can behave as a control of nonprecipitated material in the assay of choice.
8. Wash the pellet by adding 1 mL of modified lysis buffer, mix and rock at 4°C for 5 min.
9. Centrifuge in a microcentrifuge at maximum speed (12,000–14,000 rpm) for 30 s to pellet agarose,
remove supernatant and add 1 mL of wash buffer 1 to pellet, mix and rock at 4°C for 5 min.
10. Centrifuge in a microcentrifuge at maximum speed (12,000–14,000 rpm) for 30 s to pellet
agarose, remove and discard supernatant.
11. Wash the pellet a second time by adding 1 mL of wash buffer 2, mix and rock at 4°C for 5 min.
12. Centrifuge in a microcentrifuge at maximum speed (12,000–14,000 rpm) for 30 s to pellet
agarose, remove and discard supernatant.
13. The final pellet should then be suspended in a suitable assay buffer (if enzyme activity is to be
measured) and/or in sample buffer for analysis by SDS-PAGE (for immunoblotting). Immunoblot
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analysis should be performed on a portion of the immunoprecipitated pellet and the IP supernatant
to confirm appropriate IP of caveolin and to assess which proteins have been coprecipitated.
3.3. Disruption of Lipid Rafts and Caveolae
Using β-cyclodextrin, a cholesterol-binding agent, one can remove cholesterol from the
plasma membrane of living cells and disrupt both lipid rafts and caveolae. Cholesterol depletion is toxic to cells over time, thus one must be careful to optimize exposure to β-cyclodextrin
as well as control for cell toxicity or stress. The method below describes using a 60-min treatment period (which have been found to be appropriate in several cell types). However, it is
strongly suggested to perform a treatment time-course and determine cell viability (e.g., using
trypan blue exclusion) in order to optimize the treatment for each cell type. The most critical
control, which is described in Subheading 3.3.4., involves the replenishment of cholesterol in
cells following its extraction. This condition will control for nonspecific effects of β-cyclodextrin treatment and indicate the reversibility of any observed effects. Thus, all assays should be
conducted in vehicle treated control cells, β-cyclodextrin treated cells, and cells that are treated
with β-cyclodextrin followed by cholesterol replenishment. This method can be used before
almost any type of signal transduction assay to ascertain the role of lipid rafts and caveolae in a
given response.
1. Check that cells are at least 70% confluent. Aspirate medium and wash three times with warm
(37°C) PBS. On the last wash, be sure to remove all PBS by tilting the plate at a steep angle for
30 s and then aspirating all liquid. This step will ensure the lysis buffer is not overly diluted.
2. To one plate or set of plates apply 15 mL of warm DMEH containing β-cyclodextrin (MBCD
media) to each 150-mm plate, so that it covers the entire monolayer. Incubate cells at 37°C for
60 min. A CO2-enriched environment (i.e., cell culture incubator) is not needed because of the
buffering of this media with HEPES.
3. To a separate plate or set of plates apply 15 mL of warm DMEH containing MBCD vehicle
(MBCD vehicle media) to each 150-mm plate following aspiration (step 1), so that it covers the
entire monolayer. Incubate cells at 37°C for 60 min. This step will remove membrane cholesterol, disrupting lipid rafts, and caveolae.
4. To a third plate or set of plates apply 15 mL of warm MBCD Media to each 150-mm plate following aspiration (step 1), so that it covers the entire monolayer. Incubate cells at 37°C for 60 min.
Aspirate MBCD media, wash cells three times with warm PBS (as in step 1), then add 15 mL of
warm maintenance media containing β-cyclodextrin/cholesterol complexes (MBCD-cholesterol
media). Incubate cells at 37°C for 60 min.
5. Aspirate media from cells, wash three times with warm PBS (as in step 1), and conduct assay
of choice. Positive controls for this method include performing sucrose density centrifugation
(see Subheading 3.1.4.) and/or assaying for cholesterol content.
4. Notes
1. Complementary approaches to defining caveolae and lipid rafts must be considered. Caveolins
appear to also act as scaffolding proteins that can bind multiple signaling proteins; thus, caveolins may act to organize a signaling pathway or regulate signaling activity (3). Therefore, IP of
caveolin proteins and expression of peptides that interfere with the caveolin-binding motif are
useful approaches for defining the role of caveolins in organizing signal-transduction cascades
(3). Overexpression or knockout of caveolins have also been used to examine the physiological
role of these proteins (32). However, altering caveolin expression should not be considered a
pure probe of the compartmentation or organization of a signaling pathway in caveolae, because
caveolins also act as direct regulators of several signal transduction pathways (7).
2. In the nondetergent fractionation method, the ultrasonic disruption of cells is the most critical
step, as this is the point at which membrane lipids are dissolved but lipid raft material remains
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3.
4.
5.
6.
7.
8.
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intact. Thus, the power of the sonication step is critical. Too much power can disrupt the raft
structure, resulting in little material floating up in the sucrose gradient. Too little power can
result in insufficient dissolution of nonraft lipids, resulting in raft material not being sufficiently
freed from “heavy” material, which also results in less material floating up in the fractionation.
Therefore, the power setting used will depend on the make and model of the ultrasonic disruptor used and may need to be optimized for individual cell types. In the authors’ experience, the
sonication time and rest period should not be significantly altered.
Each method for defining lipid rafts and caveolae has advantages and disadvantages.
Experimentalists should consider these factors when choosing the experimental approaches they
will use to answer their particular biological question. For example, the nondetergent fractionation
of cells retains certain proteins in lipid raft fractions that are often lost in detergent-based methodologies (33,34). However, detergent-based approaches allow for measuring protein function, such
as enzyme activity, whereas the high-pH and -energy sonication of the nondetergent methods
generally impair protein function. It is generally desirable to use a combination of different,
complementary approaches in order to define signal transduction in lipid rafts and caveolae.
Nonionic detergents other than Triton X-100, including NP-40, octylglucoside, CHAPS, Lubrol,
and Brij 98, can be used to solubilize cells and isolate lipid raft and caveolar domains (35). In
addition, some investigators have used concentrations of Triton X-100 lower than 1% in protocols similar to that described in Subheading 3.1.2.
The method described here utilizes a discontinuous gradient of sucrose. However, continuous gradients of sucrose or of Optiprep Sigma-Aldrich, St. Louis, MO. Catalog # D1556 (described in
refs. 33 and 36) are capable of resolving proteins and structures with intermediate buoyancies.
Other rotors can be used for the sucrose density centrifugation, including a Beckman SW41Ti
rotor with 12-mL buckets. In this case, 2 mL of cell homogenate is mixed with 2 mL of 90%
sucrose and 4 mL of 35% sucrose and 4 mL of 5% sucrose is layered on top. The rotation speed
is adjusted to maintain equivalent g-force (~39,000 rpm). Fractions are collected in 1 mL
aliquots to yield 12 fractions. Other rotors can be used and the Beckman rotor resources web
page (http://www.beckman.com/resourcecenter/labresources/centrifuges/rotorcalc.asp) is useful
for calculating the appropriate rotational speed.
Each fractionation from a sucrose-density centrifugation should be carefully analyzed for markers
of certain cellular organelles that can contaminate the buoyant fractions. Immunoblot analysis of
fractions from the 5% sucrose/35% sucrose interface (fractions numbered 2 and 3 from the method
described in 3.1.4.) should contain the bulk of caveolin isoform immunoreactivity. At the same
time, these fractions should largely exclude markers of clathrin-coated pits (such as adaptin-β) and
Golgi apparatus (such as mannosidase II). One can also confirm the appropriateness of a fractionation by examining the total protein in each fraction. The buoyant fractions from most cells should
contain approx 5% of the total cellular protein. When immunoblot analysis of fractions is planned,
it is best to add SDS-PAGE sample buffer to each fraction and to denature at (70°C for 10 min
immediately after collecting the gradient. This will ensure more reproducible results when storing
frozen samples for extended periods. For detection of low-abundance proteins, samples can also be
concentrated in a speed-vac (or similar type) concentrator Thermo Fisher Scientific, Waltham, MA
before addition of sample buffer. However, the fractions from the bottom of the gradient will not
concentrate as well because of the presence of higher concentrations of sucrose. Dialysis can also
be used to remove sucrose and to concentrate the samples.
IP efficiency can be maximized by carefully selecting the most effective agarose bead conjugate
based on the primary antibody being used. Protein A has high affinity for human, rabbit, guinea
pig, and pig immunoglobulin G’s (IgG). Protein G has high affinity for human, horse, cow, pig,
and rabbit IgG’s. When using mouse IgG’s, both protein A and protein G have moderate affinity. However, protein A and protein G have further differences in affinities for the subclasses of
IgG’s. For more detailed information on the different affinities of protein A and protein G, refer
to the manufacturer’s product information sheet.
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References
1 Shaul, P. W. and Anderson, R. G. (1998) Role of plasmalemmal caveolae in signal transduction.
1.
Am. J. Physiol. 275(5 Pt 1), L843–L851.
2 Ostrom, R. S. and Insel, P. A. (2004) The evolving role of lipid rafts and caveolae in G protein2.
coupled receptor signaling: Implications for molecular pharmacology. Br. J. Pharmacol. 143(2),
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