- Email: [email protected]
Chapter Six Analyzing Lipid Raft Dynamics during Cell Apoptosis
C H A P T E R
S I X
Analyzing Lipid Raft Dynamics during Cell Apoptosis Walter Malorni,* Tina Garofalo,† Antonella Tinari,‡ Valeria Manganelli,† Roberta Misasi,† and Maurizio Sorice† Contents 126 128 128 130 130 131 131 131 132 133 133 133 134 134 134 134 135 136 136 137 137 139
1. Introduction 2. The Contribution of Biochemical Approaches 2.1. Coimmunoprecipitation 2.2. Protocol 2.3. Coimmunoprecipitation from cell pellet 2.4. Coimmunoprecipitation from DRM fractions 2.5. Ganglioside analysis in immunoprecipitate samples 2.6. Reagents 2.7. Procedure 2.8. Isolation of mitochondria 2.9. Procedure 2.10. Detergent solubilization of isolated mitochondria 2.12. Coimmunoprecipitation from isolated mitochondria 3. The Contribution of Morphological Analyses 3.1. Light microscopy 3.2. Protocol 3.3. Postembedding electron microscopy 3.4. Fixation 3.5. Embedding medium 3.6. Embedding procedure 3.7. Postembedding procedure References
* { {
Department of Drug Research and Evaluation, Istituto Superiore di Sanita`, Rome, Italy Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy Department of Technology and Health, Istituto Superiore di Sanita`, Rome, Italy
Methods in Enzymology, Volume 442 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)01406-7
#
2008 Elsevier Inc. All rights reserved.
125
126
Walter Malorni et al.
Abstract Increasing lines of evidence suggest a role for lipid rafts, glycosphingolipidenriched microdomains, in cell life and death. This chapter describes in brief the methods used to analyze raft interactions with proteins involved in apoptosis. This chapter focuses mainly on coimmunoprecipitation methods, which represent a useful tool in analyzing raft dynamics during apoptosis. Glycosphingolipid analysis in the immunoprecipitates is performed by thin-layer chromatography. Moreover, methods for the analysis of mitochondrial raft-like microdomains are also described. Detergent (Triton X-100)-insoluble material from isolated mitochondria can be analyzed by Western blot. Further insights can also come from both light and electron microscopy analyses. These can provide useful information as concerning lipid raft distribution at the cell surface or in the cell cytoplasm. Paradigmatic micrographs are shown. The combined use of all these different approaches appears to be mandatory for analyzing the role of lipid raft dynamics during apoptosis.
1. Introduction Several investigations have been carried out since the late 1990s in order to address the function of lipid rafts in cell life and death. It is commonly accepted that the plasma membrane is a complex system in which constituents are organized in small lipid/protein domains, known as ‘‘lipid rafts.’’ Membrane rafts are a specific type of lipid domains enriched in sphingolipids, including gangliosides (GSLs), sphingomyelin, and cholesterol, that result from tight hydrophobic interactions among these molecules, leading to the spontaneous formation of aggregates that separate from glycerophospholipids in cell membranes (Simons et al., 1997). Because of their lipid composition, the physical state of these domains is supposed to be similar to a liquid-ordered (Lo) phase, structurally and dynamically distinct from the rest of the lipid bilayer, which is, in turn, assumed to be in a liquiddisordered phase (Ipsen et al., 1987). On the basis of the peculiar biophysical and biochemical properties of lipid rafts, a number of possible interactions with various subcellular structures have been suggested. It is well known that these lipid microdomains may function to concentrate or segregate different proteins to form a glycosignaling domain. Several studies described a role for rafts in a variety of cellular processes, such as cell signaling, lipid and protein sorting, membrane organization and trafficking, immune response, and cell death (Malorni et al., 2007). Indeed, a large variety of proteins have been detected in these microdomains, including tyrosine kinase receptors (EGF-R), G proteins, Src-like tyrosine kinases (lck, lyn, fyn), protein kinase c isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins, adhesion molecules, and the death-inducing signaling complex. Thus, a general function of lipid
127
Lipid Rafts in Cell Apoptosis
rafts in signal transduction may be to allow the lateral segregation of proteins within the plasma membrane, providing a mechanism for the compartmentalization of signaling components, concentrating certain components in lipid rafts, including those of importance in apoptosis, and excluding others (Simons et al., 1997). Several studies indicate that lipid rafts could play a role in the apoptotic cascade. Although they are detected in various cell types, their role has been studied mostly in lymphocytes undergoing apoptosis induced by CD95/Fas. In particular, it was proposed that rafts are the primary site of action of the enzyme sphingomyelinase, which catalyzes the hydrolysis of sphingomyelin, resulting in ceramide formation; ceramides deriving from sphingomyelin hydrolysis are essential mediators of apoptotic signals originating from CD95/Fas (Grassme´ et al., 2001). Moreover, a role for gangliosides as structural components of the multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway has been reported (Garofalo et al., 2003). Although lipid rafts are considered ubiquitous constituents of plasma membrane, recent lines of evidence also indicated that they are present on organelles, including the Golgi apparatus and a subcompartment of the endoplasmic reticulum. Raft-like microdomains have also been detected on mitochondrial membranes after CD95/Fas triggering (Garofalo et al., 2005). Formation of a multimolecular complex that includes VDAC-1, Bcl-2 family, and fission proteins, for example, h-Fis, has been demonstrated. Thus, the complex may represent preferential sites where some key reactions can take place and can be catalyzed, leading to either survival or death of T cells. Different methods for studying lipid rafts in cell apoptosis are reported in Table 6.1.
Table 6.1 Methods used for studying lipid rafts in apoptosis
Apoptosis
Lipid rafts
Electron microscopy TUNEL Annexin V Activity of caspases DNA laddering Mitochondrial membrane potential Sucrose gradient analysis Coimmunoprecipitation FRET Single particle tracking Fluorescence correlation spectroscopy Scanning confocal microscopy Electron microscopy
128
Walter Malorni et al.
2. The Contribution of Biochemical Approaches In the last years, numerous different techniques have been considered for studying the association of apoptosis-related proteins with lipid rafts. The use of nonionic detergent extraction to generate low-density detergent-resistant membranes (DRMs) has had a major role in implicating rafts in cellular functions (Brown et al., 2000). Although this treatment may disrupt lipid– lipid interactions, a minor fraction of cell membranes is preserved and can be isolated as DRMs. Because detergent extraction may also disrupt several lipid– protein interactions, only a few proteins, interacting strongly with highly ordered domains, retain their association with lipids and are recovered in DRMs (Schuck et al., 2003). Essentially, all of these proteins have a saturated hydrocarbon chain modification, as a membrane anchor inserted into the raft domain of the outer plasma membrane leaflet and many transmembrane proteins require palmitoylation for their association to lipid rafts. Accordingly, isolation of DRMs represents a valuable tool for the analysis of raft-associated proteins involved in cell apoptosis and a useful starting point for defining membrane subdomains and/or protein–ganglioside interaction. Applying a variety of detergents may affect lipid–protein interactions (Schuck et al., 2003). Specific protein–lipid interactions within rafts have been studied preferentially by coimmmunoprecipitation experiments (Garofalo et al., 2002, 2003) or by fluorescence resonance energy transfer (FRET) (Edidin, 2003). In addition, many new approaches for detecting heterogeneity in cell membranes have emerged that rely on the distinct diffusion characteristics or enhanced proximity between raft components. Single particle tracking (SPT) has enabled us to measure the diffusion characteristics of GPI-anchored proteins. Fluorescence correlation spectroscopy, which combines different evaluations of biophysical properties of the plasma membrane, can contribute to better analyze the dynamics of raft components in living cells. Some of these approaches are of widespread use in laboratory practice (mainly coimmunoprecipitation experiments), whereas others are specifically employed under certain experimental conditions. Coimmunoprecipitation has become the tool of choice for analyzing the dynamics of raft components and their functional interaction with proteins involved in cell apoptosis (Fig. 6.1).
2.1. Coimmunoprecipitation The starting material can be (i) a cell pellet or (ii) the DRM fraction, containing lipid rafts, isolated by sucrose density-gradient ultracentrifugation after Triton X-100 (TX-100) solubilization at 4 , according to Iwabuchi et al. (2000).
129
Lipid Rafts in Cell Apoptosis
A
1 mm B
0.5 mm C
Caspase-8 immunoprecipitates
GM3
GM1
Anti-GM3
CTxB
Figure 6.1 (A) Immunogold transmission electron microscopy showing the typical clustered distribution of gold particles, corresponding to ganglioside-enriched microdomains, on cell plasma membrane of a T cell. (B) Immunogold labeling of GD3 in CEM cells induced to apoptosis by treatment with anti-CD95/Fas. Cells were prepared following the standard embedding procedure. Ultrathin sections, picked up on gold grids, were labeled using an anti-GD3 MoAb (1:30) as the primary antibody and, subsequently, with an antimouse IgM-10 nm gold conjugated (1:10). Note the distribution of GD3 molecules, which appear specifically localized on mitochondrial membranes. (Inset) The well-arranged distribution of gold particles at higher magnification.
130
Walter Malorni et al.
Detergents Although TX-100 is the most widely used detergent for the purification of lipid rafts, other detergents have been used for this purpose, including Brij96, Brij98, CHAPS, Triton X-14, and Lubrol WX (Schuck et al., 2003). The use of detergents to solubilize the cell membrane leads to the generation of small micelles and large particles that can be separated by centrifugation at low temperature (4 ) for 17 to 18 h at 200,000g in a 5 to 35% linear discontinous gradient of sucrose (Iwabuchi et al., 2000). Before starting solubilization, make sure all lysing/solutions are prechilled on ice, as it has been also shown that the amount of lipids in the plasma membrane raft is increased at low temperature.
2.2. Protocol 2.2.1. Cultured cells Both adherent cell lines and cells growing in suspension can be examined. 2.2.2. Reagents
Lysis buffer: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 mg of leupeptin/ml, 1 mM sodium orthovanadate Protein A/G acrylic or magnetic beads
2.3. Coimmunoprecipitation from cell pellet 1. Collect the culture supernatant, centrifuge at 300g for 10 min, and wash twice with ice-cold phosphate-buffered saline (PBS). 2. After removing the supernatant, resuspend the pellet in 1 ml ice-cold lysis buffer. 3. Transfer the lysate to a 1-ml homogenization tube and set on ice for at least 20 to 30 min. 4. Homogenize the samples using a tight-fitting Dounce pestle (20 strokes), maintaining the tube on ice at all times. 5. Transfer the lysate to a 1.5-ml tube and centrifuge for 5 min at 1300g to remove nuclei and large cellular debris. 6. Determine the protein concentration in the supernatant fraction using the Bio-Rad kit (Bio-Rad, Richmond, CA) with bovine serum albumin (BSA) as standard. (C) Tcells, undergoing apoptosis induced by CD95/Fas, were immunoprecipitated with anticaspase-8. The immunoprecipitates were analyzed for the presence of ganglioside molecules byTLC immunostaining using anti-GM3 MoAb or cholera toxin, B subunit (CTxB) (from Garofalo et al., 2003).
Lipid Rafts in Cell Apoptosis
131
7. Mix the supernatant fraction with protein A/G acrylic beads (Sigma Chemical Co., St. Louis, MO), stirring by a rotary shaker for 2 h at 4 to preclear nonspecific binding. 8. Centrifuge the lysate for 1 min at 500g at 4 . 9. Collect the supernatant. 10. Incubate with specific antibodies for specific binding with proteins, stirring by a rotary shaker for 2 h at 4 . 11. Add protein A/G acrylic beads for 1 h. 12. Centrifuge the sample for 1 min at 500g at 4 . 13. Remove the supernatant. Resuspend the pellet in 0.5 ml of H2O and transfer it into a 15-ml tube for ganglioside extraction.
2.4. Coimmunoprecipitation from DRM fractions Detergent-resistant membranes, obtained as described (Iwabuchi et al., 2000), are subjected to coimmunoprecipitation following the procedure starting from step 7.
2.5. Ganglioside analysis in immunoprecipitate samples Immunoprecipitation samples are subjected to GSL analysis by the highperformance thin-layer chromatography (HPTLC) technique. HPTLC has become the tool of choice for the identification and quantification of GSLs in small amounts of sample extracted from the plasma membrane of T cells, based on the fact that the HPTLC plate has a high-resolution property for glycosphingolipid separation. Of importance, the detection of glycosphingolipids after HPTLC separation is achieved by using specific colorimetric reagents or specific anti-GSLs monoclonal antibodies, followed by peroxidase-conjugated secondary antibodies. The latter represents an important method used to identify with high sensitivity small amounts (10 ng) of protein-associated ganglioside.
2.6. Reagents 1. HPTLC aluminium-backed silica gel 60 (20 20) plates (Merck, Darmstadt, Germany) 2. Solvent system for TLC (chloroform:methanol:0.25% aqueous KCl (5:4:1) (v:v:v) 3. Resorcinol-HCl reagent. Resorcinol (200 mg) in 20 ml of distilled water is mixed with 80 ml of concentrated HCl and 0.25 ml of 0.1 M CuSO4 4. 0.5% (w/v) solution of poly(isobutyl methacrylate) beads (Polyscience, Warrington, PA) dissolved in hexane (PIM solution) 5. 0.1% (w/v) BSA/PBS 6. Monoclonal antibodies against glycosphingolipids
132
Walter Malorni et al.
7. Secondary antibody, horseradish peroxidase-conjugated antimouse immunoglobulins 8. Enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Life Science Ltd., Buckinghamshire, UK)
2.7. Procedure Gangliosides from immunoprecipitates are extracted according to the method of Svennerholm et al. (1980), with minor modifications. 2.7.1. Ganglioside extraction 1. Extract the immunoprecipitate in chloroform:methanol:water (4:8:3) (v:v:v) and centrifuge twice at 1500g for 30 min at room temperature. 2. Collect the supernatant and add water, resulting in a final chloroform: methanol:water ratio of 1:2:1.4 (v:v:v). 3. Centrifuge the gradient at 1500g for 30 min at room temperature. 4. Collect the upper phase containing polar glycosphingolipids. 5. Purify the upper phase of salts and low molecular weight contaminants using Bond elute C18 columns (Superchrom, Milan, Italy). According to Williams and McCluer, 1980. 2.7.2. TLC Immunostaining 1. Resuspend the ganglioside extract in 10 ml of chloroform:methanol 2:1 (v:v) and separate on HPTLC aluminum-backed plates. 2. After HPTLC, dry the plate thoroughly with a hair dryer to remove the organic solvent. 3. Soak gently the dried plate for 90 s in 0.5% PIM solution in a glass box of appropriate size. 4. Remove the plate from the PIM solution and air dry. 5. Incubate the plate with a buffer containing 5% BSA/PBS for 30 min at room temperature to block nonspecific absorption of antibodies. 6. Remove the blocking solution and wash the plate gently for 10 min with 0.5% BSA/PBS with two changes of the buffer. 7. Add the first antibody (anti-GSLs) diluted with 0.1% BSA/PBS for 1 h at room temperature. 8. Wash the plate with 0.5% BSA/PBS with three changes of the buffer and then incubate it with the peroxidase-labeled secondary antibody solution for 1 h at room temperature. 9. Wash with 0.5% BSA/PBS in the same way. 10. Using ECL, visualize the positive reaction.
Lipid Rafts in Cell Apoptosis
133
2.8. Isolation of mitochondria 2.8.1. Reagents and buffers Mitochondria isolation buffer (MIB): 220 mM mannitol, 68 mM sucrose, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 10 mM HEPES-KOH (pH 7.4), 0.1% (w/v) BSA, supplemented with a cocktail of protease inhibitors 2.8.2. Equipment Glass Dounce homogenizer (15 ml) with a tight Teflon pestle Glass Dounce homogenizer (2 ml) with a glass pestle (B type)
2.9. Procedure Preparation of cytosolic extracts Cytosolic extracts are obtained according to Bossy-Wetzel et al. (2000). Cells are harvested by centrifugation at 200g for 10 min at 4 , washed twice with cold PBS, pH 7.4, and resuspended in 3 volumes of cold cytosolic extraction buffer. After incubation on ice for 30 min, cells are disrupted by homogenization with a glass Dounce homogenizer and a tight glass pestle, applying 50 strokes. Nuclei and unaffected cells are removed by centrifugation at 800g for 10 min at 4 in an Eppendorf centrifuge. Supernatants are transferred to new Eppendorf tubes and centrifuged further at 22,000g for 30 min. Supernatants are then used for detergent solubilization.
2.10. Detergent solubilization of isolated mitochondria Reagents and buffers Extraction buffer: 25 mM HEPES, pH 7.5, 0.15 M NaCl, 1% Triton X-100, and 100 U/ml kallikrein/aprotinin Solubilization buffer: 50 mM Tris-HCl, pH 8.8, 5 mM EDTA, and 1% SDS 2.11. Procedure Supernatants containing isolated mitochondria are detergent solubilized according to Skibbens et al. (1989). Briefly, mitochondria are lysed with 1 ml of extraction buffer for 20 min on ice. Lysates are collected and centrifuged for 2 min in a Brinkmann microfuge at 10,000g at 4 . Supernatants, containing Triton X-100 soluble material, are collected; pellets are centrifuged a second time (30 s) in order to remove the remaining soluble material. Pellets are then solubilized in 100 ml of solubilization buffer. DNA is sheared by passage through a 22-gauge needle. Both Triton X-100-soluble and -insoluble material are then analyzed by Western blot for specific proteins.
134
Walter Malorni et al.
2.12. Coimmunoprecipitation from isolated mitochondria Isolated mitochondria are subjected to coimmunoprecipitation following the procedure reported earlier.
3. The Contribution of Morphological Analyses Numerous different morphological techniques have been considered for studying the complex sequence of structural modifications of lipid rafts during cell apoptosis. These are mainly qualitative analyses that can be carried out by means of light (LM) and electron microscopy (EM) techniques. Morphometric analyses, essentially performed by LM, can also be carried out. The use of these different technical approaches can provide information on the surface or intracellular localization of lipid rafts (Fig. 6.1).
3.1. Light microscopy The aim of immunocytochemistry is to localize antigens by labeling them with specific antibodies. Analyzing lipid raft dynamics in cell apoptosis, antiganglioside antibodies may be considered a good tool. However, we recommend that biochemical analysis be used to confirm that a particular ganglioside is present in the cell or tissue under test. 3.1.1. Reagents Available antiganglioside antibodies are summarized by Schwarz et al. (2000). 3.1.2. Fixation The major problem to be taken into consideration when using antiganglioside antibodies is the choice of fixative (Schwarz et al., 2000). For detection of lipid rafts on the cell surface, incubation with antibodies prior to fixation (prefixation) with 4% formaldehyde for 30 min at room temperature yields reproducible patterns of immunofluorescence. Alternatively, in order to detect raft-like microdomains associated with intracellular organelles (i.e., mitochondrion), lymphocytic cells may be postfixed with 4% paraformaldehyde in PBS for 30 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature (Garofalo et al., 2005).
3.2. Protocol 1. Soak in Hanks’ balanced salt solution. 2. Incubate with anti-GSLs MoAb for 1 h (times and temperatures of incubation with primary antibodies are empirical and need to be determined for each antibody and cell type) at 4 .
Lipid Rafts in Cell Apoptosis
135
3. Wash three times in PBS, pH 7.4. 4. Fixate with 4% paraformaldehyde in PBS for 30 min (times and temperatures of incubation with primary antibodies are empirical and need to be determined for each antibody and cell type) at room temperature. 5. Wash three times in PBS, pH 7.4. 6. Stain with FITC-conjugated secondary antibody for 45 min (times and temperatures of incubation with primary antibodies are empirical and need to be determined for each antibody and cell type) at 4 . 7. Wash three times in PBS, pH 7.4. 8. Counterstain with Texas red-conjugated antiprotein antibody. 9. Wash three times in PBS, pH 7.4. 10. Mount in 0.1 M Tris-HCl, pH 9.2, containing 60% glycerol (v:v). 11. Acquire images through a confocal laser-scanning microscope. The green (FITC) and red (Texas red) fluorophores are excited simultaneously at 488 and 518 nm and are observed by two different detectors. Before image acquisition, samples are scanned at different filter conditions to choose a setting that reduces the overlap of emission spectra with a maximal signal-to-noise ratio. Then, acquired images are processed by subtracting a scaled version of green from red series, and vice versa. The scale factor (bleed-through factor) is determined by scanning single-stained samples in a dual fluorescence scanning configuration. Samples are counterstained with Texas red fluorophore, which reduces fluorescence overlapping greatly. Serial optical sections are assembled in depth-coding mode. Acquisition and processing are carried out using appropriate confocal software.
3.3. Postembedding electron microscopy There are two most common procedures that allow one to visualize cellular antigens with transmission electron microscopy: preembedding and postembedding techniques. Preembedding is used to label surface antigens only. It consists of an antigen–antibody reaction on living cells, that is, before the embedding and sectioning procedure. In contrast, the postembedding technique should be used when an internal antigen would be labeled. It consists of an antigen-antibody reaction in ultrathin sections, that is, after the embedding and sectioning procedure. For both of these methods, subcellular antigens recognized by primary antibodies can be localized and visualized with a transmission electron microscope (TEM) using gold-conjugated secondary antibodies to provide areas of high electron density. Colloidal gold can be coupled either to protein A, a protein from Staphylococcus cell walls that binds the Fc portion of immunoglobulins, or to a secondary antibody.
136
Walter Malorni et al.
Preservation of excellent structural details and, at the same time, maintenance of antigenic sites reactivity are the hardest but primary concerns for immune gold labeling. In order to preserve antigenicity, conventional electron microscopy procedure should be modified. In fact, few antigens survive to the routine fixation and embedding procedures. The tendency is to use a gentler aldehyde fixation, combined with low temperature embedding, that has been shown to better preserve the antigenicity of biological samples (Gonzalez et al., 1997; Scala et al., 1992) but, to some extent, this procedure compromises the ultrastructural features of the cells.
3.4. Fixation The reactivity and the stability of diverse antigens differ so widely from each other that no standardized method of fixation can be used for all EM immunocytochemistry experiments. Initially, it would be routinely worthwhile to assess the preparation of the sample. In fact, it is important to check if the antigen to be labeled is resistant to the fixation procedures and if the sample ultrastructural features could be preserved. Milder fixation regimens normally required to retain antigenicity can actually result in the loss of ultrastructural details. Depending on the chemical nature of such an antigen, successful immunolabeling may be obtained with routine preparations. Unfortunately, for most antigens the standard procedure results in a complete, or at least severely impaired, loss of antigenicity. This means that a range of preparative procedures, varying in their degree of fixation strength, is recommended to optimize the compromise between ultrastructural preservation and immunolabeling efficiency. In our experience, proteins are affected more profoundly by the standard embedding procedures, whereas lipid antigens are more likely to be preserved (Garofalo et al., 2005).
3.5. Embedding medium Biological specimens are embedded in resin in order to allow thin sectioning. Many different resin formulas are currently available and all these mixtures have been used for postembedding labeling (Bogers et al., 1996; Brorson, 1998). For immunocytochemistry purposes, acrylic resins that polymerize under ultraviolet light (UV) at 4 are preferred. Epoxy resins are exposed at 65 for polymerization with the possible denaturing effects of heat. Furthermore, acrylic resins have low viscosity and are hydrophilic, thus enhancing the subsequent immunolabeling on the sections.
Lipid Rafts in Cell Apoptosis
137
3.6. Embedding procedure 1. Fixation: antigens are quite commonly glutaraldehyde sensitive. Hence the variation of the concentration of this fixative has to be established experimentally for every antigen under consideration. Split the sample into three lots and fix as follows. Sample 1: 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2; 0.1 M ) Sample 2: 1% glutaraldehyde in sodium cacodylate buffer (pH 7.2; 0.1 M ) Sample 3: 4% paraformaldehyde þ 0.01% or 0.25% glutaraldehyde in sodium cacodylate buffer (pH 7.2; 0.1 M) for 20 min at room temperature or overnight at 4 2. Wash in 0.1 M sodium cacodylate buffer three to four times for 5 min each. 3. Postfixation for sample 1 only: postfix in 1% OsO4 in sodium cacodylate buffer for 30 to 60 min and carefully wash in sodium cacodylate buffer three to four times for 5 min each. Although most antigens are osmium tetroxide sensitive, some of them may resist this mild osmium fixation, thus improving preservation of ultrastructural details, especially membranes and membranous structures. 4. Dehydrate in graded series of ethanol solutions (50, 70, 95, and 100% twice) for 10 min each. 5. Resin embedding: Embed sample 1, which is OsO4 treated, in Epoxy resin–absolute ethanol mixtures (1:2; 1:1; 2:1; absolute) at room temperature and proceed as for the standard embedding procedure. Embed the other samples in acrylic resin as follows: withdraw absolute ethanol and add resin. The time and conditions of each acrylic resin are different; see the data sheet of the chosen resin. For example, Unicryl resin is directly added as absolute: leave the sample to infiltrate with resin for 4 h with at least two changes of resin and leave overnight at 4 . 6. Polymerization: Polymerize sample 1 at 65 for 48 h. Place Unicryl samples in closed gelatin capsules with fresh resin. Leave them to polymerize by suspension in a rack held 35 cm above a UV light lamp for 72 h.
3.7. Postembedding procedure Sections of about 80 nm are picked up on inert 200 mesh grids, such as gold or nickel ones. For all incubations, grids are placed on 30-ml droplets on Parafilm within a petri dish. Buffer rinses are carried out by floating grids on 50-ml droplets. Take great care not to wet the opposite grid face while transferring grids from one solution to another.
138
Walter Malorni et al.
1. For epoxy resin sections only, etching with 0.5% sodium metaperiodate for 5 to10 min at room temperature is required to allow exposure of antigenic sites. 2. To avoid nonspecific background labeling, a blocking step in PBS containing 1% BSA as blocking agent for 20 to 30 min at room temperature is recommended. 3. Incubate in specific primary antibody for 1 h at room temperature or overnight at 4 in a humidified chamber. The latter is preferable, as it appears to produce lower levels of nonspecific binding. To determine the optimal conditions for labeling with minimum background, the concentration of the primary antibody should be determined experimentally by serial dilution in PBS containing 1% BSA. Polyclonal antibody dilutions are usually 1:10, 1:100, 1:1000, and 1:10,000. Monoclonal antibody dilutions are normally between 1:5 and 1:30. Concurrent incubations should be performed in order to confirm the specific labeling of the sections:
Omit the primary antibody and replace with PBS containing 1% BSA for checking the secondary antibody. Replace the primary antibody with normal nonimmune serum, obtained from the same animal, for checking the primary antibody. 4. Wash in PBS containing 1% BSA several times for 5 min each. 5. Incubate with gold-conjugated secondary antibody diluted 1:10 to 1:50 in PBS containing 1% BSA for 1 h at room temperature. The usual range of gold probe size for TEM is 5 to 20 nm. Probes 5 nm in size have the advantage of improving spatial resolution over the tissue. However, because of their size, they present some disadvantage in the observation at the EM. 6. Wash in PBS containing 1% BSA several times for 5 min each. 7. Fix grids in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 15 min at room temperature. 8. Wash grids twice in distilled water for 5 min each. 9. Staining is recommended for acrylic resin sections (5 min in a saturated solution of aqueous uranyl acetate and 1 min in lead citrate). Staining is instead facultative for epoxy resin sections. To avoid insoluble precipitates of lead carbonate over the sections, lead citrate staining should be carried out in a covered petri dish containing sodium hydroxide pellets.
Lipid Rafts in Cell Apoptosis
139
REFERENCES Bogers, J. J., Nibbeling, H. A., Deelderm, A. M., and van Marck, E. A. (1996). Quantitative and morphological aspects of Unicryl versus Lowicryl K4M embedding in immunoelectron microscopic studies. J. Histochem. Cytochem. 44, 43–48. Bossy-Wetzel, E., and Green, D. R. (2000). Assay for cytochrome c release from mitochondria during apoptosis. Methods Enzymol. 322, 235–242. Brorson, S. H. (1998). Antigen detection on resin sections and methods for improving the immunogold labeling by manipulating the resin. Histol. Histopathol. 13, 275–281. Brown, D. A., and London, E. (2000). Structure and function of sphingolipid and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224. Edidin, M. (2003). The state of lipid rafts: From model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283. Garofalo, T., Giammarioli, A. M., Misasi, R., Tinari, A., Manganelli, V., Gambardella, L., Pavan, A., Malorni, W., and Sorice, M. (2005). Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells. Cell Death Differ. 12, 1378–1389. Garofalo, T., Lenti, L., Longo, A., Misasi, R., Mattei, V., Pontieri, G. M., Pavan, A., and Sorice, M. (2002). Association of GM3 with Zap-70 induced by T cell activation in plasma membrane microdomains: GM3 as a marker of microdomains in human lymphocytes. J. Biol. Chem. 227, 11233–11238. Garofalo, T., Misasi, R., Mattei, V., Giammarioli, A. M., Malorni, W., Pontieri, G. M., Pavan, A., and Sorice, M. (2003). Association of the death-inducing signaling complex with microdomains after triggering through CD95/Fas: Evidence for caspase-8-ganglioside interaction in T cells. J. Biol. Chem. 278, 8309–8315. Gonzalez Santander, R., Martinez Cuadrado, G., Gonzalez-Santander Martinez, M., Monteagudo, M., Martinez Alonso, F. J., and Toledo Lobo, M. V. (1997). The use of different fixatives and hydrophilic embedding media (Historesin and Unicryl) for the study of embryonic tissues. Microsc. Res. Tech. 36, 151–158. Grassme´, H., Jekle, A., Riehle, A., Schwarz, H., Berger, J., Sandhoff, K., Kolesnick, R., and Gulbins, E. (2001). CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem. 276, 20589–20596. Ipsen, J. H., Karlstrom, G., Mouritsen, O. G., Wennerstrom, H., and Zuckermann, M. J. (1987). Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta 905, 162–172. Iwabuchi, K., Handa, K., and Hakomori, S. I. (2000). Separation of glycosphingolipidenriched microdomains from caveolar membrane characterized by presence of caveolin. Methods Enzymol. 312, 488–494. Malorni, W., Giammarioli, A., Garofalo, T., and Sorice, M. (2007). Dynamics of lipid raft components during lymphocyte apoptosis: The paradigmatic role of GD3. Apoptosis 12, 941–949. Scala, C., Cenacchi, G., Ferrari, C., Pasquinelli, G., Preda, P., and Manara, G. C. (1992). A new acrylic resin formulation: A useful tool for histological, ultrastructural, and immunocytochemical investigations. J. Histochem. Cytochem. 40, 1799–1804. Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A., and Simons, K. (2003). Resistance of cell membranes to different detergents. Proc. Natl. Acad. Sci. USA. 100, 5795–5800. Schwarz, A., and Futerman, A. H. (2000). Immunolocalization of gangliosides by light microscopy using anti-ganglioside antibodies. Methods Enzymol. 312, 179–187. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572.
140
Walter Malorni et al.
Skibbens, J. E., Roth, M. G., and Matlin, K. S. (1989). Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts. J. Cell Biol. 108, 821–832. Svennerholm, L., and Fredman, P. A. (1980). A procedure for the quantitative isolation of brain gangliosides. Biochim. Biophys. Acta 617, 97–109. Williams, M. A., and McCluer, R. H. (1980). The use of Sep-Pak C18 cartridges during the isolation of gangliosides. J. Neurochem. 35, 266–269.
S I X
Analyzing Lipid Raft Dynamics during Cell Apoptosis Walter Malorni,* Tina Garofalo,† Antonella Tinari,‡ Valeria Manganelli,† Roberta Misasi,† and Maurizio Sorice† Contents 126 128 128 130 130 131 131 131 132 133 133 133 134 134 134 134 135 136 136 137 137 139
1. Introduction 2. The Contribution of Biochemical Approaches 2.1. Coimmunoprecipitation 2.2. Protocol 2.3. Coimmunoprecipitation from cell pellet 2.4. Coimmunoprecipitation from DRM fractions 2.5. Ganglioside analysis in immunoprecipitate samples 2.6. Reagents 2.7. Procedure 2.8. Isolation of mitochondria 2.9. Procedure 2.10. Detergent solubilization of isolated mitochondria 2.12. Coimmunoprecipitation from isolated mitochondria 3. The Contribution of Morphological Analyses 3.1. Light microscopy 3.2. Protocol 3.3. Postembedding electron microscopy 3.4. Fixation 3.5. Embedding medium 3.6. Embedding procedure 3.7. Postembedding procedure References
* { {
Department of Drug Research and Evaluation, Istituto Superiore di Sanita`, Rome, Italy Department of Experimental Medicine, "Sapienza" University of Rome, Rome, Italy Department of Technology and Health, Istituto Superiore di Sanita`, Rome, Italy
Methods in Enzymology, Volume 442 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)01406-7
#
2008 Elsevier Inc. All rights reserved.
125
126
Walter Malorni et al.
Abstract Increasing lines of evidence suggest a role for lipid rafts, glycosphingolipidenriched microdomains, in cell life and death. This chapter describes in brief the methods used to analyze raft interactions with proteins involved in apoptosis. This chapter focuses mainly on coimmunoprecipitation methods, which represent a useful tool in analyzing raft dynamics during apoptosis. Glycosphingolipid analysis in the immunoprecipitates is performed by thin-layer chromatography. Moreover, methods for the analysis of mitochondrial raft-like microdomains are also described. Detergent (Triton X-100)-insoluble material from isolated mitochondria can be analyzed by Western blot. Further insights can also come from both light and electron microscopy analyses. These can provide useful information as concerning lipid raft distribution at the cell surface or in the cell cytoplasm. Paradigmatic micrographs are shown. The combined use of all these different approaches appears to be mandatory for analyzing the role of lipid raft dynamics during apoptosis.
1. Introduction Several investigations have been carried out since the late 1990s in order to address the function of lipid rafts in cell life and death. It is commonly accepted that the plasma membrane is a complex system in which constituents are organized in small lipid/protein domains, known as ‘‘lipid rafts.’’ Membrane rafts are a specific type of lipid domains enriched in sphingolipids, including gangliosides (GSLs), sphingomyelin, and cholesterol, that result from tight hydrophobic interactions among these molecules, leading to the spontaneous formation of aggregates that separate from glycerophospholipids in cell membranes (Simons et al., 1997). Because of their lipid composition, the physical state of these domains is supposed to be similar to a liquid-ordered (Lo) phase, structurally and dynamically distinct from the rest of the lipid bilayer, which is, in turn, assumed to be in a liquiddisordered phase (Ipsen et al., 1987). On the basis of the peculiar biophysical and biochemical properties of lipid rafts, a number of possible interactions with various subcellular structures have been suggested. It is well known that these lipid microdomains may function to concentrate or segregate different proteins to form a glycosignaling domain. Several studies described a role for rafts in a variety of cellular processes, such as cell signaling, lipid and protein sorting, membrane organization and trafficking, immune response, and cell death (Malorni et al., 2007). Indeed, a large variety of proteins have been detected in these microdomains, including tyrosine kinase receptors (EGF-R), G proteins, Src-like tyrosine kinases (lck, lyn, fyn), protein kinase c isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins, adhesion molecules, and the death-inducing signaling complex. Thus, a general function of lipid
127
Lipid Rafts in Cell Apoptosis
rafts in signal transduction may be to allow the lateral segregation of proteins within the plasma membrane, providing a mechanism for the compartmentalization of signaling components, concentrating certain components in lipid rafts, including those of importance in apoptosis, and excluding others (Simons et al., 1997). Several studies indicate that lipid rafts could play a role in the apoptotic cascade. Although they are detected in various cell types, their role has been studied mostly in lymphocytes undergoing apoptosis induced by CD95/Fas. In particular, it was proposed that rafts are the primary site of action of the enzyme sphingomyelinase, which catalyzes the hydrolysis of sphingomyelin, resulting in ceramide formation; ceramides deriving from sphingomyelin hydrolysis are essential mediators of apoptotic signals originating from CD95/Fas (Grassme´ et al., 2001). Moreover, a role for gangliosides as structural components of the multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway has been reported (Garofalo et al., 2003). Although lipid rafts are considered ubiquitous constituents of plasma membrane, recent lines of evidence also indicated that they are present on organelles, including the Golgi apparatus and a subcompartment of the endoplasmic reticulum. Raft-like microdomains have also been detected on mitochondrial membranes after CD95/Fas triggering (Garofalo et al., 2005). Formation of a multimolecular complex that includes VDAC-1, Bcl-2 family, and fission proteins, for example, h-Fis, has been demonstrated. Thus, the complex may represent preferential sites where some key reactions can take place and can be catalyzed, leading to either survival or death of T cells. Different methods for studying lipid rafts in cell apoptosis are reported in Table 6.1.
Table 6.1 Methods used for studying lipid rafts in apoptosis
Apoptosis
Lipid rafts
Electron microscopy TUNEL Annexin V Activity of caspases DNA laddering Mitochondrial membrane potential Sucrose gradient analysis Coimmunoprecipitation FRET Single particle tracking Fluorescence correlation spectroscopy Scanning confocal microscopy Electron microscopy
128
Walter Malorni et al.
2. The Contribution of Biochemical Approaches In the last years, numerous different techniques have been considered for studying the association of apoptosis-related proteins with lipid rafts. The use of nonionic detergent extraction to generate low-density detergent-resistant membranes (DRMs) has had a major role in implicating rafts in cellular functions (Brown et al., 2000). Although this treatment may disrupt lipid– lipid interactions, a minor fraction of cell membranes is preserved and can be isolated as DRMs. Because detergent extraction may also disrupt several lipid– protein interactions, only a few proteins, interacting strongly with highly ordered domains, retain their association with lipids and are recovered in DRMs (Schuck et al., 2003). Essentially, all of these proteins have a saturated hydrocarbon chain modification, as a membrane anchor inserted into the raft domain of the outer plasma membrane leaflet and many transmembrane proteins require palmitoylation for their association to lipid rafts. Accordingly, isolation of DRMs represents a valuable tool for the analysis of raft-associated proteins involved in cell apoptosis and a useful starting point for defining membrane subdomains and/or protein–ganglioside interaction. Applying a variety of detergents may affect lipid–protein interactions (Schuck et al., 2003). Specific protein–lipid interactions within rafts have been studied preferentially by coimmmunoprecipitation experiments (Garofalo et al., 2002, 2003) or by fluorescence resonance energy transfer (FRET) (Edidin, 2003). In addition, many new approaches for detecting heterogeneity in cell membranes have emerged that rely on the distinct diffusion characteristics or enhanced proximity between raft components. Single particle tracking (SPT) has enabled us to measure the diffusion characteristics of GPI-anchored proteins. Fluorescence correlation spectroscopy, which combines different evaluations of biophysical properties of the plasma membrane, can contribute to better analyze the dynamics of raft components in living cells. Some of these approaches are of widespread use in laboratory practice (mainly coimmunoprecipitation experiments), whereas others are specifically employed under certain experimental conditions. Coimmunoprecipitation has become the tool of choice for analyzing the dynamics of raft components and their functional interaction with proteins involved in cell apoptosis (Fig. 6.1).
2.1. Coimmunoprecipitation The starting material can be (i) a cell pellet or (ii) the DRM fraction, containing lipid rafts, isolated by sucrose density-gradient ultracentrifugation after Triton X-100 (TX-100) solubilization at 4 , according to Iwabuchi et al. (2000).
129
Lipid Rafts in Cell Apoptosis
A
1 mm B
0.5 mm C
Caspase-8 immunoprecipitates
GM3
GM1
Anti-GM3
CTxB
Figure 6.1 (A) Immunogold transmission electron microscopy showing the typical clustered distribution of gold particles, corresponding to ganglioside-enriched microdomains, on cell plasma membrane of a T cell. (B) Immunogold labeling of GD3 in CEM cells induced to apoptosis by treatment with anti-CD95/Fas. Cells were prepared following the standard embedding procedure. Ultrathin sections, picked up on gold grids, were labeled using an anti-GD3 MoAb (1:30) as the primary antibody and, subsequently, with an antimouse IgM-10 nm gold conjugated (1:10). Note the distribution of GD3 molecules, which appear specifically localized on mitochondrial membranes. (Inset) The well-arranged distribution of gold particles at higher magnification.
130
Walter Malorni et al.
Detergents Although TX-100 is the most widely used detergent for the purification of lipid rafts, other detergents have been used for this purpose, including Brij96, Brij98, CHAPS, Triton X-14, and Lubrol WX (Schuck et al., 2003). The use of detergents to solubilize the cell membrane leads to the generation of small micelles and large particles that can be separated by centrifugation at low temperature (4 ) for 17 to 18 h at 200,000g in a 5 to 35% linear discontinous gradient of sucrose (Iwabuchi et al., 2000). Before starting solubilization, make sure all lysing/solutions are prechilled on ice, as it has been also shown that the amount of lipids in the plasma membrane raft is increased at low temperature.
2.2. Protocol 2.2.1. Cultured cells Both adherent cell lines and cells growing in suspension can be examined. 2.2.2. Reagents
Lysis buffer: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 mg of leupeptin/ml, 1 mM sodium orthovanadate Protein A/G acrylic or magnetic beads
2.3. Coimmunoprecipitation from cell pellet 1. Collect the culture supernatant, centrifuge at 300g for 10 min, and wash twice with ice-cold phosphate-buffered saline (PBS). 2. After removing the supernatant, resuspend the pellet in 1 ml ice-cold lysis buffer. 3. Transfer the lysate to a 1-ml homogenization tube and set on ice for at least 20 to 30 min. 4. Homogenize the samples using a tight-fitting Dounce pestle (20 strokes), maintaining the tube on ice at all times. 5. Transfer the lysate to a 1.5-ml tube and centrifuge for 5 min at 1300g to remove nuclei and large cellular debris. 6. Determine the protein concentration in the supernatant fraction using the Bio-Rad kit (Bio-Rad, Richmond, CA) with bovine serum albumin (BSA) as standard. (C) Tcells, undergoing apoptosis induced by CD95/Fas, were immunoprecipitated with anticaspase-8. The immunoprecipitates were analyzed for the presence of ganglioside molecules byTLC immunostaining using anti-GM3 MoAb or cholera toxin, B subunit (CTxB) (from Garofalo et al., 2003).
Lipid Rafts in Cell Apoptosis
131
7. Mix the supernatant fraction with protein A/G acrylic beads (Sigma Chemical Co., St. Louis, MO), stirring by a rotary shaker for 2 h at 4 to preclear nonspecific binding. 8. Centrifuge the lysate for 1 min at 500g at 4 . 9. Collect the supernatant. 10. Incubate with specific antibodies for specific binding with proteins, stirring by a rotary shaker for 2 h at 4 . 11. Add protein A/G acrylic beads for 1 h. 12. Centrifuge the sample for 1 min at 500g at 4 . 13. Remove the supernatant. Resuspend the pellet in 0.5 ml of H2O and transfer it into a 15-ml tube for ganglioside extraction.
2.4. Coimmunoprecipitation from DRM fractions Detergent-resistant membranes, obtained as described (Iwabuchi et al., 2000), are subjected to coimmunoprecipitation following the procedure starting from step 7.
2.5. Ganglioside analysis in immunoprecipitate samples Immunoprecipitation samples are subjected to GSL analysis by the highperformance thin-layer chromatography (HPTLC) technique. HPTLC has become the tool of choice for the identification and quantification of GSLs in small amounts of sample extracted from the plasma membrane of T cells, based on the fact that the HPTLC plate has a high-resolution property for glycosphingolipid separation. Of importance, the detection of glycosphingolipids after HPTLC separation is achieved by using specific colorimetric reagents or specific anti-GSLs monoclonal antibodies, followed by peroxidase-conjugated secondary antibodies. The latter represents an important method used to identify with high sensitivity small amounts (10 ng) of protein-associated ganglioside.
2.6. Reagents 1. HPTLC aluminium-backed silica gel 60 (20 20) plates (Merck, Darmstadt, Germany) 2. Solvent system for TLC (chloroform:methanol:0.25% aqueous KCl (5:4:1) (v:v:v) 3. Resorcinol-HCl reagent. Resorcinol (200 mg) in 20 ml of distilled water is mixed with 80 ml of concentrated HCl and 0.25 ml of 0.1 M CuSO4 4. 0.5% (w/v) solution of poly(isobutyl methacrylate) beads (Polyscience, Warrington, PA) dissolved in hexane (PIM solution) 5. 0.1% (w/v) BSA/PBS 6. Monoclonal antibodies against glycosphingolipids
132
Walter Malorni et al.
7. Secondary antibody, horseradish peroxidase-conjugated antimouse immunoglobulins 8. Enhanced chemiluminescence (ECL) Western blotting detection reagents (Amersham Life Science Ltd., Buckinghamshire, UK)
2.7. Procedure Gangliosides from immunoprecipitates are extracted according to the method of Svennerholm et al. (1980), with minor modifications. 2.7.1. Ganglioside extraction 1. Extract the immunoprecipitate in chloroform:methanol:water (4:8:3) (v:v:v) and centrifuge twice at 1500g for 30 min at room temperature. 2. Collect the supernatant and add water, resulting in a final chloroform: methanol:water ratio of 1:2:1.4 (v:v:v). 3. Centrifuge the gradient at 1500g for 30 min at room temperature. 4. Collect the upper phase containing polar glycosphingolipids. 5. Purify the upper phase of salts and low molecular weight contaminants using Bond elute C18 columns (Superchrom, Milan, Italy). According to Williams and McCluer, 1980. 2.7.2. TLC Immunostaining 1. Resuspend the ganglioside extract in 10 ml of chloroform:methanol 2:1 (v:v) and separate on HPTLC aluminum-backed plates. 2. After HPTLC, dry the plate thoroughly with a hair dryer to remove the organic solvent. 3. Soak gently the dried plate for 90 s in 0.5% PIM solution in a glass box of appropriate size. 4. Remove the plate from the PIM solution and air dry. 5. Incubate the plate with a buffer containing 5% BSA/PBS for 30 min at room temperature to block nonspecific absorption of antibodies. 6. Remove the blocking solution and wash the plate gently for 10 min with 0.5% BSA/PBS with two changes of the buffer. 7. Add the first antibody (anti-GSLs) diluted with 0.1% BSA/PBS for 1 h at room temperature. 8. Wash the plate with 0.5% BSA/PBS with three changes of the buffer and then incubate it with the peroxidase-labeled secondary antibody solution for 1 h at room temperature. 9. Wash with 0.5% BSA/PBS in the same way. 10. Using ECL, visualize the positive reaction.
Lipid Rafts in Cell Apoptosis
133
2.8. Isolation of mitochondria 2.8.1. Reagents and buffers Mitochondria isolation buffer (MIB): 220 mM mannitol, 68 mM sucrose, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 10 mM HEPES-KOH (pH 7.4), 0.1% (w/v) BSA, supplemented with a cocktail of protease inhibitors 2.8.2. Equipment Glass Dounce homogenizer (15 ml) with a tight Teflon pestle Glass Dounce homogenizer (2 ml) with a glass pestle (B type)
2.9. Procedure Preparation of cytosolic extracts Cytosolic extracts are obtained according to Bossy-Wetzel et al. (2000). Cells are harvested by centrifugation at 200g for 10 min at 4 , washed twice with cold PBS, pH 7.4, and resuspended in 3 volumes of cold cytosolic extraction buffer. After incubation on ice for 30 min, cells are disrupted by homogenization with a glass Dounce homogenizer and a tight glass pestle, applying 50 strokes. Nuclei and unaffected cells are removed by centrifugation at 800g for 10 min at 4 in an Eppendorf centrifuge. Supernatants are transferred to new Eppendorf tubes and centrifuged further at 22,000g for 30 min. Supernatants are then used for detergent solubilization.
2.10. Detergent solubilization of isolated mitochondria Reagents and buffers Extraction buffer: 25 mM HEPES, pH 7.5, 0.15 M NaCl, 1% Triton X-100, and 100 U/ml kallikrein/aprotinin Solubilization buffer: 50 mM Tris-HCl, pH 8.8, 5 mM EDTA, and 1% SDS 2.11. Procedure Supernatants containing isolated mitochondria are detergent solubilized according to Skibbens et al. (1989). Briefly, mitochondria are lysed with 1 ml of extraction buffer for 20 min on ice. Lysates are collected and centrifuged for 2 min in a Brinkmann microfuge at 10,000g at 4 . Supernatants, containing Triton X-100 soluble material, are collected; pellets are centrifuged a second time (30 s) in order to remove the remaining soluble material. Pellets are then solubilized in 100 ml of solubilization buffer. DNA is sheared by passage through a 22-gauge needle. Both Triton X-100-soluble and -insoluble material are then analyzed by Western blot for specific proteins.
134
Walter Malorni et al.
2.12. Coimmunoprecipitation from isolated mitochondria Isolated mitochondria are subjected to coimmunoprecipitation following the procedure reported earlier.
3. The Contribution of Morphological Analyses Numerous different morphological techniques have been considered for studying the complex sequence of structural modifications of lipid rafts during cell apoptosis. These are mainly qualitative analyses that can be carried out by means of light (LM) and electron microscopy (EM) techniques. Morphometric analyses, essentially performed by LM, can also be carried out. The use of these different technical approaches can provide information on the surface or intracellular localization of lipid rafts (Fig. 6.1).
3.1. Light microscopy The aim of immunocytochemistry is to localize antigens by labeling them with specific antibodies. Analyzing lipid raft dynamics in cell apoptosis, antiganglioside antibodies may be considered a good tool. However, we recommend that biochemical analysis be used to confirm that a particular ganglioside is present in the cell or tissue under test. 3.1.1. Reagents Available antiganglioside antibodies are summarized by Schwarz et al. (2000). 3.1.2. Fixation The major problem to be taken into consideration when using antiganglioside antibodies is the choice of fixative (Schwarz et al., 2000). For detection of lipid rafts on the cell surface, incubation with antibodies prior to fixation (prefixation) with 4% formaldehyde for 30 min at room temperature yields reproducible patterns of immunofluorescence. Alternatively, in order to detect raft-like microdomains associated with intracellular organelles (i.e., mitochondrion), lymphocytic cells may be postfixed with 4% paraformaldehyde in PBS for 30 min at room temperature and then permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature (Garofalo et al., 2005).
3.2. Protocol 1. Soak in Hanks’ balanced salt solution. 2. Incubate with anti-GSLs MoAb for 1 h (times and temperatures of incubation with primary antibodies are empirical and need to be determined for each antibody and cell type) at 4 .
Lipid Rafts in Cell Apoptosis
135
3. Wash three times in PBS, pH 7.4. 4. Fixate with 4% paraformaldehyde in PBS for 30 min (times and temperatures of incubation with primary antibodies are empirical and need to be determined for each antibody and cell type) at room temperature. 5. Wash three times in PBS, pH 7.4. 6. Stain with FITC-conjugated secondary antibody for 45 min (times and temperatures of incubation with primary antibodies are empirical and need to be determined for each antibody and cell type) at 4 . 7. Wash three times in PBS, pH 7.4. 8. Counterstain with Texas red-conjugated antiprotein antibody. 9. Wash three times in PBS, pH 7.4. 10. Mount in 0.1 M Tris-HCl, pH 9.2, containing 60% glycerol (v:v). 11. Acquire images through a confocal laser-scanning microscope. The green (FITC) and red (Texas red) fluorophores are excited simultaneously at 488 and 518 nm and are observed by two different detectors. Before image acquisition, samples are scanned at different filter conditions to choose a setting that reduces the overlap of emission spectra with a maximal signal-to-noise ratio. Then, acquired images are processed by subtracting a scaled version of green from red series, and vice versa. The scale factor (bleed-through factor) is determined by scanning single-stained samples in a dual fluorescence scanning configuration. Samples are counterstained with Texas red fluorophore, which reduces fluorescence overlapping greatly. Serial optical sections are assembled in depth-coding mode. Acquisition and processing are carried out using appropriate confocal software.
3.3. Postembedding electron microscopy There are two most common procedures that allow one to visualize cellular antigens with transmission electron microscopy: preembedding and postembedding techniques. Preembedding is used to label surface antigens only. It consists of an antigen–antibody reaction on living cells, that is, before the embedding and sectioning procedure. In contrast, the postembedding technique should be used when an internal antigen would be labeled. It consists of an antigen-antibody reaction in ultrathin sections, that is, after the embedding and sectioning procedure. For both of these methods, subcellular antigens recognized by primary antibodies can be localized and visualized with a transmission electron microscope (TEM) using gold-conjugated secondary antibodies to provide areas of high electron density. Colloidal gold can be coupled either to protein A, a protein from Staphylococcus cell walls that binds the Fc portion of immunoglobulins, or to a secondary antibody.
136
Walter Malorni et al.
Preservation of excellent structural details and, at the same time, maintenance of antigenic sites reactivity are the hardest but primary concerns for immune gold labeling. In order to preserve antigenicity, conventional electron microscopy procedure should be modified. In fact, few antigens survive to the routine fixation and embedding procedures. The tendency is to use a gentler aldehyde fixation, combined with low temperature embedding, that has been shown to better preserve the antigenicity of biological samples (Gonzalez et al., 1997; Scala et al., 1992) but, to some extent, this procedure compromises the ultrastructural features of the cells.
3.4. Fixation The reactivity and the stability of diverse antigens differ so widely from each other that no standardized method of fixation can be used for all EM immunocytochemistry experiments. Initially, it would be routinely worthwhile to assess the preparation of the sample. In fact, it is important to check if the antigen to be labeled is resistant to the fixation procedures and if the sample ultrastructural features could be preserved. Milder fixation regimens normally required to retain antigenicity can actually result in the loss of ultrastructural details. Depending on the chemical nature of such an antigen, successful immunolabeling may be obtained with routine preparations. Unfortunately, for most antigens the standard procedure results in a complete, or at least severely impaired, loss of antigenicity. This means that a range of preparative procedures, varying in their degree of fixation strength, is recommended to optimize the compromise between ultrastructural preservation and immunolabeling efficiency. In our experience, proteins are affected more profoundly by the standard embedding procedures, whereas lipid antigens are more likely to be preserved (Garofalo et al., 2005).
3.5. Embedding medium Biological specimens are embedded in resin in order to allow thin sectioning. Many different resin formulas are currently available and all these mixtures have been used for postembedding labeling (Bogers et al., 1996; Brorson, 1998). For immunocytochemistry purposes, acrylic resins that polymerize under ultraviolet light (UV) at 4 are preferred. Epoxy resins are exposed at 65 for polymerization with the possible denaturing effects of heat. Furthermore, acrylic resins have low viscosity and are hydrophilic, thus enhancing the subsequent immunolabeling on the sections.
Lipid Rafts in Cell Apoptosis
137
3.6. Embedding procedure 1. Fixation: antigens are quite commonly glutaraldehyde sensitive. Hence the variation of the concentration of this fixative has to be established experimentally for every antigen under consideration. Split the sample into three lots and fix as follows. Sample 1: 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2; 0.1 M ) Sample 2: 1% glutaraldehyde in sodium cacodylate buffer (pH 7.2; 0.1 M ) Sample 3: 4% paraformaldehyde þ 0.01% or 0.25% glutaraldehyde in sodium cacodylate buffer (pH 7.2; 0.1 M) for 20 min at room temperature or overnight at 4 2. Wash in 0.1 M sodium cacodylate buffer three to four times for 5 min each. 3. Postfixation for sample 1 only: postfix in 1% OsO4 in sodium cacodylate buffer for 30 to 60 min and carefully wash in sodium cacodylate buffer three to four times for 5 min each. Although most antigens are osmium tetroxide sensitive, some of them may resist this mild osmium fixation, thus improving preservation of ultrastructural details, especially membranes and membranous structures. 4. Dehydrate in graded series of ethanol solutions (50, 70, 95, and 100% twice) for 10 min each. 5. Resin embedding: Embed sample 1, which is OsO4 treated, in Epoxy resin–absolute ethanol mixtures (1:2; 1:1; 2:1; absolute) at room temperature and proceed as for the standard embedding procedure. Embed the other samples in acrylic resin as follows: withdraw absolute ethanol and add resin. The time and conditions of each acrylic resin are different; see the data sheet of the chosen resin. For example, Unicryl resin is directly added as absolute: leave the sample to infiltrate with resin for 4 h with at least two changes of resin and leave overnight at 4 . 6. Polymerization: Polymerize sample 1 at 65 for 48 h. Place Unicryl samples in closed gelatin capsules with fresh resin. Leave them to polymerize by suspension in a rack held 35 cm above a UV light lamp for 72 h.
3.7. Postembedding procedure Sections of about 80 nm are picked up on inert 200 mesh grids, such as gold or nickel ones. For all incubations, grids are placed on 30-ml droplets on Parafilm within a petri dish. Buffer rinses are carried out by floating grids on 50-ml droplets. Take great care not to wet the opposite grid face while transferring grids from one solution to another.
138
Walter Malorni et al.
1. For epoxy resin sections only, etching with 0.5% sodium metaperiodate for 5 to10 min at room temperature is required to allow exposure of antigenic sites. 2. To avoid nonspecific background labeling, a blocking step in PBS containing 1% BSA as blocking agent for 20 to 30 min at room temperature is recommended. 3. Incubate in specific primary antibody for 1 h at room temperature or overnight at 4 in a humidified chamber. The latter is preferable, as it appears to produce lower levels of nonspecific binding. To determine the optimal conditions for labeling with minimum background, the concentration of the primary antibody should be determined experimentally by serial dilution in PBS containing 1% BSA. Polyclonal antibody dilutions are usually 1:10, 1:100, 1:1000, and 1:10,000. Monoclonal antibody dilutions are normally between 1:5 and 1:30. Concurrent incubations should be performed in order to confirm the specific labeling of the sections:
Omit the primary antibody and replace with PBS containing 1% BSA for checking the secondary antibody. Replace the primary antibody with normal nonimmune serum, obtained from the same animal, for checking the primary antibody. 4. Wash in PBS containing 1% BSA several times for 5 min each. 5. Incubate with gold-conjugated secondary antibody diluted 1:10 to 1:50 in PBS containing 1% BSA for 1 h at room temperature. The usual range of gold probe size for TEM is 5 to 20 nm. Probes 5 nm in size have the advantage of improving spatial resolution over the tissue. However, because of their size, they present some disadvantage in the observation at the EM. 6. Wash in PBS containing 1% BSA several times for 5 min each. 7. Fix grids in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 15 min at room temperature. 8. Wash grids twice in distilled water for 5 min each. 9. Staining is recommended for acrylic resin sections (5 min in a saturated solution of aqueous uranyl acetate and 1 min in lead citrate). Staining is instead facultative for epoxy resin sections. To avoid insoluble precipitates of lead carbonate over the sections, lead citrate staining should be carried out in a covered petri dish containing sodium hydroxide pellets.
Lipid Rafts in Cell Apoptosis
139
REFERENCES Bogers, J. J., Nibbeling, H. A., Deelderm, A. M., and van Marck, E. A. (1996). Quantitative and morphological aspects of Unicryl versus Lowicryl K4M embedding in immunoelectron microscopic studies. J. Histochem. Cytochem. 44, 43–48. Bossy-Wetzel, E., and Green, D. R. (2000). Assay for cytochrome c release from mitochondria during apoptosis. Methods Enzymol. 322, 235–242. Brorson, S. H. (1998). Antigen detection on resin sections and methods for improving the immunogold labeling by manipulating the resin. Histol. Histopathol. 13, 275–281. Brown, D. A., and London, E. (2000). Structure and function of sphingolipid and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224. Edidin, M. (2003). The state of lipid rafts: From model membranes to cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283. Garofalo, T., Giammarioli, A. M., Misasi, R., Tinari, A., Manganelli, V., Gambardella, L., Pavan, A., Malorni, W., and Sorice, M. (2005). Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells. Cell Death Differ. 12, 1378–1389. Garofalo, T., Lenti, L., Longo, A., Misasi, R., Mattei, V., Pontieri, G. M., Pavan, A., and Sorice, M. (2002). Association of GM3 with Zap-70 induced by T cell activation in plasma membrane microdomains: GM3 as a marker of microdomains in human lymphocytes. J. Biol. Chem. 227, 11233–11238. Garofalo, T., Misasi, R., Mattei, V., Giammarioli, A. M., Malorni, W., Pontieri, G. M., Pavan, A., and Sorice, M. (2003). Association of the death-inducing signaling complex with microdomains after triggering through CD95/Fas: Evidence for caspase-8-ganglioside interaction in T cells. J. Biol. Chem. 278, 8309–8315. Gonzalez Santander, R., Martinez Cuadrado, G., Gonzalez-Santander Martinez, M., Monteagudo, M., Martinez Alonso, F. J., and Toledo Lobo, M. V. (1997). The use of different fixatives and hydrophilic embedding media (Historesin and Unicryl) for the study of embryonic tissues. Microsc. Res. Tech. 36, 151–158. Grassme´, H., Jekle, A., Riehle, A., Schwarz, H., Berger, J., Sandhoff, K., Kolesnick, R., and Gulbins, E. (2001). CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem. 276, 20589–20596. Ipsen, J. H., Karlstrom, G., Mouritsen, O. G., Wennerstrom, H., and Zuckermann, M. J. (1987). Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim. Biophys. Acta 905, 162–172. Iwabuchi, K., Handa, K., and Hakomori, S. I. (2000). Separation of glycosphingolipidenriched microdomains from caveolar membrane characterized by presence of caveolin. Methods Enzymol. 312, 488–494. Malorni, W., Giammarioli, A., Garofalo, T., and Sorice, M. (2007). Dynamics of lipid raft components during lymphocyte apoptosis: The paradigmatic role of GD3. Apoptosis 12, 941–949. Scala, C., Cenacchi, G., Ferrari, C., Pasquinelli, G., Preda, P., and Manara, G. C. (1992). A new acrylic resin formulation: A useful tool for histological, ultrastructural, and immunocytochemical investigations. J. Histochem. Cytochem. 40, 1799–1804. Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A., and Simons, K. (2003). Resistance of cell membranes to different detergents. Proc. Natl. Acad. Sci. USA. 100, 5795–5800. Schwarz, A., and Futerman, A. H. (2000). Immunolocalization of gangliosides by light microscopy using anti-ganglioside antibodies. Methods Enzymol. 312, 179–187. Simons, K., and Ikonen, E. (1997). Functional rafts in cell membranes. Nature 387, 569–572.
140
Walter Malorni et al.
Skibbens, J. E., Roth, M. G., and Matlin, K. S. (1989). Differential extractability of influenza virus hemagglutinin during intracellular transport in polarized epithelial cells and nonpolar fibroblasts. J. Cell Biol. 108, 821–832. Svennerholm, L., and Fredman, P. A. (1980). A procedure for the quantitative isolation of brain gangliosides. Biochim. Biophys. Acta 617, 97–109. Williams, M. A., and McCluer, R. H. (1980). The use of Sep-Pak C18 cartridges during the isolation of gangliosides. J. Neurochem. 35, 266–269.