Association of the death-inducing signaling complex with microdomains after triggering through CD95/Fas. Evidence for caspase-8-ganglioside interaction in T cells.

In this investigation we show that the death-inducing signaling complex (DISC) associates with glycosphingolipid-enriched microdomains (GEM) upon CD95/Fas engagement. We primarily analyzed the ganglioside pattern and composition of GEM after triggering through CD95/Fas and observed that GM3 is the main ganglioside constituent of GEM. Stimulation with anti-CD95/Fas did not cause translocation of gangliosides within or from the GEM fraction. Scanning confocal microscopy showed that triggering through CD95/Fas induced a significant GM3-caspase-8 association, as revealed by nearly complete colocalization areas. Coimmunoprecipitation experiments demonstrated that GM3 and GM1 were immunoprecipitated by anti-caspase-8 only after triggering through CD95/Fas. This association was supported by the recruitment of caspase-8, as well as of CD95/Fas, to GEM upon CD95/Fas engagement, as revealed by the analysis of linear sucrose gradient fractions. It indicates that the DISC associates with GEM; no changes were observed in the distribution of caspase-9. The disruption of GEM by methyl-beta-cyclodextrin prevented DNA fragmentation, as well as CD95/Fas clustering on the cell surface, demonstrating a role for GEM in initiating of Fas signaling. These findings strongly suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in CD95/Fas receptor-mediated apoptotic pathway.

Gangliosides, sialic acid-containing glycosphingolipids, are ubiquitous constituents of cell plasma membranes (1). However, each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 1 represents the main ganglioside constituent of cell plasma membrane (2), where it is concentrated in glycosphingolipidenriched microdomains (GEM) (3,4). The presence of tyrosine kinase receptors, mono-(Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (Lck, Lyn, Fyn), PKC isozymes, glycosylphosphatidylinositol-anchored proteins (5,6) and, after T cell activation, the Syk family kinase Zap-70 (7) prompts these portions of the plasma membrane to be considered as "glycosignaling domains" (8,9). Recently, we studied the glycosphingolipid composition of these specialized portions of plasma membrane in human peripheral blood lymphocytes (7), demonstrating by high performance thin layer chromatography, gas chromatographic and mass spectrometric analysis that GM3 represents one of the main markers of microdomains in these cells. Minor components are sialosyl paragloboside (NeuAc-nLac 4 Cer), migrating between GM3 and GM1, sialosyl lactohexaosyl ceramide, and disialoganglioside GD3 (2,10,11). Gangliosides may be involved in modulating signal transduction, mainly by interaction with specific signal transducer molecules detected in these domains (8), such as p56 lck (12) or Zap-70 (7). In addition, we demonstrated an association of GM3 and GD3 gangliosides with the cytoskeletal protein ezrin, induced by CD95/Fas-mediated apoptosis in lymphoblastoid T cells (13).
Apoptosis is a type of cell death characterized by chromatin condensation, DNA fragmentation, and membrane blebbing (14). Triggering of cell apoptosis is strictly regulated by ligandreceptor systems, including Fas ligand-CD95/Fas (15,16). The binding of CD95/Fas by its ligand results in trimerization of the receptor, recruitment of Fas-associated death domain (FADD) protein to the death domain of CD95, and binding of caspase-8 to the death-effector domain of FADD (17). This process induces the formation of the DISC. Binding and activation of caspase-8 results in transmission of the activation signal to other caspases, in particular caspase-3, involvement of mitochondria with release of cytochrome c and membrane depolarization, and release of apoptosis-inducing factor (18). Two types of cells have been classified on the basis of the CD95/Fas signaling (19). Type-1 cells show a rapid recruitment of the DISC to the receptor resulting in the activation of caspase-8, whereas type-2 cells, including lymphoblastoid T cells (i.e. CEM) (20), have slow apoptotic kinetics depeding on mitochondrial activation. Evidence suggests that many receptors aggre-* This work was supported by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MIUR) and Ateneo 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ gate in distinct plasma membrane microdomains or rafts (21). This notion is supported by the finding that disruption of microdomains prevents clustering of many receptors, including TNF-R (22) and CD95/Fas (23).
On the basis of these considerations and following the observation that the main gangliosides present in GEM associate with ezrin-CD95 complex after the receptor triggering (13), we decided to analyze whether GEM were involved in the initiation of Fas/CD95 signaling.
Thus, in this investigation we analyzed the ganglioside pattern of GEM after CD95/Fas engagement and then we focused on the interaction of the death-inducing signaling complex with these domains.
Ganglioside Extraction and Analysis by High Performance Thin Layer Chromatography (HPTLC)-Ganglioside extraction was performed according to the method of Svennerholm and Fredman (25), with minor modifications. Briefly, glycosphingolipids were extracted twice in chloroform:methanol:water (4:8:3) (v:v:v) and subjected to Folch partition by the addition of water resulting in a final chloroform: methanol:water ratio of 1:2:1.4. The upper phase, containing polar glycosphingolipids, was purified of salts and low molecular weight contaminants using Bond Elut-C18 columns, 3 ml (Superchrom, Harbor City, CA), according to the method of Williams and McCluer (26). The eluted glycosphingolipids were dried down and separated by HPTLC, using silica gel 60 HPTLC plates (Merck, Darmstadt, Germany). Chromatography was performed in chloroform:methanol:0.25% aqueous KCl (5:4:1) (v:v:v). Plates were then air-dried and gangliosides visualized with resorcinol (27).
Alternatively, the ganglioside extract was run on HPTLC aluminumbacked silica gel 60 (20 ϫ 20) plates (Merck). Plates were soaked in a 0.2% solution of polyisobutylmethacrylate in hexane for 90 s, air-dried, and incubated in blocking solution consisting of 3% albumin (Sigma) in phosphate-buffered saline (PBS) pH 7.4, for 1 h at room temperature. The blocking solution was removed and replaced by washing buffer (PBS). The plates were then incubated for 1 h at room temperature with HRP-conjugated cholera toxin, B subunit (CTxB, Sigma). Immunoreactivity was assessed by chemiluminescence reaction using the ECL Western blocking detection system (Amersham Biosciences, Buckinghamshire, UK).
Isolation and Analysis of GEM Fraction-GEM fraction from lymphoblastoid CEM cells, either untreated or treated with anti-CD95/Fas (250 ng/ml for 1 or 2 h at 37°C), was isolated as described previously (28). Briefly, 2 ϫ 10 8 cells were suspended in 1 ml of lysis buffer containing 1% Triton-X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM NaVO 4 , and 75 units of aprotinin and allowed to stand for 20 min. The cell suspension was mechanically disrupted by Dounce homogenization (10 strokes). The lysate was centrifuged for 5 min at 1300 ϫ g to remove nuclei and large cellular debris. The supernatant fraction (postnuclear fraction) was subjected to sucrose density gradient centrifugation, i.e. the fraction was mixed with an equal volume of 85% sucrose (w/v) in lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA). The resulting diluent was placed at the bottom of a linear sucrose gradient (5-30%) in the same buffer and centrifuged at 200,000 ϫ g for 16 -18 h at 4°C in a SW41 rotor (Beckman Instruments). After centrifugation, the gradient was fractionated, and 11 fractions were collected starting from the top of the tube. All steps were done at 0 -4°C. The amount of protein in each fraction was first quantified by Bio-Rad protein assay (Bio-Rad GmbH, Munchen, Germany).
Finally, the GEM fraction from untreated and CD95/Fas-stimulated cells was subjected to ganglioside extraction, as reported above.
Analysis of GM3-Caspase-8 Colocalization by Scanning Confocal Microscopy-CEM cells, either untreated or treated with anti-CD95/Fas (250 ng/ml for 1 h at 37°C), were fixed with 4% formaldehyde in PBS for 30 min at 4°C and labeled with anti-caspase-8 MoAb (Upstate Biotechnology) for 1 h at 4°C, followed by addition (30 min at 4°C) of Texas Red-conjugated anti-mouse IgG (Calbiochem). After three washes in PBS, cells were incubated with GMR6 anti-GM3 MoAb (Seikagaku Corp., Chuo-ku, Tokyo, Japan) (29) for 1 h at 4°C, followed by three washes in PBS and addition (30 min at 4°C) of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (Sigma). In parallel experiments, cells were stained with anti-GM3 MoAb before fixing the cells. Cells were finally washed three times in PBS and resuspended in glycerol/Tris-HCl (pH 9.2). The images were acquired through a confocal laser scanning microscope Leica, equipped with an argon ion laser. Simultaneously, the green (FITC) and the red (Texas Red) fluorophores were excited at 488 and 518 nm. Acquisition of single FITC-stained samples in dual fluorescence scanning configuration did not show contribution of green signal in red. Images were collected at 512 ϫ 512 pixels. Gangliosides were extracted in chloroform/methanol/water. The plate was stained with resorcinol (ganglioside-specific stain). Lane A, gangliosides obtained from total CEM cells; lane B, standard gangliosides GM3, GM1, GD3, GD1a, GD1b, and GT1b. b, TLC immunostaining of the ganglioside extract with HRP-conjugated CTxB. Gangliosides were separated using HPTLC aluminium-backed silica gel plates. Immunoreactivity was assessed by chemiluminescence reaction using the ECL Western blocking detection system. Lane A, gangliosides obtained from CEM cells; lane B, standard GM1.

FIG. 2. Ganglioside distribution in CEM cells sucrose gradient fractions.
Lymphoblastoid CEM cells were lysed in lysis buffer, and the supernatant fraction (postnuclear fraction) was subjected to sucrose density gradient. After centrifugation the gradient was fractionated, and each gradient fraction was recovered. Gangliosides were extracted in chloroform-methanol-water. The plate was stained with resorcinol (ganglioside-specific stain). A, untreated CEM cells; B, anti-CD95/Fastreated (250 ng/ml for 2 h at 37°C) CEM cells.
The immunoprecipitates were subjected to ganglioside extraction as reported above. The ganglioside extract was split into two aliquots. A portion was separated by HPTLC, using silica gel 60 HPTLC plates (Merck) and stained with resorcinol (27). Another portion was run on HPTLC aluminum-backed silica gel 60 (20 ϫ 20) plates (Merck), as reported above. The plates were immunostained for 1 h at room temperature with HRP-conjugated CTxB (Sigma) or, alternatively, with GMR6 anti-GM3 MoAb (Seikagaku Corp.) and then HRP-conjugated anti-mouse IgM (Sigma). Immunoreactivity was assessed by chemiluminescence reaction using the ECL Western blocking detection system (Amersham Biosciences).
Detergent Solubilization-Lymphoblastoid CEM cells, either untreated or treated with anti-CD95/Fas (250 ng/ml for 5 min, 30 min or 1 h at 37°C), were detergent solubilized according to Skibbens et al. (30). Briefly, cells were lysed on ice with 1 ml of extraction buffer (25 mM HEPES (pH 7.5), 0.15 NaCl, 1% Triton X-100, and 100 kallikrein units/ml aprotinin) for 20 min on ice. Lysates were collected and centrifuged for 2 min in a Brinkmann microfuge at 12,000 rpm at 4°C. Sovranatants, containing Triton X-100-soluble material, were collected; pellets were undertaken to a second centrifugation (30 s) to remove the remaining soluble material. The pellets were then solubilized in 100 l of buffer containing 50 mM Tris-HCl (pH 8.8), 5 mM EDTA, and 1% SDS. DNA was sheared by passage through a 22-gauge needle. Both Triton X-100-soluble and -insoluble material were analyzed by Western blot as described above.
Immunoblotting Analysis of GEM Fraction-All the fractions obtained as reported above were subjected to 10% sodium dodecyl sulfatepolyacrilamide gel electrophoresis (SDS-PAGE). The proteins were electrophoretically transferred to nitrocellulose membrane (Bio-Rad) and then, after blocking with PBS, containing 1% albumin, probed with anticaspase-8 MoAb (Upstate Biotechnology), anti-caspase-9 polyclonal Ab (Upstate Biotechnology), or anti-CD95/Fas MoAb (Medical & Biological Laboratory). Bound antibodies were visualized with HRP-conjugated anti-mouse or anti-rabbit IgG (Sigma) and immunoreactivity assessed by chemiluminescence reaction using the ECL Western blocking detection system (Amersham Biosciences). As a control for nonspecific reactivity, parallel blots were performed as above, using an anti-mouse IgG (Sigma).
Densitometric scanning analysis was performed by Mac OS 9.0 (Apple Computer International) using NIH Image 1.62 software. The density of each band in the same gel was analyzed, values were totaled, and then the percent distribution across the gel was detected.
Immunofluorescence and Intensified Video Microscopy-CD95/Fas distribution was analyzed by immunofluorescence in cells treated with anti-CD95/Fas (250 ng/ml for 2 h at 37°C) or in cells preincubated with 5 mM methyl-␤-cyclodextrin (M␤CD) (20 min at 37°C) in 10 mM HEPES, either treated or untreated with anti-CD95/Fas. Control and treated cells were collected by centrifugation, attached to glass coverslips precoated with polylysine, and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. After washing in the same buffer, cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. Samples were incubated at 37°C for 30 min with polyclonal antibodies to CD95 (Santa Cruz Biotechnology, Santa Cruz, CA). Cells were then incubated with FITC-conjugated anti-rabbit IgG (Sigma). After washing, all samples were mounted with glycerol/PBS (2:1) and observed with a Nikon Microphot fluorescence microscope. Images were captured by a color chilled 3CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan). Normalization and background subtraction were performed for each image. fixed with cold 70% ethanol in PBS for 1 h at 4°C. After centrifugation at 200 ϫ g for 10 min at 4°C, cells were washed once in PBS. The pellet was resuspended in 0.5 ml PBS, 50 l of RNase (Type I-A, Sigma, 10 mg/ml in PBS) was added, followed by 50 g/ml propidium iodide (Sigma) in PBS. The cells were incubated in the dark at room temperature for 15 min and kept at 4°C until measured. In parallel samples the amount of cholesterol was evaluated as described previously (32). Free cholesterol was quantitated from TLC plates by densitometric scanning and comparison with standard. The density of the bands used to quantitate cholesterol concentration fell within the linear range of compound concentration versus absorbance. A trypan blue exclusion test was performed to evaluate the viability of the cultures.

RESULTS
Ganglioside Pattern of Lymphoblastoid CEM Cells-We primarily analyzed the ganglioside pattern of lymphoblastoid CEM cells. Gangliosides were extracted in chloroform:methanol:water and separated in HPTLC as reported above. Five main resorcinol-positive bands were detected: a main GM3 comigrating double band, a band migrating between GM3 and GM1, a GM1 comigrating band, one comigrating with GD3, and one with GD1a (Fig. 1a). The GM3 double band is due to the heterogeneity of fatty acid composition, as described previously (7,33).
The identity of the band comigrating with GM1 was verified by HPTLC immunostaining using the CTxB. The analysis revealed that in these cells the band was immunostained by the CTxB (Fig. 1b), indicating that it is GM1.
Ganglioside Pattern of GEM after Triggering through CD95/ Fas-We investigated the ganglioside composition of sucrose gradient fractions obtained from lymphoblastoid CEM cells in the absence or in the presence of triggering through CD95/Fas. The resorcinol-positive bands were present mainly in the fraction 5 that, under our experimental condition, corresponds to GEM and, to a less extent, in the fractions 4, 6, and 7. About 90% of ganglioside content present in the total cell extract was recovered in the fractions 4 -7. No bands were detectable in the Triton X-100-soluble fractions 10 and 11.
The comparative analysis of the ganglioside pattern of sucrose gradient fractions revealed that triggering through CD95/Fas did not modify the distribution pattern of gangliosides (Fig. 2B).
Analysis of the Association of GM3 with Caspase-8 after Triggering through CD95/Fas-To study the possible association of caspase-8 with GEM after triggering through CD95/Fas, we analyzed, by scanning confocal microscopy, the distribution of caspase-8 and GM3 (Fig. 3). As expected, in untreated cells caspase-8 staining was mostly diffuse in the cytoplasm (A, panel 1), whereas after stimulation, it appeared uneven and punctate near the plasma membrane (A, panel 2), indicating that the protein translocates mostly in correspondence of specific membrane domains. This change of localization pattern is generally associated with protein activation (17). The results revealed that most of the cells showed an uneven signal distri- FIG. 5. A, lymphoblastoid CEM cells, either untreated or treated with anti-CD95/Fas (250 ng/ml for 5 min, 30 min, or 1 h at 37°C), were detergent-solubilized as described under "Experimental Procedures." Both Triton X-100-soluble and -insoluble materials were analyzed by Western blot and probed with anti-caspase-8 MoAb followed by incubation with HRP-conjugated anti-mouse IgG. Immunoreactivity was assessed by chemiluminescence reaction. B, caspase-8 distribution in control CEM sucrose gradient fractions. CEM cells were lysed in lysis buffer, and the supernatant fraction (postnuclear fraction) was subjected to sucrose density gradient. After centrifugation the gradient was fractionated, and each gradient fraction was recovered and analyzed by Western blotting with anti-caspase-8 MoAb. C, caspase-8 distribution in stimulated CEM (anti-CD95/Fas, 250 ng/ml for 1 h at 37°C) sucrose gradient fractions. CEM cells were lysed in lysis buffer, and the supernatant fraction was subjected to sucrose density gradient. After centrifugation the gradient was fractionated, and each gradient fraction was recovered and analyzed by Western blotting with anti-caspase-8 MoAb. D, caspase-9 distribution in control CEM sucrose gradient fractions. CEM cells were lysed in lysis buffer, and the supernatant fraction was subjected to sucrose density gradient. After centrifugation the gradient was fractionated, and each gradient fraction was recovered and analyzed by Western blotting with anti-caspase-9 polyclonal Ab. E, caspase-9 distribution in stimulated CEM (anti-CD95/Fas, 250 ng/ml for 1 h at 37°C) sucrose gradient fractions. CEM cells were lysed in lysis buffer, and the supernatant fraction was subjected to sucrose density gradient. After centrifugation the gradient was fractionated, and each gradient fraction was recovered and analyzed by Western blotting with anti-caspase-9 polyclonal Ab. bution of ganglioside molecules over the cell surface, either costitutively (B, panel 1) or after treatment with anti-CD95/Fas (250 ng/ml) for 2 h at 37°C (B, panel 2). To determine the possible association between caspase-8 and GM3, we superimposed the double immunostaining of anti-caspase 8 and anti-GM3 in the absence or in the presence of triggering through CD95/Fas. In the absence of cell stimulation, GM3 and caspase-8 showed weak colocalization (C, panel 1). This finding suggests that GM3 and caspase-8 are not associated in untreated CEM. After cell stimulation through CD95/Fas, the merged image of anti-CD95/Fas and anti-GM3 staining revealed brown yellow areas, resulting from overlap of green and red fluorescence, which correspond to nearly complete colocalization areas (C, panel 2). Therefore, cell triggering through CD95/Fas preferentially promotes translocation of caspase-8 in selective membrane microdomains in which GM3 is highly enriched.
Coimmunoprecipitation of Gangliosides and Caspase-8 after Triggering through CD95/Fas-To verify whether caspase-8 may interact with gangliosides, cell-free lysates from anti-CD95/Fas-treated and untreated cells were immunoprecipitated with the anti-caspase-8 MoAb, followed by protein Gacrylic beads. Acidic glycosphingolipids were extracted from the caspase-8 immunoprecipitates and analyzed by HPTLC analysis followed by either resorcinol staining or immunostaining. The analysis revealed a main GM3 comigrating double band and a GM1 comigrating band after triggering through CD95/Fas (Fig. 4, lane A). No resorcinol-positive bands were detected in the immunoprecipitates from untreated cells (Fig.  4, lane B). TLC immunostaining revealed that the extract from the immunoprecipitates from anti-CD95/Fas-treated cells were immunostained by both anti-GM3 MoAb (Fig. 4, lane D) and B subunit cholera toxin (Fig. 4, lane F). On the contrary, no bands were detected in the immunoprecipitates from untreated cells (Fig. 4, lanes E and G), as well as in control samples immunoprecipitated with a mouse IgG with irrelevant specificity, under the same condition (Fig. 4, lane H).
Recruitment of Caspase-8 to GEM after Triggering through CD95/Fas-To analyze the distribution of caspase-8, we inves-tigated the presence of this protein in both Triton X-100-insoluble and -soluble fractions after different incubation times with anti-CD95/Fas. As shown in Fig. 5A, caspase-8 was almost enterely soluble in Triton X-100. However, the protein became progressively less soluble in the detergent after triggering through CD95/Fas. By 1 h, about 90% was insoluble. To better clarify the distribution of the protein, GEM fractions of CEM cells, obtained by a 5-30% linear sucrose gradient, in the absence or in the presence of anti-CD95/Fas (250 ng/ml for 1 h at 37°C), were analyzed. The results revealed that in nonstimulated cells caspase-8 was present in fractions 6 -10, but was almost completely absent in the fraction 5, corresponding to GEM (Fig. 5B). After triggering through CD95/Fas, caspase-8 was detected in the detergent-insoluble fraction 5 and in fractions 4, 6, 7, and 8, but not in fractions 9 and 10 ( Fig. 5C), indicating that caspase-8 recruited to GEM upon CD95/Fas cross-linking. On the contrary, caspase-9 was detected in fractions 6 -11 in both anti-CD95/Fastreated (Fig. 5E) and untreated (Fig. 5D)

cells.
Distribution of CD95/Fas in Sucrose Density Gradient Fractions-To analyze the distribution of CD95/Fas, we primarily investigated the presence of this protein in both Triton X-100insoluble and -soluble fractions after different incubation times with anti-CD95/Fas. As shown in Fig. 6A, the receptor was mainly soluble in Triton X-100 and became progressively less soluble in the detergent after triggering through CD95/Fas. To better clarify the distribution of the protein, we also investigated the presence of CD95/Fas in the GEM fractions of CEM cells in the absence or in the presence of cross-linking with the antibody (250 ng/ml for 1 h at 37°C). The results revealed that in control cells only a small amount of CD95/Fas was detectable in the fraction 5, corresponding to GEM (5% of the total content, as revealed by densitometric analysis) (Fig. 6B). In cells treated with the antibody, CD95/Fas content in fraction 5 increased to 12% of the total content (Fig. 6C). It indicated that the antibody triggering induced CD95/Fas enrichment in GEM fraction, suggesting that GEM represent the plasma membrane sites from which CD95/Fas initiates signaling cascade upon binding to its ligand.

Effect of M␤CD on CD95/Fas Distribution and CD95/Fasinduced Apoptosis-
To analyze the contribution of GEM in CD95/Fas-induced apoptosis, we analyzed Fas distribution and DNA fragmentation after triggering through CD95/Fas of cells pretreated with 5 mM M␤CD (20 min at 37°C), which is known to induce cholesterol efflux from plasma membrane and, consequently, GEM desruption (9). The total cellular cholesterol concentration was about 60% that found in normal cells. Consistent with previous papers (13,34), immunoflorescence analysis showed that CD95/Fas might be detected at one pole of control CEM cells (Fig. 7a), and it was markedly polarized in anti-CD95 MoAb-treated cells (Fig. 7c). The disruption of lipid rafts by M␤CD led to the loss of cell polarity with redistribution of CD95/Fas molecule all over the CEM cells (Fig. 7b) and prevented the receptor polarization observed in anti-CD95 MoAb-treated cells (Fig. 7d). As expected, DNA staining with propidium iodide of anti-Fas treated cells followed by cytofluorimetric analysis showed a subdiploid peak of fluorescence, consistent with apoptosis. Fig. 7e shows that M␤CD greatly prevented the CD95/Fas-triggered apoptosis, suggesting that GEM play a key role in CD95/Fas signaling cascade, leading to programmed cell death.
In all samples the percentage of necrotic cells was less than 2%, as a result of trypan blue exclusion test. DISCUSSION In this investigation we provide evidence that in CEM lymphoblastoid T cells the death-inducing signaling complex associates with GEM upon CD95/Fas engagement. This finding extends our preliminary observation that in the same cells gangliosides GM3 and GD3 coimmunoprecipitated with the cytoskeletal protein ezrin (13), which, in turn, was complexed with CD95/Fas (34) after triggering through anti-Fas Ab, but not after treatment with staurosporine (13). Here, we primarily analyzed whether triggering through CD95/Fas modified the ganglioside pattern and composition of GEM. In agreement with our previous reports on human peripheral blood lymphocytes (3, 7), we observed that GM3 is the main ganglioside constituent of GEM and can be considered a marker of these specialized portions of plasma membrane in CEM lymphoblastoid T cells. Interestingly, we revealed by CTxB staining that also GM1 is present in these cells, although as a minor constituent. Moreover, stimulation with anti-CD95/Fas did not cause translocation of gangliosides within or from the GEM fraction.
Following our observation that T cell activation induced Zap-70-GM3 interaction within GEM (7) and the recent studies that pointed out the role of GEM in initiating of CD95/Fas triggered cell death in mouse thymocytes (35) or in lymphoblastoid T cells (13,23), we analyzed the possible association of DISC components with GM3 during CD95/Fas-triggered apoptosis. Scanning confocal microscopic observations showed the presence of GM3 clusters, revealing the existence on the cell surface of GEM with GM3 molecule concentration either in the absence or in the presence of triggering through CD95/Fas. These observations are consistent with previously reported thermodynamic results (36) and with our immunoelectron microscopic (3) and immunofluorescence (7) data in human peripheral blood lymphocytes. We now demonstrate by scanning confocal microscopy the association of caspase-8 with GEM, as revealed by nearly complete colocalization areas between caspase-8 and GM3 after triggering through CD95/Fas. Interestingly, T cell stimulation does not modify the ganglioside distribution, revealing that CD95/Fas engagement does not promote a redistribution of GEM, but induces a preferential translocation of DISC to discrete microdomains of cell plasma membrane in which it associates with GM3. This association was supported by coimmunoprecipitation experiments which demonstrated that not only GM3 but also GM1 were immunoprecipitated by anti-caspase 8 after triggering through CD95/Fas. In addition, the analysis of linear sucrose gradient fractions further clarified the recruitment of caspase-8, as well as of Fas, to GEM upon CD95/Fas engagement. Thus, we provide evidence that CD95/Fas triggering induces the lateral organization of rafts, bringing the CD95/Fas receptor together with GEM and demonstrating that the DISC associates with GEM. This finding strongly suggests a role for GEM in triggering of T cell apoptosis, since binding and activation of caspase-8 results in transmission of the activation signal to other caspases and involvement of mitochondria. Indeed, the binding of CD95/Fas by its ligand results in trimerization of the receptor, recruitment of FADD to the death domain of CD95 and binding of procaspase-8 to the death-effector domain of FADD (17). As a control, we show that caspase-9 did not modify its distribution pattern after triggering through CD95/Fas, which is consistent with the cytoplasmic localization of this caspase.
The key role of GEM in initiating of Fas signaling gained further support from the demonstration that disruption of GEM prevents DNA fragmentation as well as CD95/Fas clustering on the cell surface. These conclusions are fully in agree- M␤CD and then with anti-CD95/Fas, 250 ng/ml for 2 h at 37°C; e, DNA fragmentation studied by propidium iodide staining followed by flow cytometric analysis. It was evaluated in control cells, in cells treated with anti-CD95/Fas (250 ng/ml for 2 h at 37°C), or in cells preincubated with 5 mM M␤CD, either treated or untreated with anti-CD95/Fas. ment with the observations in mouse thymocytes (35), or in human CD4ϩ cells (37), and are supported by the demonstration that CD95/Fas clustering and signaling were mediated by ceramide-rich membrane rafts (23,38). Here, we show that apoptosis was prevented not only by neutralization of surface ceramide or inhibition of ceramide release (38), but also by cholesterol depletion on cell plasma membrane. These observations reveal an additional difference in the apoptotic pathways of type-1 and type-2 cells, since CD95/Fas signaling is GEMindependent in type-1 cells (20).
These findings strongly suggest a role for gangliosides as structural components of the membrane multimolecular signaling complex involved in cell apoptosis pathways. In our previous work (7) we demonstrated their involvement in T cell activation; the present one deals with cell death proneness. Hence, to understand the regulation pathways supervising both cell activation (and proliferation) and, on the opposite side, cell death by apoptosis could provide useful information on the subcellular mechanisms influencing T cell fate. The present work, according to literature (39), allows us to hypothesize that microdomains might represent a sort of "closed chamber," where specific key reactions can take place, including the phosphorylation of tyrosine kinases (8,9) and of the membrane/cytoskeletal linker ezrin (13,34), hijacking T cells toward their survival or death. This could also be of relevance in the elucidation of the regulatory mechanisms underlying cell death process in terms of both resistance and susceptibility.