TCR activation through cholesterol-depleted lipid rafts

Cellular Signalling 19 (2007) 1404 – 1418 www.elsevier.com/locate/cellsig

Full CD3/TCR activation through cholesterol-depleted lipid rafts ☆ Alexandre K. Rouquette-Jazdanian, Claudette Pelassy, Jean-Philippe Breittmayer, Claude Aussel ⁎ Institut National de la Santé et de la Recherche Médicale (INSERM) Unit 576, IFR 50, Hôpital de l'Archet I, 151 Route de Saint Antoine de Ginestière, B.P. 79, 06202 Nice Cedex 3, France Received 6 December 2006; received in revised form 11 January 2007; accepted 14 January 2007 Available online 23 January 2007

Abstract Exogenous bacterial sphingomyelinase (SMase) and C6-Ceramides (C6-Cer) considerably lower buoyant cholesterol on sucrose densitygradient (at least 55% less cholesterol). In opposition, short C2-Cer fails to displace buoyant cholesterol. Note that neither SMase nor C6-Cer delocalize raft markers (Lck, LAT, CD55, and GM1). They are still anchored in ceramides-rich/cholesterol-poor domains, demonstrating that cholesterol is not necessary for their buoyancy. SMase-treated cells, i.e. cells exhibiting cholesterol-depleted rafts, optimally transmit CD3-induced phosphorylations (tyrosine, threonine, and serine). SMase, that extracts and partially displaces buoyant cholesterol, does not inhibit PLCγ1–LAT interaction, Vav 1 phosphorylation, the actin polymerization, IL-2 and NF-κB (EMSA and luciferase assays) activation, and CD25 up-regulation (RT-PCR and cytometry) at all. Nevertheless, Ca2+ influx and diacylglycerol (palmitoyl-DAG and arachidonoy-DAG) production are lowered. The drop of CD3-induced Ca2+ influx is due to a strong plasma membrane depolarization because of Cer. The decreased DAG level is a consequence of the drop of intracellular Ca2+ that is a cofactor for the PLCγ1. In conclusion, our study challenges the real role of cholesterol-rich rafts in CD3/TCR signaling and suggests that other membrane domains than cholesterol-rich rafts can optimally transmit CD3/TCR signals. © 2007 Elsevier Inc. All rights reserved. Keywords: Sphingomyelin (SM); Sphingomyelinase (SMase); C6-Ceramides (C6-Cer); T lymphocyte activation; Rafts; Detergent-resistant membranes (DRMs)

1. Introduction In 1972, Singer and Nicolson proposed “the fluid mosaic model of the structure of cell membranes” [1]. In 1995, Jacobson revisited this model [2], and in 1997, rafts were born [3]. Immunologists [4] explain that rafts are platforms from which the CD3/TCR signals emanate. They are enriched in cholesterol, sphingomyelin (SM), glycosylphosphatidylinositol-anchored proteins (GPI-APs), gangliosides, Src kinases and LAT. As reminded by Viola's group in 2002, the raft conception for TCR signaling is only a theory. During 2003, Munro spoke ironically and printed that rafts were elusive or illusive. In 2004, Glebov and Nichols hammered it in since they demonstrated ☆ This work was supported by INSERM. Alexandre Rouquette-Jazdanian is a recipient of a doctoral fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche and from the ARC. ⁎ Corresponding author. INSERM U 576, Hôpital de l'Archet I, 151, Route Saint Antoine de Ginestière, B.P. 79, 06202 Nice Cedex 3, France. Tel.: +33 492 15 77 02; fax: +33 492 15 77 09. E-mail address: [email protected] (C. Aussel).

0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2007.01.015

that “lipid raft proteins have a random distribution during localized activation of T cell receptor”. During the Keystone Symposium on Lipid Rafts and Cell Function, 2006, the participants defined membrane rafts. There is one consensus: rafts are enriched both in cholesterol and SM. Pike [5] wrote (1) that “…enrichment in cholesterol and sphingolipids was readily adopted as a characteristic of membrane rafts…” and (2) that “detergent-resistance is an artificial and highly subjective approach that can induce the formation of membrane domains and hence does not provide physiologically relevant information” [6] “this approach (i.e. DRM isolation)… is no longer viable”. It is extremely paradoxical to juxtapose these two ideas in the same report since the clues for the enrichment in cholesterol come directly from DRM isolation. Indeed, in vivo studies with fluorescent cholesterol molecules gave contradictory results [7,8] and cholesterol staining in living cells with Filipin III also pose problems [9]. So, we must never forget that the amount of buoyant cholesterol, i.e. cholesterol enrichment, depends both on the technique purification of DRMs and on the choice of the detergent [10].

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In our previous study [11], we demonstrated that Jurkat cells contain two distinct cholesterol pools: a fast pool and a slow pool. We found that the fast pool could correspond to rafts. This crucial result raises the question of the long m-β-CD-treatments frequently observed in the literature. That is the reason why we treat lymphocytes for only 2 min We demonstrated that short m-β-CD treatment specifically extracts buoyant cholesterol and only buoyant cholesterol, and this is in contrast with standard m-βCD treatment that extracts buoyant cholesterol, cholesterol from non-raft plasma membrane, intracellular cholesterol, GPI-APs, GM1, non-raft proteins such as CD45 (this study), Lck, LAT, and [3H]palmitic acid-labeled lipids. If standard m-β-CD treatment inhibits T cell activation, short m-β-CD treatment does not. Other publications also emphasized the serious problems met with m-βCD [12–15]. We also demonstrated that cholesterol oxidase (COase) oxide buoyant cholesterol into Triton X-100-soluble cholestenone, leading to cholesterol-rich raft disruption. Our results demonstrated that COase treatment does not inhibit TCR signaling either in Jurkat cells or in T CD4+ lymphocytes [11]. In this study, we change our orientation: we take an interest in the other lipid component of lipid rafts, i.e. SM. We demonstrate that exogenous sphingomyelinase (SMase) induces the release of buoyant cholesterol and that C6-Ceramides displace it. It is important to point the fact that neither SMase nor C6Ceramides delocalize CD55 and GM1 (external leaflet), Lck and LAT (internal leaflet). These four molecules are still anchored in ceramides-rich/cholesterol-poor domains, demonstrating again that cholesterol is not necessary for the buoyancy of raft markers. SMase-treated cells, i.e. cells exhibiting cholesterol-depleted rafts, optimally transmit CD3 signals. Nevertheless, we also demonstrate that Ca2+ influx and diacylglycerol (palmitoyl-DAG and arachidonoy-DAG) production are lowered after SMase treatment because of plasma membrane depolarization due to channels formed by ceramides. Combined all together, our results clearly establish that buoyant cholesterol is not necessary to TCR activation. To our knowledge, only one study uses exogenous SMase to disrupt rafts in Jurkat cells [16]. The authors use 10 mU/ml of SMase for 30 min whereas we only use 0.25 mU/ml for 5 min Their inappropriate conditions, 40× in concentration and 6× in time of incubation, will be discussed. 2. Materials and methods 2.1. Cells The human leukemic T cell line Jurkat JE 6.1 was obtained from American Type Culture Collection (Manassas, VA). Jurkat cells were grown in RPMI 1640 supplemented with 10% (vol/vol) heat inactivated fetal calf serum, 50 U/ml of penicillin G sodium, 50 μg/ml of streptomycin sulfate, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES (Life Technologies, Rockville, MD). Cells were maintained at densities between 8 × 105 and 1 × 106 in a humidified incubator under 5% CO (Heraeus, Hanau, Germany).

2.2. Cell treatments Cell treatment used for our purpose were as mentioned below (except when precised in the figure legends). Jurkat JE 6.1 cells were washed in RPMI 1640 plus 10 mM HEPES at 37 °C to remove the fetal calf serum that contains lipids. SMase: 0.25 mU/ml for 5 min at 37 °C; C2- and C6-Ceramides: 20 μM for 1 h at

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37 °C; m-β-CD: 10 mM for either 2 or 10 min at 37 °C; COase: 0.5 U/ml for 30 min at 37 °C.

2.3. Antibodies and reagents Rabbit polyclonal antibody (Ab) anti-ZAP-70 (LR, sc-574), rabbit polyclonal Ab anti-PLCγ1 (1249, sc-81), goat polyclonal p-MEK-1/2 (Ser 218/Ser 222) (sc7995), mouse monoclonal antibody (mAb) anti-CD45 (35-Z6, sc-1178, IgG1), mAb anti-CD3-ζ (6B10.2, sc-1239, IgG1), mAb anti-Vav 1 (E-4, sc-17831, IgG1), and goat polyclonal Ab anti-CD55 (C-20, sc-7064) were purchased from Santa Cruz Biotechnology Inc. (Santa-Cruz, CA). Rabbit polyclonal phospho-p44/42 Map Kinase (p-Erk-1/2) (Thr 202/Tyr 204) was purchased from Cell Signaling Technology. mAb anti-phosphotyrosine (4G10, IgG2b,κ, 05−321), rabbit polyclonal Ab anti-p56Lck (IgG, 06-583), and rabbit polyclonal Ab antiLAT (IgG, 06-807) were from Upstate Biotechnology (Lake Placid, NY). mAb anti-CD3 (X3, IgG2a) was produced in our laboratory. Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (GAR-HRP), HRP-labeled rabbit anti-goat IgG (RAG-HRP), rabbit anti-mouse (RAM) IgG coupled to HRP (RAM-HRP), and RAM-FITC were from Rockland (Gilbertsville, PA). Streptavidin-HRP were purchased from Dako (Glostrup, Denmark). Sphingomyelin phosphodiesterase (Sphingomyelinase, SMase, EC 3.1.4.12) from Staphylococcus aureus, methyl-βcyclodextrin (m-β-CD), FITC-phalloidin, biotin-labeled Cholera Toxin B subunit (CTB), H2O2, o-phenylenediamine (OPD) tablets, protein G-Sepharose beads, gramicidin, pepstatin, leupeptin, chymostatin, and α-phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (Saint-Louis, Mo). α2-macroglobulin was purchased from Roche (Indianapolis, IN). Indo-1-AM, 3,3′-dipentyloxacarbocyanine (DioC5(3)), and bis(1,3-dibutylbarbituric acid)trimethine oxonol (DiBaC4) were from Molecular Probes (Leiden, The Netherlands). CD25-PE (clone M-A251, IgG1), and apoptosis detection kit (annexin V-PE, 7-AAD) were purchased from Pharmingen (San Diego, CA). D-(e)-C2-ceramide (C2-ceramide) and D-(e)-C6-ceramide (C6-ceramide) were obtained from Euromedex (Mundolsheim, France). Proteinase K and RNase were from Roche Molecular Biochemicals. [methyl-3H]choline chloride (2.22–3.14 TBq/mmol), [1α, 2α(n)-3H]cholesterol (1.3–1.85 TBq/mmol), [9, 10(n)-3H]palmitic acid (37 MBq/mmol), and [5, 6, 8, 9, 11, 12, 14, 15-3H]arachidonic acid (5.55–8.51 TBq/mmol) were purchased from Amersham (Arlington Heights, IL).

2.4. Viability measurement The viability of Jurkat cells after the different treatments (SMase, C6ceramides, COase, and m-β-CD) was performed using 2 complementary techniques: the Trypan-Blue exclusion method and the annexin V-PE/7-aminoactinomycin D (7-AAD) staining. See [11].

2.5. DNA fragmentation DNA fragmentation was accomplished using a combination of published protocols [17,18]. Jurkat cells (1 × 106) in RPMI 1640, 10 mM HEPES, 37 °C were treated or not with either C2-Ceramides (20 μM) or C6-Ceramides (20 μM) for either 1 h or 6 h. Jurkat cells were washed, collected, then lysed in 200 μl of lysis buffer containing 10 mM Tris, pH 7.5, 1 mM EDTA, and 0.2% Triton X100. Lysates were treated with 100 μg/ml RNase for 45 min and then incubated with 100 μg/ml proteinase K for 45 min at 37 °C. Cellular DNA was isopropanol-precipitated, dried, resuspended in Tris–EDTA buffer (10 mM Tris and 5 mM EDTA, pH 7.5), and incubated for 30 min at 55 °C. DNA was analyzed by electrophoresis on 1.4% agarose gels impregnated with ethidium bromide and visualized by UV transillumination.

2.6. DRM isolation DRM isolation was accomplished exactly according to already published protocols [11,19].

2.7. Glycerophospholipids and SM content analysis Jurkat cells were washed then incubated for 16 h in a HEPES saline buffer (HSB), pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 1 mM Na2HPO4,

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12 H20, 2.5 mM glucose, 20 mM HEPES, 5 mM MgCl2, 1 mM CaCl2, and 0.1% BSA at 37 °C in the presence of either [3H]palmitic acid or [3H]choline chloride. We previously showed that these conditions resulted in an efficient metabolic labeling of lipids within rafts [20]. Lipids were extracted and analyzed from either (a) whole cells or (b) fractions obtained after ultracentrifugation. (a) SMase-treated cells and control cells (Fig. 2A) were rapidly sedimented, supernatants were discarded and cell lipids were extracted with chloroform/methanol according to Bligh and Dyer [21] then separated by monodimensional thin-layer chromatography (TLC) on plates LK6D Silica Gel 60 A (Whatman Inc., Clifton, NJ) in a solvent system composed of chloroform/methanol/acetic acid/water (75/45/12/3). Authentic phospholipid standards (Sigma) were run in parallel and detected with iodide vapors. Radioactivity in lipid spots was determined by using an automatic linear radiochromatography analyzer, Tracemaster 20 (Berthold) equipped with an 8 mm window and the integration software supplied by the manufacturer. (b) An aliquot (50 μl) of each different fraction obtained after ultracentrifugation on sucrose density-gradient were extracted and analyzed as described above (Fig. 2B).

2.8. Ceramide quantification Lipids from Jurkat cells pre-labeled with [3H]palmitic acid as described above and treated with SMase were extracted then separated by the twodimensional TLC procedure described by Bodennec et al. [22]. The plates were developed first with a solvent system composed of chloroform/methanol (50/5) (v/v) then dried and developed again with hexane/diethyl ether/acetic acid (80/ 20/1) (v/v/v) then with heptane/diisopropyl ether/acetic acid, (60/40/4) (v/v/v). Quantification of radioactivity was done as described above.

2.9. DAG kinase assays Exogenous C2-Ceramides and C6-Ceramides (Fig. 2C), and total endogenous cellular ceramide levels (Fig. 4I) were quantified by the DAG kinase assay as 32P incorporated upon phosphorylation of ceramide to ceramide-1phosphate (C-1-P) by DAG kinase from Escherichia coli [23]. Jurkat cells (80 ± 106) were washed twice with ice-cold PBS. DRM isolation was performed and the whole 9 fractions were harvested. Lipids from 50 μl-aliquots were extracted with 1 ml of chloroform/methanol/hydrochloric acid (1N) (100/100/1, v/v/v), 170 μl of buffered saline solution (135 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 5.5 mM glucose, and 10 mM HEPES, pH 7.2), and 30 μl of 100 mM EDTA. The lipids of the organic phase were transferred to a new glass tube and dried under a stream of N2. Lipid extracts were then subjected to mild alkaline hydrolysis (0.1 M KOH in methanol for 1 h at 37 °C) to remove glycerophospholipids. 500 μl of chloroform, 270 μl of buffered saline solution, and 30 μl of 100 mM EDTA were added. After drying the organic phase with N2, in vitro phosphorylation of extracted ceramides was performed as described by the manufacturer (RPN 200 kit, Amersham Pharmacia Biotech). 1 μCi of [γ-32P] ATP (4000 Ci/mmol) was used to start the reaction. After 30 min at RT, the reaction was stopped by extraction of lipids with 1 ml of chloroform/methanol/ hydrochloric acid (1N) (100/100/1, v/v/v), 170 μl of buffered saline solution, and 30 μl of 100 mM EDTA. The lower organic phase was dried under N2. The samples were resuspended in 30 μl of chloroform/methanol (95/5, v/v) and spotted on plates LK6D Silica Gel 60 A. C-1-P was resolved by TLC using chloroform/methanol/acetic acid (75/25/5, v/v/v) as solvent and migrated as a single spot at RF = 0.25. Linearity of the assay was established using purified C16-ceramide (Sigma). Radioactivity in lipid spots was determined by using an automatic linear radiochromatography analyzer, Tracemaster 20 (Berthold) equipped with an 8 mm window and the integration software supplied by the manufacturer.

2.10. CD25 staining Jurkat cells were washed then re-suspended in pre-warmed culture medium at a cellular concentration of 1 × 106/ml. Cell suspension was dispensed in triplicate sets into flat-bottom 96-well plates in the volume of 200 μl/well. Jurkat

cells (1 × 106) treated or not with SMase were stimulated or not by soluble antiCD3 mAb (2 μg/ml) for 20 h. Then Jurkat cells were washed then incubated for 30 min in the dark at 4 °C with CD25-PE according to the manufacturer's instructions. Cells were washed again and fixed with 0.37% paraformaldehyde. The mean fluorescence intensity of 5000 cells was determined by flow cytometry (FACScan, Becton Dickinson, Mountain View, CA).

2.11. Immunoblot analysis 2.11.1. Phospho-tyrosine immunoblotting For Fig. 4A, Jurkat cells (80–100 × 106) pre-treated or not with SMase were stimulated or not for 3 min with 2 μg/ml of mAb anti-CD3 and DRMs were prepared as already detailed. 50 μl-aliquots of each sucrose density-gradient fraction were solubilized in 50 μl 2X Hoessli buffer (150 mM Tris–HCl [pH 8.5], 20% glycerol, 5 mM EDTA, 5% SDS, 10% β-mercaptoethanol). Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Saint Quentin en Yvelines, France). Membranes were blocked for 2 h at RT in a blocking buffer containing 5% (wt/vol) bovine serum albumin (BSA) in TBS (10 mM Tris–HCl, 140 mM NaCl, pH 7.4). They were subsequently incubated with 4G10 diluted 1000-fold as recommended by the manufacturer, in TBS plus 1% BSA (wt/vol) for 2 h at RT. The membranes were extensively washed in TBS containing 0.4% (vol/vol) Tween-20. 4G10 signals were detected with RAM-HRP, followed by enhanced-chemiluminescence reagents (ECL) (Amersham, UK) according to manufacturer's instructions. 2.11.2. Phospho-threonine, -serine For Fig. 4C, Jurkat cells (2 × 106) pre-treated or not with SMase were stimulated or not for 3 min with 2 μg/ml of mAb anti-CD3. Cells were washed and lysed in 100 μl of ice-cold lysis buffer (1% Nonidet P-40 (NP-40) in 25 mM HEPES, 150 mM NaCl, 5 mM EDTA, 10 mM sodium pyrophosphate, 5 mM Na3VO4, and 10 mM NaF) supplemented with a mixture of protease inhibitors (1 mM PMSF, 100 U/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 2 mg/ ml chymostatin, and 5 mg/ml α2-macroglobulin). Whole cell lysates (WCLs) were solubilized in a denaturing sample buffer and then resolved by 10% SDSPAGE under reducing conditions. Proteins were transferred onto PVDF membranes. Membranes were blocked for 2 h at RT in a blocking buffer containing 5% (wt/vol) bovine serum albumin (BSA) in TBS (10 mM Tris–HCl, 140 mM NaCl, pH 7.4). They were subsequently incubated with either anti-pMEK-1 (Ser 218/Ser 222) or anti-phospho-p44/42 Map Kinase (anti-p-ERK-1/2) (Thr 202/Tyr 204) diluted as recommended by the manufacturer, in TBS plus 1% BSA (wt/vol) for 2 h at RT. The membranes were extensively washed in TBS containing 0.4% (vol/vol) Tween-20. Signals were revealed respectively with RAG-HRP and GAR-HRP, followed by enhanced-chemiluminescence reagents (ECL) (Amersham, UK) according to manufacturer's instructions. 2.11.3. Protein immunoblotting For Figs. 3A, 4B, and J, Jurkat cells (80–100 × 106) pre-treated as indicated in each legend were stimulated or not for 3 min with 2 μg/ml of mAb anti-CD3 and DRMs were prepared as already detailed. 50 μl-aliquots of sucrose densitygradient fractions were solubilized in 50 μl 2× Hoessli buffer (150 mM Tris– HCl [pH 8.5], 20% glycerol, 5 mM EDTA, 5% SDS, 10% β-mercaptoethanol) and then resolved by 10% SDS-PAGE. PVDF membranes were blocked for 2 h at RT in a blocking buffer containing 5% (wt/vol) non-fat dry milk in TBS. Then they were incubated for 1 h with the appropriated primary antibody that was diluted in TBS plus 1% (wt/vol) non-fat milk using the dilution recommended by the suppliers. Detection was performed with HRP-conjugated Ab anti-species followed by ECL. 2.11.4. Immunoprecipitation (IP) For Fig. 4D, Jurkat cells (5 × 106) were lyzed as detailed. Cleared lysates were incubated for 4 h at 4 °C with anti-LAT Abs and then for 2 h with protein G-Sepharose beads. The pellets were washed three times with ice-cold lysis buffer containing 0.2% NP-40 and resuspended in SDS denaturing buffer. Eluted immunoprecipitates were blotted with anti-LAT and anti-PLCγ1 Abs as already detailed. For Fig. 4J, the protocol was the same except that cleared lysates were incubated with anti-Vav 1 and protein G-Sepharose beads. Eluted immunoprecipitated were blotted with mAb 4G10, membranes were stripped and re-blotted with mAb anti-Vav 1 as already detailed.

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2.12. Luciferase assays Jurkat cells (5 × 105) were transiently transfected by electroporation (320 V, 960 μF) with 10 μg of either a luciferase reporter gene controlled by a minimal thymidine kinase promoter and six reiterated κB sites (κBx6 thymidine kinase luc) or IL-2 promoters. At 36 h after transfection, cells were stimulated by mAb anti-CD3 (2 μg/ml). Cells were washed twice in phosphate-buffered saline, pH 7.2, and lysed in 100 μl of reporter lysis buffer (Promega). Luciferase activity was assayed by luminometry (Lumat, EG and G Berthold) using the Promega luciferase assay system. Luciferase activity was determined in triplicate and expressed as fold increase relative to basal activity seen in untreated unstimulated cells. Normalization of transfection efficiency was done using a co-transfected β-galactosidase expression vector (pEF/β-galactosidase). For the β-galactosidase assay, 20 μl of the supernatants were incubated at 37 °C in 150 μl of assay buffer containing 60 mM Na2HCO, 80 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, 10 mM dithiothreitol (DTT), and 60 μg of o-nitrophenyl-βD-galactopyranoside, until a yellow color developed. Absorbance was measured at 400 nm in a spectrophotometer.

2.13. Cholesterol analysis, DAG measurement, surface receptor detection by measurement of HRP activity, measurement of changes in cytosolic Ca2+ concentration, measurement of membrane potential, and measurement of F-actin See [11].

2.14. Semi-quantitative Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Electrophoretic Mobility Shift Assays (EMSAs) See [24].

3. Results 3.1. In terms of cell viability, the hydrolyze of SM by SMase levels really is a better strategy to disrupt lipid rafts than cholesterol modification From a lipid point of view, conventional lipid rafts are said to be highly enriched in cholesterol, SM, and gangliosides. As a consequence, manipulation of either cholesterol, SM, or ganglioside contents logically offers three distinct possibilities to disorganize rafts. Nevertheless, cholesterol extraction with mβ-CD largely became, for many biologists, the most preferred way to disrupt lipid rafts. In our former study [11], we unambiguously demonstrated that the classical m-β-CD treatment (10 mM, 10 min at 37 °C) is not specific at all of buoyant cholesterol and is toxic, therefore we opted for the specific oxidation of buoyant cholesterol into Triton X-100-soluble cholestenone with COase (0.5 U/ml, 60 min). In the present study, we decided to change the direction of our investigations. Actually, to disrupt conventional lipid rafts, we prefer to hydrolyze SM by SMase or to add exogenous C6Ceramides (C6-Cer), instead of modifying cholesterol (extraction or oxidation). Fig. 1A clearly shows the great interest of such a choice. Indeed, m-β-CD and COase reveal being very toxic compare to SMase. As shown by Fig. 1A, SMase (from 0.05 to 1 mU/ml for 4 days) does not affect cell viability at all. At least three articles demonstrate that ceramides displace cholesterol from ordered domains due to the tight interactions between SM and cholesterol. Indeed, ceramides are able to compete with cholesterol for

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association with these ordered domains in bilayer membranes [25–27]. Thus, we decided to use C2- and C6-Ceramides to disrupt lipid rafts. The problem is that these three studies have been performed with vesicles and not with living cells and exogenous ceramides can be very toxic as shown by Fig. 1A and B. Exogenous C2- and C6-Ceramides begin to be proapoptotic from a 2 h treatment. At time 2 h, C2-and C6-Ceramides start to induce apoptosis as shown by phosphatidylserine (PtdSer) externalization (annexin V-PE staining) and 7-AAD incorporation. At time 6 h, JE 6.1 cells treated with C2- or C6Ceramides are completely dead as shown by DNA fragmentation (Fig. 1B). Thus, we decided not to treat with C2- and C6Ceramides beyond 1 h since at time 1 h, cells are 100% viable. 3.2. Optimal SMase treatment: 0.25 mU/ml for 5 min There are several possibilities to metabolically label SM molecules: actually, it is possible to use radioactive serine, sphingosine, fatty acids, or choline chloride. To analyze SM level after SMase treatment, we chose at least 2 of the 4 radioactive tracers: palmitic acid and choline. In a previous article [20], we demonstrated that SM species containing palmitate, in sharp contrast with polyunsaturated fatty acid-labeled SM, were preferentially found into DRMs. The advantage of choline is that it also allows the labeling of PtdCho, as palmitate does. SM belongs to the sphingolipid family, whereas PtdCho belongs to the glycerophospholipid family. SM and PtdCho are structural homologues, thus the study of PtdCho level indicates if the treatment used to modulate SM is specific or not. We performed kinetics and doses–responses (data not shown) of SMase. Jurkat cells pre-labeled at isotopic equilibrium with either [3H]palmitic acid or [3H]choline chloride were left untreated or incubated with 0.25 mU/ml of SMase for different periods of time varying from 0 to 30 min. The analysis of lipids extracted from whole cells indicates SM hydrolysis is maximal at time 5 min and reaches 40% of total cellular SM independently of the radioactive tracer used (Fig. 2A). When higher SMase concentrations (0.5 and 1 mU/ml of SMase) are used for times longer than 5 min (from 5 min to 4 days), SM hydrolysis never exceed 40% (data not shown). In conclusion, 0.25 mU/ml of SMase for 5 min maximally hydrolyzes SM without affecting cell viability. 3.3. SMase converts SM-rich/cholesterol-rich rafts into ceramides-rich/cholesterol-poor domains Jurkat cells pre-labeled with [3H]palmitic acid were treated or not with SMase (0.25 mU/ml, 5 min). Then PNS preparations were fractionated on a sucrose density-gradient. The analysis of the 9 fractions was performed by TLC and it shows that DRMs (fractions 2 + 3) are highly enriched in [3H]palmitic acid-labeled SM (Fig. 2B), confirming previous data [20]. As obtained in Fig. 2A, SMase hydrolyzes 40% of total SM (to calculate it, one has to consider that fraction 9 makes 3 ml), but note that it hydrolyzes more than 80% of SM found into DRMs. [3H] palmitic acid-labeled SM found into Triton X-100-soluble fractions (fractions 8 + 9) remains intact (Fig. 2B). Interestingly,

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Fig. 1. Viability of Jurkat treated with the different reagents that modify lipid raft composition. (A) Jurkat pre-treated or not with the different reagents for the indicated time at the indicated concentration were co-stained with annexin V-PE/7-AAD. Percentage of viable cells is shown in Fig. 1A. This graph is representative of 3 different experiments. The values represent means ± S.E.M (where no visible, errors bars are within the symbols). (B) Exogenous ceramides induce DNA fragmentation after a long time of incubation and they are absolutely not pro-apoptotic molecules when incubated for only 1 h. Jurkat (1 × 106) were left untreated or incubated with either C2-Ceramides (20 μM) or C6-Ceramides (20 μM) for either 1 or 6 h. DNA fragmentation was visualized on agarose gels stained with BET.

[3H]palmitic acid-labeled ceramides produced by the hydrolysis of SM are buoyant (Fig. 2B). In our previous article [11], we used COase (0.5 U/ml, 60 min) to oxide cholesterol into cholestenone. We found that cholestenone is Triton X-100soluble, which is in opposition with [3H]palmitic acid-labeled ceramides that are Triton X-100-insoluble. Thus SMase converts SM-rich rafts into ceramides-rich domains that are not solubilized by Triton X-100. Then, we investigated the distribution of [3H]palmitic acid-labeled PtdCho and [3H]palmitic acid-labeled PtdEtn in ceramides-rich domains. As expected, SMase does not affect the level of these two glycerophospholipids. Fig. 2B shows that their distribution is the same in SM-rich rafts and in ceramides-rich domains. Finally, we investigated both cholesterol level and cholesterol distribution in SMase-treated cells. We show that SMase treatment induces the release of 15% of total cholesterol. It is recovered in the incubation medium. After DRM purification (Fig. 2D), we observe that cholesterol release occurs only in DRMs: 55% of buoyant cholesterol is released after a 5 min

treatment. In conclusion, SMase converts SM-rich/cholesterolrich rafts into ceramides-rich/cholesterol-poor domains. 3.4. Exogenous C6-Ceramides displace buoyant cholesterol and modestly induce its extraction. C2-Ceramides and sphingosine fails to do so Alanko et al. [27] studied how different membrane intercalators could affect raft stability and cholesterol distribution. They show that sterols can easily be displaced from ordered domains by a variety of saturated, single-chain (hexadecenol and hexadecyl amide) and double-chain (ceramides and dihydroceramides) intercalators with a small polar group as a common denominator. We demonstrated, as they did, that among the single-chain intercalators, sphingosine (10 μM, 1 h) fails to displace cholesterol from DRMs (data not shown). Anyway, sphingosine is a bad reagent for that purpose since it appeared to be very toxic at low concentrations for short time incubations. We also used permeant C2- and C6-Ceramides (in both cases,

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20 μM for 1 h) to perturb raft stability (Fig. 2D). Neither SM nor the major glycerophospholipids (PtdCho, PtdEtn, PtdSer, and PtdIns) are affected by the treatments with C2- or C6-Ceramides: their levels and their DRM localizations remain unchanged (not shown). On the other hand, C6-Ceramides efficiently displace cholesterol from DRMs. After C6-Ceramides-treatment, the cholesterol from the buoyant fractions is found in heavy fractions and a modest proportion of it is also released in the incubation medium. In opposition, C2-Ceramides is absolutely inefficient to displace cholesterol from DRMs. As a consequence, its utilization in this optic has been stopped. We measured that SMase (0.25 mU/ml for 5 min at 37 °C) produced 8.3545 ≈ 8.4 pmol of long-chain ceramides (80 × 106 cells in 10 ml). And we added 200 nmoles of short-chain C6Cer to the Jurkat cells (80 × 106 cells in 10 ml). Finally, there are about 23 800 more exogenous added short-chain C6-Cer than naturally produced long-chain ceramides by SMase (for further details as for the calculations, please read the Supplemental data in which everything is carefully explained).

[32]. The GM1s with sphingosine acylated with C14-, C16-, C18-, C22-, or C24 FAs were similarly abundant in DRMs. GM1s acylated with C18:1, C22:1, or C24:1 were less abundant than those acylated with saturated FAs of the same length. C2-Cer do not partition to a different membrane fraction (even though their tropism for DRMs is weaker than the one of C6-Cer). What is crucial for the localization of ceramides within DRMs is the degree of saturation of the FAs. For instance C18:1 (see structure in Supplemental data) have a poor tropism for DRMs, contrary to C18:0. Therefore saturated C2-Cer and C6Cer both localize into DRMs. Furthermore, what is important for the displacement of cholesterol by ceramides is the length of the FAs. One has to note that C2 FA = 1.43 Å, C6 FA = 7.6 Å, C18 FA = 26 Å, and the length of cholesterol is 17.5 Å (see Supplemental data). C2-Cer is ineffective in cholesterol depletion because it is much shorter than C6-Cer and especially C18:0. C6-Cer (20 μM) displaces cholesterol after 1 h treatment whereas natural C18:0, produced by bacterial SMase, does the same after only 5 min.

3.5. Localization of added C6-Cer and C2-Cer

3.6. SMase and C6-Ceramides do not modify the distribution of raft markers

In order to measure the distribution of C2-Cer and C6-Cer (raft fractions versus non-raft fractions), we have to label or tag C2-Cer and C6-Cer. For this purpose, we can choose between radioactivity and fluorescence, but things get quite difficult. Indeed, on one hand radioactive ceramides (C2- and C6-) cannot be purchased, so we have to synthesize them [28–30], on the other hand only fluorescent C6-Cer actually exists (NBD or BODIPY dye series), whereas C2-Cer does not (fluorochrome is too large for C2-Cer). As a consequence, we opted for DAG kinase assay because it was complicated to measure the distribution of radioactive C2Cer and C6-NBD-Cer, and the results appeared to be nonhomogeneous (radioactivity for C2-Cer versus fluorescence for C6-Cer). To do so we followed Alicia Bielawska's procedure [31]. One has to note that we have already used this technique in a former publication [24]. The results can be seen in Fig. 2C. Short C2-Cer have a certain tropism for DRMs. As a matter of fact, there are more C1-P in DRMs than in heavy fractions (but keep in mind that fractions 2 + 3 = 2 ml, whereas fractions 8 + 9 = 4 ml). Remarkably there are substantially more C6-Cer in DRMs than in heavy fractions. This is in agreement with Susanna Nybond's conclusions [26]. She explains that the C2-Cer resembles sphingosine more than it does a natural ceramide. The effects of the C2Cer are very similar to the effects observed with sphingosine (displacement of cholesterol with ceramides), suggesting that C2-Cer co-exist with the cholesterol in the SM-rich domain. According to her, all tested ceramides (C2-, C4-, C6-, C8-, C10-, C12- and, C14-) appeared to partition into SM-rich domains, their effect on the cholesterol composition of the SM-domains varied markedly with the N-linked acyl chain length. Only medium (C6) and long-chain ceramides are able to displace cholesterol from SM-rich domains. Miroslawa Panasiewicz et al. showed that the ceramide moieties of the GM1s determined their occurrence in DRMs

Fig. 3A shows the distribution of Lck and LAT both in control cells and in SMase-treated cells. Lck and LAT are still anchored in ceramides-rich/cholesterol-poor domains since SMase does not affect the buoyancy of the two acylated signaling proteins. Furthermore, C6-Ceramides, that displace buoyant cholesterol, do not modify the distribution of the two molecules (data not shown). In conclusion, as we demonstrated in our precedent article using gentle m-β−CD procedure (10 mM, 2 min) or COase (0.5 U/ml, 30 min) [11], we demonstrate again with SMase (0.25 mU/ml, 5 min) and C6-Ceramides (20 μM, 1 h) that cholesterol-dependence of the proteins involved in TCR signaling must not be taken as an established fact. SMase (0.25 mU/ml, 5 min), C6-Ceramides (20 μM, 1 h), and m-β-CD (10 mM, 10 min) represent three different ways to considerably lower cholesterol in DRMs. We calculated the percentage of buoyant cholesterol after the following treatments: SMase, C6-Cer, C2-Cer, m-β-CD (2 min), m-β-CD (10 min), and COase. The results of these calculations are shown in Fig. 3B. Fig. 3C allows the comparison of these treatments with other molecules than cholesterol. Jurkat cells were incubated at 4 °C either with primary mAbs against the glycosylphosphatidylinositol-anchored protein (GPI-AP) CD55 or the non-raft marker CD45, or with CTB-biotin that binds the monosialoganglioside GM1. Then cells were stained with secondary mAb conjugated to HRP or streptavidin-HRP. DRM purification was performed and the fractions were assayed for peroxidase activity to determine the residency of the chosen raft markers on control, SMase-, C6-ceramides-(data not shown), and m-β-CD-treated Jurkat. We confirm, see [11], that m-β-CD treatment (10 mM, 10 min) does not allow the expected Triton X-100-solubilization of raft markers but extracts them dramatically. We also demonstrate that the extraction of non-cholesterol molecules

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does not concern only molecules that are present in DRMs since m-β-CD extracts 30% of CD45. As shown in Fig. 3B, CD55 and GM1 reside in ceramides-rich/cholesterol-poor domains as well as in SM-rich/cholesterol-rich rafts. Furthermore, the distribution of CD55 and GM1 is the same in control cells and in cells incubated with C6-Ceramides that displace buoyant

cholesterol (data not shown). These results argue, once again, against the cholesterol-dependence of raft markers. To conclude, if DRMs equal rafts then the raft markers Lck, LAT, CD55, and GM1 are anchored in a subtype of raft that is not enriched in cholesterol. Its Triton X-100-insolubility is not due to cholesterol–SM interactions but may be due to

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interactions between ceramides or phosphorylcholine ester of ceramides i.e. SM. 3.7. Raft cholesterol is not necessary for full transduction of CD3-generated tyrosine-phosphorylations The objective of this experiment was to define whether buoyant cholesterol is required or not for the transduction of the CD3-induced signals. Jurkat cells pre-treated or not with 0.25 mU/ml of SMase for 5 min were activated or not by CD3 X3 mAb. After DRM purification, tyrosine-phosphorylated proteins were detected by immunoblotting in both of the fraction B (DRMs) and the fraction E (Triton X-100-soluble material). Fig. 4A shows that the pattern of phosphorylation is the same for control and SMase-treated cells. SMase treatment does not prevent phosphorylations of CD3-activated cells. Arrows show that the CD3-induced phosphorylations of PLCγ1, ZAP-70, Lck, LAT, and CD3-ζ are optimal in cells exhibiting ceramidesrich/cholesterol-poor domains. Furthermore, C6-ceramides treatment (20 μM, 1 h) that displaces buoyant cholesterol, does not prevent the full propagation of the CD3-induced phosphorylations (data not shown). In conclusion, the propagation of the CD3induced signals occurs in a raft cholesterol-independent way. As a consequence, we confirm the results obtained both in Jurkat cells and in human T CD4+ lymphocytes that were treated with COase (0.5 mU/ml, 30 and 60 min). They clearly establish that raft cholesterol is not required for the propagation of the CD3-induced tyrosine-phosphorylations. 3.8. ERK-1/2 and MEK-1 are not recruited into DRMs after the engagement of CD3 JE 6.1 cells were left untreated (Control) or stimulated with either CD3 alone (data not shown) or CD3 + RAM for 3 min at 37 °C. DRM purification was performed as usual and the presence of both PLCγ1, ERK-1/2, and MEK-1 (fractions A–E) was detected by immunoblot analysis (Fig. 4B). Concerning the stimulation, the two conditions: CD3 alone or CD3 + RAM give the same results. In both cases, PLCγ1 is recruited in fraction A and especially in fraction B in CD3-stimulated JE 6.1, whereas ERK-1/2 and MEK-1 are absolutely not after a 3 min – (CD3 + RAM) – stimulation.

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Since CD3-induced tyrosine-phosphorylations normally occur from cholesterol-depleted Jurkat cells, we were interested in studying whether serine-phosphorylations and threoninephosphorylations were also optimal in cholesterol-depleted cells. For that purpose, we studied the phosphorylations of MEK-1 (Ser 218/Ser 222) and ERK-1/2 (p44/42 MAP Kinase (Thr 202/Tyr 204). We conducted only WCLs and not DRMs since we demonstrated that ERK-1/2 and MEK-1 are never recruited into DRMs after CD3 engagement. WCLs were subjected to SDS-PAGE and immunoblotted with phospho-MEK-1 and phospho-ERK-1/2. As shown in Fig. 4C, phospho-MEK-1 and phospho-ERK-1/2 exhibit the same intensity in control cells and in cholesterol-depleted cells. This important result clearly shows for the first time that cholesterol-rich rafts, contrary to preconceived ideas, are not necessary for full propagation of CD3-induced serine-, and threonine-phosphorylations. 3.9. Cytosolic PLCγ1 does associate with LAT in cholesteroldepleted cells As expected, PLCγ1 is immunoprecipitated from the CD3activated Jurkat but not from the resting cells (Fig. 4D). Remarkably, cholesterol elimination by SMase (0.25 mU/ml, 5 min) or by C6-Ceramides (20 μM, 1 h) (not shown), does not prevent the binding of PLCγ1 on LAT, thus it shows that cholesterol-rich rafts are not necessary for the binding of LAT on PLCγ1 upon CD3 engagement. The LAT molecule is a 36–38 kDa integral membrane protein. Zhang W. et al., wrote that “LAT in the GEM fractions was predominantly the p36 form. Two prominent forms of LAT, p36 and p38, were detected in Triton-soluble fractions [33]”. Consequently the identity of the upper band in the Triton X100-soluble material is the p38 form of LAT which is absent from the raft fractions. 3.10. Inhibition of CD3-induced Ca2+ fluxes by SMase Then we investigated whether CD3-induced Ca2+ fluxes were affected or not by SMase treatment. SMase (0.25 mU/ml) was added to the medium immediately before the CD3-stimulation. Fig. 4E shows that SMase dramatically inhibits CD3induced Ca2+ fluxes. SMase treatment decreases the plateau level of Ca2+ but does not affect the Ca2+ release from

Fig. 2. Effects of exogenous SMase on the composition of lipid rafts. (A) SM hydrolysis by SMase (0.25 mU/ml) is maximal at 5 min. Jurkat pre-labeled with either [3H]palmitic acid or [3H]choline were washed then treated (closed squares) or not (open squares) with 0.25 mU/ml of SMase for different periods of time varying from 0 to 30 min. Lipids were extracted and analyzed as explained in the Materials and methods section. These graphs are representative of 3 different and independent experiments. The values represent means±S.E.M. (where no visible, errors bars are within the symbols). (B) SMase specifically hydrolyzes SM from rafts into buoyant ceramides. DRM isolation was performed with Jurkat pre-labeled with [3H]palmitic acid and incubated (closed squares) or not (open squares) with SMase. Lipids from the 9 fractions were extracted and analyzed as explained in the Materials and methods section. These graphs are representative of 3 different and independent experiments. (C) Distribution of exogenous C2- and C6-ceramides. Jurkat were washed then incubated with C2- or C6-ceramides (20 μM, 1 h). DRM isolation was performed. Lipids were extracted and, after mild alkaline hydrolysis, were subjected to phosphorylation by the DAG kinase in the presence of [γ-32P]ATP as described in the Materials and methods section. The resulting C-1-P were separated by TLC. These graphs are representative of 3 different and independent experiments. The values represent means±S.E.M. (D) SMase and C6-Cer lower cholesterol level only in DRMs. Cholesterol is displaced from DRMs to heavy fractions but is also released in the incubation medium. Jurkat pre-labeled with [3H]cholesterol were left untreated (open squares) or incubated with either SMase (0.25 mU/ml, 5 min) (closed squares) or C2-Cer (20 μM, 1 h) (closed squares), or C6-Cer (20 μM, 1 h) (closed squares). DRM purification was achieved. The 9 fractions were assayed for radioactivity by liquid scintillation. These graphs are representative of 3 different and independent experiments. The values represent means±S.E.M (where no visible, errors bars are within the symbols). Supernatants from either control cells or SMase-, C2-Cer, and C6-Cer-treated cells were collected then centrifuged to remove cells. Cell-free supernatants were also assayed for radioactivity by liquid scintillation to quantify [3H]cholesterol released in the incubation medium by these two treatments. These graphs are representative of 3 different and independent experiments. The values represent means±S.E.M.

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Fig. 3. SMase does not modify the distribution of raft markers. (A) SMase does not modify the distribution of raft markers (Lck and LAT) in the internal leaflet. Jurkat were left untreated (Control) or incubated with SMase. DRM purification was achieved. The 9 fractions were pooled and the pooled fractions (A–E) were immunoblotted with the indicated antibodies. Data represent one of 3 similar experiments. (B) Buoyant cholesterol contents after different treatments. Jurkat prelabeled with [3H]cholesterol were left untreated or incubated with different reagents. DRM purification was achieved. The 9 fractions were assayed for radioactivity by liquid scintillation. Percentage of buoyant cholesterol was measured for each treatment. These graphs are representative of 3 different and independent experiments. The values represent means ± S.E.M (where no visible, errors bars are within the symbols). (C) SMase does not modify the distribution of raft markers (CD55 and GM1) in the external leaflet. Jurkat stained with the indicated primary antibody (CD55 or CD45) or CTB-biotin were washed then incubated with the appropriated secondary antibody (or streptavidin) all conjugated to HRP. Then Jurkat cells were washed then left untreated (open squares) or incubated with either SMase (closed squares) or 10 mM m-β-CD for 10 min (closed circles) at 37 °C in a serum-free medium. DRM purification was achieved. 50 μl-aliquots were assayed for peroxidase activity. These graphs are representative of 3 different and independent experiments. The values represent means ± S.E.M. (where no visible, errors bars are within the symbols).

intracellular stores. C6-Ceramides (20 μM, 1 h) induces the same kind of inhibition (data not shown). These results are in agreement with the fact that the phosphorylation status of PLCγ1 is not changed (Fig. 4A). Further experiments demonstrated that SMase (0.25 mU/ml) and C6-Ceramides (20 μM) cause a strong plasma membrane depolarization (Fig. 4F). Retention of either DioC5(3) or DiBaC4 (not shown) was used to monitor membrane polarization. Jurkat cells pre-loaded with either the cationic- or the anionic-probe were treated or not

with either SMase, C6-ceramides, or gramicidin (a Na+ ionophore that translocates Na+ from the medium to the cell interior, used as a positive control). Reductions in mean fluorescence for DioC5(3) or increases for DiBaC4 indicate a membrane depolarization. As shown in Fig. 4F, SMase and C6-Ceramides cause a strong plasma membrane depolarization. Because membrane potential is known to be an essential mean to regulate Ca2+ movements, Fig. 4F explains the inhibition of calcium mobilization observed in Fig. 4E.

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3.11. SMase inhibits the CD3-induced DAG production In Jurkat JE 6.1 cells, labeled until isotopic equilibrium with either [ 3 H]palmitic- or [ 3 H]arachidonic acid, the engagement of the complex CD3-TCR results in the generation of both [3 H]palmitoyl- or [3 H]arachidonoyl-DAG that peaked at time 2 min then returned to near basal level after a 15 min period. Our result shows that a 5 min pre-treatment with SMase (0.25 mU/ml) totally impairs the both of the CD3induced arachidonoyl-DAG and the palmitoyl-DAG production (Fig. 4G). Both arachidonoyl-DAG and palmitoyl-DAG are considerably lowered by C6-Ceramides (20 μM, 1 h) (data not shown). SMase treatment inhibits CD3-induced Ca2+ fluxes and DAG production. This is due to the fact that SMase depolarizes plasma membrane potential. There is quite a simple explanation for this, i.e. ceramides form channels throughout plasma membrane [34,35]. One could believe that rafts regulate plasma membrane potential because SMase, that disrupts rafts, depolarizes plasma membrane potential. If it was true, COase treatment, that also disrupts cholesterol-rich rafts, would also depolarize membrane potential, which is absolutely not the case. It means that rafts do not regulate membrane potential. SMase-induced plasma membrane depolarization is a side effect of ceramides that form channels in the plasma membrane. In this article we show that COase treatment affects neither the level nor the distribution of SM (Fig. 4H) and ceramides (Fig. 4I). Thus COase does not form channels through plasma membrane, so it does not depolarize plasma membrane and therefore does not inhibit CD3-induced Ca2+ mobilization. Both arachidonoyl-DAG and palmitoyl-DAG are not diminished by COase treatment, because CD3-induced Ca2+ fluxes are not diminished by COase. In conclusion, COase specifically converts cholesterol-raft into Triton X-100-soluble cholestenone, that does not interact anymore with SM, leading to raft disruption (without channels formation), whereas SMase converts SM-rich/ cholesterol-rich rafts into ceramides-rich/cholesterol-poor domains (with channels creation). These two methods, used to disrupt lipid rafts (SMase and COase), work in a completely different way. That is the reason why SMase inhibits CD3-induced Ca 2+ fluxes and DAG production whereas COase does not at all. 3.12. Vav 1 is recruited into DRMs after the engagement of CD3 JE 6.1 cells were left untreated (Control) or stimulated with either CD3 alone (data not shown) or CD3 + RAM for 3 min at 37 °C. DRM purification was performed as usual and the presence of Vav 1 (fractions A–E) was detected by immunoblot analysis (Fig. 4J). Concerning the stimulation, the two conditions: CD3 alone or CD3 + RAM give the same results. In both cases, Vav 1 is recruited in fraction A and especially in fraction B in CD3-stimulated JE 6.1. Jurkat cells pre-treated or not with 0.25 mU/ml of SMase for 5 min were activated or not by CD3 X3 mAb. After DRM purification, Vav 1 was immunoprecipitated from both of the

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fraction B (DRMs) and the fraction E (Triton X-100-soluble material). Then Vav 1 was immunoblotted with 4G10. Fig. 4J shows that the phosphorylation of Vav 1 is the same for control and SMase-treated cells. SMase treatment does not prevent Vav 1 phosphorylation of CD3-activated cells. Stripped membranes were re-blotted with mAb anti-Vav 1 to control equal loading. Furthermore, C6-ceramides treatment (20 μM, 1 h) that displaces buoyant cholesterol does not prevent Vav 1 phosphorylation after CD3 ligation. In conclusion, Vav 1 phosphorylation occurs in a raft cholesterol-independent way. 3.13. Cholesterol-raft elimination by SMase does not inhibit cytoskeletal rearrangements under CD3 ligation at all Since CD3-induced phosphorylations of MEK-1 and ERK1/2 (Fig. 4C), and Vav 1 (Fig. 4J) optimally occur in cholesterolraft-depleted Jurkat JE 6.1 cells by SMase treatment, we were interested in studying whether CD3-induced polymerization of actin filaments was also optimal in cholesterol-raft-depleted cells. We examined the organization of the actin cytoskeleton following CD3 (3 min) cross-linking in control and in cholesterol-raftdeficient Jurkat. As shown in Fig. 4K, CD3 cross-linking induces an increase of F-actin (1.60-fold augmentation of RFI) that is not inhibited by cholesterol-raft elimination with SMase at all. We have a non-significant increase of RFI in SMase-treated JE 6.1 cells, which goes from 1.60 to 1.85. In conclusion, our results unambiguously demonstrate that cholesterol-raft-depleted cells optimally propagate the TCR signaling cascades involved in actin rearrangement. C6-Ceramides treatment gives the same results than SMase. 3.14. Cholesterol-raft elimination by SMase does not inhibit the genes involved in TCR signaling at all The engagement of CD3 is known to activate nuclear translocation of NF-κB in Jurkat cells. JE 6.1 cells were left untreated or incubated with 0.25 mU/ml of SMase for 5 min. Translocation of NF-κB was visualized by EMSAs after CD3 stimulation (2 μg/ml, 3 min) by its binding to a radioactive probe containing κB sites from the Igκ promoter. CD3 translocates NF-κB in SMase-treated cells as well in control cells (Fig. 4L). This result demonstrates that CD3 signals that activate NF-κB can be fully transmitted from ceramides-rich/cholesterol-poor domains. NF-κB activation was also measured in Jurkat cells transfected with a reporter luciferase gene under the control of NFκB (Fig. 4M). In total accordance with EMSAs, NF-κB activation is optimal in SMase-treated cells, indicating that cholesterol-rafts are not required for NF-κB activation by CD3. Another transcription factor also activated in TCR signaling was studied both in control cells and in cholesterol-raft-depleted cells thanks to SMase treatment (0.25 mU/ml, 5 min). Fig. 4M clearly indicates that IL-2 activation is equivalent in untreated cells and in cholesterol-raft-depleted cells with SMase. These results unambiguously demonstrate that CD3 signals that activate NF-κB and IL-2 can be fully transmitted in the absence of buoyant cholesterol.

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Fig. 4. Ceramides-rich/cholesterol-poor domains full transmit CD3 signals. (A) Ceramides-rich/cholesterol-poor domains do transmit CD3-induced tyrosinephosphorylations. Jurkat were incubated or not with SMase. Then cells were stimulated or not with 2 μg/ml of mAb anti-CD3 for 3 min DRM isolation was performed. The pooled fractions B and E were subjected to SDS-PAGE then transferred onto PVDF membranes. Fig. 4A shows the CD3-induced phosphorylations of fractions containing the Triton X-100-soluble material (on the left) and both raft-Lck and raft-LAT (on the right). Data represent 1 of 3 similar experiments. (B) MEK-1 and ERK-1/2 are not recruited into DRMs after CD3 stimulation (contrary to PLCγ1). Jurkat were left untreated (Control) or stimulated with CD3 (2 μg/ml) + RAM (1 μg/ml) for 3 min. DRM purification was achieved. Fractions A to E were immunoblotted with mAb anti-PLCγ1, mAb anti-ERK-1/ 2, and mAb anti-MEK-1. Data represent 1 of 3 similar experiments. (C) ERK-1/2 and MEK-1 are full phosphorylated by CD3 in SMase-treated JE 6.1. Jurkat pretreated or not with SMase were stimulated or not with 2 μg/ml of mAb anti-CD3 X3 for 3 min. Whole cell lysates (WCLs) were immunoblotted with p-ERK-1/2 or p-MEK-1. Data represent 1 of 3 similar experiments. (D) PLCγ1 optimally binds to CD3-activated LAT after SMase treatment. Jurkat were incubated or not with SMase. Then cells were stimulated or not with 2 μg/ml of mAb anti-CD3 for 3 min. DRM isolation was performed. Immunoprecipitation of LAT was performed in fractions B and E. Presence of LAT and PLCγ1 was detected by immunoblot analysis. Data represent 1 of 3 similar experiments. (E) SMase inhibits CD3-induced [Ca2+]i elevation and thapsigargin-induced [Ca2+]i elevation. Indo-1-loaded Jurkat were left untreated (open squares) or treated with SMase (closed squares). At time 0, 2 μg/ml of mAb anti-CD3 X3 or thapsigargin were added and cytosolic Ca2+ concentration was assayed by flow cytometry. Each curve is representative of three independent determinations. Relative variations of ratios for given time and condition never exceeded 5%. (F) SMase and C6-ceramides decrease membrane potential. Jurkat pre-loaded with DioC5(3) were treated or not with either SMase, C6-Cer or gramicidin (1 μM, 5 min). Histograms represent DioC5 MCF (mean channel fluorescence units) ± S.E.M of 3 different experiments. (G) SMase inhibits the CD3-induced DAG production. Jurkat pre-labeled with either [3H]palmitic acid or [3H]arachidonic acid were left untreated (open squares) or pre-treated with SMase (closed squares). At time 0, 2 μg/ml of cross-linked CD3 mAb X3 were added and the DAG production was measured. These graphs are representative of 3 different and independent experiments. The values represent means ± S.E.M. (H) COase does not affect SM distribution. DRM isolation was performed with Jurkat pre-labeled with [3H]palmitic acid and incubated (closed squares) or not (open squares) with SMase. Lipids from the 9 fractions were extracted and analyzed as explained in the Method section. This graph is representative of 3 different and independent experiments. (I) COase does not affect ceramide distribution. Jurkat were incubated or not with SMase or COase. DRM purification was achieved and C-1-P were measured. (J) Vav 1 is recruited into DRMs after CD3 stimulation and is optimally phosphorylated in SMase and CD3-treated cells. Jurkat were left untreated (Control) or stimulated with CD3 for 3 min. DRM purification was achieved. Fractions A to E were immunoblotted with mAb anti-Vav 1. Data represent 1 of 3 similar experiments. Jurkat were incubated or not with SMase. Then cells were stimulated or not with 2 μg/ml of mAb anti-CD3 for 3 min. DRM isolation was performed. Immunoprecipitation of Vav 1 was performed in fractions B and E. Fractions B and E were subjected to SDS-PAGE then transferred onto PVDF membranes. Presence of phosphorylated Vav 1 was detected by immunoblot analysis with 4G10. Then, membranes were stripped and reblotted with mAb anti-Vav 1 to check equal loading. Data represent 1 of 3 similar experiments. (K) SMase-treated cells normally reorganize actin filaments under CD3 ligation. Jurkat pre-treated or not with SMase were stimulated or not with CD3 (2 μg/ml) for 3 min. Permeabilized cells were stained with phalloidin-FITC. F-actin fluorescence was determined by cytometric analysis. The data are reported as the ratio of the mean of the fluorescence intensity of stimulated cells to control cells. The data are representative of 3 different and independent experiments. The values represent means ± S.E.M. (L) SMase-treated JE 6.1 cells full promote NF-κB activation by CD3. Jurkat cells pre-treated or not with SMase were treated or not with CD3 (2 μg/ml) for 3 min. The total cell extracts were then incubated with a radioactive double-stranded oligonucleotide encompassing the κB site of the Igκ promoter. Complexes were then separated by non-denaturing electrophoresis followed by autoradiography. Data represent 1 of 3 similar experiments. (M) Jurkat were pre-treated or not with SMase, then cells were transfected with 10 μg of luciferase reporter gene. At 36 h after transfection (Jurkat cells were maintained for 36 h in a medium containing SMase), cells were treated or not with CD3 (2 μg/ml) for 3 min. Luciferase activity was measured. Data represent 1 of 3 similar experiments. The values represent means ± S.E.M. (N) SMase does not inhibit CD25 up-regulation in CD3-stimulated Jurkat. Jurkat were left untreated or incubated with SMase. Then cells were washed, and then they were stimulated or not with CD3 (2 μg/ml) for 20 h. RNA was extracted and reverse transcription-PCR was performed using primers amplifying either CD25 or βactin. Data represent 1 of 3 similar experiments. Jurkat were treated and stimulated in the same way and surface expression of CD25 was measured by flow cytometry. Data represent 1 of 3 similar experiments. The values represent means ± S.E.M.

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3.15. SMase treatment does not inhibit CD3-induced CD25 up-regulation JE 6.1 cells pre-treated or not with SMase were stimulated or not by mAb anti-CD3 for 20 h. Then RT-PCRs for CD25 and βactin were performed. As shown by Fig. 4N, SMase treatment does not inhibit the synthesis of the CD25 mRNA under CD3 ligation. It means that the engagement of the CD3/TCR complex through ceramides-rich/cholesterol-poor domains emits signals that are optimal and equivalent to those emitted from conventional rafts. To determine whether cell surface expression of CD25 correlates with CD25 mRNA, cytometric analysis was performed. In total accordance with RT-PCRs, cytometry confirms that SMase treatment does not inhibit CD3-induced CD25 up-regulation at all. Ceramides-rich/cholesterol-poor domains transduce CD3 signals that allow CD25 up-regulation at plasma membrane. 4. Discussion In 1998, the work of Xavier [4] refutes previous studies since it establishes, for the first time, that CD3 signals through specialized domains that will be later termed rafts. Before that year, these domains, that were called glycolipid-based domains [36] or detergent-insoluble complexes [37], were thought to be enriched in GPI-APs and gangliosides only. CD3 was used as a negative control [37,38]. Deckert et al. [38] found that, in lymphoid cells, nystatin inhibits the Ab-induced internalization of CD59 while the CD3-TCR internalization pathway is unaffected. Stulnig et al. [37] lowered cellular cholesterol content by blocking endogenous cholesterol biosynthesis by lovastatin. According to their results, treatment of cells with 2 μM lovastatin for a 3-day period decreases cellular cholesterol content by 30%. One has to note that m-β-CD (10 mM, 10 min) extracts about 35% of cellular cholesterol content. They found that lowering cholesterol by lovastatin suppresses calcium response via GPI-APs (CD59 and CD48) by about 50%, whereas stimulation via CD3 is only minimally affected (inferior to 10%). In 2005, Lagerholm et al. wrote that “putative lipid raft membrane domains have been postulated to exist based in large part on the results that a significant fraction of the membrane is detergent-insoluble and that molecules facilitating key membrane processes like signal transduction are often found in the detergentresistant membrane fraction. Yet, the in vivo existence of lipid rafts remains extremely controversial because, despite being sought for more than a decade, evidence for their presence in intact cell membranes is inconclusive” [39]. Let us examine the exact meaning of “significant fraction of the membrane is detergentinsoluble”. For that purpose, we will precise the notion of detergentresistance and we will study the conclusions we can draw from it. Detergent-resistance is a complex notion. For example, Madore et al. [40] demonstrated that Triton X-100-complexes can be further differentiated using Brij 96. Röper et al. [41] showed that prominin is soluble in Triton X-100 but remains insoluble in another nonionic detergent, Lubrol WX. Schuck et al. [10] extensively studied the resistance of cell membranes (MDCK and Jurkat cells) to different detergents (1% Tween 20, 1% Brij 58, 0,5% Lubrol WX,

1% Brij 98, 0,5% Brij 96, 1% Triton X-100, and 4% CHAPS). As emphasized in their excellent work “Detergent treatment leads to coalescence of detergent-resistant cholesterol-sphingolipid microdomains, which come together to form aggregates or fuse into continuous membrane sheets”. As a consequence, conventional cholesterol-rafts are thought to result from the coalescence of segregated DRMs units [42]. Because rafts are isolated as heterogeneous mixture of coalescent DRMs, it is not evident to interpret results on protein and lipid composition of DRMs subsets. Indeed, “(…) the presence of proteins or lipids in DRMs obtained with a particular detergent does not necessarily indicate an association with the same domains in the native membrane”. [10]. Keeping this remark in mind, we will focus on some examples of proteins that co-fractionate using Triton X-100, but nevertheless, extensive researches have demonstrated that they do not originate from the same starting structure. All together, these studies underscore the expanding complexity of DRMs. Using Triton X100 extraction method, Zeng et al. [43] showed that the scavenger receptor CD36 and the caveolin-1 are in the same fraction indeed, nevertheless they did not conclude that CD36 was in caveolae. Actually, their study clearly demonstrated that Triton X-100 extraction does not allow discrimination between rafts and caveolae when present in the same cell and that CD36 utilize a lipid raft pathway that does not require caveolin-1. The GPI-APs (mainly CD59, CD55, CD48, CDw90/Thy-1, and the prion protein/PrP) are frequently used as positive controls for raft purification by the majority of authors, depending on the cell type. One has to note that GPI-APs are almost ever used indiscriminately as if they were equal. Nevertheless, Madore et al. [40] have demonstrated a structural diversity of the domains occupied by functionally different GPI-APs. In fact, the authors have shown that Thy-1 and PrP are localized in different subdomains. Furthermore, Millán et al. [44] have demonstrated that CD59 and the monosialoganglioside GM1 cluster in different membrane subdomains of Jurkat cells. According to Schade et al. [45], there are Lck-rafts and LAT-rafts that are distinct in resting human peripheral blood T cells (hPBTs). Muñoz et al. [46] have demonstrated the existence of at least three types of raft subsets: (1) CD38-rafts (enriched in CD38, CD3-ζ, and Lck), (2) TCR/CD3-rafts (enriched in CD3-ζ, CD3-ε, and Lck), and (3) LAT-rafts (enriched both in LAT and Lck). In conclusion, until today, the residence of the CD3/TCR complex, raft markers, the members of the Src kinase family, and the adaptor LAT in a homogeneous and native structure is still elusive. These different subsets of DRMs exhibit different sensitiveness to cholesterol depletion. Detergents differ considerably in their ability to enrich cholesterol over glycerophospholipids [10]. For example, 25% of the cellular cholesterol is Triton X100-insoluble [47], whereas only 15% of cellular cholesterol is found in the large CHAPS-DRMs that contain the caveolar markers [48]. Furthermore, one has to note that prominin, confined to microvilli, is present in a membrane microdomain that is more sensitive to cholesterol removal than that containing PLAP [41]. Tetraspan microdomains are distinct from conventional lipid rafts as they enrich MHC class II molecules carrying a selected set of peptide antigens [49]. They are characterized by the CDw78 determinant, which is a marker of activation on B cells. The authors demonstrated that m-β-CD leaves CDw78

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microdomains unchanged. Interestingly, Schade et al. [45] showed that cholesterol extraction with m-β-CD (10 mM, 5 min, 37 °C) destabilizes the Lck-microdomains, while LAT-rafts remain intact. Glycosignaling domains (GSDs) are functional membrane units that are enriched in GM3, Src kinases, Rho A, and focal adhesion kinase (FAK) [50,51]. They are involved in cell-adhesion and signal transduction leading to enhanced mouse melanona B16 motility and invasiveness. One has to note that GSDs are not enriched in cholesterol, nonetheless they are most often mixed up with conventional cholesterol-rafts. Superrafts exist in the brush border membrane of enterocytes [52,53]. They harbor the GPI-linked alkaline phosphatase and the transmembrane aminopeptidase N, whereas annexin 2, a conventional raft-resident protein, is essentially absent. Superrafts define a membrane unit highly resistant to detergent solubilization that exists at physiological temperature, nonetheless superrafts are cholesterol-independent DRMs. According to the authors, galectin-4 functions as a raft stabilizer in place of cholesterol. Marwali et al. [54] showed that the plasma membrane of primary T cells and the T cell lines T28 and EL4 has a very different lipid composition. GM1 level is approximately 10 to 100-fold higher in splenic T cells than in those cell lines, whereas cholesterol level is 10-fold lower. Consequently, DRMs isolated from splenic T cells with Triton X-100 are not disrupted by m-β-CD and thus, m-β-CD-treatment did not change the distribution of any of the molecules tested by the authors [54], including Thy-1 and GM1. m-β-CD extracts cholesterol from Triton X-100-resistant membranes without affecting the buoyant properties of Thy-1 and GM1 in P1798 murine T lymphoma cells [12]. Triton X-100-resistance of GM1 is more complex as it seems at first sight. The occurrence of GM1 in DRMs depends on its ceramide moiety. Depletion of 73% of cellular cholesterol with m-β-CD significantly affects the recovery in DRMs of GM1s acetylated or acylated with C8 or unsaturated fatty acids but not GM1 acylated with C18, C22, or C24 saturated fatty acids [32]. Although sphingolipids are largely restricted to the outer leaflet, several observations suggest that rafts are present in the inner leaflet and that rafts in the two leaflets are coupled. First DRMs have a bilayer appearance when isolated [47] and are enriched in dually acylated cytofacial membrane proteins, such as Src family kinases and G protein α subunits. Second, Src family kinases can co-redistribute when cell surface GPI-anchored proteins or gangliosides are clustered using antibodies or toxins [55,56]. How these rafts might be coupled together is very poorly understood. It is possible that the long sphingolipid acyl chains present in the outer leaflet domains may help organize and stabilize phospholipid domains in the inner leaflet. In the SM molecule there is often a large mismatch in length between the N-acyl chain and the sphingosine backbone. Such a chain mismatch may lead to interdigitation between the hydrocarbons of the two opposing monolayers which form the lipid bilayer [57]. Interdigitation of opposing monolayers could promote Lo domains: the mixing of such asymmetric SM with other symmetric lipids in the plasma membranes of cells would be highly non-ideal and would lead to the formation of lateral domain [58]. SM-induced monolayer coupling has been observed in model membranes [57].

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According to what we know, only one study uses exogenous SMase to disrupt raft in Jurkat cells [16]. The authors use 10 mU/ml of SMase for 30 min whereas we only use 0.25 mU/ ml for 5 min Thus it represents a inappropriate condition (40× in concentration and 6× in time of incubation) since our conditions are largely sufficient to strongly modify the lipid content of rafts, and they allow the shift from SM/cholesterol-rich rafts to Cer-rich/cholesterol-poor domains. Some people could argue that exogenous SMase only gains access to the outer leaflet. It would mean that Cer-rich/cholesterol-poor domains would only be restricted to this outer leaflet. Thus the inner leaflet would not be modified and it would explain why Lck and LAT are still buoyant in SMase-treated cells and it would justify the fact that the CD3-induced Tyr-phosphorylations are not diminished in SMase-treated JE 6.1. But we definitely disagree with this way of thinking. First, the cholesterol content of the outer leaflet is considerably lowered, nonetheless CD55 and GM1 are still buoyant. Thus, the cholesterol-dependence of the raft markers must be severely re-evaluated. Second, the two leaflets are coupled thanks to SM interdigitations as explained just above. Disorganization of the outer leaflet must logically have consequential effects on the organization of the inner leaflet and the buoyancy of Lck and LAT remains problematic. As a consequence, optimal CD3-induced Tyr-phosphorylations in SMasetreated cells are an unexpected result. Sphingolipids greatly influence membrane permeability to small ions and other small non-electrolytes [59] because their saturated acyl chains prevent the acyl chain rearrangements that are necessary for the transit across the bilayer [60,61]. Ceramides have the capacity to modify the permeability barrier of cell membranes that results in enhanced solute efflux [62]. Consequently, as we demonstrated, SMase causes plasma membrane depolarization and thus leads to a drop in the CD3-induced Ca2+ fluxes. Because CD3-induced Ca2+ influx by the calcium release activated channel (CRAC) is largely dependent on the membrane potential, we conclude that the observed inhibition of the Ca2+ movements is a consequence of the CRAC inhibition. Considering that PLCγ1 activity requires Ca2+ ions and that the enzyme binding to the lipid interface, used as substrate, necessitates Ca2+ [63], the reduction in the Ca2+ level into the cytoplasm is likely to result in lowering the PLC γ1 activity and thus leads to the observed inhibition of DAG production. It appears that the transformation of SM/cholesterol-rich rafts into Cer-rich/cholesterol-poor domains by exogenous SMase inhibits CD3-induced second messengers without interference with the early phosphorylation events implicated in T cell activation. In conclusion, our results clearly show that buoyant cholesterol is not crucial for the stabilization and the functioning of rafts involved in the CD3/TCR signaling. They support the existence of another kind of Lo domain that would not be enriched in cholesterol and that would contain SM, ceramides, the Src kinases and the CD3/TCR complex in the native plasma membrane. Acknowledgments The authors would like to thank Dr. Laurence Lamy, Dr. Isabelle Foucault, Romain Gallais, and Frank Leporati.

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