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Ceramide Selectively Displaces Cholesterol from Ordered Lipid Domains (Rafts)

Ceramide Selectively Displaces Cholesterol from Ordered Lipid Domains (Rafts) THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 11, Issue of March 12, pp. 9997–10004, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Ceramide Selectively Displaces Cholesterol from Ordered Lipid Domains (Rafts) IMPLICATIONS FOR LIPID RAFT STRUCTURE AND FUNCTION* Received for publication, September 8, 2003, and in revised form, November 14, 2003 Published, JBC Papers in Press, December 29, 2003, DOI 10.1074/jbc.M309992200 Megha and Erwin London‡ From the Department of Biochemistry and Cell Biology and Department of Chemistry, Stony Brook University, State University of New York, Stony Brook, New York 11794-5215 Ceramide is a membrane lipid involved in a number of (3–9). Ceramide-triggered apoptosis is of particular interest crucial biological processes. Recent evidence suggests because it seems to play an important role in the sensitivity of that ceramide is likely to reside and function within tumor cells to chemotherapy and radiotherapy (7, 10, 11) and in lipid rafts; ordered sphingolipid and cholesterol-rich the development of atherosclerosis (12). Ceramide location lipid domains believed to exist within many eukaryotic within lipid rafts is an important factor in ceramide action (3, cell membranes. Using lipid vesicles containing co-exist- 5, 6, 13). Lipid rafts are liquid ordered state membrane do- ing raft domains and disordered fluid domains, we find mains rich in cholesterol and saturated polar lipids (usually that natural and saturated synthetic ceramides displace sphingolipids). They can co-exist with disordered fluid state sterols from rafts. Other raft lipids remain raft-associ- membrane domains rich in unsaturated lipids (14, 15). Ceram- ated in the presence of ceramide, showing displacement ide both stabilizes and associates strongly with lipid rafts (16, is relatively specific for sterols. Like cholesterol-con- 17). It can also induce the formation of unusually large raft taining rafts, ceramide-rich “rafts” remain in a highly domains (“platforms”) in plasma membranes (3). ordered state. Comparison of the sterol-displacing abil- In this study, we found that natural ceramides or ceramides ities of natural ceramides with those of saturated dig- with long saturated acyl chains, and analogous diglycerides, lycerides and an unsaturated ceramide demonstrates efficiently displace cholesterol from rafts in model membranes. that tight lipid packing is critical for sterol displace- Other raft-associating molecules were not displaced from rafts ment by ceramide. Based on these results, and the fact by ceramide or diglyceride. A model for the origin of this dis- that cholesterol and ceramides both have small polar placement phenomenon is proposed. This model may provide headgroups, we propose that ceramides and cholesterol insights into how various molecules, including proteins, partic- compete for association with rafts because of a limited ipate in raft formation. Displacement of sterols from rafts is capacity of raft lipids with large headgroups to accom- modate small headgroup lipids in a manner that pre- likely to have a variety of important consequences for raft vents unfavorable contact between the hydrocarbon function. For example, displacement of cholesterol from rafts groups of the small headgroup lipids and the surround- can explain the displacement of cholesterol from plasma mem- ing aqueous environment. Minimizing the exposure of branes upon ceramide generation, and may have implications cholesterol and ceramide to water may be a strong driv- for the association of cholesterol-bound proteins with rafts. ing force for the association of other molecules with EXPERIMENTAL PROCEDURES rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft Materials and Sample Preparation—Unlabeled lipids (including sphingosine-based ceramides) were purchased from Avanti Polar Lipids structure and function, altering liquid ordered proper- (Alabaster, AL). Dehydroergosterol (DHE) and 1,6-diphenyl-1,3,5- ties as well as molecular composition. In this regard, hexatriene (DPH) were purchased from Sigma-Aldrich. 1-palmitoyl-2- certain previously observed physiological processes (3-(diphenylhexatrienyl)propanoyl)-phosphatidylcholine (DPH-PC) was may be a result of displacement. In particular, a direct purchased from Molecular Probes (Eugene, OR). 22-(Diphenylhexatrie- connection to the previously observed sphingomyeli- nyl)docosyltrimethylammonium, (long chain TMADPH, LcTMADPH) nase-induced displacement of cholesterol from plasma was a kind gift from G. Duportail and D. Heissler (Universite ´ Louis membranes in cells is proposed. Pasteur, Strasbourg). Lipids and probes were stored dissolved in etha- nol at 20 °C. Concentrations were determined by dry weight. Radio- labeled dioleoylphosphatidylcholine (DOPC) and dipalmitoylphoshati- dylcholine (DPPC) were purchased from Amersham Biosciences Ceramide is a membrane lipid involved in many biological (Piscataway, NJ). Radiolabeled palmitoyl (C16:0) ceramide was pur- processes. Sphingomyelinase action can result in a rapid in- chased from American Radiolabeled Chemicals (St. Louis, MO). Lipid crease in ceramide levels in which a large fraction of cellular (including radiolabeled lipid) purity was confirmed by TLC. Acetyl- sphingomyelin is converted to ceramide (1, 2). Sudden in- K W L AL W K -amide (LW peptide) was purchased from Invitrogen 2 2 8 8 2 2 creases in ceramide levels due to the action of sphingomyeli- (Carlsbad, CA) and purified as described previously (18). nase upon the plasma membrane have important effects upon Multilamellar vesicles (MLV) were prepared at a concentration of bacterial pathogenesis, cholesterol homeostasis, and apoptosis The abbreviations used are: DHE, dehydroergosterol; PBS, phos- * This work was supported by National Institutes of Health Grant phate-buffered saline; SM, sphingomyelin; cer, ceramide; DRM, de- GM 48596. The costs of publication of this article were defrayed in part tergent-resistant membrane; DPH, 1,6-diphenyl-1,3,5-hexatriene; by the payment of page charges. This article must therefore be hereby DOPC, dioleoylphosphatidylcholine; DPPC, dipalmitoylphoshatidylcho- marked “advertisement” in accordance with 18 U.S.C. Section 1734 line; DPH-PC, 1-palmitoyl-2-(diphenylhexatrienyl)-propanoyl-phospha- solely to indicate this fact. tidylcholine; LcTMADPH, 22-(diphenylhexatrienyl)docosyltrimethyl- ‡ To whom correspondence should be addressed. Tel.: 631-632-8564; ammonium; MLV, multilamellar vesicles; 12 SLPC, 1-palmitoyl-2-(12- Fax: 631-632-8575; E-mail: [email protected]. doxyl)-stearoyl-phosphatidylcholine; SUV, small unilamellar vesicles. This paper is available on line at http://www.jbc.org 9997 This is an Open Access article under the CC BY license. 9998 Ceramide and Implications for Raft Structure 500 M lipid in PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7) as lipid dioleoylphosphatidylcholine (DOPC). Previous studies described previously (19). Dried lipid mixtures were dispersed in buffer have shown that such vesicles form bilayers containing co- at 70 °C using a multitube vortexer for 15 min and cooled to 25 °C. existing ordered “raft” domains rich in DPPC and sterol and Small unilamellar vesicles (SUV) at a concentration of 50 M lipid in non-raft regions rich in DOPC (19 –22). In the first series of PBS were prepared by ethanol dilution as described previously (20). experiments, the insolubility of ordered domains in Triton Lipids mixed in ethanol were diluted slightly more than 50-fold in PBS buffer heated to 70 °C, briefly vortexed, incubated at 70 °C for about 5 X-100 at 23 °C was used to evaluate raft association. A DRM min, re-vortexed, and cooled to 25 °C. pellet was isolated by centrifugation, and then the amount of Fluorescence Measurements—Fluorescence was measured on a SPEX the molecule of interest that was DRM-associated was meas- Fluorolog 3. Excitation and emission wavelength sets used (in nm) ured (19, 23, 24). This value was normalized to the fraction of were: (358, 427) for DPH and LcTMADPH, (358, 436) for DPH-PC, (324, total lipid that was detergent insoluble, i.e. the fraction of total 376) for DHE, and (280, 340) for LW peptide. Fluorescence intensity in background samples lacking fluorophore was subtracted when signifi- lipid found in DRM. Using multilamellar vesicles, which scat- cant. Unless otherwise noted fluorophore concentrations were 0.1 mol% ter much more light than detergent-solubilized material, the for LcTMADPH and DPH-PC and 1.5 mol% for LW peptide fraction of undissolved lipid could be estimated from the % Insolubility Assay of Raft Content—The detergent-resistant mem- optical density (%OD) remaining after addition of Triton X-100. brane (DRM) were isolated by insolubility at 23 °C as described previ- Although detergent insolubility of lipids is sensitive to a num- ously (19). The DRM fraction from a 1-ml MLV sample was collected ber of variables (25, 26) previous studies have shown that after2hof incubation in 0.5%(w/v) Triton X-100 by pelleting at 14,000 rpm for 15 min in an Eppendorf 5415C tabletop centrifuge. The fluo- under the conditions in our experiments the level of detergent- rescence of 970 –990 l of supernatant and of the pellet suspended in insolubility reflect the amount of lipids and other molecules 975 l of PBS were compared. For calculating % pelleting corrections associated with ordered domains reasonably well (19, 20, 27). were made for differences in fluorophore intensity (quantum yield) in Detergent insolubility was measured for radiolabeled DPPC, lipid and Triton X-100. Radioactive tracers were used at 0.02– 0.1 Ci DOPC, C16:0 ceramide, and cholesterol, and also for DPH-PC, per sample. Average values and the range for duplicate experiments were calculated. (In most cases, for these and the fluorescence studies a fluorescently labeled phosphatidylcholine, DHE, a fluores- described below, additional experiments that are not reported gave cent sterol with properties similar to those of cholesterol (28), similar results.) and LcTMADPH, a derivative of DPH attached to a trimethyl- Fluorescence Quenching Assays—Quenching of fluorophores incorpo- amino-terminated C22:0 hydrocarbon chain (29). Previous rated into SUV was measured at 23 °C as described previously (17, 20). studies have shown that the affinity for ordered domains is Fluorescence (F) of samples containing 12SLPC was normalized (F )to highest for LcTMADPH, relatively high in the case of DPPC, that in samples in which DOPC replaced 12SLPC. Control experiments confirmed that quenching of DHE was not affected by the length of time ceramide and cholesterol, lower but still significant for that samples were incubated after preparation, or by whether MLV or DPH-PC, and least for DOPC (16, 17, 21, 22, 24, 29). In rea- SUV samples were prepared. Average values and the range for dupli- sonable agreement with this order, Fig. 1, A and B show that in cate experiments were calculated. The temperature dependence of the absence of ceramide relative association with DRM (given quenching was used to evaluate the thermal stability of rafts as de- by the ratio % pelleting/% OD) decreased in the order scribed previously (17, 19). Samples containing 0.3 mol% LcTMADPH were heated at about 3 °C per min, and fluorescence periodically LcTMADPHceramideDPPCcholesterolDHEDPH- measured during heating. Average values for duplicate experiments PCDOPC. These measurements were repeated using samples were calculated. in which almost half of the unlabeled DPPC was replaced by an Polarization Measurements—Anisotropy measurements were made equimolar amount of stearoyl (C18:0) ceramide. Fig. 1, A and B at 23 °C using a SPEX Glan-Thompson automated polarizer accessory. show that in the presence of C18:0 ceramide both LcTMADPH SUV samples were prepared as described above except that 1mol% and C16:0 ceramide remained strongly associated with DRM, DPH or LcTMADPH was used. Background intensities were negligible. Average values and the range for duplicate experiments were while DPPC association with DRM actually increased. In con- calculated. trast, the presence of ceramide resulted in the strong displace- Measurement of DHE to LcTMADPH Energy Transfer—SUV sam- ment of both cholesterol and DHE from DRM. (In a control ples were prepared with either LcTMADPH or with LcTMADPH plus experiment, it was found that C16:0 ceramide displaced cho- DHE. Samples containing DHE were composed of 12:12:61:7.5:7.5 (mol: lesterol at least as well as C18:0 ceramide (data not shown).) mol) DPPC:cer:DOPC:DHE:cholesterol (F )or 85:7.5:7.5 DOPC:D- raft HE:cholesterol (F ). Samples lacking DHE were composed of 12: DPH-PC was moderately displaced from the DRM by ceramide. homog 12:61:15 (mol:mol) DPPC:cer:DOPC:cholesterol (F ) or 85:15 DOPC: raft Fig. 1, C and D show that both cholesterol and DHE exhibited cholesterol (F ). Sensitized LcTMADPH emission was measured at homog a progressive displacement as ceramide concentration was in- excitation 324 nm and emission 427 nm. Energy transfer was measured creased. Sterol displacement did not parallel the ceramide de- from the fractional increase of LcTMADPH emission in the presence of pendence of the overall fraction of lipids within DRM, which DHE. Fractional increase in LcTMADPH fluorescence is given by ([F / tended to remain relatively constant as judged by % OD. F ]  1) and the relative fractional increase is given by ([F /F ]  1)/([F /F ]  1). raft raft homog homog Detection of Displacement of Sterol from Rafts by Ceramide No correction was made for quenching of DHE by LcTMADPH when using Fluorescence Quenching—DRM isolated by use of Triton corrections for background fluorescence (from samples lacking LcT- X-100 can overestimate or underestimate raft levels, and % OD MADPH) were made. We estimate correction for this would increase the is a crude measure of total raft levels (25, 26). For these enhancement of LcTMADPH fluorescence we measured by a factor of reasons, studies were also carried out using fluorescence one-quarter to one-third under conditions in which the bilayer was homogeneous. This should have little effect on the results obtained quenching. Fluorescent probes were incorporated into vesicles because the correction would be the same, and thus cancel out, for containing both raft-forming lipids and 1-palmitoyl 2-(12-doxy- samples with and without DPPC/ceramide under conditions in which l)stearoyl PC (12SLPC), a fluorescence quenching lipid that, the bilayer was homogeneous. Average values from duplicates like DOPC, is relatively excluded from rafts (20). 12SLPC is a were calculated. short-range quencher that has to be in near contact with a RESULTS fluorescent molecule in order to quench its fluorescence (30, 31). When rafts and non-raft regions co-exist in 12SLPC-con- Detection of Displacement of Sterol from Rafts by Ceramide using a Detergent Insolubility Assay—The effect of ceramide taining vesicles the intensity of fluorescence from a membrane- bound probe depends on whether it associates with rafts or not. upon association of various molecules with ordered lipid do- mains was measured starting with model membrane vesicles In mixtures of DPPC (or sphingomyelin) with cholesterol and containing sterol and a 1:1 mixture of the saturated lipid di- 12SLPC, rafts have a low % of 12SLPC while the remainder of palmitoylphosphatidylcholine (DPPC) and the unsaturated the bilayer, which is in a disordered fluid state, is enriched in Ceramide and Implications for Raft Structure 9999 FIG.1. Ceramide-induced displace- ment of molecules from rafts assayed by insolubility in Triton X-100 at 23 °C. Samples lacking ceramide were composed of 1:1:0.35 (mol:mol) DPPC: DOPC:sterol dispersed in PBS. In sam- ples containing cer, DPPC was replaced by equivalent mole amounts of cer. Mol% cer shown is always relative to the total lipid concentration. Sterol was cholesterol except in samples containing DHE in which a 1:1 mol:mol cholesterol:DHE mix- ture was used. A and B, normalized con- tent of fluorescent probes (A) or radioac- tive lipids (B) in detergent-insoluble fraction in absence (open bars) or pres- ence (shaded bars) of 18 mol% C 18:0 cer. Abbreviations: DP, DPPC, DO, DOPC, Ch, cholesterol. C and D, effect of ceram- ide concentration upon displacement of DHE (C)or[ H]cholesterol (D) from the detergent-insoluble fraction. Symbols: squares, % OD remaining after detergent addition; triangles, % insoluble radioac- tivity or fluorescence; circles, % pellet/% OD. The % pellet/% OD equals the ratio of the % of the radioactive tracer or fluores- cent probe in the Triton X-100 insoluble pellet fraction to the % of unsolubilized lipid as judged by OD at 400 nm. The average and range of duplicate samples are shown in this and most of the follow- ing figures. In most cases, additional ex- periments that are not shown gave simi- lar results. 12SLPC (19, 20, 32). A molecule that associates with rafts cence showed a much smaller increase in quenching as ceram- fluoresces relatively strongly, because there is a good chance it ide concentration was increased, indicating a more modest will not be next to a 12SLPC molecule. A molecule that remains extent of displacement. Overall, the fluorescence quenching in the non-raft regions of the bilayer will fluoresce weakly experiments are in agreement with those based upon because the chance it will be next to a 12SLPC is high. There- detergent insolubility. fore, the level of quenching will reflect the degree of association Utilizing quenching, ceramide-induced displacement of ste- with rafts (18, 20). rol from rafts was also observed when brain sphingomyelin Fig. 2A shows the ceramide dependence of quenching of the (SM) and a brain ceramide mixture were substituted for DPPC fluorescence of DHE, LcTMADPH, DPH-PC, and LW peptide and C18:0 ceramide, respectively (Fig. 2B). This shows that (acetyl-K W L AL W K -amide), a synthetic transmembrane displacement is similar for analogous chemically defined and 2 2 8 8 2 2 polypeptide that does not associate with lipid rafts (18). The natural lipid mixtures. Comparison of the ability of different y-axis gives the difference between fluorescence in raft-contain- sphingolipids to displace sterol (Fig. 2C) showed that partial ing samples and that in control samples forming homogeneous substitution of SM for DPPC had no effect on quenching of bilayers, i.e. lacking rafts. (Without subtraction of control val- DHE fluorescence, whereas partial substitution of a cerebro- ues, the inherently different quenching sensitivity of different side mixture for DPPC increased quenching, but to a much types of fluorescent groups would partially obscure differences lesser degree than substitution of DPPC by C18:0 ceramide. between their raft association.) In raft-containing samples The behavior of cerebroside-containing mixtures suggests that lacking ceramide (i.e. 0% ceramide values in Fig. 2A), fluores- sterols associate with cerebroside-rich rafts to much higher cence was most weakly quenched relative to the homogeneous degree than to ceramide-rich rafts but to a lesser degree than control samples for LcTMADPH, consistent with the strong those formed by SM or DPPC, consistent with previous association of LcTMADPH with lipid rafts. In contrast, the Trp studies (17). Comparison of Diglyceride and Ceramide Behavior—The ef- fluorescence of the peptide was more strongly quenched in raft-containing samples than in the control samples. This re- fect of ceramide and diglyceride on raft stability and sterol displacement were compared. Both of these lipids contain two flects the relative exclusion of LW peptide from rafts, as found previously (18). Quenching levels were intermediate for hydrocarbon chains and relatively small polar headgroups, but they differ in the details of their polar headgroup structure. In DPH-PC and DHE, showing that they associate with rafts to a significant degree. one experiment, their effects on raft stability were compared when added to mixtures of DPPC, 12SLPC, cholesterol, and Partial substitution of DPPC with ceramide had little effect on quenching of LcTMADPH and peptide fluorescence, showing LcTMADPH. As noted above, when rafts are present they bind LcTMADPH so that its fluorescence is only weakly quenched the locations of these molecules was not greatly altered by ceramide. (The small changes observed in their quenching at by 12SLPC (17, 20). However, at temperatures above that at which rafts are stable all lipids mix in a relatively homogene- high ceramide concentrations may reflect a small change in raft association or ceramide-induced displacement of some of ous fashion, and thus quenching increases because LcT- MADPH molecules come into increased contact with 12SLPC the residual raft-associated 12SLPC from rafts.) In contrast, as ceramide concentrations increased a large and progressive in- molecules. The temperature dependence of quenching can be used to monitor the thermal stability of rafts. The midpoint of crease in the quenching of DHE fluorescence was observed, confirming DHE displacement from rafts. DPH-PC fluores- a quenching versus temperature curve (raft “melting” temper- 10000 Ceramide and Implications for Raft Structure whereas C18:1 ceramide only induced weak displacement rel- ative to C18:0 ceramide. Together these results suggest that acyl chain saturation is a more important factor than the details of the polar headgroup structure for both raft stabiliza- tion and sterol displacement. (Whether natural diglycerides would affect raft structure or formation is unclear. Preliminary studies indicate that 1-palmitoyl 2-oleoyl glycerol can displace cholesterol from rafts, although to a lesser extent than dipalmitoylglycerol. ) Ceramide-rich Domains Are Highly Ordered—Fluorescence anisotropy measurements were made on samples containing LcTMADPH and DPH incorporated into various lipid bilayers to probe the effect of ceramide upon lipid order (Fig. 4A). As expected, for both probes anisotropy values were high in vesi- cles composed solely of lipids forming ordered states (DPPC or DPPC/cholesterol) and low in vesicles composed of lipids exist- ing in the disordered liquid state (DOPC or DOPC/cholesterol). In vesicles composed of DPPC, DOPC, and cholesterol mixtures under conditions in which both ordered and disordered do- mains co-exist (see above) Fig. 4A shows anisotropy of LcT- MADPH fluorescence remained as high as that in fully ordered bilayers regardless of ceramide concentration. Since LcT- MADPH partitions strongly into ordered phases this indicates that the more ordered domains remain highly ordered in the presence of ceramide. DPH, which partitions into both ordered and disordered phases, gave fluorescence with intermediate anisotropy values in vesicles containing both ordered and dis- ordered states. There was a decrease in DPH anisotropy in the presence of ceramide. This decrease can be explained by partial displacement of DPH from ceramide-rich ordered domains. Partial displacement of DPH by ceramide was confirmed by quenching experiments (data not shown). Effect of Sterol Concentration upon Displacement of Sterol from Rafts by Ceramide and upon Displacement of Ceramide from Rafts by Sterol—Because sterol levels in the plasma mem- brane are high, the effect of cholesterol concentration upon displacement by ceramide was assessed (Fig. 4B). Even at a concentration as high as 40 mol% cholesterol, the presence of 18 mol% ceramide was able to displace the great majority of cholesterol from rafts. We were also interested to see if sterol could displace ceramide from rafts. Fig. 4C shows that choles- terol can displace some C16:0 ceramide from ordered domains both when ceramide is present in trace amounts (open bars)or when it forms a large (18 mol%) fraction of the lipid bilayer (closed bars). This indicates sterol and ceramide are competing FIG.2. Ceramide-induced displacement of molecules from for association with rafts. However, displacement of ceramide rafts at 23 °C assayed by fluorescence quenching. A, effect of by cholesterol was much less effective than displacement of C18:0 cer concentration upon quenching of various fluorophores. Sam- ples contained 1:1:0.35 (mol:mol) [DPPCcer]:[12SLPC or DOPC]:s- cholesterol by ceramide. For example, upon introduction of 25 terol dispersed in PBS. Sterol was cholesterol except in samples con- mol% cholesterol into the model membrane vesicles, ceramide taining DHE in which a 1:1 cholesterol:DHE mixture was used. concentrations in the ordered domains only decreased by one- Symbols: triangles, LcTMADPH; squares, DPH-PC; diamonds, LW pep- third when samples contained 18 mol% C18:0 ceramide. This tide; circles, DHE. B, effect of sphingolipids upon quenching of DHE. Samples contained 1:1:0.35 (mol:mol) [DPPCsphingolipid]:[12SLPC should be contrasted to the roughly 4-fold decrease of choles- or DOPC]:DHE dispersed in PBS. Symbols: squares, SM; triangles, terol concentration within ordered domains induced by the mixed cerebrosides; circles, C18:0 ceramide. C, comparison of displace- introduction of 18 mol% ceramide into bilayers containing 25 ment using natural and synthetic lipids. squares, 1:1:0.35 [brain mol% cholesterol (Fig. 4C). The observation that ceramide dis- SMbrain cer]:[12SLPC or DOPC]:DHE; circles, 1:1:0.35, places cholesterol more efficiently than cholesterol displaces [DPPCC18:0 cer]:[12SLPC or DOPC]:DHE. ceramide confirms that ceramide has a much stronger affinity ature) is a measure of raft stability (17, 19). Fig. 3, A and B for ordered domains than cholesterol. show that substitution of 4 mol%, 9 mol%, or 18 mol% DPPC Energy Transfer Analysis of Domain Organization—To con- with an equimolar amount of either C18:0 ceramide or 1,2- firm that sterol displacement was not a sample preparation dipalmitoylglycerol-stabilized rafts. The degree of stabilization artifact, we used energy transfer to examine whether sterol by the C18:0 ceramide and dipalmitoylglycerol was similar. In and raft-forming lipids were present in the same vesicles. In contrast, unsaturated oleoyl (C18:1) ceramide decreased raft homogeneous control bilayers containing DOPC, sterol, and stability (Fig. 3A). We next compared the ability of dipalmitoyl- LcTMADPH, energy transfer from DHE to LcTMADPH signif- glycerol and ceramide to displace sterols from rafts. As shown in Fig. 3, C and D dipalmitoylglycerol was able to displace sterol from rafts even more efficiently than C18:0 ceramide, Megha and E. London, unpublished observations. Ceramide and Implications for Raft Structure 10001 FIG.3. Effect of different ceramides and diglyceride upon ordered domain stability and sterol displacement from rafts. A and B, quenching assay of raft stability. The temperature dependence of quenching of the fluorescence of LcTMADPH was measured in samples composed of A, 1:1:0.35 [DPPCcer]:[12SLPC or DOPC]:cholesterol dispersed in PBS. Samples contained 0% (diamonds), 4% (squares), 9% (filled triangles), or 18 mol% C18:0 cer (filled circles), or contained 9% (open triangles) or 18% C18:1 cer (open circles). B, 1:1:0.35 [DPPCdiglyceride]:[12SLPC or DOPC]:cholesterol dispersed in PBS. Samples contained: 0% (diamonds), 4% (squares), 9% (triangles), or 18 mol % dipalmitoylglycerol (circles). C and D, comparison of sterol displacement from rafts by ceramide and diglyceride at 23 °C. C, insolubility in Triton X-100 assay of displacement of radioactive cholesterol from rafts by C18:0 cer (circles), or dipalmitoylglycerol (squares). Samples composed of 1:1:0.35 [DPPC(cer or diglyceride)]: DOPC:cholesterol dispersed in PBS. D, fluorescence quenching assay of displacement of DHE by ceramide and diglyceride. Displacement of DHE from rafts by C18:0 cer (circles), C18:1 cer (triangles), or dipalmitoylglycerol (squares). Samples composed of 1:1:0.175:0.175 [DPPC(cer or diglyceride)]:[12SLPC or DOPC]:DHE:cholesterol dispersed in PBS. DAG, diglyceride. icantly enhanced LcTMADPH fluorescence. The increase in may be also energetically driven by the fact that the small polar LcTMADPH emission in the presence of DHE was somewhat headgroup (the OH) of cholesterol is insufficient to shield the temperature dependent, decreasing from a 42% increase at sterol rings from water, and tight packing with other lipids 20 °C to a 26% increase at 45 °C. At low temperatures the allows the sterol rings to hide under an “umbrella” formed by increase in LcTMADPH fluorescence in bilayers containing lipids with large polar headgroups (33). Because ceramides also DPPC and ceramide was much less than that in the control have a small polar headgroup we propose a similar phenome- samples (Fig. 4D). This was expected because, as shown by the non applies to them, and that the limited ability of normal studies above, LcTMADPH should be located in the DPPC/ polar lipids to simultaneously shield hydrocarbon groups on ceramide-rich rafts while the displaced DHE should be located both ceramides and cholesterol from water results in a compe- in disordered fluid domains. However, upon melting of the rafts tition between ceramide and sterol for inclusion in ordered at high temperature, energy transfer approached levels ob- domains. An analogous model has been proposed to explain the served in control samples lacking raft-forming lipids. This ability of ceramide in disordered fluid domains to increase the should not have been observed if there was one set of vesicles binding of cholesterol to a cytolysin protein (46). The observa- containing raft-forming lipids and LcTMADPH and a separate tion that DPH-PC, which has a large polar headgroup (but also population containing DHE. Instead, this indicates that the a rather bulky fluorophore-labeled acyl chain, was partially raft-forming lipids and DHE were in a single set of vesicles. displaced from rafts by ceramide suggests the possibility that Additional experiments showed that the raft melting tem- especially tight packing of hydrocarbon chains in ceramide- perature detected by energy transfer was close to that detected containing ordered domains relative to those containing satu- by 12SLPC quenching of LcTMADPH fluorescence (Fig. 4D), rated lipid and cholesterol can also contribute to displacement indicating that the lipid composition of the bulk sample and the by squeezing out molecules that cannot pack as well as ceram- vesicles containing DHE was similar. If the DHE had been in a ide. On the other hand, hydrogen bonding appears to be less different subset of vesicles than the raft-forming lipids, energy important for displacement. Diglycerides have a very different transfer should have shown a very different thermal depend- set of hydrogen bonding groups than do ceramide, and yet a ence than that exhibited by quenching of LcTMADPH. saturated diglyceride displaced sterol more effectively than a DISCUSSION ceramide with a similar hydrocarbon chain structure. An alternative explanation of displacement is that ceramide Origin of the Displacement of Sterol from Ordered Domains forms cholesterol-poor ordered domains that are separate from by Ceramide—What is the origin of sterol displacement from rafts by ceramide? Both cholesterol and natural ceramides the ordered domains formed by DPPC (or SM). When ceramide is substituted for DPPC there would be a lower amount of the have hydrocarbon structures allowing tight packing with phos- pholipids and/or sphingolipids. Tight packing involving sterols DPPC-rich ordered domains into which sterol could incorpo- 10002 Ceramide and Implications for Raft Structure FIG.4. Assay of vesicle properties in the presence of cer. A, effect of inclusion of cer in rafts upon fluorescence anisotropy of 1 mol% DPH (black bars) or 1 mol% LcTMADPH (striped bars)at23 °C. Ceramide-containing samples were composed of 1:1:0.35 [DPPCcer]:DOPC:cholesterol dispersed in PBS. For calibration purposes, anisotropy values in vesicles in the liquid disordered (DOPC  15 mol % cholesterol), and gel (DPPC) states at 23 °C are shown. Also shown: DPPC  15 mol % cholesterol. Abbreviations: DP, DPPC; DO, DOPC; Ch, cholesterol. B, effect of cholesterol concentration on its displacement by C18:0 cer measured by insolubility in Triton X-100 at 23 °C. Samples composed of 1:1 [DPPCcer]:DOPC with various mol fractions of cholesterol. Shaded bars, 18 mol% cer; open bars, 0% cer. C, displacement of [ H]C16:0 ceramide from rafts as a function of cholesterol concentration as measured by insolubility in Triton X-100. Shaded bars, 18 mol% C18:0 cer; open bars, 0% C18:0 cer. D, comparison of the effect of temperature upon energy transfer from DHE to LcTMADPH and upon DHE quenching. Squares, the ratio of the fractional increase in LcTMADPH fluorescence due to energy transfer from DHE in vesicles composed of 12:12:61:7.5:7.5 (mol:mol) DPPC:cer:DOPC:DHE:cholesterol relative to the fractional increase in energy transfer in vesicles composed of 85:7.5:7.5 DOPC:DHE:cholesterol. Triangles, ratio of LcTMADPH fluorescence (F) in vesicles composed of 12:12:30.5:30.5:15 (mol:mol) DPPC:cer:DOPC:12SLPC:cholesterol to fluorescence (F ) of vesicles composed of 12:12:61:15 (mol:mol) DPPC:cer:DOPC:cholesterol. Samples also contained 1 mol % LcTMADPH. Vesicles were dispersed in PBS. rate. The observations that increasing ceramide concentration levels reach 18 mol%. These values are comparable to those induces only a gradual increase in raft melting temperature, reached in the plasma membrane under physiological condi- and that melting remains a single step process, argue against tions. In cases of bacterial infections, viral infection and tumor multiple types of ordered domains. A separate ceramide-rich necrosis factor action it has been found that 40% of all-cellular phase would likely give rise to a separate melting event at a SM is digested by ceramide-producing cellular SMase in as distinct temperature. Furthermore, inclusion of ceramide in- little as a few minutes (1, 34, 35). It has also been shown that creased the fraction of DPPC associated with ordered domains for each molecule of SM digested roughly one molecule of cer- while displacing sterol (Fig. 1B). This would not be expected if amide appears (2, 34). Thus, we can approximate ceramide ceramide and DPPC formed separate ordered domains, but levels in the plasma membrane. If we estimate that the plasma would if ceramide and DPPC interact in a single type of mixed membrane lipid is typically 15% SM, and that almost all SM is ordered domain. Finally, the partial displacement of ceramide in the outer leaflet of the membrane, then about 30% of the from ordered domains by cholesterol is much easier to ration- outer leaflet of the plasma membrane is SM. In that case 40% alize if ceramide and cholesterol are competing for inclusion in digestion by SMase means that 12% of the plasma membrane a single type of ordered domain. outer leaflet lipid is converted to ceramide. This number is only Physiologically Relevant Ceramide Levels Displace Choles- a lower estimate because it ignores the existence of SM pools in terol from Plasma Membranes in Cells and Are Equivalent to internal membranes [lysosomes, endosomes, Golgi]. If one-half Values Sufficient to Displace Cholesterol from Rafts—Our data of cellular SM is in internal membranes, and thus insensitive show that displacement of cholesterol from rafts is significant to SMase, then there would be 80% conversion of plasma mem- (roughly 50%) when cer comprises 9 mol% of the total bilayer brane SM to ceramide by SMase. This would double the value lipid and that displacement can be nearly complete when cer for ceramide levels produced in the outer leaflet to 24 mol%. Ceramide and Implications for Raft Structure 10003 Several other studies have shown that SMase digestion also of the stratum corneum of skin, which are primarily composed leads to displacement of cholesterol from the plasma mem- of a cholesterol/ceramide mixture (44). The tendency of both brane (1, 2, 4, 9, 36). There appears to be a direct relationship cholesterol and ceramide to avoid excessive hydration presum- between these events. The rate of cholesterol displacement ably contributes functionally to the action of the stratum cor- neum as a barrier to water. from the plasma membrane follows the rate of SM digestion without a significant time lag, and plasma membrane choles- The tendency of cholesterol and ceramide to avoid exposure to water also might be a driving force for the association of terol levels recover at the same rate that SM is resynthesized other types of molecules with rafts. It could contribute, along from ceramide (2, 36). Even the step in which internalized with saturated acyl chain packing interactions, to the raft- cholesterol is esterified follows ceramide generation within association of molecules that can cover the bilayer surface, such minutes (1, 2, 9). as carbohydrate anchors of GPI-anchored proteins, or glyco- Detailed data about the relationship between ceramide gen- sphingolipids with large headgroups. It could also contribute to eration and plasma membrane cholesterol levels was very re- the ability of protein domains that can cover hydrophobic sites cently presented by Al-Makdissy et al. (36). These investigators on the bilayer surface to bind to rafts (or to any membranes directly assayed both SM and cholesterol levels in isolated with high cholesterol or ceramide levels). plasma membrane. Digestion of 25% of plasma membrane SM In this regard, it should be noted that such interactions led to a loss of 50% of plasma membrane cholesterol, similar to should be enhanced at high cholesterol levels. It has been what we would predict based on the data in this report. In proposed that the ability of phospholipids to form a complex addition, the authors measured DPH anisotropy to measure with cholesterol (45) or to maintain a spatial distribution that membrane fluidity. They found DPH anisotropy dropped when prevents contact of cholesterol hydrocarbon with water (33) ceramide was produced. In other words, DPH found itself in a would become overwhelmed at high cholesterol concentrations. more disordered lipid environment upon ceramide generation. For all these reasons, it will be important in future studies to The connection between DPH anisotropy and ceramide levels is investigate the behavior of ceramide-rich rafts in cells. We plan direct, as anisotropy values returned to higher values in par- to undertake studies both of their lipid composition, protein allel with the reconversion of ceramide to SM (36). Our anisot- composition, and physical behavior by examining the behavior ropy data shows a similar relationship between DPH anisot- of detergent-resistant membranes in normal and SMase- ropy and ceramide levels. 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Ceramide Selectively Displaces Cholesterol from Ordered Lipid Domains (Rafts)

London, Erwin
Journal of Biological Chemistry – Mar 1, 2004
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 11, Issue of March 12, pp. 9997–10004, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Ceramide Selectively Displaces Cholesterol from Ordered Lipid Domains (Rafts) IMPLICATIONS FOR LIPID RAFT STRUCTURE AND FUNCTION* Received for publication, September 8, 2003, and in revised form, November 14, 2003 Published, JBC Papers in Press, December 29, 2003, DOI 10.1074/jbc.M309992200 Megha and Erwin London‡ From the Department of Biochemistry and Cell Biology and Department of Chemistry, Stony Brook University, State University of New York, Stony Brook, New York 11794-5215 Ceramide is a membrane lipid involved in a number of (3–9). Ceramide-triggered apoptosis is of particular interest crucial biological processes. Recent evidence suggests because it seems to play an important role in the sensitivity of that ceramide is likely to reside and function within tumor cells to chemotherapy and radiotherapy (7, 10, 11) and in lipid rafts; ordered sphingolipid and cholesterol-rich the development of atherosclerosis (12). Ceramide location lipid domains believed to exist within many eukaryotic within lipid rafts is an important factor in ceramide action (3, cell membranes. Using lipid vesicles containing co-exist- 5, 6, 13). Lipid rafts are liquid ordered state membrane do- ing raft domains and disordered fluid domains, we find mains rich in cholesterol and saturated polar lipids (usually that natural and saturated synthetic ceramides displace sphingolipids). They can co-exist with disordered fluid state sterols from rafts. Other raft lipids remain raft-associ- membrane domains rich in unsaturated lipids (14, 15). Ceram- ated in the presence of ceramide, showing displacement ide both stabilizes and associates strongly with lipid rafts (16, is relatively specific for sterols. Like cholesterol-con- 17). It can also induce the formation of unusually large raft taining rafts, ceramide-rich “rafts” remain in a highly domains (“platforms”) in plasma membranes (3). ordered state. Comparison of the sterol-displacing abil- In this study, we found that natural ceramides or ceramides ities of natural ceramides with those of saturated dig- with long saturated acyl chains, and analogous diglycerides, lycerides and an unsaturated ceramide demonstrates efficiently displace cholesterol from rafts in model membranes. that tight lipid packing is critical for sterol displace- Other raft-associating molecules were not displaced from rafts ment by ceramide. Based on these results, and the fact by ceramide or diglyceride. A model for the origin of this dis- that cholesterol and ceramides both have small polar placement phenomenon is proposed. This model may provide headgroups, we propose that ceramides and cholesterol insights into how various molecules, including proteins, partic- compete for association with rafts because of a limited ipate in raft formation. Displacement of sterols from rafts is capacity of raft lipids with large headgroups to accom- modate small headgroup lipids in a manner that pre- likely to have a variety of important consequences for raft vents unfavorable contact between the hydrocarbon function. For example, displacement of cholesterol from rafts groups of the small headgroup lipids and the surround- can explain the displacement of cholesterol from plasma mem- ing aqueous environment. Minimizing the exposure of branes upon ceramide generation, and may have implications cholesterol and ceramide to water may be a strong driv- for the association of cholesterol-bound proteins with rafts. ing force for the association of other molecules with EXPERIMENTAL PROCEDURES rafts. Furthermore, displacement of sterol from rafts by ceramide is very likely to have marked effects upon raft Materials and Sample Preparation—Unlabeled lipids (including sphingosine-based ceramides) were purchased from Avanti Polar Lipids structure and function, altering liquid ordered proper- (Alabaster, AL). Dehydroergosterol (DHE) and 1,6-diphenyl-1,3,5- ties as well as molecular composition. In this regard, hexatriene (DPH) were purchased from Sigma-Aldrich. 1-palmitoyl-2- certain previously observed physiological processes (3-(diphenylhexatrienyl)propanoyl)-phosphatidylcholine (DPH-PC) was may be a result of displacement. In particular, a direct purchased from Molecular Probes (Eugene, OR). 22-(Diphenylhexatrie- connection to the previously observed sphingomyeli- nyl)docosyltrimethylammonium, (long chain TMADPH, LcTMADPH) nase-induced displacement of cholesterol from plasma was a kind gift from G. Duportail and D. Heissler (Universite ´ Louis membranes in cells is proposed. Pasteur, Strasbourg). Lipids and probes were stored dissolved in etha- nol at 20 °C. Concentrations were determined by dry weight. Radio- labeled dioleoylphosphatidylcholine (DOPC) and dipalmitoylphoshati- dylcholine (DPPC) were purchased from Amersham Biosciences Ceramide is a membrane lipid involved in many biological (Piscataway, NJ). Radiolabeled palmitoyl (C16:0) ceramide was pur- processes. Sphingomyelinase action can result in a rapid in- chased from American Radiolabeled Chemicals (St. Louis, MO). Lipid crease in ceramide levels in which a large fraction of cellular (including radiolabeled lipid) purity was confirmed by TLC. Acetyl- sphingomyelin is converted to ceramide (1, 2). Sudden in- K W L AL W K -amide (LW peptide) was purchased from Invitrogen 2 2 8 8 2 2 creases in ceramide levels due to the action of sphingomyeli- (Carlsbad, CA) and purified as described previously (18). nase upon the plasma membrane have important effects upon Multilamellar vesicles (MLV) were prepared at a concentration of bacterial pathogenesis, cholesterol homeostasis, and apoptosis The abbreviations used are: DHE, dehydroergosterol; PBS, phos- * This work was supported by National Institutes of Health Grant phate-buffered saline; SM, sphingomyelin; cer, ceramide; DRM, de- GM 48596. The costs of publication of this article were defrayed in part tergent-resistant membrane; DPH, 1,6-diphenyl-1,3,5-hexatriene; by the payment of page charges. This article must therefore be hereby DOPC, dioleoylphosphatidylcholine; DPPC, dipalmitoylphoshatidylcho- marked “advertisement” in accordance with 18 U.S.C. Section 1734 line; DPH-PC, 1-palmitoyl-2-(diphenylhexatrienyl)-propanoyl-phospha- solely to indicate this fact. tidylcholine; LcTMADPH, 22-(diphenylhexatrienyl)docosyltrimethyl- ‡ To whom correspondence should be addressed. Tel.: 631-632-8564; ammonium; MLV, multilamellar vesicles; 12 SLPC, 1-palmitoyl-2-(12- Fax: 631-632-8575; E-mail: [email protected]. doxyl)-stearoyl-phosphatidylcholine; SUV, small unilamellar vesicles. This paper is available on line at http://www.jbc.org 9997 This is an Open Access article under the CC BY license. 9998 Ceramide and Implications for Raft Structure 500 M lipid in PBS (10 mM sodium phosphate, 150 mM NaCl, pH 7) as lipid dioleoylphosphatidylcholine (DOPC). Previous studies described previously (19). Dried lipid mixtures were dispersed in buffer have shown that such vesicles form bilayers containing co- at 70 °C using a multitube vortexer for 15 min and cooled to 25 °C. existing ordered “raft” domains rich in DPPC and sterol and Small unilamellar vesicles (SUV) at a concentration of 50 M lipid in non-raft regions rich in DOPC (19 –22). In the first series of PBS were prepared by ethanol dilution as described previously (20). experiments, the insolubility of ordered domains in Triton Lipids mixed in ethanol were diluted slightly more than 50-fold in PBS buffer heated to 70 °C, briefly vortexed, incubated at 70 °C for about 5 X-100 at 23 °C was used to evaluate raft association. A DRM min, re-vortexed, and cooled to 25 °C. pellet was isolated by centrifugation, and then the amount of Fluorescence Measurements—Fluorescence was measured on a SPEX the molecule of interest that was DRM-associated was meas- Fluorolog 3. Excitation and emission wavelength sets used (in nm) ured (19, 23, 24). This value was normalized to the fraction of were: (358, 427) for DPH and LcTMADPH, (358, 436) for DPH-PC, (324, total lipid that was detergent insoluble, i.e. the fraction of total 376) for DHE, and (280, 340) for LW peptide. Fluorescence intensity in background samples lacking fluorophore was subtracted when signifi- lipid found in DRM. Using multilamellar vesicles, which scat- cant. Unless otherwise noted fluorophore concentrations were 0.1 mol% ter much more light than detergent-solubilized material, the for LcTMADPH and DPH-PC and 1.5 mol% for LW peptide fraction of undissolved lipid could be estimated from the % Insolubility Assay of Raft Content—The detergent-resistant mem- optical density (%OD) remaining after addition of Triton X-100. brane (DRM) were isolated by insolubility at 23 °C as described previ- Although detergent insolubility of lipids is sensitive to a num- ously (19). The DRM fraction from a 1-ml MLV sample was collected ber of variables (25, 26) previous studies have shown that after2hof incubation in 0.5%(w/v) Triton X-100 by pelleting at 14,000 rpm for 15 min in an Eppendorf 5415C tabletop centrifuge. The fluo- under the conditions in our experiments the level of detergent- rescence of 970 –990 l of supernatant and of the pellet suspended in insolubility reflect the amount of lipids and other molecules 975 l of PBS were compared. For calculating % pelleting corrections associated with ordered domains reasonably well (19, 20, 27). were made for differences in fluorophore intensity (quantum yield) in Detergent insolubility was measured for radiolabeled DPPC, lipid and Triton X-100. Radioactive tracers were used at 0.02– 0.1 Ci DOPC, C16:0 ceramide, and cholesterol, and also for DPH-PC, per sample. Average values and the range for duplicate experiments were calculated. (In most cases, for these and the fluorescence studies a fluorescently labeled phosphatidylcholine, DHE, a fluores- described below, additional experiments that are not reported gave cent sterol with properties similar to those of cholesterol (28), similar results.) and LcTMADPH, a derivative of DPH attached to a trimethyl- Fluorescence Quenching Assays—Quenching of fluorophores incorpo- amino-terminated C22:0 hydrocarbon chain (29). Previous rated into SUV was measured at 23 °C as described previously (17, 20). studies have shown that the affinity for ordered domains is Fluorescence (F) of samples containing 12SLPC was normalized (F )to highest for LcTMADPH, relatively high in the case of DPPC, that in samples in which DOPC replaced 12SLPC. Control experiments confirmed that quenching of DHE was not affected by the length of time ceramide and cholesterol, lower but still significant for that samples were incubated after preparation, or by whether MLV or DPH-PC, and least for DOPC (16, 17, 21, 22, 24, 29). In rea- SUV samples were prepared. Average values and the range for dupli- sonable agreement with this order, Fig. 1, A and B show that in cate experiments were calculated. The temperature dependence of the absence of ceramide relative association with DRM (given quenching was used to evaluate the thermal stability of rafts as de- by the ratio % pelleting/% OD) decreased in the order scribed previously (17, 19). Samples containing 0.3 mol% LcTMADPH were heated at about 3 °C per min, and fluorescence periodically LcTMADPHceramideDPPCcholesterolDHEDPH- measured during heating. Average values for duplicate experiments PCDOPC. These measurements were repeated using samples were calculated. in which almost half of the unlabeled DPPC was replaced by an Polarization Measurements—Anisotropy measurements were made equimolar amount of stearoyl (C18:0) ceramide. Fig. 1, A and B at 23 °C using a SPEX Glan-Thompson automated polarizer accessory. show that in the presence of C18:0 ceramide both LcTMADPH SUV samples were prepared as described above except that 1mol% and C16:0 ceramide remained strongly associated with DRM, DPH or LcTMADPH was used. Background intensities were negligible. Average values and the range for duplicate experiments were while DPPC association with DRM actually increased. In con- calculated. trast, the presence of ceramide resulted in the strong displace- Measurement of DHE to LcTMADPH Energy Transfer—SUV sam- ment of both cholesterol and DHE from DRM. (In a control ples were prepared with either LcTMADPH or with LcTMADPH plus experiment, it was found that C16:0 ceramide displaced cho- DHE. Samples containing DHE were composed of 12:12:61:7.5:7.5 (mol: lesterol at least as well as C18:0 ceramide (data not shown).) mol) DPPC:cer:DOPC:DHE:cholesterol (F )or 85:7.5:7.5 DOPC:D- raft HE:cholesterol (F ). Samples lacking DHE were composed of 12: DPH-PC was moderately displaced from the DRM by ceramide. homog 12:61:15 (mol:mol) DPPC:cer:DOPC:cholesterol (F ) or 85:15 DOPC: raft Fig. 1, C and D show that both cholesterol and DHE exhibited cholesterol (F ). Sensitized LcTMADPH emission was measured at homog a progressive displacement as ceramide concentration was in- excitation 324 nm and emission 427 nm. Energy transfer was measured creased. Sterol displacement did not parallel the ceramide de- from the fractional increase of LcTMADPH emission in the presence of pendence of the overall fraction of lipids within DRM, which DHE. Fractional increase in LcTMADPH fluorescence is given by ([F / tended to remain relatively constant as judged by % OD. F ]  1) and the relative fractional increase is given by ([F /F ]  1)/([F /F ]  1). raft raft homog homog Detection of Displacement of Sterol from Rafts by Ceramide No correction was made for quenching of DHE by LcTMADPH when using Fluorescence Quenching—DRM isolated by use of Triton corrections for background fluorescence (from samples lacking LcT- X-100 can overestimate or underestimate raft levels, and % OD MADPH) were made. We estimate correction for this would increase the is a crude measure of total raft levels (25, 26). For these enhancement of LcTMADPH fluorescence we measured by a factor of reasons, studies were also carried out using fluorescence one-quarter to one-third under conditions in which the bilayer was homogeneous. This should have little effect on the results obtained quenching. Fluorescent probes were incorporated into vesicles because the correction would be the same, and thus cancel out, for containing both raft-forming lipids and 1-palmitoyl 2-(12-doxy- samples with and without DPPC/ceramide under conditions in which l)stearoyl PC (12SLPC), a fluorescence quenching lipid that, the bilayer was homogeneous. Average values from duplicates like DOPC, is relatively excluded from rafts (20). 12SLPC is a were calculated. short-range quencher that has to be in near contact with a RESULTS fluorescent molecule in order to quench its fluorescence (30, 31). When rafts and non-raft regions co-exist in 12SLPC-con- Detection of Displacement of Sterol from Rafts by Ceramide using a Detergent Insolubility Assay—The effect of ceramide taining vesicles the intensity of fluorescence from a membrane- bound probe depends on whether it associates with rafts or not. upon association of various molecules with ordered lipid do- mains was measured starting with model membrane vesicles In mixtures of DPPC (or sphingomyelin) with cholesterol and containing sterol and a 1:1 mixture of the saturated lipid di- 12SLPC, rafts have a low % of 12SLPC while the remainder of palmitoylphosphatidylcholine (DPPC) and the unsaturated the bilayer, which is in a disordered fluid state, is enriched in Ceramide and Implications for Raft Structure 9999 FIG.1. Ceramide-induced displace- ment of molecules from rafts assayed by insolubility in Triton X-100 at 23 °C. Samples lacking ceramide were composed of 1:1:0.35 (mol:mol) DPPC: DOPC:sterol dispersed in PBS. In sam- ples containing cer, DPPC was replaced by equivalent mole amounts of cer. Mol% cer shown is always relative to the total lipid concentration. Sterol was cholesterol except in samples containing DHE in which a 1:1 mol:mol cholesterol:DHE mix- ture was used. A and B, normalized con- tent of fluorescent probes (A) or radioac- tive lipids (B) in detergent-insoluble fraction in absence (open bars) or pres- ence (shaded bars) of 18 mol% C 18:0 cer. Abbreviations: DP, DPPC, DO, DOPC, Ch, cholesterol. C and D, effect of ceram- ide concentration upon displacement of DHE (C)or[ H]cholesterol (D) from the detergent-insoluble fraction. Symbols: squares, % OD remaining after detergent addition; triangles, % insoluble radioac- tivity or fluorescence; circles, % pellet/% OD. The % pellet/% OD equals the ratio of the % of the radioactive tracer or fluores- cent probe in the Triton X-100 insoluble pellet fraction to the % of unsolubilized lipid as judged by OD at 400 nm. The average and range of duplicate samples are shown in this and most of the follow- ing figures. In most cases, additional ex- periments that are not shown gave simi- lar results. 12SLPC (19, 20, 32). A molecule that associates with rafts cence showed a much smaller increase in quenching as ceram- fluoresces relatively strongly, because there is a good chance it ide concentration was increased, indicating a more modest will not be next to a 12SLPC molecule. A molecule that remains extent of displacement. Overall, the fluorescence quenching in the non-raft regions of the bilayer will fluoresce weakly experiments are in agreement with those based upon because the chance it will be next to a 12SLPC is high. There- detergent insolubility. fore, the level of quenching will reflect the degree of association Utilizing quenching, ceramide-induced displacement of ste- with rafts (18, 20). rol from rafts was also observed when brain sphingomyelin Fig. 2A shows the ceramide dependence of quenching of the (SM) and a brain ceramide mixture were substituted for DPPC fluorescence of DHE, LcTMADPH, DPH-PC, and LW peptide and C18:0 ceramide, respectively (Fig. 2B). This shows that (acetyl-K W L AL W K -amide), a synthetic transmembrane displacement is similar for analogous chemically defined and 2 2 8 8 2 2 polypeptide that does not associate with lipid rafts (18). The natural lipid mixtures. Comparison of the ability of different y-axis gives the difference between fluorescence in raft-contain- sphingolipids to displace sterol (Fig. 2C) showed that partial ing samples and that in control samples forming homogeneous substitution of SM for DPPC had no effect on quenching of bilayers, i.e. lacking rafts. (Without subtraction of control val- DHE fluorescence, whereas partial substitution of a cerebro- ues, the inherently different quenching sensitivity of different side mixture for DPPC increased quenching, but to a much types of fluorescent groups would partially obscure differences lesser degree than substitution of DPPC by C18:0 ceramide. between their raft association.) In raft-containing samples The behavior of cerebroside-containing mixtures suggests that lacking ceramide (i.e. 0% ceramide values in Fig. 2A), fluores- sterols associate with cerebroside-rich rafts to much higher cence was most weakly quenched relative to the homogeneous degree than to ceramide-rich rafts but to a lesser degree than control samples for LcTMADPH, consistent with the strong those formed by SM or DPPC, consistent with previous association of LcTMADPH with lipid rafts. In contrast, the Trp studies (17). Comparison of Diglyceride and Ceramide Behavior—The ef- fluorescence of the peptide was more strongly quenched in raft-containing samples than in the control samples. This re- fect of ceramide and diglyceride on raft stability and sterol displacement were compared. Both of these lipids contain two flects the relative exclusion of LW peptide from rafts, as found previously (18). Quenching levels were intermediate for hydrocarbon chains and relatively small polar headgroups, but they differ in the details of their polar headgroup structure. In DPH-PC and DHE, showing that they associate with rafts to a significant degree. one experiment, their effects on raft stability were compared when added to mixtures of DPPC, 12SLPC, cholesterol, and Partial substitution of DPPC with ceramide had little effect on quenching of LcTMADPH and peptide fluorescence, showing LcTMADPH. As noted above, when rafts are present they bind LcTMADPH so that its fluorescence is only weakly quenched the locations of these molecules was not greatly altered by ceramide. (The small changes observed in their quenching at by 12SLPC (17, 20). However, at temperatures above that at which rafts are stable all lipids mix in a relatively homogene- high ceramide concentrations may reflect a small change in raft association or ceramide-induced displacement of some of ous fashion, and thus quenching increases because LcT- MADPH molecules come into increased contact with 12SLPC the residual raft-associated 12SLPC from rafts.) In contrast, as ceramide concentrations increased a large and progressive in- molecules. The temperature dependence of quenching can be used to monitor the thermal stability of rafts. The midpoint of crease in the quenching of DHE fluorescence was observed, confirming DHE displacement from rafts. DPH-PC fluores- a quenching versus temperature curve (raft “melting” temper- 10000 Ceramide and Implications for Raft Structure whereas C18:1 ceramide only induced weak displacement rel- ative to C18:0 ceramide. Together these results suggest that acyl chain saturation is a more important factor than the details of the polar headgroup structure for both raft stabiliza- tion and sterol displacement. (Whether natural diglycerides would affect raft structure or formation is unclear. Preliminary studies indicate that 1-palmitoyl 2-oleoyl glycerol can displace cholesterol from rafts, although to a lesser extent than dipalmitoylglycerol. ) Ceramide-rich Domains Are Highly Ordered—Fluorescence anisotropy measurements were made on samples containing LcTMADPH and DPH incorporated into various lipid bilayers to probe the effect of ceramide upon lipid order (Fig. 4A). As expected, for both probes anisotropy values were high in vesi- cles composed solely of lipids forming ordered states (DPPC or DPPC/cholesterol) and low in vesicles composed of lipids exist- ing in the disordered liquid state (DOPC or DOPC/cholesterol). In vesicles composed of DPPC, DOPC, and cholesterol mixtures under conditions in which both ordered and disordered do- mains co-exist (see above) Fig. 4A shows anisotropy of LcT- MADPH fluorescence remained as high as that in fully ordered bilayers regardless of ceramide concentration. Since LcT- MADPH partitions strongly into ordered phases this indicates that the more ordered domains remain highly ordered in the presence of ceramide. DPH, which partitions into both ordered and disordered phases, gave fluorescence with intermediate anisotropy values in vesicles containing both ordered and dis- ordered states. There was a decrease in DPH anisotropy in the presence of ceramide. This decrease can be explained by partial displacement of DPH from ceramide-rich ordered domains. Partial displacement of DPH by ceramide was confirmed by quenching experiments (data not shown). Effect of Sterol Concentration upon Displacement of Sterol from Rafts by Ceramide and upon Displacement of Ceramide from Rafts by Sterol—Because sterol levels in the plasma mem- brane are high, the effect of cholesterol concentration upon displacement by ceramide was assessed (Fig. 4B). Even at a concentration as high as 40 mol% cholesterol, the presence of 18 mol% ceramide was able to displace the great majority of cholesterol from rafts. We were also interested to see if sterol could displace ceramide from rafts. Fig. 4C shows that choles- terol can displace some C16:0 ceramide from ordered domains both when ceramide is present in trace amounts (open bars)or when it forms a large (18 mol%) fraction of the lipid bilayer (closed bars). This indicates sterol and ceramide are competing FIG.2. Ceramide-induced displacement of molecules from for association with rafts. However, displacement of ceramide rafts at 23 °C assayed by fluorescence quenching. A, effect of by cholesterol was much less effective than displacement of C18:0 cer concentration upon quenching of various fluorophores. Sam- ples contained 1:1:0.35 (mol:mol) [DPPCcer]:[12SLPC or DOPC]:s- cholesterol by ceramide. For example, upon introduction of 25 terol dispersed in PBS. Sterol was cholesterol except in samples con- mol% cholesterol into the model membrane vesicles, ceramide taining DHE in which a 1:1 cholesterol:DHE mixture was used. concentrations in the ordered domains only decreased by one- Symbols: triangles, LcTMADPH; squares, DPH-PC; diamonds, LW pep- third when samples contained 18 mol% C18:0 ceramide. This tide; circles, DHE. B, effect of sphingolipids upon quenching of DHE. Samples contained 1:1:0.35 (mol:mol) [DPPCsphingolipid]:[12SLPC should be contrasted to the roughly 4-fold decrease of choles- or DOPC]:DHE dispersed in PBS. Symbols: squares, SM; triangles, terol concentration within ordered domains induced by the mixed cerebrosides; circles, C18:0 ceramide. C, comparison of displace- introduction of 18 mol% ceramide into bilayers containing 25 ment using natural and synthetic lipids. squares, 1:1:0.35 [brain mol% cholesterol (Fig. 4C). The observation that ceramide dis- SMbrain cer]:[12SLPC or DOPC]:DHE; circles, 1:1:0.35, places cholesterol more efficiently than cholesterol displaces [DPPCC18:0 cer]:[12SLPC or DOPC]:DHE. ceramide confirms that ceramide has a much stronger affinity ature) is a measure of raft stability (17, 19). Fig. 3, A and B for ordered domains than cholesterol. show that substitution of 4 mol%, 9 mol%, or 18 mol% DPPC Energy Transfer Analysis of Domain Organization—To con- with an equimolar amount of either C18:0 ceramide or 1,2- firm that sterol displacement was not a sample preparation dipalmitoylglycerol-stabilized rafts. The degree of stabilization artifact, we used energy transfer to examine whether sterol by the C18:0 ceramide and dipalmitoylglycerol was similar. In and raft-forming lipids were present in the same vesicles. In contrast, unsaturated oleoyl (C18:1) ceramide decreased raft homogeneous control bilayers containing DOPC, sterol, and stability (Fig. 3A). We next compared the ability of dipalmitoyl- LcTMADPH, energy transfer from DHE to LcTMADPH signif- glycerol and ceramide to displace sterols from rafts. As shown in Fig. 3, C and D dipalmitoylglycerol was able to displace sterol from rafts even more efficiently than C18:0 ceramide, Megha and E. London, unpublished observations. Ceramide and Implications for Raft Structure 10001 FIG.3. Effect of different ceramides and diglyceride upon ordered domain stability and sterol displacement from rafts. A and B, quenching assay of raft stability. The temperature dependence of quenching of the fluorescence of LcTMADPH was measured in samples composed of A, 1:1:0.35 [DPPCcer]:[12SLPC or DOPC]:cholesterol dispersed in PBS. Samples contained 0% (diamonds), 4% (squares), 9% (filled triangles), or 18 mol% C18:0 cer (filled circles), or contained 9% (open triangles) or 18% C18:1 cer (open circles). B, 1:1:0.35 [DPPCdiglyceride]:[12SLPC or DOPC]:cholesterol dispersed in PBS. Samples contained: 0% (diamonds), 4% (squares), 9% (triangles), or 18 mol % dipalmitoylglycerol (circles). C and D, comparison of sterol displacement from rafts by ceramide and diglyceride at 23 °C. C, insolubility in Triton X-100 assay of displacement of radioactive cholesterol from rafts by C18:0 cer (circles), or dipalmitoylglycerol (squares). Samples composed of 1:1:0.35 [DPPC(cer or diglyceride)]: DOPC:cholesterol dispersed in PBS. D, fluorescence quenching assay of displacement of DHE by ceramide and diglyceride. Displacement of DHE from rafts by C18:0 cer (circles), C18:1 cer (triangles), or dipalmitoylglycerol (squares). Samples composed of 1:1:0.175:0.175 [DPPC(cer or diglyceride)]:[12SLPC or DOPC]:DHE:cholesterol dispersed in PBS. DAG, diglyceride. icantly enhanced LcTMADPH fluorescence. The increase in may be also energetically driven by the fact that the small polar LcTMADPH emission in the presence of DHE was somewhat headgroup (the OH) of cholesterol is insufficient to shield the temperature dependent, decreasing from a 42% increase at sterol rings from water, and tight packing with other lipids 20 °C to a 26% increase at 45 °C. At low temperatures the allows the sterol rings to hide under an “umbrella” formed by increase in LcTMADPH fluorescence in bilayers containing lipids with large polar headgroups (33). Because ceramides also DPPC and ceramide was much less than that in the control have a small polar headgroup we propose a similar phenome- samples (Fig. 4D). This was expected because, as shown by the non applies to them, and that the limited ability of normal studies above, LcTMADPH should be located in the DPPC/ polar lipids to simultaneously shield hydrocarbon groups on ceramide-rich rafts while the displaced DHE should be located both ceramides and cholesterol from water results in a compe- in disordered fluid domains. However, upon melting of the rafts tition between ceramide and sterol for inclusion in ordered at high temperature, energy transfer approached levels ob- domains. An analogous model has been proposed to explain the served in control samples lacking raft-forming lipids. This ability of ceramide in disordered fluid domains to increase the should not have been observed if there was one set of vesicles binding of cholesterol to a cytolysin protein (46). The observa- containing raft-forming lipids and LcTMADPH and a separate tion that DPH-PC, which has a large polar headgroup (but also population containing DHE. Instead, this indicates that the a rather bulky fluorophore-labeled acyl chain, was partially raft-forming lipids and DHE were in a single set of vesicles. displaced from rafts by ceramide suggests the possibility that Additional experiments showed that the raft melting tem- especially tight packing of hydrocarbon chains in ceramide- perature detected by energy transfer was close to that detected containing ordered domains relative to those containing satu- by 12SLPC quenching of LcTMADPH fluorescence (Fig. 4D), rated lipid and cholesterol can also contribute to displacement indicating that the lipid composition of the bulk sample and the by squeezing out molecules that cannot pack as well as ceram- vesicles containing DHE was similar. If the DHE had been in a ide. On the other hand, hydrogen bonding appears to be less different subset of vesicles than the raft-forming lipids, energy important for displacement. Diglycerides have a very different transfer should have shown a very different thermal depend- set of hydrogen bonding groups than do ceramide, and yet a ence than that exhibited by quenching of LcTMADPH. saturated diglyceride displaced sterol more effectively than a DISCUSSION ceramide with a similar hydrocarbon chain structure. An alternative explanation of displacement is that ceramide Origin of the Displacement of Sterol from Ordered Domains forms cholesterol-poor ordered domains that are separate from by Ceramide—What is the origin of sterol displacement from rafts by ceramide? Both cholesterol and natural ceramides the ordered domains formed by DPPC (or SM). When ceramide is substituted for DPPC there would be a lower amount of the have hydrocarbon structures allowing tight packing with phos- pholipids and/or sphingolipids. Tight packing involving sterols DPPC-rich ordered domains into which sterol could incorpo- 10002 Ceramide and Implications for Raft Structure FIG.4. Assay of vesicle properties in the presence of cer. A, effect of inclusion of cer in rafts upon fluorescence anisotropy of 1 mol% DPH (black bars) or 1 mol% LcTMADPH (striped bars)at23 °C. Ceramide-containing samples were composed of 1:1:0.35 [DPPCcer]:DOPC:cholesterol dispersed in PBS. For calibration purposes, anisotropy values in vesicles in the liquid disordered (DOPC  15 mol % cholesterol), and gel (DPPC) states at 23 °C are shown. Also shown: DPPC  15 mol % cholesterol. Abbreviations: DP, DPPC; DO, DOPC; Ch, cholesterol. B, effect of cholesterol concentration on its displacement by C18:0 cer measured by insolubility in Triton X-100 at 23 °C. Samples composed of 1:1 [DPPCcer]:DOPC with various mol fractions of cholesterol. Shaded bars, 18 mol% cer; open bars, 0% cer. C, displacement of [ H]C16:0 ceramide from rafts as a function of cholesterol concentration as measured by insolubility in Triton X-100. Shaded bars, 18 mol% C18:0 cer; open bars, 0% C18:0 cer. D, comparison of the effect of temperature upon energy transfer from DHE to LcTMADPH and upon DHE quenching. Squares, the ratio of the fractional increase in LcTMADPH fluorescence due to energy transfer from DHE in vesicles composed of 12:12:61:7.5:7.5 (mol:mol) DPPC:cer:DOPC:DHE:cholesterol relative to the fractional increase in energy transfer in vesicles composed of 85:7.5:7.5 DOPC:DHE:cholesterol. Triangles, ratio of LcTMADPH fluorescence (F) in vesicles composed of 12:12:30.5:30.5:15 (mol:mol) DPPC:cer:DOPC:12SLPC:cholesterol to fluorescence (F ) of vesicles composed of 12:12:61:15 (mol:mol) DPPC:cer:DOPC:cholesterol. Samples also contained 1 mol % LcTMADPH. Vesicles were dispersed in PBS. rate. The observations that increasing ceramide concentration levels reach 18 mol%. These values are comparable to those induces only a gradual increase in raft melting temperature, reached in the plasma membrane under physiological condi- and that melting remains a single step process, argue against tions. In cases of bacterial infections, viral infection and tumor multiple types of ordered domains. A separate ceramide-rich necrosis factor action it has been found that 40% of all-cellular phase would likely give rise to a separate melting event at a SM is digested by ceramide-producing cellular SMase in as distinct temperature. Furthermore, inclusion of ceramide in- little as a few minutes (1, 34, 35). It has also been shown that creased the fraction of DPPC associated with ordered domains for each molecule of SM digested roughly one molecule of cer- while displacing sterol (Fig. 1B). This would not be expected if amide appears (2, 34). Thus, we can approximate ceramide ceramide and DPPC formed separate ordered domains, but levels in the plasma membrane. If we estimate that the plasma would if ceramide and DPPC interact in a single type of mixed membrane lipid is typically 15% SM, and that almost all SM is ordered domain. Finally, the partial displacement of ceramide in the outer leaflet of the membrane, then about 30% of the from ordered domains by cholesterol is much easier to ration- outer leaflet of the plasma membrane is SM. In that case 40% alize if ceramide and cholesterol are competing for inclusion in digestion by SMase means that 12% of the plasma membrane a single type of ordered domain. outer leaflet lipid is converted to ceramide. This number is only Physiologically Relevant Ceramide Levels Displace Choles- a lower estimate because it ignores the existence of SM pools in terol from Plasma Membranes in Cells and Are Equivalent to internal membranes [lysosomes, endosomes, Golgi]. If one-half Values Sufficient to Displace Cholesterol from Rafts—Our data of cellular SM is in internal membranes, and thus insensitive show that displacement of cholesterol from rafts is significant to SMase, then there would be 80% conversion of plasma mem- (roughly 50%) when cer comprises 9 mol% of the total bilayer brane SM to ceramide by SMase. This would double the value lipid and that displacement can be nearly complete when cer for ceramide levels produced in the outer leaflet to 24 mol%. Ceramide and Implications for Raft Structure 10003 Several other studies have shown that SMase digestion also of the stratum corneum of skin, which are primarily composed leads to displacement of cholesterol from the plasma mem- of a cholesterol/ceramide mixture (44). The tendency of both brane (1, 2, 4, 9, 36). There appears to be a direct relationship cholesterol and ceramide to avoid excessive hydration presum- between these events. The rate of cholesterol displacement ably contributes functionally to the action of the stratum cor- neum as a barrier to water. from the plasma membrane follows the rate of SM digestion without a significant time lag, and plasma membrane choles- The tendency of cholesterol and ceramide to avoid exposure to water also might be a driving force for the association of terol levels recover at the same rate that SM is resynthesized other types of molecules with rafts. It could contribute, along from ceramide (2, 36). Even the step in which internalized with saturated acyl chain packing interactions, to the raft- cholesterol is esterified follows ceramide generation within association of molecules that can cover the bilayer surface, such minutes (1, 2, 9). as carbohydrate anchors of GPI-anchored proteins, or glyco- Detailed data about the relationship between ceramide gen- sphingolipids with large headgroups. It could also contribute to eration and plasma membrane cholesterol levels was very re- the ability of protein domains that can cover hydrophobic sites cently presented by Al-Makdissy et al. (36). These investigators on the bilayer surface to bind to rafts (or to any membranes directly assayed both SM and cholesterol levels in isolated with high cholesterol or ceramide levels). plasma membrane. Digestion of 25% of plasma membrane SM In this regard, it should be noted that such interactions led to a loss of 50% of plasma membrane cholesterol, similar to should be enhanced at high cholesterol levels. It has been what we would predict based on the data in this report. In proposed that the ability of phospholipids to form a complex addition, the authors measured DPH anisotropy to measure with cholesterol (45) or to maintain a spatial distribution that membrane fluidity. They found DPH anisotropy dropped when prevents contact of cholesterol hydrocarbon with water (33) ceramide was produced. In other words, DPH found itself in a would become overwhelmed at high cholesterol concentrations. more disordered lipid environment upon ceramide generation. For all these reasons, it will be important in future studies to The connection between DPH anisotropy and ceramide levels is investigate the behavior of ceramide-rich rafts in cells. We plan direct, as anisotropy values returned to higher values in par- to undertake studies both of their lipid composition, protein allel with the reconversion of ceramide to SM (36). Our anisot- composition, and physical behavior by examining the behavior ropy data shows a similar relationship between DPH anisot- of detergent-resistant membranes in normal and SMase- ropy and ceramide levels. 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