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Triton X-100-Resistant Bilayers: Effect of Lipid Composition and Relevance to the Raft Phenomenon Jesu´s Sot, M. Isabel Collado, Jose´ L. R. Arrondo, Alicia Alonso, and Fe´lix M. Gon˜i* Unidad de Biofı´sica (CSIC-UPV/EHU), and Departamento de Bioquı´mica, Universidad del Paı´s Vasco, Aptdo. 644, 48080 Bilbao, Spain Received September 3, 2001. In Final Form: January 2, 2002 The molecular basis for the existence of the so-called “detergent-resistant membranes” has been explored. With that aim, vesicles composed of phosphatidylcholine, sphingolipid, and cholesterol were treated with the nonionic detergent Triton X-100 either at 4 °C or at 37 °C and tested for solubilization using turbidity and centrifugation methods. Bilayer fluidity was systematically measured as fluorescence anisotropy of a diphenylhexatriene derivative of phosphatidylcholine. Putative sphingomyelin-cholesterol interactions were explored using IR spectroscopy. The combined experimental evidence clearly indicates that these lipid mixtures are solubilized more easily at 4 °C than at 37 °C, that an increased membrane fluidity does not correlate with an easier solubilization, and that sphingomyelin-cholesterol interactions are essential for insolubility. Sphingolipids by themselves do not hinder detergent solubilization, and some of them, e.g., gangliosides, actually increase bilayer solubility in the presence of detergents. At least with some lipid compositions, there is a range of detergent concentrations at which partial solubilization occurs concomitantly with major changes in bilayer architecture (lysis and reassembly). Moreover, a nonsolubilized residue of composition phosphatidylcholine/sphingomyelin/cholesterol of ca. 1:1:1 (mole ratio) is recovered by centrifugation after detergent treatment of vesicles with very different original lipid compositions. These observations do not preclude the presence of liquid-ordered domains in the cell membrane but support the idea that the detergent-resistant membranes obtained after detergent treatments may well be the result of bilayer partial solubilization and reassembly, instead of corresponding precisely to structures preexisting in the cell membrane.
Introduction Membrane proteins that are anchored to the lipid bilayer through glycosyl-phosphatidylinositol (GPI) have been known for years to be resistant to solubilization by certain detergents, such as Triton X-100.1 Further studies have shown that detergent insolubility is associated with the presence of both cholesterol and sphingolipids in the membrane.2-4 Moreover, Brown and Rose5 introduced the idea of detergent-insoluble microdomains existing within the lipid bilayer into which domains the GPI anchors would be inserted. Following a different line of thought, Simons and co-workers have proposed the existence of the socalled “rafts”, which are microdomains rich in sphingolipid and cholesterol involved in numerous cellular functions from membrane traffic and cell morphogenesis to cell signaling.6,7 Rafts and detergent-insoluble domains were related at a rather early stage in these studies,8 and at present, both terms are used virtually as synonyms despite their rather different origins and conceptual meanings. The phenomenon of detergent insolubility has been tentatively explained on the basis of a number of physicochemical principles. Since GPI-bound alkaline phos* To whom correspondence should be addressed. Tel: +34-94601.26.25. Fax: +34-94-464.85.00. E-mail:
[email protected]. (1) Hooper, N. M.; Turner, A. J. Biochem. J. 1988, 250, 865-869; Hooper, N. M. Mol. Membr. Biol. 1999, 16, 145-156. (2) Cerneus, D. P.; Ueffing, E.; Posthuma, G.; Strous, G. J.; van der Ende, A. J. Biol. Chem. 1993, 268, 3150-3155. (3) Hanada, K.; Nishijima, M.; Akamatsu, Y.; Pagano, R. E. J. Biol. Chem. 1995, 270, 6254-6260. (4) Schroeder, R. J.; Ahmed, S. N.; Zhu, Y.; London, E.; Brown, D. A. J. Biol. Chem. 1998, 273, 1150-1157. (5) Brown, D. A.; Rose, J. K. Cell 1992, 68, 533-544. (6) Simons, K.; Ikonen, E. Nature 1997, 387, 569-572. (7) Simons, K.; Ikonen, E. Science 2000, 290, 1721-1726. (8) Parton, R. G.; Simons, K. Science 1995, 269, 1398-1399.
phatase was insoluble in Triton X-100 at 0 °C but became soluble at 37 °C,2 it was suggested that the high gel-fluid transition temperature Tc of sphingolipids, as compared to glycerophospholipids, was responsible for the insolubility.9 Later, the origin of detergent-insoluble cell membranes was proposed to reside in the formation of detergent-insoluble, liquid-ordered lipid phases in membranes (see ref 10 for a review). Harder and Simons,11 on the basis of a variety of biophysical studies,12-14 suggested the preferential interaction between sphingolipids and cholesterol as a reason for raft formation and stability. The presence of lipid domains in membranes has been the object of debate for years, although the available evidence appears to confirm their existence even in pure lipid systems.15 However, some of the principles that have been invoked to explain the observation of detergentresistant microdomains seem to be at odds with the available experimental data. Thus, against the presumed insolubility of the high-Tc sphingolipids in Triton X-100 are the observations by Hertz and Barenholz16 that, in PC/SM mixtures, the higher the SM proportion the smaller the detergent concentration required for solubilization. (9) Schroeder, R.; London, E.; Brown, D. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12130-12134. (10) London, E.; Brown, D. A. Biochim. Biophys. Acta 2000, 1508, 182-195. (11) Harder, T.; Simons, K. Curr. Opin. Cell Biol. 1997, 9, 534-542. (12) Smaby, J. M.; Monsen, M.; Kulkarni, V. S.; Brown, R. E. Biochemistry 1996, 35, 5696-5704. (13) Bittman, R.; Kasireddy, C. R.; Mattjus, P.; Slotte, J. P. Biochemistry 1994, 33, 11776-11781. (14) Ferraretto, A.; Pitto, M.; Palestini, P.; Masserini, M. Biochemistry 1997, 36, 9232-9236. (15) Dietrich, C.; Bagatolli, L. A.; Volovyk, Z. N.; Thompson, N. L.; Levi, M.; Jacobson, K.; Gratton, E. Biophys. J. 2001, 80, 1417-1428. (16) Hertz, R.; Barenholz, Y. J. Colloid Interface Sci. 1977, 60, 188195.
10.1021/la011381c CCC: $22.00 © 2002 American Chemical Society Published on Web 02/21/2002
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Also, the presence of glycosphingolipids in detergentresistant membranes does not agree with the spontaneous tendency of many of these compounds to form micelles in water (see, e.g., ref 17). Moreover, despite recent advances,18 the detection of liquid-ordered phases in cell membranes is not easy. In view of these uncertainties, we have performed a series of studies with pure lipids and lipid mixtures under various conditions, with the aim of broadening the basis for detergent insolubility. Thus, our studies on the solubilization by Triton X-100 of different phosphatidylcholines in the gel state19 revealed that the gel state, per se, does not imply insolubility. In fact, in some cases, e.g., dimyristoylphosphatidylcholine, solubilization in the gel state required less detergent than in the liquid crystalline state. Also, with pure egg sphingomyelin (Tc ≈ 40 °C), solubilization in the gel state occurs at lower detergent concentrations than in the fluid state.20 In the same work, we were able to confirm that, in PC/SM mixtures, SM made the system more easily amenable to solubilization by Triton X-100. Also, relevant in the context of detergent-resistant membranes, we found that in liposomes containing SM and cholesterol, cholesterol at higher than 10 mol % gave rise to detergent insolubility, particularly at temperatures above Tc of SM. Finally, an IR spectroscopic examination of SM/Ch liposomes revealed a strong effect of cholesterol on the amide carbonyl group of SM, suggestive of hydrogen bonding between both molecules.20 Very recently, we examined an extensive series of PC/SM and PC/SM/Ch mixtures with spin-labeled PC or SM.21,22 In the binary PC/SM mixtures, SM-rich gel phase domains were seen to coexist with PC-rich fluid domains. In ternary systems, cholesterol appeared to stabilize the coexistence of gel phase and liquid-ordered domains, while PC attenuated the interaction between SM and cholesterol.22 Our present contribution includes solubilization studies of ternary mixtures egg PC/sphingolipid/cholesterol by Triton X-100 at 4 and 37 °C. Solubilization data, obtained from systematic turbidity and centrifugation studies, have been complemented with IR and fluorescence anisotropy measurements. The experimental evidence provides clear indications that the above mixtures are more easily solubilized at 4 °C than at 37 °C, that sphingolipids by themselves do not hinder detergent solubilization, and that sphingomyelin-cholesterol interactions are essential for insolubility. Materials and Methods Triton X-100 (regular, batch 48H0208) was purchased from Sigma (St. Louis, MO). Egg-yolk phosphatidylcholine (containing 34% C 16:0, 32% C 18:1, 18% C 18:2, and 16% others) and eggyolk sphingomyelin (85% C 16:0) were grade I from Lipid Products (South Nutfield, UK). Cholesterol and GM3 ganglioside were from Sigma. PC-DPH was from Molecular Probes (Alabaster, AL); galactosylceramide (cerebroside) was a kind gift from Dr. G. Fidelio (Co´rdoba, Argentina). All lipids were >98% pure as supplied and were used without further purification. All other reagents were of analytical grade. The lipids were dissolved in chloroform and mixed as required, and the solvent was evaporated exhaustively. Large unilamellar (17) Maggio, B. Prog. Biophys. Mol. Biol. 1994, 62, 55-117. (18) Ge, M.; Field, K. A.; Aneja, R.; Holowka, D.; Baird, B.; Freed, J. H. Biophys. J. 1999, 77, 925-933. (19) Patra, S. K.; Alonso, A.; Gon˜i, F. M. Biochim. Biophys. Acta 1998, 1373, 112-118. (20) Patra, S. K.; Alonso, A.; Arrondo, J. L. R.; Gon˜i, F. M. J. Liposome Res. 1999, 9, 247-260. (21) Veiga, M. P.; Alonso, A.; Gon˜i, F. M.; Marsh, D. Biochemistry 2000, 39, 9876-9883. (22) Veiga, M. P.; Arrondo, J. L. R.; Gon˜i, F. M.; Alonso, A.; Marsh, D. Biochemistry 2001, 40, 2614-2622.
Langmuir, Vol. 18, No. 7, 2002 2829 vesicles were prepared by the extrusion method with filters that were 0.1 µm in diameter.23 The final lipid concentration was measured in terms of lipid phosphorus. Vesicle size was estimated by quasi-elastic light scattering using a Malvern Zeta-Sizer spectrometer. Vesicle preparations were homogeneous judging from the polydispersity measurements that were in the 0.050.20 range. The average diameter of the vesicles was in the 120170 nm range with the higher values occurring in the presence of cholesterol. Lipids were hydrated in 10 mM HEPES, 150 mM NaCl, and pH 7.4 buffer. Liposome suspensions were mixed with the same volumes of the appropriate detergent solutions, in the same buffer. Final lipid concentration was always 1 mM. Both liposomes and detergent had been previously equilibrated at the desired temperature. The mixtures were left to equilibrate for 1 h at the appropriate temperature, and solubilization was assessed from the changes in turbidity.24,25 Turbidity was measured as absorbance at 500 nm in a Cary Bio 3 spectrophotometer, equipped with thermoregulated cell holders. Turbidity values were normalized by setting 100% as the turbidity of the LUV suspension, 1 mM in lipid in the absence of detergent, while 0% turbidity corresponded to pure buffer. Under these conditions, D50 corresponds to the total detergent concentration producing a 50% decrease in suspension turbidity. This value is obtained from a plot of supension turbidity vs detergent concentration. Total, rather than “effective”,26 Triton X-100 concentrations have been used for convenience. This simplification is acceptable because lipid concentration is kept constant at 1 mM in all measurements. For IR measurements, the appropriate lipid mixtures were dried and resuspended in 20 mM HEPES buffer, pD 7.4, in D2O buffer to a final lipid concentration of 80 mM in terms of SM. The samples were placed in a Harrick cell (Ossining, NY) equipped with CaF2 windows and 50 µm spacers. Spectra were recorded in a Nicolet Magna II 550 spectrometer, equipped with an MCT detector. A Nicolet Rapid Scan software was used. The samples were heated at a constant rate of 1 °C min-1. At the appropriate temperatures, 304 interferograms were collected and treated to obtain the spectra with a nominal resolution of 2 cm-1. Anisotropy of PC-DPH fluorescence was measured as follows. Lipids and probe were mixed in a ratio of 250:1 in organic solvent; then the solvent was evaporated, and the mixture was vacuumdried for at least 2 h in the dark. The vesicles were prepared in HEPES buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). Fluorescence anisotropy was recorded in a SLM-AMINCO MC200 spectrofluorometer equipped with calcite polarizers. The instrument software computes anisotropies from averages of five measurements for each experimental point, automatically correcting for the G factor. PC-DPH fluorescence was excited at 360 nm; emission was recorded at 430 nm. To avoid light scattering and inner filter effects, fluorescence anisotropy was measured on increasingly diluted samples. Only when anisotropy values remained constant with further dilution were they recorded. Under certain conditions, subsolubilizing detergent concentrations give rise to large multilamellar structures. The latter were separated from unilamellar vesicles and micelles by lowspeed centrifugation (13 000× g, 4 °C, 20 min) in a standard Eppendorff centrifuge. Detergent solubilization of LUV was assayed by centrifugation as follows. Detergent-treated vesicle suspensions were centrifuged at 510 000× g (4 °C, 2 h) in a Beckman TLA 120.2 rotor. Lipid phosphorus27 was assayed in the supernatant and pellet. Solubilization was defined as the percentage of lipid phosphorus in the supernatant. For quantitative analysis of the lipid compositions in nonsolubilized residues, after high-speed centrifugation, the pellets were extracted with chloroform/methanol (2:1). The organic phase was concentrated and separated on thin-layer chromatography Silica Gel 60 plates, using successively the solvents chloroform/methanol/water (60: (23) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161-168. (24) Partearroyo, M. A.; Urbaneja, M. A.; Gon˜i, F. M. FEBS Lett. 1992, 302, 138-140. (25) Gon˜i, F. M.; Alonso, A. Biochim. Biophys. Acta 2000, 1508, 5168. (26) Lichtenberg, D. Biochim. Biophys. Acta 1985, 821, 470-478. (27) Bottcher, C. S. F.; van Gent, C. M.; Fries, C. Anal. Chim. Acta 1961, 1061, 297-303.
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Figure 2. The influence of lipid composition on Triton X-100 solubilization of vesicles composed of PC, SM, and Ch at different PC concentrations. Solubilization is expressed as D50, which is the detergent concentration required to decrease the original suspension turbidity by 50%. All mixtures had the general composition PC/SM/Ch (N:1:1, mole ratio), with N varying from 2.6 to 9.0. Data derived from experiments is as shown in Figure 4. Mean values (S.D. (n ) 3). Measurements were performed at 4 °C (b) or at 37 °C (O). For description of regions I, II, and III, see main text.
Figure 1. The influence of bilayer lipid composition on Triton X-100 solubilization of pure lipid vesicles. Percent vesicle suspension turbidity (% A500) is plotted as a function of detergent concentration. 100% was defined as the turbidity of a surfactantfree vesicle suspension. Solubilization was assessed as a decrease in turbidity. Lipid concentration was 1mM. The experiment was performed at 4 °C (b) or at 37 °C (O). Bilayer lipid composition is written on the upper right angle of each panel. 30:5 by volume) in the same direction, then petroleum ether/ ethyl ether/acetic acid (60:40:1 by volume) for the whole plate. After the organic phase was charred with an H2SO4 reagent, the spot intensities were quantified by comparison with lipid standards with a dual wavelength TLC scanner CS-930 Shimadzu from Shimadzu Corp. Occasionally, lipid phosphorus was determined on scraped chromatographic spots, and the results were in good agreement with the scanner method.
Results Solubilization of PC/SM/Ch Mixtures. Detergentresistant membranes have been described as containing PC, sphingolipid, and cholesterol. In a first series of experiments, the effects of a sphingolipid (egg sphingomyelin) and cholesterol on the solubility of bilayers, composed basically of egg PC, were tested. Egg PC bilayers are completely solubilized by Triton X-100 at detergent/ lipid ratios close to 2:1.28 In our experiments, solubilization was assayed by the turbidity method, with separate sets of samples incubated and measured either at 4 °C or at 37 °C. Results from representative experiments are shown in Figure 1. The PC/Ch (4:1 mole ratio) mixture was easily solubilized at 4 °C but required higher detergent concentrations at 37 °C (Figure 1A). D50, the total detergent concentration producing a 50% decrease in the original turbidity, is a useful parameter to compare solubilization under varying conditions.19,20 For the PC/Ch (4:1) mixture, D50 values at 4 °C and at 37 °C were, respectively, 0.9 and 2.7 mM. At 37 °C, subsolubilizing detergent concentrations produced a notorious increase in turbidity. This phenomenon will be discussed below. (28) Dennis, E. A. Adv. Colloid Interface Sci. 1986, 26, 155-175.
Mixtures of PC and SM could be solubilized at all proportions, both at 4 and 37 °C.16,20 An example (PC/SM, 3:2 mole ratio) is shown in Figure 1B, D50 values at 4 and 37 °C being, respectively, 0.5 and 1.3 mM. Inclusion of DPPC in the mixture was justified because DPPC has a gel-to-fluid transition temperature at 41 °C, which is very close to that of egg SM (≈ 40 °C, our own unpublished observations). The results in Figure 1C show that egg PC/DPPC/Ch (3:1:1) bilayers could be solubilized both at 4 °C (D50 ) 4.2 mM) and at 37 °C (D50 ) 2.4 mM). The mixture PC/SM/Ch (3:1:1) was equally tested at both temperatures. A representative experiment is shown in Figure 1D. Subsolubilizing concentrations of Triton X-100 produced a remarkable increase in turbidity at 37 °C (note the Y-axis values in Figure 1D). Solubilization at 4 °C had a high D50 (3.5 mM) which is similar to that of egg PC/DPPC/Ch (3:1:1), but at 37 °C, 50% solubilization was not achieved even at the highest Triton X-100 concentration tested, 5 mM. The results in Figure 1 suggest that the joint presence of SM and Ch in a bilayer renders it resistant to solubilization by Triton X-100, particularly at 37 °C, and that the high gel-fluid transition temperature of SM is not the main reason to explain the detergent resistance. The latter is clear from comparing the PC/ SM/Ch and PC/DPPC/Ch mixtures (Figures 1C and 1D). In a previous study,20 we had suggested that a specific SM/Ch interaction, perhaps hydrogen bonding, could be responsible for that effect. In a further series of experiments, we varied the egg PC proportion in the bilayer while keeping constant a SM/Ch equimolar ratio, i.e., PC/SM/Ch (N:1:1 mole ratio), N varying from 2.7 to 9.0. In Figure 2, values of D50 at 4 and 37 °C are plotted as a function of PC molar fraction in the bilayers XPC. It should be noted that, in all cases, 50% solubilization at 4 °C required less detergent than at 37 °C. Moreover, the plots in Figure 2 revealed three regions of PC/SM/Ch (N:1:1) compositions with respect to solubilization by Triton X-100, namely, region I of mixtures that could not be solubilized even at a detergent/lipid molar ratio of 5 (5 mM detergent in our studies), corresponding to N e 3, or XPC e 0.60, region II of mixtures that could be solubilized but required higher detergent concentrations than pure PC (3 < N < 5.5, 0.60 < XPC < 0.73), and
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Figure 3. PC-DPH fluorescence anisotropy in bilayers containing PC, SM, and Ch. Lipid mixtures had the general composition PC/SM/Ch (N:1:1). Data are plotted as a function of the PC molar fraction in the bilayers XPC.
region III of mixtures that were solubilized under similar conditions than pure PC (N g 5.5, XPC g 0.73). D50 values for pure egg PC are 1.9 (4 °C) and 2.1 (37 °C).19 To obtain a further understanding of these phenomena, the fluidity of the bilayers at 4 and 37 °C, in the absence of detergents, for 0.30 < XPC 5 73.0 PC/Cb/GM3/Ch (2:1:1:2) [4 °C] 0.084 0.15 0.40 2.7 48.7 [37 °C] 0.074 0.092 0.40 3.3 39.8 a Average values of 3-4 measurements. Errors were within (15% of the mean.
presence of sphingolipids of various kinds in those systems. In the present series of experiments, we have examined the effects of totally or partially substituting the SM for other polar sphingolipids in the PC/SM/Ch (3:1:1) sample that is highly insoluble especially at 37 °C (Figure 1D). The effects of substituting SM with galactosylceramide (cerebroside, Cb) or GM3 ganglioside (GM3) are summarized in the data in Table 1. Representative experiments are shown in Figure 5. Both Cb and GM3 are seen to facilitate membrane solubilization by Triton X-100, with GM3 ganglioside being more effective than cerebroside in promoting solubilization (see, e.g., Figures 5B and 5C). Xu et al.35 have shown that Cb had a weaker tendency than SM to form ordered lipid domains with cholesterol. The Origin of the Increase in Turbidity. With most lipid mixtures used in the present study, subsolubilizing detergent concentrations were seen to induce an increase in the vesicle suspension turbidity (Figures 1 and 5). The phenomenon was studied in more detail for the series of compositions PC/SM/Ch (N:1:1) whose solubilization parameters are shown in Figure 2. The phenomenon was significant only at XPC < 0.75 and N < 6, i.e., in what we called regions I and II of the plot in Figure 2. The extent of the increase was larger at 4 °C than at 37 °C and was achieved at lower detergent concentrations at the lower temperature (data not shown). The observation of increased turbidities, by detergent concentrations lower than those producing solubilization, was first made by Alonso et al.29 These authors observed that the increase in turbidity was caused by a remarkable increase in size of the vesicles and attributed the phenomenon to the “lysis and reassembly” of the original (unilamellar) vesicles.30 Similar observations have been made afterward in different lipid-detergent systems and were also explained by the lysis and reassembly mechanism (see, e.g., ref 31). (29) Alonso, A.; Villena, A.; Gon˜i, F. M. FEBS Lett. 1981, 123, 200204. (30) Alonso, A.; Sa´ez, R.; Villena, A.; Gon˜i, F. M. J. Membr. Biol. 1982, 67, 55-62. (31) Edwards, K.; Almgren, M. J. Colloid Interface Sci. 1991, 147, 1-21.
Figure 5. The influence of sphingolipid nature on the solubilization of bilayers containing PC, sphingolipid, and cholesterol. Experimental details as in Figure 1. Cb, cerebroside (galactosylceramide); GM3, GM3 ganglioside. Experiments at 4 °C (b) and at 37 °C (O).
In our systems, centrifugation experiments demonstrated that the turbidity was due to the presence of large structures and not, for example, the formation of highly scattering micelles. Detergent-treated vesicle suspensions that had been incubated at 4 °C were centrifuged at low speed, i.e., 13 000× g for 20 min, at 4 °C. The turbidity of the supernatants was then measured and found to be nearly zero. Representative experiments for the PC/SM/ Ch (3:1:1) and (7:1:1) samples are shown in Figure 6. Solubilization Assessed by Centrifugation. Differential Solubilization. The above results suggested that vesicle growth through lysis and reassembly could occur concomitantly or even while competing with solubilization. To clarify this aspect of detergent-vesicle interaction, detergent-treated suspensions were centrifuged at high forces (510 000× g, 2 h, 4 °C) to physically separate the solubilized from the nonsolubilized fractions. Two vesicle compositions were selected for these experiments, namely, PC/SM/Ch (3:1:1) and PC/SM/Ch (7:1:1), respectively, representing regions I and III of Figure 2. Only experiments at 4 °C were performed, because no lipidic material was sedimented at 37 °C under the above conditions even in the absence of detergent. Lipid phosphorus was assayed in the pellets and supernatants after centrifugation, and the proportion of lipid phosphorus in the supernatants was considered to be a measure of membrane solubilization. Solubilization assessed by the centrifugation assay is shown in Figure 7. The 7:1:1 sample (Figure 7B) showed a similar solubilization profile when measured by turbidity and by centrifugation. D50 measured by centrifugation was 1.8 vs 1.4 from the turbidity measurements. This is probably representative of all region III samples, with little increase in turbidity and D50 e 2. The 3:1:1 sample, however, showed a very different centrifugation profile when measured by one or another
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Figure 6. The turbidity of detergent-treated vesicle suspensions before centrifugation (O) and their supernatants after centrifugation (b). The original lipid compositions are given at the upper right angle of each panel. Experiments were performed at 4 °C.
Figure 7. Pure lipid vesicle solubilization by Triton X-100 assessed by centrifugation. The percent lipid phosphorus recovered in the pellet after centrifuging the detergent-treated membranes is taken as the fraction of solubilized membrane (see main text for experimental details). (A) PC/SM/Ch, 3:1:1 mole ratio. (B) PC/SM/Ch, 7:1:1 mole ratio. Experiments at 4 °C.
method (Figure 7A). Turbidity measurements at 4 °C showed that the suspension reached 50% of the original turbidity at 4 mM Triton X-100 (Figure 1 and Table 1), while centrifugation data yielded a D50 of ca. 0.8, i.e., even lower than the corresponding data for the 7:1:1 sample. Therefore, the centrifugation data show that for region I
Langmuir, Vol. 18, No. 7, 2002 2833
Figure 8. Lipid composition of the nonsolubilized fractions of detergent-treated vesicles. The nonsolubilized fraction corresponds to the pellets of the experiment described in Figure 7. Percent distributions of PC (b), SM (O) , and Ch (1) are given for the pellets obtained after treatment with different Triton X-100 concentrations. Experiments at 4 °C.
samples, solubilization occurs concomitantly with increase in vesicle size, i.e., increase in turbidity. Both turbidity and centrifugation assays have inherent limitations for the study of membrane solubilization by detergents. In the turbidity assay, solubilization may be masked by an increase in vesicle size, as discussed above. Centrifugation has, among other problems, the requirement of separately assaying each of the membrane components for precise quantitation of solubilization, since not all of them are necessarily solubilized in parallel. This was explored for the PC/SM/Ch system by analyzing the pellets containing the nonsolubilized fraction in the experiments in Figure 7. The pellets were resuspended and washed twice in detergent-free buffer, and the lipids were extracted in chloroform/methanol (2:1). The organic extracts were applied to thin-layer chromatography plates so that the three lipids, PC, SM, and Ch, could be separated and quantitated by densitometry, as detailed under Methods. The results of pellet analysis are shown in Figure 8. For both lipid compositions, the pellet became depleted in PC and relatively enriched in SM and PC as solubilization progressed. For example, at 2 mM Triton X-100, the nonsolubilized fraction of the original 7:1:1 sample contained PC/SM/Ch at an approximate molar ratio of 0.7:1:1 and the pellets from the 3:1:1 sample contained PC/SM/Ch at ca. 0.8:1:1.2, which is relatively close to the 1:1:1 ratio that some authors consider paradigmatic of detergent-resistant membranes. Discussion Our studies on the solubilization by Triton X-100 of pure lipid bilayers composed of egg PC, egg SM, and cholesterol allow us to infer certain rules that govern the formation of detergent-resistant membranes. Together with our previous studies on this subject,19-22 the present results show that (a) both SM and cholesterol are required
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to resist solubilization, (b) membrane fluidity is not an important factor in this respect, (c) solubilization is easier at lower temperatures, and (d) molecular interactions between SM and cholesterol are involved in the resistance to Triton X-100 solubilization. In addition, the issue of whether detergent-resistant domains exist in the absence of surfactant also deserves some comment. Both Sphingomyelin and Cholesterol Are Required for Insolubility. In previous studies,19,20 we have taken as a criterion, for insolubility of a given bilayer composition, the fact that at a total detergent/lipid molar ratio of 5, the suspension turbidity remains equal or above 50% of its original value (i.e., in the absence of detergents). This is a rather stringent criterion since, e.g., the turbidity of pure egg PC bilayers decreases to less than 10% of the original value at a total Triton X-100/PC molar ratio of 2.24,28 According to this criterion, the data in Figure 1 show that insolubility (at 37 °C) occurs only when both SM and cholesterol are present. DPPC, despite its Tc gel-to-fluid transition temperature being very close to that of SM, i.e., ≈41 °C, cannot substitute for the sphingolipid (Figure 1C). The combined molar fraction of SM and cholesterol (XSM+Ch) must be at least 0.30 to detect some decreased solubility in relation to that of pure PC (Figure 2). Sphingolipids by themselves do not contribute to detergent insolubility. Mixtures of PC and SM of natural origin at all proportions can be solubilized by Triton X-100, and solubilization is easier at the higher SM proportions.16,20 Moreover glycosphingolipids, whose presence in rafts and detergent-resistant membranes is often invoked, in fact greatly increase the solubility of bilayers containing SM and cholesterol (Figure 5). This is rather easily understood on the basis of (a) the principle that solubilization consists of the formation of phospholipiddetergent mixed micelles32 and (b) the “molecular shape theory” of Israelachvili et al.33 Both PC and SM would be “cylindrical” lipids, thus favoring spontaneously the lamellar organization, while glycosphingolipids, and particularly gangliosides, would have a “conical” shape, thus favoring the formation of micelles in cooperation with Triton X-100. The fact that egg SM is more easily solubilized than egg PC, both being cylindrical lipids, has a different explanation. According to the model by Helenius and Simons,34 solubilization is preceded by a process of detergent insertion in the bilayer. Only when the latter becomes saturated with detergent does formation of lipiddetergent mixed micelles start. Perhaps, egg SM forms much more densely packed bilayers than egg PC because of the saturated hydrocarbon chains, as shown by their respective Tc values of ≈ 41 vs ≈ -10 °C. The higher packing density of SM leads to a smaller capacity to accommodate Triton X-100 molecules while keeping the lamellar organization and thus to an easier solubilization. Membrane Fluidity Is Not Critical. As mentioned in the Introduction, some authors have suggested that at a given temperature, lipids at or near the gel phase would be less prone to solubilization by Triton X-100 than those in the liquid crystalline phase and that this would explain the insolubility of SM-rich bilayers. In fact the opposite is true, as discussed in the preceding paragraph. In a (32) Dennis, E. A. Arch. Biochem. Biophys. 1974, 165, 764-773; RicoLattes, I.; Gouzy, M. F.; Andre-Barres, C.; Guidetti, B.; Lattes, A. Biochimie 1998, 80, 483-487; Almgren, M. Biochim. Biophys. Acta 2000, 1508, 146-163. (33) Israelachvili, J. N.; Marcelja, S.; Horn, R. G. Q. Rev. Biophys. 1980, 13, 121-200. (34) Helenius, A.; Simons, K. Biochim. Biophys. Acta 1975, 415, 2979.
Sot et al.
previous paper,19 with bilayers consisting of pure phosphatidylcholines, we showed that for chain lengths below C16, bilayers in the gel phase were more easily solubilized than those in the fluid phase. The data in this paper also point in the same direction. With the single exception of the PC/DPPC/Ch (3:1:1) mixture, all other systems are more easily solubilized at 4 °C than at 37 °C (Figures 1 and 5, Table 1). (This is at odds with the observations of protein solubilization from sphingolipid-rich membranes at 37 °C but not at 4 °C; see, e.g., ref 2. Such behavior cannot be explained on the basis of lipid properties alone.) Moreover, when bilayer fluidity is measured in terms of anisotropy of PC-DPH fluorescence (Figure 3), anisotropy decreases, i.e., fluidity increases as the mole fraction of PC in the PC/SM/Ch mixture is increased, and solubilization becomes easier. Thus, in some cases, an increased fluidity means an easier solubilization, while the opposite holds in other cases. The PC/SM/Ch samples 1:1:1 at 37 °C and 7:1:1 at 4 °C are interesting, because the former is totally insoluble while the latter can be easily solubilized, and both display the same PC-DPH anisotropy. In summary, no clear correlation is found between bilayer fluidity and solubilization by Triton X-100. Sphingomyelin-Cholesterol Interactions Are Relevant. In recent papers, we have shown that SM interacts with cholesterol in bilayers, presumably through hydrogen bonding between the 3β-OH of cholesterol and the carbonyl group of SM, and that SM/Ch mixtures cannot be solubilized by Triton X-100.20,22 Similar results have been found by London and co-workers.35 In addition, Li et al.,36 using a Langmuir film balance approach, have found that cholesterol lowers the in-plane elasticity of SM bilayers more than that of PC bilayers and suggest that the decrease in interfacial elasticity may be related to the resistance to solubilization by Triton X-100. Radhakrishnan et al.,37 also using lipid monolayers, observed formation of stoichiometric cholesterol-sphingomyelin complexes. The data in Figure 4 confirm the above but, in addition, show that egg PC weakens and eventually abolishes the interaction between SM and cholesterol and concomitantly makes the bilayers amenable to solubilization by Triton X-100. Moreover, in systems such as PC/ SM/Ch (7:1:1), at detergent concentrations producing partial solubilization, the insoluble fraction is enriched in SM and cholesterol (Figure 8B). Thus, we conclude that, in our systems, the molecular interactions between SM and cholesterol are directly linked to detergent insolubility. The Liquid-Ordered Phases. Cholesterol (but not all sterols35), when mixed with many kinds of phospholipids, gives rise to bilayers in a state that is characterized by both a fast translational diffusion and a high degree of molecular order in the hydrocarbon chains, the liquidordered state.38 Mixtures of sphingomyelin and cholesterol are also known to give rise to liquid-ordered phases.22,39 Bilayers in the liquid-ordered state have been proposed to be highly resistant to bilayer solubilization.10,40 Although we have not specifically addressed this point in the present (35) Xu, X.; London, E. Biochemistry 2000, 39, 843-849; Xu, X.; Bittman, R.; Duportail, G.; Heissler, D.; Vilcheze, C.; London, E. J. Biol. Chem. 2001, 276, 33450-33456. (36) Li, X. M.; Momsen, M. M.; Smaby, J. M.; Brockman, H. L.; Brown, R. E. Biochemistry 2001, 40, 5954-5963. (37) Radhakrishnan, A.; Li, X. M.; Brown, R. E.; McConnell, H. M. Biochim. Biophys. Acta 2001, 1511, 1-6. (38) Ipsen, J. H.; Karlstro¨m, G.; Mouritsen, O. G.; Wennerstro¨m, H.; Zuckermann, M. J. Biochim. Biophys. Acta 1987, 905, 162-172. (39) Sankaram, M. B.; Thompson, T. E. Biochemistry 1990, 29, 10670-10675. (40) Brown, D. A.; London, E. J. Membr. Biol. 1998, 164, 103-114.
Triton X-100-Resistant Bilayers
paper, our published41 and unpublished observations show that, in fact, liquid-ordered phases are also detergent resistant. However, the results in Figures 1C and 1D show that by keeping a constant mole fraction of egg PC (XPC ) 0.60) and cholesterol (XCh ) 0.20), bilayers in which DPPC was the third component were more easily solubilized than those containing SM, particularly at 37 °C. This suggests that given roughly similar proportions of liquid-ordered lipids, SM/Ch interactions increase the resistance to solubilization by Triton X-100. In cell membranes, liquid-ordered microdomains may arise in those regions where XCh g 0.20,38,41 and those domains will probably be detergent resistant. Nevertheless, for reasons of conceptual accuracy, we would suggest that the relevant membrane fractions be referred to as “liquidordered microdomains” in the native cell and as detergentresistant membranes only after detergent has been effectively used. Detergent-Resistant Domains before and after Detergent Addition. Dietrich et al.15 have shown in an elegant way that equimolar mixtures of dioleoylphosphatidylcholine, brain sphingomyelin, and cholesterol (a system very similar to ours) give rise to bilayers containing coexisting liquid-ordered and liquid-disordered domains. In parallel, many examples are known of nonsolubilized proteolipid fractions that are collected after detergent treatments of membranes. However, the relationship between the membrane domains existing in the absence of detergent and the detergent-resistant membrane fractions is not straightforward. Giocondi et al.,42 on the basis of in situ imaging of Triton X-100-treated cell membranes by atomic force microscopy, suggest that membrane microdomains can arrange themselves into larger detergent-resistant membranes during Triton X-100 treatment. Our observations also indicate that the detergentresistant residue may be very different from the microdomains in the native membrane. Subsolubilizing detergent concentrations lead to marked increases in turbidity (Figures 1 and 5), that are reflecting large changes in bilayer architecture, namely, lysis and reassembly of the vesicles. The morphological changes taking place under these conditions have been described in our early papers.29,30 At this subsolubilizing stage, part of the lipid gives rise to very large structures that can be easily sedimented (Figure 6), while a fraction of the lipid is solubilized (Figure 7). Under conditions (4 °C, 2 mM Triton X-100) leading to partial solubilization, the nonsolubilized residue has a composition close to PC/SM/Ch (1:1:1), irrespective of the composition of the native membrane (Figure 8). In view of this series of complex and profound (41) Sa´ez-Cirio´n, A.; Alonso, A.; Gon˜i, F. M.; McMullen, T. P. W.; McElhaney, R. N.; Rivas, E. Langmuir 2000, 16, 1960-1968; Alonso, A.; Sa´ez, R.; Villena, A.; Gon˜i, F. M. J. Membr. Biol. 1982, 67, 55-62. (42) Giocondi, M. C.; Vie´, V.; Lesniewska, E.; Goudonnet, J. P.; Le Grimellec, C. J. Struct. Biol. 1982, 131, 38-43.
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changes induced by the detergent, it cannot be ascertained that the resulting detergent-resistant membranes correspond precisely to structures pre-existing in the bilayer. While accepting the existence of liquid-ordered domains in the cell or model membrane, the nonsolubilized fraction recovered after detergent treatments corresponds most probably to partially solubilized and reassembled elements whose structure and properties may or may not resemble those in the living cell. Conclusions When pure lipid bilayers in the form of large unilamellar vesicles, containing phosphatidylcholine, sphingolipids, and cholesterol, are treated with Triton X-100 in the 0-5 range of total surfactant/lipid ratios, at 4 or 37 °C, it is observed that (i) solubilization requires less detergent at 4 °C than at 37 °C, (ii) sphingolipids by themselves do not hinder detergent solubilization, and some of them actually facilitate solubilization, (iii) two independent factors, namely, the presence of liquid-ordered domains in the membrane and the molecular interactions between sphingomyelin and cholesterol, concur in inducing resistance to Triton X-100 solubilization, and (iv) detergentresistant membranes obtained after detergent treatment may be the result of detergent-induced bilayer partial solubilization and reassembly, instead of corresponding to structures existing in the native membrane. Acknowledgment. This work was supported in part by Grants PB 96/0171 from DGICYT (Spain), PI 1999/7 from the Basque Government, and G03/98 from the University of the Basque Country. J.S. and M.I.C. were supported by the Basque Goverment. Abbreviations A0 ) apparent absorbance at 500 nm (turbidity) of a lipid vesicle suspension in the absence of detergent Amax ) maximum absorbance at 500 nm of a detergenttreated vesicle suspension Cb ) cerebroside (galactosylceramide) Ch ) cholesterol D50 ) detergent concentration producing a 50% decrease in membrane suspension turbidity Dmax ) detergent concentration producing maximum turbidity when added to a membrane suspension DPPC ) dipalmitoylphosphatidylcholine GM3 ) GM3 ganglioside GPI ) glycosyl phosphatidylinositol LUV ) large unilamellar vesicles PC ) phosphatidylcholine PC-DPH ) 2-(3-(diphenylhexatrienyl) propanoyl)-1hexadecanoyl -sn-glycero-3-phosphorylcholine SM ) sphingomyelin Tc ) gel-fluid transition temperature of a phospholipid bilayer. LA011381C
