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Published as doi: 10.1096/fj.06-7809com.
 (The FASEB Journal. 2007;21:1714-1723.)
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Plant sterols in "rafts": a better way to regulate membrane thermal shocks

Johannes G. Beck, Damien Mathieu, Cécile Loudet, Sébastien Buchoux and Erick J. Dufourc1

UMR 5248 CBMN, CNRS-Université Bordeaux1-ENITAB, IECB, 33607 Pessac Cedex, France

1Correspondence: UMR5248 CBMN CNRS-Université Bordeaux1-ENITAB, IECB 2 rue Robert Escarpit, 33607 Pessac, France. E-mail: e.dufourc{at}iecb.u-bordeaux.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Specialized lipid domains (rafts) that are generally enriched in sterols and sphingolipids, are most likely present in cell membranes of animals, plants and fungi. While cholesterol and ergosterol are predominant in vertebrates and fungi, plants possess complex sterol profiles, dominated by sitosterol and stigmasterol in Arabidopsis thaliana. Fully hydrated model membranes of composition approaching those found in rafts of mammals, fungi and plants were investigated by means of solid-state 2H-NMR, using deuterated dipalmitoylphosphatidylcholine (2H62-DPPC). The dynamics of such membranes was determined through measuring of membrane ordering or disordering properties. The presence of the liquid-ordered, lo, phase, which may be an indicator of rigid sterol-sphingolipid domains, was detected in all binary or ternary mixtures of all sterols investigated. Of great interest, the dynamics of ternary mixtures mimicking rafts in plants (phytosterol/glucosylcerebroside/DPPC), showed a lesser temperature sensitivity to thermal shocks, on comparing to systems mimicking rafts in mammals and fungi. This effect was particularly marked with sitosterol. The presence of an ethyl group branched on the alkyl chain of sitosterol and stigmasterol is proposed as reinforcing the membrane cohesion by additional attractive van der Waals interactions with the alkyl chains of sphingolipids and phospholipids. As a side result, the elevated resolution of NMR spectra in the presence of sitosterol also suggests domains of smaller size than with other sterols. Finally, the role of phytosterols in maintaining plant membranes in a state of dynamics less sensitive to temperature shocks is discussed.— Johannes G. Beck, Damien Mathieu, Cécile Loudet, Sébastien Buchoux and Erick J. Dufourc. Plant sterols in "rafts": a better way to regulate membrane thermal shocks.


Key Words: 2H-NMR • cholesterol • ergosterol • sitosterol • stigmasterol • sphingomyelin • glucosylcerebroside • dipalmitoylphosphatidylcholine • order parameters


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN EUKARYOTIC CELLS, STEROLS REGULATE biological processes and sustain the domain structure of cell membranes. While cholesterol is the major sterol of vertebrates, ergosterol plays a key role in fungi. Plants usually possess more complex sterol compositions. Cholesterol, a 24-demethyl sterol; campesterol, a 24-methyl sterol; stigmasterol and sitosterol, two 24-ethyl sterols, are major constituents of the sterol profiles of plant species. Sterol-C24-methyltransferases are important for plant growth and development (1 2 3) and alkylate the side chain on the D ring of sterols by two successive methylation steps on C-24 (4 , 5) . Therefore, typical phytosterols like sito- and stigmasterol possess additional alkyl groups on C-24. Brassinosteroids are structurally modified sterols that are involved in the postembryonic growth of plants whereas other phytosterols are important in the embryonic growth (3 , 6) . Sterols are critical for the formation of liquid ordered (lo) lipid domains (lipid rafts) that are supposed to play an important role in fundamental biological processes like signal transduction, cellular sorting, cytoskeleton reorganization and infectious diseases (7 , 8) . In plants, specialized lipid domains are involved in the polarized growth of pollen tube and root hair (9) and the asymmetric growth of plant cells is in general due to the asymmetric distribution of membrane components. Although most studies about lipid rafts focused on cholesterol dependent mammalian systems, some investigations suggest that similar domains exist in plants (10) and fungi (11) . Interestingly, Xu and London (12 , 13) showed that phase separation in lo and ld phase can also occur with stigmasterol and sitosterol, two typical phytosterols.

Depending on temperature and composition, lipid bilayers can posses liquid-disordered (ld), liquid-ordered (lo) and solid-ordered (so) phases. ld phases are untightly packed, observed at high temperatures and usually enriched in lipids with unsaturated acyl chains. Both, the lateral diffusion of the lipids and the disorder of their acyl chains are high. They are often termed "fluid" or L{alpha} phases. The tightly packed lo phases, which are thought to depict the physical state of rafts, are generally enriched in sterols, sphingolipids and lipids with saturated acyl chains. While the lipids keep their high lateral diffusion, their acyl chains are strongly ordered. They are also termed as ß phases. Solid ordered phases, so, are usually composed of saturated lipid chains, they exclude proteins (14) , they are encountered at low temperatures and exhibit low lateral diffusion (15) . They are often termed "gel-phases" or Lß, and seem unadjusted to convey membrane in/out biological processes. The presence of lo and ld domains in the binary DPPC/cholesterol system was intensively studied and resulted in several partial phase diagrams that indicated lo/ld coexistence (16 17 18 19 20) . The coexistence of lo and ld domains was preferentially observed when sterols and lipids with saturated or unsaturated acyl chains were present. By fluorescence quenching techniques and detergent insolubility experiments (12 , 13) it was found that the order of sterol-enriched domains depends strongly on the structure of the partitioning sterol. Mongrand and coworkers (10) obtained detergent resistant membranes (DRMs) from plasma membranes isolated from tobacco leaves and BY2 cells and showed that these domains were greatly enriched in glucosylceramide, as well as in a mixture of stigmasterol, sitosterol, 24-methylcholesterol and cholesterol. Recent results (21) indicate strong correlations between sterol structure and the shape of formed lipid domains. As the nondestructive investigation of the physical properties of biomembranes in vivo appeared difficult, rafts are operationally defined by their detergent insolubility. Although the correlations between rafts and DRMs are not absolutely clear (22) , the comparison to spectroscopic experiences suggested that DRMs report lo domains reliably (12 , 13 , 23) . Other common methods for the investigation of lipid domains in model membranes are confocal fluorescence microscopy and fluorescence correlation spectroscopy (21) , wide-field fluorescence microscopy (24) , fluorescence-quenching (12 , 23 , 25) , fluorescence resonance energy transfer (26) , single particle tracking (27) , EPR spin labeling (28) , 2H- and 31P-NMR (11 , 20 , 29 , 30) as well as differential scanning calorimetry (17 , 20) and electron microscopy (EM) or atomic force microscopy (31) .

Among all techniques, solid-state 2H-NMR spectroscopy is one of the very few noninvasive methods that reports on ensemble properties and on time and space averaged orientations of molecules or molecular bonds in lipid bilayers, i.e., so-called order parameters. 2H-NMR allows not only the determination of bilayer states (e.g., lamellar, hexagonal, cylindrical, cubic, etc.), but also a more precise description of the dynamics of deuterated lipid molecules in lamellar liquid-crystalline states. Such dynamics may in turn be converted into valuable bilayers information such as membrane thickness (32 33 34 35 36) and membrane protrusion (37 38 39 40) that are important parameters for cell function. In this paper, we examined by means of 2H-NMR spectroscopy several fully hydrated binary (two lipid components) and ternary (three lipid components) membrane systems made of 1,2-Dipalmitoyl-2H62-sn-Glycero-3-Phosphocholine (DPPC-2H62) and cholesterol, ergosterol, sitosterol, stigmasterol, sphingomyelin and glucosylcerebroside (Fig. 1 ). Mixtures of sito- or stigmasterol with glucosylcerebroside and a saturated chain lipid (DPPC) are chosen as models for plant membranes of "raft" composition whereas their counterparts for fungi and vertebrates were obtained by substitution of phytosterol with ergosterol and cholesterol, respectively.


Figure 1
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  		Figure 1. Planar molecular structures of sterols and sphingolipids used in this study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample preparation
Dipalmitoyl-2H62-sn-Glycero-3-Phosphocholine (DPPC-2H62) (synthetic), sphingomyelin (Brain, Porcine) and cholesterol (wool grease) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Glucosylcerebroside (plant) was obtained from Larodan Lipids (Malmö, Sweeden). Sitosterol and stigmasterol were provided by Sigma-Aldrich (St. Louis, MO, USA) and ergosterol by Fluka (Buchs, Switzerland). Pure DPPC-2H62 multi lamellar vesicles (MLVs) were prepared by homogenization of lipids in excess ultra pure water provided by an ELGA water purification system. Several freeze-thaw cycles and shaking in a vortex mixer were performed to ensure sample equilibrium. MLVs that contained more than one lipid were prepared by cosolubilization in lipophilic solvents, solvent evaporation, removal of solvent traces by lyophilization from a water suspension and then hydration as described above. One sample contained DPPC-2H62 as sole lipid; four samples containing a binary mixture of 70 mol % DPPC-2H62 and 30 mol % sterol were prepared with cholesterol, stigmasterol, sitosterol and ergosterol. To improve the approximation to biological lo phases, four ternary systems with 20% DPPC-2H62, 40% sterol and 40% sphingolipid were prepared with sitosterol and glucosylcerebroside, stigmasterol and glucosylcerebroside, ergosterol and glucosylcerebroside as well as with cholesterol and sphingomyelin. For further comparison, binary systems that contained no sterol were investigated additionally. They contained similar quantities of DPPC-2H62 and sphingolipids and were prepared with sphingomyelin or glucosylcerebroside as a sphingolipid component.

NMR spectroscopy
NMR was performed on a Bruker Avance 300 spectrometer operating at 46 MHz for deuterium. The phase-cycled quadrupolar echo sequence (41) was used with {pi}/2 pulse widths of 4.5 µs and 50 µs delays between the pulses. The recycle delay was 1.5 s and for most spectra 2K acquisitions were recorded. Signal detection was performed in quadrature on a spectral width of 500 kHz. Temperature was regulated to ± 1°C and stabilized for 30 min before the acquisitions begun. To guarantee equivalent thermal histories of the individual samples, the temperature was always increased stepwise in between spectra acquisition.

Data analysis
Due to the relative complexity of obtained spectra, which result in a superposition of subspectra received from the chemically and dynamically nonequivalent and nonsimilarly oriented deuterons of both DPPC acyl chains, the direct information that can be obtained is qualitative. A more quantitative description of the bilayer state is obtained by evaluating the spectral moments (42 , 43) . First moments were calculated using a C2+ home made routine (Buchoux S., unpublished) inserted in the Microcal Origin software to which Bruker NMR data were exported using a proper subroutine. To avoid zero values for spectra symmetrical relative to the origin, a sign reversal was used for negative frequencies. When spectra are axially symmetric (lo and ld phases) M1 may be used to directly estimate the chain average carbon-deuterium, SCD by:


Formula 1

(1)
Because the palmitic chains are perdeuterated in DPPC, we used angular brackets to represent the average over all labeled positions. AQ is the static quadrupolar coupling constant (167 kHz, (44) ). If peaks in spectra can be attributed to individual C-2H bonds, the corresponding C-2H bond order parameters, SCD, can be obtained from measurement of quadrupolar splittings, {Delta}{nu}Q, in between the so-called 90° orientations of normal to bilayers:

Formula 2(2)
Because it is far easier to measure quadrupolar splittings in magnetic field oriented spectra (29) we calculated oriented-like spectra from powder (Pake) spectra that are obtained from unoriented liposomes (MLVs). The algorithm (de-Pake-ing) developed by Bloom and coworkers (45 , 46) was used, except that we chose to calculate oriented-like spectra for normal to bilayers oriented at 90° with respect to the external magnetic field direction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiments were conducted on both binary and ternary lipid systems and as a consequence, results will be presented accordingly.

Binary systems
Figure 2 shows selected spectra observed for fully hydrated binary lipid systems that contained 30 mol % of cholesterol (A), stigmasterol (B), sitosterol (C) or ergosterol (D) and 70 mol % DPPC-2H62. All 2H-NMR powder spectra were obtained with 2 to 5°C temperature intervals over the temperature range 5 to 55°C. All spectra show similar lineshapes characteristic of nonoriented systems with axial symmetry due to the fast rotational dynamics of the DPPC molecule around its long axis that is aligned with the bilayer normal. This is true except for the lower temperature (10–15°C) spectra where loss of axial symmetry is detected. As for the pure lipid (spectra not shown), where axially asymmetric lineshapes are detected below 40°C, this is characteristic of solid-ordered phases. When the temperature is increased from 10–15 to 55°C, the binary DPPC-2H62/sterol model membrane spectra maintain their axial symmetry while reducing their global width. This is the manifestation of the well known temperature-driven disordering effect. It is noteworthy that the spectral width at 55°C is much higher than that of pure DPPC-2H62 lipids at equivalent temperature. This is again the well-documented signature, as in the case of cholesterol, of a liquid-ordered phase. Clearly, all sterols produce the same effect. A closer look at the thermal variation shows that cholesterol-containing membrane spectra maintain a maximum constant width (measured at the low intensity shoulders) of ~120 kHz from 15°C to 35°C, and then decrease in spectral width gradually above. A similar behavior is observed for other sterols, the domain for which constant width of ca. 120 kHz is detected depends on the sterol structure: stigmasterol effect is observed between 20–25°C, sitosterol between 15–25°C, ergosterol between 20–30°C. According to this criterion, plant sterols are less capable of maintaining constant high ordering when the binary model membrane is subjected to temperature variations. Interestingly, above 25°C all spectra of the DPPC-2H62/sitosterol sample show a number of individual sharp peaks that appear less resolved in spectra of the other binary systems. Binary mixtures of DPPC-2H62/sphingomyelin and DPPC-2H62/glucosylcerebroside were also recorded and showed little difference with the thermal behavior of pure DPPC-2H62 (not shown).


Figure 2
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  		Figure 2. 2H-NMR powder spectra of binary DPPC-2H62/sterol samples containing 30 mol % cholesterol (A), 30 mol % stigmasterol (B), 30 mol % sitosterol (C) and 30 mol % ergosterol (D). Each spectrum is the average of 2K acquisitions. Lorentzian line broadening prior Fourier Transformation is 100 Hz.

Figure 3 shows the first moments calculated from 2H-NMR powder spectra of binary samples that contained DPPC-2H62/sphingomyelin (1/1), DPPC-2H62/glucosylcerebroside (1/1), DPPC-2H62/sitosterol (7/3), DPPC-2H62/ergosterol (7/3) and DPPC-2H62/stigmasterol (7/3). To "translate" first moments into average chain ordering the quantity 2*<SCD>chain was also plotted on a double-y axis. The 2 factor accounts here for the fact that the average orientation of almost all C-D bonds is 90° with respect to the lipid long axis. This statement is not entirely true for the methyl terminal and for deuterons at the 2' position of the sn-2 chain, where an additional geometrical factor must be used, but the approximation allows gross comparison of ordering for the entire chain. More accurate description can be obtained from de-Paked spectra (vide infra). Using this definition allows to easily compare completely disordered systems (2*<SCD>chain=0) to fully rigid systems (2*<SCD>chain=1). First moments are plotted with lines without symbols for pure DPPC-2H62 (solid line) and for the sample with cholesterol (dashed line) for a better readability in all three panels. The DPPC-2H62 order-disorder transition is clearly depicted by the thermal variation of M1, the so-ld transition temperature, Tm, occurring between 35 and 37.5°C. As earlier reported, cholesterol induces disorder for temperatures lower that Tm and increases order above, which is the signature of the lo phase (29 , 47 48 49 50 51 52) . This effect has for consequence a quasi-vanishing of the DPPC-2H62 phase transition, the thermal variation becoming almost linear on the studied temperature range. Thus, stigmasterol, sitosterol and ergosterol obviously have a strong ordering effect on DPPC-2H62 above the phase transition temperature of pure DPPC-2H62. At temperatures below the phase transition of DPPC-2H62, stigmasterol and sitosterol have a marked disordering effect whereas ergosterol produces a weaker result. Between 10 and 50°C, the acyl chain order in the DPPC-2H62/ergosterol (7/3) system is higher compared to that in similar systems with stigmasterol or sitosterol. In the binary system containing ergosterol the total C-2H bond order of the deuterated acyl chains decreases almost linearly with increasing temperature. Remarkably, the decrease of the total C-2H bond order in the stigmasterol/sitosterol containing systems has a more sigmoid character. In the 25 to 45°C temperature range, the total C-2H bond order of DPPC-2H62 for systems with 30% stigmasterol/sitosterol is in between that of pure DPPC-2H62 and that of the (7/3) mixture with cholesterol. The spectral moments calculated for equally concentrated binary systems containing DPPC-2H62 with either glucosylcerebroside or sphingomyelin, resemble those obtained from the pure lipid sample. The obvious solid-to-liquid phase transition that occurs at ca. 30°C in these binary systems is much broader (ca. 10°C) compared to that of the pure phospholipid whose phase transition takes place in between 35 and 37.5°C. Within the whole temperature range the total DPPC-2H62 bond order of the system with glucosylcerebroside is higher compared to that with sphingomyelin. At temperatures above the solid-to-liquid phase transition of pure DPPC-2H62, glucosylcerebroside markedly increases ordering whereas sphingomyelin has a little disordering action.


Figure 3
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  		Figure 3. First moments of 2H-NMR spectra (membrane ordering) vs. temperature. First moments of pure DPPC-2H62 spectra (line without symbols) and of DPPC-2H62/cholesterol (7/3) spectra (dashed line without symbols) are shown in all diagrams. A: ({blacksquare}): DPPC-2H62/glucosylcerebroside (1/1); ({square}): DPPC-2H62/sphingomyelin (1/1); B: ({blacksquare}): DPPC-2H62/ergosterol (7/3); C: ({blacksquare}): DPPC-2H62/stigmasterol (7/3); ({square}): DPPC-2H62/sitosterol (7/3). On double Y axis is plotted twice the chain order parameter as obtained from equation 1 of text.

Spectral depaking was performed on spectra recorded at 30, 35, 40, 45, 50 and 55°C. Figure 4 shows depaked spectra of binary DPPC-2H62/sterol systems that contained 30% cholesterol (A), stigmasterol (B), sitosterol (C) and ergosterol (D). All deconvoluted spectra presented in Fig. 4 show a large peak doublet at the outer verges and one or several smaller doublets next to the centers of spectra. The quadrupolar splittings that correspond to the large peaks, the widths of the large peaks and their shapes depend strongly on sterol structure and sample temperature. At 30 and 35°C a doublet of doublets, made of relatively sharp peaks, appeared close to ±5 kHz for depaked spectra of all investigated DPPC-2H62/sterol systems. In the case of DPPC-2H62/sitosterol the small doublet only fused when the temperature was increased above 50°C, whereas this fusion was already observed at 40°C for the other three DPPC/sterol systems. The resolution of a number of small individual peaks that appeared in between the large peaks at the outer verges and the sharp peaks next to the centers of the spectra also depends on the sterol component. In the depaked spectra of the binary DPPC-2H62/sitosterol system, these peaks and all other peaks were within the whole temperature range better resolved than in the depaked spectra of the other DPPC/sterol systems. Because DPPC-2H62/sterol systems show the presence of lo states (at 30°C or above) and pure DPPC-2H62, DPPC-2H62/sphingomyelin and DPPC-2H62/glucosylcerebroside that of ld states (above 37.5°C), depaking could be applied successfully. The deconvolution procedure is only reliable when applied to axially symmetric spectra as in lo and ld states. C-2H bond order parameters (SCD), which are proportional to corresponding quadrupolar splittings (see eq. 2 ), can therefore be attributed to specific deuterons. It must be noted that some depaked spectra are better resolved than others. Assignment for low S/N spectra is indeed harder but can nonetheless be made straightforwardly for the plateau positions and for few positions near the methyl terminal (16 to 14–12). Other positions are extrapolated. This is accounted for in our plots by larger error bars, we also refer to probable order parameters to indicate that some assignments may be uncertain. Based on the data of Seelig and coworkers (36) and that of Douliez and coworkers (32 , 37) , we therefore attributed SCD order parameters, as obtained from our depaked spectra, to the probable chain positions, k, of the corresponding Ck-D bonds of DPPC-2H62 acyl chains. In certain cases both chain positions could be assigned, because there are not completely equivalent (36) ; but for most cases, and especially at high temperatures, the same SCD was found for equivalent k positions on both sn-1 and sn-2 chains. In Fig. 5 are plotted the chain segment order parameters as a function of the labeled carbon position, k, for some binary systems investigated (DPPC/glucosylcerebroside, DPPC/sitosterol) and for some temperatures of interest (30, 40 and 55°C). We also plotted data for DPPC at 40 and 55°C for reference but not at 30°C because this pure one-lipid component system is in the so state (axially asymmetric) at this temperature and hence de-Pake-ing cannot be applied. All graphs give rise to so-called "bilayer order parameter profiles" that depict the minute dynamics of the bilayer hydrophobic interior. As mentioned when dealing with spectral moments (vide supra) the quantity 2 * SCD was plotted on the y axis; again, the factor 2 accounting for the fact that the average orientation of almost all C-D bonds is 90° with respect to the lipid long axis, except for the position 2 on the sn-2 chain and for the methyl terminal (vide supra). Position 2 for both chains was however hardly detectable and not plotted in Fig. 4 . Within the 30–55°C temperature range SCD on acyl chain positions from 3 to 12, so-called "plateau" positions, have relatively high order in the DPPC-2H62/sitosterol system (2*SCD ~0.58 at 55°C to ~0.86 at 30°C). A similar behavior is seen in the presence of stigmasterol (data not shown). For pure DPPC-2H62 and DPPC-2H62/glucosylcerebroside (1/1) the corresponding "plateau" positions bonds are much less ordered and 2*SCD range from ~0.38 for the first system at 55°C to ~0.52 for the second system at 40°C. This confirms that the DPPC-2H62/sitosterol (7/3) system is mainly in a lo state and that pure DPPC-2H62 and DPPC-2H62/glycosylcerebroside systems are in ld states, when heated just above their melting transition temperatures.


Figure 4
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  		Figure 4. Depaked spectra of A) DPPC-2H62/cholesterol (7/3); B) DPPC-2H62/stigmasterol (7/3); C) DPPC-2H62/sitosterol (7/3); D) DPPC-2H62/ergosterol (7/3). The incomplete symmetry of the depaked spectra is due to residual acoustic ringing of the probe.


Figure 5
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  		Figure 5. Probable SCD order parameters of DPPC-2H62 chains (average of two chains) as a function of the labeled carbon position, k. A) pure DPPC-2H62; B) DPPC-2H62/glucosylcerebroside (1/1); C) DPPC-2H62-sitosterol (7/3). ({blacktriangleup}): 30°C, ({square}): 40°C, ({blacksquare}): 55°C

Ternary systems
In addition to the binary systems presented above, we examined ternary systems that were taken as representative models of rafts found in plants, fungi and vertebrates. Fully hydrated liposomes that contained 20 mol% DPPC-2H62 with 40 mol% sphingolipid and 40 mol% sterol were thus prepared. Plant "rafts" compositions included glucosylcerebroside and stigmasterol or sitosterol; fungi models were made of glucosylcerebroside and ergosterol whereas the "raft" model for vertebrates was made with sphingomyelin and cholesterol. Again spectra showed the lineshape and width characteristic of lo phases, i.e., axially symmetric shape and large quadrupolar splittings of ca. 50 kHz at ambient temperature (not shown). Of interest is the temperature sensitivity of systems: whereas cholesterol/sphingomyelin/DPPC shows a marked decrease in spectral width above 40°C, plant sterols-containing systems maintained a rather elevated width even at 55°C (powder spectra not shown, but it can be seen on dePaked spectra, Fig. 7 ). Ergosterol/glucosylcerebroside/DPPC systems showed an in between behavior. The quite unusual thermal behavior of plant "raft" mixtures is shown in Fig. 6 where the first moment for DPPC powder spectra in sitosterol/glucosylcerebroside/DPPC and stigmasterol/glucosylcerebroside/DPPC systems is plotted against temperature. M1 for pure DPPC is also shown with a solid line as a reference. The thermal variation is quasi linear for both systems, within the experimental error, and goes from a membrane ordering of 0.8 at 5°C to 0.5 at 55°C. This demonstrates the regulatory effect of both plant sterols that dampen down the abrupt thermal variation undergone by pure DPPC or DPPC/glucosylcerebroside systems. Selected dePaked spectra for the four ternary systems investigated are presented in Fig. 7 . Another interesting feature for ternary sample spectra consists in a relatively high resolution of individual peaks that are detected above 25°C, especially in the case of plant sterols. The large peaks at the outer verges and the sharper small peaks next to the center, that had been described for dePaked spectra of DPPC-2H62/sterol (7/3) systems, also appeared in the dePaked spectra of ternary DPPC-2H62/sterol/sphingolipid (1/2/2) systems (except that containing ergosterol). Overall, the dePaked spectra of the ternary systems resembled more those of the binary DPPC-2H62/sitosterol (7/3) system than those of the three other binary DPPC-2H62/sterol (7/3) systems. Despite of these overall similarities in terms of resolution, one distinguishes nonetheless marked differences in the thermal variation of the peaks. This is better seen when considering plots of individual C-2H bond order parameters as a function of labeled carbon position, k, on the acyl chains and temperature, Fig. 8 . The chain segments order parameters again show the characteristic bilayer dynamical profile, with high order near the glycerol backbone, "plateau" for positions 3–10, and low order at the bilayers center (chain ends). It is clearly seen that plateau positions are less temperature dependent for sitosterol-containing systems that for other systems. One may calculate {Delta}SCD, the difference between order parameters at 30°C and 55°C and find values of 0.16 and 0.26 for sitosterol and cholesterol containing systems, respectively. Stigmasterol and ergosterol-containing ternary systems show a similar value of 0.19. The corresponding value obtained on the binary system DPPC/sitosterol (Fig. 5) is 0.26. Of interest are also the variations for positions near the bilayers center, i.e., labeled carbons 13, 14, 15 (position 16 is a less good reporter because of its additional rotation around its C3 axis). Again the thermal amplitude of SCD on the 30–55°C range scales as 0.13 ± 0.04, 0.18 ± 0.03, 0.23 ± 0.04 for sitosterol, stigmasterol/ergosterol, cholesterol containing systems, respectively. One remarks also the abrupt decrease in all order parameters, above 40°C, in the case of cholesterol- and ergosterol-containing systems; at converse, phystosterols promote a more gradual decrease, as already seen in Fig. 6 .


Figure 7
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  		Figure 7. Depaked spectra of fully hydrated liposomes of: A) DPPC-2H62/cholesterol/sphingomyelin (1/2/2); B) DPPC-2H62/stigmasterol/glucosylcerebroside (1/2/2); C) DPPC-2H62/sitosterol/glucosylcerebroside (1/2/2); D) DPPC-2H62/ergosterol/glucosylcerebroside.


Figure 6
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  		Figure 6. Thermal dependence of plant "rafts" first spectral moment (membrane ordering). First moments of pure DPPC-2H62 spectra (line without symbols) are also shown as reference. (•): DPPC-2H62/glucosylcerebroside/sitosterol (1/2/2)); ({circ}): DPPC-2H62/glucosylcerebroside/stigmasterol (1/2/2).


Figure 8
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  		Figure 8. Probable SCD order parameters of DPPC-2H62 chains (average of two chains) as a function of the labeled carbon position, k. A) DPPC-2H62/cholesterol/sphingomyelin, (1/2/2); B) DPPC-2H62/stigmasterol/glucosylcerebroside (1/2/2); C) DPPC-2H62/sitosterol/glucosylcerebroside (1/2/2); D) DPPC-2H62/ergosterol/glucosylcerebroside. ({blacktriangleup}): 30°C, ({square}): 40°C, ({blacksquare}): 55°C


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Two major findings come out from our experimental study: 1) the observation of membrane ordering typical of the presence of lo states for all sterol systems investigated. 2) A lesser temperature dependence of membrane dynamics for systems modeling "rafts" in plants. These two points will be discussed sequentially and replaced in the context of domain formation in fungi and plants.

Liquid-ordered states with ergosterol, sitosterol and stigmasterol
In all binary or ternary systems investigated, the lamellar gel-to-fluid phase transition (so-ld transition) is almost or completely abolished in the presence of sterols. The effect is spectacular with sitosterol in the "raft" mixture DPPC/glucosylcerebroside/sitosterol where a smooth linear decrease of membrane ordering is observed from 5°C to 55°C. For other sterols, especially in binary mixtures where the sterol is present at 30 mol%, there is still a sigmoid variation, suggesting the possible coexistence of lo and ld phases exchanging rapidly at the NMR time scale (µs). It is good here to replace our finding in the context of phase diagrams that have been published in the last decades for DPPC/cholesterol (16 , 17 , 19 , 20) . Although the authors diverge a little on phase boundaries they all agree that lo phases exist and may exchange with ld phases especially at elevated temperatures. Because 2H-NMR spectra sample ensemble dynamics at the microsecond timescale and because the exchange between lo and ld states is fast in our conditions (one detects only one average spectrum), it is still difficult to tell whether our spectra are characteristic of pure lo phases or depict an intermediate lo-ld state. For instance, examination of membrane ordering at high temperature (55°C) shows that the average chain order is of 0.3 in the absence of sterol and 0.5 in its presence. The difference is huge in terms of dynamics and implies that if a fast exchange situation occurs in these conditions (vide infra), the pure lo phase must have a larger order. From our data it is however difficult to place boundaries between lo and ld phases. Without going into the debate, we will restrict our discussion to commenting changes in ordering. Lipid systems that undergo a so-ld transition are encountered with all synthetic systems (mostly phosphatidylcholines) and also with natural lipids such as sphingolipids or cardiolipins (53) . In our study with binary mixtures of DPPC with either sphingomyelin or glucosylcerebroside, the transition is preserved, although broadened and happens between a state of ordering of 0.8–0.9 in the so phase to 0.4–0.3 in the ld state. Interestingly exchanging a choline headgroup by a sugar group leads to a marked ordering increase (15%) in the ld phase. As a common effect of all sterols, the low-temperature order is moderately lowered by 5–10% whereas the high temperature order is markedly increased by 30–40%. As it has been proposed earlier this is clearly the sterols signature as regulators of membrane dynamics. In other words, in the absence of sterol the temperature gradient of membrane ordering is 0.014 order parameter units per °C whereas it dampers down by half to 0.007 in the presence of 30 mol% sterol.

Plant sterols as better temperature regulators in lipids mixtures of "raft" composition
In ternary systems made of saturated chain PC/sphingolipid/sterol to mimic raft composition for mammalian, fungi and plant membranes our NMR results show that plant raft models are less sensitive to temperature variations, at least in the range 5–55°C. Nonetheless, cholesterol is also capable of maintaining a liquid-ordered state in the whole range but the order parameter at high temperatures decreases faster than that of the ternary system containing sitosterol, stigmasterol and ergosterol. Of interest is also the fact that the dynamics of positions near the chain end, i.e., 13, 14, 15, are also much temperature sensitive with cholesterol. At this level of the discussion it is noteworthy to remind that the position of cholesterol in the membrane has been determined independently by neutron diffraction using deuterium labeled cholesterol and molecular dynamics in the binary system DMPC/cholesterol (54) . Cholesterol was found perpendicular to the membrane plane with the hydroxyl group at the level of the glycerol backbone region and the short aliphatic chain facing the phospholipids chains near the bilayer center. Although our systems are more complex than that of the above cited study one might reasonably assume that the aliphatic chains of all four sterols of the study face lipid chain positions 10–16 of DPPC and similar positions for the sphingolipid chains. In other words, the difference we observe in membrane dynamics in the presence of ergosterol, and phytosterols may be due to a peculiar effect of the sterol alkyl chains that differ in size, branching and unsaturation from the cholesterol sidechain. Compared to cholesterol, ergosterol has an additional methyl group and the two phytosterols additional ethyl groups branched on C-24. Ergosterol and stigmasterol also have a double bond at position 22–23. The presence of an additional ethyl group may reinforce the attractive van der Waals interactions leading to more membrane cohesion and therefore less temperature sensitivity. The role played by the trans double bond at C22-C23 is unclear and could induce more disorder than order, comparable to the effect of unsaturations in lipid fatty acyl chains. However it is also known that trans double bonds induce less disorder than cis conformers (53) . It is also worth noting that the sugar sphingolipid induced a little ordering effect on DPPC bilayers. So the observed effect of less membrane dynamics sensitivity with respect to high temperature may be related to a subtle interplay of glucosylcerebroside action and favorable sterol acyl chain van der Waals interactions. Combination of all effects leads to the following ranking for adaptability to thermal shocks: Sitosterol/Glucer > Stigmasterol/Glucer ~ Ergosterol/Glucer > Cholesterol/Sphingomyelin.

The reason for the enormous resolution differences between depaked spectra obtained with binary or ternary systems containing phytosterols and especially sitosterol remains to be elucidated. Recently, Hsueh et al. (11) observed a significant broadening of peaks in depaked 2H-NMR spectra of a DPPC-2H31/ergosterol (75/25) system when the temperature was increased from 41 to 43°C and correlated this peak broadening to the coexistence of lo and ld phases and to the exchange of lipids between these phases. In our study, the binary systems DPPC-2H62/cholesterol (7/3) and DPPC-2H62/ergosterol (7/3) or the ternary DPPC-2H62/glucosylcerebroside/ergosterol (1/2/2) composition led to much broader lines as compared with the binary or ternary systems containing phytosterols. The linewidth in the investigated lipid systems also depends on the rate of lipid exchange between lo and ld phases, which might coexist within a large temperature range. If we suppose a strong acceleration of the lipid exchange between lo and ld domains, we expect well-resolved spectra that show the averaged state of the lipid molecules between lo and ld states. At the contrary, slowing down of interphase lipid exchange would lead to line broadening. Very small domains that possess long interphase borderlines relative to the total domain surface would favor rapid lipid exchange rates between lo- and ld-phase. In contrast, huge domains, which possess short interphase borderlines relative to the total domain surface, would favor low lipid exchange rates between lo- and ld-phases. As a consequence, our results suggest that domains of smaller size would be promoted in the presence of phytosterols and especially with sitosterol. Interestingly, Xu et al. (12) showed that sitosterol-induced domain formation led to anomalously low detergent insoluble fractions, i.e., domains were almost completely dissolved when they were treated with Triton-X 100, at 23°C. The comparison of this result with fluorescence quenching, which indicated stronger domain forming capability of sitosterol compared to cholesterol at 23°C, let the authors conclude that domains induced by sitosterol were not as tightly packed as domains formed by other sterols. Caution must be taken here, our results show that ordering is similar at ambient temperature, in all ternary systems investigated, but that there are rapid lipid exchange rates between lo- and ld-phase leading to well resolved depaked spectra in the case of sitosterol. We believe that this is what the above authors attribute to lesser packing in between domains.

Biological implications
In plant cells, enzymes transfer alkyl groups to the C-24 of sterols. If we suppose that the relative activities of the different branches of the plant sterol biosynthesis are regulated, the concentrations of major sterols in plants, like sitosterol, stigmasterol, and cholesterol could be controlled. In fact, mutations of enzymes involved in the biosynthesis of sterols in plants led to various different phenotypes of Arabidopsis thaliana, which were reviewed extensively by Schaller (2 , 3) . This shows the importance of equilibrated sterol concentrations for plant growth and development. As described above, our data on ternary systems show clearly that the temperature sensitivity of "raft"-like model membranes with phytosterols and glucosylcerebroside is lower compared to the temperature sensitivity of the system with cholesterol and sphingomyelin. One may at this level of the discussion comment on the relevance of our probe, deuterated DPPC, and say that it may not account for the presence of unsaturated lipid chains as found in most membranes. This true in general but it is also well demonstrated that in rafts there is a tendency to segregate saturated chains, as those of sphingomyelin or glucosylcerebroside. So the choice of a fully saturated lipid probe (the only available at the time of the study) is sound and, in addition, it is present at only 20% in ternary systems. Returning to our main finding it appears that model membranes mimicking plant plasma membranes seem to be less temperature sensitive than those of animals. This suggests that cell membrane components like sitosterol, stigmasterol and glucosylcerebrosides, which are typical of plants, are produced in order to extend the temperature range in which membrane-associated biological processes can take place. This observation is well in accordance with the fact that plants have to face higher temperature variations than animals that usually can either regulate their body temperature or change their location in order to avoid extreme heat or coldness. Recently, Schrick and coworkers (6) found that several Arabidopsis sterol biosynthesis mutants showed a deficiency of cellulose in embryonic and postembryonic tissues, whereas pectin and sugar content was unchanged. Sitosterol-ß-glucoside (55) seems to serve as primer for plant cellulose biosynthesis. Therefore and although the observed reduction of cellulose (6) cannot directly be attributed to a lack of sterols as structurally important constituents in plasma membranes, the differential effect of sitosterol on membrane model systems implicates strongly that sitosterol might also be structurally important for cellulose synthesis and other membrane associated biological processes.


   ACKNOWLEDGMENTS
 
CNRS, University Bordeaux 1 and the French Ministry of Research and Education (AC CNRS DRAB) are thanked for research funding and Aquitaine region for NMR equipment. J.G.B. is grateful to the Friedrich-Ebert-Foundation for a fellowship. We would also like to thank Patrick Moreau (CNRS UMR 5200) for stimulating discussions on plant rafts.

Received for publication November 27, 2006. Accepted for publication January 11, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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