An X-ray diffraction study of model membrane raft structures Peter J Quinn1 and Claude Wolf2 Biochemistry Department, King’s College London, UK ´ ´ ER7-Faculte de Medecine-UPMC, APLIPID, Universite Paris 6, France Keywords lipid rafts; liquid-ordered phase; membrane rafts; sphingomyelin; X-ray diffraction Correspondence P J Quinn, Biochemistry Department, King’s College London, 150 Stamford Street, London SE1 9NH, UK Fax: +442078484500 Tel: +442078484408 E-mail: p.quinn@kcl.ac.uk (Received July 2010, revised September 2010, accepted September 2010) doi:10.1111/j.1742-4658.2010.07875.x Protein sorting and assembly in membrane biogenesis and function involves the creation of ordered domains of lipids known as membrane rafts The rafts are comprised of all the major classes of lipids, including glycerophospholipids, sphingolipids and sterol Cholesterol is known to interact with sphingomyelin to form a liquid-ordered bilayer phase Domains formed by sphingomyelin and cholesterol, however, represent relatively small proportions of the lipids found in membrane rafts and the properties of other raft lipids are not well characterized We examined the structure of lipid bilayers comprised of aqueous dispersions of ternary mixtures of phosphatidylcholines and sphingomyelins from tissue extracts and cholesterol using synchrotron X-ray powder diffraction methods Analysis of the Bragg reflections using peak-fitting methods enables the distinction of three coexisting bilayer structures: (a) a quasicrystalline structure comprised of equimolar proportions of phosphatidylcholine and sphingomyelin, (b) a liquid-ordered bilayer of phospholipid and cholesterol, and (c) fluid phospholipid bilayers The structures have been assigned on the basis of lamellar repeat spacings, relative scattering intensities and bilayer thickness of binary and ternary lipid mixtures of varying composition subjected to thermal scans between 20 and 50 °C The results suggest that the order created by the quasicrystalline phase may provide an appropriate scaffold for the organization and assembly of raft proteins on both sides of the membrane Co-existing liquid-ordered structures comprised of phospholipid and cholesterol provides an additional membrane environment for assembly of different raft proteins Introduction Cell membranes, once regarded as uniform structures, are now yielding up a complexity that is required to explain the multiplicity of tasks they are reputed to perform One particular function that demands a highly specific assembly of membrane components is the receipt and transmission of molecular signals from one side of the membrane to the other Current think- ing favours the, so-called, raft hypothesis, which postulates that the signalling elements are segregated and assembled in ordered lipid domains in the membrane [1–6] This membrane heterogeneity is rationalized on the basis that, for the efficient operation of a signalling system, the protein components must be closely associated and organized in such a way that structural Abbreviations brainSM, bovine brain sphimgomyelin; egg-PtdCho, hen egg-yolk phosphatidylcholine; GPI, glycerylphosphorylinsitol; SAXS, small-angle (1°–14°) X-ray scattering; WAXS, wide-angle (12°–30°) X-ray scattering FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4685 Membrane raft structure P J Quinn and C Wolf changes accompanying the generation of a signal are coupled to the transducing elements responsible for execution of the response [7] Critical tests of the hypothesis have largely been aimed at characterizing the forces that govern the creation of lipid rafts rather than identifying the way in which the signalling complexes are assembled within the structure [8] The main obstacle to progress has been the use of unreliable methods to isolate membrane rafts The conventional protocol, irrespective of the type of membrane, has been to recover a membrane fraction that survives dissolution by Triton X100 treatment at °C The integrity of this method has recently been challenged [7] and alternative methods based on a milder detergent treatment that is more compatible with physiological conditions have been developed [9] The resulting membrane raft fraction retains properties consistent with an arrangement of constituents expected of its biological progenitor Using such methods, it has been possible to demonstrate that subpopulations of raft vesicles which contain predominantly one surface antigen or another can be separated by immunoadsorption Moreover, analyses of the composition of these subpopulations show that they contain different proportions of specific polar lipids [10] The fatty acid substituents attached to cerebrosides and sphingomyelins also differ and represent products of different metabolic pools; they are consequently remodelled via different pathways One remarkable feature of the lipid analysis is the relatively high proportion (20–30%) of monounsaturated polar lipids Moreover, the proportion of polyunsaturated molecular species of phospholipids, particularly phosphatidylinositols, increases following the activation of raft proteins [11] These findings appear contrary to the idea that cholesterol preferentially forms liquidordered phases with saturated molecular species of phospholipid [12] Taken together, these results support not only the notion that the rafts are truly domains present in the parental membrane, but also that the lipids are distinct in each raft population The results also infer that membrane lipids may fulfil more specific functions in the segregation and assembly of protein components in the raft domains than hitherto contemplated Many attempts have been made to model membrane lipid rafts, some of which are focused on gel-phase separation of lipid mixtures comprised of molecular species that differ in the temperature of their transition between gel and liquid–crystal phases [13,14] The relevance of these studies was underscored by the fact that molecular species of sphingomyelin found in membranes and enriched in membrane raft fractions 4686 exhibited order–disorder transitions poised around physiological temperatures Attention switched to cholesterol when it was reported that the condensing effect of sterols on phospholipids, particularly sphingomyelins, created a bilayer phase that has properties intermediate between a gel and a liquid–crystal phase, referred to as a liquid-ordered phase [15,16] Cholesterol is known to be a prominent lipid component of membrane rafts irrespective of the isolation method [6,17] A third type of lipid enriched in membrane rafts are the glycosphingolipids [18] Because the molecular species of sphingolipids are characterized by a high proportion of long N-acyl fatty acids (C-22 to C-26) it was suggested that these lipids may act to couple the two leaflets of the bilayer by interdigitation of the long chain fatty acid from one side to the other of the structure [19,20] Other suggested functions of these asymmetric lipids have been to stabilize highly curved membrane domains formed transiently in the process of membrane budding and fusion during progress along the secretory pathway [21], or to increase hydrocarbon packing density to impede the permeability of small solute molecules [22] More recent molecular dynamics simulation studies are more equivocal on this point and although long hydrocarbon chains are able to penetrate the opposing monolayers of fluid bilayers, the terminal region of the chain appears to be localized in the centre of the bilayer [23] Other experimental and thermodynamic arguments have also cast doubt on the action of long-chain molecular species of lipids in coupling domains of bilayer structures [24,25] The role of these long-chain molecular species has now been reassessed in the light of the action of these asymmetric sphingolipids to form stoichiometric complexes with phospholipids that have the properties of a quasicrystalline phase [26,27] We have undertaken an examination of the phase behaviour of ternary mixtures containing representatives of all the lipid classes identified in membrane raft preparations Phospholipids of biological extraction were used so that a range of molecular species of phosphatidylcholines and sphingomyelins are present The thermotropic phase behaviour was examined in multilamellar dispersions at temperatures spanning the physiological range to characterize the miscibility of the different lipids under conditions in which mammalian membrane rafts are likely to form The use of synchrotron X-ray powder diffraction methods is able to provide detailed information on phase coexistence in complex bilayers as well as on coupling of the two monolayers of the bilayer, an essential feature in the formation of a membrane raft FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS P J Quinn and C Wolf Membrane raft structure Results Thermotropic phase behaviour of ternary mixtures To characterize the thermotropic phase behaviour of ternary mixtures of egg-phosphatidylcholine (PtdCho) ⁄ brain sphingomyelin (SM) ⁄ cholesterol, aqueous dispersions equilibrated at 20 °C were subjected to initial heating scans to 50 °C and subsequent cooling scans to 20 °C at 2°Ỉmin)1 The intensity of scattered X-rays was recorded simultaneously in the small-angle (SAXS = 1°–14°) and wide-angle (WAXS = 12°–30°) scattering regions during the scans The results obtained from an initial heating scan of a ternary mixture comprised of egg-PtdCho ⁄ brainSM ⁄ cholesterol in molar proportions 80 : 10 : 10 are presented in Fig 1A Two series of reflections in the SAXS region can be detected and they are in the order : ⁄ : ⁄ (only the first two-orders are shown), indicating that all structures are lamellar Within each order of Bragg reflection more than one lamellar phase is present; this is particularly evident from the second-order reflections in which overlapping peaks are obvious The absence of a sharp WAXS peak indicates that no gel or crystal phases are present in the mixture [28] The scattering intensity profiles were subject to a peak fitting analysis to characterize the coexisting lamellar phases The SAXS data were best fitted by three Gaussian + Lorentzian curves as seen in Fig S1C,D The fit of two peaks to the Bragg peak is shown for comparison in Fig S1A,B The relationship between d-spacings of the three individual peaks and temperature is plotted in Fig 1B The fact that discrete lamellar reflections can be deconvolved from the scattering bands means that the two leaflets of each of the respective bilayer structures are coupled An analysis of the scattering intensity profiles recorded during a subsequent cooling scan (see Figs S2 and S3) indicates that the changes observed in lamellar d-spacings (Fig 1B) during the heating scans are completely reversible with no significant temperature hysteresis This is consistent with the absence of any structural alteration in the bilayer or thickness of the A C Fig Characterization of egg-PtdCho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10 An overview of small- and wide-angle X-ray scattering intensity profiles recorded from an aqueous dispersion of egg-PtdCho ⁄ brainSM ⁄ cholesterol in molar proportions 80 : 10 : 10, recorded during a heating scan at 2°Ỉmin)1 between 20 and 50 °C, is shown in (A) as the scattering intensity profiles from the first two orders of lamellar repeat structures and wide-angle scattering profiles (B) Lamellar d-spacings (C) Scattering intensities (D) Peak shape (scattering amplitude ⁄ full-width at half maximum intensity) of the first-order lamellar structures (E) WAXS d-spacings (F) WAXS scattering intensities B D E F FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4687 Membrane raft structure P J Quinn and C Wolf hydration layer characterizing the dimensions of the lamellar unit cell The scattering intensities of the three peaks and an index of the peak sharpness (peak amplitude ⁄ full width at half maximum intensity) are presented in Fig 1C,D, respectively Unlike lamellar d-spacings, the decrease in scattering intensity observed during the initial heating scan is not reversed during the subsequent cooling scan (Fig S1B,C) Likewise, the simultaneous broadening of these peaks is not reversed on cooling This suggests that the size, but not the structure as judged by lamellar d-spacing, of the scattering arrays decreased during heating from the equilibration temperature to $ 35 °C as a consequence of the fragmentation of the scattering units into smaller, possibly less well-ordered, arrays Heating to higher temperatures appears to have no additional effect on the arrangement of the scattering units, therefore, a reliable indication of the relative amounts of lamellar structure in the deconvolved peaks contributing to the overall scattering intensity can be obtained at 38 °C It is noteworthy that the parameters of the peak of greatest d-spacing, which contributes least to the total scattering intensity, are relatively constant during the temperature scans This may indicate that A B C the arrangement and presentation of the scattering units in this lamellar structure not change significantly with temperature A peak-fitting analysis of the WAXS intensity profiles was undertaken and the results are presented in Fig 1E,F There is no evidence of a sharp peak at $ 0.42 nm to indicate the presence of a gel phase A minor peak located at a d-spacing of 0.45 nm can be deconvolved from the scattering profiles recorded at temperatures < 30 °C during the initial heating scan, but this peak becomes indistinguishable from a broad scattering band at $ 0.463 nm typical of disordered hydrocarbons at higher temperatures A ternary mixture containing higher proportions of brainSM and cholesterol was then examined and the results are presented in Fig The scattering intensity patterns recorded in the SAXS and WAXS regions during the initial heating scan from 20 to 50 °C from the ternary mixture comprised of egg-PtdCho ⁄ brainSM ⁄ cholesterol, 60 : 20 : 20, are presented in Fig 2A The SAXS intensity peaks in this mixture are best fit by only two Gaussian + Lorentzian curves, in contrast to the mixture shown in Fig Lamellar d-spacings, scattering intensity profiles and peak D E 4688 F Fig Characterization of egg-PtdCho ⁄ brainSM ⁄ cholesterol; 60 : 20 : 20 An overview of small- and wide-angle X-ray scattering intensity profiles recorded from an aqueous dispersion of egg-PtdCho ⁄ brainSM ⁄ cholesterol in molar proportions 60 : 20 : 20, recorded during a heating scan at 2°Ỉmin)1 between 20 and 50 °C, is shown in (A) as the scattering intensity profiles from the first two orders of lamellar repeat structures and wide-angle scattering profiles (B) Lamellar d-spacings (C) Scattering intensities (D) Peak shape of the second-order of the lamellar structures (E) WAXS d-spacings (F) WAXS scattering intensities FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS P J Quinn and C Wolf Membrane raft structure shapes derived from analysis of the thermal scans are shown to be distinct in Fig 2B,C,D, respectively It can be seen that the peak of shortest d-spacing observed in the mixture comprised of 80 : 10 : 10 (Fig 1B) is absent from this ternary mixture Moreover, the remaining two lamellar phases have correspondingly greater lamellar d-spacings than those observed in the mixture shown in Fig The temperature-dependent change in scattering intensity is considerably less marked, suggesting that the scattering units are more stable when the proportions of brainSM and cholesterol in the mixture are increased relative to eggPtdCho The Bragg peaks also tend to be sharper The dominant scattering peak in the WAXS region is shifted to shorter d-spacings indicating that increased proportions of brainSM and cholesterol bring about a closer packing in the hydrocarbon region of the bilayers The effect of increasing only the proportion of brainSM in the ternary mixture is exemplified by the behaviour of a mixture comprised of egg-PtdCho ⁄ brainSM ⁄ cholesterol, 10 : 80 : 10 shown in Fig The scattering intensity profiles in the SAXS region show the first two orders of reflection of lamellar phases and the presence of a relatively sharp WAXS peak at 0.42 nm, indicating that a gel phase forms on equilibration at 20 °C This WAXS peak coexists with a relatively weak scattering band at $ 0.47 nm which can no longer be distinguished from the main peak at a dspacing at 0.44–0.45 nm upon heating above $ 32 °C The changes observed in the SAXS ⁄ WAXS profiles are consistent with a progressive replacement of a gel phase of brainSM and the disappearance of a small proportion of a coexisting highly disordered lamellar phase with a homogeneous liquid-ordered phase of dspacing 0.44 nm during heating to 32 °C At higher temperatures, a new lamellar phase of greater d-spacing appears but represents only a relatively minor component of the overall scattering intensity It can be concluded from analysis of the behaviour of this ternary mixture that the properties of the major constituent of the mixture, namely, long N-acyl chain molecular species of sphingomyelin, tend to dominate the temperature-dependent structural parameters of the bilayers Assignment of lamellar structures The next task was to establish the identity of the coexisting lamellar phases in ternary mixtures contain- A C Fig Characterization of egg-PtdCho ⁄ brainSM ⁄ cholesterol; 10 : 80 : 10 An overview of small- and wide-angle X-ray scattering intensity profiles recorded from an aqueous dispersion of egg-PtdCho ⁄ brainSM ⁄ cholesterol in molar proportions 10 : 80 : 10, recorded during a heating scan at 2°Ỉmin)1 between 20 and 50 °C, is shown in (A) as the scattering intensity profiles from the first two orders of lamellar repeat structures and wide-angle scattering profiles (B) Lamellar d-spacings (C) Scattering intensities (D) Peak shape of the first-order lamellar structures (E) WAXS d-spacings (F) WAXS scattering intensities B D E F FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4689 Membrane raft structure P J Quinn and C Wolf ing relatively high proportions of the fluid host phospholipid, egg-PtdCho, which are representative of the lipid composition of mammalian membrane extracellular leaflet embedding the raft microdomains Three possible lamellar structures comprised of varying proportions of lipids are brainSM ⁄ egg-PtdCho, brainSM ⁄ cholesterol or egg-PtdCho ⁄ cholesterol; a ternary complex of the three lipids is excluded as implausible in this analysis because most ternary mixtures are comprised of more than one bilayer component Figure shows a method of assigning the composition of the different lamellar phases on the basis of the relationship between lamellar d-spacing and temperature The result of a peak-fitting analysis of the SAXS intensity profile recorded from the ternary mixture comprised of egg-PtdCho ⁄ brainSM ⁄ cholesterol, 80 : 10 : 10, at 38 °C is presented in Fig 4A The profile can be seen to be best fit by three Gaussian + Lorentzian peaks which are shown in Fig 4B together with the difference between the observed and calculated fit to the data (Fig 4C) (see Fig S1) Peak represents $ 10% of the total scattering from the first-order Bragg reflections The d-spacings of this peak coincide closely with d-spacing recorded from a binary mixture of egg-PtdCho ⁄ brainSM in equimolar proportions recorded under the same conditions (Fig 4D) It is known that gel-phase separation occurs in this binary mixture when equilibrated at 20 °C [29], however, there is no evidence that gel-phase separation occurs in this ternary mixture (Fig 1E) The presence of 10 mole% cholesterol in the ternary mixture apparently hinders formation of a gel phase by brainSM in this mixture Assignment of peak to a structure of pure brainSM can also be excluded on this evidence The fit of peak to brainSM ⁄ cholesterol mixtures was considered from the respective dimensions of the unit cell (d-spacings) and peak shape parameter representing the order of the diffracting units The effect of varying proportions of cholesterol in binary mixtures with brainSM is presented in Fig 5A It can be seen that the d-spacing of brainSM bilayers at 38 °C is 8.3 nm and this is progressively reduced by increasing the proportions of cholesterol (Fig 5C) An equimolar proportion of cholesterol would be required to reduce the d-spacing to that observed for peak (6.8 nm) in Fig 4C The assignment of peak as comprised of egg-PtdCho and $ 20 mole% cholesterol (Fig 5D) cannot be excluded on this criterion Other evidence presented below, however, indicates that cholesterol is not a constituent of peak The scattering intensity of peak contributes $ 30% to the total intensity of the first-order Bragg 4690 A B C D Fig Assignment of lamellar structures An analysis of the ternary mixture of egg-PtdCho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10 recorded at 38 °C (A) Fit of scattering intensity from the first-order Bragg reflection (d) to three Gaussian + Lorentzian area curves (s) (B) Peak deconvolution from the scattering intensity profile shown in (A) (C) Difference between observed and calculated fits to the data (D) Lamellar d-spacings as a function of temperature recorded during heating scans at 2°Ỉmin)1 d, peak 1; s, egg-PtdCho ⁄ brainSM, 50 : 50; j, peak 2; h, egg-PtdCho ⁄ cholesterol, 70 : 30; m, peak 3; D, egg-PtdCho peaks of the mixture shown in Fig The d-spacing of peak in Fig 4B coincides closely with bilayers formed from a binary mixture of egg-PtdCho and proportions of cholesterol of $ 25 mole% (Fig 5D) The effect of cholesterol on d-spacings of egg-PtdCho bilayers is complex The presence of only 10 mole% FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS P J Quinn and C Wolf Membrane raft structure A B C D Fig Binary mixtures of phospholipid and cholesterol Small-angle X-ray scattering intensity patterns of binary mixtures of (A) brainSM and (B) egg-PtdCho with the indicated mol% cholesterol at 37 °C (C) and (D) show the respective relationships between lamellar d-spacing and mol% cholesterol cholesterol causes a dramatic increase in d-spacing because of the full extension and vertical orientation of the hydrocarbon chains of the phospholipid in the bilayer Increasing proportions of cholesterol up to 30 mole% result in a progressive decrease in repeat spacing because of hydration effects at the bilayer– water interface An assignment of peak to a binary mixture of brainSM and cholesterol can be excluded on the basis of lamellar d-spacings > nm at 38 °C [30] Assuming peak is comprised of phospholipid and 25 mole% cholesterol, the contribution of the peak to the total scattering from the ternary mixture is calculated to be 25% This is close to the observed proportion of the total scattering from peak in the ternary mixture Assignment of the dominant peak (peak 3) representing $ 60% of total scattering at 38 °C in the ternary mixture egg-PtdCho ⁄ brainSM ⁄ cholesterol, 80 : 10 : 10, was made by comparison with bilayers of pure egg-PtdCho, the most abundant phospholipid in the mixture As can be seen from Fig 4C, the d-spacing of the pure phospholipid is $ 0.5 nm less at equivalent temperatures than observed for peak Because the d-spacing is less than binary mixtures containing high proportions of cholesterol in egg-PtdCho, it follows that the increase in d-spacing of peak must be caused by the presence of proportions of cholesterol < 10 mole% That peak is comprised predominantly of egg-PtdCho is also evident from the absence of this peak in ternary mixtures containing lower proportions of egg-PtdCho, as demonstrated in the ternary mixture consisting of egg-PtdCho ⁄ brainSM ⁄ cholesterol, 60 : 20 : 20 examined in Fig Thus, assignment of peak 3, as judged by d-spacing, can be made as eggPtdCho containing a relatively small proportion of cholesterol Further evidence consistent with assignment of peak to a liquid-ordered lamellar phase comprised of an equimolar proportion of egg-PtdCho and brainSM was obtained by relating the relative mass of brainSM in ternary mixtures of egg-PtdCho ⁄ brainSM ⁄ cholesterol to the scattering contribution from peak to the total scattering intensity recorded at 38 °C A peak of lamellar repeat of 6.7–6.8 nm could be deconvolved from first-order Bragg reflections in 12 ternary mixtures examined; no peak at this position was observed in mixtures with proportions of cholesterol exceeding 50 mole% There was no correlation between the scattering intensity of this peak and the relative mass of egg-PtdCho, brainSM, cholesterol or any binary combinations of the lipids in the mixtures (Fig S4) However, if the contribution to the scattering of the peak was limited to a mass of brainSM equivalent to the proportion of egg-PtdCho in those ternary mixtures where the mol% of brainSM exceeds that of egg-PtdCho, a correlation is obtained A plot of the relationship between the scattering intensity of the peak and the mass of equimolar proportions of brainSM ⁄ FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4691 Membrane raft structure P J Quinn and C Wolf Fig Relationship between brainSM and scattering intensity Correlation between relative scattering intensity of peak in Fig from the first-order Bragg reflection (d-spacing 6.7–6.8 nm) and mass of brainSM + equimolar egg-PtdCho in different ternary mixtures of the two phospholipids and cholesterol recorded at 38 °C Mixtures with proportions of brainSM greater than egg-PtdCho were taken as equimolar to the proportion of egg-PtdCho in the mixture egg-PtdCho in different ternary mixtures is shown in Fig A third method to investigate the assignments of composition of peaks and was to compare relative electron densities through the bilayer repeat structures The results of such calculations are summarized in Fig It can be seen that relative electron density distributions across the bilayer repeats calculated at 38 °C for peaks and are different By contrast, the thickness of the bilayers and the water layers of peak are almost identical to those calculated for bilayers consisting of an equimolar mixture of egg-PtdCho and brainSM The bilayer thickness of peak is significantly greater, and the water layer significantly less, than calculated for peak Resolution of the electrondensity calculation for peak was relatively low because only three orders of reflection were detected in this mixture Nevertheless, the thickness of the bilayer and water layer are almost identical to the parameters calculated from a binary mixture comprised of eggPtdCho and 30 mole% cholesterol, a characteristic Lo phase [31] where three orders of reflection were used in the calculation Discussion The lipids identified in rafts isolated from biological membranes differ from those of the parent membrane, 4692 Fig Electron-density calculations Relative electron density profiles were calculated through the lamellar repeats of peak and peak recorded at 38 °C from the data in Fig 1A Relative electron densities calculated from binary mixtures of brainSM ⁄ egg-PtdCho in equimolar proportions and egg-PtdCho ⁄ 30 mole% cholesterol at 38 °C are shown for comparison but such results need to be regarded with some caution at this stage The reason is that reliable methods of isolating rafts have not generally been employed The size of domains in living cell membranes is defined as between 10 and 200 nm [32], and vesicles derived from rafts occupying areas of the parent membrane of this order would be between and 30 nm in diameter This is at odds with the size of vesicles isolated as detergent-resistant membranes Estimates of the size of detergent-resistant membrane vesicles prepared from rat brain indicate a relatively homogeneous population of unilamellar vesicles of diameter ranging from 130 to 240 nm [33] This corresponds to an average domain diameter in the parent membrane in the order of 600 nm, somewhat larger than areas envisaged for membrane raft domains Subpopulations of these vesicles can be separated by immunoadsorption methods containing different surface antigens, which argues against the fusion and amalgamation of domains in the parent membrane Electron microscopy examination of these vesicles indicates that the prion protein (PrPc) and thymusderived antigen (Thy-1) associated with these raft preparations are generally clustered together and occupy only a small fraction of the vesicle membrane [9] This suggests that the rafts are not homogeneous FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS P J Quinn and C Wolf bilayers of lipids in liquid-ordered phase but that an organization is imposed on the proteins that causes their association within the structure The question of whether membrane proteins or lipids alone or together play a part to bring about the segregation of raft components is a moot point It is known that successful delivery of plasma membrane raft proteins from the Golgi in yeast depends on the biosynthesis of ergosterol and sphingolipids [34] Genetic screening of defective mutants indicated a lack of a functional fatty acid elongating system for synthesis of long-chain molecular species of sphingolipids [35], or a defect of dihydrosphingosine C4 hydroxylase in the biosynthesis of phytosphingosine [36] may be responsible One possible mechanism for organizing proteins in the liquid-ordered phase is by homotypic interactions between the proteins themselves or interactions mediated by intermediary proteins An example of the latter is the clustering of Pma1p, the plasma membrane H+-ATPase of yeast, in raft lipid domains This has been shown to require a peripheral membrane protein, Ast1, in the endoplasmic reticulum, a process that is an essential step in the transfer of the raft protein to the plasma membrane [37] In the case of the N+ ⁄ H+ antiporter in yeast (Nha1p), however, the sorting signal apparently resides in the hydrophobic domain of the membrane [38] and sphingolipid is essential for retention of the protein in the plasma membrane [39] There is also a strong possibility that the lipid anchors that tether proteins to membrane rafts may interact in a specific way with the lipids forming the raft Clearly, there is scope for different methods of organizing proteins within membrane rafts, but it is not easy to envisage how the specificity required to bring about clustering of one type of receptor protein on one side of the membrane, and co-localizing this with specific lipid-anchored proteins on the opposite side of the membrane, can occur simply within a liquidordered phase of polar lipid and cholesterol On the basis of the evidence obtained in this study, such specificity can be proposed The structures formed by binary mixtures of long N-acyl molecular species of sphingolipids and phospholipids consist of stoichiometric complexes of : phosphatidylethanolamines [27] and : phosphatidylcholine [26] A phase with hydrocarbon chain spacings consistent with a liquidordered quasicrystalline phase, formed from equimolar proportions of egg-PtdCho and brainSM [29], has been identified in this study in ternary mixtures with cholesterol We propose that the structure formed by long N-acyl fatty acid molecular species of sphingolipids Membrane raft structure and phospholipids creates a matrix that is coupled across the raft membrane According to such a model, glycosphingolipids based on molecular species of galactosylceramides with long N-acyl fatty acids and phosphatidylcholines would reside in the outer monolayer in mammalian plasma membrane These domains are coupled with glucosylceramides and phosphatidylethanolamines in the cytoplasmic leaflet These structures form a matrix into which GPI-anchored receptor proteins are interpolated on the outer surface and are coupled with corresponding lipid-anchored effecter proteins located in the cytoplasmic leaflet The specificity of these interactions may involve the sugar residues of the glycosphingolipids and the domains of the proteins, or the configuration of the lipid anchor, or both That activity-associated remodelling of lipid anchors is a recognized process in raft function [40,41] suggests that the configuration of the raft anchors is a likely candidate The lipid matrix model of the membrane raft structure can be formulated according to a two-stage process of molecular assembly Cartoons of the structures comprising the model and their relationship to bilayer spacings are presented in Fig Liquid-ordered domains comprised of more saturated molecular species of phospholipid and cholesterol serve to exclude most membrane proteins and accommodate those proteins required for raft function Glycosphingolipids with long N-acyl fatty acid chains and phospholipids form a quasicrystalline matrix acting to concentrate and organize the protein components into a functional complex in the raft It is reported that cholesterol is excluded from such phases [42] The remodelling of lipid anchors takes place in the liquid-ordered domain in a manner that allows them to interpolate into the matrix component of the raft membrane The intimate association between receptors and effectors brought about by their integration into the matrix is an essential feature designed to facilitate the transmission of molecular signals generated on one side of the matrix to the other The model of raft structure we propose fits current knowledge of the lipid composition of membrane rafts obtained without detergent treatment Eighty-three molecular species of membrane lipid have been identified and quantified in highly purified raft preparations from yeast [43] Glycosphingolipids with almost exclusively long-chain hydroxylated N-acyl substituents [C26:0(OH)] are present in equimolar proportions with di-unsaturated molecular species of phosphatidylcholine and would be expected to form a quasicrystalline bilayer structure The remaining phospholipids are dominated by phosphatidylinositol with a saturated FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4693 Membrane raft structure P J Quinn and C Wolf Fig Cartoons of the molecular composition of the different structures proposed for the lipid matrix model of membrane raft structure and their relationship to the Bragg spacings of ternary mixtures of egg-PtdCho ⁄ brainSM ⁄ cholesterol Other evidence is reviewed in Quinn [6] fatty acid acylated to the C-1 position of the glycerol This would form a liquid-ordered phase with a composition comprised of 38 mole% ergosterol The order of the lipids in the membrane rafts as measured by spectroscopic studies using C-Laudan is consistent with the tight packing of acyl chains in the raft model membrane Materials and methods Lipids Egg-yolk phosphatidylcholine (egg-PtdCho, 715 Da), bovine brain sphingomyelin (brainSM, 788 Da) and cholesterol (387 Da) were purchased from Sigma (Sigma-Aldrich, St Quentin-Fallavier, France) A complete lipid analysis of each phospholipid was performed by ESI-tandem MS [29] and the data are presented in Table S1 4694 Sample preparation Samples for X-ray diffraction examination (Table S2) were prepared by dissolving lipids in warm (45 °C) chloroform ⁄ methanol (2 : 1, v ⁄ v) and mixing them in the desired proportions (denoted as molar ratios in binary mixtures) The organic solvent was subsequently evaporated under a stream of oxygen-free dry nitrogen at 45 °C and any remaining traces of solvent were removed by storage under high vacuum for days at 20 °C Dry lipids were hydrated with an equal mass of water This was sufficient to fully hydrate egg-PtdCho [44] and brainSM [45], respectively The lipids were stirred thoroughly with a thin needle, sealed under argon, and annealed by 50 thermal cycles between 20 and 65°C, ensuring a complete mixing of phospholipids Samples were stored under argon at a temperature not below °C X-Ray diffraction examination was performed after h sample equilibration at 20 °C and after careful stirring before transfer into the sample cell In order to check FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS P J Quinn and C Wolf for possible dehydration or demixing of the components, various control measurements were undertaken such as checking for reversibility of phase behaviour during subsequent heating and cooling cycles The samples were also checked for the absence of SAXS and WAXS diffraction peaks from crystals of cholesterol The mean transition temperatures of lipids were found in the expected range documented in the data bank Lipid Data Bank (LDB; http://www.lipidat.tcd.ie/) A list of the samples examined in this study are tabulated in the Supporting information Synchrotron X-ray diffraction measurements X-Ray diffraction measurements were performed on beamline 2.1 at the Daresbury Laboratory The X-ray wavelength was 0.154 nm with a beam geometry of $ 0.2 · 0.5 mm in a mica sandwich cell with a surface of · mm and a path length of 0.5 mm Simultaneous SAXS and WAXS intensities were recorded so that a correlation could be established between the mesophase repeat spacings and the packing arrangement of acyl chains The SAXS intensity was recorded using a 2D RAPID area detector and the signal was circularly integrated to give a 1D pattern The WAXS intensity was recorded with a 1D HOTWAXS detector The sample to SAXS detector distance was 1.5 m and calibration of d-spacings was performed using silver behenate (d = 5.838 nm) Wide-angle X-ray scattering intensity profiles were calibrated using the diffraction peaks from high-density polyethylene [46] The measurement cell was mounted on a programmable temperature stage (Linkam, Tadworth, UK) and the temperature was monitored by a thermocouple inserted directly into the lipid dispersion (Quad Service, Poissy, France) The set-up, calibration and facilities available on Station 2.1 are described comprehensively in the website; http://www.srs ac.uk/srs/stations/station2.1.htm Data reduction and analysis were performed using originpro8 software (OriginLab Corp., Northampton, MA, USA) Analysis of X-ray diffraction data The small-angle X-ray scattering-intensity profiles were analysed using standard procedures [47] Polarization and geometric corrections for line-width smearing were assessed by checking the symmetry of diffraction peaks in the present camera configuration using a sample of silver behenate The orders of reflection could all be fitted by Gaussian + Lorentz symmetrical (Voigt) functions with fitting coefficients greater than R2 = 0.99 Deconvolution is consistent with the sample to detector distance used [48] It was noted that scattering intensity from some lamellar repeat structures decreased significantly during heating samples equilibrated at 20 °C up to $ 35 °C and remained constant irrespective of the temperature for the duration of a heating and cooling cycle Because the changes in d-spac- Membrane raft structure ings were completely reversible, the changes in scattering intensity are consistent with a decrease in the size and order within the diffracting units from that achieved during temperature equilibration (see methods of powder diffraction analysis in the Supporting information) The scattering intensity data from the first four orders of the Bragg reflections from the multilamellar liposomes were used to construct electron density profiles [49] After correction of the raw data by subtraction of the background scattering from both water and the sample cell, each of the Bragg peaks was fitted by a Lorentzian+Gaussian area (Voigt) distribution by peak fitting performed using peakfit 4.12 (Systat Software Inc., Bangalore, India) Details of the peak fitting procedure are described in the Supporting information The square root of integrated peak intensity I(h) is used to determine the form factor F(h) of each respective reection: p Fhị ẳ h Ihị 1ị where h = order of peak reflection, I(h) = integrated intensity of each respective reflection The electron density profile is calculated by the Fourier synthesis: X qzị ẳ ặ Fhịcos2phz=dị 2ị d = dspacing = dpp + dW (dpp: phosphate-phosphate bilayer thickness, dW: hydration layer) at a resolution of d ⁄ 2hmax $ 12 nm for four orders The phase sign of each diffraction order is either positive or negative for a centro symmetric electron-density profile for lamellar phases The phasing choice was made by inspection of all possible phase combinations; for all bilayers examined the phase combination () ) + )) of signs uniquely provides the expected electron density profile with the minimum density appropriately located at the bilayer centre, the maxima at the two electron-rich interfaces and the hydration layer density (0.33e) ⁄ A3) at the intermediate value on the relative electron density scale All other phase combinations result in aberrant distributions Deconvolution of the WAXS intensity peaks was also undertaken using peakfit software No corrections for polarization or geometric factors were necessary with the HOTWAXS detector Scattering in the WAXS region originates predominantly from hydrocarbon chains of the phospholipids with a diffuse scattering contribution from the polar head-groups The presence of cholesterol in the bilayer is known to impose an orientation of the chains normal to the bilayer plane [50] Acknowledgements The authors are grateful to Dr Gunter Grossman for assistance in setting up beamline 2.1 at the Daresbury FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4695 Membrane raft structure P J Quinn and C Wolf Laboratory Drs Galya Staneva and Lin Chen are thanked for providing assistance in preparing samples The work was aided by a grant of beamtime (DL49098) and funds provided by the Human Science Frontier Program (RGP0016 ⁄ 2005C) 14 References Brown DA & London E (1998) Function of lipid rafts in biological membranes Annu Rev Cell Dev Biol 14, 111–136 Vereb G, Szollosi J, Matko J, Nagy P, Farkas T, Matyus A, Waldmann TA & Damjanovich S (2003) Dynamic yet structured: the cell membrane three decades after the Singer–Nicoloson model Proc Natl Acad Sci USA 100, 8053–8058 Edidin M (2003) The state of lipid rafts: from model membranes to cells Annu Rev Biophys Biomol Struct 32, 257–283 Jacobson K, Mouritsen OG & Anderson RGW (2007) Lipid rafts: a crossroad between cell biology and physics Nat Cell Biol 9, 7–14 Owen DM, Williamson D, Rentero C & Gaus K (2009) Quantitative microscopy: protein dynamics and membrane organization Traffic 10, 962–971 Quinn PJ (2010) A lipid matrix model of membrane raft structure Prog Lipid Res 49, 390–406 Inglemo-Torres MM, Gaus K, Herms A, GonzalezMerino E, Kassan A, Bosch M, Grewal T, Tebar F, Enrich C & Pol A (2009) Triton X-100 promotes a cholesterol-dependent condensation of the plasma membrane Biochem J 420, 373–381 Kenworthy AK (2008) Have we become overtly reliant on lipid rafts? Talking point on the involvement of lipid rafts in T-cell activation EMBO Rep 9, 531–535 Chen X, Jen A, Warley A, Lawrence MJ, Quinn PJ & Morris RJ (2009) Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition Biochem J 417, 525–533 10 Brugger B, Graham C, Leibrecht I, Mombelli E, Jen A, Wieland F & Morris R (2004) The membrane domains occupied by glycosylphosphatidylinositol-anchored prion protein and Thy-1 differ in lipid composition J Biol Chem 279, 7530–7536 11 Han X, Smith NL, Sil D, Holowka DA, McLafferty FW & Baird BA (2009) IgE receptor-mediated alteration of membrane-cytoskeleton interactions revealed by mass spectrometric analysis of detergent-resistant membranes Biochemistry 48, 6540–6550 12 Quinn PJ & Wolf C (2009) The liquid-ordered phase in membranes Biochim Biophys Acta 1788, 33–46 13 McMullen TPW, Lewis RNAH & McElhaney RN (2009) Calorimetric and spectroscopic studies of the 4696 15 16 17 18 19 20 21 22 23 24 25 26 27 effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylglycerol bilayer membranes Biochim Biophys Acta 1788, 345–357 Staneva G, Chachaty C, Wolf C, Koumanov K & Quinn PJ (2008) The role of sphingomyelin in regulating phase coexistence in complex lipid model membranes: competition between ceramide and cholesterol Biochim Biophys Acta 1778, 2727–2739 Ipsen JH, Karlstrom G, Mouritsen OG, Wennerstrom H & Zuckermann MJ (1987) Phase equilibria in the phosphatidylcholine–cholesterol system Biochim Biophys Acta 905, 162–172 Sankaram M & Thompson TE (1990) Modulation of acyl chain order by cholesterol A solid-state 2H nuclear magnetic resonance study Biochemistry 29, 10676–10684 Persaud-Sawin D-A, Lightcap S & Harry GJ (2009) Isolation of rafts from mouse brain tissue by a detergent-free method J Lipid Res 50, 759–767 Prinetti A, Loberto N, Chigorno V & Sonnino S (2009) Glycosphingolipid behaviour in complex membranes Biochim Biophys Acta 1788, 184–193 Bar LK, Barenholz Y & Thompson TE (1997) Effect of sphingomyelin composition on the phase structure of phosphatidylcholine–sphingomyelin bilayers Biochemistry 36, 2507–2516 Pinto SN, Silva LC, de Almeida RFM & Prieto M (2008) Membrane domain formation, interdigitation, and morphological alterations induced by very long chain asymmetric C24:1 ceramide Biophys J 95, 2867–2879 Schneiter R, Brugger B, Amann CM, Prestwich GD, Epand RF, Zelling G, Weiland FT & Epand RM (2004) Identification and biophysical characterization of a verylong-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes Biochem J 381, 941–949 Toulmay A & Schneiter R (2007) Lipid-dependent surface transport of the proton pumping ATPase: a model to study plasma membrane biogenesis in yeast Biochimie 89, 249–254 Niemela PS, Hyvonen MT & Vattulainen I (2009) Atom-scale molecular interactions in lipid raft mixtures Biochim Biophys Acta 1788, 122–135 Almeida PFF (2008) Thermodynamics of lipid interactions in complex bilayers Biochim Biophys Acta 1788, 72–85 Collins MD (2008) Interleaflet coupling mechanisms in bilayers of lipids and cholesterol Biophys J 94, L32– L34 Quinn PJ (2009) Long N-acyl fatty acids on sphingolipids are responsible for miscibility with phospholipids to form liquid-ordered phase Biochim Biophys Acta 1788, 2267–2276 Feng Y, Rainteau D, Chachaty C, Yu Z-W, Wolf C & Quinn PJ (2004) Characterisation of a quasi-crystalline FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS P J Quinn and C Wolf 28 29 30 31 32 33 34 35 36 37 38 39 40 phase in codispersions of phosphatidylethanolamine and glucocerebroside Biophys J 86, 2208–2217 Tardieu A, Luzzati V & Reman FC (1973) Structure and polymorphism of hydrocarbon chains of lipids – study of lecithin–water phases J Mol Biol 75, 711–733 Quinn PJ & Wolf C (2009) Hydrocarbon chains dominate coupling and phase coexistence in bilayers of natural phosphatidylcholines and sphingomyelins Biochim Biophys Acta 1788, 33–46 Quinn PJ & Wolf C (2009) Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol Biochim Biophys Acta 1778, 1877– 1889 Chachaty C, Rainteau D, Tessier C, Quinn PJ & Wolf C (2005) Building up of the liquid-ordered phase formed by sphingomyelin and cholesterol Biophys J 88, 4032–4044 Pike LJ (2009) The challenge of lipid rafts J Lipid Res 50, S323–S328 Chen X, Lawrence MJ, Barlow DJ, Morris RJ, Heenan RK & Quinn PJ (2009) The structure of detergent-resistant membrane vesicles from rat brain cells Biochim Biophys Acta 1788, 477–483 Proszynski TJ, Klemm RW, Gravert M, Hsu PP, Gloor Y, Wagner J, Kozak K, Grabner H, Walzer K, Bagnat M et al (2006) A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast Proc Nat Acad Sci USA 102, 17981–17986 Oh CS, Toke DA, Mandala S & Martin CE (1997) EL02 and EL03 homologues of the Saccharomyces cerevisiae EL01 gene, function in fatty acid elongation and are required for sphingolipid formation J Biol Chem 272, 17376–17384 Haak D, Gable K, Beeler T & Dunn T (1997) Hydroxylation of Saccharomyces cerevesiae ceramides requires Sur2p and Scs7p J Biol Chem 272, 29704–29710 Bagnat M, Chang A & Simons K (2001) Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast Mol Cell Biol 12, 4129– 4138 Mitsui K, Kamauchi S, Nakamura N, Inoue H & Kanazawa H (2004) A conserved domain in the tail region of the Saccharomyces cerevisiae Na+ ⁄ H+ antiporter (Nha1p) plays important roles in localization and salinity resistant cell-growth J Biochem 135, 139– 148 Mitsui K, Hatakeyama K, Matsushita M & Kanazawa H (2009) Saccharomyces cerevisiae Na+ ⁄ H+ antiporter Nha1p associates with lipid rafts and requires sphingolipid for stable localization to the plasma membrane J Biochem 145, 709–720 Kinoshita T, Fujita M & Maeda Y (2008) Biosynthesis, remodeling and functions of mammalian GPI-anchored proteins: recent progress J Biochem 144, 287–294 Membrane raft structure 41 Iwanaga T, Tsutsumi R, Noritake J, Fukata Y & Fukata M (2009) Dynamic protein palmitoylation in cellular signaling Prog Lipid Res 48, 117–127 42 Bjorkqvist YJE, Brewer J, Bagatolli LA, Slotte JP & Westerlund B (2009) Thermotropic behavior and lateral distribution of very long chain sphingolipids Biochim Biophys Acta 1788, 1310–1320 43 Klemm RW, Ejsing CS, Surma MA, Kaiser H-J, Gerl MJ, Sampaio JL, De Robillard Q, Ferguson C, Proszynski TJ, Shevchenko A et al (2009) Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network J Cell Biol 185, 601–612 44 Jendrasiac GL, Smith RL & McIntosh TJ (1997) The effect of phloretin on the hydration of egg phosphatidylcholine multilayers Biochim Biophys Acta 1329, 159–168 45 Shipley GG, Avecilla LS & Small DM (1974) Phase behaviour and structure of aqueous dispersions of sphingomyelin J Lipid Res 15, 124–131 46 Addink EJ & Beintema J (1961) Polymorphism of crystalline polypropylene Polymer 2, 185–187 47 Zhang R, Suter RM & Nagle JF (1994) Theory of the structure factor of lipid bilayers Phys Rev E 50, 5047– 5060 48 Yao T & Jinno H (1982) Polarization factor for the X-ray-powder diffraction method with a single-crystal monochromator Acta Crysallogr A 38, 287–288 49 McIntosh TJ (1978) Effect of cholesterol on structure of phosphatidylcholine bilayers Biochim Biophys Acta 513, 43–58 50 Ladbrooke BD, Williams DM & Chapman D (1968) Studies of lecithin-cholesterol-water interactions by differential scanning calorimetry and X-ray diffraction Biochim Biophys Acta 150, 333–340 Supporting information The following supplementary material is available: Fig S1 First-order Bragg scattering intensity peak (•) recorded from the mixed aqueous dispersion of eggPtdCho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10 at 38 °C Fig S2 (A) Lamellar d-spacing, (B) scattering intensity and (C) amp ⁄ FWHM for a dispersion of egg-PtdCho ⁄ brainSM ⁄ cholesterol; 80 : 10 : 10, equilibrated for h at 20 °C during an initial heating and subsequent cooling scan at 2°Ỉmin)1 Fig S3 (A) Lamellar d-spacing, (B) scattering intensity and (C) amp ⁄ FWHM for a dispersion of egg-PtdCho ⁄ brainSM ⁄ cholesterol; 60 : 20 : 20, equilibrated for h at 20 °C during an initial heating and subsequent cooling scan at 2°Ỉmin)1 Fig S4 Relationship between the per cent scattering intensity observed in the peak of lamellar d-spacing of $ 6.7 nm deconvolved from ternary mixtures of FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS 4697 Membrane raft structure P J Quinn and C Wolf egg-PtdCho, egg-SM and cholesterol in varying proportions and (A) mass of egg-PtdCho, (B) mass brainSM, and (C) mass cholesterol Table S1 Fatty acid analysis of egg phosphatidylcholine and bovine brain sphingomyelin Table S2 Mixtures examined in this study This supplementary material can be found in the online version of this article 4698 Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 4685–4698 ª 2010 The Authors Journal compilation ª 2010 FEBS ... FEBS 4693 Membrane raft structure P J Quinn and C Wolf Fig Cartoons of the molecular composition of the different structures proposed for the lipid matrix model of membrane raft structure and their... remodelling of lipid anchors is a recognized process in raft function [40,41] suggests that the configuration of the raft anchors is a likely candidate The lipid matrix model of the membrane raft structure... parent membrane of this order would be between and 30 nm in diameter This is at odds with the size of vesicles isolated as detergent-resistant membranes Estimates of the size of detergent-resistant