Cholesterol–Ceramide Interactions in Phospholipid and Sphingolipid

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Cholesterol-ceramide interactions in phospholipid and sphingolipid bilayers as observed by positron annihilation lifetime spectroscopy and molecular dynamics simulations Aritz B. Garcia-Arribas, Eneko Axpe, Jon I. Mujika, David Merida, Jon V Busto, Jesús Sot, Alicia Alonso, Xabier Lopez, Jose Angel Garcia, Jesus M. Ugalde, Fernando Plazaola, and Felix M. Goni Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00927 • Publication Date (Web): 09 May 2016 Downloaded from http://pubs.acs.org on May 15, 2016

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Cholesterol-ceramide interactions in phospholipid and sphingolipid bilayers as observed by positron annihilation lifetime spectroscopy and molecular dynamics simulations

Aritz B. García-Arribas*†§, Eneko Axpe*‡, Jon Iñaki Mujika*¶||, David Mérida‡, Jon V. Busto†§, Jesús Sot†§, Alicia Alonso†§, Xabier Lopez¶||, Jose Ángel García#, Jesus M. Ugalde¶||, Fernando Plazaola‡ and Félix M. Goñi†§. †

Biofisika Institute (CSIC, UPV/EHU), 48080, Bilbao, Spain. Departamento de Bioquímica, University of the Basque Country (UPV/EHU), 48080, Bilbao, Spain. ‡ Department of Electricity and Electronics, University of the Basque Country (UPV/EHU), Leioa, Basque Country, Spain. ¶ Kimika Fakultatea, Euskal Herriko Unibertsitatea (UPV/EHU), Donostia, Euskadi, Spain. || Donostia International Physics Center (DIPC), Donostia, Basque Country, Spain. # Department of Applied Physics II, University of the Basque Country (UPV/EHU), Leioa, Basque Country, Spain. §

* = These authors contributed equally to this work

Keywords: lipid bilayers, cholesterol, ceramide, gel phase, PALS, molecular dynamics

Short title: Cholesterol-ceramide interaction in phospholipid bilayers

Correspondence should be addressed to: Félix M. Goñi Biofisika Institute (CSIC, UPV/EHU) Leioa 48940 Basque Country Spain e-mail: [email protected]

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ABSTRACT Free volume voids in lipid bilayers can be measured by Positron Annihilation Lifetime Spectroscopy (PALS). This technique has been applied, together with differential scanning calorimetry and molecular dynamics (MD) simulations, to study the effects of cholesterol (Chol) and ceramide (Cer) on free volume voids in sphingomyelin (SM) or dipalmitoylphosphatidylcholine (DPPC) bilayers. Binary lipid samples with Chol were studied (DPPC:Chol 60:40, SM:Chol 60:40 mol ratio) and no phase transition was detected in the 20 - 60 ºC range, in agreement with calorimetric data. Chol-driven liquid-ordered phase showed an intermediate free volume void size as compared to gel and fluid phases. For SM and SM:Cer (85:15 mol:mol) model membranes measured in the 20 - 60 ºC range the gel-to-fluid phase transition could be observed with a related increase in free volume, which was more pronounced for the SM:Cer sample. MD simulations suggest a hitherto unsuspected lipid tilting in SM:Cer bilayers but not in pure SM. Ternary samples of DPPC:Cer:Chol (54:23:23) and SM:Cer:Chol (54:23:23) were measured and a clear pattern of free volume increase was observed in the 20 - 60 ºC because of the gel-to-fluid transition. Interestingly, MD simulations showed a tendency of Cer to change its distribution along the membrane to make room for Chol in ternary mixtures. The results suggest that the gel phase formed in these ternary mixtures is stabilized by Chol-Cer interactions.


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INTRODUCTION Ceramides (Cer) are one of the most relevant sphingolipids 1-3 (lipids structurally derived from sphingosine) because of their role as signaling molecules in biologically relevant processes such as programmed cell death 4-5. Effects on lipid membranes such as bilayer permeation 6 or lipid flip-flop 7 have been reported for long-chain ceramides (C16 or longer) and they could be related to their biological functions. Of the many different ceramides (because of different chain length, degree of unsaturation, nature of the sphingoid base) 8-11, the one most widely used in lipid biophysics is ceramide C16:0 because of the abundance of C16:0 sphingolipids from which Cer can be generated enzymatically 12. One relevant Cer precursor is sphingomyelin (SM), since Cer is an endproduct of the sphingomyelinase attack on SM, and Cer molecules exhibit a preferential interaction with SM, giving rise to SM- and Cer-enriched domains that would be phase-segregated causing membrane heterogeneity 13-16. The existence of Cer-enriched domains is important in the context of membrane heterogeneity and it would also be related to the “lipid raft hypothesis” (i.e. transient lipid structures in a different phase that would be enriched in cholesterol and sphingolipids such as SM) 17-19 . Thus Cer have the ability to drastically change the dynamic properties of biomembranes and the diffusion of lipids and proteins in the bilayer, which is in turn related to the generation and dynamics of free volume voids 20-21. Cer-enriched domains have been described in cell membranes, e.g. in induced hot-cold hemolysis of erythrocytes 22 or in the mitochondrial outer membrane of mammalian cells upon irradiation stress, and are reportedly essential for apoptosis through the intrinsic pathway 23. Ceramide interactions with cholesterol are relevant as cholesterol has been reported to interfere with cell death in tumour cells 24-25. Interestingly, cholesterol has also been reported to interfere with Cer-driven domain generation in model membranes, with reports pointing to a Cer vs. Chol displacement depending on the composition and Cer:Chol mol ratio 26-31. In a recent work we demonstrated that ceramide C16:0 was able to increase the mean size of the free volume voids inside DPPC model lipid bilayers 20. This work included direct measurements of the size and distribution of the free volume holes by Positron Annihilation Lifetime Spectroscopy (PALS) that were compared with additional data from Differential Scanning Calorimetry (DSC) and Molecular Dynamics (MD) simulations. In the present study we have characterized the effects of ceramide C16:0 in cholesterol-containing samples in the presence of DPPC or SM. In addition, we have evaluated how free volume voids are affected when both ceramide C16:0 and cholesterol are present at saturating conditions (gel ternary mixture) 26. Our results show that ceramide C16:0 increases free volume voids in the range 20 ºC - 60 ºC and that ternary mixtures feature a different free volume void pattern when compared to phospholipid-ceramide and/or phospholipid-cholesterol mixtures, suggesting the existence of a lipid phase that depends on Cer-Chol interactions to be stable. The emerging picture of phospholipid-ceramide-cholesterol interactions might be relevant to understand hitherto unexplored aspects of membrane lipid dynamics.


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MATERIALS AND METHODS Chemicals Egg-yolk ceramide (Cer, 85% 16:0 Cer), 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), Cholesterol (Chol) and egg-yolk sphingomyelin (SM, 85% 16:0 SM) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Lipids are depicted in Chart 1.

Chart 1: Lipid molecules involved in the study.

Membrane preparation Multilamellar vesicles (MLVs) were prepared by initially mixing the appropriate amount of lipids dissolved in chloroform/methanol (2:1, v/v). The samples were dried by evaporating the solvent under a stream of nitrogen, then placing them under high vacuum for 2 h. The samples were then hydrated in purified water (lipid:water 40:60 w/w for PALS, 1 mM lipid in buffer for DSC), helping dispersion by stirring with a glass rod and finally extruding the solutions between two syringes through a narrow tubing (0.5 mm internal diameter, 10 cm long) 150 times. The procedure was performed at a temperature well above that of the gel-fluid phase transitions for all compositions. Differential Scanning Calorimetry The measurements were performed in a VP-DSC high-sensitivity scanning microcalorimeter (MicroCal, Northampton, MA, USA). Both lipid and buffer solutions were degassed prior to loading into the appropriate cell in the form of MLVs. MLVs were prepared as described previously, but in assay buffer (NaCl 150 mM, 20 mM PIPES, 1 mM EDTA) and at a 1 mM concentration. 0.5 ml at 1 mM total 4 ACS Paragon Plus Environment

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lipid concentration was loaded into the calorimeter, performing 3-5 heating scans at a 45ºC/h rate, between 20 and 70ºC for all samples. Phospholipid concentration was determined as lipid phosphorus, and used together with data from the last scan, to obtain normalized thermograms. The software Origin 7.0 (MicroCal), provided with the calorimeter, was used to determine the different thermodynamic parameters from the scans. The software PeakFit (Systat Software Inc., Chicago, IL, USA) was used for endotherm deconvolution.

Positron annihilation lifetime spectroscopy (PALS) measurements An ORTEC (Oak Ridge, TN, USA) fast-fast coincidence system was employed for PALS measurements; two BC-422 Saint Gobain (Hiram, OH, USA) plastic scintillators and two Hamamatsu Photonics (Tokyo, Japan) H1949-50 photo multipliers were placed in a vertical position designed for measuring biological samples as described elsewhere 21 inside of a Radiber (Barcelona, Spain) FFD-1402 industrial refrigerator. The full width at half maximum intensity was around 260 ps. An Eurotherm (United Kingdom) 3508 programmable temperature control system equipped with a variable power supply provided by SALICRU (Bilbao, Spain), a 100W FIREROD® cartridge heater from Watlow Europe (Kronau, Germany), and a TC S.A. (Madrid, Spain) PT-100 CS5 (1|5) temperature sensor was utilized for controlling the temperature of the samples. The PT-100 and the heater were installed inside the biosample holder. The fabrication of the sealed positron source of 15 µCi was described in detail in previous articles 20-21. The source was sandwiched between two identical samples. Around 3 ⋅10 6 counts/spectrum were analysed by the LT_polymers program . The positron source contribution (31.55%, 0.382 ns) was subtracted and 3 lifetime components were obtained. The longest-lived lifetimes were linked with the orthoPositronium lifetimes. These lifetimes presented distributions, and the o-Ps mean were utilized for the free volume hole size calculations. The Tao-Eldrup model equation was employed to calculate the average free volume hole radius 33-34 as in previous PALS experiments in lipid membranes 20, 35:   2πR   R 1 −1 τ o−Ps = 2 1− + sin    R0    R0 2π 32

where R0 = R + ∆R , ∆R being an empirical parameter fitted to 1.66 Å 36. The average free volume void size was evaluated within a spherical approximation: 4 Vf = π R 3 . 3 By using the Tao-Eldrup formula, we quantify the mean size of the free volume holes. This way, we also quantify the distribution of the size of the free volume holes detected by PALS.

Molecular Dynamics Simulations A total of four model bilayers with SM as their main constituent were studied by atomistic molecular dynamics simulations: i) SM alone, ii) SM + C16:0 ceramide, iii) SM + cholesterol and iv) the SM + C16:0 ceramide + cholesterol ternary system. 5 ACS Paragon Plus Environment

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In addition two DPPC-based model bilayers were also studied: DPPC + cholesterol and DPPC + C16:0 ceramide + cholesterol. In order to analyze the thermal phase transition, each system was simulated at four temperatures: 20 ºC, 30 ºC, 37 ºC and 52 ºC. The final structure of the previously studied DPPC in solution 20, which included 128 lipids, was taken as the initial structure. The united atom GROMOS lipid force field, including the parametrization scheme proposed by Berger was employed for the lipids 37. For C16:0 ceramide, the parameters described in our previous work were used 20. The same parameters were chosen to describe the fatty acid and sphingosine group of SM, while the parameters for the phosphocholine group were taken from DPPC. The parameters for cholesterol were taken from the web page of the Biocomputing group at the University of Calgary 38. The Gromacs package (version 4.5.3) 39-40 was employed to run all molecular dynamics simulations. Periodic boundary conditions were considered by defining an orthorhombic cell. The system was solvated by adding a total of 5726 SPC model water molecules. All simulations were carried out under isothermal–isobaric ensemble (NPT) conditions using Nose-Hoover temperature (at 20 ºC, 30 ºC, 37 ºC or 52 ºC) 41 and Parrinello-Rahman pressure coupling (1 atm) 42. The temperature of the lipids and the solvent were independently coupled. Long-range electrostatics were calculated using the smooth particle mesh Ewald (PME) method 43, with a fourthorder spline and 0.12 nm grid spacing. A cut-off of 10 Å was defined for the electrostatics and van der Waals non-bonded interaction and neighbor-list. All bond lengths were constrained with Linear Constraint Solver (LINCS) 44, allowing an integration time step of 2 fs. A total run of 100 ns was carried out for the production of each simulation. Based on these molecular dynamics simulations, the electron densitity profiles and the distribution of voids in each system were calculated following the protocol used previously in Axpe, et al. 20.


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RESULTS

Effects of cholesterol on DPPC and DPPC:Cer:Chol ternary mixtures First the effect of cholesterol on DPPC-based membranes was studied. The results of cholesterol incorporation into DPPC or DPPC:Cer can be viewed in Figure 1 and Table 1. Each sample has a unique free volume void pattern, which is in accordance with ours and other previous reports as each sample reflects different phases or phase patterns along the 10ºC – 60ºC temperature range 26. The mean free volume hole size values represent the average size (in Å3) of unoccupied spaces, i.e. an arithmetic mean of the size of every single free volume hole detected by PALS. The 60:40 phospholipid:Chol mol ratio corresponds to maximum saturation of the system by Chol molecules without affecting membrane integrity. 35, 45

Table 1: Mean void volumes (A3) of the samples at different temperatures

T (ºC) 10 13 15 20 25 30 32 34 35 37 38 39 40 41 43 44 45 50 55 60

DPPC* 98 ± 1 106 ± 2 102 ± 1 104 ± 2 113 ± 1 115 ± 2 143 ± 2 149 ± 1 161 ± 1 192 ± 1

DPPC:Chol 100 ± 7 111 ± 11 126 ± 11 146 ± 5 170 ± 5

DPPC:Cer* 117 ± 2 113 ± 2 137 ± 2 147 ± 2 151 ± 2 192 ± 2 205 ± 1

DPPC:Cer:Chol 109 ± 5 113 ± 6 141 ± 7 185 ± 5 207 ± 4 225 ± 5 227 ± 4

SM 109 ± 2 113 ± 2 108 ± 2 110 ± 2 110 ± 2 111 ± 2 110 ± 2 118 ± 2 122 ± 2 133 ± 2 136 ± 2 167 ± 2

SM:Cer 108 ± 2 109 ± 2 129 ± 2 135 ± 2 146 ± 2 153 ± 2 -

SM:Chol 100 ± 4 106 ± 8 112 ± 4 120 ± 6 126 ± 2 132 ± 4 -

* DPPC and DPPC:Cer data reproduced from our previous report Axpe, et al. 20


SM:Cer:Chol 110 ± 5 107 ± 7 115 ± 7 127 ± 7 164 ± 8 201 ± 5 214 ± 4 219 ± 2

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DPPC:Chol (60:40 mol ratio) does not exhibit any phase transition because of the homogenous Lo phase that is present (also checked by DSC, Figure 2) and the free volume increases linearly with temperature. Furthermore, and particularly at high T, DPPC:Chol free volume is intermediate between that of a gel phase and of a fluid phase of pure DPPC, in agreement with other biophysical properties of Lo phases 46. The DPPC:Cer:Chol ternary mixture (54:23:23 mol ratio, that represents a system saturated in both Cer and Chol 27) exhibits a clear increase in free volume along the range 20 – 60 ºC in agreement with DSC data presented in Busto, et al. 26 (also plotted in Figure 2). The fact that, along the whole range of temperatures, the free volume detected for DPPC:Cer:Chol is higher than that of the other DPPC-based samples (Figure 1) points to a different phase being present, in agreement with previous reports on this system 26, 47. This evidence supports the idea of a homogenous gel phase being formed, rich in both Chol and Cer.

240 Mean free volume (Å3)

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Figure 1. Free volume void size dependence with temperature for DPPC-based samples. Data from PALS for DPPC (black, as reported in Axpe, et al. 20), DPPC:Cer 85:15 (grey, as reported in Axpe, et al. 20), DPPC:Chol 60:40 (blue) and DPPC:Cer:Chol 54:23:23 (red). DPPC and DPPC:Cer have been reproduced here from our previous report 20.


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Figure 2. DSC thermograms of DPPC-based samples. Differential Scanning Calorimetry (DSC) representative thermograms of DPPC, DPPC:Chol (60:40 mol ratio), DPPC:Cer (85:15 mol ratio) and DPPC:Cer:Chol (54:23:23 mol ratio) multilamellar vesicles. Y axis shows heat capacity (Cp) in kcal/molºC. Dotted lines represent peak fitted endotherms. DPPC Cp values have been divided by a factor of 3 for clarity. DPPC and DPPC:Cer (85:15 mol ratio) are taken from Axpe, et al. 20, while DPPC:Cer:Chol is taken from Busto, et al. 26.

Sphingomyelin-based mixtures PALS experiments were performed on SM-based samples. The results are shown in Figure 3 and Table 1 for SM, SM:Cer (85:15 mol ratio), SM:Chol (60:40 mol ratio) and SM:Cer:Chol (54:23:23 mol ratio). As shown for DPPC, each sample shows different free volume properties. Differences between SM and DPPC can be seen for pure lipids as free volume void size stays constant for SM up to 40 ºC. This is in accordance with DSC reports that show that egg SM has a transition at ~ 38 ºC without any pretransition 15. DSC for SM:Cer shows that, in the same way as DPPC:Cer, at 60 ºC the sample is in the fluid state and does not exhibit any further phase transition (Figure 4a). The comparison between the integrated DSC data and the PALS results shows a 5 ºC drift towards lower temperatures of the broad transition as seen by PALS (Figure 4b), which is in accordance with our previous findings with DPPC:Cer mixtures 20. The small drift in the 30 – 40 ºC range was attributed in our previous work to phospholipid molecules transitioning at lower T than Cer, taking into account a previous NMR study 48. This experiment confirms that PALS follows the phospholipid transition (in this case SM) while DSC is directed by Cer transition.


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Figure 3. Free volume void size dependence with temperature. PALS data for SM (black), SM:Cer 85:15 (grey), SM:Chol 60:40 (blue) and SM:Cer:Chol 54:23:23 (red).

SM:Chol samples do not exhibit any phase transition (Figure 4a), as was the case for DPPC:Chol (Fig. 1), but differ from the latter in the slope (Figure 5). The DPPC-containing mixture exhibits a dependence with temperature, which would make DPPC:Chol closer than SM:Chol to a fluid phase at higher temperatures. Ternary mixture experiments also render a unique free volume hole size pattern (Figures 1 and 3) and reflect a transition in accordance with DSC data (Figures 2 and 4a). Interestingly, a comparison of the transitions of both DPPC- and SM-based ternary mixtures by PALS (Figure 6a) shows a similar trend but with a noticeable difference in temperature, in good agreement with DSC data as the DPPCbased ternary mixture starts transitioning at lower temperatures (Figure 6b). This supports the idea that although both ternary mixtures exhibit a gel phase, some differences may be observed between both of them because of the intrinsic properties of each phospholipid (DPPC or SM). Despite differences between the ∆H of both ternary mixtures (∆H of the DPPC-based composition nearly doubles the enthalpy of the SM-based one, Figure 6b), PALS profiles almost overlap at the end of the transition, with free volume voids of ~220 Å3 (Figure 6a, Table 1) for both samples.


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Figure 4. DSC data from SM-based samples. (A) Representative thermograms of the different SM-based samples. Dotted lines represent peak fitted endotherms. (B) Comparison of integrated thermogram (grey solid line) and PALS mean free volume void sizes (black dashed line) for SM:Cer (85:15 mol ratio).


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Figure 5. Comparison of PALS results for liquid-ordered samples. DPPC:Chol (grey dots, black line), SM:Chol (blue dots, pink line). Solid lines represent linear fittings: DPPC:Chol y = 1.54 x + 75.9 (R2 = 0.985) and SM:Chol y = 0.65 x + 93.2 (R2 = 0.998).

Figure 6. Comparison of PALS results (A) and DSC integrated thermograms (B) for 54:23:23 ternary mixtures. DPPC:Cer:Chol (black), SM:Cer:Chol (grey).


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Molecular Dynamics Simulations MD simulations were carried out at four temperatures for the different lipid mixtures under study, which were used to analyzed various parameters, including: i) Representative bilayer snapshots (Figure 7, S1, S2) and electron density diagrams (Figures 8, 9, S3), ii) tilt angles of SM chains (Figure S4, Table S1) and Cer chains (Figure S5, Table S1), iii) deuterium order parameter (Figure S6), iv) area per lipid (Table S2), v) Cer – Chol relative positions (Figure S7), vi) lipid-to-lipid distance (Table S3), vii) radial distribution function (Figure S8) and viii) computed average voids' size (Figure S9). For pure SM snapshots, an increased disorder is seen at 37 ºC and above (see Figure 7a), confirmed by a wider distribution of the angles computed (Fig. S4 & S5) and lower -SCD values (Fig. S6) in agreement with our previous report for DPPC (where a more detailed explanation for –SCD calculation can be found) 20. The area per lipid value also increases with the temperature, going from 49.44 Å2 at 20 ºC to 55.19 Å2 at 52 ºC (Table S2). All these observations are in line with the gel to the fluid state transition pointed by our DSC data in Figure 4a. When Cer is present in the SM system (Figure 7b) at 20 ºC and 30 ºC the SM lipids exhibit a higher degree of tilting (Table S1). In tilted bilayers the thickness should be lower than in non-tilted ones. Lipid tilting could explain why DPPC:Cer domains are reportedly thicker than SM:Cer domains 47. This points to Cer as a tilting-inducing molecule even in nonDPPC bilayers, although the tendency towards lipid tilting decreases at higher temperatures (Table S1, Fig. S4 & S5). In addition, in agreement with DPPC:Cer mixtures investigated in Axpe, et al. 20, the formation of Cer-enriched domains is not observed clearly. This is not surprising, as the description of the system by a united atom force field limits the number of molecules included in the system and the length of the simulations, making difficult the simulation of long-term events, such as the formation of local domains. The methodology employed, however, allows us to perform a detail description of interactions into a molecular level. Snapshots of SM:Chol (Fig. S1a) and DPPC:Chol (Fig. S1b) show that there is almost no temperature effect, because the mixture is the liquid-ordered phase at the four studied temperatures (20 ºC, 30 ºC, 37 ºC and 52 ºC) without any transition in that range. The computed order parameters (Fig. S6) are also less affected by the temperature and show a larger ordering than the pure SM system, in agreement with previous studies 49. Interestingly, the lipid tilt angles and order parameters computed indicate that this increment on the lipid ordering induced by cholesterol does not lead to their tilting. For both ternary mixtures (Fig. S2a for SM:Cer:Chol & S2b for DPPC:Cer:Chol) at 52 ºC the gel-fluid transition is in process (according to the DSC data in Figure 2 and 4). Even so, an increased chain disorder is observed at that temperature for both ternary compositions. The electron density diagrams for pure SM:Cer (Figure 8a) closely resemble those reported for DPPC:Cer 20. If we compare these with pure SM electron density diagrams (Fig. S3a), we cannot detect any increase in bilayer thickness (unlike the case for DPPC and DPPC:Cer 20, 47), in agreement with the possibility of lipid tilting (experimental reports point to SM:Cer exhibiting even lower thickness than pure SM 47 ). Electron density diagrams for SM:Chol (Figure 8b) show that, unlike ceramide, cholesterol is clearly located within each monolayer, and leaves the interleaflet plane 13 ACS Paragon Plus Environment

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unoccupied. DPPC:Chol electron density diagrams (Fig. S3b) show that this effect is similar in DPPC and SM. This behavior is of interest as both Chol and Cer are highly hydrophobic molecules that tend to intercalate between lipid tails but it seems that Cer is less amphipathic, thus has a higher tendency to occupy the nodal plane. For ternary mixture electron density diagrams (Figure 9) one of the most interesting features is that the density profile of Cer changes because of the presence of Chol. In order to facilitate the comparison, the electron densities of Cer and Chol in their corresponding binary system and in the SM:Cer:Chol ternary system are plotted in Figure S7. In the ternary mixtures Cer density profile looks similar to that of Chol, which is a good indication for Chol-Cer interaction 26, 47. For the SM-based ternary mixture (Fig. 9) at 20 ºC, there is a peak of maximum Cer density at -20 Å / 20 Å, while in SM:Cer (Fig. 8a) the density of Cer at that position is much smaller. This may indicate that in the SM:Cer binary system (blue lines in Figure S7a) the Cer molecules are distributed along the entire hydrophobic part of the membrane, including the nodal plane, particularly at the highest temperatures. However, when cholesterol is included (red lines in Figure S7a), the Cer density decreases in the nodal plane. If we compare the electron density profile of the DPPC-based ternary at 20 ºC with our recently published DPPC:Cer diagram 20 we observe the same trend: there is almost no ceramide at -20 Å / 20 Å at the same temperature, which means that Cer molecules again tend to change their placement because of Chol. The electron density pattern for Chol molecules looks similar for binary and ternary mixtures, meaning that Chol molecules are not changing position despite the presence of Cer. As previously suggested 26, 47, this speaks in favor of a direct Cer-Chol interaction. In order to confirm these observations, the radial distribution functions (g(r)) between the different molecule types were computed during each simulation (see Figure S8). Among them, two clear peaks can be observed in the g(r) computed between the Cer lipid chains and Chol, located ~ 5 Å and 8 Å, particularly at the lowest T. Moreover, the g(r) between SM and Cer lipid chains shows more clear peaks when cholesterol is present. Based on all these results, Cer molecules seem to accommodate their location to optimize their interaction with Chol. In order to quantify the displacement of Cer observed in electron density diagrams due to the inclusion of cholesterol, we have computed the distance between the groups that could govern the interactions: the amide group of SM, the hydroxyl group of Chol and the amide and hydroxyl of Chol. SM-Cer binaries are reportedly stabilized by NSM to OHCer interactions 5, SM-Chol through NSM to OHChol 50, and finally the ternary system could be stabilized by NCer to OHChol 26. The results are included in Table S3, and show that the distance between NCer and OHChol is in the range where an interaction is perfectly possible, and the three interactions present in the ternary mixture show a comparable distance, which may point to a stabilization of the bilayer as a combined effect of the three interactions at the same time. Another interesting result is that distances between the different groups do not show major changes despite the increase in T or a phase transition, despite an increase in area per lipid caused by the loosening of lipid tails (Table S2). In addition, SM-Cer distance tends to be smaller in the ternary sample than in the binary, particularly at 20 ºC and 52 ºC. This result is in line with the displacement observed in the electron density diagrams of Cer (Figure S7), and confirms that Cer molecules move towards the polar part of the membrane when cholesterol is present. However, a close inspection of the distribution of the distances points to different possible lipid orientations as, in some 14 ACS Paragon Plus Environment

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cases, more than one peak is observed (Fig. S10 & S11). As these distributions are in some cases of high complexity, results in Table S3 are presented as average values ± standard deviation for the complete data sets. The possibility of different conformations coexisting in these lipids phases to optimize lipid-lipid interactions cannot be discarded, and should be addressed in further studies. The free volume located in each system was computed following the procedure reported in Axpe, et al. 20 and are shown by dashed lines in the electron density diagrams. Moreover, the average void size was computed for the SM containing systems, which is qualitatively comparable with the PALS data (Figure 3). The values computed in the pure SM system capture the dependence of the voids' size on the temperature, as higher the temperature, higher the average void size. The results also confirm the higher rigidity of the system in which cholesterol is present (SM:Chol and SM:Cer:Chol), as the void size is less affected by the temperature. The only discrepancy arises for the SM:Cer system, where the average void size does not change substantially with the temperature, unlike the trend observed in Figure 3. This discordance again points to the fact that the domain separations were not observed during the MD simulations (see above).


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Figure 7. Final snapshot of SM (A) and SM:Cer (B) molecular dynamics simulations. Temperatures are indicated for each snapshot. Ceramide molecules are depicted in black.


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Figure 8. Electron density profiles (in e/Å3) along the Z axis calculated for the last 10 ns of the molecular dynamics simulations. SM + Cer (A) and SM + Chol (B) systems at four temperatures (from top to bottom): 20 ºC, 30 ºC, 37 ºC and 52 ºC. Colour legend: SM (solid black), SM heads (solid red), SM tails (solid green), water (solid blue) and Cer (A) or Chol (B) (solid orange for either case). The relative free cell distribution with respect to the Z axis length computed in the last three snapshots extracted from the simulations are shown in the dashed lines.


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Figure 9. Electron density profiles (in e/Å3) along the Z axis calculated for the last 10 ns of the molecular dynamics simulations. DPPC + Chol + Cer (A) and SM + Chol + Cer (B) systems at four temperatures (from top to bottom): 20 ºC, 30 ºC, 37 ºC and 52 ºC. Colour legend: Phospholipid (either DPPC or SM) (solid black), phospholipid heads (solid red), phospholipid tails (solid green), water (solid blue), Cer (solid orange) and Chol (solid brown). The relative free cell distribution with respect to the Z axis length computed in the last three snapshots extracted from the simulations are shown in the dashed lines.

DISCUSSION

Binary systems For cholesterol-containing samples, a direct comparison of PALS results between SM:Chol and DPPC:Chol (Figure 5) shows that they exhibit different free volume growth trends over temperature. This is interesting as both samples are in the same Lo phase and no phase transitions are observed at that T range 51-52 both having even comparable nanomechanical resistance 47. This is in agreement with reports that point to cholesterol having a preferential interaction with SM rather than DPPC 50, 5355 . According to PALS data, SM-based Lo phase has a smaller free volume at most 18 ACS Paragon Plus Environment

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temperatures and its dependence with temperature is lower, which would speak in favor of a higher intermolecular packing than DPPC-based Lo phase. Interestingly, the free volume of DPPC:Chol at higher temperatures (45 ºC – 60 ºC) is close to the free volume of pure DPPC at the same temperature range, especially at 45ºC, just after the main gel-fluid transition. This information points to the DPPC:Chol Lo phase having a free volume pattern closer to a fluid phase, while SM:Chol Lo phase would rather resemble a gel phase from that point of view. PALS experiments were performed in MilliQ pure water while DSC experiments were in buffer and this could be a source of discrepancy when comparing data due to the reported effect of ions on hydrogen bonding 56-57, but an additional control experiment was performed. General polarization (GP) of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) is indicative of lipid fluidity and membrane order and Laurdan GP was measured on both Lo samples (buffered and non-buffered) at different temperatures. Similar results were obtained for buffered and non-buffered lipid samples (Fig. S12). Cer increases free volume voids in sphingomyelin-based bilayers, and molecular dynamics simulations show an increased degree of lipid tilting in SM:Cer when compared to DPPC:Cer, which could explain the reduced thickness of the former and the lower nanomechanical resistance of the latter 47. In addition, as discussed in Axpe, et al. 20, the presence of Cer increases the size of the free volume voids mainly at the nodal plane, but at the same time it has a tendency to occupy the lipid tails region and the nodal plane because of its hydrophobicity (Figure 8a). This may seem contradictory, as the presence of more lipid molecules at the nodal plane should induce a reduction in free volume, but for DPPC:Cer an increase in bilayer thickness (as compared to pure DPPC) was detected in Axpe, et al. 20 in agreement with other published data 47, which could mean that lipid leaflets would be drifting away from each other generating more space at the nodal plane, despite the presence of Cer. For SM:Cer a slightly different scenario occurs as thickness is smaller than SM 47 probably because of the clear lipid tilting (Fig. 7b). This could mean that lipid tails from each leaflet could still be drifting away from each other, as Cer also increases free volume voids in SM bilayers (Fig. 3), but the overall thickness is decreased as lipid tilting overcompensates the latter effect.

Ternary systems As for ternary phospholipid:Cer:Chol 54:23:23 mixtures, PALS results show that the free volume void average size changes with temperature differently from the binary mixtures (phospholipid:Chol or phospholipid:Cer) and from the pure phospholipids, in agreement with AFM 47 and DSC 26. Furthermore, these PALSdetected trends are very similar in both DPPC- and SM-based ternary mixtures, suggesting that the same kind of phase is present, despite the drift in temperature (Figure 6a) that also appears when comparing integrated DSC thermograms for both samples (Figure 6b). Previous AFM reports show that both DPPC- and SM-based ternaries had a similar bilayer thickness and roughness, but the nanomechanical resistance pattern displayed some differences 47. PALS data on bilayer free volume voids is in accordance with nanoindentation experiments and speak in favor of the capacity of these techniques to resolve differences in the nano-scale biophysical properties. In summary, these results underline that the ternary mixtures at 54:23:23 ratio exist in a gel phase with different and unique properties as compared to phospholipid/Chol, phospholipid/Cer binaries and pure phospholipids. 19 ACS Paragon Plus Environment

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Further interesting feature is that calorimetric transitions of these ternary mixtures tend to appear at higher temperatures than the increase in free volume voids detected by PALS (Fig. 6). For the DPPC-based mixture, PALS shows a transition in the 20 ºC - 55 ºC (Tm = 35 ºC) range while with DSC the range is 40 ºC - 70 ºC. For the SM-based mixture the corresponding data are PALS: 35 ºC - 55 ºC (Tm = 45 ºC) and DSC: 50 ºC - 70 ºC. As previously stated, the SM-based ternary mixture starts melting at higher temperatures than the DPPC-based mixture according to both techniques, but it is worth noting that for these two samples thermotropic transitions seem to happen 20ºC above the changes in free volume detected by PALS, while binary Cer-containing samples had shown a much better correlation (SM:Cer in Fig. 4b and DPPC:Cer in Axpe, et al. 20). In addition, both ternary mixtures reach almost the same values for free volume voids at 60 ºC despite the differences at lower temperatures. This could be explained by a different degree of cooperativity in the transition, although the fact that the SM-based transition seems to be less endothermic should also be taken into account (Fig 6b). Ternary samples exhibit larger free volume voids than binary ones, or than pure phospholipids. The DPPC-based ternary mixture exhibits an enhanced size of the free volume voids along the complete temperature range, specially from 30ºC onwards, while for the SM-based mixture there is a remarkable increase in free volume from 40ºC onwards, surpassing the SM:Cer binary sample at 45ºC (Fig. 3). These ternary samples exhibit a homogenous phase enriched in both Cer and Chol 26, 47 that would be stabilized by direct Cer-Chol interactions. Indeed, molecular dynamics simulations point to the possibility of an accommodation of Cer to optimize interactions with Chol (Fig. 9). Therefore, we assume that the increase in free volume voids is related to this positive interaction and could be due to a cooperative effect between both Cer and Chol molecules. This opens the field to further studies regarding the existence of this cooperation between Cer and Chol under different circumstances, i.e. other model systems such as lipoprotein bilayers, or cell membranes. CONCLUSION The inclusion of ceramide or cholesterol has noticeable and specific effects on the size of the free volume voids in fluid phospholipid bilayers. Cholesterol gives rise to a liquid-ordered phase without any thermotropic transition in the 20 ºC – 60 ºC range, and decreases the average void size as compared to the pure phospholipid in the fluid phase. Moreover, free volume voids are larger for DPPC:Chol than for SM:Chol. In the case of SM:Cer, the mean size of the free volume voids increases with respect to pure SM, more notably at temperatures over 30 ºC, and MD simulations point to lipid tilting in the presence of Cer. The combination of both Cer and Chol at saturating concentrations (54:23:23 phospholipid:Cer:Chol mol ratio) shows an even sharper free volume size increase in the same temperature range. This would support the idea of a ternary phase occurring in the presence of equimolar amounts of Cer and Chol. Moreover according to MD simulations this phase could be stabilized by direct Chol – Cer interactions as ceramide molecules displace their position 1 - 2 Å towards the polar region of each leaflet to accommodate cholesterol in order to optimize the interaction. These findings are relevant in the context of


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membrane domain segregation and sphingolipid-driven signaling processes such as programmed cell death. ASSOCIATED CONTENT Supporting information Supporting material available: Summary of lipid tilt data (Table S1). Area per lipid simulation results (Table S2). Lipid-to-lipid distance (Table S3), final snapshot of SM:Chol (60:40) (A) and DPPC:Chol (60:40) (B) molecular dynamics simulations (Fig. S1). Final snapshot of SM:Cer:Chol (54:23:23) (A) and DPPC:Cer:Chol (54:23:23) (B) molecular dynamics simulations (Fig. S2). Electron density profiles (in e/Å3) along the Z axis calculated for the last 10 ns of the molecular dynamics simulations (SM and DPPC:Chol) (Fig. S3). Normalized probability distribution of SM tilt angles (Fig. S4). Normalized probability distribution of Cer tilt angles (Fig. S5). Chain order parameter calculation (Fig. S6). Electron density of Cer and Chol in their binary systems and in the ternary system (Fig. S7). Radial distribution functions between the center of masses of different molecule types (Fig. S8). Average free volume voids calculated by simulations (Fig. S9). Histograms of the NSM-HOCer distance in the SM:Cer and SM:Cer:Chol systems calculated by MD simulations (Fig. S10). Histograms of the NSM-HOChol and NCer-HOChol distances in the SM:Chol and SM:Cer:Chol systems calculated by MD simulations (Fig. S11). LAURDAN general polarization data of Chol binaries in presence or absence of buffer (Fig. S12).This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author [email protected]

ACKNOWLEDGEMENTS EA and AGA gratefully acknowledge the Basque Department of Education, Universities and Research funding (IT-443-10). JIM recognizes the Basque Government (GIC IT588-13) and the Spanish Ministerio de Economía y Competitividad (MINECO) (CTQ2012-38496-C05-01 and CTQ2012-38496-C05-04). This work was also supported in part by grants from the Spanish Government (MINECO BFU 2012-36241 to F.M.G. and BFU 2011-28566 to A.A.) and the Basque Government (IT849-13 to F.M.G: and IT838-13 to A.A.). The SGI/IZOSGIker UPV/EHU is also acknowledged for computational resources.

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We have studied the effect of cholesterol (Chol) and ceramide (Cer) on phospholipid bilayers by positron annihilation lifetime spectroscopy, differential scanning calorimetry and molecular dynamics simulations. Ceramide increases free volume void size in sphingomyelin (SM) and the combination of both ceramide and cholesterol at saturating conditions (54:23:23 phospholipid:Cer:Chol mol ratio) shows an even sharper free volume size enhancement at increasing temperatures. Molecular dynamic simulations show Cer-induced lipid tilting in SM/Cer mixtures and a ~5 Å displacement of Cer when Chol is present, because of Cer-Chol interactions. These findings are relevant in the context of membrane domain segregation and sphingolipid-driven signaling processes such as programmed cell death.


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Cholesterol–Ceramide Interactions in Phospholipid and Sphingolipid

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