Nanosized Phase Segregation of Sphingomyelin and

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Nanosized Phase Segregation of Sphingomyelin and Dihydrosphingomyelin in Unsaturated Phosphatidylcholine Binary Membranes without Cholesterol Tomokazu Yasuda, J. Peter Slotte, and Michio Murata Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02637 • Publication Date (Web): 16 Oct 2018 Downloaded from http://pubs.acs.org on October 22, 2018

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Nanosized

Phase

Segregation

of

Sphingomyelin

and

Dihydrosphigomyelin in Unsaturated Phosphatidylcholine Binary Membranes without Cholesterol

Tomokazu Yasuda,†,§,‡ J. Peter Slotte,§,* and Michio Murata†,*

†

Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043,

Japan §

Biochemistry, Faculty of Science and Engineering, Åbo Akademi University, Tykistökatu 6A, FIN-

20520 Turku, Finland

Present address: Research Foundation Itsuu Laboratory, C1232, Kanagawa Science Park R&D Building, 3-2-1 Sakado, Takatsu-ku, Kawasaki, Kanagawa, Japan ‡

*Correspondence: [email protected], [email protected]

1 ACS Paragon Plus Environment

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ABSTRACT In this study, we applied fluorescence spectroscopy, differential scanning calorimetry (DSC), and 2H nuclear magnetic resonance (NMR) to elucidate the properties of nanoscopic segregated domains in stearoylsphinomyelin (SSM)/dioleoylphosphatidylcholine (DOPC) and stearoyldihydrosphigomyelin (dhSSM)/DOPC binary membranes. The results obtained from fluorescence measurements suggest the existence of gel-like domains with high fluidity in both SSM and dhSSM macroscopic gel phases. DSC thermograms showed that DOPC destabilizes SM-rich gel-like domains to a much lesser extent compared to the same amount of cholesterol. It was also found that stable lateral segregation occurs without cholesterol, indicating that SSM itself undergoes homophilic interactions to form small gel-like domains. 2H NMR experiments disclosed differences in the temperature-dependent ordering of SSM/DOPC and dhSSM/DOPC bilayers; the dhSSM membrane showed less miscibility with the DOPC fluid phase, higher thermal stability, and tighter packing. In addition, the NMR results suggest the formation of mid-sized gel-like aggregates consisting of dhSSM. These differences could be accounted for by homophilic interactions, as previously reported (Yasuda et al. Biophys. J. 2016, 110, 431-440). In the absence of cholesterol, the moderately strong sphingomyelin (SM)/SM affinity results in the formation of small gel-like domains, while the stronger dhSSM/dhSSM affinity leads to larger gel-like domains. Considering the similar physicochemical features of SSM and dhSSM, the present results suggest that the formation of nanosized domains of SM is better characterized by homophilic interactions than by SM–cholesterol interplay. These effects are considered important to the ordered domain formation of SMs in biological membranes.


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INTRODUCTION Biological membranes are complex bilayer structures made up of a variety of partially saturated and unsaturated phospholipids, glycosphingoipids, and cholesterol (Chol), in which diverse membrane proteins are embedded. These membranes often show lateral heterogeneity in terms of both lipids and proteins. Such heterogeneity is assumed to play an important role in the regulation of protein functions within the bilayer and in the juxtamembrane region. One particular type of lipid-induced lateral heterogeneity is lipid rafts.1-4 The biological role of lipid rafts has sparked intense research interest because these heterogeneous domains can participate in various cellular processes, such as protein assembly, signal transduction, and microbial infections.5-10 Lipid rafts are transient nanosized aggregates of lipids, typically consisting of sphingolipids and Chol.7,11,12 They are believed to exist in a liquidordered phase, where lateral domain segregation is strongly influenced by lipid–lipid interactions, such as the packing of acyl chains (or the hydrophobic portions of Chol), charge distribution in headgroups, and hydrogen bonding.13-16 The co-existence of liquid-ordered (Lo) and liquid-disordered (Ld) domains has been successfully demonstrated in artificial lipid bilayers consisting of only three components, including sphingomyelin (SM), unsaturated phosphatidylcholine (PC), and Chol, by fluoresce microscopic experiments.17-21 Such ternary mixtures, therefore, have been used to investigate the contribution of lipid–lipid interactions to phase segregation and domain formation. Other analytical methods, such as fluorescence-lifetime analysis21, 22 and 2H nuclear magnetic resonance (NMR),23-25 have been used with these ternary systems to examine their mobility and ordering at the atomistic level. Using site-specific deuterated stearoyl-SMs (SSMs) in a ternary membrane consisting of SSM/dioleoylphosphatidylcholine (DOPC)/Chol (1:1:1), we could observe two sets of quadrupole splitting patterns for the Lo and Ld phases, which revealed detailed order profiles for an acyl chain and lipid distributions in each phase specifically and


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simultaneously.25 It is surprising that the quadrupole profile of SSM in the Lo phase of the ternary system is almost identical to that in the SSM/Chol binary mixture, although about 15 mol% of DOPC is present in the Lo phase.25 This result suggests that SSM is not affected by the disordering effects of DOPC, and it is probably separated from DOPC through the formation of small clusters in the Cholcontaining Lo phase. Moreover, several studies using modalities with different spatial and temporal resolutions26-30 have recently disclosed that nanoscopic heterogeneity occurs even in the SM–Chol binary membrane, which had previously been thought of as a pure Lo phase. We also suggested the presence of transient Chol-poor gel-like domains at the nanometer scale, which could be regarded as SM nano-clusters.31 Dihydro-sphingomyelins (dhSMs), which lack a trans Δ4 double bond in the sphingosine base, are dominant SM species in eye-lens cells.32,33 These studies also suggested that stable bilayers formed by dhSMs and Chol are responsible for the long-term stability of the human lens membrane against the oxidation of lipid constituents and their metabolic changes. Another group has recently reported that dhSM in cells possibly inhibits viral (HIV-1)–cell membrane fusion. 34 Saturated C18-acyl dhSM, stearoyldihydrosphingomyelin (dhSSM, Figure 1A), is known to undergo macroscopic lateral segregation in fluid DOPC bilayers, even in the absence of Chol.35 A similar phenomenon in macroscopic phase separation could also be observed with comparable SSM/DOPC bilayers, although the shape of the domain was not well defined. It is possible that dhSSM tends to form a more ordered and thermally stable phase due to stronger homophilic hydrogen bonding as compared to SSM molecules.36-39 In previous studies, it was observed that the more flexible 3-OH group of dhSM has a higher chance of forming hydrogen bonds compared to the sterically restricted 3-OH group of normal SMs. Hence, it can strengthen lipid–lipid interactions.32,40 In view of these findings, it is particularly important to examine the domain formation of SMs in


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unsaturated PC-containing bilayers in the absence of Chol,17,18,41 which enables us to focus more on SM–SM interactions42,43 in the formation of SM nano-clusters. Considering the similarities and dissimilarities between SM and dhSM, it may be a good idea to investigate stable dhSM–dhSM interactions in detail to gain a better understanding of the SM clusters, which are sometimes too shortlived to be detected by NMR and other modalities. In our previous studies,35,40 we examined macroscopic phase separations, as shown in the phase diagrams in Figure 2C and 2D using binary membranes with high contents of SSM and dhSSM,35 and the overall membrane properties mainly using oleoyl-SM.40 Thus, in this study, we used specific molecular probes for SSM and dhSSM to investigate the effects of the homophilic interactions of SMs or dhSMs on the nanoscopic lateral phase using DOPC-containing binary systems. Our experimental approach, which included the use of domainselective fluorescent reporter molecules as shown in Figure 1B, disclosed the changes occurring in lateral bilayer dynamics. The thermal stability of the SM-rich gel phase was examined using differential scanning calorimetry (DSC). To measure the bilayer order in the fluid phase, we adopted site-specific deuterated SSMs and dhSSMs for 2H NMR measurements at different temperatures and lipid composition ratios. These experiments can reveal the small but significant differences between SSM and dhSSM in homophilic interactions that influence the properties and sizes of lateral gel-like domains, even in the absence of the ordering effects of Chol.


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Figure 1. Chemical structures of compounds used in this study. (A) stearoylsphingomyelin (SSM) and dihydro-stearoylsphingomyelin (dhSSM) and their deuterated analogues. (B) lipid-mimicking fluorescent probes (tPA-SM, tPA-dhSM, O-tPA-PC, and 18:1-DPH-PC)

MATERIALS AND METHODS Materials DOPC, 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (O-lyso-PC), and D-erythro-sphingosylphosphorylcholine (lyso-SM) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Chol was obtained from Sigma-Aldrich (St, Louis, MO, USA). SSM was purified from bovine brain SM (Avanti


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Polar Lipids) by reverse-phase high-performance liquid chromatography (HPLC) using methanol as the eluent. For fluorescence spectroscopy experiments, fluorescent PL probes—18:1-DPH-PC, 1-oleoyl-2tPA-sn-3-glycero-phosphatidylcholine (O-tPA-PC), N-tPA-sphingomyelin (tPA-SM), and N-tPAdihydrosphingomyelin (tPA-dhSM)—were prepared by coupling propionyl-DPH or trans-parinaric acid (tPA) to the respective lyso compounds, as described previously.44-46 All phospholipids were dissolved in argon-purged methanol, stored at –20 °C, and warmed to an ambient temperature before use. The water used for fluorescence experiments was purified by reverse osmosis, followed by passage through a Millipore UF Plus water purification system (Millipore, Billerica, MA). For 2H NMR experiments, 10’,10’-d2-SSM was synthesized as previously reported.47,48 Finally, 10’,10’-d2-dhSSM was prepared from 10’,10’-d2-SSM by hydrogenation using a palladium catalyst on carbon (Sigma-Aldrich, St. Louis, MO, USA).

Steady-state fluorescence anisotropy: DPH-PC and tPA-SM Multilamellar vesicles (MLVs) for fluorescence anisotropy measurements were prepared by mixing 300 nmol of total lipids at the desired molar ratio and using a fluorescent probe to achieve a final concentration of 1 mol%. After mixing, the solvent was evaporated under a stream of nitrogen. The dry lipid films were hydrated with 2 mL of argon-purged Milli-Q water in a water bath at 80 °C for 30 min. Finally, the samples were vortex-mixed and sonicated for 10 min in a FinnSonic M3 Bath Sonicator (FinnSonic Oy, Finland) at 80 °C. The samples were cooled to room temperature before fluorescence measurements. Steady-state fluorescence measurements were performed in quartz cuvettes on a PTI Quanta Master spectrofluorometer (Photon Technology International, Lawrenceville, NJ, USA) operating in the T-format. Fluorescence emission was recorded continuously at 405 nm (tPA-SM and tPA-dhSM) or 430 nm (18:1-DPH-PC) after excitation at 305 nm and 360 nm, respectively. The samples


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were heated at a rate of 2 °C/min from 20 °C to 65 °C. Steady-state anisotropy, r, was determined as described previously49 using the Felix32 software (Photon Technology International).

Time-resolved fluorescence of tPA-SM and O-tPA-PC For time-resolved fluorescence lifetime measurements, MLVs (0.15 mM final lipid concentration) were prepared at the desired compositions by including 1 mol% O-tPA-PC, tPA-SM, or tPA-dhSM fluorophores, as described earlier. The fluorescence decay of tPA-PLs was recorded at temperatures between 10 °C and 60 °C using a FluoTime 200 spectrofluorimeter with a PicoHarp 300E timecorrelated single photon counting (TCSPC) module (PicoQuant GmbH, Berlin, Germany). The fluorophores were excited with a 298-nm LED laser source (PLS300; PicoQuant), and the emission was collected at 405 nm. During measurements, the samples were maintained at a constant temperature by continuous stirring. These measurements were performed three times for each sample composition. Data acquisition and analyses were performed with the FluoFit Pro software (PicoQuant). The obtained emission decay curves as described in Figure S1 were fitted to two or three different lifetime components (whichever gave the best unbiased residual plots) and χ-squared closest to one. The average lifetime was calculated as described in Lakowicz.49

DSC measurements MLVs for DSC were prepared from either SSM or dhSSM with 10, 30, and 50 mol% DOPC to achieve a final lipid concentration of 0.8–8 mM. All the samples were stored at 4 °C and degassed for 10 min using a ThermoVac instrument (MicroCal, Northampton, MA, USA) before being loaded into the VP-DSC instrument (Malvern, Northampton, MA). Five up and down scans were conducted at a temperature gradient of 1 °C/min (the testing temperature was in the range of 5 °C to 50 °C). The fifth


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upscan is shown in the figures reported in this study (results section). The ORISIN software (OriginLab Northampton, MA) was used for data analysis, and the enthalpy values obtained from triplicate experiments were normalized to the SM concentration in the sample.

Sample preparation and 2H NMR measurements Sample preparation and 2H NMR measurements were conducted in a manner similar to that reported in our previous work.47,48 In this study, mixtures of 10’-d2-SSM or 10’-d2-dhSSM with 10, 30, and 50 mol% DOPC were dissolved in MeOH-CHCl3, and the solvent was removed in vacuo over 12 h. The dried membrane films were hydrated with ~1 mL of water and vigorously vortexed at 70 °C to form MLVs. After being freeze-thawed, each suspension was lyophilized, rehydrated with deuterium-depleted water to 50% moisture (w/w), and freeze-thawed several times. Subsequently, the samples were transferred to individual 5-mm glass tubes (Wilmad, Vineland, NJ), which were sealed with epoxy glue. 2H

NMR spectra were recorded on a 300-MHz CMX300 spectrometer (Chemagnetics, Agilent, Palo

Alto, CA, USA) fitted with a 5-mm 2H static probe (Otsuka Electronics, Osaka, Japan) using a quadrupolar echo sequence. The 90° pulse width was set at 2.5 μs, the interpulse delay was 30 μs, and the repetition rate was 0.6 s. The sweep width was 250 kHz and the number of scans was ~100,000. The quadrupolar splitting values were essentially measured with single experiments, as the high reproducibility of these values has been widely recognized, e.g., two separate measurements for MLVs containing these 2H-labeled lipids always showed the deviation in the splitting values within 0.5 kHz.

3. RESULTS 3.1. Fluorescence anisotropy of mixed bilayers containing SM and unsaturated PC To evaluate the melting of SM-rich ordered phases in DOPC-containing binary systems, we first


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measured the steady-state fluorescence anisotropy of SSM/DOPC (5:5) and dhSSM/DOPC (5:5) bilayers as a function of temperature. We used three reporter probes: tPA-SM, tPA-dhSM, and a DPHconjugated PL (18:1-DPH-PC). The first two probes are known to partition preferentially into an ordered phase (POPC/PSM bilayers: Kp So/Ld 1.7),50 whereas the latter shows a strong preference for a disordered phase over a solid ordered phase (POPC/PSM bilayers: Kp So/Ld ≈ 0.31).46 Therefore, the anisotropy values shown in blue and red traces in Figure 2 predominantly report the acyl chain order in SM-rich ordered and DOPC-rich disordered environments, respectively. The monotonic decrease in the anisotropy values of 18:1-DPH-PC (red traces) with an increasing temperature was relatively small in both bilayers, although the anisotropy at 20 °C was lower in dhSSM/DOPC bilayers (0.17) than in SSM/DOPC bilayers (0.20; Figure 2, red traces). The melting of the SM-rich ordered phase could not be clearly sensed by 18:1-DPH-PC. On the other hand, the anisotropy values in the ordered phase (blue traces) followed the trend of a biphasic curve at low temperatures, which suggests that the ordered domains sensed by tPA-SM gradually melted when the temperature increased. The inflection point indicates that the end temperature of the ordered phase transition was 35 °C for SSM/DOPC bilayers and 45 °C for dhSSM/DOPC bilayers. These values are lower than the phase transition temperatures of the pure lipid bilayers between the gel and Ld phases (SSM: 44 °C, dhSSM: 52 °C).35,51 Thus, we next examined lipid mobility separately for the gel phase and fluid phase using fluorescence probes mimicking SSM (dhSSM) and DOPC, respectively.


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Figure 2. Fluorescence anisotropy of SSM/DOPC (5:5) bilayers (A) and dhSSM/DOPC (5:5) bilayers (B) as a function of temperature. The anisotropy in SSM (or dhSSM)-rich ordered domains (blue traces, upper) is determined using tPA-SM or tPA-dhSM, and the anisotropy in DOPC-rich disordered domains (red traces, lower) is determined using 18:1-DPH-PC. According to the previously reported phase diagrams for SSM/DOPC (C) and dhSSM/DOPC (D),35 these binary membranes show a gel/Ld mixed phase at a temperature close to the melting point of their pure bilayers. The boundary between gel/Ld and Ld was determined by fluorescence observations for macroscopic phase separation.

3.2. Fluorescence lifetime of mixed bilayers containing SM and unsaturated PC We next measured the tPA fluorescence lifetimes to examine the behavior of the SM-rich ordered


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phase and DOPC-rich disordered phase with an increasing temperature. The tPA lifetime is highly sensitive to the lateral packing properties of lipids in the vicinity of the membrane.23,52-54 In this study, tPA-based SM derivatives, tPA-SM/tPA-dhSM, and an unsaturated-PC derivative, O-tPA-PC, were used to detect ordered/gel domains and disordered/fluid domains, respectively, because the latter probe has a strong preference for the fluid phase (POPC/PSM bilayers: Kp So/Ld 0.3545), as in the case of 18:1-DPHPC. Figures 3A and 3B show the average lifetimes of the domain-specific probes in binary lipid mixtures as a function of temperature. The circles indicate the fluorescence lifetime of tPA-SM or tPAdhSM and the triangles indicate the fluorescence lifetime of O-tPA-PC. The average fluorescence lifetimes of SM-based probes were markedly higher than that of O-tPA-PC in binary lipid bilayers; this clearly suggests that SM-based probes and O-tPA-PC selectively report from SM-rich and DOPC-rich domains, respectively. The difference between the average lifetimes of ordered and fluid domains disappeared at 35 °C in SSM/DOPC and 45 °C in dhSSM/DOPC bilayers; these temperatures are similar to the end temperatures of SM-rich phase melting in a fluorescence anisotropy analysis (Figure 2). Moreover, to explore the influence of DOPC on the formation of ordered domains in both bilayers (the miscibility of DOPC in the SM-rich ordered phase), we measured the fluorescence lifetimes in the pure SSM and dhSSM bilayers, as shown by the squares in Figure 3A and 3B. A comparison with DOPCcontaining bilayers indicates that the fluorescence lifetimes in pure lipid bilayers are similar to those in DOPC-containing binary mixtures at lower temperatures. However, when the temperature is 0–15 °C below the end temperature of melting, the average lifetimes of both binary bilayers were significantly shorter than of pure bilayers. This observation indicates that DOPC enhances the fluidity of the bilayers; in other words, DOPC is miscible with SSM or dhSSM in this temperature range. In Figure 3C, longer lifetime components (τ1), which are largely attributed to the gel/ordered phase,52,53 were found to be greater than 30 ns below the end temperature of melting for each of the


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bilayers, indicating the existence of a gel phase, as previously suggested;55,56 the τ1 values of tPA-SM at 30 °C and of tPA-dhSM at 40 °C were 45 ns and 42 ns, respectively. It is generally accepted that SM/unsaturated-PC bilayers show macroscopic gel/Ld phase segregation in the temperature range of 0– 10 °C below the Tm (35–45 °C for SSM in this study) of a pure SM membrane (Figure 2C).17,18,35 However, the end temperatures of ordered-phase melting—35 °C for SSM and 45 °C for dhSSM (Figures 2, 3A, and 3B)—imply that the gel phase in this study could differ from the macroscopic gel phase observed by fluorescence microscopy. This is because the gel phase of SSM in the SSM/DOPC (5:5) system that completely melts at 35 °C, as shown in Figures 2 and 3, was not observed in the fluorescence imaging experiments at 40 °C.17,18 In other words, the reported macroscopic gel phase in SM/DOPC bilayers is not perfectly homogeneous, but it contains a coexisting gel-like phase and fluid Ld phase in this temperature range just below Tm; these points will be discussed later on in this report. Moreover, Figure 3C reveals that the fluorescence lifetimes of dhSSM bilayers are longer than of SSM bilayers in the tested temperature range. Further, temperature-dependent changes in the 1 value over the range of 10–30 °C are lesser for tPA-dhSSM than are those for tPA-SSM. These results indicate that the relative abundance of gel-like domains in dhSSM/DOPC bilayers is not highly temperature-dependent, suggesting that the gel-like domains of dhSSM exhibit higher order and thermal stability. These differences could be attributed to the stronger homophilic interactions of dhSSM compared with those of SSM.


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A

B

C

Figure 3. Detection of membrane order using the average fluorescence lifetimes of domain-selective probes as a function of temperature. Panel A shows the data corresponding to pure SSM and


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SSM/DOPC (5:5) bilayers and panel B shows the data corresponding to pure dhSSM and dhSSM/DOPC (5:5) bilayers. The circles show the lifetime of tPA-SM and tPA-dhSM (reporting the order in ordered domains) and the triangles show the lifetime of O-tPA-PC (reporting the order in disordered phase areas) in DOPC-containing bilayers. The squares show the lifetime of tPA-SM and tPA-dhSM in pure SM and dhSSM bilayers, respectively. Panel C shows the longer lifetime component (τ1) of tPA-SM fluorescence in SM/DOPC bilayers and tPA-dhSM fluorescence in dhSSM/DOPC bilayers as a function of temperature. The average ± standard deviation (SD) values of three experiments are presented (error bars may be smaller than the symbols).

3.3. DSC analysis of mixed bilayers containing SM and unsaturated PC To examine further the influence of DOPC on lateral segregation and the thermal stability of the gel phase in SSM and dhSSM bilayers, we recorded their DSC thermograms. Samples containing different molar ratios (10, 30, and 50 mol%) of SSM or dhSSM were subjected to several up and down DSC scans. Figure 4 indicates that SSM and dhSSM in all the samples formed gel phases in DOPC bilayers, for which the corresponding melting profiles were relatively broad and uncooperative. Therefore, the inclusion of DOPC in both bilayers resulted in destabilizing the gel phase, as indicated by the reduction in the end temperature of melting. This tendency was more significant as the DOPC content increased. In DOPC bilayers at 50 mol% SSM or dhSSM, the end temperatures of gel-phase melting were similar values to those obtained by fluorescent measurements (Figures 2 and 3). This is because DOPC apparently attenuates the homophilic interactions of SM molecules and the resultant formation of gel domains. However, as shown in Table 1, reductions in the transition enthalpy (H) values caused by the addition of DOPC were less significant than the reductions caused by Chol,39,48 and the enthalpies of gel-phase melting in both SM bilayers at three different compositional ratios are somewhat similar.


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Therefore, the destabilization effect of DOPC on the gel-like domain is thought weaker than the homophilic affinity between SM molecules. In addition, we conducted a detailed comparison of the SSM and dhSSM thermograms. Deconvolution of the thermograms disclosed multiple components and complex melting behavior (Figure 4). The thermograms suggest that the three SSM bilayers may contain four (or more) different components that melt successively. In SSM-containing systems, some components showed a relatively narrow peak width, implying the presence of more cooperative gel domains, whereas dhSSM-based systems showed three major components with relatively broad endothermic peaks. Although we cannot determine lipid composition for each component or assign which component in the thermogram of dhSSM corresponds to which one in that of SSM, these components probably reflect laterally segregated gel phase consisting of SM and DOPC with different ratios. Their melting temperature is thought to rise with increasing SSM (or dhSSM) content in the phase. As shown in Table 1, the H values of the dhSSM systems were a little larger than those of the SSM systems, which also supported the concept of a thermally stable dhSSM gel domain. Moreover, the enthalpy of SSM gel phase melting is almost similar in different compositional ratios including that of pure SSM, while the enthalpy in dhSSM membrane slightly increase with increasing the ratio of dhSSM in the membrane. This small difference is attributed to the relative abundance of gel-like domains as well as the size of gel-like domains; the size of larger domains of dhSSM is gradually reduced with increasing concentrations of DOPC particularly over 50 mol%.


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TABLE 1. Transition enthalpies of SSM and dhSSM gel-phase melting in different lipid compositions. The average values calculated from two samples are reported, and the variation was less than 10%. *In all bilayer systems, enthalpy values were calculated for the main transition per molar quantity of SSM or dhSSM. Thermograms for unitary bilayers of SSM and dhSSM are shown in Figure S2.

Composition

H (kcal/mol)*

SSM/DOPC (1:9)

7.2 ± 0.2

(3:7)

7.6 ± 0.2

(5:5)

7.6 ± 0.2

(10:0)

7.6 ± 0.1

dhSSM/DOPC (1:9)

8.6 ± 0.4

(3:7)

9.0 ± 0.2

(5:5)

9.7 ± 0.1

(10:0)

9.8 ± 0.1

　　

　　


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Figure 4. DSC analysis of gel-phase melting in DOPC-based MLVs with different compositions. Panel A corresponds to bilayers based on SSM, while Panel B corresponds to bilayers based on dhSSM. The MLVs were subjected to a temperature scan of 1 °C /min, and each trace represents the fifth upscan. The


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thermograms were deconvoluted to analyze the possible components present during the complex gelmelting process. The line colors and styles do not necessarily imply identical phases in different panels.

3.4. Static 2H NMR spectra To explore lateral heterogeneity at different temporal and spatial resolutions, we recorded the 2H NMR spectra of selectively deuterated SSM and dhSSM, each bearing a 10’,10’-d2-stearoyl group in 50 mol% of DOPC-containing binary systems. The splitting width of Pake doublet signals precisely report the order parameter (Smol) of the 2H-labeled segment of lipids. We chose the C10 position of the acyl group to monitor the changes in the chain order caused by interactions with neighboring lipid molecules.47,48 The order parameters at the C10’ positions of SSM and dhSSM in 50 mol% DOPC bilayers were calculated to be ～0.20 through the tested temperature, which were a little smaller than that of pure SSM bilayers (0.25)47, suggesting that chain packing is slightly disturbed by the inclusion of DOPC in the bilayers. In this experiment, we aimed to detect the formation of gel-like and/or Lo domains on the basis of the spectral changes from a Pake doublet to a highly broad signal upon changing the temperature. In Figures 5E and 5I, the broad signals, which are characteristic of a gel phase, prominently overlapped with a typical Pake doublet at 20 °C and 30 °C; these temperatures are ~15 degrees lower than the end melting temperatures of SSM/DOPC (5:5) and dhSSM/DOPC (5:5), respectively. These observations suggest the coexistence of gel-like domains and fluid areas. At higher temperatures, the SSM bilayers did not show the broad pattern at a temperature 5 degrees lower (30 °C, Figure 5D) than the melting temperature, while dhSSM still showed a broad pattern at 40 °C (Figure 5G). Moreover, a comparison between the SSM and dhSSM bilayers suggested a remarkable difference in the temperature dependence of splitting; however, it would be difficult to simply compare the bilayers, because their lateral segregation behavior differs at the same temperature.34 Figure 5J reveals that the quadrupolar splitting 19 ACS Paragon Plus Environment

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width of SSM (open circles) increased moderately as the temperature decreased, while that of dhSSM (closed circles), which is significantly smaller than that of SSM, remained constant in the temperature range of 30 °C to 50 °C. These differences could be attributed to various factors, such as the abundance of SM molecules in the macroscopic fluid phase, the exchange rate of lipids between gel-like and fluid domains, and size of the gel-like domains. These factors will be discussed in more detail in the following section. To analyze the effect of gel-like domains on the temperature dependence and NMR spectra, we recorded the 2H NMR spectra of SSM and dhSSM at 30 mol% and 10 mol% in DOPC (Figures 5K and 5L). The SSM acyl chain in DOPC-based bilayers, in which macroscopic lateral phase separation was not observed, exhibited a linear decrease in the Pake doublet width as the temperature increased. This observation could be accounted for by a temperature-dependent increase in SSM mobility in the fluid phase, as well as by a decrease in the fractional ratio of SSM in gel-like domains as the temperature rises. In the dhSSM bilayers, a different propensity was observed. As the fractional ratio of dhSSM in DOPC bilayers was increased, the temperature dependency of dhSSM mobility became weaker in the range of 30 °C to 50 °C, which was prominent for the 50% and 30% dhSSM systems (Figure 5L).


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Figure 5. (A-I) Static 2H NMR spectra of 10’,10’-d2-SSM and 10’,10’-d2-dhSSM in 50 mol% DOPC


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bilayers as a function of temperature. (J) Quadrupolar coupling Δν (kHz) of SSM (open circles) and dhSSM (closed circles) in the presence of 50 mol% DOPC as a function of temperature. (K) Quadrupolar coupling Δν (kHz) at lower concentrations of SSM (10 and 30 mol%) in DOPC bilayers as a function of temperature. (L) Quadrupolar coupling Δν (kHz) at lower concentrations of dhSSM (10 and 30 mol%) in DOPC bilayers as a function of temperature.

4. DISCUSSION In this study, we aimed to elucidate the nanosized lateral segregation occurring in SSM/DOPC and dhSSM/DOPC binary systems using fluorescence, DSC, and 2H NMR experiments, which selectively report the properties of SM-rich and DOPC-rich domains. Several previous studies have examined Cholcontaining SM membranes and shown that nanoscopic lateral segregation occurs even in binary systems.26-31 These studies suggested that Chol induces a small lateral segregation by fragmenting large SM-rich domains. Here we also imply that, as is the case with Chol, DOPC destabilizes SM gel-phase by showing that SMs forms nanosized gel-like domains mainly due to their homophilic interplay in Chol-free membranes. We further focus on the homophilic interactions of SMs using SSM-DOPC binary systems without touching the role of Chol. The effects of Chol on the lateral segregation of lipid bilayers are remarkable, including the fragmentation of large gel domains to enhance the fluidity of membranes below a melting point and the higher stability of hydrocarbon chains throughout the temperature range of this study. Needless to say, these characteristic effects of Chol have also been confirmed in this study; the 2H NMR results revealed a great difference in the chain ordering of SSM between the presence and absence of Chol, where SSM–Chol binary membranes usually showed much larger splitting width around 50 kHz in the temperature range in Figure 5.48 Our previous study35 has shown that SSM/DOPC and dhSSM/DOPC bilayers induced macroscopic


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phase segregation corresponding to the mixed fluid/gel phase even at around Tm. In the present study, fluorescence and DSC results revealed that gel/ordered domains melted at a temperature lower than the Tm of pure lipids (Figures 2, 3, and 4). In particular, the fluorescence lifetime results in Figure 3 indicate that in the temperature range of 0–10 °C below Tm, the ordered phase with a long lifetime almost disappeared. The previous and present observations suggest that SM-rich gel-like domains probably coexist with highly fluid areas in the macroscopic gel phase within this temperature range, although fluorescence lifetime measurements that should have a nanosecond time scale and a spatial resolution of tens of nanometers failed to detect these gel-like domains. It would be reasonable to consider that these domains, in which a rapid exchange of lipids occurs, are transient nanosized aggregates. Next, we discuss the 2H NMR results of 10’,10’-d2-SSM and 10’,10’-d2-dhSSM. A comparison of the splitting widths with fluorescence lifetimes indicates that the temperature dependence for the SM/DOPC (5:5) membranes was largely similar between them (Figures 3 and 5). Both fluorescence lifetime and NMR measurements indicated that the effects of temperature on the ordering of dhSSM molecules are significantly lesser than on SSM. The gel-phase melting of SSM and dhSSM is not prominent in the 2H NMR spectra as compared with fluorescence anisotropy and DSC results since Pake doublet peaks largely represent highly mobile SM molecules in Ld phase and those in interfacial Ld/gel area while those in gel phase usually do not show prominent doublet signals but very broad peaks that look like a concave-down baseline such as those in Figures 5E, 5G, 5H and 5I. On the other hand, the first spectral moments (M1) of d2-SSM and d2-dhSSM in 50 mol% DOPC, which reflect signals of the lipids in gel phase, clearly demonstrated the gel-phase melting at around similar temperatures to those revealed by the fluorescence anisotropy and DSC data (Figure S3). There is a considerable difference in the width of 2H signal splitting between SSM and dhSSM at 30 °C (Figure 5D and 5I), which could be attributed to the abundance of SM molecules in the


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macroscopic fluid phase. As the splitting width of Pake doublets largely reflects the SSM/DOPC (or dhSSM/DOPC) ratio in the fluid phase, the content of dhSSM in the fluid phase is thought smaller than that of SSM. Second, the temperature dependence of the splitting width differs between SSM and dhSSM (Figure 5J). This is probably caused by the difference in their gel-melting temperature and the size of the gel-like domains. In the range of 30–50 °C, where the splitting width of dhSSM in the dhSSM/DOPC (5:5) membrane showed no temperature dependence, gel and/or ordered domains are thought to be formed, while the area and size of the domains decrease as the temperature increases. Between 30 °C and 40 °C, the formation of the dhSSM gel phase is apparent from the enthalpy changes in the DSC thermograms (Figure 4B). Between 40 °C and 50 °C, despite the absence of endothermic peaks in the DSC thermograms, the longer fluorescence lifetime of tPA-dhSM (Figure 3C), corresponding to the gel-like phase, decreases from 41.7 ns to 5.8 ns (Table. S1E), suggesting that a phase transition from gel to Ld occurs over this temperature range. Gel-phase formation is also supported by the appearance of a gel-like broad signal in the 2H NMR spectrum of dhSSM at 40 °C (Figure 5G). In this temperature range, a reduction in the dhSSM gel-phase ratio leads to an increase in the content and order of dhSSM in the DOPC-rich fluid phase, which partly enhances the order of lipids to compensate for the temperature-dependent decrease in the splitting width. Moreover, the fast lipid exchange rate between the gel-like domains and fluid area and the reduction in the gel domain size also have a similar effect on the Pake doublet width. 31 On the other hand, the SSM/DOPC (5:5) membrane melted completely at 33 °C (Figure 4A), indicating that macroscopic gel domains were not formed in the temperature range of 30–50 °C. Moreover, the longer lifetime component of tPA-SM disappears at 35 °C (Figure 3C), thus leading to a larger temperature dependence of acyl chain ordering (Figure 5J). The rate of reduction in the splitting width of SSM from 30 °C to 45 °C is quite close to that of DOPC in a similar binary system (Figure S4).57 This observation further suggests that no detectable phase 24 ACS Paragon Plus Environment

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separation occurs in the SSM binary systems at the NMR timescale, and the domain size of the SSM gel phase in this temperature range is quite small, probably in the nanometer range, as discussed previously for SSM–Chol bilayers.31 To examine closely how the temperature-dependent mobility of SSM and dhSSM was influenced by gel-like domains, we next measured the quadrupole splitting width at lower concentrations of SSM and dhSSM (10 mol% and 30 mol%, Figure 5K and 5L) in DOPC bilayers. Plots of the splitting width of SSM at the lower contents as a function of temperature, in which macroscopic phase separation was not apparently observed, showed a nearly linear correlation up to the melting point (Figure 5K). On the other hand, 30 mol% dhSSM membranes (Figure 5L) showed a small temperature dependence compared to dhSSM/DOPC (1:9) bilayers. These characteristic properties of the temperature-dependent splitting width probably reflect the features of gel-like SM domains in the DOPC fluid phase. In both SSM/DOPC (5:5) and dhSSM/DOPC (5:5) bilayers, a broad signal overlapping with a typical Pake doublet was observed at the temperature 15 degrees below the end point of melting (20 °C and 30 °C, respectively as shown in Figure S5E and S5I), indicating that gel-like and fluid phases coexist. We deduce that under these conditions, the domains in both SSM and dhSSM bilayers are large enough to distinguish gel-like domains from fluid areas during the NMR timescale (i.e., ~10 μs). However, at a temperature 5 degrees below the end point of melting, SSM (30 °C) in the same bilayers shows a clear Pake doublet, while the dhSSM signal (40 °C) still exhibits overlapping Pake doublet and broad signals. These observations could be accounted for by the difference in the domain sizes between SSM and dhSSM as follows (a similar discussion has been reported in our previous paper).31 In this SSM/DOPC (5:5) system, the Pake doublet width became larger when temperature was raised from 20 to 30 °C (Figures 5D and 5E) while the gel phase significantly occurred in this temperature range (Figure 4A). This result suggested that the exchange speed of SSM between gel and fluid phases was higher at 30 °C 25 ACS Paragon Plus Environment

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and high enough to regenerate a typical Pake signal in the spectrum where the concave down of a baseline disappeared. We deduced that this characteristic behavior of the 2H NMR signal can be translated to changes in the following parameters of nanosized domains: the size and fractional abundance of gel domains, and the exchange speed of lipid between gel domains and fluid areas. One possible account is as follows; under the conditions in Figure 5, the domains should be small enough to allow the lipids to go back and forth between gel domains and fluid areas on the microsecond time scale, when considering the slow exchange (diffusion) speed of SSM below Tm and a relatively small fraction of the gel area at 30 °C as suggested by the DSC data (Figure 4A, bottom). The size could be a few nanometers as estimated by previous result.31 On the other hand, dhSSM showed a broad pattern even at 40 °C, where the diffusion of dhSSM should be fast. This difference implies that dhSSM tends to stay inside a large gel-like domain during the NMR time scale. This notion suggests that the size of dhSSM domains is significantly larger than that of SSM domains. Particularly, the difference is evident when the dhSSM/DOPC ratios are high in the binary systems; in other words, large gel-like domains occurring in dhSSM-abundant membranes, such as dhSSM/DOPC (5:5) and (3:7), lead to higher stability in acyl chains, as shown in Figure 5J and 5L. The formation of large domains in dhSSM-rich membranes is further suggested by the enthalpy change (H) on gel-phase melting (Table 1); the increase in H for dhSSM-DOPC (5:5) is likely due to larger (and/or stable) domains of dhSSM, where a larger fraction of dhSSM molecules can be confined without going out to the surrounding DOPC-rich areas. On the other hand, H on SSM gel-phase melting is quite similar among the three compositional ratios. This observation can be attributed to the lower abundance of gel phase and the smaller gel domains in SSM membranes. Previous studies have implied that SSM is densely packed to form nanosized gel-like domains or nano-clusters in a homophilic manner.31,40 The observations in the present study suggest that gel-like


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domains formed by dhSSM are larger in size, more tightly packed, and thermally more stable compared with those of SSM. This difference between SSM and dhSSM could be explained by the small but significant variance in their homophilic affinity, particularly in the intermolecular hydrogen bonding capability, as suggested by previous studies; it has been reported that dhSSM forms stronger intermolecular hydrogen bonds than SSM.35-40 Hydrogen bonding is thought responsible for the strong SM–SM interplay reported in previous studies 35,58; in the Chol-induced Lo phase, the amide group of SM has an orientation suitable for the formation of an intermolecular hydrogen bond network. The present results suggested that a possible stronger hydrogen bond by dhSSM-dhSSM results in the formation of larger domains compared with those by SSM. It is reasonable to assume that domains size is greatly influenced by the strength of the intermolecular hydrogen bonds and by the efficiency of colipids, such as Chol and DOPC in this study in fragmenting gel-phase domains of SMs. The present results reveal that DOPC has lower efficiency than Chol, but their basic roles in domain formation do not differ greatly. Regarding the role of the homophilic interaction of SMs in biomembranes, the observed lipid dynamics and properties of gel-like domains of SSM are expected to be grossly similar to those of lipid rafts in cellular membranes, as the size of lipid rafts is thought less than a few tens of nanometers.3,9,59-62 In conclusion, we examined the nanoscopic lateral segregation of SM-DOPC binary systems using fluorescence experiments, DSC analysis, and 2H NMR spectroscopy at different temporal and spatial resolutions. The results of the fluorescence experiments disclosed the formation of gel-like domains with relatively high fluidity in both SSM/DOPC and dhSSM/DOPC membranes, even in the absence of Chol. The 2H NMR spectra of SSM/DOPC and dhSSM/DOPC bilayers indicated significant differences in the characteristic properties of the gel-like domains. These experiments demonstrate that a small difference in the structure40 gives rise to measureable differences in acyl chain packing and nanoscale lateral


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segregation, which are largely attributable to the intrinsic homophilic interaction of SM rather than interplay with other colipids (Chol).

AUTHOR CONTRIBUTIONS T.Y., J.P.S., and M.M. designed the research study; T.Y. and J.P.S. performed the experiments. All the authors analyzed the obtained data and contributed to article writing.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXX/acs.langmuir. YYYY. Time-course data for fluorescence intensity changes; Tables of Time-resolved fluorescence decays; DSC thermograms of unitary bilayers of SSM and dhSSM; Quadrupolar splitting width of d2-SSM and d2-dhSSM in DOPC bilayers; The first spectral moment (M1) of d2-SSM and d2dhSSM in DOPC bilayers; Spectral simulations of 2H NMR spectra of of d2-SSM and d2-dhSSM in Figure 5; Static 2H NMR spectra of d2-DOPC in SSM bilayers as a function of temperature (PDF).


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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS Financial support was generously provided by Grant-In-Aids for Scientific Research (S) (No. 16H06315) from the Japan Science and Technology Agency (JST) (to M.M.), Academy of Finland (to J.P.S.), and Sigrid Juselius Foundation (to J.P.S.). T.Y. is partly supported by the “International Collaboration Promotion Program” of Osaka University and by the Japan Science and Technology Agency (JST) ERATO Lipid Active Structure Project (JPMJER1005).

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