Morphology and dynamics of domains in ergosterol or cholesterol containing membranes

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Morphology and dynamics of domains in ergosterol or cholesterol containing membranes Arturo Galván-Hernándeza, Naritaka Kobayashib, Jorge Hernández-Cobosa, Armando Antillóna, ⁎ Seiichiro Nakabayashic, Iván Ortega-Blakea, a

Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México, Av. Universidad s/n, Col. Chamilpa, Cuernavaca, Morelos, 62210, Mexico Division of Strategic Research and Development, Graduate School of Science and Engineering, Saitama University, Japan c Department of Chemistry, Faculty of Science, Saitama University, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Atomic force microscopy Supported lipid bilayers Cholesterol Ergosterol Quantum mechanical calculations Molecular dynamics

The effect of cholesterol and ergosterol on supported lipid bilayers composed of 1-Palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) and egg sphingomyelin (eSM) in a 1/1 M ratio was studied using atomic force microscopy. The addition of ergosterol or cholesterol to these membranes considerably modifies both the structure and the dynamics of the domains present in them. The height of the eSM enriched domains increases with concentration of both sterols, but more markedly with ergosterol. The height of the POPC enriched domains increases with concentration in a similar manner for both sterols. This effect is larger for eSM than for POPC when ergosterol, not cholesterol, is present. Domain coverage increases with both sterols at 5 mol% but decreases at 20 mol% and almost disappears at 40 mol%. The size of the eSM enriched domains decreases with sterol concentration, more markedly with cholesterol. Bilayer rupture forces show that overall stiffness increases with the addition of 5 mol% cholesterol, but only for the eSM enriched domains with ergosterol at the same concentration. At larger sterol concentrations the stiffness of both regions becomes reduced. At 40 mol% sterol concentration, both membranes present the same rupture force value. To gain mechanistic insight into these observations we performed Quantum Mechanical calculations and Molecular Dynamics simulations of the sterol molecules. We found that conformational freedom for the sterol molecules is quite different. This difference might be behind the observed phenomena. Finally, the different action of sterols on membrane properties is related to the sterol-dependent ionophoretic activity of polyene antibiotics.

1. Introduction The cell membrane plays an important role in many biological processes [1–4]. The occurrence of lipid rafts or lipid domains within the membrane, that are thought to occur due to molecular interactions between sphingolipids, glycerophospholipids and cholesterol (Chol) [5–8] has changed the way we consider interactions between the membrane and other molecules. [5–8]. There is evidence that lipid rafts are binding sites for proteins [9,10], and they have recently been observed in living cells using fluorescent sphingomyelin analogs [11]. Other experiments in cells have shown that sphingomyelin enriched rafts are not cholesterol enriched [12–14]. In model membranes, raft-

like domains originated by phase segregation have been found to be detergent resistant [15]; the target of toxins, like alpha hemolysin [16] and Chol-dependent [17]. These models, including Giant Unilamellar Vesicles (GUV) and Supported Lipid Bilayers (SLB), help to better understand the physicochemical properties of membranes and their interaction with molecules of interest [18,19]. Phase segregation appears in binary mixtures of lipids having one saturated and one unsaturated lipid [20,21]. This segregation is affected by sterols and the mechanism by which this occurs is still unclear, though it is generally assumed to be caused by effects on membrane ordering and condensation. This is particularly important when it comes to the different effects that sterols have on saturated and

Abbreviations: POPC, 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; eSM, egg sphingomyelin; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC, 1,2dipalmitoyl-sn-glycero-3-phosphocholine; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; Erg, Ergosterol; Chol, Cholesterol; AFM, Atomic Force Microscopy; SM, sphingomyelin; FM-AFM, Frequency Modulation Atomic Force Microscopy; AmB, Amphotericin B; Nys, Nystatin; SLB, Supported Lipid Bilayer; GUV, Giant Unilamellar Vesicles; LUV, Large Unilamellar vesicle; SUV, Small Unilamellar Vesicle; DPBS, Dulbecco's Phosphate Buffered Saline; FvD, Force versus Distance ⁎ Corresponding author. E-mail address: [email protected] (I. Ortega-Blake). https://doi.org/10.1016/j.bbamem.2019.183101 Received 10 June 2019; Received in revised form 2 October 2019; Accepted 24 October 2019 0005-2736/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Arturo Galván-Hernández, et al., BBA - Biomembranes, https://doi.org/10.1016/j.bbamem.2019.183101

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permeabilization of the membrane by Nys is related to its effects on membrane properties and organization. These results, along with electrophysiological studies of single channel [38,39], suggest that sterol dependency of the action of polyene antibiotics could be due to membrane biophysical and physicochemical properties rather than a direct chemical interaction with the polyene. Encouraged by this hypothesis, and the apparent similarity between Erg and Chol, we aim to determine any differences in the membrane structures and dynamics produced by these sterols. Previous studies using AFM have shown that bilayers of POPC and SM at equimolar ratios form well defined gel-phase domains [40,41]. Hence, we want to observe the effects of adding sterols to this equimolar mix. According to de Almeida et al. [42], at this ratio increasing amounts of Chol lead to a phase transition from ld + so to lo + ld + so and, at even higher Chol content, to lo + ld or lo. Additionally, it was found that in the lo + ld region of a POPC/sterol mix, Nys showed the highest mean conductance compared to the other existing phases [39]. We are thus interested in determining the effects produced by on membrane structure in this mixed region for the ternary mixture of POPC/eSM/sterol. Over the past twenty years AFM has been used to study biological systems [43–45]. Model membranes like SLB composed of different lipid mixtures have been studied by AFM due to the high lateral resolution this technique offers. For instance, the evaluation of the mechanical properties of cells by AFM has proven useful to differentiate healthy cells from neoplastic cells [46–49]. Topographic images of cells and model membranes allow us to study changes in morphology due to toxins, mutations or lipid composition [32,40,50,51]. In particular, AFM has proven an important tool in the study of raft-like domains and phase segregation in model membranes [15,52,53]. SLBs studied by AFM have allowed for the observation of membrane morphology [52], evaluation of mechanical properties [54] and following the interaction of molecules with membranes [16]. A liquid-environment frequency modulation AFM (FM-AFM) [55] with atomic resolution has been developed [56,57] during the past decade in the last decade, and opened a new window into the study of atomic-scale biological systems with a pico Newton-order loading force to the samples [58,59]. An FM-AFM study observed differences in the molecular arrangement of SLBs in gelphase composed of DPPC/Chol at 0, 5 and 50 mol% Chol concentration [60]. The same study showed a decrease in the rupture force of a DPPC bilayer when Chol was added at 50 mol%. Other AFM studies have shown slight changes in the lipid content of the membranes can have a large impact in phase segregation. A recent study shows that changing the unsaturated DOPC lipid for the mixed alkyl chain POPC in a mixture with sphingomyelin and Chol dramatically changes membrane morphology [40]. Macroscopic raft-like domains present in the threecomponent membrane DOPC/brain-sphingomyelin/Chol change in a gradual and continuous way into nanoscopic raft-like domains as DOPC is substituted by POPC. This dependence of membrane morphology on composition has also been reported by other techniques (e.g. fluorescence studies [61]). Hence, AFM appears a most adequate technique to study the mechanical and structural differences between lipid bilayers containing Erg or Chol. To do so, we performed AFM studies of eSM enriched domains in lipid bilayers composed of POPC, eSM and Erg or Chol and compared their morphology and other molecular properties to determine differences. We used POPC and SM due to their biological relevance. POPC mixtures with brain SM were shown to be sensitive to Chol concentration [40,41] and so we expected POPC/eSM mixtures to also be sensitive to sterol, which proved correct. The differences in relative height, coverage and size of the eSM enriched regions are presented, as are the Force versus Distance (FvD) curves that allow for the determination of the rupture force and therefore comparison of mechanical properties. Finally, high quality ab-initio calculations and Molecular Dynamics simulations were performed to gain insight into the molecular mechanisms behind these phenomena.

unsaturated lipids. The addition of ergosterol (Erg) to 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) bilayers produced filament-like structures on both gel and liquid ordered phases, as observed by atomic force microscopy (AFM) of SLBs [22]. The addition of Chol produces much larger domain-like gel-phase regions, spotted with small liquid-ordered regions on DPPC Langmuir-Blodgett monolayers imaged with AFM [23]. Both sterols lead to the appearance of liquid ordered phase domains in a gel-phase membrane, but with different domain morphologies. Hsueh et al. [24] showed, by Deuterium Nuclear Magnetic Resonance and Differential Scanning Calorimetry, that Erg is less effective than Chol at inducing liquid ordered domains in gel and liquid disordered DPPC membranes. In membranes presenting liquid disordered phase or coexistence of ordered and disordered liquid phase, Erg is more effective than Chol at ordering chains. Hung et al. [25] used X-ray Scattering and Grazing-Angle Scattering to investigate the condensing effect of Erg and Chol on membranes of: a saturated lipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); a mixed alkyl lipid, 1Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); and an unsaturated lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). They found that Erg has no condensing effect, and thus no membrane thickening on POPC and DOPC membranes, whereas Chol does. For DMPC they found that Erg has a smaller effect on chain-ordering than Chol. These results are in contradiction with previous studies [26]. Hung et al. [25] suggest that binding of Erg or Chol to lipids is quite distinct: it resembles hydrophobic-matching for Chol but not for Erg. On the other hand Pencer et al. [27] using small-angle neutron scattering measurements on pure DMPC unilamellar vesicles which contained either Chol, Erg or lanosterol showed that all three sterols increase bilayer thickness in a similar fashion. But these sterols produce different thermal area expansion coefficients, lanosterol having a higher value followed by Chol and finally Erg, implying a different condensing effect induced by each sterol. There is no condensing effect of Erg in membranes of unsaturated lipids, POPC or DOPC. In a saturated lipid bilayer (DPPC) Erg is more effective in ordering lipid chains, but there are mixed results for another saturated lipid bilayer (DMPC) there are mixed results. Similar results have been reported for ternary mixtures of DOPC, DPPC and Erg or Chol. Both sterols promote the appearance of domains of an ordered liquid phase [28] on either gel or liquid disordered bilayers. This domain formation has been observed by AFM in Erg containing mixtures [29] and in many cases for Chol containing ones, see for example a review by El Kirat et al. [20]. In summary, there are differences between Chol and Erg effects on either saturated or unsaturated lipids, but the existing results are still unclear and some are contradictory. SLBs which present phase segregation have served as a model to study the interaction of a wide variety of proteins, peptides and drugs with the lipid membrane [30–33]. This is particularly the case for polyene antibiotics, like Amphotericin B (AmB) and Nystatin (Nys), which are selective to the different sterols present in the membrane. This distinction, which presents more ionophoretic activity for ergosterol containing membranes than for cholesterol containing ones, allows for the therapeutic use of polyenes. However, they still have very serious side effects [34], which limit their therapeutic use in spite of their strong potency. We therefore need to better understand the mechanisms of action of these antimycotics. The activity of polyenes leads to the leakage of the plasma content, mainly K+, leading to cell death [34]. Polyenes show a stronger activity in Erg containing membranes, such as in fungal cells, compared to Chol containing ones, such as in mammalian cells. It has been suggested that cell membrane biophysical properties can be critical to this selectivity [35]. Hence, raft-like domains could be crucial. In fact, gel-phase domains have been observed in the plasma membrane of Saccharomyces cerevisiae [36]. These domains were found to be sphingolipid enriched but not Erg enriched. Another recent study shows [37] that Nys incorporates into gel-phase domains of a POPC membrane with a saturated lipid, concluding that 2

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2. Materials and methods

sample of a collection of very similar images. We searched for membrane areas with holes (regions of the mica surface surrounded by membrane) for each composition and attained these by adjusting incubation time. This was done in order to measure both POPC and eSM enriched membrane thickness. All topographic images shown were processed with XEI software from Park Systems to remove tilt by leveling, using a first-order function obtained from one line of the topography and then subtracting it from the rest of the lines. In some cases, a bow appeared due to the piezoelectric performing large displacements. In such cases a secondorder flattening process was used. When the eSM enriched domain coverage is high, these domains have to be excluded from the plane of reference in order to obtain an appropriate flatten process. For low coverage of eSM enriched domain the image can be flattened without this consideration. No further processing was done. Height histograms were generated from topographic data using Grace (version 5.1.22) [65] and Gaussian functions were fitted using Gnuplot (Version 5.0 patchlevel 6). Domain area coverage and height difference were obtained from the fit parameters. Five height histograms were used for each composition. Frequency modulation AFM (FM-AFM) measurements were performed by a home-built FM-AFM system with an ultra-low noise optical beam deflection sensor [56,66,67]. A cantilever is oscillated at its resonance frequency with a phase-locked loop (PLL) circuit (OC4, SPECS). The PLL circuit is also used for keeping an oscillation amplitude of the cantilever at a constant value and detecting a frequency shift of its resonance frequency, induced by a tip-sample interaction force. The tip-sample distance was regulated with a customized AFM controller (ARC2, Oxford Instruments). NCHAuD silicon cantilevers purchased from Nanoworld were used. Their nominal resonance frequency and spring constant were 150 kHz and 42 N/m, respectively. Personnel involved in the preparation of the samples for contact-AFM also prepared the samples for FM-AFM. Some materials had to be substituted due to differences in sample loading for the FM-AFM equipment. This included a larger mica substrate and no Nunclon cell culture dish. Volumes were adjusted to the new surface area, and 75 μl of CaCl2 were added to a freshly cleaved mica followed by 125 μl of liposome emulsion. Incubation times were kept unchanged.

2.1. Chemicals POPC, Chol and egg SM were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Erg and Dulbecco's Phosphate Buffered Saline (DPBS) were purchased from Sigma-Aldrich (Toluca, México). The lipids in powder form and their stock solutions in chloroform or chloroform/methanol 8/2 v/v for egg SM were kept at −20 °C. 2.2. Liposome preparation Small unilamellar vesicles (SUV) of POPC/eSM/Chol and POPC/ eSM/Erg mixtures were prepared by adding the appropriate amounts of stock solutions into a round bottom flask (4321A-5) from Pyrex (NY, USA). The solvent was evaporated using an R-II rotary evaporator from Büchi (Flawil, Switzerland) in a two-step cycle consisting of 45 min at 400 mbar at 40 °C and 45 min at 72 mbar at 40 °C to ensure solvent and water evaporation. Repressurization was done using 99% pure nitrogen. The dried lipid films were rehydrated to a final concentration of 0.25 mg/ml with ultrapure water and placed in an ultrasonication bath for 1 h (Ultrasonic bath 8890 from Cole-Parmer, IL, USA). Vesicle yield is higher in pure water so no ionic force was used in this preparation. 2.3. Supported lipid bilayers The liposomal suspension was either used the same day or one day after preparation, in which case the suspension was stored at 4 °C. To prepare SLB, grade V-5 muscovite mica disks of 9.9 mm in diameter from SPI Supplies (West Chester, PA, USA) were used as substrate. The disks were glued with double sided tape (Tuk, México) onto a Nunclon cell culture dish from Thermo Scientific (153066) purchased from Sigma Aldrich (Toluca, México). SLB were prepared using the vesicle fusion method [62–64] as follows: Liposome suspension was heated to 40 °C prior to incubation, as well as 4 mM CaCl2 solution. 20 μl of CaCl2 were added to a freshly cleaved mica followed by 80 μl of liposome emulsion. The presence of divalent cations is important to ensure bilayer adsorption to the mica surface. The cell culture dish with the mica and the emulsion was placed on a hotplate (Heidolph MR Hei-Standard, Schwabach, Germany) at 40 °C. The sample was washed three times. The first and second times the sample was washed with three volumes of 50 μl of ultrapure water. On the third one the sample was washed with five volumes of 50 μl of DPBS in order to have a standard ionic solution in the preparation. The elapsed time between each wash varied depending on the lipid mixture. Higher sterol concentrations required 15 min to obtain good membrane coverage, while 10 min was enough for the lowest concentration of sterol. The sample was then left to cool down at room temperature (21 °C) for 1 h prior to AFM imaging.

2.5. Force spectroscopy AFM can be used to obtain mechanical parameters of desired samples. Force vs Distance (FvD) curves were obtained and analyzed. When performing a FvD curve, the tip approached the sample until it made contact, then the cantilever was further displaced downward, increasing the load force applied to the sample and, finally, when a maximum desired load force was reached the tip is retracted. All throughout this process, the force at each displacement value was computed, generating a curve as that presented in Fig. 1, where the red trace is the approach curve and the blue trace is the retraction curve. The FvD curves were generated by using the displacement values recorded by the z-Scan and the force values measured at each point. The analysis of these curves was done following the description in OvalleGarcía et al. [68]. Force versus distance measurements were performed to characterize the mechanical properties of membranes composed of the different lipid mixtures. The curves were taken in a 2D matrix of 256 individual curves over different area sizes. Approach velocities were kept under 100 nm/ s. Approach and retraction curves were composed of 4096 points each. At a certain load force between the contact point and the maximum force load, the supported lipid bilayer cannot withstand the pressure applied and it allows for tip penetration. This particular load force is called breakthrough force or rupture force and is characterized by a short discontinuity in the FvD curve, see Fig. 1. Cantilevers with a nominal spring constant of 600 mN/m (MSNL-10-F from Bruker) were used to obtain FvD curves due to the their high sensitivity, which

2.4. Atomic force microscopy images of supported lipid bilayers An XE-Bio Atomic Force Microscope from Park Systems, South Korea, was used to obtain topographic images of SLB and Force versus Distance curves. XEP and XEI software from Park Systems were used to obtain and analyze data respectively. MSNL-10 silicon nitride cantilevers from Bruker AFM Probes, CA, USA, were used. The cantilevers available on each holder chip had nominal spring constants of 10, 60 and 600 mN/m. All cantilevers used were calibrated using the Thermal Method routine built into the XEI software. Cantilevers were immersed into the aqueous sample and left to stabilize for at least 15 min until fluctuations in the laser were not present. Topographic images were obtained at 21 ± 1 °C using cantilevers of nominal value of 10 mN/m. Images are 256 pixels × 256 pixels and 5 × 5 μm2 in size and were taken at a frequency between 2 and 3.5 Hz and load forces lower than 1 nN. For each lipid composition at least five different areas of each same sample were scanned. Images presented correspond to one typical 3

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Fig. 1. Example of a force versus distance curve. Force versus distance curve measured on a Supported Lipid Bilayer composed of POPC/eSM 1/1 mol/mol. The red trace represents the approach curve. The blue trace represents the retraction curve. The contact point can be identified around 160 nm. A membrane rupture, indicated by a discontinuity, can be observed around 15 nN.

for each system. Cycles of heating and cooling between 303.15 and 0 K were performed with a time period of 2.5 ns for each process. The NoseHoover thermostat was used to control the temperature. To explore the configurational space of the molecules we performed NVT molecular dynamics simulations at 303.15 K using the Nose-Hoover thermostat to control the temperature. A total time of 40 ns were simulated with a time step of 2 fs. Configurations were saved every 0.2 ps to the trajectory file. These configurations were subsequently analyzed using the Ensemble Match and Ensemble Cluster tools of the USCF Chimera program version 1.13.1 [80] which also generate the images for the clustering results. Finally, 3D molecular structure images were generated with the MOLDEN package [81].

allowed to clearly resolve the rupture forces. Each cantilever was calibrated before use. Analysis of the FvD curves was done using a routine written with Matlab (MathWorks) to obtain the rupture force values. Matlab was also used to generate the corresponding force histogram plots. The FvD curves were transformed to the Force-Separation scheme (FvS). This was accomplished by the operation S = Displacement + Force / (calibrated cantilever constant). In this scheme the signal of the rupture process is enhanced. By taking the derivative of the force under this scheme, the highest peak yields the rupture force and the width yields the penetration depth into the membrane. Penetration was used to discriminate and discard false rupture signals. Penetration values larger than 6 nm and smaller than 1.5 nm were considered false ruptures.

3. Results 2.6. Height-height correlation function 3.1. Topographic images Topographic data obtained were used to calculate the weighted height pair distribution function:

Topographic images of POPC/eSM 1/1 mol/mol supported membranes where obtained at different sterol content in contact mode and using the cantilever MSNL-10-C with nominal spring constant of 10 mN/m. Chol and Erg were added at 5 mol%, 20 mol% and 40 mol%. Fig. 2 shows typical AFM topographic images for the different lipid mixtures. In this figure, images of membrane patches with an area of 25 μm2 present three distinct zones. The mica surface (black regions), the bilayer surface (brown regions) and domains on the bilayer surface (yellow regions). We assume that the bilayer region corresponds to the POPC enriched membrane whereas the domain regions correspond to the eSM enriched membrane. There are also distinct morphologies. At zero sterol there are large domains ~8 μm2, and at 5 mol% sterol there is some resemblance to the previous case, but the domains for Chol presence seem to be more elongated. At 20 mol%, differences in the morphology of the domains are more marked. The domains for both sterols are much smaller than in the previous cases, which is in agreement with the results of Ho et al. [40]. Additionally, there is a strong difference between Chol and Erg: in the former, domain coverage is very small, whereas for the latter they are very well defined and still in abundance. At 40 mol% sterol, the membrane seems to be homogeneous for both sterols. In this latter case, domains could be too small to be distinguishable at this resolution. In order to check this, membrane patches of 1 μm2 and 40 mol% sterol concentration were scanned. The results show that Erg still produces domain formation but the height difference between the POPC and the eSM enriched regions is smaller than in the previous cases, see Fig. 3. The size of these domains is reduced in comparison to those at 5 and 20 mol% by a factor of 5. In the case of Chol it is difficult to observe clear domain formation even at this resolution. As shown in Fig. 2, Chol and Erg produce quite a distinct effect on the structure of the SLB. A better comparison can be obtained by generating distribution plots of the height of the above membrane patches with respect to the mica surface. Fig. 4 shows the histograms corresponding to the images in Fig. 2. At zero sterol, the eSM enriched

〈 (h (→ x ) × h (→ x +→ r 〉 − 〈h (→ x ) 〉〈h (→ x +→ r )〉 Ghh (r ) = σ2 → where → x is any specific point in the image and r is a displacement vector, brackets denote an ensemble average over the radial distance r, and σ is the standard deviation of the whole collection of values in the AFM grid. This function was computed with an in house program. Grace (version 5.1.22) was used to generate the height - height radial distribution function plots. 2.7. Quantum mechanical calculations High quality ab-initio calculations with the 6–31 + G* basis set were performed for Chol, Erg and Coprosterol molecules using the Gaussian 09 revision D. 01 package [69]. Two levels of calculation were used to include the electron correlation energy, the Möller-Plesset perturbation method up to the second order (MP2) and DFT calculations with the B3LYP functional adding the D3 version of Grimme's dispersion with the original D3 damping function (GD3) [70]. In order to evaluate the effect of the solvents in the configurations and relative energies obtained, we performed calculations in the presence of solvents using the Self Consistent Reaction Field (SCRF) method with the Polarizable Continuum Model (PCM) [71]. 2.8. Molecular dynamics simulations Molecular Dynamics simulations were performed using the Gromacs package version 5.0.2 [72–74] with either the CHARMM36 Lipid Force Field [75,76] or Slipids force field [77–79]. Single molecule simulations were performed in all cases without periodic boundary conditions. For energy minimizations we used the steepest descent method, with a maximum force criterion for convergence of 0.1 kJ mol−1 nm−1. For the simulated annealing, we simulated a total of 40 ns with a 2 fs step 4

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concentrations. The averages were computed from the parameters of fitted Gaussian functions, one function for each peak of the histograms presented in Fig. 4. Chol does not have a marked effect on the average Δh of the eSM enriched domains, but Erg clearly increases this value for 5 and 20 mol%. Regarding percentual area coverage of the eSM enriched regions: 5 mol% sterol increases the coverage with respect to 0 mol%, whereas 20 mol% diminishes the coverage with respect to 5 mol%. In the case of Chol the area coverage nearly vanishes at 20 mol %. At 40 mol% area coverage is barely appreciable for both sterols and thus not plotted. An independent-sample t-test of equal variance was performed to compare the effect of addition of Erg or Chol on relative height and domain coverage. The P-values for height difference for both 5 mol% and 20 mol% are P < .001 (P = .000073 and P = .000035 respectively) which shows a significant difference in the between effect of both sterols. The P-values for the domain coverage show a slightly different effect for 5 mol% sterol (P = .192) while for 20 mol% sterol the difference is quite significant (P = .00000052). In Fig. 6 we present line profiles of all regions of the bilayer for the different compositions. We point out that areas with holes correspond to the mica surface. The figure shows height differences and domain sizes, emphasizing their morphological differences. Here we can clearly see there is a non-linear response of membrane thickness to sterol concentration. This effect is discussed further down. The ordering effect of Erg on the eSM enriched domains produces an increase in the height difference with respect to the POPC enriched domain of ~100% compared to the sterol-free membrane. But Chol only increases it by ~15%. Since this result was obtained with contact AFM, it could be that the tip, even with a small force, is squashing the membrane and affecting the responses of the different regions. In order to check this, we repeated some experiments with Frequency Modulation Atomic Force Microscopy (FM-AFM). This technique has negligible action on the membrane surface [58,66,82]. In Fig. 7, FMAFM topographic images and their corresponding height profile of a POPC/eSM bilayer having Erg at 0 mol% and 20 mol% are presented. Images of eSM enriched regions were taken in areas of 1 × 1 μm2, an area much smaller than that in the contact-AFM case (5 × 5 μm2). It can be seen that there was indeed some squashing of the membrane produced by contact AFM. The height difference values are in fact larger in the FM-AFM experiments, 1.47 nm and 2.37 nm, in comparison to contact-AFM values, 1.00 nm and 1.94 nm. Another difference between contact-AFM and FM-AFM images surges when there is no sterol. What appeared to be large eSM enriched domains in the contact-AFM images are now shown to be clusters of smaller domains.

Fig. 2. AFM topographic images of POPC/eSM and Erg or Chol SLBs. A corresponds to 0 mol% sterol; B1, B2 and B3 correspond to 5 mol%, 20 mol% and 40 mol% Erg; C1, C2, and C3 correspond to 5 mol%, 20 mol% and 40 mol% Chol. Scan size 5 μm × 5 μm, z range is 10 nm. Scale bars are 1 μm.

region's thickness is ~3.75 nm and the POPC enriched region's thickness is ~2.8 nm: thus, the relative height (Δh) is ~1 nm. There is a similar profile with 5 mol% of Chol, ~2.9 nm in the POPC enriched region and ~4.2 nm in the eSM enriched region: thus, Δh is ~1.3 nm. For Erg at 5 mol%, the effect of increasing height of the eSM enriched domains is more marked: now Δh is around 2 nm (from ~2.9 nm to ~4.7 nm in the corresponding regions), indicating a larger ordering effect. At 20% sterol, the difference between Chol and Erg effects is more dramatic. The bilayer with Chol shows negligible presence of eSM enriched domains, whereas the one with Erg has well defined domains, with a thickness of ~8 nm: the relative height is again ~2 nm. On the other hand, the thickness of the POPC enriched regions for both sterols is similar (5.9 nm for Erg and 5.6 nm for Chol). At 40%, both sterols present very small and not very well defined domains, as shown in Fig. 3. Hence, the corresponding histograms have a single peak with very similar thickness for both membranes (~5.3 nm). Previous results by Fidorra et al. [21] for the 1/5 Ceramide/POPC/Chol mixture showed a reduction of the Δh from 1.2 nm in the sterol-free to 0.7 nm at 15 mol % Chol. This is not in agreement with present results, but one has to consider that the amount of ceramide in their case was very small. In Fig. 5 we present average Δh between the two regions and average area coverage of the eSM enriched domains for different sterol

3.2. Height-height correlation function It is clear that the two sterols have different effects on the morphology of the mixture. At first sight it seems that the domain size is also different as a result of the sterol presence. Domain size can be estimated by computing the height-height correlation function Ghh(r), defined in the methods section [83]. In brief, the Ghh(r) function shows correlation (positive values) or anticorrelation (negative values) between the height of a data point and the height of the surrounding points at a distance r. When the topographic image has domain structure the Ghh(r) will cross the 0 value at a certain r0. This r0 corresponds to the average radius of a region with high auto-correlation: it could be an eSM enriched region or a POPC enriched region depending on which mixture has the largest coverage. Since this parameter is statistical in nature, a collection of 5 patches of 25 μm2 were used to compute the height-height correlation function for each mixture. The results are presented in Fig. 8, where very well-defined domains with an average radius of ~1.8 μm appear for the mixture without sterol. The mixture with 5 mol% Erg content presents better defined domains due to a larger height difference but with a reduction in their radius, ~1.4 μm. On the other hand, 5 mol% Chol shows a similar reduction but with less defined domains. Addition of 20 mol% of each sterol produces very 5

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Fig. 3. AFM topographic images and height profiles of POPC/eSM and Erg or Chol SLBs at 1 × 1 μm2 scan size. 40 mol% Erg (A1) or Chol (B1). (A2) and (B2) show height profiles corresponding to the topographic image. Two profile lines are shown for Erg (red and green), while there are three for Chol (red, green and blue). Z range is 400 pm. Scale bar is 200 nm.

Fig. 4. Height histograms of POPC/eSM and Erg or Chol SLBs. Height values are relative to the mica substrate. First peak in all images corresponds to the POPC enriched region and the second peak corresponds to eSM enriched region. Histograms were obtained from the topographic data of Fig. 2. (A) shows the histograms for a sterolfree membrane containing only POPC/eSM at equimolar ratios. (B) POPC/eSM 1/ 1 mol/mol and 5 mol% sterol, (C) POPC/ eSM 1/1 mol/mol and 20 mol% sterol and (D) POPC/eSM 1/1 mol/mol and 40 mol% sterol. Erg corresponds to the blue lines and Chol to the red ones.

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Fig. 5. Average relative height difference and percentual area coverage of the POPC and eSM enriched domains of the POPC/eSM 1/1 mol/mol membrane at different sterol concentrations. (A) Relative height difference of eSM enriched domains with respect to POPC enriched domains. (B) Area coverage of the eSM enriched domains. Averages and standard deviation were obtained from five AFM topographic images for each lipid composition. Scan size was P values of t-test: *P = .000073; 5 × 5 μm2. **P = .000035; ***P = .192; ****P = .00000052.

mixture of saturated and unsaturated lipids, mainly at 20 mol%, which is biologically meaningful. Differences in membrane dynamics could also appear. One effect of the changes in membrane dynamics is that in lateral pressure and, therefore, in the membrane rupture force. Lateral pressure is mainly due to interaction between the phospholipid bodies and should prevent penetration into the bilayer. It is thus possible to estimate the lateral pressure by the resistance of the membrane to rupture. Even if measurements of the rupture force by AFM are not direct measurements of lateral pressure, they are still related [84]. Considering a lateral pressure of 1000 bar [85,86] applied on a semispherical AFM tip with a radius of ~7 nm we can compute the resulting force, which amounts to ~30 nN. A force of this magnitude can be applied to the supported bilayer in order to rupture it. Measurements of this rupture force were performed for the different systems, at the lowest possible velocity (30–50 nm/s) in order to have a quasi-static measurement. The low limit of tip speed is dictated by the need to avoid lipid adhesion to it. The results of force rupture measurements for the different systems are presented in Fig. 10. These values are similar to those obtained for a milk fat globule membrane model [87] and for a DPPC and Chol SLB [60]. As expected, the rupture force is considerably larger in the eSM enriched domains. In some cases, sterol addition increases the rupture force in agreement with the results of Ollila et al. [85]. Our results show that Erg, at low and medium concentrations, produces a larger increase in rupture force compared to Chol in eSM enriched domains, but it has no effect on the POPC enriched region. On the other hand, Chol stiffens both regions at low sterol concentration. In order to get more detail regarding force distribution, we plotted with different colors the forces computed in the POPC enriched, eSM enriched and homogeneous regions, see Fig. 10. We can see that, at zero sterol, the rupture forces are ~5 nN and ~10 nN for the POPC and eSM

different domain morphology. The Erg containing mixture that still has some well-defined domains in the topographic image (Fig. 2) shows the presence of small domains of ~0.5 μm radius. In the Chol containing membranes, the topographic image shows some small domains but the Ghh(r) shows a radius of ~1.5 μm. Looking back at the topographic image (Fig. 2) we can see small eSM domains and low coverage. This indicates that the 1.5 μm radius corresponds to the average size of the POPC enriched regions and not the eSM enriched one, as in the former case. At 40 mol% sterol no appreciable domains appear: we have what resembles a homogeneous surface given the resolution used. When smaller patches at this sterol concentration were scanned, the Ghh(r) of the Erg containing patch showed some domain evidence which is not present in the corresponding Chol containing patch (results not shown). The dispersion shown in Ghh(r) at long distances is due to the fact that no periodic conditions were used in its computation. We can further use this analysis on the high-resolution images obtained with FM-AFM on patches of 1 μm2. Fig. 9 presents the Ghh(r) corresponding to the FM-AFM images from Fig. 7(A) and (B). It is clear that, in this case, domain size is smaller than those in Fig. 8, where AFM mode prevents this resolution. The black trace corresponds to the sterol-free membrane and presents well defined oscillations from correlated to anticorrelated values reflecting small domains in coincidence with the topographic image. The red trace, corresponding to 20 mol% Erg concentration, presents a Ghh(r) showing small domains similar to the previous case, but also one large domain. The latter could be due to lack of resolution, since we cannot see if small domains are clustered. 3.3. Rupture force Clearly Erg and Chol have produced different morphologies in a 7

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Fig. 6. Height profiles and the corresponding topographic images of POPC/eSM 1/1 SLBs at different sterol concentration. (A) Sterol free membrane: B1, B2 and B3 correspond to 5 mol%, 20 mol% and 40 mol% Erg; C1, C2, and C3 correspond to 5 mol%, 20 mol% and 40 mol% Chol. Regions were the mica substrate can be seen were used to obtain the height of the POPC enriched domain. The height was measured using average cursors built into the XEI software. Red cursors measure the height between mica and the POPC enriched domain. Green cursors measure the height between POPC and eSM enriched domains, except for the case of 40 mol% Chol were eSM enriched domains are not present or not well resolved. AFM scan size was 5 × 5 μm2. Z ranges from 5 to 9 nm.

region in spite of the presence of small eSM enriched domains in the topographic images, Fig. 2. The likely cause for this is that the eSM domains are too small. Small sizes, coupled with technical issues such as image drifting, could cause difficulty when puncturing these domains. It is interesting to note that, for Erg containing membranes, the eSM enriched domain rupture force is reduced when going from 5 to 20 mol% and increased when going from 0 to 5 mol%. At 40 mol%, as expected, there is a single peak for both sterols at ~5 nN associated with a quasi-homogeneous membrane. Further analysis of Fig. 10 shows an overlap in the histograms of the

enriched domains respectively. At 5 mol% Chol, the force distribution shows a general stiffening, the POPC enriched domain goes to ~8 nN, and the eSM enriched domain to ~11 nN. At 5 mol% Erg the rupture forces are ~4 nN for the POPC enriched domain and ~15 nN for the eSM enriched domain. It is interesting to note that the addition of Erg has no effect on the POPC enriched region. At 20 mol% sterol the difference is more marked. The Erg mixture presents two clear peaks, one corresponding to the POPC enriched domain with ~4 nN, and the other to the eSM enriched domain with ~11 nN. In the Chol mixture there is a single peak at ~4 nN corresponding to the value of the POPC enriched

Fig. 7. Frequency Modulation Atomic Force Microscopy topographic images of POPC/eSM 1/1 mol/mol SLBs containing or not Erg. (A) sterol free membrane and (B) 20 mol% Erg. Their corresponding height profiles are presented in (C) and (D) respectively. Height profiles are the average of 5 lines contained within the dotted red lines. Relative height (Δh) is calculated between the eSM enriched and the POPC enriched domains. Images were filtered using a Gaussian filter of size 3 × 3 to better observe the different domains. Scale bar is 200 nm.

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domains, as coverage is not uniform. 3.4. Molecular insight In order to look into the possible causes of the different effects Erg and Chol have on the membrane, we performed Quantum Calculations and Molecular Dynamics simulations of Erg and Chol molecules. The common expectation that sterol molecules are similar, producing a universal behavior that differs only in degree [88,89] has been contested [25,26,90,91]. The expectation of similarity comes from their chemical formulas, but their reported 3D structures are quite distinct [92,93]. For instance, the double bond in the Erg alkyl chain could make the molecule more rigid, keeping it in the plane of the B, C and D rings. Rigidity will also be increased by the two double bonds of the B ring. The alkyl chain of Chol can twist more freely from the ring plane. The coprosterol molecule shows a dramatic departure from the planar geometry, lacking the double bond in the alkyl chain, but also both double bonds on ring B, allowing the rotation of ring A and, therefore, of the OH group. Not surprisingly, coprosterol does not induce order at all [91]. This suggests that geometrical flexibility could be behind the observed differences in the ordering of the membrane. The above considerations have encouraged studies regarding the geometrical freedom of different sterol molecules. Bukiya et al. [94] optimized 3D structures of Chol and epicholesterol with the MMFF94 force field [95] and the dielectric constant set at 3, a value characteristic of the lipid bilayer hydrophobic core. The reported lowest energy conformer of Chol is far from the idea of a planar molecule: it is boat shaped and therefore would be expected to be quite distinct from a purported planar Erg molecule. A similar result, but with less marked differences in the lowest energy conformers was reported by Baginski et al. [96]. However a Molecular Dynamics simulation with the GROMOS96 force field in vacuum [97] predicts that, at 300 K, both sterols exist in stretched and bent conformations. The discrepancies can be due to the environment, either vacuum or a dielectric constant equal to 3. This, however, could also be due to differences in the employed force fields. Because of this, we decided to revisit these studies with high quality ab-initio calculations. The lowest energy configurations of the sterol molecules were obtained starting from simulated annealing with the CHARMM36 force field followed by an optimization at the B3LYP level with a large basis set using Gaussian 09 [69]. We considered Chol, Erg and also coprosterol, since they were found to have quite distinct effects in the Xu et al. works [90,91]. The resulting conformers at the ab-initio level are presented in Fig. 11. As expected, the geometries are quite distinct. Chol has a twisted alkyl chain, Erg is linear, and Coprosterol has a very marked boat shape. The marked differences in the geometry of this latter sterol vis-à-vis the previous two can explain the observed experimental result [90,91], ruling out any ordering effect produced by it. We did not consider further analysis of coprosterol. The correlation between its structure and its ordering effect encouraged us to study in more detail the geometrical freedom of Erg and Chol, which could give us an initial insight into the molecular basis of the experimental differences observed in this work. Of course, the rotational freedom of the molecules depends not only on the minimum energy conformation but also on the relative energy of different conformers. In Supplementary Tables S1 and S2 we considered the previous minimal energy conformers and other stable rotamers reported in the literature. These conformers where minimized to produce local minima yielding a conformer landscape for both sterols. Supplementary Table S1 shows a convergence of the ab-initio predictions, giving confidence in the QM results. We added the results corresponding to two empirical force fields, CHARMM36 [75,76] and Slipids [79,98] to Supplementary Table S1 to assess their performance compared to the ab-initio results, since we will be using them to study the rotamer freedom of the sterols. Supplementary Table S2 presents the corresponding relative energies of the different Erg conformers.

Fig. 8. Height - height radial correlation function (Ghh(r)) for the different composition of POPC/eSM 1/1 mol/mol and sterols. (A) sterol free, (B) 5 mol% sterol, (C) 20 mol% sterol and (D) 40 mol% sterol. Blue line corresponds to free sterol. Black lines correspond to Chol. and red lines correspond to Erg containing patches.

Fig. 9. Height - height correlation function from the FM-AFM images of POPC/ eSM 1/1 mol/mol SLBs presented in Fig. 7. Black trace corresponds to sterol free membrane and the trace corresponds to 20 mol% Erg concentration.

rupture force values. This can be due to the difficulty of defining points at the interphase between domains. In order to elucidate this, we looked carefully at the location of the overlapped values. We found that data points assigned as POPC enriched domain that presented rupture forces higher than ~5 nN belonged exclusively to the borderlines. Due to image drifting, it is difficult to define the borderline region. There are also borderline points with rupture force values that correspond to those of POPC enriched regions in the eSM domains. However, there are also points with this characteristic well inside the eSM enriched domains. This led us to believe that there are POPC enriched domains within the eSM enriched domain that are not distinguishable at the resolution of the topographic images, but can be detected in FvD experiments. Of course, one could think that this effect is, again, just consequence of image drifting, or of very disperse values in the rupture force measurement. However, drifting is not as large as to place the tip outside a large eSM enriched domain. Additionally, the misplaced low rupture force values coincide with the values of the POPC enriched regions, suggesting that this is not an artifact. Furthermore, FM-AFM images, Fig. 7, show that what appeared to be a single large eSM enriched domain in the contact-AFM images is really a cluster of much smaller domains. This could explain the presence of rupture forces characteristic of the POPC enriched domain within the eSM enriched 9

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Fig. 10. Bilayer rupture force histograms obtained from POPC/eSM and Erg or Chol SLBs. 2D force spectroscopy map of 256 individual FvD curves were obtained for each lipid mixture. (A) Sterol-free POPC/eSM 1/1 mol/mol. (B1), (B2) and (B3) POPC/eSM/Erg at 5 mol%, 20 mol% and 40 mol% respectively. (C1), (C2) and (C3) POPC/eSM/Chol at 5 mol%, 20 mol% and 40 mol% respectively. The rupture forces obtained were cataloged as belonging to the POPC enriched domains (red), the eSM enriched domains (blue) or the homogeneous phase (green) by correlating the 2D force spectroscopy map and the corresponding topographic image.

Table S1 shows that the ab-initio results present less marked differences in energy than the empirical force fields values. Meaning there is greater conformational freedom at the ab-initio level, which suggests the force fields should be refined. Most geometries obtained for Chol are twisted, mainly towards the rough face, and also one turning the first ring towards the soft face. This result is really surprising, as only at the ab-initio level there is one linear conformer with a low relative energy. Contrary to Chol, the Erg relative energies predicted by the empirical force fields (Supplementary Table S2) do not have a clear correlation with the ab-initio values, highlighting the difficulty of constructing an Erg force field. In striking difference to Chol, the

In order to compare conformers we defined simple geometries based on the positions of the A ring or the tail with respect to the B, C and D rings moiety (nucleus) and also with respect to the methyl-containing face (rough) or the opposite face (soft). Linear (L), where all ring and tail atoms are mainly in the nucleus plane and along the nucleus line. (TR), where the alkyl chain is twisted towards the rough face, or to the smooth face (TS). (AR), where the first ring is twisted to the rough face, or to the soft face (AS). The conformers geometries are shown in Supplementary Figs. S1–S4, where only figures corresponding to the B3LYP level are included because they are very similar for all levels of calculations. 10

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Fig. 11. Minimum energy configurations optimized at the B3LYP/6–31 + G* level of Erg, Chol and Coprosterol. Erg has a linear geometry whereas Chol has a twisted alkyl tail and Coprosterol has a boat shape. 3D images were generated using the Molden package [81]. Arrows indicate double bonds.

differences in the figure correspond to the clustering of the rotamers along the alkyl chain done using UCSF Chimera [80]. This clustering also shows the three more abundant clusters and their percentage of occurrence (Fig. 12 A and B). It is clear that Erg is almost linear, except for a small percent of rotamers. Chol, on the other hand, presents rotamers almost equally distributed between linear and bent conformations. It is interesting to note that, for Erg, there is a link between rings A and B for all geometries predicted by the CHARMM36 force field, possibly a rarity. The equivalent analysis for the Slipids force field is presented in Fig. 12 B. Chol does not have a cluster of linear rotamers and is mainly twisted towards the soft face, whereas Erg linear rotamers have an abundance of 44% and some are twisted towards both sterol faces contrary to CHARMM36, which predicts no twisting at all towards the soft face. In spite of the differences, both force fields predict that Erg rotamers can be considered closer to a linear geometry, whereas those of Chol are mainly twisted. The difference presented above can be related to the findings that Erg is more capable that Chol of inducing order in the membranes of saturated lipids. Erg has been found to conduce to larger increases in the membrane thickness than Chol [99,100]. An MD simulation of Chol in a Stearoyl Sphingomyelin bilayer [101] shows an increase in membrane thickness very similar to the one found here. There are no MD simulations of Erg and eSM bilayers, but there is one on the effect of Erg and Chol on a DPPC membrane [99]. We found a closer interaction of phospholipids with Erg, inducing strong packing of the lipids and a higher proportion of trans lipid conformers. Thus, the results of a thicker membrane produced by Erg can be expected. Regarding the analysis of rotational freedom, we can expect that interaction of the mostly planar Erg with eSM will be strongly favored and induce stronger packing. On the other hand, Chol interacts with both lipids in a similar manner and henceforth fails to induce such a strong packing.

geometries of the Erg conformers with the lowest energies are all linear. Only Slipids predicts a TS geometry with an energy not far from the lowest value. The general picture obtained at the ab-initio level and with two empirical force fields is quite clear. The Erg molecule is mostly linear, whereas Chol is the opposite, mainly twisted. The lowest energy conformer of Chol is not as twisted as predicted by previous calculations [94]. This could be due to the difference in the dielectric constant of the media. In order to check this, we computed the energies of the previous geometries in a continuum PCSMD model [71] with n-hexadecane as a model of membrane core and chloroform, a common solvent. We found that the solvent effect did not modify the order of the conformers. Moreover, reoptimization of geometries in the presence of the solvent did not produce appreciable differences with the vacuum results: the greatest rmsd obtained was 0.013 Å. In order to determine more clearly the conformational flexibility at 30 °C, we considered MD simulations of Chol and Erg in vacuum. An analysis of the conformational freedom of Erg and Chol has been done previously [96,97,99]. Baginsky et al. [96] concluded that, even if there are some differences, they are not significative. A similar conclusion was reached by Baran and Mazerski [97]. From an analysis of the trans/ gauche distribution Cournia et al. [99], concluded that the location of the Erg double bond in the sterol alkyl tail is important and it results in conformational differences that affect the structural properties of the membrane. It is difficult to envisage the difference in the behavior of rotamers of both sterols from analysis of histograms or dihedral angles. A better idea can be obtained from overlapping on the B, C and D rings 100 snap shots taken each 0.2 ps. Such images, corresponding to the CHARMM36 force field, are shown in Fig. 12 A. As can be seen, the rotamer spectra are indeed quite distinct for both sterols. Chol sweeps its alkyl tail towards both the rough and smooth faces of the molecule, whereas Erg does not sweep towards the smooth face. The color 11

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Fig. 12. Superposition of sterol conformers occurring on a vacuum simulation at 30 °C for Erg and Chol performed with CHARMM36 (A) and Slipids (B) force fields. Clustering was performed using UCSF Chimera program. Red is the most populated cluster followed by green, purple and blue. The percentage of occurrence is shown close to each geometry.

ordering effect. The Chol molecule seems to interact equally with both the saturated and unsaturated lipids. However, due to its twisted geometry, it does not have an ordering effect as substantial as that of Erg. At 5 mol%, Erg mainly partitions to the eSM enriched region, ordering it and increasing its thickness and stiffness in contrast to the POPC enriched region, where there are no significant changes. At the same concentration, Chol seems to partition equally into both regions without producing a significant increase in thickness, but with a condensing effect that increases stiffness. At 20 mol%, Erg also partitions to the POPC enriched region, increasing thickness. But the stiffness of the eSM regions is now reduced with respect to the 5 mol% value. This could be due to the onset of a hindering effect of the hydrophobic tailtail interaction, which is responsible for lateral pressure [84,86]. This hindering effect also seems to appear in the POPC enriched region. At 20 mol%, Chol produces a similar increase in thickness for both regions and a marked reduction in stiffness due to its twisted geometry. At 40 mol%, both sterols end up in an almost homogeneous liquid ordered phase [42], producing a condensed membrane. These membranes present an increased thickness but similar stiffness with respect to the sterol free case. The differences in membrane structure produced by both sterols lead to differences in their structural and mechanical properties, as well

Furthermore, our analysis shows that the twisting of the Erg tail is done towards the rough face (more markedly in the CHARMM36 force field), increasing the possibility of a parallel conformation between esM and Erg.

4. Discussion Our AFM study of supported lipid bilayers containing POPC, egg sphingomyelin (eSM) and Chol or Erg found that sterols reduce the size of eSM enriched domains, in agreement with the work of Ho et al. [40]. More importantly, our results show that there are marked differences on the comparative effect of Chol vs Erg —particularly in the height difference of the two domains—and this does not agree with previous reports on different properties [24–27]. In order to gain molecular insight and propose an explanation of the differences observed in the topographic images and the rupture force histograms, we performed ab-initio calculations and MD simulations of the sterol molecules and concluded that the molecular flexibility of both sterols is quite distinct and could explain the observed results. Erg is a predominantly planar molecule, whereas Chol is mostly a twisted molecule. The Erg molecule may interact more favorably with the saturated lipid (eSM), mainly with the planar soft face, and has strong 12

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medium concentrations of Erg or Chol would imply very different adsorption surfaces. The Erg containing membrane will facilitate the cleavage of the interfacial regions and the insertion of molecules. This finding agrees with the hypothesis that selectivity of polyenes for Erg containing membranes vis-à-vis Chol containing ones is due to membrane structural differences [35,38,39] rather than the direct interaction with sterol molecules.

as in the biological processes occurring in them. It has long been suggested that non-specific physical interactions and lipid packing are more important in determining membrane protein stability than direct interactions with lipids [102–104]. Also, that the binding of amphipathic peptides to lipid bilayers or the distribution of proteins into membrane domains is dictated by membrane properties [105–107]. The presence of Chol or Erg in membranes is determinant for the action of polyenes. As a matter of fact, their selective action in Erg containing membranes is the reason behind their therapeutic use. It is normally accepted said selectivity is based on the distinct interaction of each sterol with the aqueous conducting barrel formed by the polyene molecules [108–110]. However, there are alternative models; for instance, the more effective withdrawal of Erg from the membrane vis-àvis Chol [111], or the effect of membrane structure in the activity of polyene channels [38,112]. This latter model has been supported by the existence of a correlation between polyene activity and the membrane phase; liquid ordered, mixed phase or liquid disordered [39]. This idea is also supported by the recent observation of the formation and stabilization of Nystatin aqueous pores in the presence of highly ordered membrane domains [37]. Hence, finding that Erg and Chol have substantially different effects on the formation of ordered domains is relevant for the understanding of polyene mode of action. It has been proposed that polyene adsorption should be occurring in the interphase between the ordered and disordered domains [39]. The present results support this hypothesis since Erg will produce a more marked interphase due to increased height and lateral pressure differences between these domains. Our results show that structural differences produced by Erg and Chol can be behind the selectivity of polyenes. Understanding the molecular mechanism of polyene action is important because of: i) the resurgence of fungal infections in immunocompromised patients with HIV or in chemotherapy treatment [113–115]; ii) the emergence of resistant strains of fungal infections [116,117], iii) acute mycosis in organ transplant and iv) aging population [118]. One should be cautious in extending the results in SLB to cell membranes but Ranz [119], in a comparative Molecular Dynamics study of SLB vs unsupported bilayers, found an effect produced by the support. The water trapped between the support and the bilayer produces membrane ripples, but the presence of domains and their behavior was not affected. We think the present results are relevant for unsupported bilayers as well as the action of polyenes in cell membranes.

Author contribution AGH contributed to the conception and design of the study, experimental work in AFM and FM-AFM and acquisition of data, analysis and interpretation of data and drafting of the article. NK contributed to experimental work in FM-AFM and acquisition of data, analysis and interpretation of data, and the drafting and revision of the article. JHC contributed to the theoretical work, molecular dynamics simulations, analysis and interpretation of data, and the drafting and revision of the article. AA contributed to the theoretical work, analysis and interpretation of data, and the drafting and revision of the article. SN contributed to the design of the study, analysis and interpretation of data, and revised the article for important intellectual content and final approval of the submitted version. IOB contributed to the design of the study, analysis and interpretation of data, revised the article for important intellectual content and final approval of the submitted version. Transparency document The Transparency document associated this article can be found, in online version. Acknowledgements This work was supported by the Universidad Nacional Autónoma de México (UNAM)[DGAPA-PAPIIT-IG100920]; the Consejo Nacional de Ciencia y Tecnología (CONACyT) through grant PEI-252300; the “Takeda Science Foundation”. Clúster Híbrido de Supercómputo Xiuhcoatl-CINVESTAV and MiztliUNAM for computational resources. We thank professor Marité Cárdenas for helpful discussions on the preparation of the supported lipid bilayers on a mica substrate. Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311.

5. Conclusions We have shown the effects of Chol and Erg on the structure and dynamics of an equimolar POPC/eSM membrane. Both sterols have important effects on the size, height and stiffness of the membrane domains but, most importantly, these effects are quite distinct. Erg favors an interaction with eSM, producing very marked effects on thickness, ordering and stiffness of the eSM enriched domain, whereas only at high concentration it increases the thickness of the POPC enriched domains. Chol, on the other hand, does not differentiate between eSM and POPC enriched domains and has a considerably less marked effect than Erg. This proved there are considerable differences between the morphology and dynamics of membranes with Erg or Chol. The Quantum Calculations and Molecular Dynamics simulations of the sterol molecules led us to conclude that molecular flexibility might be behind the observed differences. This presumption is justified by the observations on coprosterol, a strongly boat-shaped structure with no ordering effect whatsoever on the membrane. A quasi-planar Erg molecule seems to interact favorably with the saturated tails of eSM, straightening them. Its interaction with the unsaturated tail of POPC is less favorable and thus has mild effects. A twisted Chol molecule interacts equally with both lipids and has a less marked effect on observed membrane properties. The observed difference in membrane structure and dynamics at

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