Ceramide-Induced Lamellar Gel Phases in Fluid Cell Lipid Extracts

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Ceramide-induced lamellar gel phases in fluid cell lipid extracts Aritz B. Garcia-Arribas, Hasna Ahyayauch, Jesús Sot, Pablo L Lopez-Gonzalez, Alicia Alonso, and Felix M. Goni Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01579 • Publication Date (Web): 03 Aug 2016 Downloaded from http://pubs.acs.org on August 18, 2016

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Ceramide-induced lamellar gel phases in fluid cell lipid extracts* Aritz B. García-Arribas†§, Hasna Ahyayauch†§, Jesús Sot†§, Pablo L. López-González†§, Alicia Alonso†§ and Félix M. Goñi†§.

†

§

Biofisika Institute (CSIC, UPV/EHU), 48940 Leioa, Spain. Departamento de Bioquímica, University of the Basque Country (UPV/EHU), 48940 Leioa, Spain.

Keywords: lipid bilayers, cholesterol, ceramide, gel phase, red blood cell, AFM

Short title: Ceramide gel phases in fluid lipid extracts

* In memoriam Vittorio Luzzati (1923-2016), humanist, teacher and friend Correspondence should be addressed to co-corresponding authors: Félix M. Goñi Biofisika Institute (CSIC, UPV/EHU) Leioa 48940 Basque Country Spain e-mail: [email protected] Aritz B. García-Arribas Biofisika Institute (CSIC, UPV/EHU) Leioa 48940 Basque Country Spain e-mail: [email protected] 1 ACS Paragon Plus Environment

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ABSTRACT The effects of increasing amounts of palmitoylceramide (pCer) on human red blood cell lipid membranes have been studied using atomic force microscopy of supported lipid bilayers, in both imaging (bilayer thickness) and force spectroscopy (nanomechanical resistance) modes. Membranes appeared homogenous with pCer concentrations up to 10 mol% because of the high concentration of cholesterol (Chol) present in the membrane (~ 45 mol%). However, the presence of pCer at 30 mol% gave rise to a clearly distinguishable segregated phase with a nanomechanical resistance 7fold higher than the continuous phase. These experiments were validated using differential scanning calorimetry. Furthermore, Chol depletion of the bilayers caused lipid domain generation in the originally homogenous samples, and Chol-depleted domain stiffness significantly increased with higher amounts of pCer. These results point to the possibility of different kinds of transient and non-compositionally constant, complex gel-like phases present in RBC lipid membranes rich in both pCer and Chol, in contrast to the widespread opinion about the displacements between pCer-enriched “gellike” domains and liquid-ordered “raft-like” Chol-enriched phases. Changes in the biophysical properties of these complex gel-like phases governed by local modulation of pCer:Chol ratios could be a cell mechanism for fine tuning the properties of membranes as required.

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INTRODUCTION Ceramides constitute a class of sphingolipids involved in signalling cascades as bioactive molecules. They are N-acyl derivatives of sphingosine [(2S, 3R, 4E)-2-amino4-octadecen-1,3-diol]. Although for many decades they were considered merely structural elements, some years ago with the discovery of the sphingolipid signalling pathway 1-3 the interest in these lipids was renewed and very soon ceramide, sphingosine and other related compounds became established as lipid second messengers or metabolic signals. Long-chain ceramides (>14 carbons in the N-acyl chain) have been related to cell death 4-5 and they are known to cause drastic changes in the biophysical properties of the membranes, enhancing flip-flop motion, solute efflux 67 , free volume void size increase 8 and domain segregation 9. Ceramide-enriched domains have also been described in red blood cells 10 and mitochondrial outer membranes 11. Ceramide domains could be crucial for the apoptotic effect of long chain ceramides, such as palmitoylceramide (pCer) 12. It has been reported that pCer and cholesterol (Chol), due to both having large hydrophobic parts and only –OH polar heads, are able to displace each other from lipid domains in membranes. On the one hand, ceramide is able to form domains by recruiting sphingomyelin or other phospholipids from the liquid-ordered phases, thereby displacing Chol 13-17, while shortchain ceramides are reportedly unable to do so 18. On the other hand, the generation of domains seems to be modulated by the concentration of Chol 19-23, which could indeed interfere with their apoptotic activity. Interestingly, tumour cells are reported to exhibit higher Chol levels in the outer mitochondrial membrane and Chol can contribute to chemotherapy resistance 24-25. The possibility has been proposed of the generation of ternary structures where both pCer and Chol (in the presence of a third lipid) can coexist and interact positively 19 . Indeed, attractive interactions of pCer and Chol and the formation of ternary phases enriched in both lipids had already been considered in previous studies 20-21. The biophysical properties of this ternary phase would be intermediate between those of a liquid-ordered Chol-driven structure and a gel-like ceramide-enriched domain. These observations were made with model synthetic membranes to resemble specific local situations that could transiently give rise to ternary phases in an actual membrane cell. The latter would be complex gel-like phases enriched in both ceramide and Chol, with additional lipids present. In this study, we aim to further characterize these complex phases in a more physiologically-oriented model. We use lipid extracts from red blood cells (RBC) due to their reportedly high Chol content, 40 - 53 mol% 2628 . Membranes reconstituted from these lipid extracts have been studied by atomic force microscopy to check both the presence of domains and the nanomechanical resistance of the system upon increasing concentrations of pCer (10-30 mol%) and upon partial Chol depletion with β-methylcyclodextrin (βMCD). We demonstrate that complex gel-like phases appear under some conditions and that ceramide-enriched domains (generated for instance at 30% pCer) still stiffen upon Chol depletion, suggesting that these phases feature Chol as well (and therefore would be enriched in both ceramide and Chol). This speaks on behalf of the complexity of the possible lipid domains, and points to a wide range of possible metastable arrangements between ceramide and Chol in the form of complex gel-like phases with different degrees of enrichment of one over the other. 3 ACS Paragon Plus Environment

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Such domain coexistence could be a useful tool for cells to fine tune their nanomechanical resistance (and possibly other biophysical properties).

MATERIALS AND METHODS Chemicals C16:0 ceramide (palmitoylceramide, pCer) was purchased from Avanti Polar Lipids (Alabaster, AL, USA) and β-methylcyclodextrin was purchased from SigmaAldrich (St Louis, MO, USA). Buffers used for the experiments were ‘RBC Buffer’ (32 mM HEPES, 125 mM NaCl, 1 mM MgSO4, 1 mM CaCl2, 5 mM KCl , 5 mM glucose, pH 7.2), ‘osmotic shock buffer’ (acetic acid 1.2 mM, MgSO₄ 4 mM, pH 3.2) and ‘assay buffer’ (20 mM PIPES, 1 mM EDTA, 150 mM NaCl, pH 7.4). Blood management and RBC isolation Human blood was collected from healthy donors and stored in EDTA tubes (BD Vacutainer Systems, Franklin Lakes, NJ). Red blood cells were isolated by sequential centrifugations at 1700 g for 10 min at 4ºC in RBC Buffer, until a transparent and colorless supernatant was achieved. RBC lipid extraction Lipids were extracted following a modification of the Bligh & Dyer method 29-30. Briefly, RBC undergo osmotic stress with the osmotic shock buffer and are centrifuged at 31000 x g for 15 min at 4ºC. The process is repeated until a colorless supernatant is observed. Pellets are resuspended in the minimum possible volume of assay buffer and distributed in 250 µL aliquots, to which 250 µL 0.6 M perchloric acid are added. The mixture is centrifuged at 14000 x g for 15 min and supernatants are discarded. Pellets are transferred to extraction tubes and 2.5 mL cold chloroform:methanol (2:1) is added 31 . Tubes are stirred for 15 min. 5 mL cold 0.1M HCl is added and tubes are vortexed again. Finally, tubes are centrifuged at 1700 x g for 20 min and three phases can be observed. The lower phase is the organic solvent that includes the lipid extract, which is carefully transferred to a flask. Supported Planar Bilayer (SPB) formation SPB were prepared on high V-2 quality scratch-free mica substrates (AshevilleSchoonmaker Mica Co., Newport News, VA) previously attached to round 24-mm glass coverslips by the use of a two-component optical epoxy resin (EPO-TEK 301-2FL, Epoxy Technology Inc., Billerica, MA). SPB are prepared by the vesicle adsorption method 32-33. Multilamellar vesicles (MLVs) were initially prepared by mixing the appropriate amounts of lipids in chloroform/methanol (2:1, v/v) stock solutions, including the RBC lipid extract and the appropriate amount of pCer to achieve the desired composition for each sample. For this purpose RBC lipid extracts were assayed for lipid phosphate 34 and an additional 45% Chol was assumed 27 to calculate the concentration of the total RBC lipid extract stocks. Samples were then dried by evaporating the solvent under a stream of nitrogen and placing them under vacuum for 2 4 ACS Paragon Plus Environment

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h. The samples were then hydrated in assay buffer and vortexed at a temperature above that of the sample lipids highest phase transition. After complete lipid detachment from the bottom of the test tube, formed MLVs were introduced in a FB-15049 (Fisher Scientific Inc., Waltham, MA) bath sonicator and kept at 70 ºC for 1 h. In this way a proportion of small unilamellar vesicles (SUVs) were generated. Thereafter, 120 µl assay buffer containing 3 mM CaCl2 were added onto previously prepared 1.2 cm2 freshly cleaved mica substrate mounted onto a BioCell coverslip-based liquid cell for atomic force microscopy (AFM) measurements (JPK Instruments, Berlin, Germany). 60 µl sonicated vesicles were then added on top of the mica. Divalent cations such as Ca2+ or Mg2+ have been described as enhancers of the vesicle adsorption process onto mica substrates 35. Final lipid concentration was 150 µM. Vesicles were left to adsorb and extend for 30 min keeping the sample temperature at 60 ºC. In order to avoid sample evaporation and ion concentration, after the first 5 min the buffer was constantly exchanged with assay buffer without CaCl2 at 60 °C for the remaining time. Another 30 min were left for the samples to equilibrate at room temperature, discarding the nonadsorbed vesicles by washing the samples 10 times with assay buffer without CaCl2, in order to remove from the solution the remaining Ca2+ cations which are reported to drastically affect the breakthrough force (Fb) results in lipid bilayer nanoindentation processes 36 The efficiency of rinsing processes to obtain proper and clean supported lipid bilayers has been reported 37. This extension and cleaning procedure allowed the formation of bilayers that did not cover the entire substrate surface. The presence of lipid-depleted areas helped with the quantification of bilayer thicknesses and the performance of proper controls for force-spectroscopy measurements. Planar bilayers were then left to equilibrate at room temperature for 1 h prior to measurements in order to avoid the presence of possible artifacts as segregated domains appear at high temperatures (over the Tm) 38 and could still be present at lower temperatures if the cooling process was too fast (> 1 ºC/min) 35. Finally, the BioCell was set to 23ºC to start the AFM measurements. The possible effects of the mica support on the SPBs will be mentioned in the Discussion section.

βMCD extraction of Chol β-methylcyclodextrin was prepared in assay buffer to a 2.5 mM final concentration and heating the system over the lipids main transition (up to 60ºC) to achieve maximum Chol extraction 20. Differential Scanning Calorimetry (DSC) The measurements were performed in a VP-DSC high-sensitivity scanning microcalorimeter (MicroCal, Northampton, MA, USA). Both lipid and buffer solutions were degassed prior to loading into the appropriate cell in the form of MLVs. MLVs were prepared as described previously, but in assay buffer (NaCl 150 mM, 20 mM PIPES, 1 mM EDTA) and at a 1 mM concentration. 0.5 ml at 1 mM total lipid concentration was loaded into the calorimeter, performing 3-5 heating scans at a 45ºC/h rate, between 20 and 105 ºC for all samples. Phospholipid concentration was determined as lipid phosphorus 34, and used together with data from the last scan, to obtain normalized thermograms. The software Origin 7.0 (MicroCal), provided with the calorimeter, was used to determine the different thermodynamic parameters from the scans. The software PeakFit (Systat Software Inc., Chicago, IL, USA) was used for endotherm deconvolution. 5 ACS Paragon Plus Environment

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AFM imaging Planar bilayer topography was performed under contact mode AFM scanning (constant vertical deflection) in a NanoWizard II AFM (JPK Instruments, Berlin, Germany). For proper measurements the AFM was coupled to a Leica microscope and mounted onto a Halcyonics Micro 40 anti-vibration table (Halcyonics, Inc., Menlo Park, CA, USA) and inside an acoustic enclosure (JPK Instruments). The BioCell liquid sample holder (JPK Instruments) was used in order to control the assay temperature at 23 ºC. V-shaped MLCT Si3N4 cantilevers (Bruker, Billerica, MA, USA) with nominal spring constants of 0.1 or 0.5 N/m were used for bilayer imaging, always keeping the minimum possible force (0.5 – 1 nN). 512 x 512 pixel resolution images were collected at a scanning rate between 1 and 1.5 Hz and line-fitted using the JPK Data Processing software as required prior to topography-related data collection. In this regard, bilayer thicknesses were calculated by cross-section height analysis (n=30-100) from no less than 3 images of at least 3 independent sample preparations with individual cantilevers. Force spectroscopy Prior to imaging, V-shaped MLCT Si3N4 cantilevers (Bruker, Billerica, MA, USA) with nominal spring constants of 0.1 or 0.5 N/m were individually calibrated in a lipid-free mica substrate in assay buffer using the thermal noise method. After proper bilayer area localization by means of AFM topography, force spectroscopy was performed at a speed of 1 µm/sec in bilayer areas not smaller than 500 x 500 nm in the form of 10x10 or 15x15 grids. Force steps were determined for each of the indentation curves as reproducible jumps within the extended traces. The resulting histograms were generated from at least 3 independent sample preparations with at least 3 independently calibrated cantilevers (n=350-1800). Control indentations were always performed in lipid-free areas before and after bilayer indentations to ascertain the formation of a single bilayer and the absence of artifacts or debris on the tip, assessed by the lack of any force-distance step on both trace and retrace curves.

RESULTS RBC lipid extracts in supported lipid bilayers present a homogenous surface RBC lipid extracts were extended into mica supports as supported planar bilayers (SPB) and measured under the AFM. Figure 1 shows bilayers discontinuously supported on the mica. Despite the high amount of different lipids present in RBC membranes 26-28, 39-41 SPB revealed no phase segregation (Figure 1a), with a single bilayer thickness value of 5.58 ± 0.20 nm (Figure 1b, Table 1). This value would be slightly below thickness values reported for gel phases in previous reports 42-46, but would be thicker than a liquid-disordered phase 42, 47 and could indeed be a liquidordered phase.


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Figure 1. Ceramide effect on bilayer topography. Representative contact mode AFM height images of the following supported planar bilayers: control RBC lipid extract [a], + 10 mol% pCer [c], + 30 mol% pCer [e], and the respective cross-sections from the dotted lines [b, d, f]. Scale bars: 2 µm.


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Table 1. Summary of bilayer thicknesses obtained from cross-section analysis of AFM images. Average ± SD, (n = 25 – 100). Bilayer Thickness (nm) SPB

SPB + βMCD (2.5 mM)

5.58 ± 0.20 -

5.62 ± 0.22 continuous 6.79 ± 0.28 domain

RBC Extract + 10 mol% pCer 5.53 ± 0.19

5.45 ± 0.15 continuous

RBC Extract

RBC Extract + 30 mol% pCer

5.57 ± 0.20 5.91 ± 0.18

6.11 ± 0.19

domain

5.30 ± 0.12 continuous 6.01 ± 0.12 domain

To test the nanomechanical resistance of bilayers, force spectroscopy experiments were performed. Nanoindentation curves reveal a single pattern (thus confirming the homogeneity of the sample) with a breakthrough force (Fb) value of 2.49 ± 0.68 nN (Figure 2a, Table 2). This nanomechanical resistance would be too weak for a standard gel phase 8, 36, 44, 46, but would be within the range of a liquid-disordered phase 46, 48 . The possibility of a liquid-ordered (Lo) phase 49 cannot be underestimated as the Chol content is so high, but the observed nanomechanical resistance would be too low for an Lo phase. Amongst the liquid-ordered phases studied in previous reports, such as pSM:Chol 7:3 and DPPC:Chol 7:3 mol ratio 36, 45, main Fb values were in the range of ~ 20 nN or higher, and other reports point to other Lo phases with nanomechanical resistance in the range of 10 nN 48. Our hypothesis is that the RBC phase is either a very ordered fluid phase (with a high thickness) or a very weak liquid-ordered phase (with a low nanomechanical resistance). This is close to the case of DOPC:Chol where, despite the high amount of Chol, a proper Lo phase is not achieved but rather an exceptionally ordered liquid-disordered phase is formed 50. As for the present work, we will consider this phase as a “fluid phase”.


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Figure 2. Ceramide effect on bilayer nanomechanical resistance. Representative AFM tip nanoindentation curves on the following supported planar bilayers: control RBC lipid extract [a], + 10 mol% pCer [b], + 30 mol% pCer continuous phase [c] and segregated domains [c’]. N = 300 – 1000. Continuous and discontinuous lines represent extension and retraction traces respectively. Inset in b shows in detail the bilayer breakthrough event.


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Table 2. Summary of breakthrough forces obtained from AFM measurements in the force spectroscopy mode. Average ± SD, (n = 300 – 1000). Breakthrough Force Fb (nN) SPB SPB + βMCD (2.5 mM) RBC Extract

2.49 ± 0.68 -

2.18 ± 0.47 9.27 ± 1.80

continuous domain

RBC Extract + 10% pCer

2.09 ± 0.24 -

2.30 ± 0.40 14.67 ± 2.75

continuous domain

RBC Extract + 30% pCer

1.52 ± 0.41 10.93 ± 1.49

4.62 ± 0.87 21.52 ± 1.65

continuous domain

Addition of 10 mol% pCer does not generate segregated domains Vesicles containing RBC lipid extract plus 10 mol% pCer were prepared and extended to generate supported planar bilayers, of composition phospholipid/Chol/pCer (approx. 50 : 40 : 10. Working with a cellular extract, the precise composition is difficult to establish). Again, a homogenous single phase is detected by AFM with no phase segregation (Figure 1c). This was also the case for a 20 mol% pCer sample (data not shown). Bilayer thickness is 5.53 ± 0.19 nm (Figure 1d, Table 1). This reveals that the presence of 10% pCer has no effect on bilayer thickness when compared to controls (p = 0.33). However, the nanomechanical resistance is 2.09 ± 0.24 nN (Figure 2b, Table 2). The difference between this value and the Fb value for the SPB without pCer (2.49 ± 0.68 nN), albeit small, is statistically significant (p = 0.0001) which indicates that ceramide, although solubilized into the fluid phase 21, still has a slight but noticeable effect on bilayer nanomechanical stability. However, as the values are almost within the standard deviation range, care must be taken when statistically comparing the results, as force spectroscopy experiments feature a large population of curves (n = 300 – 1000 for each result) and this could overestimate differences. Despite these concerns, the effect on nanomechanical resistance would be a weakening one, which in turn discards the possibility of a gel phase being present under these conditions, as gel phases would exhibit a higher nanomechanical resistance than fluid phases. Lipid-tip adhesion in the retraction curves (Figure 2a,b, dashed lines) seems to be smaller in the 10% pCer curves, which also points to some kind of difference in the nanomechanical properties of the bilayers. The interpretation of these phenomena is complex because on the one hand more fluid bilayers tend to exhibit stronger lipid-tip adhesion 51 (which would point to the 0 mol% pCer SPB being more fluid), but on the other hand we found that the nanomechanical resistance of 10% pCer-containing SPB was smaller than control RBC extract SPB (which would point to the opposite, i.e. the 10 mol% pCer-containing SPB being more fluid). Our hypothesis is that, as bilayer thickness remains unaffected, the same kind of lipid phase is present in both control and 10% pCer, although some biophysical properties may be slightly affected by the presence of pCer. Other highly hydrophobic lipids (such as DAG) have been reported to affect bilayers while unable to segregate and 10 ACS Paragon Plus Environment

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even preventing pCer segregation 16. In addition, the fact that Chol concentration in the sample decreases as a result of pCer addition could also be significant, as Fb is highly dependent on Chol concentration 36. pCer generates segregated gel domains at 30% The preparation of SPB with 30 mol% pCer (phospholipid/Chol/pCer approximate composition: 40 : 30 : 30), revealed the existence of segregated domains, in contrast to 10 mol% pCer (Figure 1e). These domains are easily identified by an increased thickness (5.91 ± 0.18 nm) when compared to the continuous phase (5.57 ± 0.20 nm) (Figure 1f, Table 1). The thickness of the domains is comparable to that of pCer-enriched domains in DPPC bilayers 45. Meanwhile the continuous phase exhibits the same thickness as the control (p = 0.842) and 10 mol% pCer-containing bilayers (p = 0.511). In terms of nanomechanical resistance, both phases are also clearly distinguishable: 1.52 ± 0.41 nN for the continuous phase and 10.93 ± 1.49 nN for the domains (Figure 2c-c’, Table 2). Histograms are shown in Figure 3. The difference in Fb value between the 30 mol% pCer continuous phase and the 10 mol% pCer bilayer is statistically significant (p < 0.0001) despite the constant thickness, suggesting the same effect that we previously described when comparing the control and the 10 mol% pCer bilayers, thereby confirming the trend as higher pCer amounts seem to weaken the fluid phases. In addition, these results show that some pCer is still present in the continuous phase, although probably at a very small ratio 52. Interestingly, there is no tip-sample adhesion events in either phase (Figure 2c-c’, dashed lines), contrary to the observations in control and 10 mol% pCer (Figure 2a,b, dashed lines). In addition, as will be explained below, the domains also attract Chol into them (as Chol extraction stiffens the domains, Figure 3c’ vs Figure 6c’), so the continuous phase can have a smaller amount of Chol than the homogenous phases of control and 10 mol% pCer samples, thus exhibit an increased fluidity.


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Figure 3. Bilayer breakthrough force (Fb) histograms. Force step distribution from AFM tip indentation curves of control RBC lipid extract (a), +10 mol% pCer (b), +30 mol% pCer (continuous phase) (c), +30 mol% (segregated domains) (c’).

In turn, the domains exhibit a sharp increase in nanomechanical resistance, by about 7-fold, and the Fb value suggests the presence of a gel phase, in which the majority of pCer would be present. This would be unexpected, as the presence of domains is reportedly prevented (or modulated) by high ratios of Chol 20-21, 23, which is our case for RBC lipid extracts. However, two possible scenarios can explain this situation: (i) there is enough pCer to displace Chol and form ceramide-enriched domains in gel phase or (ii) as both pCer and Chol occur in a high proportion, the gel domain that is detected contains both lipids, as the ternary gel phases described in previous reports 19, 45 with intermediate properties between a Lo (Chol-enriched) and a gel (ceramide-enriched) phase. Note that the second scenario can be possible due to the lipid composition (both lipids are saturating the system). However, the presence of fluid phase-inducing lipids in the extract is a factor that was not evaluated in our previous reports (DPPC:Chol:pCer and pSM:Chol:pCer 54:23:23 ternary mixtures 19, 45) and could also serve as an explanation. Differential Scanning Calorimetry validates AFM data To complement these experiments, samples were also measured by DSC. This procedure gives further information as it is a non-supported, multilamellar vesicle experiment. Figure 4 shows the different thermograms for the samples. As expected, no clear transitions are detected either in the RBC extract or in the +10 mol% pCer sample, 12 ACS Paragon Plus Environment

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in agreement with AFM data as no phase segregation is observed in these samples. In the + 30 mol% pCer sample, two transitions are detected. Note that these transitions are less endothermic than pure lipid transitions (e.g. ∆H = 8.7 kcal/mol for DPPC 53), but the transitions detected for ternary phospholipid:pCer:Chol phases reportedly share this feature 19. The lower T transition is detected at 40.1 ºC and reflects the gel-fluid transition of the segregated gel phase detected in AFM experiments. The second transition, at 92.7 ºC, is most probably caused by remnants of pure pCer that are not incorporated into the vesicles, as pure pCer has an endothermic transition at 93.2 ºC 47. This is somehow expected as a high amount of Chol in the vesicles would definitely impede a perfect mixing of pCer into the sample. The heat associated to the pure pCer transition is small compared with the reported enthalpy of pure pCer transition (∆H = 2.24 kcal/mol 47, versus 0.24 kcal/mol in our case), suggesting that the quantity of nonincorporated pCer should be very low. Furthermore, it is extremely unlikely that pCer non-lamellar remnants would affect the AFM experiment as they would not extend on the mica substrate (as we extend at 60 ºC and the Tm for them is 92.69 ºC) and aggregates would be washed away during the rinsing process.

Figure 4. Representative DSC thermograms of the different samples. Dashed grey lines represent peak fitted endotherms. The thermodynamic parameters for the sample containing 30 mol% pCer are: (lower T peak) midpoint transition temperature Tm = 40.1 ºC, width at midheight WMH = 14 ºC, transition enthalpy ∆H = 0.8 kcal/mol; (higher T peak) midpoint transition temperature Tm = 92.7 ºC, width at mid-height WMH = 6.8 ºC, transition enthalpy ∆H = 0.24 kcal/mol. Enthalpies are computed per mol of total ceramide in the sample.


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Chol depletion promotes the generation and/or stiffening of gel domains To further characterize the effects of the presence of Chol in the system and elucidate whether the 30 mol% pCer domains are more similar to the SM-pCer-rich gel phases or to the Chol and Cer-containing complex gel-like phases, we performed the same experiments after Chol extraction with β-methylcyclodextrin (βMCD). This reagent is in common use for Chol management, both for removal from or delivery to cell membranes and model lipid membranes 20, 54-57. The samples were first analysed (obtaining the data already presented) and then the SPB were treated with 2.5 mM βMCD, quickly heating the sample up to 60ºC and leaving it to cool down to room temperature (23ºC). Imaging results and the respective cross sections for Chol-depleted bilayers are shown in Figure 5 for control RBC extracts (a-b), 10 mol% pCer (c-d) and 30 mol% pCer (e-f). First, the clear increase in bilayer defects points to an effective depletion of Chol, as the three samples had > 30 mol% Chol and the absence of this lipid would explain the generation of these defects, there being less lipid in the bilayer after extraction. More importantly, the three samples exhibit domains, in agreement with current knowledge about the effects of a saturating Chol ratio in preventing the formation of gel segregated domains. However, thickness results reveal that domains in the absence of pCer are significantly thicker than their pCer-containing counterparts: 6.79 ± 0.28 nm without pCer, versus 6.11 ± 0.19 nm and 6.01 ± 0.12 nm for 10 mol% and 30 mol% pCer respectively (Figure 1b,d,f, Table 1).


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Figure 5. Chol depletion effect on bilayer topography. Representative contact mode AFM height images of the following supported planar bilayers after Chol extraction with βMCD: RBC lipid extract [a], + 10% pCer [c], + 30 % pCer [e], and the respective dotted-line crosssections [b,d,f]. Scale bar: 2 µm.

The reasoning behind this evidence is that, in control RBC extracts, Chol depletion generates domains made of sphingomyelin (SM), mainly C16:0, C24:0 and C24:1 in human RBC lipids 28, 41, which at room temperature would be in gel phase. However the presence of pCer after Chol depletion would enhance ceramide concentration in the domain, which in the presence of SM would lead to a lower thickness and a higher nanomechanical resistance (checked with pSM:pCer mixtures in García-Arribas, et al. 45). The difference in thickness between 10 mol% pCer and 30 mol% pCer domains after Chol depletion is statistically significant (p = 0.015), which, when compared to our studies with pure pSM:pCer mixtures in García-Arribas, et al. 45 (where no difference was observed in thickness after increasing pCer content), would 15 ACS Paragon Plus Environment

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imply that these phases are not compositionally or stoichometrically constant and are not made purely of SMs and pCer. Chol depletion of 30 mol% pCer-containing SPB also affects domain thickness with a statistically significant increase in the thickness (p = 0.015). This evidence supports that the 30 mol% pCer domains (Figure 1e) contain indeed Chol and speaks in favour of a complex gel-like phase present before the extraction, changing to a ceramide-rich gel phase with pCer predominance after extracting Chol. Force spectroscopy results (Figure 6) are in good agreement with thickness data (Table 2) and decisively support our hypothesis. Domains appearing after Chol extraction have Fb values of 9.27 ± 1.80, 14.67 ± 2.75 and 21.52 ± 1.65 nN for 0 mol%, 10 mol% and 30 mol% pCer respectively (Table 2). Histograms are shown in Figure 7. An increase in Fb is observed when pCer is present, in accordance with our previous data 45, but it also increases with pCer ratio, which does not occur for pure pSM:pCer domains 45. These results confirm that the gel phases obtained are not compositionally constant. Furthermore, the increase in nanomechanical resistance for 30 mol% pCer domains after Chol extraction (Fb value raises from 10.93 to 21.52 nN) confirms that the domains were, as previously suggested, complex gel-like phases, as Chol depletion significantly affects both bilayer thickness (Table 1) and nanomechanical resistance (Table 2).


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Figure 6. Chol depletion effect on bilayer nanomechanical resistance. Representative AFM tip nanoindentation curves on the following supported planar bilayers after Chol depletion with 2.5 mM βMCD: RBC lipid extract [a-a’], + 10% pCer [b-b’], + 30 % pCer [c-c’]. Notation: continuous phase x, segregated domain x’. N = 300 – 1000. Continuous and discontinuous lines represent extension and retraction traces respectively.


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Figure 7. Bilayer breakthrough force (Fb) histograms after Chol depletion. Force step distribution from AFM tip indentation curves from RBC lipid extract (a-a’), +10% pCer (b-b’), +30% pCer (c-c’) after Chol depletion with βMCD. Notation: continuous phase x, segregated domain x’.


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DISCUSSION The visualization of a homogenous phase in the native human RBC lipid extract SPB is interesting because several recent reports point to the possibility of lipid domain segregation in erythrocyte membranes of living human RBC 55, 58. However, reports of RBC lipid GUV imaged by fluorescence show no phase segregation, in agreement with our SPB experiments 41. Besides, direct visualization of lipid domains in living cells poses several issues that have been discussed in the literature, in particular how the presence of domains would be affected by other factors such as: (i) the degree of cell attachment to the support 55, (ii) Chol content of the membranes (which we confirmed in our study), (iii) the presence of cytoskeleton 55 and (iv) effects of the fluorescent group when lipids have been modified (or when lipid-fluorescent derivatives such as BODIPY-lipids are being used). These considerations, as well as the intrinsic limitations of the use of SPB (support effects 59, and the lack of lipid asymmetry, as SPB are formed from lipid vesicles), make both approaches difficult to compare and could explain the discrepancies between results. Even the use of different supports (mica, silicon oxide) can affect results and generate further discrepancies 60, but the use of the same support (mica) throughout this work and in our previous experiments should allow a reasonable understanding across the experiments. Furthermore, previous comparative reports between SPB and GUV imaging show a good agreement between each other in terms of ceramide-induced segregation 19, 45. Another source of variability could be the use of different methods for lipid extraction from cells 61, which indeed could explain the different data of mol% Chol of the human RBC (from 40% to 53%). The generation of domain segregation due to ceramide had already been reported in human living RBC 62, but our results confirm that a significant amount of pCer (>10%) is required for the generation of domains due to the effect of Chol in the absence of membrane proteins or cytoskeleton. 10 mol% pCer is not able to generate any kind of phase segregation distinguishable by AFM imaging in our SPB, however a slight effect on nanomechanical resistance was detected, which could be caused by the reduction in mol% Chol (from 45% to 40%) caused by pCer addition. This could also speak in favour of the force spectroscopy techniques as being more informative for lipid phase characterization than imaging techniques. Cholesterol depletion in the 10 mol% pCer sample yields domains that are significantly stiffer than non-pCer Chol-depleted domains. A situation of complete Chol depletion would resemble the POPC/pSM/pCer system that has been well-characterized in the form of a phase diagram 63. Taking into account that human RBC lipids contain 12 mol% SM (mostly C16:0, C24:0 and C24:1 41) corresponding to 26 mol% of total phosphate 27, and about 40 mol% in low T-melting lipids (including PC, PE and PI) 41, the presence of 10 mol% pCer would be enough to form gel-like domains enriched in pCer after Chol extraction, which are stiffer than the domains seen without pCer. These results are in agreement and validate the phase diagram in Castro, et al. 63: a complete Chol depletion of the 10 mol% pCer sample would mean removal of 40 mol% of the total lipid and the ratios should be recalculated to 65 mol% “fluid” (low T-melting) lipids, 18 mol% SM, 17 mol% pCer, a situation reflected by the diagram as fluid + ceramide-rich gel domains. Again, this can be interpreted in terms of the above phase diagram, assuming total Chol extraction, grouping all fluid lipids behaving as POPC and all the different SM in the extract behaving as pSM.


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POPC/Chol/pCer phase diagrams are a useful tool for understanding our complex system 21. To this aim we assume that all non-Chol non-pCer lipids present resemble POPC (although as previously stated 26% of phospholipids are SM 27). The 10 mol% pCer sample (approx. 50:40:10 phospholipid:Chol:pCer) does not exhibit any gel domains, in agreement with the phase diagram reported by Castro, et al. 21. A 20 mol% pCer sample (approx. 45:35:20 phospholipid:Chol:pCer) would be also homogenously fluid according to the diagram, and this has been shown to be the case (data not shown). For 30 mol% pCer, the sample (approx. 40:30:30 phospholipid:Chol:pCer) does show a segregated phase in agreement with the same phase diagram, although the size of the domains would be larger than predicted by that report. However, this is probably caused by the presence of SM in the sample, but not in the diagram by Castro, et al. 21 As for the nature of the 30 mol% pCer domains, our results suggest that they exist in a complex gel-like phase, enriched in both pCer and Chol, in the same fashion as the ternary gel phases discussed in García-Arribas, et al. 45. The fact that the domains increase their stiffness upon Chol depletion confirms this hypothesis. Moreover, the differences that we detect between the nanomechanical resistances of the domains after Chol removal at different mol% pCer ratios provide further information. First, these results confirm that pCer has been adequately incorporated into the samples and extended on the SPB (which is also supported by DSC experiments). Second, and more important, the comparison between 30 mol% and 10 mol% pCer, Chol-depleted domains suggests that 10 mol% pCer, Chol-depleted domains are probably a complex gel-like phase as well, as ceramide-enriched domains are stoichometrically constant and their nanomechanical resistance does not change with the mol% pCer, which would be the case if both were pCer-enriched 45. This could be explained due to the Chol depletion being incomplete, which in turn would point to the possibility of 30 mol% pCer, Chol-depleted domains being another complex gel-like phase (enriched in both pCer and Chol) and not allowing Chol to be completely removed, but the latter statement cannot be confirmed. Partial Chol extraction is possible due to inefficient access to Chol for βMCD when domains arise. Another interesting issue is the possibility of different lipids in the extract being selectively recruited to the segregated phases, depending on the concentration of pCer, Chol, or even the pCer:Chol ratio of the different samples. This possibility provides an alternative perspective on the interpretation of our data. For the 30 mol% pCer sample, a reduction in the stiffness of the continuous phase is detected, probably because of the capacity of the segregated gel phase to recruit high Tm lipids such as SM into the gel phase. When the sample undergoes cholesterol depletion the domains become more pCer-enriched, meaning that probably other lipids (or Chol remnants) are displaced to the continuous phase causing a global rise in nanomechanical resistance in both phases (Table 2) and the possibility of the pCer-enriched domain being able to recruit more high-Tm lipids (particularly SM) after Chol extraction should also be taken into consideration. These assumptions imply that cholesterol depletion not only alters the properties of the phases directly, but also changes the distribution of each lipid between phases, thus affecting their properties indirectly as well. The observed reduction in domain thickness after Chol depletion could be explained as a progressive displacement of SM out of the domains as pCer concentration increases (Table 1). pSM gel phases are thicker than pSM:pCer domains 45 and, perhaps more importantly, there are other long-chain SM present in the RBC 20 ACS Paragon Plus Environment

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extract (C24:0 and C24:1) 41. The latter are probably an integral part of Chol-depleted domains in 0 mol% pCer which would explain their higher thickness but would be rapidly excluded if pCer were present as pCer would interact preferentially with pSM, causing a sharp decrease in domain thickness. Displacements of long-chain SM would not increase fluid phase thickness (Table 1) probably because of the increased miscibility of SM at lower Cer concentrations. In a previous report we showed that ternary mixtures of phospholipid:Chol:pCer (54:23:23 mol ratio) formed homogenous ternary gel phases which after Chol depletion with βMCD showed phase segregation in GUV, as seen with the fluorescent probe DiIC18 20, but the nature of this phase coexistence could not be studied and could feature either ceramide-enriched gel-like or complex gel-like phases with both ceramide and a certain amount of Chol, due to incomplete or inefficient depletion. The continuous fluorescently-marked phase would become a Lo phase. However βMCD in vesicles (GUV in that study) would yield higher amounts of Chol extraction as no support impedes the access, thus the possibility of partial Chol extraction seems less plausible. Recent reports point to Chol being more or less difficult to be cyclodextrin-extracted depending on the phase 64 and this should also be taken into consideration. Interestingly, the use of βMCD for lipid delivery (the opposite procedure to ours, previously saturating βMCD with another lipid) has indeed been described as giving rise to asymmetrical bilayers in GUV 57 and SPB 56. However, this should not be the case in this work as we use bare βMCD and, more importantly, the images of Choldepleted bilayers (Figure 5) show membrane defects caused by the Chol extraction process, thus affecting both lipid leaflets of the bilayer. Sample heating to 60ºC for Chol extraction also makes SPB more fluid and enhances proper lipid mixing, therefore any asymmetrical bilayer formation can be discarded. All results considered, we can assure that at least two complex gel-like phases appear in this study: one in 30 mol% pCer domains and the other in 10 mol% pCercontaining, partially Chol-depleted domains. The comparison between these two phases provides some insights about their biophysical properties. The differences in nanomechanical resistance and bilayer thickness speak in favour of a modulation of these properties depending on the Chol:pCer ratio of enrichment within the domains. As opposed to pCer 45, Chol has been reported to alter nanomechanical resistance in a dosedependent way 36, 65, and this would also be possible for the complex gel-like phases with different Chol:pCer ratios. In addition, Chol:pCer ratio could also govern the capacity of segregated gel-like phases to recruit or exclude specific high-Tm lipids such as sphingomyelins, therefore modulating the properties of domains in an additional indirect manner.

CONCLUSION The above results show that, in contrast to the widespread idea of ceramideenriched domains as highly-packed gel phases that exclude Chol, with constant stoichiometry and properties independent on pCer concentration, pCer can induce complex phases in the presence of Chol depending on the lipid ratio, thereby forming multiple-component gel-like phases, in an arrangement resembling ternary gel phases (phospholipid/Chol/Cer) with biophysical properties intermediate between those of the 21 ACS Paragon Plus Environment

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phospholipid-Cer gel and the phospholipid-Chol Lo phases. However, further studies are needed to define the Chol:pCer ratios needed to transform ceramide-enriched gel domains, or Lo phases, into complex gel-like domains. Moreover, complex gel-like phase properties could possibly be fine-tuned within cell membranes by locally controlling Chol:pCer ratios, perhaps through sphingomyelinase action on sphingomyelin, and the latter could be relevant in the context of sphingolipid signalling, membrane platform formation and membrane trafficking.

AUTHOR INFORMATION Corresponding Authors [email protected] & [email protected]

ACKNOWLEDGEMENTS AGA was a predoctoral student supported by the Basque Government and later by the University of the Basque Country (UPV/EHU). This work was also supported in part by grants from the Spanish Government (FEDER/MINECO BFU 2015-66306-P to F.M.G. and A.A.) and the Basque Government (IT849-13 to F.M.G: and IT838-13 to A.A.).


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Langmuir

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We have studied lipid bilayers obtained from human red blood cell extracts, and the effect of incorporating a long-chain ceramide (palmitoyl ceramide, pCer) into them. The bilayers have been examined using atomic force microscopy and differential scanning calorimetry. Gel-like phase domains are generated by the presence of pCer at high mol ratios (around 30 mol% pCer) and cause an increase in bilayer thickness and nanomechanical resistance. Furthermore, cholesterol (Chol) depletion of the system causes the domains to stiffen even more, demonstrating that Chol was an integral part of the domains and that no Cer-Chol displacement events were present. These experiments speak in favour of the possibility of lipid gel domains enriched in both Chol and pCer in cellular environments.


Ceramide-Induced Lamellar Gel Phases in Fluid Cell Lipid Extracts

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