Atomic Force Microscopy Characterization of Palmitoylceramide and

Article pubs.acs.org/Langmuir

Atomic Force Microscopy Characterization of Palmitoylceramide and Cholesterol Effects on Phospholipid Bilayers: A Topographic and Nanomechanical Study Aritz B. García-Arribas, Jon V. Busto, Alicia Alonso, and Félix M. Goñi* Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, P.O. Box 644, 48080 Bilbao, Spain S Supporting Information *

ABSTRACT: Supported planar bilayers (SPBs) on mica substrates have been studied at 23 °C under atomic force microscopy (AFM)-based surface topography and force spectroscopy with two main objectives: (i) to characterize palmitoylceramide (pCer)induced gel (Lβ) domains in binary mixtures with either its sphingolipid relative palmitoylsphingomyelin (pSM) or the glycerophospholipid dipalmitoylphosphorylcholine (DPPC) and (ii) to evaluate effects of incorporating cholesterol (Chol) into the previous mixtures in terms of Cer and Chol cooperation for the generation of lamellar gel (Lβ) phases of ternary composition. Binary phospholipid/pCer mixtures at XpCer < 0.33 promote the generation of laterally segregated micron-sized pCer-rich domains. Their analysis at different phospholipid/pCer ratios, by means of domain thickness, roughness, and mechanical resistance to tip piercing, reveals unvarying AFM-derived features over increasing pCer concentrations. These results suggest that the domains grow in size with increasing pCer concentrations while keeping a constant phospholipid/pCer stoichiometry. Moreover, the data show important differences between pCer interactions with pSM or DPPC. Gel domains generated in pSM/pCer bilayers are thinner than the pSM-rich surrounding phase, while the opposite is observed in DPPC/pCer mixtures. Furthermore, a higher breakthrough force is observed for pSM/pCer as compared to DPPC/pCer domains, which can be associated with the preferential pCer interaction with its sphingolipid relative pSM. Cholesterol incorporation into both binary mixtures at a high Chol and pCer ratio abolishes any phospholipid/pCer binary domains. Bilayers with properties different from any of the pure or binary samples are observed instead. The data support no displacement of Chol by pCer or vice versa under these conditions, but rather a preferential interaction between the two hydrophobic lipids.

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INTRODUCTION Lipid-driven membrane domain formation as a molecular reorganization preceding various cell signaling events is currently a widely accepted concept.1 Sphingosine-based lipids are potent lateral segregation inducers. The sphingolipid sphingomyelin (SM) exhibits a strong affinity for cholesterol (Chol) in model and biological membranes,2,3 and this interaction may be at the origin of raft formation.4−6 Other important sphingolipids regarding membrane domain formation are N-acylsphingosines or ceramides (Cer).7 Endogenous ceramide levels are very low (around 0.1−1 mol % of total phospholipid);8 however, an important increase to concentrations up to 10 mol % can be achieved in apoptotic cells.9 Because of their low hydrophilicity, the most frequent longchain Cer are seldom found in the cytosol but rather in cell membranes, where they can be generated by different pathways10 including the sphingomyelinase enzymatic degradation of sphingomyelin11 and the conversion of sphingosine into ceramide, either by ceramide synthase in the salvage pathway12 or by the combined effect of neutral ceramidase and thioesterase.13 For the past 15 years, model system studies have provided multiple data regarding Cer behavior in membranes. © 2015 American Chemical Society

Amidst other effects, very low (2−3 mol %) Cer concentrations are sufficient to drive lateral segregation in the presence of a variety of membrane phospholipids.14−17 In order to keep hydrophobic Cer away from water contact at the membrane interface, these domains present increased intermolecular packing and thus reduced molecular motion. The strong segregation capacity of Cer in model membranes has suggested that a similar lateral separation may occur in cell membranes. In this respect, ceramide-rich structures have been already observed for instance in erythrocyte ghost membranes upon hot−cold hemolysis18 and in mitochondrial outer membranes of mammalian cells upon irradiation.19 An additional interesting phenomenon is Cer interaction with Chol. Important SM sources in plasma membranes are the Chol-based liquid-ordered domains. Sphingomyelinase activity within those membrane regions arises as a putative mechanism for ceramide generation, thus leading to Cer and Chol accumulation in defined areas.20 On that account, four major Received: October 13, 2014 Revised: January 29, 2015 Published: February 18, 2015 3135

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quality scratch-free mica substrates (Asheville-Schoonmaker Mica Co., Newport News, VA) previously attached to round 24 mm glass coverslips by the use of a two-component optical epoxy resin (EPOTEK 301-2FL, Epoxy Technology Inc., Billerica, MA). Two different procedures were followed. Pure phospholipid and binary phospholipid/Chol mixtures were prepared using the vesicle adsorption method.31 Multilamellar vesicles (MLVs) were initially prepared by mixing the appropriate amounts of synthetic pure lipids in chloroform/methanol (2:1, v/v) solutions, including 0.4 mol % DiIC18. Samples were then dried by evaporating the solvent under a stream of nitrogen and placing them under vacuum for 2 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 °C for 1 h. In this way a proportion of small unilamellar vesicles (SUVs) were generated. Thereafter, 120 μL of assay buffer containing 3 mM CaCl2 was 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 of sonicated vesicles was 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.32 The final lipid concentration was 150 μM. Vesicles were left to adsorb and extend for 30 min, keeping the sample temperature at 70 °C. 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 70 °C for the remaining time. Another 30 min was 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 remaining Ca2+ cations from the solution which are reported to drastically affect the breakthrough force (Fb) results of lipid bilayer nanoindentation processes.33 The efficiency of rinsing processes to obtain proper and clean supported lipid bilayers has been reported.34 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 forcespectroscopy 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)35 and could still be present at lower temperatures if the cooling process was too fast (>1 °C/ min).32 Finally, the BioCell was set to 23 °C to start the AFM measurements. Samples prepared under this procedure were formed of either pure phospholipids or phospholipid:Chol samples at 70:30 mole ratios (also referred through the test as +30 mol % Chol). Supported Planar Bilayer (SPB) Preparation through SpinCoating. pCer-containing samples were prepared using the spincoating procedure,36 as they presented very low vesicle-adsorption efficiency due to the presence of gel phases with higher transition temperature. Lipids were directly dissolved at the desired ratio in an isopropanol/hexane/H2O (3:1:1) solution at 10 mM total lipid concentration, including 0.4 mol % DiIC18. 20 μL of the lipid mixture was directly pipetted onto a freshly cleaved mica substrate and rotated at 3000 rpm for 40 s using a KW-4A spin-coater (Chemat Technology Inc., Northridge, CA). The substrate was then left under vacuum overnight, mounted onto the BioCell, and hydrated with 400 μL of assay buffer. Multiple bilayers were then generated. The temperature was raised to 70 °C for 30 min, the buffer being constantly exchanged with buffer at 70 °C in order to avoid dehydration. The sample was then washed throughout with assay buffer at 70 °C. Bilayers nondirectly adhered to the mica substrate were thus discarded, and the one on top of the mica was left to perform the desired measurement. Finally, the Biocell was set to 23 °C, and the planar bilayers were left to equilibrate for 1 h prior to measurements. Samples prepared under this procedure contained phospholipid:pCer at 90:10, 80:20, or 70:30 mole ratios (also referred through the test as +10 mol

situations have been described. (i) Cer and Chol have been observed to directly compete for their respective interaction with SM, Cer displacing Chol at medium−low cholesterol concentrations21−23 and higher proportions of Chol abolishing SM/Cer domain generation.24,25 (ii) Chol concentration dependent Cer domain solubilization has been observed in PC- but not SM-containing membranes.26 (iii) The formation of pure Cer−Chol complexes has been proposed as well,27 and this has led to the generation of antibodies against Cer/Cholrich domains in cell membranes.28 (iv) We have recently characterized lamellar gel phases of ternary phospholipid/Cer/ Chol mixtures that are likely to be stabilized by Cer−Chol interactions through direct hydrogen bonding.29 In our earlier reports25,29 we have shown that in ternary mixtures based on pSM or DPPC plus a high proportion of (pCer + Chol) distinct gel lamellar phases are being formed. Our interpretation is that high cholesterol concentrations prevent phospholipid/pCer domain generation; instead, pCer− Chol interactions are established, allowing the ternary phases to be organized. Nonetheless, the reported possibility25 that small binary phospholipid/pCer nanodomains would be formed at those lipid ratios cannot be dismissed. As stated previously, the average ceramide concentrations expected to occur in both normal and apoptotic cells are below some of those used in the present study; however, the use of higher concentrations in the previous and present work intends to represent putative nanoand/or microdomains in which ceramide concentrations could be very high.7 The biophysical characterizations used in our previous studies combined spectroscopic, fluorescence, and calorimetric methodologies. These techniques do not allow to directly ascertain the formation of nanodomains in model membranes; thus, other high-resolution approaches are needed. Atomic force microscopy (AFM) is a scanning probe microscopy that allows very high-resolution analysis of supported materials including supported model membranes.30 The present contribution is focused on the characterization of pCer and Chol effects on phospholipid-based supported planar bilayers by means of AFM-based approaches. Contact mode AFM imaging has been undertaken in order to study bilayer topography, looking at possible lateral segregation effects through bilayer thickness and roughness analysis. Moreover, force spectroscopy has been applied to examine nanomechanical properties in response to AFM-tip-mediated bilayer piercing. Four different lipid systems have been studied at 23 °C, namely, (i) pure pSM or DPPC bilayers in a gel (Lβ) phase, (ii) binary phospholipid/pCer mixtures in a segregated gel (Lβ) phase, (iii) binary phospholipid/Chol mixtures in a liquidordered (Lo) phase, and (iv) ternary phospholipid/pCer/Chol bilayers.

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MATERIALS AND METHODS

Chemicals. N-Palmitoyl-D-erythro-sphingosylphosphorylcholine (pSM), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Npalmitoyl-D-erythro-sphingosine (pCer), and cholesterol (Chol) were purchased from Avanti Polar Lipids (Alabaster, AL). The lipophilic fluorescent probe 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC18) was purchased from Molecular Probes (Eugene, OR). Naphtho[2,3-a]pyrene (NAP) was purchased from Sigma-Aldrich Co. (St. Louis, MO). Assay buffer solution was 20 mM PIPES, 1 mM EDTA, and 150 mM NaCl, pH 7.4. All other reagents were of the highest commercially available purity. Supported Planar Bilayer (SPB) Preparation through the Vesicle Adsorption Method. SPBs were prepared on high V-2 3136

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Langmuir % pCer, +20 mol % pCer, or +30 mol % pCer, respectively) and phospholipid:pCer:Chol samples at 54:23:23 mole ratio. 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 antivibration table (Halcyonics, Inc., Menlo Park, CA) 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 °C. V-shaped MLCT Si3N4 cantilevers (Veeco Ins., Plainview, NY) 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 × 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 = 300−600) from no fewer than three images of at least three independent sample preparations with individual cantilevers. Roughness average (Ra) was analyzed in all images by surface analysis on 1−5 μm2 areas (n = 50−300). Epifluorescence Microscopy. Direct AFM-coupled inverted epifluorescence microscopy was performed in a Leica DMI 4000B microscope (Leica Microsystems, Wetzlar, Germany) using an appropriate filter cube for DiIC18 fluorochrome (excitation filter HQ545/30×, dichroic mirror Q570LP and emission filter HQ610/ 75m) (Chroma Tech., Bellows Falls, VT). Images were acquired using a 40×/0.60 LD objective (Leica Microsystems) with a high-resolution ORCA-R2 digital CCD camera (Hamamatsu Photonics, Shizuoka, Japan). Force Spectroscopy. Prior to imaging, V-shaped MLCT Si3N4 cantilevers (Veeco Ins., Plainview, NY) 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 and direct epifluorescence microscopy, force spectroscopy was performed at a speed of 1 μm/s in not less than 500 × 500 nm2 bilayer areas in the form of 10 × 10 or 15 × 15 grids. The stiffer 0.5 N/m cantilever was used for the high breakthrough force yielding pCer-containing SPBs. 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 three independent sample preparations with at least three independently calibrated cantilevers (n = 350−1800). Control indentations were always performed in lipidfree 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.

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Figure 1. Topographic AFM height images of the SPBs under study. Representative contact mode AFM height images of the following supported planar bilayers: pSM (a), pSM:Chol (70:30) (b), pSM:pCer:Chol (54:23:23) (c), DPPC (d), DPPC:Chol (70:30) (e), and DPPC:pCer:Chol (54:23:23) (f). Scale bars: 1 μm.

showed that the domains excluded DiIC18 fluorescent probe, suggesting the formation of phospholipid/pCer-rich bilayers in a gel-like phase as previously described.16 As for domain thickness, bilayer topography showed a different behavior in pSM/pCer and DPPC/pCer bilayers. pCer-rich domains in pSM/pCer bilayers showed a reduction in thickness of approximately 0.3−0.4 nm with regard to the pure pSM (Figure 3 and Table 1). This is clearly observed in both the topographic image and its cross section in Figure 2, where a lower thickness is observed for the domain as compared to the surrounding DiIC18-containing pSM-rich phase. In contrast, in DPPC-based bilayers pCer promoted the generation of segregated domains with a thickness increased by around 0.25−0.3 nm for 10−30 mol % pCer-containing SPBs (Figure 3 and Table 1). We next analyzed the roughness average (Ra) for the pure bilayers and all the pCer-containing domains. Roughness average is the arithmetic mean of absolute values of roughness ordinates, i.e., ups and downs in the profiles, and was calculated automatically by the software for any given bilayer region of an image. pSM and DPPC bilayers rendered roughness average values of 0.081 ± 0.039 and 0.072 ± 0.024 nm, respectively. As expected, pCer-rich domains in all samples showed a clear decrease in roughness (Figure 3 and Table 1), which is generally associated with an increased intermolecular packing and/or bilayer order. Both phospholipid/pCer domain thickness and roughness values were similar at all pCer concentrations tested (Table 1). Force Spectroscopy of Binary Phospholipid/PCer Domains. Force spectroscopy-based indentations were then performed on pCer-rich domains in order to estimate the

RESULTS

Topography of Binary Phospholipid/PCer Domains. Contact mode AFM imaging was initially performed on 0−30 mol % pCer-containing pSM or DPPC supported planar bilayers to study the effect of pCer and its dependence on the phospholipid/pCer ratio. Pure pSM and DPPC bilayers showed height-homogeneous extensions, with no phase segregation in agreement with the generation of bilayers in a lamellar gel (Lβ) phase at 23 °C25 (Figure 1). The pure lipid SPBs presented an average bilayer thickness of 5.77 nm (pSM) and 5.68 nm (DPPC) (Table 1), in the range of published values for AFMimaged phospholipid-based bilayers in a gel phase.37−39 pSM thickness value is in the range between the thicknesses of interdigitated and noninterdigitated gel phases of C18:0 SM observed at 39 °C in previous studies.40 pCer incorporation into any of the phospholipid-based bilayers induced the generation of branched micron-sized segregated domains (Figure 2). AFM-coupled direct epifluorescence microscopy 3137

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Table 1. AFM Studies of Phospholipid/Ceramide/Cholesterol Supported Lipid Bilayers (Mole Ratios Are Indicated for Each Lipid Mixture) breakthrough force (nN) pSM pSM:pCer 90:10b pSM:pCer 80:20b pSM:pCer 70:30b pSM:Chol 70:30 pSM:pCer:Chol 54:23:23 DPPC DPPC:pCer 90:10b DPPC:pCer 80:20b DPPC:pCer 70:30b DPPC:Chol 70:30 DPPC:pCer:Chol 54:23:23

24.63 52.98 55.20 60.03 9.55 32.01 16.22 41.93 43.31 26.18 9.39 20.66

± 3.76a ± 4.70 ± 3.80 ± 5.52a ± 1.22a ± 3.08a ± 2.18a ± 6.05 ± 3.78 ± 2.81a ± 2.58a ± 4.65a

38.86 ± 4.31a

23.35 ± 3.01a

37.19 ± 3.17a 20.07 ± 4.06a 39.44 ± 5.22a

thickness (nm) 5.77 5.43 5.31 5.37 6.58 5.24 5.68 5.99 5.94 5.94 6.32 5.32

± ± ± ± ± ± ± ± ± ± ± ±

0.21 0.29 0.18 0.21 0.32 0.26 0.29 0.20 0.16 0.21 0.22 0.26

Ra (nm) 0.081 0.046 0.041 0.044 0.079 0.063 0.072 0.049 0.044 0.040 0.077 0.059

± ± ± ± ± ± ± ± ± ± ± ±

0.039 0.015 0.011 0.015 0.025 0.019 0.024 0.012 0.012 0.013 0.020 0.015

a

Breakthrough force data from ref 29. Numbers in bold represent main Fb values. bData obtained from the dominant pCer-enriched domains within the corresponding SPBs.

Figure 2. Topography of pCer-enriched domains. AFM height images of (A) pSM-pCer (70:30) and (B) DPPC-pCer (80:20) supported planar bilayers. Lowest black areas correspond to the mica substrate. (C, D) Cross sections from the dashed lines in (A, B). (E, F) Epifluorescence images from (A, B). DiIC18 is excluded from pCerenriched domains. Scale bars: 10 μm.

Figure 3. Thickness and roughness of pSM-pCer and DPPC-pCer supported planar bilayers. (A) Bilayer thickness for ceramide-enriched domains in binary mixtures with increasing pCer/phospholipid ratio. Thickness measurements have been made by cross-section height analysis (n = 300−600) of the AFM images taken from each sample, using three different samples for each mixture. Bars represent mean values, and error lines represent standard deviation. **p < 0.0001. (B) Roughness average (Ra) for ceramide-enriched domains in binary mixtures with increasing pCer/phospholipid ratio. Roughness measurements have been made by surface area analysis (n = 50− 300) of AFM images taken from each sample, using three different samples for each mixture. Bars represent mean values, and error lines represent standard deviation. **p < 0.0001.

nanomechanical bilayer resistance to tip piercing (Figure 4). Typically the AFM-tip indentation process on planar bilayers gives a force−distance record with an ideally clear 4−6 nm jump in the extension trace,41 depending on the thickness of the bilayer which in turn depends on the type of lamellar phase. This jump reflects the exact force at which tip penetration through the bilayer occurs and is commonly known as the breakthrough force (Fb). In the present case Fb measurements (Figure 4) will allow to follow pCer-driven changes in bilayer order or intermolecular packing, thus to test whether pCer maintains its rigidifying character in mica-supported planar bilayers as it does in monolayers and liposomes.25 For the binary mixtures showing phase coexistence we specifically

quantified the properties of the pCer-enriched domains, as a major goal of the present study was to directly compare the 3138

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Figure 4. AFM tip indentation curves on binary phospholipid/pCer mixtures. Representative force curves from pSM- (top) and DPPCbased (bottom) supported planar bilayers (SPBs). Samples are pSM (a), pSM/pCer (90:10) (b), pSM/pCer (80:20) (c), pSM/pCer (70:30) (d), DPPC (e), DPPC/pCer (90:10) (f), DPPC/pCer (80:20) (g), and DPPC/pCer (70:30) (h). Continuous and discontinuous lines represent extension and retraction traces, respectively.

Figure 5. Bilayer breakthrough force (Fb) histograms. Force step distribution from AFM tip indentation curves in pSM- (left) and DPPC-based (right) supported planar bilayers. Samples were pSM (a) (n = 543), pSM/pCer (90:10) (b) (n = 1666), pSM/pCer (80:20) (c) (n = 1544), pSM/pCer (70:30) (d) (n = 1384), DPPC (e) (n = 447), DPPC/pCer (90:10) (f) (n = 1776), DPPC/pCer (80:20) (g) (n = 933), and DPPC/pCer (70:30) (h) (n = 997). Continuous lines represent Gaussian fittings (each histogram is based on 3 samples analyzed with 3 independent tips).

properties of ceramide-enriched domains in the presence and absence of cholesterol in order to assess the formation of new phases of ternary composition. Furthermore, as at high pCer concentrations the non-pCer-enriched area was covering a very minor area, obtaining statistically reliable roughness and force spectroscopy values was not feasible. Figure 5 shows Fb histograms for the pure sphingolipids and binary phospholipid/pCer SPBs; the data are detailed in Table 1. Under our conditions, DPPC bilayer piercing rendered an individual jump (Figure 4e) centered at 16 ± 2 nN. This is in agreement with previously reported data.33,42 A different behavior was observed for pSM, which displayed two reproducible smooth steps (Figure 4a) centered at 24 ± 4 and 38 ± 4 nN, respectively (Table 1). For these situations where two reproducible events were detected, we quantified the two events but considered the higher value as the “main Fb” value (see Discussion section). pSM and DPPC were also prepared by both SUV adsorption and spin-coating, and no significant differences were detected in the main Fb values regardless the methodology (Supporting Information Figure S1A). As to the authors’ knowledge no previous Fb values have been reported for pure pSM bilayers, we relate the increase in mechanical resistance to a stronger intermolecular packing of pSM over DPPC bilayers due to intermolecular hydrogen bonding.43,44 The absence of a clear jump but instead the presence of two smooth steps is at present understood as the tip crossing through the outer and inner monolayers.41 This issue is not commonly observed and/or taken into account in bilayer indentation studies and will be discussed later. In the present study, we quantified every single reproducible jump or smooth step in all the analyzed SPBs.

pCer incorporation into both kinds of phospholipid-based bilayers resulted in an important increase in Fb due to the appearance of pCer-rich domains. In binary mixtures with pSM, the domains exhibited single bilayer crossings at 53−60 nN for mixtures with 10−30 mol % pCer (Figure 4). 10 and 20 mol % pCer-containing bilayers showed comparable values (Table 1), while those with 30 mol % pCer, a concentration near the compositional triple point,16 exhibited a slight increase in Fb. These results point to binary pSM/pCer domains being compositionally constant, independent from the initial pSM/ pCer mol ratio during SPB preparation. In the case of DPPC, pCer-rich domains showed a similar important increase in the breakthrough force from 16 to 43 nN between pure DPPC and 10−20 mol % pCer-containing mixtures (Figure 4). A markedly different behavior was observed at 30 mol % pCer, with two reproducible steps centered at 26 and 37 nN (Figure 7h) that could be caused by domain destabilization due to the high pCer concentration. However, the excellent Fb matching of 10 and 20 mol % pCer-containing DPPC bilayers would again support the generation of stoichiometrically similar domains regardless of the initial ratio during preparation, but the domain size was indeed dependent on the phospholipid:pCer mol ratio (Supporting Information Figure S3). Imaging and Nanomechanical Resistance of Binary Phospholipid/Chol SPBs. Next, we studied binary phospholipid/Chol mixtures with Chol concentrations giving rise to homogeneous bilayers in a liquid-ordered (Lo) phase. 30 mol % Chol-containing pSM- and DPPC-based SPBs were prepared. 3139

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with a reorientation of phospholipid molecules with respect to the plane of the membrane as previously discussed.25 Interestingly, a morphologically uniform behavior was observed in both pSM- and DPPC-based mixtures in contrast to the pCer effect. SPB roughness analysis showed almost equivalent values for the phospholipid/Chol mixtures as compared to pure phospholipid bilayers (Figure 6B and Table 1). AFM-tip indentations on phospholipid/Chol (70:30 mole ratio) planar bilayers gave surprisingly similar patterns in the presence of either pSM or DPPC (Table 1). Both force−distance traces (Figure 7b,f) showed a smooth step centered around 10 nN

AFM imaging of both binary mixtures at those proportions generated homogeneous bilayers with non-height-detectable phase segregation (Figure 1), in contrast to the above-described data for pCer at same proportions. This is a predicted issue as XChol > 0.25 in the pSM/Chol binary mixture is known to give rise to a single liquid-ordered phase.25 Besides, the same phospholipid:Chol mole ratio was present in the ternary phospholipid/pCer/Chol mixtures, which is important due to the reported effect of this ratio on the nanomechanical resistance, as bilayers become more resistant with increasing cholesterol concentrations.33,45 As compared with pure pSM and DPPC, the two phospholipid/Chol (70:30 mole ratio) mixtures showed a significant increase in bilayer thickness (Figure 6A).

Figure 7. Representative AFM tip indentation curves. Representative force curves from pSM- (top) and DPPC-based (bottom) supported planar bilayers (SPBs). Samples are pSM (a), pSM/Chol (70:30) (b), pSM/pCer/Chol (54:23:23) (c), pSM/pCer (70:30) (d), DPPC (e), DPPC/Chol (70:30) (f), DPPC/pCer/Chol (54:23:23) (g), and DPPC/pCer (70:30) (h). Continuous and discontinuous lines represent extension and retraction traces, respectively.

and a marked jump at 23 ± 3 (pSM/Chol) and 20 ± 4 nN (DPPC/Chol) (Table 1). As previously reported for binary DPPC/Chol mixtures, DPPC bilayers showed an increased mechanical resistance upon Chol incorporation.33 The notable analogy of the two phospholipid/Chol indentation patterns indicates a related nanomechanical resistance to tip piercing under the present conditions. pSM/Chol SPBs prepared by SUV adsorption and spin-coating were compared, and no significant differences were observed, neither in the Fb values nor in the nanomechanical resistance pattern (Supporting Information Figure S1B). Ternary Phospholipid/PCer/Chol Mixtures. Chol incorporation into the phospholipid/pCer mixtures were examined to look for possible displacement or cooperation effects in the ternary mixtures. We prepared phospholipid/pCer (70:30 mol ratio) mixtures to which 30 mol % Chol was added in order to have both pCer and Chol saturating the system at the same time, and the resulting phospholipid/pCer/Chol (54:23:23 mole ratio) supported planar bilayers were analyzed. Both pSM- and DPPC-based ternary mixtures gave rise to homogeneous bilayers from the point of view of both height (Figure 1) and DiI-staining.25 However, as recently reported,29

Figure 6. Thickness and roughness of pSM- and DPPC-based pCer/ Chol mixtures (SPBs). (A) Bilayer thickness for binary mixtures with cholesterol or pCer and for the ternary mixtures at a 54:23:23 phospholipid/pCer/cholesterol mole ratio. Thickness measurements have been made by cross-section height analysis (n = 300−600) of the AFM images taken from each sample, using three different samples for each mixture. Bars represent mean values, and error lines represent standard deviation. **p < 0.0001, *0.001 < p < 0.0001. (B) Roughness average (Ra) for binary mixtures with cholesterol or pCer and for the ternary mixtures at a 54:23:23 phospholipid/pCer/cholesterol mole ratio. Roughness measurements have been made by surface area analysis (n = 50−300) of AFM images taken from each sample, using three different samples for each mixture. Bars represent mean values, and error lines represent standard deviation. **p < 0.0001.

The cross-section analysis of pSM/Chol bilayers resulted in an approximate 0.8 nm thicker bilayer, as compared with pure phospholipid, while a 0.6 nm increase was observed in DPPC/ Chol mixtures (Table 1). As height reduction could be expected when shifting from gel (Lβ) to less densely-packed liquid-ordered (Lo) phases, the observed increase is associated 3140

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step process has been shown for decoupled bilayers, that is, bilayers where the distal (away from the solid support) and proximal (close to the solid support) monolayers are not perfectly coupled.41,47,48 In such a situation, which is highly dependent on the solid support and on the temperature during bilayer preparation, a two-step bilayer piercing has been described.41 We relate our two-step force−distance curves to a similar situation, i.e., piercing through the two monolayers of putatively decoupled bilayers. Our AFM setup does not allow heating at temperatures above 67 °C, while the broad, hightemperature-melting transitions of some of the lipid mixtures under study may extend beyond that temperature. In particular, the observation that the highest pCer-containing binary mixture presented a two-step bilayer piercing while those with lower pCer concentrations displayed a single step would support the hypothesis of a possible bilayer decoupling, as DPPC:pCer 70:30 would not be totally fluid at 67 °C.49 However, the observation of a two-step piercing in the case of pure pSM bilayers, which were prepared at temperatures well-above its phase transition temperature, suggests that temperature may not be the only factor affecting the two steps. Further studies directly comparing monolayer and bilayer systems will help to ascertain the origin of the dual signals. Possible artifacts due to tip (or SPB) debris can be discarded due to the high reproducibility of the quantified double steps under different tip and/or sample preparations (Supporting Information Figure S1). Furthermore, control force−distance curves were performed before and after bilayer piercing on lipid-depleted areas to confirm the absence of any force step, thus of tip contamination, and the presence of a single bilayer. In addition, recent reports from F. Sanz’s group showed that conventional (nonforce clamp) force curves performed in multiple bilayers did not exhibit the same clear step-by-step patterns as our own curves,50 which helped to discard multiple bilayers as the origin of the double-step signals. Breakthrough force (Fb) for all two-step curves was defined as the highest of the steps, in which indentation depth was similar to the bilayer thickness (Figure S2). However, the total thickness as derived from the sum of both steps was smaller than the values obtained from the height images as detailed in Table 1. The difference could be attributed to bilayer compression during the indentation process. Nanoscale Characteristics of Gel-like (Lβ) Phospholipid/pCer Domains. Up to date few studies have focused on the nanomechanical properties of ceramide-rich domains in planar bilayers. To the authors’ knowledge, the only related data are those from Zou’s laboratory on raft-mimicking quaternary DOPC/eggSM/Chol/pCer membranes.51 Interestingly, using DNP-S cantilevers with spring constants of 0.06− 0.28 N/m, they were unable to break through SM/Cer-rich domains generated in a liquid-ordered (Lo) and liquiddisordered (Ld) phase-containing SPB, not even using stiffer silicon tips (generally used for measurements in air) with applied forces up to 70 nN. These results pointed to a strong packing of ceramide-rich domains in the quaternary mixture, although they could not provide quantitative data on their mechanical resistance properties. In the present study we have characterized pCer-rich domains in binary mixtures with pSM and DPPC, respectively, quantifying their mechanical resistance to tip penetration. Using silicon nitride MLCT cantilevers with a nominal spring constant of 0.5 N/m, which as announced by the manufacturer rendered spring constants of around 0.9−1 N/m after

the two mixtures showed segregation of a small proportion of NAP-fluorescence-depleted small areas within the bilayers, potentially reflecting the generation of two ternary phases of different composition. In the present study, all the topographic and force spectroscopic analysis were performed on the predominant DiIC18- and NAP-containing bilayer regions that appeared preferentially stained by DiIC18 and NAP, as described previously.29 Cross-section analysis of pSM- and DPPC-based ternary mixtures showed average bilayer heights of 5.24 ± 0.26 and 5.32 ± 0.26 nm, respectively (Table 1). This constituted a significant reduction in bilayer thickness as compared to pure phospholipid and/or the currently studied binary mixtures (Figure 6). This bilayer thickness reduction in the ternary mixtures was statistically significant, even when compared to the closest thickness value of pSM/pCer 70:30 mole ratio (p = 0.0004). However, DiIC18 fluorescence partition allowed a direct distinction between them. As previously described, the binary pSM/pCer domains allowed only a low DiIC18 partition within them (Figure 2E), while partition into the ternary mixtures was clearly higher.29 A clear difference was as well observed between the two mixtures in the calculated bilayer roughness. Roughness analysis evidenced intermediate Ra values for both ternary mixtures as compared to tightly packed phospholipid/pCer and less-ordered phospholipid/Chol binary mixtures (Figure 6 and Table 1). Overall, the data were showing that when mixing phospholipid/pCer/Chol at 54:23:23 mole ratio, a new phase with particular topographic features was formed. Force spectroscopy of ternary pSM/pCer/Chol planar bilayers revealed a single breakthrough force at 32.01 ± 3.08 nN (Figure 7c and Table 1; see also Figure 5c in ref 29), an intermediate resistance between pSM/pCer domains in a gellike (Lβ) phase and pSM/Chol bilayers in a liquid-ordered (Lo) phase. Moreover DPPC-based ternary mixtures showed two smooth reproducible steps centered at 20.66 ± 4.65 and 39.44 ± 5.22 nN, respectively. The mechanical resistance to tip piercing was significantly higher than that of DPPC/Chol in a Lo phase but close to the one from DPPC/pCer bilayers. Nevertheless, DiIC18 unique partition into the ternary phases29 provided a clear evidence for the observed values to be related to compositionally distinct bilayers, that is, bilayers of ternary composition as a result of the recently described interaction between palmitoylceramide and cholesterol.29

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DISCUSSION AFM is currently accepted as a highly useful tool in the study of membrane properties at the nanoscale level. AFM provides valuable information regarding bilayer thickness, lateral organization, and mechanical resistance with very high resolution. In the present work, we have used a combination of AFM-based imaging and force spectroscopy in the characterization of pCer-rich domains and their response to cholesterol incorporation. Many aspects are critical when analyzing planar membranes on solid supports by AFM.46 To name but a few, the type of support, the specific geometry of the cantilever and its tip, the buffer conditions, the scanning force, and the indentation speed are factors that can largely alter the obtained results, making data correlation from different laboratories a nontrivial issue. The results include the description and quantification of unusual but fully reproducible double steps in some of our indentation curves (Figures 4 and 7). Bilayer piercing in a two3141

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of 30 mol % pCer. In DPPC/pCer (70:30 mole ratio) bilayers, and in contrast to the patterns detailed for 10−20 mol % pCercontaining mixtures (Table 1 and Figure 4), two force−distance steps were observed. The high pCer concentration and the possible noninterdigitation could give rise to less stable bilayers and possible decoupling effects leading to the different force− distance pattern. Indeed, the destabilization was not observed in the possibly coupled, thus more stable pSM/pCer domains at 30 mol % pCer. A good correspondence was found for all the analyzed nanoscale properties of pCer-rich domains in both phospholipid mixtures with pCer 0.33 mole fraction liposomes become unstable.16 This would be the case for e.g. pSM/pCer mixtures, in which a stable 2:1 stoichiometry was observed within pCer-rich domains.16,25 The present observation may also allow the detection of such binary domains in further studies with more complex lipid systems. Cholesterol Incorporation and the Generation of Lamellar Gel Phases of Ternary Compositions. As mentioned in the Introduction, recent reports have shown the generation of ceramide-enriched domains in physiological membranes exposed to various stress stimuli.19,57 Remarkably, both studies have shown an accumulation of cholesterol along with ceramide in those segregated platforms. The data open new insights into the proposed biological role of the membrane ceramide/cholesterol ratio in the generation of specific domains in cells undergoing apoptosis, not in terms of displacement effects but of direct interaction between the two lipids. The assumption would be supported by the direct observation of ceramide/cholesterol complexes with the use of specific antibodies in biological membranes28 and by our previous characterization of pCer/Chol-based ternary lamellar gel phases in model systems.29 The present work provides further understanding of Cer/ Chol-rich ternary mixtures with phospholipids. The initial characterization of the binary phospholipid/Chol mixtures rendering homogeneous liquid-ordered phases was an important step for proper data interpretation. Under our experimental conditions, both pSM- and DPPC-based SPBs in a Lo phase showed very similar topographic and mechanical properties. Although a preferential interaction has been proposed between cholesterol and sphingomyelin over phosphatidylcholine because of intermolecular hydrogen bonding,58 both phospholipid/pCer/Chol mixtures showed large bilayer extensions (Figure 1c,f). These micron-sized areas did not appear to contain small nanodomains withinan issue that was previously proposed as a possible explanation to the lack of ceramide-mediated cholesterol displacement from liquid-ordered phases at high cholesterol concentrations.25 What we observed was the formation of bilayers with particular features that did not correspond to any of the pure phospholipid and/or the binary phospholipid/pCer or

calibration in our experimental setup, we applied forces of up to 120 nN to the examined bilayers that were sufficient to break through pCer-rich domains. The binary pSM/pCer bilayers gave rise to Fb values between 53 and 60 nN. The observed high mechanical resistance constitutes convincing evidence for the previously proposed strong intermolecular packing of ceramide-rich domains in simple model membranes16 and hence to their described solid character.52 The observation of these high forces in planar bilayers is not unique, as similar Fb values have been previously detailed in a comparable sodiumcontaining solution, for instance for DPPG bilayers at 20 °C,33 which required 66 nN to be pierced but in the presence of Mg2+. In addition the possibility of these high Fb values being due to artifacts can be discarded as curves performed up to 80− 90 nN did not render any quantitative or reproducible event apart from those due to bilayer piercing (Figure 4a and Supporting Information Figure 1B). Fb values for pSM/pCer domains were high as compared with the values for lipid gel phases measured in buffer in the absence of divalent cations, which are usually in the 30−40 nN range for lipids such as DPPG or DPPS33 and 10−20 nN for DPPC.42 Cholesterol-containing samples at high phospholipid:Chol ratios such as 60:40 or 50:50 have been reported to exhibit breakthrough events at 40−70 nN (but, again, in the presence of divalent cation Mg2+, which increases Fb).53 Liquidordered Chol-driven phases have been described as intermediate states between gel and fluid phases,54 but nanomechanical studies reported higher Fb values for DPPC:Chol mixtures (>30 mol % Chol) than for DPPC.33,53 pCer could exert an effect similar to Chol since both are highly hydrophobic molecules with a tendency to intercalate between lipid hydrocarbon chains, but nanomechanical resistance would be enhanced by the gel nature of pCer-enriched phases as opposed to Chol-driven liquid-ordered ones.55 Thus, the high Fb value for pSM/pCer domains could be related to the aforementioned molecular packing and, perhaps, with the reduced thickness of pSM/pCer domains (Figure 2). This would also be in agreement with the experiment from Zou’s laboratory in which no clear breakthrough events could be detected up to 70 nN for pCer-enriched domains in their quaternary mixture51 although differences between the nature of both samples should also be taken into account. In the presence of DPPC, pCer domains showed a somewhat lower although still important mechanical resistance of around 42 nN with initial pCer concentrations of less than 30 mol %. Sphingomyelin presents an amide and a hydroxyl group at its polar headgroup that can act as both hydrogen bond donors and acceptors, allowing intermolecular H-bonds to be established.56 On the contrary, phosphatidylcholine presents only two hydrogen bond acceptor carbonyl groups. Thus, an increased intermolecular hydrogen bonding may account for the observed higher resistance of pSM/pCer domains and for a preferential ceramide affinity toward sphingomyelin. At the same time the observation of a reduced bilayer thickness in pSM/pCer domains and of an increased one in those made of DPPC/pCer, points to possible minor interdigitation effects to be at the origin of the higher mechanical resistance of pSM/ pCer domains. Thus, we could think of the thinner pSM/pCer bilayers to present a degree of lipid interdigitation, which, as previously mentioned, could explain the high Fb values obtained. If lower or no interdigitation occurred in the thicker DPPC/pCer domains, this could relate to the different indentation pattern observed for these domains in the presence 3142

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Langmuir phospholipid/Chol bilayers. The present data would thus strengthen the idea that at high pCer and Chol concentrations in a phospholipid bilayer ternary phases would be established. On the basis of our recent studies on liposomes,29 these ternary phases would be stabilized by direct interactions between pCer and Chol, the phospholipids playing a structural role for the accommodation of the highly hydrophobic lipids in a lamellar structure. Some of the analyzed features of the ternary phases were intermediate between those of the highly ordered pCer-rich gellike domains and those of the Chol-rich liquid-ordered phases. The Lo phases, hence the related raft-like domains, have been proposed to present an increased intermolecular order with high lateral motionfavorable conditions for specific proteins to colocalize and self-interact in diverse cell signaling events. The possible generation of similar ceramide-driven domains in cell membranes has been attributed to the very tight packing of ceramide-rich domains in model membranes, but this would make difficult the partition of specific proteins within. The present description of pCer and Chol cooperating to build lipid bilayers with an apparently intermediate molecular packing would support the role of ceramide in the formation of specific platforms in cell membranes, hypothetically allowing the partition of specific proteins within. Moreover, these results might as well help to understand the specific colocalization of ceramide and cholesterol in specific domains upon stress stimuli.

ACKNOWLEDGMENTS

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REFERENCES

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CONCLUSION The present study shows how palmitoyl ceramide (pCer) and cholesterol (Chol) can drastically change the nanomechanical properties of phospholipids. Results evidence that pCer in binary mixtures with phospholipids induces phase segregation with highly resistant domains, while if we increase the amount of pCer domains grow in extension but nanomechanical resistance does not significantly increase. In turn, at high ratios of both pCer and Chol a homogeneous ternary gel phase can be achieved with no phase segregation. This phase exhibits intermediate nanomechanical properties when compared to liquid-ordered and ceramide-induced segregated gel phases, suggesting that at those ratios there is no displacement between these lipids. Also, a significant reduction in bilayer thickness is observed for this ternary gel phase. This data become relevant in the context of sphingolipid signaling and membrane platform formation. ASSOCIATED CONTENT

S Supporting Information *

Comparison of SUV-derived and spin-coated SPB (Figure S1); two-step breakthrough processes (Figure S2); area of ceramideenriched domains in spin-coated samples (Figure S3). This material is available free of charge via the Internet at http:// pubs.acs.org.

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This work was supported in part by grants from the Spanish Ministry of Economy (BFU 2011-28566, BFU 2012-36241) and the Basque Government (IT838-13, IT849-13). J.V.B. was supported by the University of the Basque Country. A.G.A. was a predoctoral student supported by the Basque Government.

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Fax +34 94 601 33 60 (F.M.G.). Notes

The authors declare no competing financial interest. 3143

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