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Jan 21, 2016 - Bulgaria. ‡. Biophysical Chemistry Laboratory, Department of Physical Chemistry, Faculty of Chemistry and Pharmacy, University of Sof...
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Phospholipase A2‑Induced Remodeling Processes on LiquidOrdered/Liquid-Disordered Membranes Containing Docosahexaenoic or Oleic Acid: A Comparison Study Rayna Georgieva,† Kristina Mircheva,‡ Victoria Vitkova,§ Konstantin Balashev,‡ Tzvetanka Ivanova,‡ Cedric Tessier,#,⊥ Kamen Koumanov,† Philippe Nuss,#,⊥ Albena Momchilova,† and Galya Staneva*,† †

Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria ‡ Biophysical Chemistry Laboratory, Department of Physical Chemistry, Faculty of Chemistry and Pharmacy, University of Sofia, 1 J. Bourchier Str., 1164 Sofia, Bulgaria § Institute of Solid State Physics, Bulgarian Academy of Sciences, 72 Tsarigradsko Chaussee, 1784 Sofia, Bulgaria # Sorbonne Universites-UPMC Univ Paris 06, UMR 7203, INSERM ERL 1157, CHU St. Antoine, 27 rue Chaligny, 75012 Paris, France ⊥ Department of Psychiatry, Hôpital Saint-Antoine, AP-HP, Paris, France S Supporting Information *

ABSTRACT: Vesicle cycling, which is an important biological event, involves the interplay between membrane lipids and proteins, among which the enzyme phospholipase A2 (PLA2) plays a critical role. The capacity of PLA2 to trigger the budding and fission of liquid-ordered (Lo) domains has been examined in palmitoyl-docosahexaenoylphosphatidylcholine (PDPC) and palmitoyl-oleoylphosphatidylcholine (POPC)/sphingomyelin/cholesterol membranes. They both exhibited a Lo/liquid-disordered (Ld) phase separation. We demonstrated that PLA2 was able to trigger budding in PDPC-containing vesicles but not POPC ones. The enzymatic activity, line tension, and elasticity of the membrane surrounding the Lo domains are critical for budding. The higher line tension of Lo domains in PDPC mixtures was assigned to the greater difference in order parameters of the coexisting phases. The higher amount of lysophosphatidylcholine generated by PLA2 in the PDPC-containing mixtures led to a less-rigid membrane, compared to POPC. The more elastic Ld membranes in PDPC mixtures exert a lower counteracting force against the Lo domain bending.

1. INTRODUCTION The role of phospholipase A2 (PLA2) on various cellular processes has been extensively described, in particular on intracellular membrane trafficking (such as vesicle cycling),1 cell differentiation,2 proliferation,3 and apoptosis.4 A growing evidence is showing that major brain functions, such as memory, are regulated, in part, by PLA2,5 suggesting its involvement in the development of neurodegenerative diseases such as Alzheimer’s disease.6 Conversely, brain lipids have been shown to modulate PLA2 activity.7 Phospholipase A2 family consists in a group of enzymes that hydrolyze the ester bond at the sn-2 position in glycerophospholipids, inducing the release of a free fatty acid (FA) and lysophospholipid. In nervous cells, PLA2 was also shown to control the metabolic transformation of phospholipid (PL) molecules that contain polyunsaturated fatty acids (PUFAs).8 The most abundant PUFA in the central nervous system (CNS) is docosahexaenoic acid (DHA),9 which belongs to the omega-3 FA family. DHA is characterized by 22 carbon atoms and six double bonds. NMR data shows that the © 2016 American Chemical Society

high mobility of DHA chains creates a membrane media with a high degree of fluidity, 10 specific phase behavior, 11,12 permeability,13 and elasticity.14 Interestingly, DHA was shown to have beneficial effects in the prevention of Alzheimer’s disease15 and dementia.16 The critical role of PLA2 and DHA in neuropsychiatric diseases motivated us to search for the molecular mechanisms through which PLA2 interacts with DHA-containing lipid membranes. At the molecular level, DHA is reckoned to influence membrane phase separation,17 promoting the formation of “rafts”, which are lipid platforms known to modulate numerous membrane processes such as signal transduction,18 membrane transport,19 and protein transformation.20 Rafts domains are enriched in cholesterol and sphingolipids and contain GPI-anchored proteins.18,21 When observed using fluorescence microscopy in heterogeReceived: September 3, 2015 Revised: January 20, 2016 Published: January 21, 2016 1756

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experiments (16NS), as well as Hepes buffer, were purchased from Sigma−Aldrich. Tris buffer and silicagel TLC-plaques were purchased from Merck. 2.2. Methods. 2.2.1. Visualization of the Phospholipase A2 Action on Giant Unilamellar Vesicle (GUV) Morphology. 2.2.1.1. Electroformation of GUVs and Phase Coexistence Visualization. Giant unilamellar vesicles (GUVs) were prepared using an electroformation method that was developed by Angelova and Dimitrov.32 EggPC, POPC, and PDPC stock solutions that were used as substrate vesicles, as well as lipid mixtures containing SM and chol, had a concentration of 1 mg/mL. Lipids and lipid mixtures were dissolved in a diethyl ether:methanol:chloroform solution in a ratio of 72:10:18. Approximately 6 μL of each phospholipid, or lipid mixture, respectively, were spread onto two parallel platinum electrodes, each having a diameter of 0.8 mm, with the distance between axes being 3 mm. The solvent was evaporated and then the electrodes were placed in a temperature-controlled quartz chamber. The chamber was filled with 1.3 mL 0.5 mM Hepes buffer (pH 7.4, σ = 59 μS/cm). Immediately, an alternating voltage with a frequency of 10 Hz was applied, with the amplitude rising from 100 mV up to 400 mV over a period of 30 min. Once the 400 mV amplitude was achieved, no further increase was made and these parameters were maintained for 3−5 h, depending on the mixture. Binary and ternary mixtures need more time for GUV formation. In addition, the temperature of GUV formation varies as a function of the type of the lipid mixtures. Pure PC vesicles can be prepared at room temperature (∼25 °C), which is above the phase transition temperature. Binary and ternary lipid mixtures require a higher temperature to form vesicles. Visualization of the coexisting phases was made possible by the use of the fluorescent lipid analogue egg Rhodamine-PE that preferentially partitions into Ld phase.33 When two liquid-disordered (Ld) and liquid-ordered (Lo) phases coexist in the plane of the membrane bilayer, the bright background is assigned to the Ld phase, while the observed dark circular domains are assigned to the Lo phase. 2.2.1.2. Microscopy Observations. GUVs were observed using a Zeiss Axiovert 135 microscope equipped with 40× long-workingdistance objective lens (LD Achroplan Ph2). Observations were recorded using Zeiss AxioCam HSm CCD camera that was connected to an image recording and processing system (Axiovision, Zeiss). Vesicle shape transformations occurring after PLA2 injection near the surface of vesicle membranes were followed in phase contrast and in fluorescence by Zeiss filter set 15 (Ex/Em = 546 nm/590 nm). 2.2.1.3. Microinjection of the Phospholipase A2. The PLA2 used in our experiments with GUVs was 1 mg/mL dissolved in Hepes buffer (0.5 mM, pH 7.4), and the enzymatic activity was estimated to be ∼2 u/μL. CaCl2 was added to a concentration of 10 mM (per injection volume). Each injection volume was 150 pL and was made near the surface of GUVs by using an Eppendorf Model 5246 Transjector. Micropipettes used for injection had inner tip diameters of 0.9−1 μm and were made of borosilicate capillaries (1.2 mm outer diameter), using a microprocessor-controlled vertical puller (Model PC-10, Narishige). Conclusive data are based on the observation of at least five vesicles per mixture. This enzyme quantity was determined as the optimal PLA2 content needed to obtain, with only one injection, a vesicle shape modification on a sufficient number of vesicles of similar size. The time between two consecutive injections is ∼2 min. In order to address the intrinsic effect of CaCl2 on membrane remodeling processes, control experiments have been performed on both PCs mixtures with 10 mM CaCl2. No vesicle transformation (burst, shrinkage, or domain budding) has been observed (data not shown). 2.2.2. Quantitative Estimation of the Phospholipase A2 Activity. 2.2.2.1. Preparation of the Unilamellar Vesicles. Stock solutions for each lipid class (10 mg/mL) were prepared in chloroform. Lipid mixtures prepared with the appropriate amount of the stock solution were dried and then hydrated in 100 mM Tris buffer (pH 8) and vortex-mixed for 30 s at room temperature (23 °C) until multilamellar vesicles (MLVs) were obtained. The samples were then heated at 65 °C for 30 min and vortexed again for 30 s. Three heating (65 °C)/ cooling (23 °C) cycles have been performed to ensure uniformity in

neous model membranes, a liquid-ordered (Lo) phase is identified via a fluorescent lipid analogue and considered to mimic raft domains. The Lo phase is characterized by a high lateral mobility of lipids and dense packing of their FA chains.22 PLA2 activity was shown to be dependent not only on the nature of its lipid substrate but also the organization of the medium where the substrate lipid is located. For instance, DHA-containing domains are highly disordered, which is a physical condition that is likely to influence PLA2 activity in biological membranes. This is in accordance with the substrate theory developed by Mouritsen et al., predicting that the thermodynamic parameters of the membrane system can determine the enzymatic activity.23 In addition, PLA2 activity on phosphatidylcholine (PC) vesicles was also reported to reach a maximum value at the gel/liquid phase-transition temperature.24 Factors such as thermotropic phase transition, transmembrane osmotic pressure, lateral surface pressure, and membrane curvature have been described to impact on the enzyme’s activity.25 The nature of the surrounding lipids in membranes was also examined. In previous studies, we could demonstrate that sphingomyelin (SM) can negatively modulate PLA2 activity and that cholesterol (chol) was able to counteract this effect, restoring enzyme activity. The strong interaction between SM and chol led to the formation of rafts able to sequester the PC substrate, thus making it more available for the enzyme hydrolysis.26 Nevertheless, the overall PLA2 activity is not only dependent on the direct enzyme/substrate interaction;27,8 it also is dependent on the enzyme byproducts, which can be either messengers (such as arachidonic acid)28 or modify the membrane physical properties. It was shown that the enzyme-released lysophospholipid can cause membrane budding29,30 and modulate protein complex assembly.31 The present study aims to expand the knowledge about the interplay between PLA2 and its lipid substrate in complex membranes. We focused on the differential effect of PLA2 on biochemical and biophysical characteristics of lipid model membranes either containing DHA or oleic acid (OA) PCs. We wanted to understand the influence of the number of double bonds in the acyl chain at the sn-2 position on mixtures under PLA2 activity. A variety of methods has been used. Phase contrast and fluorescence allowed the visualization of PLA2 action on heterogeneous giant unilamellar vesicles (GUVs). The specific enzyme activity was calculated by the measurement of FA release using gas chromatography. The molecular order parameter of the coexisting lipid phases in mixtures was assessed by electron spin resonance. Ultimately, the membrane elastic moduli were calculated by using compression isotherms of lipid monolayers and bending rigidity via thermal shape fluctuation analysis.

2. MATERIALS AND METHODS 2.1. Materials. Egg yolk phosphatidylcholine (eggPC), 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC), egg-yolk sphingomyelin (SM), 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC), and the fluorescent lipid analogue L-α-phosphatidylethanolamine-N-(lissamine Rhodamine B sulfonyl) (Rhod-PE) were purchased from Avanti Polar Lipids (Alabaster, AL). The distribution of FAs in eggSM is 84% C16:0, 6% C18:0, 2% C20:0, 4% C22:0, and 4% C24:0; the distribution of FAs in eggPC is 32.7% C16:0, 32% C18:1, 17.1% C18:2, 12.3% C18:0, 2.7% C20:4, 1.1% C16:1, 0.4% C22:6, 0.3% C20:3, 0.2% C20:2, 0.2% C14:0, and 1% unknown. Cholesterol, phospholipase A2 (PLA2) from bee venom, FA-free bovine serum albumin (BSA), 16-doxyl-stearic acid spin probe for ESR 1757

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chloroform, at the air/water interface of a Langmuir trough filled with double distilled water. The surface potential−mean area (ΔV−A̅ ) isotherms were measured simultaneously by using radioactive ionizing and reference electrodes connected to the electrometer (Kriona, Bulgaria). Ten minutes were enough for the chloroform to evaporate and the surface film to form. After that, the monolayer was compressed with a constant velocity of Ub = 10 cm2 min−1. The surface pressure balance had a resolution of 0.01 mN m−1. The standard deviation of the surface potential measurements was ∼5 mV. The compression isotherms were reproducible within 2−3 Å2 molecule−1. Each isotherm represents the mean of at least five recordings. All experiments were performed at room temperature. 2.2.4.2. Calculation of Elastic Moduli of Compressibility and the Dipole Moments. Monolayer compressibility (Cs) values at the indicated experimental mixing ratio were calculated from π(A̅ ) isotherms at π = 20 mN m−1:

vesicle dispersion. The use of temperatures above the main phase transition and phase separation of lipids was chosen to obtain a homogeneous lipid mixture for the preparation of large unilamellar vesicles (LUVs). The multilamellar vesicles were then extruded with a LiposoFast small-volume extruder that was equipped with polycarbonate filters (Avestin, Ottawa, Canada). The extrusion protocol consisted in 12 extrusions through an 800 nm filter. Additional 21 extrusions were made using a 100 nm filter. To ensure a constant enzyme/substrate ratio, the phospholipid concentration was made using lipid phosphorus quantification.34 LUVs samples were kept at a temperature of 4 °C, protected from light, until use. 2.2.2.2. Phospholipase A2 Activity Assay. Addition of 10 μL CaCl2 (10 mM in the volume of each sample), 100 μL BSA (1 wt %), and 12.8 μL PLA2 (25 u) was made for each of the LUV samples (Tris buffer, at pH 8.6 (500 μL)). Control incubation samples consisted of pure PCs samples without and with PLA2 enzyme were prepared. In addition, for complex mixtures, a control condition without enzyme was also performed in parallel with the PLA2 assay. Samples (each with a final volume of 622.8 μL) were incubated at 25 °C for 5 min with rotational shaking. After incubation, the enzyme reaction was totally blocked by the addition of 500 μL of methanol. After the addition of heptadecanoic acid (C17:0) as an internal standard, lipid extraction was performed with chloroform/methanol solvent, according to the method of Bligh and Dyer.35 The organic phase obtained after extraction was concentrated and analyzed by thin layer chromatography (TLC). The lipids and fatty acids were separated on silica gel G 60 plates in a solvent system containing hexane/diethyl ether/acetic acid (90/30/1) for neutral lipids. The location of the separated fractions was visualized after revelation by iodine sprayed on the plates. The fatty acids spot was scraped and fatty acids were extracted with solvent. Methylation was performed using a 3 mL of solution methanol−acetyl chloride 50:4 (v/v) for 1 h at 75 °C.36 The fatty acid methyl esters (FAMEs) were then separated and quantified by gas chromatography equipped with a flame ionization detector. The FAMEs, dissolved in hexane, were separated by gas chromatography (Perichrome (Model Peri 2000, France) on a capillary column coated with Supelcowax 10-bound phase 9 (inner diameter of 0.32 mm, length of 30 m, film thickness of 0.25 μm) (Supelco, Bellafonte, PA) using a temperature program from 150 °C to 250 °C at a rate of 10 °C/min. Quantification was referenced to an internal standard of heptadecanoic methyl ester. The response factors of the various FAs were calculated with a weighted methyl ester calibrator (Mix 37, Supelco). 2.2.3. Investigation of the Molecular Order Using Electron Spin Resonance (ESR). In electron spin resonance (ESR) experiments, we used a 16NS spin probe, which undergoes tumbling in the fast motional regime, providing a reasonable spectral resolution over the temperature range from 17 °C to 57 °C, in steps of 5 °C. Lipid mixtures were doped with 0.1 mol % of 16NS. After evaporation of the solvent, the dry lipids were hydrated with a large excess (500 μL) of buffer consisting of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1 mM CaCl2. The lipid dispersion was centrifuged and 20 μL of the pelleted liposomes were transferred to an ESR measurement capillary cell and sealed. Continuous wave−ESR spectra were recorded at 9.5 GHz (Bruker, Model ER 200D ESR, Wissembourg, France) after an equilibration time (ca. 10 min) at each temperature set by the variabletemperature device (Bruker, Model ER4111VT). The signal was digitized by the EPRWARE software (Scientific Software Service, Bloomington, IL). The ESR parameters have been determined by least-squares spectral fittings, as previously reported37 (also see www. esr-spectsim-softw.fr). The parameter of major interest is the order parameter Szz of the Z-axis of the nitrogen hyperfine coupling tensor parallel to the C15−C17 vector of the stearoyl chain. 2.2.4. Determination of Membrane Elastic Moduli of Compressibility and Dipole Moments by Compression of Lipid Monolayers. 2.2.4.1. π−A̅ Isotherms. Surface pressure−mean area per molecule (π−A̅ ) isotherms were measured using a surface balance (KSV Chemicals, Model KSV 2200, Finland) that was equipped with platinum plate and a Teflon trough with area of 475 cm2. The monolayers were formed by spreading the lipids, dissolved in

⎛ 1 ⎞⎛ dA ⎞ Cs = ⎜ − ⎟⎜ ⎟ ⎝ A ⎠⎝ dπ ⎠ where A is the area per molecule at the indicated surface pressure. Data were expressed in terms of reciprocal values of isothermal compressibility (Cs−1), i.e., through the elastic moduli of compressibility.38 The dipole moments (μ⊥) were calculated from ΔV(A) isotherms and the Helmholtz equation, (ΔV = 4πμ⊥ Γ) where Γ is the surface density (Γ = 1/A).38 2.2.5. Measurement of the Membrane Bending Rigidity by Thermal Fluctuation Analysis. 2.2.5.1. Preparation of GUVs for Bending Elasticity Measurements: Electroformation. For GUV electroformation, depositions were made by the careful spreading of 50 μL of PC solution with a concentration of 1 g/L in chloroform− methanol (9:1) organic solvent on each indium tin oxide (ITO)coated glass electrode. In our experiments freshly prepared organic solutions of lipids (previously lyophilized and kept under vacuum at −20 °C) were used. After the complete drying of the lipid for at least 2 h under vacuum, the electroformation chamber was assembled in a way to completely fill the internal volume with double-distilled water without air bubbles. AC electric field ( f = 10 Hz) was applied to the chamber, the peak-to-peak amplitude was successively increased up 300 mV during 3 h. Thereafter, a high yield of unilamellar vesicles without visible defects was obtained within 30

eggPC/SM eggPC/SM POPC/SM POPC/SM PDPC/SM PDPC/SM

Lβ/Ld Lβ/Ld Lβ/Ld Lβ/Ld Lβ/Ld Lβ/Ld

1−4 >4 1−8 >8 1−10 >10

eggPC/SM/Chol POPC/SM/Chol POPC/SM/Chol PDPC/SM/Chol

Lo/Ld Lo/Ld Lo/Ld Lo/Ld

1 2 >2 1

(60/20/20) (60/20/20)

Lo/Ld Lo/Ld

2 >2

(40/40/20)

Lo/Ld

1

Vesicle Transformation PC burst burst intact shrinkage Binary Mixtures (50/50) intact slight shrinkage intact slight shrinkage intact slight shrinkage/vesicle undulations Ternary Mixtures (50/25/25) Lo budding and fission (Lo domains on Ld matrix) intact, no domain budding Ld shrinking (Ld domains on Lo matrix) Lo budding and fission POPC/SM/Chol (Variable Ratio) intact Ld shrinking (Lo domains on Ld and Ld domains on Lo matrix) Ld shrinking (Lo domains on Ld matrix)

Mean Time for Transformation 1.14 ± 0.22 s 2.31 ± 0.19 s

Number of Vesicles Studiedc 6 9 5

3.8 ± 0.9 min 7 25.5 ± 6.1 s 7 27.1 ± 5.7 s 7 29.5 ± 4.1 s 14.9 ± 3.2 s 30.5 ± 7.1 s 1.11 ± 0.38 s

6 5 8 5

25.4 ± 5.9 s 23.9 ± 6.6 s

5

a

The mean time for vesicle shape transformation is indicated. b0.3 m units per injection (150 pL). cNumber of vesicles with similar size needed to achieve an equivalent lipid-to-enzyme ratio.

Figure 1. Panels (a)−(f) show the image sequence in phase contrast of the effect of PLA2 on a PDPC substrate vesicle at 25 °C. The flaccid vesicle before injection of PLA2 is shown in panel (a), and vesicle shrinkage after 30 injections of the enzyme is shown in panels (b)−(f). Scale bar is 20 μm. 2.2.5.3. Observation and Registration of Vesicle Shape Fluctuations. Observations of vesicle shape fluctuations were performed by means of an Axiovert 100 inverted microscope (Zeiss, Germany) with phase contrast immediately after vesicle preparation. The observation chamber consisted of an objective glass, a coverslip, and an inert spacer 0.5 mm thick (CoverWell, Sigma−Aldrich, Inc., USA) assembled in a way to prevent water evaporation during measurements. In these experiments, the visualization of the membrane fluctuations was carried out in phase contrast regime, using an oil-immersed objective (100×, NA 1.25). Image recording was performed using a CCD camera (Model C3582, Hamamatsu Photonics, Japan). The video signal from the camera was fed to a frame grabber board (Model DT3155, Data Translation, USA, 768 × 576 8-bit pixels, pixel size: 0.106 μm/pix). For each chosen vesicle, at least several hundred images were captured once per second and

processed for calculation of the bending elasticity modulus and membrane tensions.40 The stroboscopic illumination of the observed vesicles, synchronized with the camera, permitted the fastest modes of fluctuations to be recorded and analyzed.41 2.2.5.4. Flicker Spectroscopy. The method applied in the present study for the measurement of the membrane bending elasticity is the thermal shape fluctuation analysis (or “flicker spectroscopy”) performed on nearly spherical giant vesicles.42 It consists of Legendre analysis of the autocorrelation function of the vesicle contour radius and represents one of the most-advanced and well-established methods for the measurement of the bending rigidity of lipid bilayers. The vesicles subjected to fluctuation analysis and subsequently used for deduction of the membrane bending modulus for every lipid composition studied had to satisfy several rigorous criteria of goodness.43,44 Only nearly spherical and flaccid vesicles having 1759

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Figure 2. Budding and fission of the Lo domain after PLA2 injection on a vesicle composed of PDPC/SM/chol (50/25/25) mixture at 25 °C: (a) fluorescence evidence Lo (dark)/Ld (bright) phase separation; (b−f) phase contrast images, clearly showing the budding and fission process; (g) fluorescence image showing only a bright vesicle, indicating that budding resulted from the Lo domain exclusion from the parent Lo/Ld vesicle; and (h) the budded Lo domain vesicle is still visible by phase contrast. Scale bar = 20 μm. diameters of ≥7 μm and without defects on their membranes were chosen for capture. For every membrane composition, the fluctuations of at least 20 vesicles with the above-mentioned properties were recorded and processed. During further analysis, only ∼30% of each ensemble of vesicles fulfilled the further requirements for selection, as described in detail elsewhere.44 Every reported value of the bending modulus was calculated from at least five vesicles, satisfying all requirements for goodness.

to the enzyme action (Table 1). Even before PLA2 injection, PDPC vesicles shape was flaccid, displaying significant visible undulations of the membrane bilayer. Of notice, when moving the needle in the aqueous phase, the lipid bilayer follows the needle movement (Figure 1a). This high degree of fluidity is ascribed to the presence of the six double bonds at the sn-2 position of PDPC that allows the formation of a thin and permeable disordered lipid bilayer.10,46 After repeated injections of the enzyme near to the surface of PDPC vesicles, a gradual vesicle shrinking was observed (see Figures 1b− f). The diameter of these vesicles shows a significant decrease during the time interval of ∼3 min (Figure 1f). In comparison with PDPC GUVs that remained stable after several injections, a vesicle burst was observed in POPC under the PLA2 action. 3.1.1.2. PLA2 Action on Binary PC/SM Vesicles. The addition of SM to PCs was examined, because SM is both a major component of the lipid “rafts”21 and inhibitor of the phospholipase A2 activity.26 Binary PC/SM mixtures are known to exhibit a gel/liquid phase separation in a large temperature range.47 Vesicles prepared with 50/50 (mol/mol) eggPC/SM (see Figure 2SD in the Supporting Information) and POPC/SM (see Figure 3SD in the Supporting Information) remained stable after the first PLA2 injection. After subsequent injections, slight vesicle shrinkage was observed as opposed to the immediate vesicle burst seen with eggPC and POPC vesicles (Figure 1SD). The addition of SM to PDPC led to more unstable vesicles (Figure 4SD in the Supporting Information), compared with pure PDPC vesicles (recall Figure 1). PLA2 injection on 50/50 PDPC/SM led to vesicles shrinking (either with or without bilayer undulations) after 10 injections of PLA2, while such modification was only observed after 30 injections in the control PDPC substrate vesicles. When considering the “vesicle surface area to volume” (S/V) ratio, an explanation of

3. RESULTS Data will be shown in three different sections. PLA2 action and activity on POPC- and PDPC-containing mixtures will be described. Physicochemical properties of the coexisting phases will then be presented, with an emphasis on the line tension and elastic behavior of the membrane domains. 3.1. PLA2 Action and Activity. 3.1.1. Phase Contrast and Fluorescence Microscopy Observations of the Effect of PLA2 on GUVs. 3.1.1.1. Phospholipase A2 Action on Substrate PC Vesicles. The action of PLA2 on GUVs composed of three different substrates of this enzyme has been investigated, namely, eggPC, POPC, and PDPC (see Table 1). All experiments with substrate vesicles were performed at 25 °C. Upon a single injection of PLA2 (150 pL), vesicles composed of eggPC (see Table 1, as well as Figure 1SD in the Supporting Information (panels (a)−(c)) disintegrated to small vesicles or micelles via a vesicle burst. Such burst occurred for POPC-containing vesicles after at least two injections of the enzyme (see Table 1, as well as Figure 1SD in the Supporting Information (panels (d)−(f))). Time interval between injection and vesicle burst was 1−2 s for both types of PCs. In comparison with POPC, less enzyme quantity was needed for the eggPC vesicle, because of the presence of arachidonic acid and other minor fractions of polyunsaturated FAs, both of which are preferential PLA2 substrates.45 In contrast, PDPC-containing vesicles could not burst, even after more than 30 injections, which is indicative of a resistance 1760

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Figure 3. Image sequence in fluorescence microscopy of the effect of PLA2 on a vesicle composed of POPC/SM/chol (50/25/25) mixture at 25 °C. Ld (bright) domains are visible into Lo (dark) background: (a) vesicle before injection of the enzyme; (b−f) progressive disappearance of the Ld domains after PLA2 injections. Scale bar = 10 μm.

2g). The remaining vesicle that originated from the Lo/Ld parent exhibited Ld phase characteristics only and was visible by both fluorescence and phase contrast methods (Figures 2g and 2h). This result is another example of the budding of Lo domains under PLA2 action, which is a phenomenon previously demonstrated by our team on eggPC/SM/chol vesicles.48 Nevertheless, the dynamic pattern of budding observed between eggPC/SM/chol and PDPC/SM/chol vesicles is not the same. Budding and fission of Lo domains was significantly more rapid in PDPC ternary mixtures, compared to eggPC mixtures, with a time of 1−2 s versus 13−19 s, respectively.48 In 50/25/25 POPC/SM/chol vesicles at 25 °C, percolation was observed with bright domains (Ld phase) on a dark (Lo phase) background. PLA2 addition led to a gradual diminution and complete dispersion of Ld domains after the seventh PLA2 injection (see Figures 3a−f). Different POPC/SM/chol ratios were studied in order to allow a better comparison of Lo/Ld domain patterns between POPC and PDPC mixtures. Instead, we used POPC/SM/chol ratios of 60/20/20 and 40/40/20 (mol/mol/mol), as these mixtures exhibit comparable Lo/Ld phase separation with 50/ 25/25 PDPC/SM/chol mixture (L o domains on a L d continuous phase) at which the PLA2 activity was studied. Because the PLA2 activity is temperature dependent, POPC ternary mixtures exhibiting similar phase separation as 50/25/ 25 PDPC/SM/chol at higher temperature were not appropriate. In vesicles composed of 60/20/20 and 40/40/20 POPC/SM/chol mixtures at 25 °C, Lo and Ld phases coexisted and formed large separated dark and bright regions (see Figure 4). The effect of PLA2 injection on 60/20/20 POPC/SM/chol vesicles was again a diminution and dispersion of the Ld phase.

this phenomenon can be given. Pure PDPC vesicles are shown to be flaccid while PDPC/SM ones are rigid, meaning that the S/V ratio is higher in PDPC, compared to PDPC/SM vesicles. After the enzyme action, a mean molecular area decrease is observed proportional to the LPC increase as described below in section 3.3.1. In order to accommodate the decreased S/V ratio, a membrane stretch is needed. This stretching process continues until the critical strain point is reached where a shrink or burst occurs. The gel/Ld phase coexistence in PDPC/SM vesicles can also contribute to the decreased mechanical strength of the membrane, compared to the homogeneous PDPC membrane. A high resistance to PLA2 action was observed in pure PDPC; however, in PDPC/SM vesicles, the addition of SM led to decreased bilayer stability. Nevertheless, this decreased stability does not affect the overall rank in vesicle stability after PLA2 action, showing that PDPC/SM vesicles are less affected by PLA2, compared to POPC/SM ones. 3.1.1.3. PLA2 Action on Ternary PC/SM/chol Heterogeneous Vesicles. POPC/SM/chol and PDPC/SM/chol ternary mixtures were compared to reveal the influence of chol on PLA2 action and subsequent vesicle transformation. Fluorescence microscopy was able to visualize a heterogeneous appearance in 50/25/25 PDPC/SM/chol vesicles. Two liquid phases coexisted in the plane of the membrane bilayer: the Ld phase and the Lo phase (see Figure 2a). After a single PLA2 injection near to the PDPC-containing vesicles, an immediate budding and fission of Lo domains from the parent vesicle is detected (Figure 2b−f). The newly budded vesicle formed from the Lo phase was visible with the phase contrast method (Figure 2f), but not in fluorescence (Figure 1761

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Figure 4. Image sequence in fluorescence microscopy of the effect of PLA2 on a vesicle composed of a POPC/SM/chol (60/20/20) mixture at 25 °C. Large Lo (dark) and Ld (bright) regions coexist. A shrinking of Ld phase is observed after PLA2 injections: Three small Ld domains are visible in panels (a), (b), and (c) (delineated with a dashed-line ellipse in panel (a)) but disappear in the subsequent images in panels (d)−(i) (dashed line ellipse in panel (d)). Ld domain shrinkage was also visible, as observed in panel (e) compared to panel (h) (delimited by a dashed circle). After further injections of PLA2, both the Ld phase and the Lo phase shrank (panel (i), compared to panel (c)) with a greater size reduction for Ld. The Lo/ Ld interface smooth border was lost under PLA2 action (noted by the vertical arrows in panel (f)). Scale bar = 10 μm.

injection of PLA2, only a gradual diminution and dispersion of Ld phase occurred. The PLA2 action on GUVs is summarized in Table 1. Substrate GUVs composed of PDPC exposed to PLA2 were more stable, when compared to POPC ones. The addition of SM (50 mol %) resulted in less-stable PDPC/SM vesicles, compared to pure PDPC. In contrast, the addition of SM to POPC led to increased bilayer stability under PLA2 action, compared to pure POPC. However, this did not affect the overall rank in vesicle stability after PLA2 action, showing that PDPC/SM vesicle are less affected by PLA2, compared to POPC/SM ones. In GUVs composed of PDPC/SM/chol mixtures, PLA2 was able to trigger the processes of budding and fission of Lo domains. This was not observed in POPC/SM/ chol vesicles. In the latter, the enzyme was only able to diminish

The three small Ld domains evidenced in Figures 4a, 4b, and 4c (delineated with a dashed line ellipse in Figure 4a) have disappeared in the images that follow (Figures 4d−i) (see the dashed line ellipse in Figure 4d). Ld domain shrinking was also visible as seen in Figure 4e, compared to Figure 4h (delimited by dashed circles). After further injections of PLA2, an additional shrinkage is evidenced for both the Ld and Lo phases (Figure 4i), compared to (Figure 4c), with a greater size reduction for Ld (the Ld region is marked by the dashed line delineated in Figure 4c)). The smooth Lo/Ld interfacial border was lost under PLA2 action (denoted by the vertical arrows in Figure 4f). The PLA2 action was also studied on 40/40/20 POPC/SM/ chol at 30 °C so that the size of Lo domains was small enough to enable their detachment (data not shown). After the seventh 1762

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Figure 5. PLA2 specific activity in POPC- and PDPC-containing binary and ternary mixtures. Dashed lines indicate the enzyme activity in either pure POPC or PDPC vesicles. The corresponding molar ratios for binary and ternary mixtures are as follows: PCs/SM and PCs/chol (80/20, 66/34, and 50/50) and PCs/SM/chol (55/15/30, 50/25/25, 40/40/20). Standard deviation values were calculated from three independent samples for each type of mixture. The Student’s t-test showed that the enzyme activity was significantly lower (p < 0.001, confidence interval (CI) of 0.05) for each POPC-containing mixture versus PDPC-containing mixture.

due to possible inactivation of the enzyme or aging of the lipid solutions. 3.1.2.2. PLA2 Activity on PC/SM Binary Mixtures as a Function of the Amount of SM. PLA2 activity in POPC/SM and PDPC/SM (80/20, 66/34, and 50/50 mol/mol) vesicles was compared, while varying the concentration of SM (Figure 5). SM exhibited an inhibitory effect on PLA2 activity in both PC mixtures. It is in accordance with our previous studies where SM was shown to decrease PLA2 activity in different substrate-containing vesicles.26,33 The activity of the enzyme (human recombinant cytosolic PLA2 and human type II secretory PLA2) was shown to be reduced in PC/SM containing mixtures compared to pure PC vesicles. In the present study, PLA2 activity was examined as a function of the degree of fatty acid unsaturation. The SM propensity to inhibit PLA2 activity differs depending on the type of PC species. In POPC substrate mixtures, PLA2 activity was reduced to 81% and 53%, for 150 nmol and 300 nmol upon SM addition, respectively. Increase of SM (600 nmol) did not led to any additional reduction. The effect of SM addition to PDPC mixture on PLA2 activity significantly differed with no activity change for 150 nmol SM and a reduced activity to 76% and 65% for 300 nmol and 600 nmol SM, respectively. Overall, the SM inhibitory effect on PLA2 was more pronounced for POPC than for PDPC. Molar calculation indicates that a 1/4 SM/POPC and 1/2 SM/PDPC ratios are needed to reduce the enzymatic activity by 20%. 3.1.2.3. PLA2 Activity on PC/chol Binary Mixtures as a Function of the Amount of chol. The addition of chol to both types of PCs (PCs/chol: 80/20, 66/34, and 50/50 mol/mol) also exerted an inhibitory effect on the activity of the PLA2 (Figure 5). This result is in agreement with the work of Kerwin et al.,49 who reported that sterol and steryl derivatives

and disperse the bright Ld phase, regardless to the Lo/Ld pattern (i.e., dark Lo domains on a bright Ld background or vice versa). POPC and PDPC molecules differ in the length and degree of unsaturation of the fatty acid at the sn-2 position. This difference is likely to contribute to the specific action of the PLA2 on the Lo/Ld heterogeneous vesicles. A better understanding of the action of the PLA2 on the lipid segregation and budding capability also needs a precise assessment of its intrinsic enzyme activity on both POPC and PDPC ligands when codispersed in the studied lipid mixtures. 3.1.2. PLA2 Activity Assessment. PLA2 activity assay was conducted on LUVs composed of two types of PC substrate vesicles, prepared from POPC and PDPC, respectively; these LUVs included PCs, PCs/SM, PCs/chol, and PCs/SM/chol (Figure 5). The amount of PC substrate was maintained constant (600 nmol) in all mixtures. In PCs/SM and PCs/chol vesicles, the SM and chol amount was variable (150, 300, and 600 nmol). In PCs/SM/chol vesicles, the chol species was kept constant at 300 nmol while the amount of SM was varied. The corresponding molar ratios for the binary and ternary mixtures were as follows: PCs/SM and PCs/chol (80/20, 66/34, and 50/50) and PCs/SM/chol (55/15/30, 50/25/25, 40/40/20). 3.1.2.1. PLA2 Activity on Substrate Vesicles. PLA2 activity on POPC and PDPC vesicles was compared. The enzyme displayed higher activity for PDPC as a substrate. The specific activity of PLA2 for PDPC and POPC vesicles was calculated, with values of 4.84 ± 0.25 and 3.02 ± 0.18 μmol/(min mg), respectively (see Figure 5). During each subsequent experimental series with binary and ternary mixtures, PLA2 activity was tested concomitantly on pure PC species that was then taken to be maximum activity. This procedure was performed in order to eliminate the possible changes in enzymatic activity 1763

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Table 2. Molecular Order Parameter of the Two Coexisting, Lo and Ld Phases at 17, 25, and 37 °C in POPC/SM/chol and PDPC/SM/chol Mixtures Determined by ESRa

Mixture (Molar Ratio) 50/25/25 POPC/SM/chol PDPC/SM/chol 40/40/20 POPC/SM/chol PDPC/SM/chol a

17 °C

25 °C

37 °C

Order Parameter, Szz

Order Parameter, Szz

Order Parameter, Szz

Lo phase

Ld phase

ΔSzz

Lo phase

Ld phase

ΔSzz

Lo phase

Ld phase

ΔSzz

0.296 0.302

0.160 0.128

0.136 0.174

0,259 0,28

0,138 0,113

0,121 0,167

0.230 0.259

0.121 0.103

0.109 0.156

0.339 0.348

0.163 0.114

0.176 0.234

0,28 0,336

0,13 0,106

0,15 0,23

0.245 0.309

0.116 0.087

0.129 0.222

The values of difference in orders between Lo and Ld phases are also indicated.

at neutral pH (7.4) vs at pH 8.6 for the intrinsic enzyme activity. This difference in pH can modify the absolute values of the enzyme activity but it is not significant enough to reverse the qualitative difference in PLA2 activity between POPC and PDPC mixtures, because of the high intrinsic activity of Cadependent secretory PLA2. 3.2. Physicochemical Properties of the Coexisting Phases: Investigation of the Molecular Ordering of Lipid Phases in PC/SM/chol Ternary Mixtures by ESR. ESR experiments were performed to determine the difference in the molecular ordering of PDPC and POPC-containing ternary mixtures in each of the two Lo/Ld coexisting phases. POPC/ SM/chol and PDPC/SM/chol were studied and compared at the following lipid ratios: 50/25/25 and 40/40/20 mol/mol/ mol. The spectral simulations were consistent with the coexistence of two phases characterized by 0.25 < Szz < 0.4 and 0.05 < Szz < 0.2 assigned to the Lo and Ld phases, respectively. The molecular ordering of each of the coexisting phases at 17, 25, and 37 °C as well as the difference in ordering between Lo and Ld for this temperature interval are summarized in Table 2. The PDPC mixtures exhibited a higher order of Lo phase, compared to POPC, whereas a lower order of Ld phase was measured for PDPC mixtures, compared to POPC. The difference in Lo/Ld molecular order was greater in PDPCcontaining mixtures than in POPC-containing ones. This difference was more pronounced at 37 °C than at 17 °C (60% versus 30% respectively). Note that full accounting of ESR data can be found in Georgieva et al.47 3.3. Elastic Properties. 3.3.1. Measurements of the Elastic Moduli of Compressibility by Compression Isotherms of Lipid Monolayer Films. We conducted a series of monolayer experiments in order to examine the effect of the generation of LPCa byproduct of the PLA2 hydrolysis on PCon the material properties of lipid monolayers. PCs as well as PC/LPC monolayers were studied with various LPC amounts (10, 30, 50, 70, and 90 mol %). Upon increasing the amount of LPC in mixed monolayers (POPC/LPC), the surface pressure−area (π−A) and the surface potential (ΔV−A) dependence shifted to smaller areas, compared to the isotherms of pure POPC. A similar trend was observed in PDPC monolayers (data not shown). Pure POPC and PDPC formed liquid-disordered (liquidexpanded) monolayers for all values of the surface pressure. Upon increasing the content of LPC in mixed monolayers, the values of the elastic moduli Cs−1 (mN m−1), mean surface area A̅ (Å2 molecule−1), and the dipole moments μ⊥ (mD) at a surface pressure of 20 mN m−1 decreased for both POPC and

selectively inhibited PLA2 from bovine pancreas, snake venom, and human cytoplasmic 85-kDa PLA2. Cholesterol induced a concentration-dependent inhibition of PLA2 activity in POPC/ chol mixtures where the enzymatic activity was decreased to 73%, 40%, and 24% at 150, 300, and 600 nmol chol, respectively. In PDPC/chol mixtures, no effect, a 61% decrease in enzyme activity, and a 57% decrease in enzyme activity was observed with the addition of 150, 300, and 600 nmol chol, respectively. Paralleling the SM effect on PLA2 activity, chol addition led to a higher inhibitory effect in POPC-containing vesicles than in PDPC ones. A PC/chol ratio of 1:1 was able to reduce PLA2 activity by 80% in POPC mixtures and by PDPC

and

POPC/LPC > PDPC/LPC

Differences in parameters such as pH and ionic strength could quantitatively affect the physicochemical properties of the coexisting phases. Lipid packing, mechanical, and electrical properties are altered by pH.50,51 Our measurements of the bending moduli and elastic moduli of compressibility were carried out at acidic pH (5.5) versus the enzyme activity experiments (7.4−8.6). It has been shown that the elastic area compressibility modulus was unaffected in the pH 3−9 range for PC species. However, the membrane bending modulus is dependent on pH in a sophisticated manner. The observed results have been attributed to changes in membrane surface charge density and Debye length. Because POPC and PDPC 1765

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the phases and is dependent on the elastic moduli of the raft and surrounding membrane matrix.59 The Lo/Ld miscibility temperature is also characterizing line tension. Previous experiments were able to demonstrate that Lo/Ld miscibility temperatures in PDPC-containing ternary mixtures were higher than in POPC-containing mixtures, favoring a higher Lo/Ld line tension in PDPC mixtures, compared to POPC mixtures.47 Hydrophobic mismatch may not be sufficient to fully account for line tension. We were also able to demonstrate that the hydrophobic mismatch in PC ternary mixtures47 was not able to explain the observed line tension that was rather governed by the higher Lo/Ld difference in molecular order (ΔSzz) in PDPC mixtures, compared to POPC mixtures. In the present work, we could show that the Lo and Ld Szz values were higher and lower, respectively, in PDPC mixtures, compared to POPC mixtures. 4.2. Ld Membrane Elasticity/Rigidity in POPC and PDPC Mixtures. To check the importance of elasticity, which is another key factor that triggers the Lo domain budding from the Ld matrix, the material properties of lipid layer/bilayer have been assessed by determining the elastic moduli of compressibility, as well as bending moduli of the membranes. In order to address the specific effect of LPC (one of the PLA2 byproduct) action in mixtures, we have added LPC to the substrate (POPC or PDPC) mono/bilayers. Pure PDPC layers were demonstrated to be more elastic than POPC ones. LPC addition induced significant changes in their elasticity and bending modulus, making them even more elastic and prone to shape modifications. The elastic properties of mixtures could be further discussed via the vesicle behavior after LPC generation by PLA2 in both PC mixtures. A gradual diminution and dispersion of Ld domains were observed in POPC ternary mixtures while a shrinkage and burst were observed for POPC/SM and pure POPC, respectively. In contrast, in PDPC-containing mixtures, the vesicle remained quasi-unchanged. The present data, demonstrating that LPC reduces the membrane bending rigidity, are in accordance with previous results of Zhelev,61 showing that the tensile strength of the membrane decreases as the amount of LPC increases. The addition of LPC led to a considerable decrease in the work for membrane breakdown. Although the exact mechanism of membrane failure is unknown, the membrane strength is considered to be closely related to the membrane elasticity.62 Since POPC vesicles are known to be more rigid that PDPC ones, it was expected that LPC addition would have led to more-stable POPC vesicles. GUV data could show the opposite. Elasticity methods could demonstrate a more important decrease in rigidity, as a function of LPC concentration for POPC mixtures, compared to PDPC ones. This fact could be one of the reasons for the observed resistance of PDPC vesicles, compared to POPC, upon PLA2 action, although the enzyme is more active in the former. 4.3. PLA2-Induced Budding. The absence of Lo domain budding observed in POPC mixtures after PLA2 treatment also results from the decreased activity of PLA2 on POPC, compared to PDPC ternary mixtures. Indeed, PLA2 activity was shown to be 40% less active in POPC vesicles than in PDPC vesicles. The addition of SM and chol led to further decreases in enzyme activity, with a more pronounced reduction for POPC, compared to PDPC mixtures. The lower PLA2 activity observed in the POPC-containing membranes, which leads to less LPC generated in the Ld

share the same zwitterionic polar head, our presumption is that the induced differences in bending modulus by pH alteration from acidic to neutral values would not be sufficient to reverse the observed trend between POPC and PDPC mixtures.

4. DISCUSSION The present study examined the effect of the replacement of OA-containing PC by DHA-containing PC on the line tension and elastic properties of domains in a heterogeneous model membrane and the subsequent effect of this replacement on the PLA2 membrane remodeling properties. Evidence is growing that the membrane remodeling processes are highly dependent on the lipid composition and organization of the membrane.23,55,56 It has been suggested that this function is partly achieved through PLA2 activity that enables remodeling processes such as membrane vesiculation and cycling. In the present paper, we have examined the budding and fission of domains under PLA2 activity in raftlike mixtures containing either POPC or PDPC. We studied both the biophysical properties of the identified coexisting phases as well as action and activity of PLA2 in these mixtures. All data converge in showing that the DHA-containing PC favors budding, compared to the OA-containing PC, which is a property that is partly explained by the fact that line tension associated with the Lo/Ld interphase is higher in PDPCcontaining mixtures, compared to POPC ones. PLA2 exerts its enzymatic activity on the both phases. Because the enzyme substrate (either OA or DHA) is mainly partitioned into the Ld phase, the effect of the enzyme on the Ld phase is expected to be more pronounced, compared to the effect on the Lo phase. We demonstrated that PLA2 activity is higher in PDPCcontaining mixtures, compared to POPC-containing mixtures, resulting in a greater amount of LPC present in the Ld phase in PDPC mixtures. In turn, the increase in LPC further adds elasticity to the PDPC-containing Ld phase, which also favors budding. The budding and fission of raftlike domains induced by PLA2 has been initially described by our group in giant vesicles made of eggPC/SM/chol. 48 The enzyme-induced changes in spontaneous curvature and line tension were described to provide a driving force for the budding of domains. The proposed mechanism for PLA2 action in the budding process is that the enzyme modifies the local spontaneous curvature in the membrane in both coexisting phases (Lo and Ld), inducing a decrease in length of the Lo/Ld interphase boundary. We could further demonstrate that LPC, but not the fatty acids (the other enzymatic PLA2 byproduct) was critical for the fission mechanism.29 Moreover, we demonstrated that not only LPC, but any other cone-shaped molecules such as detergents, are able to induce a similar domain budding and fission.29,57 4.1. Lo Domain Line Tension in POPC and PDPC Mixtures. In the studied mixtures, Lo domains, observed in GUVs, had the tendency to spontaneously merge each other in order to minimize the boundary length, indicative of the presence of a line tension at the phase interface.56,58 One of the main contributing factors usually proposed to account for line tension is the “height mismatch” generated at the Lo/Ld domain edge. Its maintenance has an energetic cost per unit length,58 which is why the membrane undergoes splay and tilt deformations at the boundary to avoid this contact.59,60 The line tension is generally associated with differences in the membrane thickness between the two coexisting phases in the bilayer. It increases quadratically with the difference between 1766

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Figure 7. Conditions for Lo domain budding. Schematic head-to-head comparison of PLA2 action on POPC/SM/chol (left) versus PDPC/SM/chol (right) matrix. Lipid arrangement is depicted by the Lo /Ld phase separation and the difference in order parameter (panels (A) and (B)). The lower LPC content generated by PLA2 is visible in POPC mixture (panel (C)), compared to PDPC (panel (D)). Lo domain budding is represented only for the PDPC/SM/chol mixture (panels (F) and (H)), because no budding occurred in POPC (panels (E), (G), and (I)). The Ld phase that is lessenriched with LPC (panel (E)) in the POPC mixture results in more-rigid membranes, compared to the PDPC mixture (panel (F)). Lo domain fission is visible in panel (J). Both Ld and Lo phases shrink after further PLA2 addition in POPC/SM/chol mixtures, with a greater size reduction for the Ld phase, compared to the Lo phase (panels (G) and (I)). Altogether, enzymatic activity, line tension, and elastic properties of the membrane surrounding the Lo domains are critical factors for triggering the Lo domain budding. The higher line tension of Lo domains in PDPC ternary mixtures is assigned to the greater difference in order parameters of the two coexisting phases. The lower amount of LPC generated by PLA2 in the POPC-containing mixtures led to a more rigid membrane. Thus, more-elastic Ld membranes in PDPC ternary mixtures are less prone to counteract the Lo domain bending.

considered that LPC is generated asymmetrically, mainly in the outer monolayer of the membrane, because LPC exhibits a slow flip-flopon the order of several hours, which is much too long for the time scale of our experiments.63 Altogether, the mechanism of Lo domain budding, as it is observed in our lipid mixtures, is governed by prerequisite conditions such as Lo domain line tension and Ld membrane elasticity but triggered by the PLA2-induced LPC production. 4.4. Lipid Phase Interplay and PLA2 Activity. The effect of the lipid composition of the mixtures, as well as their phase

phase, also results in less-elastic POPC membranes. During the bending of the Lo domain, counteracting forces exerted by the Ld surrounding membrane tends to resist formation of the Lo vesicle. Membrane elasticity is thus critical for budding. In the POPC mixture, despite the elastic effect of the LPC, the relatively lower amount of the enzymatic-generated LPC does not allow the process to be triggered (see Figures 7E and 7F). Conversely, the higher activity of PLA2, relative to PDPC, led to a softer membrane, favoring the protrusion of the Lo domain and its following exclusion (Figure 7 F). Furthermore, it is 1767

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found in the Lo fractions between POPC and PDPC ternary mixtures that have been reported by us previously.47 In this context, the enzyme generation of DHA or OA within the Lo domains may further favor or counteract domain budding and fission. The specific role of DHA in the Ld phase and the Lo phase, as well as the Lo/Ld PLA2 interfacial properties, represent an interesting field of research that requires further investigation.

separation, on PC availability to the PLA2 is an issue. In our experiments, the PC substrate concentration was kept constant with variation in concentration of other lipids. Despite this identical amount of PC into the mixtures, the modifications of the molar ratio between PC and the other lipid components affect not only the physicochemical properties of the mixtures but also the substrate availability to PLA2. Thus, PC availability to PLA2 is not equal in each of the coexisting phases in POPC and PDPC ternary mixtures as well as in mixtures where the molar ratio between PCs and the other lipid components varies. Thus, the observed PLA2 activity results from several parameters involving the affinity of the enzyme to the substrate, amount of substrate, lipid phase-transition temperature, composition, and order of the coexisting phases, as well as characteristics of the phase interface. Our main assumption is that the observed differences result from the length and degree of unsaturation of the FAs at the sn-2 position. When considering the differences in physicochemical properties of the coexisting phases between the two PC ternary mixtures before the exposition of the enzyme, the higher difference in the Lo/Ld order parameter was shown to be a prerequisite for domain budding and fission in PDPC/SM/chol mixtures, compared to POPC-containing mixtures. The effect of the enzyme on the mixtures is leading to increased complexity. Undeniably, the substrate quantity and affinity vary in the coexisting phases for each PC mixtures but it cannot be ignored that the enzyme byproducts such as OA, DHA, and LPC also interfere on the complex lipid interplay into the phases. Although the two types of PC ternary mixtures were chosen to exhibit similar Lo/Ld phase separation, the composition of each of the coexisting phases is different. First, in our previous work, we reported that the fraction of Lo domains in Lo/Ld POPC/ SM/chol mixtures (50/25/25, 40/40/20, 33/33/34) is ∼10%− 25% larger, compared to PDPC ones at 23 °C.47 Second, based on the similarity between SDPC (1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoholine) and PDPC, we used a ternary PC/SM/chol phase diagram with POPC64−66 or SDPC67 to estimate the relative ratio of the three lipids in each of the coexisting phases. Thus, for 50/25/25 POPC or SDPC/SM/ chol mixtures at 23 °C, the quantity of polyunsaturated PC in the Ld phase is higher, compared to the monounsaturated one, and opposite trend is observed for the Lo phase. Since DHA chains are long and polyunsaturated, it is accepted that the induced disorder favors the segregation of cholesterol into SM-rich/sterol-rich rafts, forming DHA-rich domains in the Ld phase via cholesterol exclusion.11 Thus, the observed higher affinity of PLA2 to PDPC, larger PDPC fraction in the Ld phase, and lower Ld packing compared to POPC mixtures is consistent with higher enzyme activity and increased LPC content into the Ld phase. A result consistent with our finding showing that the softer Ld phase in PDPC mixtures results from a larger LPC content, which favors Lo domain budding and fission. Consequently, the decreased enzyme activity in the POPC ternary mixtures results from the lower affinity of the enzyme for POPC, as well as decreased POPC fraction in Ld and higher Ld packing, compared to PDPC mixtures. Further understanding of this phenomenon can be drawn from recent studies on POPC or PDPC/SM/ cholesterol, showing that both PCs also partition into raftlike domains.17,47 By using published ternary phase diagrams, cited here above, we estimated that the POPC fraction within the Lo phase is ∼15% larger, compared to SDPC in the 50/25/25 ternary mixture. This ratio is in accordance with the differences

5. CONCLUSION The present work uses raftlike lipid mixtures to clearly demonstrate that line tension and elastic properties govern budding formation after the addition of phospholipase A2 (PLA2), which explains why docosahexaenoic acid (DHA)containing phosphatidylcholine (PC), but not oleic acid (OA)containing PC, are able to exhibit liquid-ordered (Lo) domain budding. The overarching concept of the present study was to examine to what extent a biological event such as vesicle cycling can be influenced by the replacement of OA by DHA in PC. We could show that this subtle difference in the chemical structure of the fatty acids leads to a unique membrane organization that, in turn, results in a strongly different effect of PLA2 on vesicle budding capability. Vesicle cycling is of high importance for cell trafficking, in particular at the synapse level. The scientific literature on this topic is scarce, with no clear explanation on how raft domains in synapse regulate the functional aspects of neurotransmission, such as vesicle formation. While rafts have been involved in neuronal protein localization, a more in-depth understanding of their role in vesicle formation can highlight the role of lipids in neurotransmission. The present study is relevant for the understanding of central nervous system (CNS) signaling, since the brain is highly enriched in DHA. Furthermore, PLA2, which is known to be a major mediator in membrane shape and trafficking, is also described to be dysfunctional in numerous neuropsychiatric disorders. The PLA2 interplay with physicochemical properties of the lipid membranes can be considered as a crossroads to the understanding of vesicle cycling, which is a major event involved in cell functioning.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b03317. Additional images series illustrating the action of PLA2 on substrate PCs and PCs/SM giant vesicles; graphical representation of the bending rigidity of POPC membranes containing various concentrations of either LPC, PDPC or both as deduced from the thermal fluctuation analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (+359 2) 9793686. Fax: (+359 2) 8723787. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 1768

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ACKNOWLEDGMENTS Financial support from the National Science Fund, Bulgaria (Grant Nos. DFNI B 02/23/2014 and DMU 03-80/2011), is gratefully acknowledged.

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ABBREVIATIONS PC = phosphatidylcholine eggPC = egg-yolk phosphatidylcholine POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine PDPC = 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine SM = egg-yolk sphingomyelin chol = cholesterol LPC = 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine OA = oleic acid DHA = docosahexaenoic acid Lo phase = liquid-ordered phase Ld phase = liquid-disordered phase PLA2 = Phospholipase A2

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DOI: 10.1021/acs.langmuir.5b03317 Langmuir 2016, 32, 1756−1770