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Thermally-induced aggregation and fusion of protein-free lipid vesicles
Colloids and Surfaces B: Biointerfaces 136 (2015) 545–552
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Thermally-induced aggregation and fusion of protein-free lipid vesicles Maitane Ibarguren a,1 , Paul H.H. Bomans b , Kepa Ruiz-Mirazo a,c , Peter M. Frederik b , ˜ a,∗ Alicia Alonso a , Félix M. Goni a
Unidad de Biofísica (Centro Mixto CSIC, UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Apto. 644, 48080 Bilbao, Spain Soft Matter CryoTEM Research Unit, Laboratory for Materials and Interface Chemistry, P.O. Box 513, 5600MB Eindhoven, The Netherlands c Logic and Philosophy of Science Department, University of the Basque Country, Spain b
a r t i c l e
i n f o
Article history: Received 3 June 2015 Received in revised form 21 August 2015 Accepted 24 September 2015 Available online 30 September 2015 Keywords: Fusion Cholesterol Non-lamellar phases Thermal fluctuations
a b s t r a c t Membrane fusion is an important phenomenon in cell biology and pathology. This phenomenon can be modeled using vesicles of defined size and lipid composition. Up to now fusion models typically required the use of chemical (polyethyleneglycol, cations) or enzymatic catalysts (phospholipases). We present here a model of lipid vesicle fusion induced by heat. Large unilamellar vesicles consisting of a phospholipid (dioleoylphosphatidylcholine), cholesterol and diacylglycerol in a 43:57:3 mol ratio were employed. In this simple system, fusion was the result of thermal fluctuations, above 60 ◦ C. A similar system containing phospholipid and cholesterol but no diacylglycerol was observed to aggregate at and above 60 ◦ C, in the absence of fusion. Vesicle fusion occurred under our experimental conditions only when 31 P NMR and cryo-transmission electron microscopy of the lipid mixtures used in vesicle preparation showed nonlamellar lipid phase formation (hexagonal and cubic). Non-lamellar structures are probably the result of lipid reassembly of the products of individual fusion events, or of fusion intermediates. A temperaturetriggered mechanism of lipid reassembly might have occurred at various stages of protocellular evolution. © 2015 Published by Elsevier B.V.
1. Introduction Membrane fusion occurs in cells whenever two vesicles coalesce giving rise to a single compartment. The “vesicles” may be two liposomes, two cells (e.g., sperm and ovum), one virus and one cell (e.g., HIV particle and T-lymphocite), or one intracellular vesicle and the plasma membrane (e.g., in the release of neurotransmitters), as examples of the multitude of membrane fusion events. In spite of the obvious biological interest and of abundant studies, the molecular mechanisms of membrane fusion are not completely understood. One debated point is the relative importance of the roles of lipids and proteins in the fusion event [1]. Several examples are known of fusion in pure lipid vesicles, but in
∗ Corresponding author at: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Spain. Fax: +34 946013360. E-mail addresses: [email protected] (M. Ibarguren), [email protected] (P.H.H. Bomans), [email protected] (K. Ruiz-Mirazo), [email protected] (P.M. Frederik), ˜ [email protected] (A. Alonso), [email protected] (F.M. Goni). 1 Present address: Laboratory of Molecular Cell Biomedicine, University of the Balearic Islands-Lipopharma Therapeutics, S L. Palma, Spain. http://dx.doi.org/10.1016/j.colsurfb.2015.09.047 0927-7765/© 2015 Published by Elsevier B.V.
all cases addition of a chemical or a biological catalyst was required [2–6]. Early [7,8] and more recent [9] studies have shown that polyethyleneglycol induces fusion of pure lipid vesicles by creating an osmotic force that drives membranes into closer contact in a dehydrated region. It is however disputable the extent to which these experiments mimic the physiological conditions in which free water exists. Many efforts have been devoted to characterizing proteins that mediate fusion [10,11]. However, we might not be able to determine the mechanism of those proteins until we understand what happens to two adjacent lipid bilayers to make them fuse. Once the mechanism of fusion in pure lipid systems is understood, the various ways by which proteins catalyze these processes in the cell may be more easily identified. Cholesterol (Chol) is found in all mammalian cells; the human erythrocyte plasma membrane contains as much as 45 mol% Chol [12]. The physiological significance of this molecule is not clear, but many studies show that it alters a number of membrane physicochemical properties [13], and appears to play a direct role in cellular fusion [14]. Chol is found to destabilize phosphatidylethanolamine (PE) and phosphatidylcholine (PC)-PE bilayers, inducing the formation of inverted hexagonal (HII ) phase in these systems [15,16]. It also reduces the amount of diacylglycerol required to induce the lamellar-hexagonal transition [17]. Chol has a paradoxical effect
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in cell membranes: it has the ability to form rigid, low-curvature raft-like patches, while still being able to promote formation of highly-curved, nonlamellar inverted hexagonal and cubic phases [18]. Chol also induces lateral phase separation by forming liquidordered domains separated from regions enriched in unsaturated PCs [19,20]. Control of the extent of lateral phase separation of unsaturated PCs may be one mechanism that cells use to modulate the spatial and temporal occurrence of fusion events [18]. Lipid polymorphism plays an essential role in membrane fusion [21]. Even if lipids in cell membranes adopt the disposition of lamellar phases [22], the mechanism of fusion of two apposed lipid bilayers necessarily requires the formation of a transient nonlamellar intermediate. This intermediate has been called the “stalk” [4,23–29]. Siegel et al. [30] emphasized the linkage between formation of “isotropic phase” and fusion, and proposed a causal relationship between the two. Nieva et al. [31] suggested a bicontinuous inverted cubic (Q224 ) phase structure formed by fusogenic lipid mixtures containing diacylglycerols (DAG). DAG is a powerful agent in restructuring lipid bilayers and cell organelles [32–34]. Low levels of DAG are surprisingly effective at stabilizing inverted phases in phospholipid systems [30,35–38]. DAG stabilizes inverted phases mainly by reducing (making more negative) the spontaneous radius of curvature of the membrane [39–41]. Searching for the simplest fusion model system, in the absence of any added catalyst, we aimed to fuse protein-free unilamellar vesicles of a defined lipid composition by the sole effect of thermal motion. In a recent work, two adjacent vesicles were heated by laser irradiating gold nanoparticles, this caused fusion with associated membrane and cargo mixing [42]. We observed that vesicles composed of a single phospholipid, Chol and DAG (at a 43:57:3 mol ratio) indeed fuse at temperatures above 60 ◦ C in the absence of protein. Chol promotes thermally-induced vesicle aggregation, while DAG is essential in the generation of inverted non-lamellar lipid phases and in the process of membrane fusion.
2. Materials and methods 2.1. Materials 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Chol were purchased from Avanti Polar Lipids (Alabaster, AL). Egg DAG was purchased from Lipid Products (South Nutfield, UK). DAG fatty acid composition was C16:0, 32%; C18:0, 12%; C18:1, 36%; C18:2, 13%; other, 7%. N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) and rhodamine B 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (Rho-PE) were supplied by Molecular Probes, Inc. (Eugene, OR). A kit for measuring cholesterol concentration was supplied by BioSystems (Barcelona, Spain).
2.3. Large unilamellar vesicle (LUV) preparation LUV of diameters 100–150 nm were prepared from MLV by the extrusion method [43] using Nuclepore filters 0.1 m pore diameter at room temperature, in 25 mM HEPES, 150 mM NaCl, pH 7.4. The final phospholipid concentration was measured in terms of lipid phosphorous [44] and the Chol amount was assayed with a commercial kit based on three coupled reactions using Chol esterase, Chol oxidase and peroxidase. When Chol and phospholipid were initially mixed at a 2:1 mol ratio, the bilayer composition after LUV formation was 57 ± 6.4 Chol and 43 ± 4.1 phospholipid. Note that, as shown by Ibarguren et al. [45] final Chol concentrations in extruded LUV may be different from the ones in the starting mixture. Throughout this paper the 43:57 DOPC:Chol mol ratio refers to measurements after LUV formation. Addition of up to 3 mol% DAG to the 43:57 mixture did not change the effective phospholipid:Chol ratio. 2.4. Thermal treatment, aggregation and lipid mixing measurements A stock of buffer was pre-incubated at the desired temperature in a water bath. Then 550 l buffer were added to a cuvette in a thermostatted setting and again left to equilibrate until the selected temperature was reached and stabilized. LUVs (≈100 l, final concentration 0.3 mM) were added at time 0 to the buffer. Addition of this LUV volume did not cause any detectable change in the temperature of the system. For all thermal treatments the spectroscopic cuvettes were covered by a lid. No measurable evaporation occurred during the experiments. Liposome aggregation was estimated as an increase in turbidity (absorbance at 400 nm) [46], measured as a function of time in a Uvikon 922 (Kontron Instruments, Milan, Italy). Lipid mixing was assayed by the resonance energy transfer method [47] using NBD-PE and Rho-PE. Vesicles containing 2% NBD-PE and 2% Rho-PE in their bilayer composition were mixed with probe-free liposomes at a 1:4 ratio. NBD emission was followed at 530 nm (excitation wavelength at 465 nm with a cut-off filter at 515 nm). 100% mixing was set after addition of 1 mM Triton X-100. Lipid bilayer fusion was assayed as mixing of inner monolayer lipids as described by Montes et al. [48]. For this assay vesicles containing 2% NBD-PE and 2% Rho-PE are briefly treated with sodium dithionite, a reagent that does not penetrate the membrane and that bleaches the fluorescence of the probes located in the outer monolayers. The resulting vesicles, containing fluorescent probes only in the inner monolayers are mixed with probe-free liposomes at 1:4 ratio and lipid mixing is monitored following standard procedures [47]. Fluorescence measurements were performed in an Aminco Bowman Series 2 luminescence spectrometer. 2.5. Vesicle contents release and leakage mechanism
2.2. Multilamellar vesicle (MLV) preparation For MLV preparation the lipids were dissolved in chloroform/methanol (2:1) and mixed in the required proportions, and the solvent was evaporated to dryness under a stream of nitrogen. Traces of solvent were removed by evacuating the samples under high vacuum for at least 2 h. The dry lipids were hydrated in 25 mM HEPES, 150 mM NaCl, pH 7.4 helping dispersion by stirring with a glass rod. The lipid concentration of the dispersions was 10% wt/wt. To ensure homogeneous dispersion the hydrated samples were pushed between two syringes through a narrow tubing (0.5 mm internal diameter, 10 cm long) 100 times at 45 ◦ C.
Vesicle efflux was measured with the ANTS:DPX system as in [47,48]. A series of measurements was carried out to find out whether partial leakage occurs as a result of an all-or-none event (some of the vesicles release all of their contents) or as a graded event (all of the vesicles release part of their contents). Briefly, a quench curve for vesicles containing varying concentrations of ANTS and DPX, always at a 3.6/1 mole ratio, was constructed. Then the extent of leakage induced by thermal fluctuations was measured at various temperatures, keeping constant lipid concentration. Samples were applied to a Sephadex G-75 column to separate the lipid vesicles; fluorescence of the eluted vesicle fraction was measured before and after detergent solubilization, and
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Fig. 1. Aggregation of vesicles composed of DOPC:Chol (43:57 mol ratio) at various temperatures, as indicated. In one case (dotted line), a mixture DOPC:Chol (60:40) was used at 90 ◦ C. Inset: Aggregation rates of vesicles composed of DOPC:Chol (43:57 mol ratio).
the percent fluorescence remaining within the vesicles calculated. Experimental values can be compared with predicted results. 2.6. Nuclear magnetic resonance 10 mM lipid in the form of MLV was transferred to 5 mm NMR tubes. Data acquisition was performed in a Bruker AV500 spectrometer (Rheinstetten, Germany) operating at 202.4 MHz for 31 P, with a 5 mm wide-band probe and a gradient in the Z-axis, at 25 ◦ C. The experiments were performed with the zgig sequence (Bruker) and a delay time of 15 s between scans. Data were processed with 1 Hz exponential factor. 2.7. Cryo-transmission electron microscopy (cryo-TEM) Samples were prepared under controlled conditions (99% humidity) in a Vitrobot (Eindhoven, The Netherlands). 3 l of a suspension containing 10 mg lipid/ml were applied on a grid. After careful spreading of the drop, excess liquid was immediately blotted with filter paper. A 3 l droplet creates a cylinder of about 0.5 mm thick on a grid and by blotting away excess liquid this cylinder is reduced to a layer of about 100 nm. After blotting, the sample was immediately plunged into liquid ethane held at its freezing point [49]. The specimen was transferred to the cryoholder of a microscope. After a thermal equilibration period (20 min), images were taken at 100 kV and low-dose conditions in a Philips CM 12 microscope (Philips, Eindhoven, The Netherlands). 3. Results and discussion 3.1. Vesicle aggregation Large unilamellar vesicles (LUV) consisting of DOPC:Chol were used in this study. Preliminary experiments were designed to find out the DOPC:Chol mixture with the minimum Chol proportion that would allow vesicle aggregation upon heating. It was found that a 43:57 DOPC:Chol mol ratio satisfied our requirements. Heat was applied to the suspension and vesicle aggregation was followed as an increase in suspension turbidity (A400 ) [50], at each temperature. As shown in Fig. 1, vesicle aggregation is observed at and
above 60 ◦ C. Bilayers containing less than 57% Chol (see the example of 40% Chol in Fig. 1, dotted line) did not undergo aggregation under our conditions. At and above 70 ◦ C the turbidity curves show a maximum, followed by a steady decrease. This is an artifact due to the formation of large size aggregates that do not conform to the Rayleigh condition of the particles being smaller than the incident light wavelength [46,50]. Equimolar mixtures of Chol and either 1palmitoyl-2-oleoyl, or 1-palmitoyl-2-arachidonoyl, or dilinoleoyl phosphatidylcholine failed to aggregate under the same conditions. 3.2. Lipid vesicle fusion Demonstration of lipid vesicle fusion usually requires the observation of both total lipid mixing and inner monolayer lipid mixing. Intervesicular lipid mixing alone does not prove lipid vesicle fusion, since lipid mixing can also be observed in the so-called “hemifusion” phenomenon [4] also known as “close apposition” [46,51,52]. In the latter, the lipid of outer monolayers of two adjoining vesicle membranes mix, however no fusion pore is formed. To overcome this problem, a technique that allows the direct observation of mixing of inner monolayer lipids can be applied [48]. Detection of intervesicular mixing of inner monolayer lipids occurs only when a fusion pore is opened, so it is a suitable method for observation of real vesicle fusion. The procedure is based on a fluorescence resonance energy transfer (FRET) technique; in this case one population of vesicles is labeled with Rho-PE/NBD-PE in the inner monolayer while the other population is not labeled. Once the inner-monolayer fusion has occurred, the fluorescence of NBD increases. The increased temperature of vesicles containing high amounts of Chol causes vesicle aggregation, but it does not induce lipid mixing at the temperature range between 20 and 90 ◦ C (Fig. 2, curves marked “no DAG”). Thus, Chol is not sufficient to produce thermally induced lipid bilayer fusion under our conditions. Small amounts of alkanes in phospholipid mixtures facilitate the formation of HII phase in lipid-water systems [36,40] by filling the interstices between cylinders so that the hydrocarbon chain packing stress decreases [53]. Low doses of hexadecane were found to increase lipid vesicle fusion induced by phospholipase C [38]. In this study, hexadecane was used to relieve hydrocarbon packing
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Fig. 3. (A) Total and (B) inner lipid mixing of LUVs composed of DOPC:Chol (43:57 mol ratio) + increasing amounts of DAG.
Fig. 2. Aggregation, total and inner lipid mixing at 70 ◦ C of LUVs composed of DOPC:Chol (43:57 mol ratio). Effect of 3 mol% DAG.
stress and promote HII phase transition. However, even in the presence of 15 mol% of hexadecane no lipid mixing (fusion) event in the mentioned lipid mixture was detected in the temperature range between 40 and 90 ◦ C (results not shown). DAG is a lipid with a truncated-cone shape that lowers the transition temperature of lamellar to non-lamellar phases [54] since it reduces the spontaneous radius of curvature of the host lipid system. The presence of only 3 mol% DAG causes extensive hemifusion and fusion in the DOPC:Chol system (Fig. 3A and B) without causing large changes in aggregation when the system is heated up (Fig. 2A). However, even in the presence of 3 mol% DAG, fusion was not observed below 70 ◦ C (Fig. S1), probably because vesicle aggregation is a pre-requisite for fusion to occur. In order to investigate the minimum concentration of DAG that produces a fusion event, liposomes containing 1, 2 or 3 mol% DAG were examined under the same conditions as in Fig. 1. 1 or 2 mol% DAG are not enough to produce either total lipid mixing or inner monolayer lipid mixing (Fig 3). It is known that DAG is a “fusogenic” lipid in cells and in enzyme-induced fusion [31,55,56]. It lowers the curvature radius
of the membrane and induces the formation of the “stalk” fusion intermediate [26,57]. Note the lag time of about 50 s in the “inner” as compared to total lipid mixing time courses (Fig. 2B and C). The lag period corresponds probably to the “hemifusion” stage. DAG by itself does not appear to promote aggregation or fusion, at least a mixture of DOPC:DAG (97: 3 mol ratio) was inactive in this respect (results not shown). The strict requirement of certain cholesterol and DAG concentrations for fusion to be observed is remarkable, and calls for the construction of phase diagrams of aqueous DOPC:Chol:DAG mixtures at different temperatures, a somewhat tedious endeavor but one that will clarify important aspects of lipid bilayer fusion and subsequent (or concomitant) reassembly of lipid structures. 3.3. Release of vesicular aqueous contents Release of vesicular aqueous contents, or vesicle leakage is frequently seen as a side effect in model membrane fusion [58]. Leakage requires the breakdown of the liposomal permeability barrier, and presumably non-lamellar phase formation may be involved. When LUV containing entrapped water-soluble fluorescent probes become permeable, the probes diffuse to the outer aqueous medium and this often leads to fluorescence changes that can be readily detected. In this study, LUVs were loaded with the fluorescence emitter/quencher couple ANTS/DPX. Upon release from the LUV, the complex dissociated and ANTS fluorescence was detected. As shown in Fig. 4, above 50 ◦ C vesicles with a high content of Chol undergo leakage of intravesicular contents. The amount of Chol was again a key parameter, since leakage did not happen when Chol was less than 57 mol%. Note that the presence of 3 mol%
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Fig. 4. Thermally induced release of aqueous contents from vesicles. (A) Vesicles composed of DOPC:Chol (43:57 mol ratio), effect of temperature (B) leakage observed at 60 ◦ C in vesicles in the presence or absence of 3 mol% DAG.
DAG, which facilitates lipid vesicle fusion, also appears to enhance the rate of release of vesicle contents (Fig. 4). Parente et al. [59] distinguished between two putative leakage mechanisms, namely the “all-or-none” and the “graded release” mechanisms (Fig. S2). Our results clearly suggest that the leakage produced by thermal fluctuations is a “graded release” in the mixture DOPC:Chol (Fig. 5A), i.e., intravesicular aqueous contents are “gradually” released from most or all the vesicles as the temperature increases. However, the mechanism of release of aqueous contents is different in the presence of 3 mol% DAG. In this case it is an all-or-none type leakage (Fig. 5B), meaning that the vesicles release all of their contents once they are heated above 50 ◦ C (i.e., under fusogenic conditions). Studies of vesicle fusion induced by peptides or proteins include cases of both leaky and non-leaky fusion (e.g., Refs. [58,60–62]) indicating that both may occur in nature. In the context of the present study, it would be interesting to design and perform experiments in which vesicle fusion was induced by a combination of thermal treatment and presence of peptides/proteins. A complex system should be expected to occur, in which protein folding-unfolding equilibria and lipid phenomena would be intertwined. However the complexity might also reveal novel aspects of membrane fusion and/or evolution. 3.4.
31 P
NMR studies
To explore the possible formation of non-lamellar phases temperature 31 P-nuclear magnetic resonance (31 P NMR) was used. This technique reveals that, for the DOPC:Chol mixture, at 55 ◦ C an isotropic signal starts to appear and by 75 ◦ C the signal is fully isotropic (Fig. S3 A). The onset of the isotropic signal (55 ◦ C) corresponds to the beginning of LUV aggregation. It is known that Chol increases the negative spontaneous curvature of DOPC [63], a trend that favors inverted phase formation. Tenchov et al. [18] observed
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Fig. 5. Leakage mechanism of the mixture (A) DOPC:Chol (43:57 mol ratio) and (B) DOPC:Chol (43:57 mol ratio) + 3 mol% DAG.
extensive formation of cubic phases at 65 ◦ C with an equimolar DOPC:Chol mixture, a phenomenon that is probably related to our observations. Perhaps under their conditions the lamellar-cubic phase formation was facilitated by the relatively high lipid concentration in their samples, about 12-fold the one used in our NMR studiesThe fluorescence results of lipid mixing show that, in the presence of DAG, fusion occurs at and above ≈60 ◦ C (Fig. 2). The NMR experiments show an isotropic signal in the temperature range between 55 and 75 ◦ C (Fig. S3 B). This isotropic peak reveals the presence of an inverted phase. It is also important to mention that there is another peak, in the same temperature range, that corresponds to a HII phase. The HII signal appears more clearly at the higher temperatures. Tenchov et al. [18] were also able to observe a HII phase by heating an equimolar DOPC:Chol mixture. The system containing DAG behaves in a similar way than pure DOPE-Me, which exists in Lo below 20 ◦ C, in HII above 75 ◦ C and displays an isotropic signal in 31 P NMR between 20 and 75 ◦ C [64]. (Note that the relatively poor signal-to-noise ratio of the spectra containing DAG is due to sample inhomogeneities associated to the tendency of these vesicles to float). 3.5. Cryo-microscopy of vesicles composed of DOPC:Chol or DOPC:Chol +3% DAG mixtures Cryotransmision electron microscopy (Cryo-TEM) is a powerful technique used to obtain images that are two-dimensional projections of particles in suspension without the use of chemical reagents for sample fixation. Thus, this technique was suitable to follow morphological changes leading to vesicle aggregation, and eventually fusion in mixtures of DOPC:Chol and DOPC:Chol + 3 mol% DAG. Fig. S4 shows cryo-TEM micrographs corresponding to LUVs composed of DOPC:Chol (43:57 mol ratio) at room temperature and at 70 ◦ C. Liposomes incubated at RT are 121 ± 13 nm in diameter (Fig. S4 A). The lipid mixture heated at
550
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Fig. 6. Cryo-TEM of DOPC:Chol LUVs (43:57 mol ratio) + 3 mol% DAG (A) room temperature, (B) 70 ◦ C, and (C) after overnight incubation at 80 ◦ C. Bar = 100 nm.´ı
70 ◦ C (Fig. S4 B) shows essentially the same structures, except that occasionally (<5% of the vesicles) some non-lamellar elements are seen (Fig. S4 B, inset). Samples containing 3 mol% DAG are shown in Fig. 6. The cryo-TEM micrographs correspond to LUVs composed of DOPC:Chol:DAG (43:57:3) at room temperature and at 70 ◦ C. Liposomes incubated at RT are ≈133 ± 30 nm in diameter, some of them being oligolamellar (Fig. 6A). At 70 ◦ C they give rise to huge aggregates with areas of HII precursors and extensive regions of QII phases (Fig. 6B). Samples incubated at 80 ◦ C for 24 h exhibit the tubular structures that are characteristic of the HII phase (Fig. 6C). These data should be interpreted in the light of the studies by Nieva et al. [31], who found a correlation between liposomal fusion induced by phospholipase C and the formation of Q224 and HII phases by the lipid mixture. We suggest, in agreement with other authors [6,26,65–67], that the products of individual fusion events assemble into QII phase lattices, i.e., that fusion is an obligatory step in the L/QII phase transition, and that DAG, which allows the formation of vast amounts of Q phase upon T-cycling, is also able to induce the localized and transient formation of cubic phase precursors during a single heating run, in the conditions under which fusion is observed (Fig. 2). Note however that Yang and Huang [68] have shown structural evidence for the formation of a “stalk”-like shape in partially hydrated diphytanoyl phosphatidylcholine, under conditions in which an orthorhombic phase is formed. Moreover, Aeffner et al. [69] found rhombohedral phases in DOPC:Chol (6:4 mol ratio) at 22 ◦ C, and relative humidity 70%. Thus the existence, under physiological conditions, of fusion intermediates leading to non-lamellar phases other than inverted hexagonal/cubic should also be considered.
lipid bilayer and trigger the formation of the fusion intermediate (stalk). The 43:57 molar mixture DOPC:Chol allows vesicle aggregation, but fusion is not observed. DAG however acts, together with Chol, as a powerful “fusogenic” lipid. Cryo-TEM shows formation of inverted phases precisely under the conditions leading to lipid vesicle fusion. The mixture DOPC:Chol + 3 mol% DAG forms an inverted cubic phase at ≈65 ◦ C, and the same mixture leads to massive vesicle aggregation and fusion. In the absence of Chol however, DAG is not able to induce inverted cubic phases, nor vesicle aggregation or fusion. In our system fusion was observed at temperatures above the mammalian physiological temperature, but that does not exclude the possibility that other Chol- and PCrich membranes, perhaps with low levels of other lipids, could give rise to fusogenic mixtures at lower temperatures. In any case, a temperature-triggered mechanism of lipid vesicle fusion might have played an important role at various stages of cellular evolution, all the way back to the origin of the first cells. At those ancient times Chol is not supposed to have been present in biological membranes yet, but other compounds, like polycyclic aromatic hydrocarbons could have provided precursor bio-membranes with similar properties [73].
4. Concluding remarks
Appendix A. Supplementary data
Lipid bilayer fusion has been observed under the simplest conditions, namely in a protein-free system, using unilamellar vesicles of a defined lipid composition. Purely lipidic vesicles were only very recently reported to fuse, induced by mechanical force stress in viscous solutions [70]. In our system fusion occurred as a result of mere thermal motion in a buffer, in the absence of any added chemical or biological catalyst. Fluid bilayers are known to expand with temperature [71,72] and this leads to the opening of the hydrophobic core in defect areas. This is probably an important factor in overcoming the energy barrier for bilayer fusion. It has been demonstrated that Chol plays an important role in lipid bilayer fusion but it is not always sufficient to destabilize the
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.09. 047.
Acknowledgements The authors are indebted to Dr. D.P. Siegel and Dr. B. Tenchov for many useful discussions. M.I. was a pre-doctoral fellow of the Basque Government. This work was supported in part by funds from the Spanish Ministerio de Ciencia e Innovación [grants No. BFU2011-28566) (A.A.) and grant No BFU2012-36241 (F.M.G.)] and from the Basque Government [(grants No. IT-849-13) (F.M.G.). and No. IT838-13 (A.A.)]
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Thermally-induced aggregation and fusion of protein-free lipid vesicles Maitane Ibarguren a,1 , Paul H.H. Bomans b , Kepa Ruiz-Mirazo a,c , Peter M. Frederik b , ˜ a,∗ Alicia Alonso a , Félix M. Goni a
Unidad de Biofísica (Centro Mixto CSIC, UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Apto. 644, 48080 Bilbao, Spain Soft Matter CryoTEM Research Unit, Laboratory for Materials and Interface Chemistry, P.O. Box 513, 5600MB Eindhoven, The Netherlands c Logic and Philosophy of Science Department, University of the Basque Country, Spain b
a r t i c l e
i n f o
Article history: Received 3 June 2015 Received in revised form 21 August 2015 Accepted 24 September 2015 Available online 30 September 2015 Keywords: Fusion Cholesterol Non-lamellar phases Thermal fluctuations
a b s t r a c t Membrane fusion is an important phenomenon in cell biology and pathology. This phenomenon can be modeled using vesicles of defined size and lipid composition. Up to now fusion models typically required the use of chemical (polyethyleneglycol, cations) or enzymatic catalysts (phospholipases). We present here a model of lipid vesicle fusion induced by heat. Large unilamellar vesicles consisting of a phospholipid (dioleoylphosphatidylcholine), cholesterol and diacylglycerol in a 43:57:3 mol ratio were employed. In this simple system, fusion was the result of thermal fluctuations, above 60 ◦ C. A similar system containing phospholipid and cholesterol but no diacylglycerol was observed to aggregate at and above 60 ◦ C, in the absence of fusion. Vesicle fusion occurred under our experimental conditions only when 31 P NMR and cryo-transmission electron microscopy of the lipid mixtures used in vesicle preparation showed nonlamellar lipid phase formation (hexagonal and cubic). Non-lamellar structures are probably the result of lipid reassembly of the products of individual fusion events, or of fusion intermediates. A temperaturetriggered mechanism of lipid reassembly might have occurred at various stages of protocellular evolution. © 2015 Published by Elsevier B.V.
1. Introduction Membrane fusion occurs in cells whenever two vesicles coalesce giving rise to a single compartment. The “vesicles” may be two liposomes, two cells (e.g., sperm and ovum), one virus and one cell (e.g., HIV particle and T-lymphocite), or one intracellular vesicle and the plasma membrane (e.g., in the release of neurotransmitters), as examples of the multitude of membrane fusion events. In spite of the obvious biological interest and of abundant studies, the molecular mechanisms of membrane fusion are not completely understood. One debated point is the relative importance of the roles of lipids and proteins in the fusion event [1]. Several examples are known of fusion in pure lipid vesicles, but in
∗ Corresponding author at: Unidad de Biofísica (CSIC, UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Spain. Fax: +34 946013360. E-mail addresses: [email protected] (M. Ibarguren), [email protected] (P.H.H. Bomans), [email protected] (K. Ruiz-Mirazo), [email protected] (P.M. Frederik), ˜ [email protected] (A. Alonso), [email protected] (F.M. Goni). 1 Present address: Laboratory of Molecular Cell Biomedicine, University of the Balearic Islands-Lipopharma Therapeutics, S L. Palma, Spain. http://dx.doi.org/10.1016/j.colsurfb.2015.09.047 0927-7765/© 2015 Published by Elsevier B.V.
all cases addition of a chemical or a biological catalyst was required [2–6]. Early [7,8] and more recent [9] studies have shown that polyethyleneglycol induces fusion of pure lipid vesicles by creating an osmotic force that drives membranes into closer contact in a dehydrated region. It is however disputable the extent to which these experiments mimic the physiological conditions in which free water exists. Many efforts have been devoted to characterizing proteins that mediate fusion [10,11]. However, we might not be able to determine the mechanism of those proteins until we understand what happens to two adjacent lipid bilayers to make them fuse. Once the mechanism of fusion in pure lipid systems is understood, the various ways by which proteins catalyze these processes in the cell may be more easily identified. Cholesterol (Chol) is found in all mammalian cells; the human erythrocyte plasma membrane contains as much as 45 mol% Chol [12]. The physiological significance of this molecule is not clear, but many studies show that it alters a number of membrane physicochemical properties [13], and appears to play a direct role in cellular fusion [14]. Chol is found to destabilize phosphatidylethanolamine (PE) and phosphatidylcholine (PC)-PE bilayers, inducing the formation of inverted hexagonal (HII ) phase in these systems [15,16]. It also reduces the amount of diacylglycerol required to induce the lamellar-hexagonal transition [17]. Chol has a paradoxical effect
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in cell membranes: it has the ability to form rigid, low-curvature raft-like patches, while still being able to promote formation of highly-curved, nonlamellar inverted hexagonal and cubic phases [18]. Chol also induces lateral phase separation by forming liquidordered domains separated from regions enriched in unsaturated PCs [19,20]. Control of the extent of lateral phase separation of unsaturated PCs may be one mechanism that cells use to modulate the spatial and temporal occurrence of fusion events [18]. Lipid polymorphism plays an essential role in membrane fusion [21]. Even if lipids in cell membranes adopt the disposition of lamellar phases [22], the mechanism of fusion of two apposed lipid bilayers necessarily requires the formation of a transient nonlamellar intermediate. This intermediate has been called the “stalk” [4,23–29]. Siegel et al. [30] emphasized the linkage between formation of “isotropic phase” and fusion, and proposed a causal relationship between the two. Nieva et al. [31] suggested a bicontinuous inverted cubic (Q224 ) phase structure formed by fusogenic lipid mixtures containing diacylglycerols (DAG). DAG is a powerful agent in restructuring lipid bilayers and cell organelles [32–34]. Low levels of DAG are surprisingly effective at stabilizing inverted phases in phospholipid systems [30,35–38]. DAG stabilizes inverted phases mainly by reducing (making more negative) the spontaneous radius of curvature of the membrane [39–41]. Searching for the simplest fusion model system, in the absence of any added catalyst, we aimed to fuse protein-free unilamellar vesicles of a defined lipid composition by the sole effect of thermal motion. In a recent work, two adjacent vesicles were heated by laser irradiating gold nanoparticles, this caused fusion with associated membrane and cargo mixing [42]. We observed that vesicles composed of a single phospholipid, Chol and DAG (at a 43:57:3 mol ratio) indeed fuse at temperatures above 60 ◦ C in the absence of protein. Chol promotes thermally-induced vesicle aggregation, while DAG is essential in the generation of inverted non-lamellar lipid phases and in the process of membrane fusion.
2. Materials and methods 2.1. Materials 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Chol were purchased from Avanti Polar Lipids (Alabaster, AL). Egg DAG was purchased from Lipid Products (South Nutfield, UK). DAG fatty acid composition was C16:0, 32%; C18:0, 12%; C18:1, 36%; C18:2, 13%; other, 7%. N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) and rhodamine B 1,2-dihexadecanoyl-sn-glycero-3phosphoethanolamine (Rho-PE) were supplied by Molecular Probes, Inc. (Eugene, OR). A kit for measuring cholesterol concentration was supplied by BioSystems (Barcelona, Spain).
2.3. Large unilamellar vesicle (LUV) preparation LUV of diameters 100–150 nm were prepared from MLV by the extrusion method [43] using Nuclepore filters 0.1 m pore diameter at room temperature, in 25 mM HEPES, 150 mM NaCl, pH 7.4. The final phospholipid concentration was measured in terms of lipid phosphorous [44] and the Chol amount was assayed with a commercial kit based on three coupled reactions using Chol esterase, Chol oxidase and peroxidase. When Chol and phospholipid were initially mixed at a 2:1 mol ratio, the bilayer composition after LUV formation was 57 ± 6.4 Chol and 43 ± 4.1 phospholipid. Note that, as shown by Ibarguren et al. [45] final Chol concentrations in extruded LUV may be different from the ones in the starting mixture. Throughout this paper the 43:57 DOPC:Chol mol ratio refers to measurements after LUV formation. Addition of up to 3 mol% DAG to the 43:57 mixture did not change the effective phospholipid:Chol ratio. 2.4. Thermal treatment, aggregation and lipid mixing measurements A stock of buffer was pre-incubated at the desired temperature in a water bath. Then 550 l buffer were added to a cuvette in a thermostatted setting and again left to equilibrate until the selected temperature was reached and stabilized. LUVs (≈100 l, final concentration 0.3 mM) were added at time 0 to the buffer. Addition of this LUV volume did not cause any detectable change in the temperature of the system. For all thermal treatments the spectroscopic cuvettes were covered by a lid. No measurable evaporation occurred during the experiments. Liposome aggregation was estimated as an increase in turbidity (absorbance at 400 nm) [46], measured as a function of time in a Uvikon 922 (Kontron Instruments, Milan, Italy). Lipid mixing was assayed by the resonance energy transfer method [47] using NBD-PE and Rho-PE. Vesicles containing 2% NBD-PE and 2% Rho-PE in their bilayer composition were mixed with probe-free liposomes at a 1:4 ratio. NBD emission was followed at 530 nm (excitation wavelength at 465 nm with a cut-off filter at 515 nm). 100% mixing was set after addition of 1 mM Triton X-100. Lipid bilayer fusion was assayed as mixing of inner monolayer lipids as described by Montes et al. [48]. For this assay vesicles containing 2% NBD-PE and 2% Rho-PE are briefly treated with sodium dithionite, a reagent that does not penetrate the membrane and that bleaches the fluorescence of the probes located in the outer monolayers. The resulting vesicles, containing fluorescent probes only in the inner monolayers are mixed with probe-free liposomes at 1:4 ratio and lipid mixing is monitored following standard procedures [47]. Fluorescence measurements were performed in an Aminco Bowman Series 2 luminescence spectrometer. 2.5. Vesicle contents release and leakage mechanism
2.2. Multilamellar vesicle (MLV) preparation For MLV preparation the lipids were dissolved in chloroform/methanol (2:1) and mixed in the required proportions, and the solvent was evaporated to dryness under a stream of nitrogen. Traces of solvent were removed by evacuating the samples under high vacuum for at least 2 h. The dry lipids were hydrated in 25 mM HEPES, 150 mM NaCl, pH 7.4 helping dispersion by stirring with a glass rod. The lipid concentration of the dispersions was 10% wt/wt. To ensure homogeneous dispersion the hydrated samples were pushed between two syringes through a narrow tubing (0.5 mm internal diameter, 10 cm long) 100 times at 45 ◦ C.
Vesicle efflux was measured with the ANTS:DPX system as in [47,48]. A series of measurements was carried out to find out whether partial leakage occurs as a result of an all-or-none event (some of the vesicles release all of their contents) or as a graded event (all of the vesicles release part of their contents). Briefly, a quench curve for vesicles containing varying concentrations of ANTS and DPX, always at a 3.6/1 mole ratio, was constructed. Then the extent of leakage induced by thermal fluctuations was measured at various temperatures, keeping constant lipid concentration. Samples were applied to a Sephadex G-75 column to separate the lipid vesicles; fluorescence of the eluted vesicle fraction was measured before and after detergent solubilization, and
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Fig. 1. Aggregation of vesicles composed of DOPC:Chol (43:57 mol ratio) at various temperatures, as indicated. In one case (dotted line), a mixture DOPC:Chol (60:40) was used at 90 ◦ C. Inset: Aggregation rates of vesicles composed of DOPC:Chol (43:57 mol ratio).
the percent fluorescence remaining within the vesicles calculated. Experimental values can be compared with predicted results. 2.6. Nuclear magnetic resonance 10 mM lipid in the form of MLV was transferred to 5 mm NMR tubes. Data acquisition was performed in a Bruker AV500 spectrometer (Rheinstetten, Germany) operating at 202.4 MHz for 31 P, with a 5 mm wide-band probe and a gradient in the Z-axis, at 25 ◦ C. The experiments were performed with the zgig sequence (Bruker) and a delay time of 15 s between scans. Data were processed with 1 Hz exponential factor. 2.7. Cryo-transmission electron microscopy (cryo-TEM) Samples were prepared under controlled conditions (99% humidity) in a Vitrobot (Eindhoven, The Netherlands). 3 l of a suspension containing 10 mg lipid/ml were applied on a grid. After careful spreading of the drop, excess liquid was immediately blotted with filter paper. A 3 l droplet creates a cylinder of about 0.5 mm thick on a grid and by blotting away excess liquid this cylinder is reduced to a layer of about 100 nm. After blotting, the sample was immediately plunged into liquid ethane held at its freezing point [49]. The specimen was transferred to the cryoholder of a microscope. After a thermal equilibration period (20 min), images were taken at 100 kV and low-dose conditions in a Philips CM 12 microscope (Philips, Eindhoven, The Netherlands). 3. Results and discussion 3.1. Vesicle aggregation Large unilamellar vesicles (LUV) consisting of DOPC:Chol were used in this study. Preliminary experiments were designed to find out the DOPC:Chol mixture with the minimum Chol proportion that would allow vesicle aggregation upon heating. It was found that a 43:57 DOPC:Chol mol ratio satisfied our requirements. Heat was applied to the suspension and vesicle aggregation was followed as an increase in suspension turbidity (A400 ) [50], at each temperature. As shown in Fig. 1, vesicle aggregation is observed at and
above 60 ◦ C. Bilayers containing less than 57% Chol (see the example of 40% Chol in Fig. 1, dotted line) did not undergo aggregation under our conditions. At and above 70 ◦ C the turbidity curves show a maximum, followed by a steady decrease. This is an artifact due to the formation of large size aggregates that do not conform to the Rayleigh condition of the particles being smaller than the incident light wavelength [46,50]. Equimolar mixtures of Chol and either 1palmitoyl-2-oleoyl, or 1-palmitoyl-2-arachidonoyl, or dilinoleoyl phosphatidylcholine failed to aggregate under the same conditions. 3.2. Lipid vesicle fusion Demonstration of lipid vesicle fusion usually requires the observation of both total lipid mixing and inner monolayer lipid mixing. Intervesicular lipid mixing alone does not prove lipid vesicle fusion, since lipid mixing can also be observed in the so-called “hemifusion” phenomenon [4] also known as “close apposition” [46,51,52]. In the latter, the lipid of outer monolayers of two adjoining vesicle membranes mix, however no fusion pore is formed. To overcome this problem, a technique that allows the direct observation of mixing of inner monolayer lipids can be applied [48]. Detection of intervesicular mixing of inner monolayer lipids occurs only when a fusion pore is opened, so it is a suitable method for observation of real vesicle fusion. The procedure is based on a fluorescence resonance energy transfer (FRET) technique; in this case one population of vesicles is labeled with Rho-PE/NBD-PE in the inner monolayer while the other population is not labeled. Once the inner-monolayer fusion has occurred, the fluorescence of NBD increases. The increased temperature of vesicles containing high amounts of Chol causes vesicle aggregation, but it does not induce lipid mixing at the temperature range between 20 and 90 ◦ C (Fig. 2, curves marked “no DAG”). Thus, Chol is not sufficient to produce thermally induced lipid bilayer fusion under our conditions. Small amounts of alkanes in phospholipid mixtures facilitate the formation of HII phase in lipid-water systems [36,40] by filling the interstices between cylinders so that the hydrocarbon chain packing stress decreases [53]. Low doses of hexadecane were found to increase lipid vesicle fusion induced by phospholipase C [38]. In this study, hexadecane was used to relieve hydrocarbon packing
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Fig. 3. (A) Total and (B) inner lipid mixing of LUVs composed of DOPC:Chol (43:57 mol ratio) + increasing amounts of DAG.
Fig. 2. Aggregation, total and inner lipid mixing at 70 ◦ C of LUVs composed of DOPC:Chol (43:57 mol ratio). Effect of 3 mol% DAG.
stress and promote HII phase transition. However, even in the presence of 15 mol% of hexadecane no lipid mixing (fusion) event in the mentioned lipid mixture was detected in the temperature range between 40 and 90 ◦ C (results not shown). DAG is a lipid with a truncated-cone shape that lowers the transition temperature of lamellar to non-lamellar phases [54] since it reduces the spontaneous radius of curvature of the host lipid system. The presence of only 3 mol% DAG causes extensive hemifusion and fusion in the DOPC:Chol system (Fig. 3A and B) without causing large changes in aggregation when the system is heated up (Fig. 2A). However, even in the presence of 3 mol% DAG, fusion was not observed below 70 ◦ C (Fig. S1), probably because vesicle aggregation is a pre-requisite for fusion to occur. In order to investigate the minimum concentration of DAG that produces a fusion event, liposomes containing 1, 2 or 3 mol% DAG were examined under the same conditions as in Fig. 1. 1 or 2 mol% DAG are not enough to produce either total lipid mixing or inner monolayer lipid mixing (Fig 3). It is known that DAG is a “fusogenic” lipid in cells and in enzyme-induced fusion [31,55,56]. It lowers the curvature radius
of the membrane and induces the formation of the “stalk” fusion intermediate [26,57]. Note the lag time of about 50 s in the “inner” as compared to total lipid mixing time courses (Fig. 2B and C). The lag period corresponds probably to the “hemifusion” stage. DAG by itself does not appear to promote aggregation or fusion, at least a mixture of DOPC:DAG (97: 3 mol ratio) was inactive in this respect (results not shown). The strict requirement of certain cholesterol and DAG concentrations for fusion to be observed is remarkable, and calls for the construction of phase diagrams of aqueous DOPC:Chol:DAG mixtures at different temperatures, a somewhat tedious endeavor but one that will clarify important aspects of lipid bilayer fusion and subsequent (or concomitant) reassembly of lipid structures. 3.3. Release of vesicular aqueous contents Release of vesicular aqueous contents, or vesicle leakage is frequently seen as a side effect in model membrane fusion [58]. Leakage requires the breakdown of the liposomal permeability barrier, and presumably non-lamellar phase formation may be involved. When LUV containing entrapped water-soluble fluorescent probes become permeable, the probes diffuse to the outer aqueous medium and this often leads to fluorescence changes that can be readily detected. In this study, LUVs were loaded with the fluorescence emitter/quencher couple ANTS/DPX. Upon release from the LUV, the complex dissociated and ANTS fluorescence was detected. As shown in Fig. 4, above 50 ◦ C vesicles with a high content of Chol undergo leakage of intravesicular contents. The amount of Chol was again a key parameter, since leakage did not happen when Chol was less than 57 mol%. Note that the presence of 3 mol%
M. Ibarguren et al. / Colloids and Surfaces B: Biointerfaces 136 (2015) 545–552
Fig. 4. Thermally induced release of aqueous contents from vesicles. (A) Vesicles composed of DOPC:Chol (43:57 mol ratio), effect of temperature (B) leakage observed at 60 ◦ C in vesicles in the presence or absence of 3 mol% DAG.
DAG, which facilitates lipid vesicle fusion, also appears to enhance the rate of release of vesicle contents (Fig. 4). Parente et al. [59] distinguished between two putative leakage mechanisms, namely the “all-or-none” and the “graded release” mechanisms (Fig. S2). Our results clearly suggest that the leakage produced by thermal fluctuations is a “graded release” in the mixture DOPC:Chol (Fig. 5A), i.e., intravesicular aqueous contents are “gradually” released from most or all the vesicles as the temperature increases. However, the mechanism of release of aqueous contents is different in the presence of 3 mol% DAG. In this case it is an all-or-none type leakage (Fig. 5B), meaning that the vesicles release all of their contents once they are heated above 50 ◦ C (i.e., under fusogenic conditions). Studies of vesicle fusion induced by peptides or proteins include cases of both leaky and non-leaky fusion (e.g., Refs. [58,60–62]) indicating that both may occur in nature. In the context of the present study, it would be interesting to design and perform experiments in which vesicle fusion was induced by a combination of thermal treatment and presence of peptides/proteins. A complex system should be expected to occur, in which protein folding-unfolding equilibria and lipid phenomena would be intertwined. However the complexity might also reveal novel aspects of membrane fusion and/or evolution. 3.4.
31 P
NMR studies
To explore the possible formation of non-lamellar phases temperature 31 P-nuclear magnetic resonance (31 P NMR) was used. This technique reveals that, for the DOPC:Chol mixture, at 55 ◦ C an isotropic signal starts to appear and by 75 ◦ C the signal is fully isotropic (Fig. S3 A). The onset of the isotropic signal (55 ◦ C) corresponds to the beginning of LUV aggregation. It is known that Chol increases the negative spontaneous curvature of DOPC [63], a trend that favors inverted phase formation. Tenchov et al. [18] observed
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Fig. 5. Leakage mechanism of the mixture (A) DOPC:Chol (43:57 mol ratio) and (B) DOPC:Chol (43:57 mol ratio) + 3 mol% DAG.
extensive formation of cubic phases at 65 ◦ C with an equimolar DOPC:Chol mixture, a phenomenon that is probably related to our observations. Perhaps under their conditions the lamellar-cubic phase formation was facilitated by the relatively high lipid concentration in their samples, about 12-fold the one used in our NMR studiesThe fluorescence results of lipid mixing show that, in the presence of DAG, fusion occurs at and above ≈60 ◦ C (Fig. 2). The NMR experiments show an isotropic signal in the temperature range between 55 and 75 ◦ C (Fig. S3 B). This isotropic peak reveals the presence of an inverted phase. It is also important to mention that there is another peak, in the same temperature range, that corresponds to a HII phase. The HII signal appears more clearly at the higher temperatures. Tenchov et al. [18] were also able to observe a HII phase by heating an equimolar DOPC:Chol mixture. The system containing DAG behaves in a similar way than pure DOPE-Me, which exists in Lo below 20 ◦ C, in HII above 75 ◦ C and displays an isotropic signal in 31 P NMR between 20 and 75 ◦ C [64]. (Note that the relatively poor signal-to-noise ratio of the spectra containing DAG is due to sample inhomogeneities associated to the tendency of these vesicles to float). 3.5. Cryo-microscopy of vesicles composed of DOPC:Chol or DOPC:Chol +3% DAG mixtures Cryotransmision electron microscopy (Cryo-TEM) is a powerful technique used to obtain images that are two-dimensional projections of particles in suspension without the use of chemical reagents for sample fixation. Thus, this technique was suitable to follow morphological changes leading to vesicle aggregation, and eventually fusion in mixtures of DOPC:Chol and DOPC:Chol + 3 mol% DAG. Fig. S4 shows cryo-TEM micrographs corresponding to LUVs composed of DOPC:Chol (43:57 mol ratio) at room temperature and at 70 ◦ C. Liposomes incubated at RT are 121 ± 13 nm in diameter (Fig. S4 A). The lipid mixture heated at
550
M. Ibarguren et al. / Colloids and Surfaces B: Biointerfaces 136 (2015) 545–552
Fig. 6. Cryo-TEM of DOPC:Chol LUVs (43:57 mol ratio) + 3 mol% DAG (A) room temperature, (B) 70 ◦ C, and (C) after overnight incubation at 80 ◦ C. Bar = 100 nm.´ı
70 ◦ C (Fig. S4 B) shows essentially the same structures, except that occasionally (<5% of the vesicles) some non-lamellar elements are seen (Fig. S4 B, inset). Samples containing 3 mol% DAG are shown in Fig. 6. The cryo-TEM micrographs correspond to LUVs composed of DOPC:Chol:DAG (43:57:3) at room temperature and at 70 ◦ C. Liposomes incubated at RT are ≈133 ± 30 nm in diameter, some of them being oligolamellar (Fig. 6A). At 70 ◦ C they give rise to huge aggregates with areas of HII precursors and extensive regions of QII phases (Fig. 6B). Samples incubated at 80 ◦ C for 24 h exhibit the tubular structures that are characteristic of the HII phase (Fig. 6C). These data should be interpreted in the light of the studies by Nieva et al. [31], who found a correlation between liposomal fusion induced by phospholipase C and the formation of Q224 and HII phases by the lipid mixture. We suggest, in agreement with other authors [6,26,65–67], that the products of individual fusion events assemble into QII phase lattices, i.e., that fusion is an obligatory step in the L/QII phase transition, and that DAG, which allows the formation of vast amounts of Q phase upon T-cycling, is also able to induce the localized and transient formation of cubic phase precursors during a single heating run, in the conditions under which fusion is observed (Fig. 2). Note however that Yang and Huang [68] have shown structural evidence for the formation of a “stalk”-like shape in partially hydrated diphytanoyl phosphatidylcholine, under conditions in which an orthorhombic phase is formed. Moreover, Aeffner et al. [69] found rhombohedral phases in DOPC:Chol (6:4 mol ratio) at 22 ◦ C, and relative humidity 70%. Thus the existence, under physiological conditions, of fusion intermediates leading to non-lamellar phases other than inverted hexagonal/cubic should also be considered.
lipid bilayer and trigger the formation of the fusion intermediate (stalk). The 43:57 molar mixture DOPC:Chol allows vesicle aggregation, but fusion is not observed. DAG however acts, together with Chol, as a powerful “fusogenic” lipid. Cryo-TEM shows formation of inverted phases precisely under the conditions leading to lipid vesicle fusion. The mixture DOPC:Chol + 3 mol% DAG forms an inverted cubic phase at ≈65 ◦ C, and the same mixture leads to massive vesicle aggregation and fusion. In the absence of Chol however, DAG is not able to induce inverted cubic phases, nor vesicle aggregation or fusion. In our system fusion was observed at temperatures above the mammalian physiological temperature, but that does not exclude the possibility that other Chol- and PCrich membranes, perhaps with low levels of other lipids, could give rise to fusogenic mixtures at lower temperatures. In any case, a temperature-triggered mechanism of lipid vesicle fusion might have played an important role at various stages of cellular evolution, all the way back to the origin of the first cells. At those ancient times Chol is not supposed to have been present in biological membranes yet, but other compounds, like polycyclic aromatic hydrocarbons could have provided precursor bio-membranes with similar properties [73].
4. Concluding remarks
Appendix A. Supplementary data
Lipid bilayer fusion has been observed under the simplest conditions, namely in a protein-free system, using unilamellar vesicles of a defined lipid composition. Purely lipidic vesicles were only very recently reported to fuse, induced by mechanical force stress in viscous solutions [70]. In our system fusion occurred as a result of mere thermal motion in a buffer, in the absence of any added chemical or biological catalyst. Fluid bilayers are known to expand with temperature [71,72] and this leads to the opening of the hydrophobic core in defect areas. This is probably an important factor in overcoming the energy barrier for bilayer fusion. It has been demonstrated that Chol plays an important role in lipid bilayer fusion but it is not always sufficient to destabilize the
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.09. 047.
Acknowledgements The authors are indebted to Dr. D.P. Siegel and Dr. B. Tenchov for many useful discussions. M.I. was a pre-doctoral fellow of the Basque Government. This work was supported in part by funds from the Spanish Ministerio de Ciencia e Innovación [grants No. BFU2011-28566) (A.A.) and grant No BFU2012-36241 (F.M.G.)] and from the Basque Government [(grants No. IT-849-13) (F.M.G.). and No. IT838-13 (A.A.)]
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