Effect of Triton X-100 on Raft-Like Lipid Mixtures: Phase Separation

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Effect of Triton X-100 on raft-like lipid mixtures: phase separation and selective solubilization Amanda C Caritá, Bruno Mattei, Cleyton C Domingues, Eneida de Paula, and Karin A. Riske Langmuir, Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 5, 2017

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Effect of Triton X-100 on raft-like lipid mixtures: phase separation and selective solubilization Amanda C. Caritá1, Bruno Mattei1,§, Cleyton C. Domingues2,†, Eneida de Paula2, Karin A. Riske1,* 1

2

Departamento de Biofísica, Universidade Federal de São Paulo, Sao Paulo, Brazil Departamento de Bioquímica e Biologia Tecidual, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil

* Corresponding author [email protected] §

Present Address: Núcleo de Pesquisas em Ciências Biológicas, Universidade Federal de Ouro Preto, Ouro Preto – MG, Brazil. † Present Address: Department of Medicine, The George Washington University, Washington, DC, USA.

Abstract Under certain conditions, biological membranes exhibit resistance to solubilization, even at high detergent concentration. These insoluble fragments are enriched in sphingolipids, cholesterol and certain proteins having a preference for more organized environments. Here we investigated the effect of the detergent Triton X-100 (TX-100) on raft-like lipid mixtures composed of POPC (palmitoyl oleoyl phosphatidylcholine, an unsaturated lipid), SM (sphingomyelin, a saturated lipid) and cholesterol, focusing the detergent-induced phase separation at sub-solubilizing concentration and the extent of solubilization at higher concentration. Giant unilamellar vesicles (GUVs) of POPC:SM:chol containing a fluorescent probe known to prefer the liquid-disordered phase were prepared and observed with fluorescence microscopy. A phase diagram constructed in the presence and absence of 0.1 mM TX-100 showed that the detergent induces macroscopic liquid-ordered/liquid-disordered (Lo/Ld) phase separation in a wide range of membrane composition, indicating that TX-100 has the ability to rearrange the lateral heterogeneity of the lipid mixture. The extent of solubilization of the POPC:SM:chol GUVs was quantified by measuring the vesicle size before and after injection of a high concentration of TX-100. In parallel, the solubilization extent of large unilamellar vesicles (LUVs) was assessed by turbidity measurements. The extent of solubilization decreases significantly as the fractions of SM and cholesterol in the mixture increase. The origin of the detergent resistance is the low partition of TX-100 in cholesterol-rich membranes, especially in SM-containing ones, as evidenced by isothermal titration calorimetry experiments on LUVs. Our results provide a guide to future research on the effects of TX-100 on raft-like lipid mixtures. 1 ACS Paragon Plus Environment

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Introduction Proteins are usually considered to be the key actors in the functionality of biological membranes, while lipids are often viewed as simple components that provide a stable structural matrix, with minor functional importance [1,2]. However, studies in the last decades have shown that lipids can be organized into functional domains [3], revealing the presence of lateral heterogeneity in the cell membrane. These domains, also called membrane rafts, are highly dynamic, with sizes in the nanometer range and offer a more attractive environment for many proteins [4-8]. Membrane rafts are postulated to be involved in a number of important cellular phenomena, such as endocytosis, signal transduction, viral infection and intracellular trafficking of proteins and lipids [3,9]. Nevertheless, the existence of rafts and their mechanisms of formation and stability are not completely elucidated [10,11]. The discovery of the rafts and their physicochemical nature are closely related to the existence of detergentresistant membranes (DRMs) [12-14], which are sphingolipid- and cholesterol-rich detergentinsoluble membrane patches, found after treatment of biological membranes with detergents. This fact is attributed mainly to the interaction between sphingomyelin (SM) and cholesterol, which creates a more condensed state, preventing the incorporation of detergent in the membrane [12]. Initially, DRMs were considered to be identical to lipid rafts due to compositional similarity. Currently, it is known that DRMs characteristics are detergent and protocol-dependent and cannot be directly associated with lipid rafts [6, 15-18]. The study of rafts in vivo has been a challenge due to the inherent complexity of biological membranes. Therefore, biomimetic lipid membranes, such as giant unilamellar vesicles (GUVs), have been widely used to elucidate fundamental properties of lateral heterogeneities in membranes. Model membranes composed of unsaturated lipids, long saturated lipids and cholesterol exhibit macroscopic separation between liquid-disordered (Ld) and liquid-ordered (Lo) phases at physiologically relevant temperatures, depending on the proportion of their components. These phases are biologically relevant, since biomembranes are usually found in the Ld phase (mainly due to the abundance of unsaturated lipids) and the Lo phase exhibits similarity with the membrane rafts due to their comparable composition [19-24]. Many ternary lipid mixtures have been studied over the last decades with several experimental approaches and ternary phase diagrams can be found in the literature containing mainly unsaturated phosphatidylcholine lipids, sphingomyelins or long saturated phosphatidylcholines, and cholesterol [23,25-32]. The phase diagrams reported are somewhat distinct, depending on the compositional differences and sensitivity of the techniques used in the experiments. For example, optical microscopy allows only the visualization of large domains (in the micrometer range), while more sensitive techniques, such as FRET (fluorescence resonance energy transfer), can detect nanoscopic domains as well [26,30]. Detergents have been widely used for solubilization of biological and model membranes due to their amphiphilic character and their ability to intercalate into lamellar structures at low concentrations and partially or fully solubilize them at higher concentrations [33,34]. The solubilization process proposed by Helenius & Simons in 1975 [35] is described in a three-stage model as the detergent-to-lipid ratio increases: In the first stage, detergents are 2 ACS Paragon Plus Environment

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incorporated in lipid bilayers; in the second stage, the solubilization starts and mixed detergent-lipid micelles coexist with saturated bilayers; the final stage happens when all lipids have been transferred to micellar structures. The solubilization process depends on several factors, such as the degree of unsaturation and/or size of the hydrophobic chains of the lipids, phase of the lipid bilayer and temperature, as well as detergent characteristics, as for example amphiphilic balance and critical micelle concentration (CMC) [14,36]. The non-ionic detergent Triton X-100 (TX-100) has been extensively used for research in the field of biological membranes due to its effective power of solubilization and relatively mild effect on the activity of isolated proteins and enzymes. TX-100 has also been employed in protein reconstitution protocols [34,37]. Several authors have investigated the interaction of this detergent with membranes [13, 38-42]. The composition and the membrane phase are dominant factors in the solubilization process. In general, the solubilization is complete for membranes in the gel and fluid (Ld) phases, although a higher concentration of TX-100 is required to solubilize bilayers in the fluid phase [43]. Otherwise, cholesterol-rich and/or Lo phase bilayers tend to exhibit resistance to solubilization [14,36,44-46]. In a previous work of our group [47], the effect of TX-100 on giant unilamellar vesicles (GUVs) composed of the lipid extract of erythrocyte membranes was investigated by phase contrast and fluorescence microscopy. This biological lipid composition was homogeneous within the optical resolution limit, as revealed by the surface distribution of a fluorescent lipid that is known to prefer the Ld phase. However, addition of TX-100 induced Lo/Ld (macroscopic) phase separation, followed by solubilization of the (fluorescent) Ld phase only, while the mother vesicle (in the Lo phase) remained insoluble. Interestingly, the same behavior was observed for GUVs composed of the ternary mixture palmitoyl oleoyl phosphatidyl choline (POPC):SM:cholesterol 4:2:4, attesting that the effect found is a result of the physicochemical properties of the lipid components. Here, we have investigated the interaction of TX-100 with giant (GUVs) and large (LUVs) unilamellar vesicles composed of the POPC:SM:cholesterol ternary mixture in a wide range of compositions at room temperature. The goal of this work was to construct a phase diagram of this mixture including the addition of TX-100 below the onset of solubilization, and to quantify the extent of solubilization for higher TX-100 concentrations, as the proportion of lipids in the ternary composition was varied. The main technique used was optical microscopy of GUVs. In parallel, turbidity and isothermal titration calorimetry (ITC) measurements were performed on LUVs. We clearly show that addition of TX-100 significantly increases the region of macroscopic Ld/Lo phase coexistence, and the presence of SM and cholesterol renders the mixture increasingly insoluble.

Materials and Methods Materials The lipids 1-palmitoyl-2-oleoyl-sn-glicero-3-phosphocholine (POPC), egg (chicken) sphingomyelin (SM) and cholesterol (chol) were obtained from Avanti Polar Lipids (Alabaster, AL). The acyl chain distribution of egg-SM comprises 16:0 (86%), 18:0 (6%), 22:0 (3%), 24:1 (3%) and others (2%). The fluorescent probe 1,1′-Dioctadecyl-3,3,3′,3′3 ACS Paragon Plus Environment

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tetramethylindocarbocyanine perchlorate (DiIC18) was from Life Technologies (Carlsbad, CA). The detergent Triton X-100 (TX-100), HEPES buffer, glucose and sucrose were purchased from Sigma-Aldrich (St. Louis, MO). The cholesterol monoreagent K083 dosage kit was obtained from BIOCLIN (Quibasa Basic Chemistry, Belo Horizonte, MG, Brazil). The water was purified by a Milli-Q system from Millipore (Billerica, MA). Preparation of giant unilamellar vesicles (GUVs) GUVs were obtained by the eletroformation method [19]. A lipid film of the desired lipid mixture was formed on the surfaces of two conductive glasses (covered by FTO-tin oxide doped with fluorine) by spreading 8 µL of a 2 mg/mL lipid solution in chloroform with a syringe needle. For fluorescence microscopy experiments, 0.5 mol% DiIC18 were added to the lipid chloroform solution. The solvent was evaporated with a stream of N2. The glasses were separated by a 1 mm-thick Teflon spacer to form a chamber, which was then filled with a 0.2 M sucrose solution. The system was sealed with clamps and an AC field of 1 V and 10 Hz frequency was applied for 1 h at 50 °C. The suspension of GUVs was then diluted approximately 10 times in a 0.2 M glucose solution and transferred to an observation chamber. The osmolarity of the solutions was checked with an osmometer Osmomat 030 of Gonotec (Berlin, Germany) and adjusted to be the same. Preparation of large unilamellar vesicles (LUVs) From a chloroform solution of the desired lipid mixture, a film was formed on the inside of a test tube by evaporating the organic solvent with a stream of N2. Then, the tube was left in vacuum for approximately 2 h, ensuring that all organic solvent was eliminated. A solution of 10 mM HEPES buffer, pH 7.4, was added to the test tube and multilamellar vesicles (MLVs) were formed by mechanical agitation of the tube (2 min vortexing). To obtain large unilamellar vesicles (LUVs), the dispersion was extruded at least 11 times through a polycarbonate membrane with 100 nm pore diameter [48]. The preparation of MLVs and the extrusion process were conducted in the fluid phase, at 50 oC. Optical microscopy experiments GUVs were observed by phase contrast and fluorescence microscopy on a Zeiss Axiovert 200 inverted microscope (Jena, Germany), equipped with a digital camera sCMOS Pco.edge 4.2 (Kelheim, Germany) or Zeiss Axiocam HSm (Jena, Germany) and 40x and 63x Ph2 air objectives. For fluorescence microscopy, the GUVs contained 0.5 mol% DiIC18 and images were achieved with illumination from a mercury lamp and a set of filters for excitation (540-552 nm) and emission (575-640 nm). Two different experimental setups were used to observe the effects of TX-100 on GUVs. In the first one, to verify the existence of phase separation, 5 µL of a suspension of GUVs prepared in 0.2 M sucrose were diluted in 95 µL of a 0.2 M glucose solution with or without 0.1 mM TX-100 and then immediately placed in a homemade observation chamber. In the second experimental setup, to follow the entire solubilization process, a 0.2 M glucose solution with 5 mM TX-100 was injected directly close to previously selected GUVs through a glass micropipette of about 5-10 µm diameter. The micropipettes were prepared using a Sutter Instrument puller capillary (Novato, CA) and

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manipulated by a micromanipulator MP-225, also from Sutter Instrument. All experiments were performed at room temperature (22-25 °C). Turbidity measurements The turbidity of suspensions of LUVs composed of POPC:SM:chol along the titration with TX-100 (0–5 TX-100/lipid molar ratio) was quantified through measurements of the optical density at λ = 600 nm using a UV-1601 Shimadzu spectrophotometer (Kyoto, Japan). A suspension of 3 mM lipid (1.5 mL) was placed in a quartz cuvette with 1 cm optical path. Absorption spectra were obtained before and 10 min after each aliquot injection (6–30 µL) of a 50 mM TX-100 stock suspension. The results are shown as relative turbidity in respect to the value before injection of TX-100. The residual turbidity at the end of each titration (Turbres) was used to quantify the insoluble fraction of each composition and to construct a color-contour diagram. ITC experiments Isothermal titration calorimetry (ITC) measurements were performed in a Microcal VP-ITC (Northampton, MA) equipment. The reference cell was filled with water and the sample cell was filled with 50 µM TX-100 solution (below the CMC). A dispersion of LUVs (20 mM lipid concentration) was loaded in the syringe. The experiment consisted of recording the power delivered to the sample cell as aliquots of 10 µL were injected every 10 min at constant temperature (22 oC). A first 0.5 µL injection was always made to reduce the volumetric error of the syringe plunger and it was discarded from the analysis. The partition coefficient K was obtained assuming a simple partition model, described in detail in ref. [49]. In this model, the extent of TX-100 bound to the lipid bilayer, Xb, is directly proportional to the concentration of free detergent, Cdet,f: =

,

= ,

eq. 1

where Clip is the lipid concentration and Cdet,b is the concentration of bound detergent, which can be expressed as:

, =

eq. 2

where Cdet is the total detergent concentration (Cdet = Cdet,f + Cdet,b). The heat (δhi) associated with each injection of lipid (δClip) into the calorimeter cell filled with TX-100 is given by

ℎ = (

)

∆!

eq. 3

where Vcell is the cell volume (1.4576 mL) and ∆H the total enthalpy change, which is obtained by summing all heat exchanged along the whole titration and dividing by the number of moles of TX-100 in the cell.

Results The ternary lipid mixtures used in this study were composed of POPC–as the unsaturated lipid, egg SM–as the saturated lipid, and cholesterol (chol). Given that cholesterol 5 ACS Paragon Plus Environment

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incorporation in a membrane is only effective up to 50 mol% of the total lipid composition and that the focus of the present study was to explore biomimetic compositions, we chose three fixed concentrations of cholesterol (20, 30 and 40 mol%) and varied the proportion of the other two components of the system at intervals of 10 mol%. Depending on the conditions, the POPC:SM:chol ternary mixture can exhibit lateral phase separation of up to three phases: Ld, Lo and gel. Here, we investigated the role of TX-100 in promoting phase separation at sub-solubilizing conditions (0.1 mM TX-100, below its CMC) and in the extent of solubilization of these mixtures at higher TX-100 concentrations using optical microscopy of GUVs and turbidity and ITC measurements on LUVs. The experiments were conducted at room temperature (22-25 °C). Phase diagram of POPC:SM:chol with and without TX-100 Fluorescence microscopy was used to verify the existence of lateral phase separation in GUVs composed of POPC:SM:chol without and with 0.1 mM TX-100, thus below its critical micelle concentration (CMC ~0.23 mM) [41]. The chosen fluorescent lipid (DiIC18) is known to prefer the Ld phase [47]. Figure 1 shows representative images of GUVs of some lipid compositions and the phase diagram of the POPC:SM:chol mixture without and with 0.1 mM TX-100.

Figure 1 – Phase diagram of the POPC:SM:chol mixture with and without 0.1 mM TX-100. The compositions explored are indicated with symbols (circles for phase coexistence with– open symbols– and without TX-100–filled symbols and squares for homogenous GUVs). The snapshots are representative images of some lipid compositions obtained in the presence of TX-100. In the absence of TX-100, only three compositions, indicated with filled circles in Figure 1, exhibited lateral phase separation. However, not all vesicles in the chamber displayed detectable domains. In fact, previous experiments with GUVs showed that this 6 ACS Paragon Plus Environment

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composition exhibited only a small region of macroscopic phase separation, and with miscibility temperatures close to room temperature [22]. On the other hand, in the presence of 0.1 mM TX-100 almost all compositions explored exhibited phase separation (indicated with circles in Figure 1). The only two exceptions were the compositions in the upper right corner of the phase diagram (indicated with grey squares), rich in SM and cholesterol and probably in the Lo phase, which exhibited homogeneous fluorescence irrespective of the presence of TX-100. When present, the domains were mostly circular, diffused freely and could coalesce after collision, resulting in larger domains. These characteristics indicate coexistence of liquid phases, namely Ld/Lo. In some GUVs in the region close to the SM corner of the phase diagram, domains with irregular shapes were also observed, which could indicate the presence of gel phase in the mixture (see snapshot 2:6:2 in Figure 1). The relative proportion of Ld and Lo fractions in the GUVs were composition-dependent. Figure 1 shows representative sequences of images along three lines in the phase diagram. The left and right columns show images as the cholesterol fraction decreases and either SM or POPC are kept constant, respectively. The sequence on the bottom shows images of GUVs along the line with 20 mol% cholesterol. Clearly, the Ld phase is predominant for POPC-rich compositions and decreases as POPC is replaced by SM. On the left, Lo domains are present in an Ld matrix. The reverse is observed on the right, in which Ld domains exist in an Lo matrix. The lateral phase separation shown in Figure 1 refers to the detection of (optically resolved) macroscopic domains and therefore do not exclude the existence of domains of sizes below the optical resolution (~ 0.3 µm). Observation of the solubilization process To visualize the whole solubilization process and quantify the extent of solubilization as a function of the membrane composition, the GUVs were subjected to a direct injection of 5 mM TX-100 through a micropipette. This concentration is high enough to allow observation of the whole solubilization process qualitatively [41,47]. The contact with detergent generated different responses, clearly dependent on the specific membrane composition. Figure 2 shows representative sequences of four lipid compositions.


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Figure 2 – Representative image sequences of GUVs of different POPC:SM:chol compositions (indicated in the figure) during injection of 5 mM TX-100 with a micropipette. The time is relative to the moment when the pipette approached each vesicle. Scale bars represent 10 µm. The first sequence (Figure 2A) shows a POPC-rich GUV (6:2:2). Note that in the first snapshot the GUV is initially spherical and homogeneous when viewed under fluorescence microscopy. Then, in the second and third snapshots, the vesicle has already a larger surface area and (dark, Lo) domains appear (4 s) and coalesce (6s). Then, the bottom side of the vesicle opens (6-11 s) and additional pores are visualized close to the open side (8 s). The solubilization process continues (11-24 s) until practically the whole vesicle is solubilized and no detectable fragment is seen (28 s). In some cases, small bilayer fragments remained and were flushed away by the injection flux. Similar behavior was observed for other compositions rich in POPC (5:3:2 and 7:2:1). This sequence of events is similar to that presented earlier by our group for vesicles composed solely of POPC in the course of their solubilization by TX-100 [41,45]. The second sequence (Figure 2B) (4:3:3) shows an example of partial vesicle solubilization. Before contact with TX-100, the GUV is homogeneously fluorescent, with no detectable lateral phase separation. Right after the contact with TX-100, phase coexistence is promoted and the (bright, Ld) domains coalesce (5-10 s). Eventually, the Ld fraction is detached (15 s) and solubilized (15-28 s) and a smaller vesicle, in the Lo phase, remains. The same behavior was previously reported for the 4:2:4 mixture and for the lipid extract of erythrocyte membranes [47]. It is noteworthy that most compositions explored here exhibited this behavior, but the proportion of soluble and insoluble fractions varied with the particular composition. Interestingly, TX-100 was shown to promote fission of the Lo domains from GUVs composed of POPC/SM/chol 6:2:2 at a low temperature (4 oC) [21]. The third sequence (Figure 2C) shows a GUV that already exhibited phase separation (2:5:3) prior to detergent injection. The Ld domains seem to coalesce after contact with TX-100 (7 s) and are then solubilized (10-15s). The sequence shown in Figure 2D is an example of a virtually insoluble vesicle (1:5:4). These vesicles were composed mainly of SM and cholesterol and exhibit Lo characteristics, as judged by the mild shape fluctuations of quasi-spherical vesicles. As the TX-100 is injected close to such GUVs, no significant changes in the vesicle size and lateral homogeneity were observed even after a long period of time (few minutes). However, it is possible to detect that a fraction of the fluorescent probes is extracted from the mixture right after contact with the TX-100 solution (2 s), which is consistent with the preference of the fluorescent lipid for more fluid environments. Quantification of the insoluble fraction of GUVs The results obtained with injection of TX-100 with a micropipette (Figure 2) showed that the extent of solubilization depends on the lipid composition of the mixture. In most cases, TX-100 induced partial solubilization of the GUV, corresponding to the Ld phase, and a smaller vesicle in the Lo phase remained insoluble after contact with TX-100. To quantify the fraction of vesicle surface area insoluble to TX-100, Xinsol, the vesicle diameter before and 8 ACS Paragon Plus Environment

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after treatment with TX-100 was measured and the insoluble fraction was defined as Xinsol = (Dafter/Dbefore)2, where Dbefore and Dafter corresponds to the vesicle diameters before and after contact with TX-100, respectively [47]. This parameter could be measured for most compositions, except for those rich in POPC (see Figure 2A), for which the insoluble fraction, if present, was very small and was therefore flushed away by the injection flux and could not be quantified. In that case, Xinsol was arbitrarily set to 0.1 and indicate the limitation of this approach. The results are shown in Figure 3. Each open symbol represents Xinsol measured for one GUV and the yellow squares indicate the average insoluble area fraction with standard deviation obtained for each composition. The compositions are distributed horizontally in the graph and are grouped according to the percentage of cholesterol in the mixture (40, 30 and 20 mol %).

Figure 3 – A) Fraction of insoluble surface area, Xinsol, measured from POPC:SM:chol GUVs. Each symbol indicates one vesicle exposed to a flux of 5 mM TX-100 from a micropipette and yellow squares indicate the mean values with standard deviation for each composition. B) GUV solubility diagram. The contour color map indicates the mean values of Xinsol. It is clear that the insoluble fraction is directly related to the composition of the vesicle. For each cholesterol fraction, Xinsol consistently decreases as POPC is replaced by SM in the mixture. Overall, the larger the cholesterol fraction, the more insoluble the mixtures become. The results shown in Figure 3A are presented as a solubility diagram in Figure 3B, in which the extent of solubilization is represented as a contour color map. Red regions of the diagram show practically insoluble compositions with Xinsol ~ 0.9-1. In the POPC-rich zone, the membrane solubility to TX-100 is significantly increased (indicated in blue). Quantification of the residual turbidity of LUVs One of the most used and suitable experimental approaches to follow the solubilization of lipid dispersions by detergents is the measurement of the sample turbidity (optical density) or light scattering along the titration of LUVs with the detergent [14,38,45,50]. Sample turbidity is high for LUVs and starts to decrease at the onset of solubilization and eventually reaches values close to zero when total solubilization into micelles occurs. This procedure 9 ACS Paragon Plus Environment

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was performed for LUVs composed of POPC:SM:chol (0-50 mol% cholesterol and POPC and SM varying in steps of 10 mol%) along titration with TX-100 up to 5 TX-100/lipid molar ratio. The optical density (turbidity) of the sample at 600 nm was recorded before and 10 min after aliquots of TX-100 were added into the lipid mixtures. The turbidity values relative to the initial value in the absence of TX-100 as a function of the TX-100/lipid molar ratio are shown for the ternary mixtures in Figure 4A. The solubilization profiles obtained with 10 mol% chol are consistent with the three-stage model [35] and show total solubilization of the mixtures. The concentration of TX-100 required for the onset and completion of the solubilization process decreases significantly as POPC is gradually replaced by SM, as expected, because less TX-100 is needed to solubilize gel phase membranes [43,51,52]. For higher cholesterol fractions (20-40 mol%), the solubilization process is not complete and sample turbidity reaches a fairly constant value at high TX-100 concentrations. This residual turbidity (Turbres) increases with the SM fraction for constant cholesterol concentration and overall with the fraction of cholesterol in the mixture, until virtually insoluble mixtures are obtained at high SM and cholesterol fractions. Figure 4B shows the complete solubility diagram of the POPC:SM:chol mixture as a contour color map of the Turbres values obtained at the end of each turbidity curve. The results obtained from turbidity of LUVs are quite similar to those obtained from the solubilization extent of GUVs (see Figure 3B). Importantly, turbidity experiments allowed the assessment of the whole diagram, whereas the approach with GUVs was limited by the resolution of the technique.


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Figure 4 – A) Relative turbidity (λ = 600 nm) of 3 mM lipid dispersion of LUVs along titration with TX-100 for different POPC:SM:chol compositions. B) LUVs solubility diagram. The contour color map indicates the mean values of residual turbidity (Turbres) obtained at the end of each curve shown in A).

Incorporation of TX-100 into LUVs According to the three-stage model [35] detergents are incorporated into the membranes in stage I (at low TX-100/lipid molar ratios). The incorporation process can be viewed as a simple partition model, in which the concentration of detergent incorporated is related to the concentration of free detergent via the partition coefficient K [49]. An uptake protocol using isothermal titration calorimetry (ITC) was employed to measure the binding constant of TX-100 to membranes of different compositions. The calorimeter cell was filled with a TX-100 solution below its CMC, aliquots of a concentrated LUV dispersion were added into the cell and the heat associated with the uptake of TX-100 into the bilayers was detected. Figure 5 shows the heat per injection (δh) as a function of the total lipid concentration in the cell for LUVs composed of POPC or SM containing increasing molar fractions of cholesterol. Incorporation of TX-100 into the bilayer is endothermic, showing that it is an entropy-driven process due to the classical hydrophobic effect. The results were well fitted with the model described in Materials and Methods and the fitting curves are shown as lines in Figure 5. The corresponding fitting parameters (K and ∆H) are given in Table 1. The results obtained are comparable to existing literature data [45,53] and confirm that the affinity of TX-100 to the lipid bilayer is highly decreased as the molar fraction of cholesterol in the membrane increases. The binding constant is nearly the same for pure POPC and SM membranes and becomes negligible for POPC:chol 6:4 and SM:chol 7:3. The magnitude of heat absorbed is higher for POPC as compared with SM.


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POPC

60

mol% cholesterol 0 10 20 30 40

δh (µcal)

50 40 30 20 10 0 0.0

15

δh (µcal)

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0.5

1.0

1.5

2.0

2.5

3.0

3.5

SM

10

5

0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0

[Lipid] (mM)

Figure 5 – Heat per injection (δh) as a function of the total lipid in the calorimeter cell. The cell was filled with 50 µM TX-100 (below its CMC) and 10 µL injections of a 20 mM lipid dispersion of LUVs were injected every 10 min. The lines correspond to the fits obtained with the simple partition model (eq. 3).

Table 1 – Fitting parameters of the curves shown in Figure 5 according to the model described in eq. 3. mol% chol K (M-1) ∆H (kcal/mol) POPC SM POPC SM 0 1500 1500 5.8 1.5 10 1000 1000 7.2 0.4 20 800 400 6.2 0.2 30 300 100 5.0 0.2 40 100 3.2 -

Discussion We conducted a study exploring the physicochemical aspects that govern the solubilization process of raft-like lipid ternary mixtures composed of POPC:SM:chol by TX100 in a wide range of compositions. Our results indicate that addition of TX-100 in the lipid 12 ACS Paragon Plus Environment

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system considerably increases the coexistence region of macroscopic phases and that the Ld phase is selectively solubilized by TX-100 whereas the Lo fraction remains insoluble. The composition of the vesicle exerts a determinant role in the solubilization process: SM and cholesterol-rich mixtures exhibit an insoluble character. Several raft-like ternary lipid mixtures have been studied and the phase diagrams reported in the literature have significant differences among them, which can be attributed to variations in composition, sensitivity of the technique used and domain size. In the present work, a systematic study of the effect of the detergent TX-100 on a ternary diagram was carried out for the first time. The purpose here was not to precisely define the boundaries of the phase diagram, since it is known that many variants may interfere with the results, but to better understand the effect of the detergent on raft-like mixtures. In reviewing the diagrams available in the literature, it is possible to observe that the lipid composition varies considerably and the contrast among the resulting diagrams is evident. Although POPC is a more biologically relevant lipid (most biological phospholipids have one unsaturated and one saturated tail), the reported diagrams are usually based on the use of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Phase diagrams containing POPC as the unsaturated component were reported to exhibit a limited region with phase separation, at least at the macroscopic level. However, when the unsaturated component is replaced by DOPC the domains become more abundant and clearly detectable with optical microscopy. This can be probably explained by the fact that DOPC has a larger molecular area (with two unsaturated tails) in comparison with POPC and a lower transition temperature [22]. Studies with GUVs show that the size and morphology of the domains can be controlled by the concentration and type of the unsaturated lipid present in the composition [54]. In fact, Heberle and coworkers [55] have demonstrated that the size of the membrane domains increases with the extent of the acyl chain unsaturation. As for the saturated component, most of the authors make use of N-palmitoyl-Dsphingomyelin (PSM) [25,26,28,31]. However, it is also possible to find diagrams with brain SM [27,32], N-oleoyl-sphingomyelin (OSM) [28], 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) [20,23] and 1,2-disteatoyl-sn-glycero-3-phosphocholine (DSPC) [29,30]. No phase diagrams with egg-SM, rich in PSM (see Materials and Methods for the acyl chain composition of egg-SM), were described so far. Therefore, the mixture that more closely resembles the one explored here is POPC:PSM:chol. The sensitivity of the technique used in the experiments is another relevant parameter in the study of phase diagrams. Techniques with higher spatial resolution (such as FRET, electronic paramagnetic resonance, fluorescence anisotropy, etc.) are able to detect the immediate environment of the marker and indicate the presence of very small domains. Therefore, a prominent presence of domains in various compositions can be justified. However, the microscopy technique requires a larger scale lipid cluster for its detection [32], which is limited by the optical resolution. Almeida and coworkers [25] studied the PSM:POPC:chol system through fluorescence anisotropy, life time and quenching. The resulting diagram at 23 °C showed the existence of domains in a wide range of compositions, with coexistence of Lo/gel phases in compositions near the PSM corner, coexistence of Lo/Ld phases near the axis of the POPC and coexistence of Lo phases/Ld/gel in the central region of the diagram. In a subsequent work, the same lipid system was used to study the size of 13 ACS Paragon Plus Environment

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domains through the FRET technique [26]. The results indicated that compositions rich in POPC have smaller domains, in the order of 20 nm. As the proportion of cholesterol increased in the mixture, the domains increased in size. Later, the same lipid system was investigated with fluorescence anisotropy and similar results were obtained: presence of domains in several compositions and coexistence of liquid phases near the axis of POPC [28]. Regarding the microscopy results, we highlight the data obtained by Veatch and Keller [23], in which fluorescence microscopy of GUVs was used to verify the presence of domains, varying the unsaturated (POPC and DOPC) and saturated (PSM and DPPC) components and the system temperature. The diagram of the PSM:POPC:chol system at 23 °C fits well with the data obtained here in the absence of detergent. However, when TX-100 is added to the mixture, the presence of domains becomes abundant and our diagram resembles those obtained from high spatial resolution techniques. Since the literature corroborates the presence of small domains near the POPC corner we conclude from the results obtained that the domains found by the microscopy technique after the addition of TX-100 probably existed previously in the vesicle, but on a submicroscopic scale. The addition of the detergent creates a quaternary system and favors a greater differentiation between the Ld and Lo phases, leading to the coalescence of submicroscopic domains. In fact, studies indicate that the addition of TX-100 does not induce the formation of new domains in the vesicles, but leads to the increase of pre-existing domains in the composition [56,57]. The relative proportion between the phases is also related to the detergent partitioning constant, which in the case of TX-100 is high in the Ld phase and greatly reduced in the presence of cholesterol, and therefore an increase in the relative area of the Ld domains is expected upon incorporation of TX-100. Thus, we conjecture that different detergents should cause different rearrangements in the heterogeneity of the mixture depending on their relative partition between the coexisting phases. In our previous work [47], GUVs made of the lipid extract of erythrocyte membranes were prepared and exposed to TX-100 at solubilizing conditions. We showed that one third of the original vesicle surface area, corresponding to the Ld fraction, was solubilized by TX-100 and a smaller insoluble vesicle remained. Surprisingly, the same solubilization extent was measured for GUVs composed of POPC:SM:chol 4:2:4, indicating that the effects promoted by TX-100 originate from elementary physicochemical features, reproducible in a simple ternary system. Here, we quantified the solubilization extent of the POPC:SM:chol mixture in a wide composition range, both with optical microscopy of GUVs and turbidity of LUVs. Direct visualization of the solubilization process showed that TX-100 selectively solubilizes the Ld fraction and the Lo part remains insoluble. The lipid bilayer resistance to TX-100 is highly correlated with the presence of cholesterol and SM. In fact, ITC results presented here and elsewhere [53] showed that the partition of TX-100 is highly suppressed as the fraction of cholesterol in the mixture increases, indicating that the interaction between TX-100 and cholesterol is unfavorable. In summary, we showed that the widely used detergent TX-100 causes a considerable lateral rearrangement of raft-like membrane compositions and that the solubilization extent is highly modulated by the membrane composition. We provide a thorough quantification of both detergent-induced domain formation/coalescence and solubilization extent for a wide range of biologically relevant lipid compositions. These 14 ACS Paragon Plus Environment

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results can serve as future guide in several studies, as for instance in designing model systems to determine the preference of a protein for environments with different order as well as to tune the resistance of a mixture to the action of detergents.

Acknowledgements The financial support of FAPESP, CNPq and INCT-FCx is acknowledged.

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(51) Patra, S. K.; Alonso, A.; Goñi, F. M. Detergent solubilisation of phospholipid bilayers in the gel state: the role of polar and hydrophobic forces. Biochim. Biophys. Acta 1998, 1373, 112-118. (52) Ribeiro, A. A.; Dennis, E. A. Effect of thermotropic phase transitions of dipalmitoylphosphatidylcholine on the formation of mixed micelles with Triton X-100. Biochim. Biophys. Acta 1974, 332, 26-35. (53) Tsamaloukas, A., Szadkowska, H., Heerklotz, H. Nonideal mixing in multicomponent lipid/detergent systems. J. Phys.: Condens.Matter. 2006, 18, 1125-1138. (54) Konyakhina, T. M.; Goh, S. L.; Amazon, J.; Heberle, F. A.; Wu, J.; Feigenson, G. W. Control of a nanoscopic-to-macroscopic transition: modulated phases in four-component DSPC/DOPC/POPC/Chol giant unilamellar vesicles. Biophys. J. 2011, 101, L8-L10. (55) Heberle, F. A.; Petruzielo, R. S.; Pan, J.; Drazba, P.; Kučerka, N.; Standaert, R. F.; Feigenson, G. W.; Katsaras, J. Bilayer thickness mismatch controls domain size in model membranes. J. Am. Chem. Soc. 2013, 135, 6853-6859. (56) Giocondi, M. C.; Vié, V.; Lesniewska, E.; Goudonnet, J. P.; Le Grimellec, C. In situ imaging of detergent-resistant membranes by atomic force microscopy. J. Struct. Biol. 2000, 131, 38-43. (57) Pathak, P.; London, E. Measurement of lipid nanodomain (raft) formation and size in sphingomyelin/POPC/cholesterol vesicles shows TX-100 and transmembrane helices increase domain size by coalescing preexisting nanodomains but do not induce domain formation. Biophys. J. 2011, 101, 2417-2425.


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Effect of Triton X-100 on Raft-Like Lipid Mixtures: Phase Separation

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