9
Charge-induced phase separation in lipid membranes Hiroki Himeno,a Naofumi Shimokawa,a Shigeyuki Komura, b David Andelman, c Tsutomu Hamada * a and Masahiro Takagi a Phase separation in lipid bilayers that include negatively charged lipids is examined experimentally. We observed phase-separated structures and determined the membrane miscibility temperatures in several binary and ternary lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine (DOPC), saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC), unsaturated charged lipid, dioleoylphosphatidylglycerol (DOPG () ), saturated charged lipid, dipalmitoylphosphatidylglycerol (DPPG () ), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, the combination of the charged head with the saturation of the hydrocarbon tail is a dominant factor in the stability of membrane phase separation. DPPG () enhances phase separation, while DOPG () suppresses it. Furthermore, the addition of DPPG () to a binary mixture of DPPC/cholesterol induces phase separation between DPPG () -rich and cholesterol-rich phases. This indicates that cholesterol localization depends strongly on the electric charge on the hydrophilic head group rather than on the ordering of the hydrocarbon tails. Finally, when DPPG () was added to a neutral ternary system of DOPC/DPPC/ cholesterol (a conventional model of membrane rafts), a three-phase coexistence was produced. We conclude by discussing some qualitative features of the phase behaviour in charged membranes using a free energy approach. Introduction One of the major components of cell membranes is their lipid bilayer composed of a mixture of several phospholipids, all having a hydrophilic head group and two hydrophobic tails. Recently, a number of studies have investigated heterogeneities in lipid membranes in relation to the lipid ra hypothesis. 1,2 Lipid ras are believed to function as a platform on which proteins are attached during signal transduction and membrane tracking. 3 It is commonly believed (but still debatable) that the ra domains are associated with phase separation that takes place in multi-component lipid membranes. 4 In order to reveal the mechanism of phase separation in lipid membranes, giant unilamellar vesicles (GUVs) consisting of mixtures of lipids and cholesterol have been used as model biomembranes. 57 In particular, studies of phase separation and membrane dynamics have been performed on such GUVs consisting of saturated lipids, unsaturated lipids and choles- terol. 8 Multi-component membranes phase separate into domains rich in saturated lipids and cholesterol, whereas the surrounding uid phase is composed largely of unsaturated lipids. The essential origin of this lateral phase separation was argued to be the hydrophobic interactions between acyl chains of lipid molecules. In the past, most of the studies have investigated the phase separation in uncharged model membranes. 911 However, bio- membranes also include charged lipids, and, in particular, phosphatidylglycerol (PG () ) is found with high fractions in prokaryotic membranes. In this respect it is worth mentioning that in Staphylococcus aureus the PG () membranal fraction is as high as 80%, whereas the Escherichia coli membrane includes 15% of PG () . 12 Although the charged lipid fraction in eukary- otic plasma membranes is lower, its sub-cellular organelles such as mitochondria and lysosome are enriched with several types of charged lipids. 13 For example, the inner membrane of mitochondria includes 20% of charged lipids such as car- diolipin (CL () ), phosphatidylserine (PS () ) and PG () . 14,15 It is indispensable to include the eect of electrostatic interactions on the phase behavior in biomembranes. To emphasize even further the key role played by the charges, we note that membranes composed of a binary mixture of charged lipids were reported to undergo phase separation induced by addition of salt, even when the two lipids have the same hydrocarbon a School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa, 923-1292, Japan. E-mail: [email protected]; Tel: +81-761-51-1670 b Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo 192-0397, Japan c Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Ramat Aviv 69978, Tel Aviv, Israel These authors contributed equally to this work. Cite this: Soft Matter, 2014, 10, 7959 Received 19th May 2014 Accepted 28th July 2014 DOI: 10.1039/c4sm01089b www.rsc.org/softmatter This journal is © The Royal Society of Chemistry 2014 Soft Matter, 2014, 10, 79597967 | 7959 Soft Matter PAPER

Charge-induced phase separation in lipid membranes - TAUandelman/reprints/169_SoftMatter_2014_10_7959.pdf · in lipid membranes in relation to the lipid ra hypothesis.1,2 Lipid ra

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Soft Matter

PAPER

Charge-induced

aSchool of Materials Science, Japan Advance

Asahidai, Nomi, Ishikawa, 923-1292, Jap

+81-761-51-1670bDepartment of Chemistry, Graduate Sch

Metropolitan University, Tokyo 192-0397, JacRaymond and Beverly Sackler School of Ph

Ramat Aviv 69978, Tel Aviv, Israel

† These authors contributed equally to th

Cite this: Soft Matter, 2014, 10, 7959

Received 19th May 2014Accepted 28th July 2014

DOI: 10.1039/c4sm01089b

www.rsc.org/softmatter

This journal is © The Royal Society of C

phase separation in lipidmembranes

Hiroki Himeno,†a Naofumi Shimokawa,†a Shigeyuki Komura,b David Andelman,c

Tsutomu Hamada*a and Masahiro Takagia

Phase separation in lipid bilayers that include negatively charged lipids is examined experimentally. We

observed phase-separated structures and determined the membrane miscibility temperatures in several

binary and ternary lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine (DOPC),

saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC), unsaturated charged lipid,

dioleoylphosphatidylglycerol (DOPG(�)), saturated charged lipid, dipalmitoylphosphatidylglycerol

(DPPG(�)), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, the

combination of the charged head with the saturation of the hydrocarbon tail is a dominant factor in the

stability of membrane phase separation. DPPG(�) enhances phase separation, while DOPG(�) suppresses

it. Furthermore, the addition of DPPG(�) to a binary mixture of DPPC/cholesterol induces phase

separation between DPPG(�)-rich and cholesterol-rich phases. This indicates that cholesterol localization

depends strongly on the electric charge on the hydrophilic head group rather than on the ordering of

the hydrocarbon tails. Finally, when DPPG(�) was added to a neutral ternary system of DOPC/DPPC/

cholesterol (a conventional model of membrane rafts), a three-phase coexistence was produced. We

conclude by discussing some qualitative features of the phase behaviour in charged membranes using a

free energy approach.

Introduction

One of the major components of cell membranes is their lipidbilayer composed of a mixture of several phospholipids, allhaving a hydrophilic head group and two hydrophobic tails.Recently, a number of studies have investigated heterogeneitiesin lipid membranes in relation to the lipid ra hypothesis.1,2

Lipid ras are believed to function as a platform on whichproteins are attached during signal transduction andmembrane trafficking.3 It is commonly believed (but stilldebatable) that the ra domains are associated with phaseseparation that takes place in multi-component lipidmembranes.4

In order to reveal themechanism of phase separation in lipidmembranes, giant unilamellar vesicles (GUVs) consisting ofmixtures of lipids and cholesterol have been used as modelbiomembranes.5–7 In particular, studies of phase separation andmembrane dynamics have been performed on such GUVs

d Institute of Science and Technology, 1-1

an. E-mail: [email protected]; Tel:

ool of Science and Engineering, Tokyo

pan

ysics and Astronomy, Tel Aviv University,

is work.

hemistry 2014

consisting of saturated lipids, unsaturated lipids and choles-terol.8 Multi-component membranes phase separate intodomains rich in saturated lipids and cholesterol, whereas thesurrounding uid phase is composed largely of unsaturatedlipids. The essential origin of this lateral phase separation wasargued to be the hydrophobic interactions between acyl chainsof lipid molecules.

In the past, most of the studies have investigated the phaseseparation in uncharged model membranes.9–11 However, bio-membranes also include charged lipids, and, in particular,phosphatidylglycerol (PG(�)) is found with high fractions inprokaryotic membranes. In this respect it is worth mentioningthat in Staphylococcus aureus the PG(�) membranal fraction is ashigh as 80%, whereas the Escherichia coli membrane includes15% of PG(�).12 Although the charged lipid fraction in eukary-otic plasma membranes is lower, its sub-cellular organellessuch as mitochondria and lysosome are enriched with severaltypes of charged lipids.13 For example, the inner membrane ofmitochondria includes 20% of charged lipids such as car-diolipin (CL(�)), phosphatidylserine (PS(�)) and PG(�).14,15 It isindispensable to include the effect of electrostatic interactionson the phase behavior in biomembranes. To emphasize evenfurther the key role played by the charges, we note thatmembranes composed of a binary mixture of charged lipidswere reported to undergo phase separation induced by additionof salt, even when the two lipids have the same hydrocarbon

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Soft Matter Paper

tail.16–18 For this charged lipid mixture, the segregation ismediated only by the electrostatic interaction between the lipidsand the electrolyte.

In related studies, Shimokawa et al.19,20 studied mixturesconsisting of neutral saturated lipid (DPPC), negatively chargedunsaturated lipid (DOPS(�)) and cholesterol. The main result isthe suppression of the phase separation due to electrostaticinteractions between the charged DOPS(�) lipids. Two otherrelevant studies are worth mentioning. Vequi-Suplicy et al.21

reported the suppression of phase separation using othercharged unsaturated lipids, and more recently Blosser et al.22

investigated the phase diagram and miscibility temperature inmixtures containing charged lipids. However, the effects ofelectric charges on the phase behaviour in lipid/cholesterolmixtures have not been addressed so far systematically.

In the present study, we investigate the physicochemicalproperties of model membranes containing various mixtures ofcharged lipids, with the hope that the study will enhance ourunderstanding of biomembranes in vivo, which are much morecomplex. We examine the electric charge effect on the phasebehaviour using uorescence microscopy and confocal laserscanning microscopy. In addition, the salt screening effect oncharged membranes is explored. We discuss these effects inthree stages starting from the simpler one. First, the phasediagram in charged binary mixtures of unsaturated and satu-rated lipids is presented. Second, we investigate the phasebehaviour in ternary mixtures consisting of saturated lipids(charged and neutral) and cholesterol. And third, we include thechange of phase behaviour when a charged saturated lipid isadded as a fourth component to a ternary mixture of neutralsaturated and unsaturated lipids and cholesterol. We concludeby discussing qualitatively the phase behaviour of chargedmembranes using a free energy modeling. The counterionconcentration adjacent to the charged membrane is calculatedin order to explore the relationship between the electric chargeand the ordering of the hydrocarbon tail.

Materials and methodsMaterials

Neutral unsaturated lipid dioleoyl-sn-glycero-3-phosphocholine(DOPC, with chain melting temperature, Tm ¼ �20 �C), neutralsaturated lipid dipalmitoyl-sn-glycero-3-phosphocholine (DPPC,Tm ¼ 41 �C), negatively charged unsaturated lipid 1,2-dioleoyl-sn-glycero-3-phospho-(10-rac-glycerol) (sodium salt) (DOPG(�),Tm ¼ �18 �C), negatively charged saturated lipid 1,2-dipalmi-toyl-sn-glycero-3-phospho-(10-rac-glycerol) (sodium salt)(DPPG(�), Tm ¼ 41 �C), and cholesterol were obtained fromAvanti Polar Lipids (Alabaster, AL). BODIPY labelled cholesterol(BODIPY–Chol) and Rhodamine B–1,2-dihexadecanoyl-sn-glyc-ero-3-phosphoethanolamine (Rhodamine–DHPE) werepurchased from Invitrogen (Carlsbad, CA). Deionized water wasobtained from aMillipore Milli-Q purication system. We chosephosphatidylcholine (PC) as the neutral lipid head and phos-phatidylglycerol (PG) as the negatively charged lipid headbecause the chain melting temperature of PC and PG lipidshaving the same acyl tails is almost identical. In cellular

7960 | Soft Matter, 2014, 10, 7959–7967

membranes, PC is the most common lipid component, and PGis highly representative among charged lipids.

Preparation of giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) were prepared by a gentlehydration method. Lipids and uorescent dyes were dissolvedin 2 : 1 (vol/vol) chloroform–methanol solution. The organicsolvent was evaporated under a ow of nitrogen gas, and thelipids were further dried under vacuum for 3 h. The lms werehydrated with 5 mL deionized water at 55 �C for 5 min (pre-hydration), and then with 200 mL deionized water or NaClsolution for 1–2 h at 37 �C. The nal lipid concentration was0.2 mM. Rhodamine–DHPE and BODIPY–Chol concentrationswere 0.1 mM and 0.2 mM, respectively.

Microscopic observations

The GUV solution was placed on a glass coverslip, which wascovered with another smaller coverslip at a spacing of ca. 0.1mm. We observed the membrane structures with a uorescentmicroscope (IX71, Olympus, Japan) and a confocal laser scan-ning microscope (FV-1000, Olympus, Japan). In the presentstudy, Rhodamine–DHPE and BODIPY–Chol were used asuorescent dyes. Rhodamine–DHPE labels the lipid liquidphase, whereas BODIPY–Chol labels the cholesterol-rich one. Astandard lter set, U-MWIG with excitation wavelength lex ¼530–550 nm and emission wavelength lem ¼ 575 nm, was usedto monitor the uorescence of Rhodamine–DHPE, and anotherlter set, U-MNIBA with lex ¼ 470–495 nm and lem ¼ 510–550nm, was used for the BODIPY–Chol dye. The sample tempera-ture was controlled with a microscope stage (type 10021, JapanHitec).

Measurement of miscibility temperature

The miscibility temperature corresponds to the boundarybetween one- and two-phase regions. It is dened as the phaseseparation point at which more than 50% of the phase-sepa-rated domains have disappeared upon heating. The tempera-ture was increased from room temperature to the desiredtemperature by 10 �C min�1, and a further delay of 5 min wasused in order to approach the equilibrium state. We thenmeasured the percentage of vesicles that were in the two-phasecoexisting region. If the percentage of such two-phase vesicleswas over 50%, the temperature was further increased by 2 �C.We continued this procedure until the percentage of two-phasevesicles decreased below 50%.

ResultsBinary lipid mixtures

First, we focus on the effect of charges on the phase separationof binary unsaturated/saturated lipid mixtures. We use neutralunsaturated lipid DOPC, neutral saturated lipid DPPC, nega-tively unsaturated lipid DOPG(�), and negatively saturated lipidDPPG(�) (see Table 1). We observed the phase separation andmeasured the miscibility temperatures in three different binarymixtures: DOPC/DPPC, DOPC/DPPG(�), and DOPG(�)/DPPC.

This journal is © The Royal Society of Chemistry 2014

Table 1 The four neutral and negatively charged lipids and their chainmelting temperatures

Neutral head (PC)Negative chargedhead (PG)

Saturated tail (DP) DPPC DPPG(�)

Tm ¼ 41 �C Tm ¼ 41 �CUnsaturated tail (DO) DOPC DOPG(�)

Tm ¼ �20 �C Tm ¼ �18 �C

Paper Soft Matter

Fig. 1(A) shows the phase behaviour in these three binarymixtures taken for three temperatures: T ¼ 22 �C, 30 �C and40 �C. Each of the images was taken by superimposing severalpictures at a slightly different focus position of the confocallaser scanning microscope. At room temperature (22 �C), all

Fig. 1 Phase behaviour in binary lipid mixtures (DOPC/DPPC, DOPC/DPPG(�), DOPG(�)/DPPC). (A) Microscopic images of the phase sepa-ration for three temperatures, 22 �C, 30 �C and 40 �C. Red and blackregions indicate unsaturated lipid-rich (Ld) and saturated lipid-rich (So)phases, respectively. (B) Phase boundary (miscibility temperature)between one-phase and two-phase regions (filled squares: DOPC/DPPG(�), filled circles: DOPC/DPPC, filled triangles: DOPG(�)/DPPC,open squares: DOPC/DPPG(�) in 10 mM NaCl, open triangles:DOPG(�)/DPPC in 10 mM NaCl).

This journal is © The Royal Society of Chemistry 2014

three mixtures exhibit a phase separation (images 7, 8, and 9).The red regions indicate the liquid-disordered phase (Ld) thatincludes a large amount of the unsaturated lipid, while the darkregions represent the solid-ordered phase (So) that is rich in thesaturated lipid. When the temperature was raised to 30 �C, thephase separation of DOPG(�)/DPPC disappears (image 6). Onthe other hand, the two other mixtures (DOPC/DPPC and DOPC/DPPG(�)) still retained the phase-separated structure (images 4and 5). As the temperature was further increased to 40 �C, theDOPC/DPPC mixture also became homogeneous (image 1),while the DOPC/DPPG(�) mixture still retained its phase-sepa-rated structure at the same temperature (image 2). Thus, theDOPC/DPPG(�) mixture shows the highest miscibility tempera-ture of all the studied systems. Note that a similar phase-sepa-rated structure was reported in binary mixtures of eggsphingomyelin (eSM)/DOPG(�).21,23

Miscibility temperatures of binary mixtures are summarizedin Fig. 1(B). The lled circles denote the neutral lipid mixture,DOPC/DPPC. We also examined charged binary mixtures of twonegatively charged lipids, DOPG(�)/DPPG(�). Miscibilitytemperatures (data not shown) were quite similar to those ofneutral DOPC/DPPC mixtures. This implies that the phaseseparation behavior is determined by the interaction betweenhydrocarbon tails in mixtures consisting of the same lipid headgroup. When the neutral unsaturated lipid (DOPC) was replacedwith the charged unsaturated lipid (DOPG(�)), the miscibilitytemperature in the DOPG(�)/DPPC mixture (denoted by lledtriangles) became lower as compared with a neutral lipidmixture, DOPC/DPPC. In other words, the phase separation issuppressed when a negatively charged unsaturated lipid isincluded. This result is consistent with previous studies per-formed on lipid mixtures containing negatively charged unsat-urated lipids.19,21–23 At higher concentrations of DPPC, phase-separated domains could not be observed for mixtures ofDOPG(�)/DPPC ¼ 20 : 80 and 10 : 90, because stable vesicleformation was prevented by the larger amount of DPPC.

We also replaced the neutral saturated lipid, DPPC, with thenegatively charged saturated lipid, DPPG(�). In the DOPC/DPPG(�) mixture, the miscibility temperature (denoted by lledsquares in Fig. 1(B)) increases signicantly as compared withthe neutral system. In particular, we can see that a maximum inthe miscibility temperature appears in the phase diagramaround 50% relative concentration of the saturated lipid.Interestingly, at DOPC/DPPG(�) ¼ 50 : 50, the miscibilitytemperature of about 44 �C was higher than 41 �C of theDPPG(�) chain melting temperature (Table 1). Thus, the phaseseparation is enhanced in mixtures containing the negativelycharged saturated lipid (DPPG(�)). This result should be con-trasted with the phase behaviour of the DOPG(�)/DPPC charged/neutral mixture. We will further elaborate on such a phasebehaviour in the Discussion section.

The phase behaviour of charged membranes is also investi-gated in the presence of salt (10 mM NaCl solution) for variouscharged/neutral mixtures. The miscibility temperatures ofDOPG(�)/DPPC and DOPC/DPPG(�) with NaCl solutions areindicated by open triangles and squares, respectively, inFig. 1(B). The phase separation was enhanced by the addition of

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salt for DOPG(�)/DPPC, which is in agreement with the previousndings.19,21 On the other hand, the phase separation of DOPC/DPPG(�) with NaCl was suppressed. It seems that the phasebehaviour in charged membranes with salt approaches that ofthe neutral mixture, DOPC/DPPC. This is consistent with thefact that salt screens the electrostatic interactions of thecharged DOPG(�) and DPPG(�) lipids.

Ternary lipid/cholesterol mixtures

In general, cholesterol prefers to be localized in the saturatedlipid-rich phase rather than in the unsaturated lipid-rich one.However, the localization of cholesterol also depends stronglyon the structure of the lipid head group.24 We investigated thelocalization of cholesterol and the resulting phase behaviour internary mixtures composed of a neutral saturated lipid, nega-tively charged saturated lipid and cholesterol, such as DPPC/DPPG(�)/Chol. The effect of the hydrocarbon tail was excludedby using lipids with the same acyl chain.

The phase behavior of DPPC/DPPG(�)/Chol mixtures for MilliQ water and NaCl aqueous solutions is summarized in Fig. 2.Although the cholesterol solubility limit in phospholipidmembranes is about 60%, we show the results for Chol > 60% toemphasize the phase boundary, especially in the case of Milli Qwater. For membranes consisting only of neutral lipids (DPPC/Chol ¼ 80 : 20), the phase separation was not observed at roomtemperature, as shown in image 1 of Fig. 2. In the DPPC/Cholbinary mixture, however, it was reported that the nanoscopicdomains are formed even though they cannot be detected usingoptical microscopes.25 On the other hand, when we replaced afraction of the DPPC with negatively charged lipid DPPG(�),DPPC/DPPG(�)/Chol ¼ 40 : 40 : 20, a stripe-shaped domain wasobserved using Rhodamine–DHPE uorescent dye as shown inimage 2 of Fig. 2. Since the stripe-shaped domain has ananisotropic shape, this is a strong indication that the domain isin the So phase. The phase behavior of DPPC/DPPG(�)/Cholmixtures in Milli Q water is summarized in the le diagram ofFig. 2. For higher concentrations of DPPC or cholesterol, two-phase vesicles were not observed or rarely observed (open

Fig. 2 Phase diagrams of DPPC/DPPG(�)/Chol mixtures in Milli Q and NaCtemperature (�22 �C). Filled, grey, and open circles correspond to systemexhibit two-phase regions. Microscopic images of GUVs are taken at com40/40/20 (image 2) in Milli Q water at 22 �C. Cross marks indicate the restable.

7962 | Soft Matter, 2014, 10, 7959–7967

circles). On the other hand, their percentage clearly increaseswith the DPPG(�) concentration (lled circles).

Three experimental ndings led us to conclude that redand dark regions in the uorescence images represent,respectively, DPPC/Chol-rich and DPPG(�)-rich phases. (i)The domain area (dark region) became larger as thepercentage of DPPG(�) was increased, as shown in Fig. 3(C).(ii) While the homogeneous phase is stable for DPPC/Cholmixtures, DPPG(�)/Chol mixtures show a phase separation.Therefore, cholesterol molecules mix easily with DPPC butnot with DPPG(�). (iii) We used BODIPY–Chol as a uorescentprobe that usually favors the cholesterol-rich phase. TheBODIPY–Chol was localized in the red regions stained byRhodamine–DHPE (the data are not shown). Although thebulky BODIPY–Chol may not behave completely like choles-terol, BODIPY–Chol is partitioned into the Chol-rich phase inall our experiments.26 In addition, we also observed the phasebehaviors without BODIPY–Chol, and the observed resultsdid not change in any signicant way. Thus, we think thatbulky BODIPY–Chol plays a rather minor role in our study.

Since most of the cholesterol is included in the DPPC/Chol-rich region, the DPPC/Chol-rich region is identied as a liquid-ordered (Lo) phase. In contrast, the DPPG(�)-rich domain is inan So phase, because its domain shape is not circular but ratherstripe-like. We also note that without cholesterol, a membranecomposed of pure DPPG(�) will be in an So phase atroom temperature (lower than its chain melting temperature,Tm ¼ 41 �C). Our results indicate that DPPG(�) tends to repelDPPC and cholesterol. In other words, the interaction betweenthe head groups of the lipids affects the localization of choles-terol. Furthermore, as the fraction of DPPG(�) of DPPC/DPPG(�)/cholesterol membranes increases, the corresponding misci-bility temperature also increases continuously (Fig. 3(A)). Forsystems with the DPPG(�) percentage of over 30%, a two-phasecoexistence was observed even above the chain meltingtemperature of DPPG(�) (Table 1). It implies that the head groupinteraction of DPPG(�) makes a large contribution to thestabilization of the phase structure. We will further discuss thispoint in the Discussion section.

l solutions (left: Milli Q, centre: NaCl 1 mM, right: NaCl 10 mM) at rooms where 60–100%, 40–60%, and 0–40% of the vesicles, respectively,position of DPPC/Chol ¼ 80/20 (image 1) and DPPC/DPPG(�)/Chol ¼

gion where the vesicles formed by the natural swelling method are not

This journal is © The Royal Society of Chemistry 2014

Fig. 3 (A) Phase diagram of DPPC/DPPG(�)/Chol mixtures for fixedChol ¼ 20%. (B) Percentage of a two-phase vesicle at 22 �C, and (C)area percentage of the So phase at 22 �C as a function of DPPG(�)/DPPC ratio for fixed Chol ¼ 20%. Filled and open squares indicate MilliQ and 10 mM NaCl solution, respectively.

Fig. 4 (A) Phase behaviour in multi-component mixtures of DOPC/DPPC/DPPG(�)/Chol. (A) Microscopy images of GUVs at compositionsof DOPC/DPPC/Chol ¼ 40/40/20 (image 1), DOPC/DPPC/DPPG(�)/Chol ¼ 40/20/20/20 (image 2), and DOPC/DPPG(�)/Chol ¼ 40/40/20(image 3) at 22 �C. Red, green, and dark regions indicate DOPC-rich(Ld), DPPC/Chol-rich (Lo), and DPPG(�)-rich (So) phases, respectively.The yellow region in image 3, which includes a large amount of DOPCand Chol, indicates an Ld phase. (B) Phase diagram of four-componentmixtures of DOPC/DPPC/DPPG(�)/Chol for fixed Chol ¼ 20% at 22 �C.Black, grey, and light grey regions denote, respectively, Lo/Ld two-phase coexistence, Lo/Ld/So three-phase coexistence, and Ld/So or Lo/So two-phase coexistence.

Paper Soft Matter

We now turn to the addition of salt and its effect on thephase behaviour. The phase-separated regions with 1 mM and10 mM of NaCl are indicated in Fig. 2. As the salt concentrationis increased, the phase separation tends to be suppressed. Thiscan be understood because DPPG(�) is screened in the presenceof salt and approaches the behaviour of the neutral DPPC. Thisobservation is qualitatively consistent with the result of DOPC/DPPG(�) mixtures shown in Fig. 1. For a xed amount of Chol ¼20%, we measured the percentage of two-phase vesicles and thearea percentage of the So phase. The results are summarized inFig. 3(B) and (C). From Fig. 3(B) we can see that the addition ofsalt decreases the percentage of domain formation. Also, thephase separation is enhanced in the region where a largeamount of DPPG(�) is included, as DPPG(�) molecules tend toexclude the cholesterol.

A further nding is shown in Fig. 3(C), where it can be seenthat the area fraction of the So phase decreases by the additionof the salt. Since salt screens the DPPG(�) charge, DPPG(�) tends

This journal is © The Royal Society of Chemistry 2014

to be incorporated into the Lo phase, similar to what is seen forneutral DPPC.

Four-component mixtures of lipid and cholesterol

From the results of ternary mixtures, we conclude that choles-terol prefers to be localized in the neutral DPPC-rich domainsrather than in the DPPG(�)-rich ones.

Next, we investigated four-component mixtures of DOPC/DPPC/DPPG(�)/Chol. Previously, a number of studies have usedthe mixtures of DOPC/DPPC/Chol as a biomimetic systemrelated to modelling of ras.8 In these mixtures, unsaturatedlipids (DOPC) form an Ld-phase, whereas domains rich insaturated lipids (DPPC) and cholesterol form an Lo-phase.Aiming to reveal the effect of charges on the Ld/Lo phase sepa-ration, we replace a fraction of the DPPC component in theDOPC/DPPC/Chol mixture with a negatively charged saturatedlipid, DPPG(�). We also screen the head group charge by addingsalt, and examined how the charged lipid, 4th component,affects phase organization of the ternary mixture.

For ternary mixtures with DOPC/DPPC/Chol ¼ 40 : 40 : 20(without the charged lipid), a phase separation is observed,Fig. 4(A1), using the Rhodamine–DHPE dye (red color) and theBODIPY–Chol dye (green color). The circular green domains arerich in DPPC and cholesterol, inferring an Lo phase, while thered region is a DOPC-rich (Ld) phase. When half of DPPC wasreplaced by the charged DPPG(�), a distinct phase separation

Soft Matter, 2014, 10, 7959–7967 | 7963

Fig. 5 (A) Fluorescence microscopy images of phase separation inDOPC/DPPC/DPPG(�)/Chol ¼ 40 : 15 : 25 : 20 hydrated by Milli Qwater (image 1) and 10 mM NaCl solution (image 2) at 22 �C. (B) Thephase diagram of four-component mixtures hydrated by Milli Q water(upper graph) and 10 mM NaCl solution (lower graph), respectively.The temperature was fixed at 22 �C. The relative ratio betweenDPPG(�) and DPPC is changed while keeping the fixed amount ofDOPC ¼ 40% and Chol ¼ 20%. Black, grey, and light grey regionsindicate the Lo/Ld two-phase coexistence, Lo/Ld/So three-phasecoexistence, and Ld/So or Lo/So two-phase coexistence, respectively.

Soft Matter Paper

(three-phase coexistence) was observed in the four-componentmixture, DOPC/DPPC/DPPG(�)/Chol ¼ 40 : 20 : 20 : 20, asshown in Fig. 4(A2). The black regions that appear inside thegreen domains contain a large amount of DPPG(�) as is the caseof ternary mixtures. Because this black region excludes anyuorescent dyes, the DPPG(�)-rich region is inferred as the Sophase. We consider that the observed three-phase coexistence isequilibrated, since the three-phase coexistence reappears atthe same temperature when the system is heated and cooledagain.

Moreover, for ternary mixtures of DOPC/DPPG(�)/Chol ¼40 : 40 : 20 without DPPC, a coexistence between So and Ldphases is observed as shown in Fig. 4(A3). The phase diagram ofDOPC/DPPC/DPPG(�) for xed Chol ¼ 20% presented inFig. 4(B) shows that the phase-separation strongly depends onthe DPPG(�) concentration. The boundary between the Lo/Soand Ld/So coexistence is not marked on the phase diagram,because from optical microscopy it was not possible to distin-guish between the Lo and Ld phases. But the region where Socoexists with either Lo or Ld is indicated as a light grey region inthe phase diagram.

Interestingly, at DOPC/DPPC/DPPG(�)/Chol¼ 40 : 15 : 25 : 20,a transition between the two-phase and the three-phase coexis-tence was driven by adding salt, as shown in the images ofFig. 5(A). In Fig. 5(B), the percentage of phase-separated vesicleshydrated with 10 mM NaCl solution is presented for a xedfraction of DOPC ¼ 40% and Chol ¼ 20%. As shown in Fig. 5(B),the phase separation changes with the DPPG(�) concentration.Without salt, the phase boundary between Lo/Ld the two-phasecoexistence and the Lo/Ld/So three-phase coexistence is posi-tioned at DPPC/DPPG(�) ¼ 25 : 15. On the other hand, in 10 mMNaCl solution, the phase boundary is DPPC/DPPG(�) ¼ 20 : 20.The phase boundary between the Lo/Ld/So three-phase coexis-tence and the Ld/So or Lo/So two-phase coexistence also dependson the salt condition: the boundaries are DPPC/DPPG(�)¼ 20 : 20(without salt) and 15 : 25 (10 mM NaCl). These results suggestthat the addition of salt affects the phase structure of DOPC/DPPC/DPPG(�)/Chol mixtures.

Discussion

One of our important results is that when neutral lipids arereplaced by charged ones, the phase separation was suppressedfor the DOPG(�)/DPPC mixtures, whereas it was enhanced formixtures of DOPC/DPPG(�). Furthermore, by adding salt, thesetwo mixtures approached the behaviour of the non-chargedDOPC/DPPC mixture. As mentioned above, it was reported inthe previous experiments19,21–23 that phase separation of othermixtures containing negatively charged unsaturated lipids wassuppressed similarly to our DOPG(�)/DPPC result. However, theenhanced phase separation for DOPC/DPPG(�) is novel andunaccounted for.

We discuss now several theoretical ideas that are related tothese empirical observations based on a phenomenological freeenergy model.19,20,27,28 The rst step is to take into account onlythe electrostatic contribution to the free energy, fel, using thePoisson–Boltzmann (PB) theory. For symmetric monovalent

7964 | Soft Matter, 2014, 10, 7959–7967

salts (e.g., NaCl), the electric potential J(z) at distance z from acharged membrane satises the PB equation:

d2J

dz2¼ 2enb

3Wsinh

eJ

kBT; (1)

where e is the electronic charge, nb the bulk salt concentration,and 3W the dielectric constant of the aqueous solution, kB theBoltzmann constant, and T the temperature. For a chargedmembrane with area fraction f of negatively charged lipids, thesurface charge density is written as s ¼ �ef/S. The cross-sectional area S of the two lipids is assumed, for simplicity, tobe the same. The PB eqn (1) can be solved analytically byimposing s as the electrostatic boundary condition, and theresulting electrostatic free energy is obtained as29

This journal is © The Royal Society of Chemistry 2014

Paper Soft Matter

fel�f� ¼ 2kBT

Sf

241�

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þ ðp0fÞ2

qp0f

þ ln

�p0fþ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1þ ðp0fÞ2

q �35;(2)

where p0 ¼ 2plBlD/S is a dimensionless parameter proportionalto the Debye screening length lD ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

3wkBT=2e2nbp

, and to1/S, while lB ¼ e2/(4p3wkBT) z 7 A is the Bjerrum length.

One essential outcome of the PB model is that for any p0, theelectrostatic free energy fel increases monotonically as a func-tion of f, and a large fraction of negatively charged lipid willincrease the free energy substantially. This implies that anycharged domain formed due to lipid/lipid lateral phase sepa-ration would cost an electrostatic energy. Hence, within the PBapproach, the phase separation in charged/neutral mixtures oflipids should be suppressed (rather than enhanced) ascompared with neutral ones. Indeed, phase diagrams calculatedby using a similar PB approach clearly showed the suppressionof the phase separation.19,20,30,31

The above argument does not explain all our experimentalndings. Mixtures containing negatively charged saturatedlipids are found to enhance the phase separation and indicatethat there should be an additional attractive mechanismbetween charged saturated lipids to overcome the electrostaticrepulsion. Indeed, the demixing temperature in the DOPC/DPPG(�) mixture (Fig. 1) was found to be even higher than thechain melting temperature of pure DPPG(�) (Tm ¼ 41 �C).Furthermore, the charged DPPG(�)/Chol binary mixturesexhibited the phase separation, whereas the neutral DPPC/Cholmixtures (see Fig. 2) did not.

The next step is to include entropic and enthalpic terms inthe free energy for a membrane consisting of a mixture ofnegatively charged and neutral lipids,

ftot ¼ kBT

S½fln fþ ð1� fÞlnð1� fÞ þ cfð1� fÞ� þ fel; (3)

where the rst and second terms in the square brackets accountfor the entropy and enthalpy of mixing between the charged andneutral lipids, respectively, while the last term, fel, is the elec-trostatic free energy as in eqn (2). As before, f is the area frac-tion of the negatively charged lipid, 1 � f is that of the neutrallipid, and c is a dimensionless interaction parameter betweenthe two lipids (of non-electrostatic origin). Note that we took forsimplicity the cross-sectional area S of the two lipids to be thesame, meaning that f can be thought of as the charged lipidmole fraction. We note that the free energy formulation as ineqn (3) was used in other studies, such as surfactant adsorptionat uid–uid interface32 or lamellar–lamellar phase transition.33

In the case of a neutral lipid mixture membrane (fel ¼ 0), thismodel leads to a lipid/lipid demixing curve with a critical pointlocated at fc ¼ 0.5, cc ¼ 2.

The phase behaviour difference between mixtures of DOPC/DPPG(�) and DOPG(�)/DPPC also suggests a specic attractiveinteraction between DPPG(�) molecules. This is not accountedfor by the PB theory of eqn (2), but the enhanced phase sepa-ration can effectively be explained in terms of an increased c-value in eqn (3) for mixtures containing DPPG(�). We plan to

This journal is © The Royal Society of Chemistry 2014

explore the origins of such non-electrostatic attractive contri-butions in a future theoretical study, and in particular, toexplore the relationship between the electrostatic surface pres-sure and the phase separation.34,35

Although DOPG(�)/DPPC and DOPC/DPPG(�) mixtures lookvery similar from the electrostatic point of view, it is worthwhileto point out some additional difference between these mixtures(beside the value of the c parameter). In particular, the phasebehavior of DOPC/DPPG(�) approaches that of the neutralDOPC/DPPC system by adding salt. Since the attractive forcebetween DPPG(�) molecules vanishes by the addition of salt, weconsider that this attractive force may be related to the chargeeffect. Because DOPG(�) has an unsaturated bulky hydrocarbontail, its cross-sectional area S is larger than that of DPPG(�) thathas a saturated hydrocarbon tail. In the literature, the cross-sectional areas of DOPG(�) and DPPG(�) are reported to be68.6 A2 (at T¼ 30 �C) and 48 A2 (at T¼ 20 �C), respectively.36 Thisarea difference affects the surface charge density s ¼ �ef/S. Asa result, the counterion concentrations near the chargedmembrane are different for DOPG(�)/DPPC as compared withDOPC/DPPG(�). Based on the PB theory, eqn (1), one can obtainthe counterion concentration n0 ¼ n+(z / 0), adjacent to themembrane

n0 ¼ nb

�p0fþ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffið p0fÞ2 þ 1

q �2

: (4)

This relationship is known as the Grahame equation,37,38 and isused in Fig. 6 to plot n0 for nb¼ 10mM. As shown in Fig. 6(A), n0sharply increases when the cross-sectional area S decreases.This tendency is signicantly enhanced at higher area fraction f

of the charged lipid. In Fig. 6(B), n0 is plotted forS¼ 50 A2 (solidline) and 70 A2 (dashed line), which to a good approximationcorrespond to the values of DPPG(�) and DOPG(�), respectively.The larger value of n0 for DPPG(�) may inuence the relativedomain stability that cannot be described by the simplecontinuum PB theory. We also speculate that the hydrogenbonds between charged head groups and water molecules canbe affected by the presence of a large number of counterions.Although this counter-ion condensation is one of the possibleexplanations for the strong attraction between DPPG(�) mole-cules, it is not enough in order to describe the underlyingmechanism completely. In addition, it is important to under-stand whether this attractive force is also observed in systemsincluding other types of charged lipids (e.g. phosphatidylserine(PS(�))). Such questions remain for future explorations.

Moreover, we found that ternary mixtures of DPPC/DPPG(�)/Chol exhibit phase separation between DPPC/Chol-rich andDPPG(�)-rich phases. This is because the strong attractionbetween DPPG(�) molecules excludes cholesterol from DPPG(�)-rich domains. In addition, the difference of the molecular tiltbetween different lipids may also affect this phase separation.The localization of cholesterol strongly depends on the molec-ular shape of membrane phospholipids. It was reported thatpolar lipids, such as DPPC, which contain both positive andnegative charges in their head group, tend to tilt due to elec-trostatic interactions between the neighboring polar lipids.39,40

Soft Matter, 2014, 10, 7959–7967 | 7965

Fig. 6 (A) The counterion concentration, n0¼ n+(z/ 0), extrapolatedto the membrane vicinity as a function of cross-sectional area per lipidf for the bulk salt concentration, nb ¼ 10 mM. The different linecolours represent f ¼ 0.25 (black), 0.5 (red), 0.75 (blue), and 1.0(green). (B) The counterion concentration at the membrane as afunction of the charged lipid concentration, for bulk salt concentra-tion, nb ¼ 10 mM. The solid and dashed lines denote S ¼ 50 A2 and 70A2, respectively.

Soft Matter Paper

The tilting produces an intermolecular space that cholesterolcan occupy. However, since the molecular orientation ofDPPG(�) is almost perpendicular to the membrane surface, itwill be unfavorable for cholesterol to occupy such a narrowspace between neighboring DPPG(�) molecules.

The three phase coexistence in four-component mixtures ofDOPC/DPPC/DPPG(�)/Chol ¼ 40 : 20 : 20 : 20 could be causedby the samemechanism. Unsaturated DOPC forms an Ld phase,whereas cholesterol, which is localized in DPPC domains, formsan Lo phase. Thus, the DPPG(�)-rich region results in an Sophase. Since the hydrocarbon tails of DPPG(�) in the So phaseare highly ordered, whereas the DOPC hydrocarbon tails in theLd phase are disordered, the So/Ld line tension is larger than theline tension of the So/Lo interface. Therefore, So domains aresurrounded by Lo domains in order to prevent a direct contactbetween So and Ld domains.

Although charged lipids in biomembranes are generallyassumed to be in the uid phase, the So phase with a largeamount of charged lipids is observed in our experiments (on 4-component mixtures). Notably, the formation of the So phasehas been reported in model membrane systems either by

7966 | Soft Matter, 2014, 10, 7959–7967

decreasing the cholesterol fraction or by increasing themembrane surface tension.7,8 Although the So phase has notbeen seen in vivo, we believe that our study on modelmembranes is meaningful and will help to reveal some impor-tant physicochemical mechanisms that underlie the phasebehaviour and domain formation of lipid membranes in vivo.The Lo domains in articial membranes can be regarded asmodels mimicking ras in biomembranes. Because most of theproteins have electric charges, sections of the proteins that havepositive charges can easily be attached to the negatively chargeddomains due to electrostatic interactions. Conversely, nega-tively charged sections of proteins are electrically excluded fromsuch domains. Thus, such charged domains may play animportant role in the selective adsorption of chargedbiomolecules.

Finally, we comment that, in all of our experiments, the saltconcentration was 10 mM. This concentration is lower than theconcentration in physiological conditions of living cells, wherethe monovalent salt concentration is about�140mM. From ourresults, we can see that screening by the salt is signicant evenfor 10 mM.19,20,30,31

Conclusions

In the present study, we investigated the phase separationinduced by negatively charged lipids. As compared to the phase-coexistence region (in the phase diagram) of neutral DOPC/DPPC mixtures, the phase separation in the charged DOPG(�)/DPPC case is suppressed, whereas it is enhanced for thecharged DOPC/DPPG(�) system. The phase behaviours of bothcharged mixtures approach that of the neutral mixture whensalt is added due to screening of electrostatic interactions. InDPPC/DPPG(�)/Chol ternary mixtures, the phase separationoccurs when the fraction of charged DPPG(�) is increased. Thisresult implies that cholesterol localization is inuenced by thehead group structure as well as the hydrocarbon tail structure.Furthermore, we observed a three-phase coexistence in four-component DOPC/DPPC/DPPG(�)/Chol mixtures, and that thephase-separation strongly depends on the amount of chargedDPPG(�).

Our ndings shed some light on how biomembranes changetheir own structures, and may help to understand the mecha-nisms that play an essential role in the interactions of proteinswith lipid mixtures during signal transduction.

Acknowledgements

Technical assistance from Ms Ryoko Sugimoto and Mr MasatoAmino is greatly appreciated. We thank Mr M. C. Blosser, Dr R.Dimova, Dr M. Hishida, Dr W. Shinoda and Dr T. Taniguchi forfruitful discussions and comments. This work was supported inpart by the Sasagawa Scientic Research Grant from The JapanScience Society and Young Scientist (B) from JSPS and onPriority Areas “Molecular Robotics” and “Spying Minority inBiological Phenomena” from MEXT and Kurata Grant from theKurata Memorial Hitachi Science and Technology Foundation.SK acknowledges support from the Grant-in-Aid for Scientic

This journal is © The Royal Society of Chemistry 2014

Paper Soft Matter

Research on Innovative Areas “Fluctuation & Structure” (grantno. 25103010), Grant-in-Aid for Scientic Research (C) grant no.24540439 from the MEXT of Japan, and the JSPS Core-to-CoreProgram “International research network for non-equilibriumdynamics of so matter”. DA acknowledges support from theIsrael Science Foundation (ISF) under grant no. 438/12.

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