Soft Matter
View Article Online

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

PAPER

Cite this: Soft Matter, 2014, 10, 7959

View Journal | View Issue

Charge-induced phase separation in lipid
membranes
Hiroki Himeno,†a Naofumi Shimokawa,†a Shigeyuki Komura,b David Andelman,cTsutomu 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

neutrallipid,

dioleoylphosphatidylglycerol

dipalmitoylphosphatidylcholine(DOPG()),saturatedcharged(DPPC),
lipid,

unsaturated

chargedlipid,

dipalmitoylphosphatidylglycerol(DPPG()), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, thecombination of the charged head with the saturation of the hydrocarbon tail is a dominant factor in thestability 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 phaseseparation 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 ofReceived 19th May 2014
Accepted 28th July 2014

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

DOI: 10.1039/c4sm01089b

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

www.rsc.org/softmatter

free energy approach.

Introduction
One of the major components of cell membranes is their lipid
bilayer composed of a mixture of several phospholipids, allhaving 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 ras are believed to function as a platform on which
proteins are attached during signal transduction andmembrane traﬃcking.3 It is commonly believed (but still
debatable) that the ra domains are associated with phaseseparation 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.5–7 In particular, studies of phase separation andmembrane dynamics have been performed on such GUVsaSchool of Materials Science, Japan Advanced Institute of Science and Technology, 1-1
Asahidai, Nomi, Ishikawa, 923-1292, Japan. E-mail: t-hamada@jaist.ac.jp; Tel:
+81-761-51-1670b

Department of Chemistry, Graduate School of Science and Engineering, Tokyo
Metropolitan University, Tokyo 192-0397, Japanc
Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University,
Ramat Aviv 69978, Tel Aviv, Israel

consisting of saturated lipids, unsaturated lipids and cholesterol.8 Multi-component membranes phase separate into
domains rich in saturated lipids and cholesterol, whereas thesurrounding uid phase is composed largely of unsaturated
lipids. 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, biomembranes 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 includes
15% of PG().12 Although the charged lipid fraction in eukaryotic 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 cardiolipin (CL()), phosphatidylserine (PS()) and PG().14,15 It is
indispensable to include the eﬀect of electrostatic interactions
on the phase behavior in biomembranes. To emphasize evenfurther 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 additionof salt, even when the two lipids have the same hydrocarbon† These authors contributed equally to this work.

This journal is © The Royal Society of Chemistry 2014

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

View Article Online

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

Soft Matter

tail.16–18 For this charged lipid mixture, the segregation ismediated only by the electrostatic interaction between the lipids
and the electrolyte.
In related studies, Shimokawa et al.19,20 studied mixturesconsisting of neutral saturated lipid (DPPC), negatively charged
unsaturated 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 in
mixtures containing charged lipids. However, the eﬀects ofelectric charges on the phase behaviour in lipid/cholesterol
mixtures have not been addressed so far systematically.
In the present study, we investigate the physicochemical
properties of model membranes containing various mixtures ofcharged lipids, with the hope that the study will enhance ourunderstanding of biomembranes in vivo, which are much more
complex. We examine the electric charge eﬀect on the phasebehaviour using uorescence microscopy and confocal laser
scanning microscopy. In addition, the salt screening eﬀect on
charged membranes is explored. We discuss these eﬀects inthree stages starting from the simpler one. First, the phase
diagram in charged binary mixtures of unsaturated and saturated 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 is
added as a fourth component to a ternary mixture of neutral
saturated and unsaturated lipids and cholesterol. We conclude
by discussing qualitatively the phase behaviour of charged
membranes using a free energy modeling. The counterion
concentration adjacent to the charged membrane is calculated
in order to explore the relationship between the electric charge
and the ordering of the hydrocarbon tail.

Materials and methods
Materials
Neutral unsaturated lipid dioleoyl-sn-glycero-3-phosphocholine(DOPC, with chain melting temperature, Tm ¼ 20  C), neutral
saturated lipid dipalmitoyl-sn-glycero-3-phosphocholine (DPPC,
Tm ¼ 41  C), negatively charged unsaturated lipid 1,2-dioleoylsn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) (DOPG(),Tm ¼ 18  C), negatively charged saturated lipid 1,2-dipalmitoyl-sn-glycero-3-phospho-(10 -rac-glycerol)(sodium
salt)(DPPG(), Tm ¼ 41  C), and cholesterol were obtained from
Avanti Polar Lipids (Alabaster, AL). BODIPY labelled cholesterol(BODIPY–Chol) and Rhodamine B–1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine(Rhodamine–DHPE)
werepurchased from Invitrogen (Carlsbad, CA). Deionized water was
obtained from a Millipore Milli-Q purication system. We chose
phosphatidylcholine (PC) as the neutral lipid head and phosphatidylglycerol (PG) as the negatively charged lipid headbecause the chain melting temperature of PC and PG lipids
having the same acyl tails is almost identical. In cellular

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

Papermembranes, PC is the most common lipid component, and PG
is highly representative among charged lipids.
Preparation of giant unilamellar vesicles
Giant unilamellar vesicles (GUVs) were prepared by a gentle
hydration method. Lipids and uorescent dyes were dissolvedin 2 : 1 (vol/vol) chloroform–methanol solution. The organic
solvent was evaporated under a ow of nitrogen gas, and thelipids were further dried under vacuum for 3 h. The lms were
hydrated with 5 mL deionized water at 55  C for 5 min (prehydration), and then with 200 mL deionized water or NaClsolution for 1–2 h at 37  C. The nal lipid concentration was
0.2 mM. Rhodamine–DHPE and BODIPY–Chol concentrations
were 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. 0.1
mm. We observed the membrane structures with a uorescent
microscope (IX71, Olympus, Japan) and a confocal laser scanning microscope (FV-1000, Olympus, Japan). In the present
study, Rhodamine–DHPE and BODIPY–Chol were used asuorescent dyes. Rhodamine–DHPE labels the lipid liquid
phase, whereas BODIPY–Chol labels the cholesterol-rich one. A
standard lter set, U-MWIG with excitation wavelength lex ¼
530–550 nm and emission wavelength lem ¼ 575 nm, was used
to monitor the uorescence of Rhodamine–DHPE, and anotherlter set, U-MNIBA with lex ¼ 470–495 nm and lem ¼ 510–550
nm, was used for the BODIPY–Chol dye. The sample temperature was controlled with a microscope stage (type 10021, Japan
Hitec).
Measurement of miscibility temperature
The miscibility temperature corresponds to the boundary
between one- and two-phase regions. It is dened as the phaseseparation point at which more than 50% of the phase-separated domains have disappeared upon heating. The temperature was increased from room temperature to the desired
temperature by 10  C min1, 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 vesicles
was over 50%, the temperature was further increased by 2  C.
We continued this procedure until the percentage of two-phasevesicles decreased below 50%.

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

This journal is © The Royal Society of Chemistry 2014

View Article Online

Paper

Soft Matter

The four neutral and negatively charged lipids and their chain
melting temperatures

Table 1

Saturated tail (DP)Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

Unsaturated tail (DO)

Neutral head (PC)

Negative charged
head (PG)

DPPC
Tm ¼ 41  C
DOPC
Tm ¼ 20  C

DPPG()Tm ¼ 41  C
DOPG()Tm ¼ 18  C

Fig. 1(A) shows the phase behaviour in these three binary
mixtures taken for three temperatures: T ¼ 22  C, 30  C and40  C. Each of the images was taken by superimposing several
pictures at a slightly diﬀerent focus position of the confocal
laser scanning microscope. At room temperature (22  C), all

Phase behaviour in binary lipid mixtures (DOPC/DPPC, DOPC/
DPPG(), DOPG()/DPPC). (A) Microscopic images of the phase separation for three temperatures, 22  C, 30  C and 40  C. Red and black
regions 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).
Fig. 1This 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) that
includes a large amount of the unsaturated lipid, while the dark
regions 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). On
the other hand, the two other mixtures (DOPC/DPPC and DOPC/
DPPG()) still retained the phase-separated structure (images 4
and 5). As the temperature was further increased to 40  C, the
DOPC/DPPC mixture also became homogeneous (image 1),
while the DOPC/DPPG() mixture still retained its phase-separated structure at the same temperature (image 2). Thus, the
DOPC/DPPG() mixture shows the highest miscibility temperature of all the studied systems. Note that a similar phase-separated structure was reported in binary mixtures of egg
sphingomyelin (eSM)/DOPG().21,23
Miscibility temperatures of binary mixtures are summarized
in Fig. 1(B). The lled circles denote the neutral lipid mixture,
DOPC/DPPC. We also examined charged binary mixtures of two
negatively charged lipids, DOPG()/DPPG(). Miscibility
temperatures (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 miscibility
temperature in the DOPG()/DPPC mixture (denoted by lledtriangles) became lower as compared with a neutral lipid
mixture, DOPC/DPPC. In other words, the phase separation issuppressed when a negatively charged unsaturated lipid is
included. This result is consistent with previous studies performed on lipid mixtures containing negatively charged unsaturated lipids.19,21–23 At higher concentrations of DPPC, phaseseparated domains could not be observed for mixtures of
DOPG()/DPPC ¼ 20 : 80 and 10 : 90, because stable vesicle
formation 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 signicantly as compared with
the neutral system. In particular, we can see that a maximum inthe miscibility temperature appears in the phase diagram
around 50% relative concentration of the saturated lipid.
Interestingly, at DOPC/DPPG() ¼ 50 : 50, the miscibility
temperature of about 44  C was higher than 41  C of the
DPPG() chain melting temperature (Table 1). Thus, the phaseseparation is enhanced in mixtures containing the negatively
charged saturated lipid (DPPG()). This result should be contrasted 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 investigated in the presence of salt (10 mM NaCl solution) for various
charged/neutral mixtures. The miscibility temperatures of
DOPG()/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 ofSoft Matter, 2014, 10, 7959–7967 | 7961

View Article Online

Soft Matter

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

salt for DOPG()/DPPC, which is in agreement with the previous
ndings.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 of
the 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 saturated
lipid-rich phase rather than in the unsaturated lipid-rich one.
However, the localization of cholesterol also depends strongly
on the structure of the lipid head group.24 We investigated the
localization of cholesterol and the resulting phase behaviour in
ternary mixtures composed of a neutral saturated lipid, negatively charged saturated lipid and cholesterol, such as DPPC/DPPG()/Chol. The eﬀect of the hydrocarbon tail was excluded
by using lipids with the same acyl chain.
The phase behavior of DPPC/DPPG()/Chol mixtures for Milli
Q water and NaCl aqueous solutions is summarized in Fig. 2.
Although the cholesterol solubility limit in phospholipid
membranes is about 60%, we show the results for Chol > 60% toemphasize the phase boundary, especially in the case of Milli Q
water. For membranes consisting only of neutral lipids (DPPC/
Chol ¼ 80 : 20), the phase separation was not observed at room
temperature, as shown in image 1 of Fig. 2. In the DPPC/Cholbinary mixture, however, it was reported that the nanoscopic
domains are formed even though they cannot be detected using
optical 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 is
in 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, twophase vesicles were not observed or rarely observed (openPapercircles). On the other hand, their percentage clearly increases
with the DPPG() concentration (lled circles).
Three experimental ndings led us to conclude that red
and 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 but
not with DPPG(). (iii) We used BODIPY–Chol as a uorescentprobe that usually favors the cholesterol-rich phase. The
BODIPY–Chol was localized in the red regions stained by
Rhodamine–DHPE (the data are not shown). Although thebulky BODIPY–Chol may not behave completely like cholesterol, BODIPY–Chol is partitioned into the Chol-rich phase in
all our experiments.26 In addition, we also observed the phasebehaviors without BODIPY–Chol, and the observed results
did not change in any signicant 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/Cholrich region, the DPPC/Chol-rich region is identied as a liquidordered (Lo) phase. In contrast, the DPPG()-rich domain is in
an So phase, because its domain shape is not circular but rather
stripe-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 repel
DPPC and cholesterol. In other words, the interaction between
the head groups of the lipids aﬀects the localization of cholesterol. Furthermore, as the fraction of DPPG() of DPPC/DPPG()/cholesterol membranes increases, the corresponding miscibility temperature also increases continuously (Fig. 3(A)). For
systems with the DPPG() percentage of over 30%, a two-phasecoexistence was observed even above the chain melting
temperature 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.

Fig. 2 Phase diagrams of DPPC/DPPG()/Chol mixtures in Milli Q and NaCl solutions (left: Milli Q, centre: NaCl 1 mM, right: NaCl 10 mM) at room
temperature (22  C). Filled, grey, and open circles correspond to systems where 60–100%, 40–60%, and 0–40% of the vesicles, respectively,
exhibit two-phase regions. Microscopic images of GUVs are taken at composition of DPPC/Chol ¼ 80/20 (image 1) and DPPC/DPPG()/Chol ¼
40/40/20 (image 2) in Milli Q water at 22  C. Cross marks indicate the region where the vesicles formed by the natural swelling method are notstable.

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

This journal is © The Royal Society of Chemistry 2014

View Article Online

Paper

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

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

Four-component mixtures of lipid and cholesterol
From the results of ternary mixtures, we conclude that cholesterol prefers to be localized in the neutral DPPC-rich domains
rather than in the DPPG()-rich ones.
Next, we investigated four-component mixtures of DOPC/
DPPC/DPPG()/Chol. Previously, a number of studies have used
the mixtures of DOPC/DPPC/Chol as a biomimetic system
related to modelling of ras.8 In these mixtures, unsaturated
lipids (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 separation, we replace a fraction of the DPPC component in the
DOPC/DPPC/Chol mixture with a negatively charged saturated
lipid, DPPG(). We also screen the head group charge by adding
salt, 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 the
BODIPY–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 was
replaced by the charged DPPG(), a distinct phase separation

Fig. 3 (A) Phase diagram of DPPC/DPPG()/Chol mixtures for ﬁxed
Chol 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 Milli
Q and 10 mM NaCl solution, respectively.

We now turn to the addition of salt and its eﬀect on thephase behaviour. The phase-separated regions with 1 mM and
10 mM of NaCl are indicated in Fig. 2. As the salt concentration
is 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 large
amount of DPPG() is included, as DPPG() molecules tend to
exclude 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

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 DOPC
and Chol, indicates an Ld phase. (B) Phase diagram of four-component
mixtures of DOPC/DPPC/DPPG()/Chol for ﬁxed Chol ¼ 20% at 22  C.
Black, grey, and light grey regions denote, respectively, Lo/Ld twophase coexistence, Lo/Ld/So three-phase coexistence, and Ld/So or Lo/
So two-phase coexistence.

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

View Article Online

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

Soft Matter(three-phase coexistence) was observed in the four-component
mixture, DOPC/DPPC/DPPG()/Chol ¼ 40 : 20 : 20 : 20, as
shown 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 any
uorescent 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 at
the same temperature when the system is heated and cooledagain.
Moreover, for ternary mixtures of DOPC/DPPG()/Chol ¼
40 : 40 : 20 without DPPC, a coexistence between So and Ldphases is observed as shown in Fig. 4(A3). The phase diagram of
DOPC/DPPC/DPPG() for xed Chol ¼ 20% presented inFig. 4(B) shows that the phase-separation strongly depends on
the 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 distinguish between the Lo and Ld phases. But the region where So
coexists with either Lo or Ld is indicated as a light grey region in
the phase diagram.
Interestingly, at DOPC/DPPC/DPPG()/Chol ¼ 40 : 15 : 25 : 20,
a transition between the two-phase and the three-phase coexistence was driven by adding salt, as shown in the images ofFig. 5(A). In Fig. 5(B), the percentage of phase-separated vesicles
hydrated with 10 mM NaCl solution is presented for a xed
fraction 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 positioned at DPPC/DPPG() ¼ 25 : 15. On the other hand, in 10 mM
NaCl solution, the phase boundary is DPPC/DPPG() ¼ 20 : 20.
The phase boundary between the Lo/Ld/So three-phase coexistence and the Ld/So or Lo/So two-phase coexistence also depends
on 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 are
replaced by charged ones, the phase separation was suppressed
for the DOPG()/DPPC mixtures, whereas it was enhanced for
mixtures of DOPC/DPPG(). Furthermore, by adding salt, these
two mixtures approached the behaviour of the non-charged
DOPC/DPPC mixture. As mentioned above, it was reported in
the previous experiments19,21–23 that phase separation of othermixtures containing negatively charged unsaturated lipids was
suppressed 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 only
the electrostatic contribution to the free energy, fel, using the
Poisson–Boltzmann (PB) theory. For symmetric monovalent7964 | Soft Matter, 2014, 10, 7959–7967

Paper(A) Fluorescence microscopy images of phase separation in
DOPC/DPPC/DPPG()/Chol ¼ 40 : 15 : 25 : 20 hydrated by Milli Q
water (image 1) and 10 mM NaCl solution (image 2) at 22  C. (B) The
phase 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 between
DPPG() and DPPC is changed while keeping the ﬁxed amount of
DOPC ¼ 40% and Chol ¼ 20%. Black, grey, and light grey regions
indicate the Lo/Ld two-phase coexistence, Lo/Ld/So three-phasecoexistence, and Ld/So or Lo/So two-phase coexistence, respectively.
Fig. 5salts (e.g., NaCl), the electric potential J(z) at distance z from acharged membrane satises the PB equation:
d2 J 2enb
eJ
;
¼
sinh
dz2
kB T
3W

(1)

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

This journal is © The Royal Society of Chemistry 2014

View Article Online

Paper

Soft Matter

qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
32

qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
1  1 þ ðp0 fÞ2
  2kB T
2
fel f ¼þ ln p0 f þ 1þ ðp0 fÞ 5;
f4
p0 f
S

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

(2)
where p0 ¼ 2plBlD/S is a dimensionless p
parameter
proportional
ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ
ﬃ
to the Debye screening length lD ¼ 3w kB T=2e2 nb , and to
1/S, while lB ¼ e2/(4p3wkBT) z 7 Å is the Bjerrum length.
One essential outcome of the PB model is that for any p0, theelectrostatic free energy fel increases monotonically as a function of f, and a large fraction of negatively charged lipid will
increase the free energy substantially. This implies that any
charged domain formed due to lipid/lipid lateral phase separation would cost an electrostatic energy. Hence, within the PB
approach, the phase separation in charged/neutral mixtures oflipids should be suppressed (rather than enhanced) as
compared with neutral ones. Indeed, phase diagrams calculated
by using a similar PB approach clearly showed the suppressionof the phase separation.19,20,30,31
The above argument does not explain all our experimental
ndings. Mixtures containing negatively charged saturated
lipids are found to enhance the phase separation and indicatethat there should be an additional attractive mechanism
between charged saturated lipids to overcome the electrostatic
repulsion. Indeed, the demixing temperature in the DOPC/
DPPG() mixture (Fig. 1) was found to be even higher than the
chain melting temperature of pure DPPG() (Tm ¼ 41  C).
Furthermore, the charged DPPG()/Chol binary mixtures
exhibited the phase separation, whereas the neutral DPPC/Cholmixtures (see Fig. 2) did not.
The next step is to include entropic and enthalpic terms in
the free energy for a membrane consisting of a mixture ofnegatively charged and neutral lipids,
ftot ¼

kB T½fln f þ ð1  fÞlnð1  fÞ þ cfð1  fÞ þ fel ;S(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 electrostatic free energy as in eqn (2). As before, f is the area fraction of the negatively charged lipid, 1  f is that of the neutral
lipid, and c is a dimensionless interaction parameter between
the two lipids (of non-electrostatic origin). Note that we took for
simplicity the cross-sectional area S of the two lipids to be thesame, meaning that f can be thought of as the charged lipid
mole 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 point
located at fc ¼ 0.5, cc ¼ 2.
The phase behaviour diﬀerence between mixtures of DOPC/
DPPG() and DOPG()/DPPC also suggests a specic attractive
interaction between DPPG() molecules. This is not accounted
for by the PB theory of eqn (2), but the enhanced phase separation can eﬀectively be explained in terms of an increased cvalue in eqn (3) for mixtures containing DPPG(). We plan toThis journal is © The Royal Society of Chemistry 2014

explore the origins of such non-electrostatic attractive contributions in a future theoretical study, and in particular, toexplore the relationship between the electrostatic surface pressure and the phase separation.34,35
Although DOPG()/DPPC and DOPC/DPPG() mixtures lookvery similar from the electrostatic point of view, it is worthwhile
to 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 neutral
DOPC/DPPC system by adding salt. Since the attractive force
between DPPG() molecules vanishes by the addition of salt, weconsider that this attractive force may be related to the charge
eﬀect. Because DOPG() has an unsaturated bulky hydrocarbon
tail, its cross-sectional area S is larger than that of DPPG() thathas a saturated hydrocarbon tail. In the literature, the crosssectional areas of DOPG() and DPPG() are reported to be
68.6 Å2 (at T ¼ 30  C) and 48 Å2 (at T ¼ 20  C), respectively.36 This
area diﬀerence aﬀects the surface charge density s ¼ ef/S. As
a result, the counterion concentrations near the charged
membrane are diﬀerent for DOPG()/DPPC as compared with
DOPC/DPPG(). Based on the PB theory, eqn (1), one can obtainthe counterion concentration n0 ¼ n+(z / 0), adjacent to themembrane
qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ2(4)
n0 ¼ nb p0 f þ ð p0 fÞ2 þ 1 :This relationship is known as the Grahame equation,37,38 and isused in Fig. 6 to plot n0 for nb ¼ 10 mM. As shown in Fig. 6(A), n0
sharply increases when the cross-sectional area S decreases.
This tendency is signicantly enhanced at higher area fraction fof the charged lipid. In Fig. 6(B), n0 is plotted for S ¼ 50 Å2 (solidline) and 70 Å2 (dashed line), which to a good approximation
correspond to the values of DPPG() and DOPG(), respectively.
The larger value of n0 for DPPG() may inuence the relative
domain stability that cannot be described by the simple
continuum 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 possible
explanations for the strong attraction between DPPG() molecules, it is not enough in order to describe the underlying
mechanism completely. In addition, it is important to understand whether this attractive force is also observed in systems
including 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 attraction
between DPPG() molecules excludes cholesterol from DPPG()rich domains. In addition, the diﬀerence of the molecular tilt
between diﬀerent lipids may also aﬀect this phase separation.
The localization of cholesterol strongly depends on the molecular 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 electrostatic interactions between the neighboring polar lipids.39,40

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

View Article Online

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

Soft Matter

Paper

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 model
membranes is meaningful and will help to reveal some important physicochemical mechanisms that underlie the phasebehaviour and domain formation of lipid membranes in vivo.
The Lo domains in articial membranes can be regarded asmodels mimicking ras in biomembranes. Because most of theproteins have electric charges, sections of the proteins that havepositive charges can easily be attached to the negatively charged
domains due to electrostatic interactions. Conversely, negatively charged sections of proteins are electrically excluded fromsuch domains. Thus, such charged domains may play animportant role in the selective adsorption of charged
biomolecules.
Finally, we comment that, in all of our experiments, the salt
concentration was 10 mM. This concentration is lower than theconcentration in physiological conditions of living cells, where
the monovalent salt concentration is about 140 mM. From ourresults, we can see that screening by the salt is signicant evenfor 10 mM.19,20,30,31

Conclusions(A) The counterion concentration, n0 ¼ n+(z / 0), extrapolated
to the membrane vicinity as a function of cross-sectional area per lipid
f for the bulk salt concentration, nb ¼ 10 mM. The diﬀerent line
colours 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 concentration, nb ¼ 10 mM. The solid and dashed lines denote S ¼ 50 Å2 and 70
Å2, respectively.
Fig. 6

The tilting produces an intermolecular space that cholesterol
can occupy. However, since the molecular orientation of
DPPG() is almost perpendicular to the membrane surface, itwill be unfavorable for cholesterol to occupy such a narrow
space between neighboring DPPG() molecules.
The three phase coexistence in four-component mixtures of
DOPC/DPPC/DPPG()/Chol ¼ 40 : 20 : 20 : 20 could be causedby the same mechanism. Unsaturated DOPC forms an Ld phase,whereas cholesterol, which is localized in DPPC domains, forms
an Lo phase. Thus, the DPPG()-rich region results in an Sophase. Since the hydrocarbon tails of DPPG() in the So phase
are highly ordered, whereas the DOPC hydrocarbon tails in the
Ld phase are disordered, the So/Ld line tension is larger than theline tension of the So/Lo interface. Therefore, So domains are
surrounded by Lo domains in order to prevent a direct contact
between So and Ld domains.
Although charged lipids in biomembranes are generally
assumed to be in the uid phase, the So phase with a large
amount of charged lipids is observed in our experiments (on 4component mixtures). Notably, the formation of the So phasehas been reported in model membrane systems either by

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

In the present study, we investigated the phase separationinduced by negatively charged lipids. As compared to the phasecoexistence 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 the
charged DOPC/DPPG() system. The phase behaviours of bothcharged mixtures approach that of the neutral mixture whensalt is added due to screening of electrostatic interactions. In
DPPC/DPPG()/Chol ternary mixtures, the phase separation
occurs when the fraction of charged DPPG() is increased. This
result implies that cholesterol localization is inuenced by thehead group structure as well as the hydrocarbon tail structure.
Furthermore, we observed a three-phase coexistence in fourcomponent DOPC/DPPC/DPPG()/Chol mixtures, and that thephase-separation strongly depends on the amount of charged
DPPG().
Our ndings shed some light on how biomembranes change
their own structures, and may help to understand the mechanisms that play an essential role in the interactions of proteins
with lipid mixtures during signal transduction.

Acknowledgements
Technical assistance from Ms Ryoko Sugimoto and Mr Masato
Amino 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 Scientic Research Grant from The Japan
Science Society and Young Scientist (B) from JSPS and on
Priority Areas “Molecular Robotics” and “Spying Minority in
Biological Phenomena” from MEXT and Kurata Grant from theKurata Memorial Hitachi Science and Technology Foundation.
SK acknowledges support from the Grant-in-Aid for ScienticThis journal is © The Royal Society of Chemistry 2014

View Article Online

Paper

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

Published on 26 August 2014. Downloaded by SCD Université Paris 7 on 24/09/2014 14:30:48.

Notes and references
1 K. Simons and J. L. Sampaio, Cold Spring Harbor Perspect.
Biol., 2011, 3, 10.
2 K. G. N. Suzuki and A. Kusumi, Trends Glycosci. Glycotechnol.,
2008, 20, 341.
3 M. Vestergaard, T. Hamada and M. Takagi, Biotechnol.
Bioeng., 2008, 99, 753.
4 R. Lipowsky and R. Dimova, J. Phys.: Condens. Matter, 2003,
15, S31.
5 T. Hamada, Y. Miura, K. Ishii, K. Yoshikawa, M. Vestergaard
and M. Takagi, J. Phys. Chem. B, 2007, 111, 10853.
6 T. Hamada and K. Yoshikawa, Materials, 2012, 5, 2292–2305.
7 T. Hamada, Y. Kishimoto, T. Nagasaki and M. Takagi, So
Matter, 2011, 7, 9061.
8 S. L. Veatch and S. L. Keller, Biophys. J., 2003, 85, 3074.
9 S. L. Veatch and S. L. Keller, Phys. Rev. Lett., 2002, 89, 268101.
10 T. Baumgart, S. T. Hess and W. W. Webb, Nature, 2003, 425,
821.
11 L. Bagotolli and P. B. S. Kumar, So Matter, 2009, 5, 3234.
12 W. Dowhan, Annu. Rev. Biochem., 1996, 66, 199.
13 M. Schlame, J. Lipid Res., 2008, 49, 1607.
14 W. Dowhan, M. Bogdanov and E. Mileykovskaya, in
Biochemistry of Lipids, Lipoproteins and Membranes, Elsevier
Press, Amsterdam, 2008, vol. 5, p. 1.
15 D. Ardail, J. P. Privat, M. Egretcharlier, C. Levrat, F. Lerme
and P. J. Louisot, J. Biol. Chem., 1990, 265, 18797.
16 T. Ito, S. Ohnishi, M. Ishinaga and M. Kito, Biochemistry,
1975, 14, 3064.
17 S. Mittler-Neher and W. Knoll, Biochim. Biophys. Acta, 1993,
1152, 259.

This journal is © The Royal Society of Chemistry 2014

Soft Matter

18 G. Denisov, S. Wanaski, P. Luan, M. Glaser and
S. McLaughlin, Biophys. J., 1998, 74, 731.
19 N. Shimokawa, M. Hishida, H. Seto and K. Yoshikawa, Chem.
Phys. Lett., 2010, 496, 59.
20 N. Shimokawa, S. Komura and D. Andelman, Phys. Rev. E:
Stat., Nonlinear, So Matter Phys., 2011, 84, 031919.
21 C. C. Vequi-Suplicy, K. A. Riske, R. L. Knorr and R. Dimova,
Biochim. Biophys. Acta, 2010, 1798, 1338.
22 M. C. Blosser, J. B. Starr, C. W. Turtle, J. Ashcra and
S. L. Keller, Biophys. J., 2013, 104, 2629.
23 S. Pataraia, Y. Liu, R. Lipowsky and R. Dimova, Biochim.
Biophys. Acta, 2014, 1838, 2036.
24 R. Bibhu, K. Sanat and V. A. Raghunathan, Adv. Planar Lipid
Bilayers Liposomes, 2010, 11, 159.
25 D. Marsh, Biochim. Biophys. Acta, 2009, 1788, 2114.
26 D. Wüstner, Chem. Phys. Lipids, 2007, 146, 1.
27 G. D. Guttman and D. Andelman, J. Phys. II, 1993, 3, 1411.
28 S. May, D. Harries and A. Ben-Shaul, Phys. Rev. Lett., 2002, 89,
268102.
29 D. F. Evans and H. Wennerström, The Colloidal Domain, John
Wiley, New York, 1999.
30 S. May, J. Phys.: Condens. Matter, 2005, 17, R833.
31 E. C. Mbamala, A. Ben-Shaul and S. May, Biophys. J., 2005,
88, 1702.
32 H. Diamant and D. Andelman, J. Phys. Chem., 1996, 100,
13732.
33 D. Harries, R. Podgornik, V. A. Parsegian, E. Mar-Or and
D. Andelman, J. Chem. Phys., 2006, 124, 224702.
34 F. Jähnig, Biophys. Chem., 1976, 4, 309.
35 E. Sackmann, Handbook of Biological Physics, Elsevier Science
B.V, 1995, vol. 1, p. 213.
36 J. H. Kim and M. W. Kim, Eur. Phys. J. E: So Matter Biol.
Phys., 2007, 23, 313.
37 D. C. Grahame, Chem. Rev., 1947, 41, 441.
38 J. N. Israelachivili, Intermolecular and Surface Forces,
Academic Press, London, 2011.
39 J. Huang and G. W. Feigenson, Biophys. J., 1999, 76, 2142.
40 A. Kurrle and P. Rieber, Biochemistry, 1990, 29, 8274.

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