Eur. Phys. J. E 25, 403–413 (2008)
DOI 10.1140/epje/i2007-10307-0

THE EUROPEAN
PHYSICAL JOURNAL E

Adhesion of binary giant vesicles containing negative spontaneous
curvature lipids induced by phase separation
Y. Sakuma1 , M. Imai1,a , M. Yanagisawa1 , and S. Komura2
1
2

Department of Physics, Ochanomizu University, Bunkyo, Tokyo 112-8610, Japan
Department of Chemistry, Tokyo Metropolitan University, Tokyo 192-0397, Japan
Received 15 November 2007 and Received in final form 30 January 2008
Published online: 17 April 2008 – c EDP Sciences / Società Italiana di Fisica / Springer-Verlag 2008
Abstract. We report the adhesion of binary giant vesicles composed of two types of phospholipids, one
has negative spontaneous curvature which tends to bend toward the head group and the other has zero
spontaneous curvature. In a homogeneous one-phase region, the giant vesicles do not adhere to each other,whereas in a coexisting two-phase region, the giant vesicles show adhesion. A fluorescence microscopeobservation reveals that the adhesion takes place through the domains rich in phospholipids having negative
spontaneous curvature. We propose a phase separation induced hemifusion model where two apposedmonolayers of adjacent vesicles are hemifused in order to reduce the bending energy of monolayers withnegative spontaneous curvature and the boundary energy between the domains and matrix. We provide astrong evidence for the hemifusion model by lipid transfer experiments.
PACS. 87.16.D- Membranes, bilayers, and vesicles – 87.16.dr Assembly and interactions – 87.14.Cc Lipids

1 Introduction
Adhesion of cell membranes is one of the elementary processes for biological phenomena such as membrane traffic, fertilization and infection by enveloped viruses. Extensive investigations have been performed to understand themembrane adhesion mechanism [1]. It has been consideredthat the cell adhesion is governed by the interplay of specific interactions, generic interactions and membrane elasticity. The specific interactions are lock and key forces between ligand and receptor molecules embedded in the cell
membranes. A well-studied example of the ligand-receptor
pair is a biotin-avidin system, which has the binding energy of ∼ 35kB T . The pairs promote the adhesion of membranes by pulling together opposing membranes in closer
contact.
In addition to the specific interactions, generic interactions including van der Waals, electrostatic, hydration andundulation interactions are present between lipid membranes. For two neutral membranes with distance l, thetotal generic interaction potential energy per unit area [2–8] is given by
Ftot (l) = Fhyd (l) + FvdW (l) + Fund (l),(1)

where the hydration energy, Fhyd (l), has the empiricalform
Fhyd (l) = Ah exp(−l/λh ),(2)ae-mail: imai@phys.ocha.ac.jp

with typical values of Ah ∼= 0.2 J/m2 and λh ∼= 0.3 nm [4].
The van der Waals interaction energy is expressed by


W 1
2
1
FvdW (l) = −
−
+
,
(3)
12π l2(l + δ)2(l + 2δ)2where W ∼= 10−22 –10−21 J is the Hamaker constant and∼δ = 5 nm denotes the membrane thickness. The undulation interaction energy in the presence of membrane tension, τ , [6] is given byτ kB T −l/lτFund = cundeκplτ = kB T /2πτ ,



lτ
l

1/4

,

(4)
(5)

where κ is the bending elasticity of a single membrane andcund is the dimensionless prefactor that determines thestrength of the fluctuation-induced-interaction [9]. The total energy given by equation (1) yields a minimum, whichcauses the adhesion of membranes.
On the other hand, an intermediate hemifusion structure is considered to be formed in the first stage of themembrane fusion [10–15]. In the hemifusion state, theouter leaflets of both bilayers merge whereas the innerleaflets and the aqueous inner contents remain independent as shown in Figure 1(a). Heuvingh and co-workers
showed a mixing of lipids between adhering vesicles using a fluorescence lipid transfer technique, which strongly

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suggests the hemifusion state [16]. From the geometrical point of view, the hemifusion state is preferable forthe lipids with negative spontaneous curvature, whichtends to bend toward the head group. Actually Yang
and Huang observed a periodic hemifused bilayer structure, the so-called rhombohedral phase, for a negative
spontaneous curvature lipid, DPhPC (1,2-diphytanoylsn-glycero-3-phosphocholin) and water system [17,18].
They measured X-ray diffraction patterns from a stackof DPhPC bilayers as a function of the relative humidity.
Decreasing the relative humidity, DPhPC showed a characteristic transition from lamellar bilayer to the rhombohedral structure with a space group of R3̄, where the hemifusion structure forms a hexagonally perforated lamellar
structure with ABCABC stacking as shown in Figure 1(b).
The observation suggests that the two apposed monolayers with a negative spontaneous curvature merge via anhourglass-shaped structure, i.e. the hemifusion.
In this study, we investigate the adhesion of giant unilamellar vesicles (GUVs) by using binary GUVs composedof negative spontaneous curvature phospholipid (NP) andzero spontaneous curvature phospholipid (ZP). Here NP
prefers the hourglass-like interbilayer structure when thetwo vesicles come into contact, whereas ZP stabilizes thebinary GUVs. On the other hand, it is known that the
mixture of phospholipids having different melting temperatures (Tm ) shows a phase separation below the miscibility transition temperature (Tc ). We used unsaturated
and saturated acyl chains to control the melting temperatures of NP and ZP. Thus NP and ZP with different melting temperatures are homogeneously mixed above Tc andform stable spherical GUVs. Below Tc , they are separated
into an NP-rich phase and a ZP-rich phase, and GUVs
may adhere through the NP-rich region. We use DPPE
(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine) with
Tm = 62 ◦ C and DPhPC having no chain melting temperature between +120 and −120 ◦ C as NPs. DPPE has asmall phosphoethanolamine head group, whereas DPhPC
has a bulky and disordered acyl tails, which gives rise to anegative spontaneous curvature. Additionally, DPPC (1,2dipalmitoyl-sn-glycero-phosphocholine; Tm = 41 ◦ C) having zero spontaneous curvature is used as a reference lipid.
These three phospholipids are mixed with ZPs (DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine; Tm = −20 ◦ C)
or DPPC) to form the binary GUVs. Thus we prepared
three different combinations of binary GUVs, i.e., two
NP/ZP pairs, DPPE/DOPC, DPhPC/DPPC and one
ZP/ZP pair, DPPC/DOPC.
In order to investigate the vesicle adhesion induced byNPs, we first address spontaneous curvatures of DPPC,
DPhPC and DPPE by examining the existence of therhombohedral phase using an X-ray diffraction technique.
Then we show the temperature-composition phase diagrams of the three types of the binary GUVs. With thehelp of the obtained phase diagrams, the adhesion behaviors are examined by a micro-manipulation technique. On
the basis of the observed results, we propose a phase separation induced hemifusion model for binary GUVs containing NP. The hemifusion model is examined by the fluorescence lipid transfer experiments and the stability of thehemifusion states is discussed in terms of the free-energy
calculation.

2 Experiments and methods
2.1 Commercial reagents
DPPE (> 99% purity), DPhPC (> 99% purity), DPPC(> 99% purity), and DOPC (> 99% purity) were obtained in a powder form from Avanti Polar Lipid, Inc.
(Alabaster, AL). All lipids were used without further purification and were stored in chloroform at −20 ◦ C until use. TR-DHPE (Texas Red 1,2-dihexanoyl-sn-glycero3-phosphoethanolamine) was obtained from Molecular
Probes (Eugene, OR), and used as a dye for a liquid disordered phase of membranes.
2.2 Preparation of giant vesicles
In this work, we prepared three different combinations of binary GUVs, DPPE/DOPC, DPhPC/DPPCand DPPC/DOPC. The compositions of the binary
GUVs were varied from 0/100 to 100/0, although the
DPPE/DOPC mixture could not form GUVs in MDPPE >60% region (MDPPE is the mole fraction of DPPE). The
GUVs were prepared by a gentle hydration method [19,20]. At first we dissolved the prescribed amount of phospholipids in 10 µl of chloroform (10 mM). In order to dye
the liquid disordered phase, TR-DHPE was added to thesolution. TR-DHPE strongly localizes in less ordered liquid phase [21]. The solvent was evaporated in a streamof nitrogen gas and the obtained lipid film was kept under vacuum for one night to remove the remaining solvent
completely. The dried lipid film was pre-warmed at 70 ◦ C,
and then the sample was hydrated with 1 ml of pure water of 70 ◦ C. During the hydration process, the lipid films
spontaneously form GUVs with diameters of 10–100 µm.
All the experiments in this study were performed using
pure water without salt.
2.3 Small-angle X-ray diffraction experiments
Following Yang and Huang’s experiments, the formations of the rhombohedral phase of DPPE, DPhPC andDPPC were examined by the small-angle X-ray diffraction(SAXD) measurements. The thin film cast from the chloroform solution was dried under vacuum for one night andthen chipped off from the substrate. The flakes of thin film
were exposed to water vapor for one hour and then immediately sealed in the SAXD cell. The degree of hydration
was controlled by keeping the hydrated sample under vacuum for 30 minutes. The SAXD measurements were performed using an apparatus (Rigaku NANO-Viewer) constructed from an X-ray rotating anode generator (18 kW,
λ = 1.54 Å), a confocal mirror, and a two-dimensional
position-sensitive proportional detector (Bruker AXS HiStar). A 1 × 1 mm2 X-ray beam irradiates the sample via

Y. Sakuma et al.: Adhesion of binary vesicles induced by phase separation

405

glass immediately. This sample cell was set on the temperature control stage (Carl Zeiss) with ±0.2 ◦ C of accuracy. We observed the phase separation process using an
inverted confocal microscope (Carl Zeiss, LSM 5) with either the laser scanning mode or the Hg lamp mode. To
avoid the domain formation before the observation, we
kept the sample temperature above the melting temperature of the phospholipids. For the observation of the phaseseparation behavior, we decreased the temperature fromthe one-phase region (60 ◦ C) to the coexisting two-phaseregion (10 ◦ C) with a step of 5 ◦ C. At each temperature weequilibrated the sample for 5 minutes and then examined
the formation of domains by the fluorescence microscopeobservation. We performed similar experiments for the binary GUVs and determined the phase separation curve.
To avoid the photo-oxidation, we paid special attention tominimize the exposure time of light.

2.5 Adhesion experiments
The adhesive nature of GUVs was examined by a micromanipulation technique. A GUV with the radius of 10–
40 µm was weakly aspirated by a micro-pipette (Eppendorf, Vacu Tip with the internal diameter, Dp , of 15 µm).
We controlled the suction pressure at P ∼ 3 Pa during
the adhesion tests, which was monitored by a pressuretransducer (Varlidyne, DP-15). The membrane tension,τ , at this suction pressure can be calculated by an expression [22]
τ = P Dp /4(1 − Dp /Dv ),(6)
Fig. 1. (a) Schematic representation of stalk intermediate
structure. (b) Hexagonally perforated lamellar structure with
ABCABC stacking (rhombohedral structure with R3̄ spacegroup). Here we shifted the positions of holes in B and C layers to show ABCABC sequence clearly. Actual arrangement ofholes is identical with the face centered cubic lattice.

pinhole collimator with three-slit system and the sample
to detector distance is about 0.80 m, which covers the magnitude of the scattering vector q = 4π sin θ/λ range from0.03 to 0.30 Å−1 . All SAXD measurements were performed
at 25 ◦ C. The obtained scattering patterns were corrected
for the nonuniformity, image distortion of the detector,
background scattering and then circularly integrated toobtain the one dimensional profile.

2.4 Phase diagrams
We prepared three types of binary vesicles with various compositions, DPhPC/DPPC = 0/100, 5/95, 10/90,
20/80, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20, 90/10,
100/0, DPPE/DOPC = 0/100, 10/90, 20/80, 30/70,
40/60, 50/50, 60/40, and DPPC/DOPC = 0/100, 20/80,
40/60, 60/40, 80/20, 100/0. The binary GUV suspension
was put on a glass plate with a silicon rubber spacer having the thickness of 0.5 mm, and then sealed with a coverwhere Dv is the spherical vesicle diameter exterior to thepipette. In our adhesion experiments the calculated tension was in the region of 1.4–4.5 × 10−2 mN/m. The effect of tension on the membranes is well investigated [23]and is classified into low- and high-tension regions. In the
low-tension region (10−3 –0.5 mN/m), the vesicle surface
increases by smoothing of thermal vesicle shape fluctuations, whereas in the high-tension region (> 0.5 mN/m)
direct surface stretch in area per lipid molecule becomessignificant. Thus in our adhesion experiments, the vesicle
membrane fluctuates around the spherical shape, whichcauses repulsive interaction to the adjacent membranes,or the so-called undulation repulsive force [24]. Thus inorder to achieve the adhesion of vesicles in the low-tension
region, the strong adhesion force is required.
Micro-manipulators (Narishige, MMO-202ND and
MN-4) with a micro-injector (Narishige, IM-9B and IM9C) system were used for positioning of the micropipettes. We applied the almost same tension (∼ 3 Pa)
to the two manipulated vesicles. Both of the aspirated
GUVs were contacted each other for 2–3 seconds and thenwe pulled the micro-pipettes away from the contact position to examine whether the adhesion takes place ornot. Adhesion test processes were monitored by a fluorescence microscope (Carl Zeiss, Akioskop) with a longdistance objective and recorded by a CCD camera (Carl
Zeiss, Axio Cam). For the non-adhesion case, the contacted vesicles are separated by pulling the micro-pipette

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The European Physical Journal Eaway as shown in Figure 2(a). On the other hand, forthe adhesion case, the contacting vesicles leave from theone side of the micro-pipette and keep contact as shownin Figure 2(b). We carried out 5 adhesion trials at eachmeasuring point. When all trials at the measuring point
showed adhesion or non-adhesion, we defined the point asadhesion point or non-adhesion point, respectively. Otherwise, we called the point as the boundary point. The
adhesion measurements were performed with descending
temperature, which agrees with the procedure of the phase
diagram construction. The adhesion behaviors were reversible against the temperature, although they had small
hysteresis of ∼ 5 ◦ C.

Fig. 2. Non-adhesion (a) and adhesion (b) behaviors observedin adhesion experiments. Scale bars indicate 15 µm.

2.6 Lipids transfer experiments
In order to examine the hemifusion state, we performed
the fluorescence lipid transfer experiments. First, we prepared a cell with two chambers. The two chambers, a stockchamber and an observation chamber, are separated by abank with a narrow channel. The fluorescence labeled andthe non-labeled vesicles were placed in the stock chamber and the observation chamber, respectively. One of
the non-labeled vesicles was held by a micro-pipette andtransferred to the observation chamber through the narrow channel. In the observation chamber, a labeled vesicle
and a non-labeled vesicle were brought into close proximity by the micro-manipulation technique. We observed
the transfer of fluorescent signal to the non-labeled vesicle by means of the fluorescence microscope (Carl Zeiss,
Akioskop) with the long-distance objective. In the lipid
transfer experiments, we used giant multi-lamellar vesicles (GMVs) for the non-labeled vesicle because unilamellar vesicles are too weak to hold on the top of a micropipette during a long movement from the stock chamberto the observation chamber through the narrow channel by
the micro-manipulation technique. We confirmed by thefluorescence microscope observation that the phase separation of the inner bilayers does not affect that of theouter bilayer.

3 Results
3.1 Spontaneous curvatures of lipids
First we address the spontaneous curvatures of DPPC,
DPhPC and DPPE by examining the existence of therhombohedral phase using the X-ray diffraction techniqueas reported by Yang and Huang [17,18]. In this study
we examined the lamellar to rhombohedral transition forthree phospholipids, DPPC with zero spontaneous curvature, and DPhPC and DPPE with negative spontaneous
curvatures. The effect of the hydration on SAXD patternsof DPPC sample at 25 ◦ C are shown in Figure 3(a). The
DPPC sample exposed to water vapor for 1 hour showed atypical diffraction pattern of the lamellar structure. Afterthe vacuum treatment for 30 minutes, the DPPC sample
kept the lamellar structure, although the lamellar peaks

were shifted to the high-q side and the intensity ratio of
the first peak to the second peak decreased considerably.
Since the bilayer has a thickness of 50 Å, the thicknessof the water layer was decreased from 11.5 Å to 7 Å by
the vacuum treatment in this case. We performed similar experiments for two NPs, DPhPC and DPPE. Sufficiently hydrated DPhPC and DPPE samples showed typical diffraction patterns for the lamellar phase as shown in
Figures 3(b) and (c), respectively. By vacuum treatment
for 30 minutes, several new diffraction peaks appeared inboth of the SAXD profiles. The peak positions predictedby the rhombohedral structure with the space group of R3̄
are marked by the triangles in Figures 3(b) and (c). Unfortunately the intensities of some peaks in the q range from
0.15 to 0.20 Å−1 were obscure, because the samples had
many defects. We summarize the new diffraction peaksof DPhPC and DPPE samples in Tables 1 and 2, respectively, with the indexation for the R3̄ space group. The
spacing dh,k,l can be expressed by the unit cell dimensions, a and c, as
1

4 (h2 + hk + k 2 )
l2
+ 2.
2
a
c

=
d2h,k,l3(7)

The pronounced peak positions in the q range of 0.05 to
0.15 Å−1 are consistent with the space group of R3̄. It
should be noted that by the subsequent vapor treatment,
the rhombohedral phase transformed into the lamellar
phase and this rhombohedral ↔ lamellar transition was
reversible against the degree of hydration. From these results we consider that DPhPC and DPPE tend to form
an hourglass-like structure by the reduction of bilayer distance, indicating that the both lipids have negative spontaneous curvatures.
3.2 Phase behavior of binary vesicles
Next we investigate phase diagrams of binary GUVs composed of NP and ZP. It is known that a binary systemcomposed of phospholipids having a high Tm and a low
Tm undergoes a phase separation between the two melting temperatures [21]. At high temperature, a binary lipid

Y. Sakuma et al.: Adhesion of binary vesicles induced by phase separation

407

Table 1. Summary of SAXD peaks of DPhPC after the vacuum treatment for 30 min. Dashes denote that definite peak
was not observed.
q (Å−1 )dobs (Å)

hkl

dcal (Å)

a (Å)

0.086

73.06

101

75.54

100.21

0.108

58.18

012

57.65

99.87

0.122

51.50

003

51.50

-

110

50.00

-

021

41.69

-

104

35.28

0.214

29.36

015

29.10

0.244

25.75

006

25.75

c (Å)

154.5

99.13
154.5

Table 2. Summary of SAXD peaks of DPPE after the vacuum
treatment for 30 min. Dashes denote that definite peak was notobserved.
q (Å−1 )dobs (Å)

hkl

dcal (Å)

a (Å)

0.082

76.59

101

76.59

99.62

0.107

58.18

012

59.81

99.38

0.114

55.12

003

55.58

0.123

51.08

110

49.84

-

021

41.87

-

104

37.38

-

015

30.94

006

27.67

0.225

Fig. 3. Effect of degree of hydration on SAXD patterns at
25 ◦ C. (a) DPPC, (b) DPhPC and (c) DPPE; closed circle profile: sufficiently hydrated sample, open diamond profile: after
vacuum treatment for 30 min. Triangles in (b) and (c) indicate
predicted peak positions from R3̄ symmetry.

mixture is in a single uniform liquid phase, and by decreasing the temperature, the system separates into thecoexisting two phases. We examined three binary systems,
DPhPC/DPPC (Tm = 41 ◦ C), DPPE (62 ◦ C)/DOPC(−20 ◦ C), and DPPC/DOPC. DPhPC does not exhibit
the chain melting temperature between +120 ◦ C and

27.93

c (Å)

166.7
99.68

166.0

−120 ◦ C and is in the disordered liquid state at room temperature [25]. The first two systems are mixture of NP and
ZP, and the third system is a mixture of two ZPs as a reference sample.
The temperature composition phase diagram for the
DPhPC/DPPC binary system is shown in Figure 4(a).
In the high-temperature region, DPhPC and DPPC mix
completely as one uniform liquid phase. By decreasing
the temperature, the binary system undergoes a phaseseparation into coexisting two liquid phases. It should be
noted that for MDPhPC < 5% (MDPhPC : mole fraction of
DPhPC) a solid-liquid coexisting phase boundary is observed and pure DPPC shows a solid phase in the regionof T < 41 ◦ C [21]. In the phase diagram we show the fluorescence micrograph images of vesicles in the one-phaseregion and the two-phase region. Here the bright region
and the dark region correspond to DPhPC-rich phase andDPPC-rich phase, respectively. In the one-phase region,
the vesicle shows a homogeneous monotonic appearance,
whereas in the coexisting liquid-liquid phase region, thevesicle has several circular liquid domains.
For the DPPE/DOPC binary system, the obtainedphase diagram is shown in Figure 4(b). It should be
noted that the binary systems with DPPE mole fraction MDPPE > 60% could not form GUVs, which may be
due to its large negative spontaneous curvature. At hightemperature, a mixture of DPPE and DOPC is in one

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Fig. 4. Phase diagram of binary GUVs. Fluorescence micrograph images in figures show the appearance of vesicle
at one-phase and two-phase region. Scale bars indicate 5 µm.
(a) DPhPC/DPPC binary system and (b) DPPE/DOPC binary system. MDPhPC and MDPPE are mole fractions of
DPhPC and DPPE, respectively.

uniform liquid phase. Between the melting temperaturesof DPPE and DOPC, the system separates into coexistingsolid and liquid phases. The immiscible boundary line approaches Tm of DPPE with the increase of MDPPE . In thecoexisting two-phase region, the vesicle has domains withcomplex shapes (Fig. 4(b)) compared with the circularliquid domains (Fig. 4(a)). The former has characteristic
morphologies for solid domains [21]. Here the dark region
and the bright region correspond to DPPE-rich phase andDOPC-rich phase, respectively.
The DPPC/DOPC binary system shows a similarphase diagram to the DPPE/DOPC binary system. At
high temperature, DPPC and DOPC mix completely in asingle homogeneous liquid phase. By decreasing the temperature, the system separates into a solid phase (rich inDPPC) and a liquid phase (rich in DOPC). The phaseboundary approaches Tm of DPPC for MDOPC → 0% andFig. 5. Adhesion diagram of binary GUVs in the composition and the temperature space, (a) DPhPC/DPPC systemand (b) DPPE/DOPC system. Circle, cross and triangle indicate adhesion, non-adhesion and boundary states, respectively.
Dashed lines are phase boundaries.

Tm of DOPC for MDOPC → 100% (MDOPC : DOPC mole
fraction). The phase behavior of the DPPC/DOPC binary
system agrees well with that in the literature [26,27].
3.3 Adhesion of binary GUVs
Based on the above phase diagrams, we examine the adhesion of the binary GUVs using the micro-manipulation
method. Before we examined the adhesion of the binary
vesicles, we confirmed that one-component GUVs composed of pure ZP (DPPC or DOPC) have no adhering
ability in the temperature region from 20 to 60 ◦ C.
The adhesion tests for the DPhPC/DPPC binary
GUVs were performed at grid points in the temperaturecomposition space shown in Figure 5(a). Circles indicatethat the GUVs adhere to each other at the corresponding
compositions and temperatures, cross symbols mean that
GUVs do not show the adhesion and triangles indicate

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409

Fig. 6. Fluorescence microscope images of adhering GUVs for (a) DPhPC/DPPC and (c) DPPE/DOPC and corresponding
confocal microscope images for (b) DPhPC/DPPC and (d) DPPE/DOPC. Scale bars indicate 10 µm.

the boundary region. The adhesion takes place only in the
two-phase region of the binary system; the adhesion/nonadhesion boundary (Fig. 5(a)) agrees well with the immiscible boundary of the DPhPC/DPPC binary vesicles(Fig. 4(a)). Figure 5(a) clearly shows that GUVs composed of pure DPhPC (MDPhPC = 100%) show no adhesion in the temperature region from 60 to 20 ◦ C. Thus theformation of DPhPC domains plays a crucial role in thevesicle adhesion. In Figure 6(a) we show a confocal fluorescence cross-section image of the adhering DPhPC/DPPCbinary vesicles dyed with TR-DHPE with a corresponding
confocal image (Fig. 6(b)). The TR-DHPE is localized in
the region rich in DPhPC and the region rich in DPPC is
recognized as dark parts. The adhering part of the GUVs
shows intense line compared with the other circumference of the GUVs. By taking into account the fact thatthe adhesion behavior of DPhPC/DPPC binary vesicles
without TR-DHPE agrees with that of DPhPC/DPPCbinary vesicles in the presence of TR-DHPE, we see thatthe fluorescence dye does not affect the present adhesion
results significantly. From these experimental results weconclude that the DPhPC/DPPC binary GUVs adhereto each other through the region rich in DPhPC, i.e. thenegative spontaneous curvature region.

We performed similar experiments for the DPPE/
DOPC binary GUVs and results of the adhesion test aresummarized in Figure 5(b). Again we obtained good agreement between the phase behavior (Fig. 4(b)) and the adhesion map (Fig. 5(b)), indicating the importance of thephase separation in the adhesion. In Figure 6(c) we show
the confocal fluorescence cross-section image of adhering
DPPE/DOPC binary GUVs dyed with TR-DHPE, where
TR-DHPE is localized in the DOPC-rich phases. The fluorescence cross-section microphotograph image and thecorresponding confocal image (Fig. 6(d)) clearly showthat GUVs adhere to each other through the dark region,
namely, DPPE solid domains with the negative spontaneous curvature.
Furthermore, we forced to separate the adhering vesicles using the micro-pipettes with high suction pressureP ∼ 500 Pa (τ ∼ 33 mN/m). In Figure 7 we show the snapshots of the forced separation experiments of adhering binary vesicles with the composition of DPPE/DOPC = 6/4
at 25 ◦ C. By pulling one of the adhering vesicles, the opposing vesicle was stretched showing an hourglass shape
as in Figure 7(b). Finally the opposing vesicle was ruptured and the adhering area was left on the pulling vesicle as a patch (Fig. 7(c)). Due to the smearing effect of

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Fig. 7. Snapshots of forced separation experiments (P ∼500 Pa) of adhering binary vesicles with the composition of
DPPE/DOPC = 6/4 at 25 ◦ C, (a) just after pulling the pipetteaway, (b) intermediate state where opposing vesicle was deformed to hourglass shape, and (c) opposing vesicle was ruptured and the adhering area was left on the pulling vesicle asa patch. Bars in (a), (b) and (c) indicates 15 µm. The enlarged
high-contrast image of the dotted-square region in (c) is shownin (d). Bar in (d) indicates 7.5 µm.

fluorescence from the surrounding matrix, the patch seems
to be bright, but the high contrast image of this patch(Fig. 7(d)) shows that its center region is occupied by thedark DPPE-rich region. Thus the adhesion through thenegative spontaneous curvature region has a strong adhesion force.
On the other hand, DPPC/DOPC binary GUVs did
not show adhesion of GUVs in the observed temperaturecomposition space, indicating that the domains with zero
spontaneous curvature have no adhesion ability even in
the coexisting two-phase region. These results clearly showthat the adhesion takes place for the binary GUVs composed of NP and ZP through domains rich in NPs and isindependent of whether the domain is in liquid or solid
phase.
3.4 Lipid transfer observation
The essential point of our observations is that the adhesion takes place only through the domains rich in NPs.
So far the membrane adhesions have been explained fromthe view points of the specific interactions using the ligand
and receptor molecules embedded in membranes or the interplay among the generic interactions [1–9,24]. Since inour case no specific molecules are added to the systems,
one of the plausible explanations is the interplay between
the generic interactions. Thus if DPPE and DPhPC havelarge adhesion energies (energy at the position of the interaction potential minima), the domains rich in DPPE
(or DPhPC) adhere with the domains on opposing vesicles. It is reported that DPPE has the adhesion energy

Fig. 8. Schematic representation of (a) parallel membrane(non-hemifusion) state, (b) hemifusion state with pancake
shape and (c) hemifusion state with spherical shape. Coordinates for free-energy calculation are shown in (b) and (c).

of −0.7 ≈ −0.8 × 10−3 J/m2 = −0.07kB T /molecule [5,7],
which is larger than that of DPPC (−0.15×10−3 J/m2 ) [5].
Although the adhesion energy of DPhPC has not been reported in the literatures, the interplay among the genericinteractions may be one model to explain the observed adhesions. In our experiments, however, the adhesion took
place only for the coexistence phase region and we neverobserved adhesion for the one component GUVs composedof pure NP (DPhPC). The coupling between generic interactions and phase separation has been addressed bytheoretical [28,29] and simulation approaches [30–32]. Komura and Andelman proposed a phenomenological modelfor the unbinding transition of multicomponent fluid membranes and showed the phase separation coupled with thebound and unbound membrane states [29]. The theoreticalphase diagram predicts that the adhesion takes place not
only in the two-phase region but also in the one-phase region, which cannot describe the observed adhesion behavior. Therefore, it is worthwhile to consider another model
to explain the experimental results. In this paper we propose a hemifusion model where the negative spontaneous
curvature domain forms the hemifusion structure with theapposing negative spontaneous curvature domain. In Figure 8 we show the schematic representations of the hemifusion models: (a) parallel membrane (non-adhesion) state,

Y. Sakuma et al.: Adhesion of binary vesicles induced by phase separation

Fig. 9. Two adhering GUVs composed of DPPE/DOPC(= 60/40) in fluorescence lipid transfer experiment,(a) phase contrast + fluorescence image and (b) fluorescence
image for adhering GUVs just after contacting. Bright GUV is
a fluorescent labeled GUV and non-labeled GUV is positioned
at the left side of the labeled GUV (detected by phase contrast
image in (a) and not seen in (b)). After 10 minutes, fluorescent signal appears in non-labeled GUV in the neighborhood of
the contacting area (indicated by an arrow) in the fluorescence
image (c).

(b) hemifusion (adhesion) state with a pancake shape and(c) hemifusion state with a spherical shape. In the hemifusion states, the outer leaflets of both vesicles’ bilayers are
merged whereas the inner leaflets remained independentand the domain boundary between NP-rich and ZP-richregions is eliminated.
In order to examine the hemifusion model, we performed fluorescence lipid transfer experiments using two
types of DPPE/DOPC binary GMVs. The first type contained fluorescent labeled lipids TR-DHPE and the second
type had no fluorescence. Two phase separating GMVs
of different types were brought into adhesion by meansof the micro-manipulation (Fig. 9(a): phase contrast +fluorescence microscope image). Just after the adhesion,
the labeled vesicle showed a fluorescent signal from thematrix rich in DOPC whereas the non-labeled GMV could
not be detected by the fluorescent microscope observation as shown in Figure 9(b). Slightly after the adhesion(several minutes), a fluorescent signal was observed in theneighborhood of the contact area on the non-labeled GMV
as shown by an arrow in Figure 9(c). Unfortunately thephoto images are somewhat obscure, because the fluorescence from the transferred lipids is fairly weak and localized to the parts of the non-labeled GMV. Then we
repeated the lipid transfer experiments several times andconfirmed the reproducibility of fluorescence lipid transfer
by eye observations.
The main argument on the lipid transfer experiments
may be the migration of fluorescent lipids between oppos-411

ing bilayers across the water layer. In this case it would
not need hemifusion. Here we estimate the diffusion constant of typical saturated phospholipids, DPPC, acrossthe water layer using a time-resolved small-angle neutronscattering (TR-SANS) technique [33]. This is based on
the fact that scattering length densities (SLDs) are significantly different between vesicles consisting of hydrogenated and deuterated phospholipids and that the lipid
exchange between these vesicles reduces the differencein SLD with time. The rough estimated diffusion constant of DPPC across the water layer at 25 ◦ C is D <1 × 10−25 m2 /s, which is quite slow compared with thetime scale of our lipid exchange experiments of ∼ 10 min.
Then we believe that the fluorescent lipids transfer to thenon-labeled vesicle through the hemifused outer leafletsand not by the migration across the water layer. Detailsof this lipid exchange experiments using TRSANS will
be described in a forthcoming paper. Thus the labeled
lipids transfer to the adhering non-labeled GMV throughthe hemifusion of the outer leaflets of the matrix. Thisstrongly supports the hemifusion model to account for theadhesion of binary GUVs containing NP.

4 Discussion
Here we discuss the stability of the hemifusion state using
a membrane elasticity model. In the hemifusion model, theadhesion may take place in order to decrease the bending
energy of the flat membrane with negative spontaneous
curvature and eliminate the boundary energy between thedomain and matrix. The total energies of adhering membranes and non-adhering membranes, which are expressedby a sum of the curvature elasticity and boundary energy,isZIF = 2κ (H − H0 )2 dS + 2σ dl,(8)where H is the mean curvature, H0 the spontaneous curvature, and σ the line tension between the domain andmatrix. As shown in Figure 8, in the hemifusion state, theouter leaflets of both vesicles’ bilayers are merged whereas
the inner leaflets remain independent. We calculate theenergy of the outer leaflets to compare the total energy
between the hemifusion state and the parallel membrane(non-hemifusion) state. It should be noted that the deformation takes place not only in the domain side (bentdomain) but also in the matrix adjacent to the domainboundary (bent matrix). For convenience we deal withthe pancake shape hemifusion case shown in Figure 8(b),where the domains have circular shape and adhesion takes
place between opposing domains with the same area. In
order to calculate the bending energy in equation (8) we
introduce a cylindrical coordinate as shown in Figure 8(b).
For a surface given in terms of the local radius, R(z), thearea element isp
dS = 2πR(z) 1 + Rz (z)2 dz,(9)where the subscript denotes partial derivative, and the412

The European Physical Journal E

mean curvature H is expressed by
H=

RRzz − 1 − Rz2.
2R(1 + Rz2 )3/2

(10)

We consider two situations according to the experiments,
one is the membranes with negative spontaneous curvature domain (H0 = −1/a) and the zero spontaneous curvature matrix (H0 = 0), and the other is the membranes
with zero spontaneous curvature domain and zero spontaneous curvature matrix.
First we deal with the NP and ZP mixture membranes.
For the hemifusion membrane case, the boundary between
domain and matrix is eliminated by the formation of thehemifusion state and the total energy is expressed by asum of the bending energies of the flat domain region,
bent domain region, and bent matrix region. For the bent
domain (Fig. 8(b)), the local radius R(z) of the domainboundary has the formp
R(z) = b + a2 − z 2(11)
and for bent matrix the boundary is expressed byp
R(z) = c − a2 − z 2 .

(12)Then the total energy is given by

!
2 2
2
πb2 arctan √b2a−a2
π
b
b
hemi
√
√
−
FNZ
= 4πκ1 2 +2κ1a2a b2 −a2
a b2 − a2!
πc2 arctan √c2a−a2
7π 2 c2√+,+2κ2 −4π+ √2a c2 −a2
a c2 −a2(13)where κ1 and κ2 are the bending rigidities of domainmonolayer with H0 = −1/a and matrix monolayer with
H0 = 0, respectively. In our experiments, the domain size(b ∼ c ∼ 5 µm) is much larger than the inter-membrane
distance (a ∼ 10 nm), then the total energy can be approximated as
hemi
FNZ
∼

b
b2
π2
(2κ1 + 7κ2 ) + 4πκ1 2 .
2
a
a

(14)

On the other hand, for the parallel membrane (nonhemifusion) case, the total energy is a sum of the bending
energy and the boundary energy. The radius of the circular domain, r, is related to the geometrical parameters aand b in the hemifused domain via the area conservation
constraintp(15)
r = 2a2 + b2 + πab .

Then the total energy is given by


p
b
b2
para
FNZ = 4πκ1 2 + π + 2 + 2πσ 2a2 + b2 + πab
a a


b2
b
(16)
∼ 4πκ1 π + 2 + 2πσb,a awhere σ is the line tension between the negative spontaneous curvature domain and the zero spontaneous curvature matrix. The formation of the hemifusion state isdetermined by the balance of the boundary energy andbending energy.
Next we address the ZP and ZP mixture membranes.
In this case the total energy for the hemifusion state isgiven by!
πb2 arctan √−aa2+b2
π 2 b2hemi√
FZZ = 2κ3 4π+ √−2a −a2 +b2
a −a2 +b2!
πc2 arctan √−aa2+c2
7π 2 c2√++ 2κ2 −4π+ √2a −a2 +c2
a −a2 +c2∼π2b(2κ3 + 7κ2 ) ,
2
a

(17)

where κ3 is the bending elasticity of domain monolayer
with H0 = 0. The total energy for the parallel membranestate is expressed by the boundary energy only and given
by
p
para
FZZ
= 2πσ ′ 2a2 + b2 + πab ∼ 2πσ ′ b,(18)

where σ ′ is the line tension between the zero spontaneous
curvature domain and matrix.
Thus the total energy difference between the hemifusion state and the parallel membrane state for NP and ZP
binary membranes is expressed by
π2 b
b
para
hemi
(2κ1 +7κ2 )∆FNZ = FNZ−FNZ= 4π 2 κ1 +2πσb−
a
2a
(19)
and for ZSCP and ZSCP binary membranes the energy
difference is given by
para
hemi
∆FZZ = FZZ− FZZ= 2πσ ′ b −π2 b(2κ3 + 7κ2 ). (20)2aFrom the literature, the bending modulus of bilayer membrane, κ, typically has a value of ∼ 10−19 J [34] (DOPC:
0.8 × 10−19 J [34], DPPC: 0.5 × 10−19 J for liquid disordered phase [35] and ∼ 1 × 10−19 J for ordered phase [36],and DPhPC: 1.2 × 10−19 J [35]) and the line tension,σ, is estimated as the order of ∼ 10−11 N [37]. For thenumerical calculations, we adopted the following values:κ1 ∼ κ2 ∼ κ3 ∼ 21 × 10−19 = 5 × 10−20 J and σ ∼ σ ′ ∼1−11= 5×10−12 N (factor 1/2 accounts for the mono2 ×10
layer membranes). For the NP and ZP binary membranes,
para
hemi
we obtained ∆FNZ = 3.8 × 10−17 J, i.e. FNZ> FNZ,
indicating that the membranes prefer to form the hemifusion state. On the other hand, for the ZP and ZP binary membranes, we obtained ∆FZZ = −86 × 10−17 J, i.e.
para
hemi
FZZ
< FZZ
, indicating that the membranes prefer the
separated membrane state. These theoretical predictions
agree well with the experimental observations. It is worthwhile to note that ∆FNZ is larger than ∆FZZ by 4π 2 κ1 b/a,which determines the sign of the total energy differences,∆FNZ and ∆FZZ . This term originates from the bendingof the negative spontaneous curvature domain. Thus the

Y. Sakuma et al.: Adhesion of binary vesicles induced by phase separation

formation of negative spontaneous curvature domain is responsible for the observed adhesion behavior.
In the model calculation described above, we adopted
the pancake shape model for the hemifusion state. Of
course we can consider other hemifusion shapes such as
spherical hemifusion shape, where the outer leaflets form
spheres with the curvature of H0 = 1/a as shown in Figure 8(c). In this case, the total energy for the sphericalhemifusion is expressed by
sp

hemi
FNZ
∼

7π 2 b
κ2 ,
2 a

(21)

where the geometrical parameters a and b are defined in
Figure 8(c). For the spherical hemifusion case, the bending energy merit of the negative spontaneous curvaturemembranes is more emphasized and the hemifusion is encouraged compared with the case of the pancake hemifusion. Thus the pancake shape is the least stable shapeto achieve the hemifusion and the hemifusion models can
explain the observed adhesion behavior.

5 Conclusion
In this study we found the adhesion of the binary GUVs
composed of the NP and ZP induced by the phase separation. In the one-phase region, the GUVs do not show
adhesion but in the coexisting two-phase region the GUVs
show adhesion through the domains rich in NPs. Basedon the experimental results we propose the phase separation induced hemifusion model where the outer leaflets ofthe bilayers merge at the boundary of the negative spontaneous curvature domains. The fluorescence lipid transfer experiment strongly supports the hemifusion model.
The model energy calculations show that the formation of
the negative curvature domain is crucial for the stabilization of the hemifusion state. In contrast to the adhesionof membrane induced by the specific interactions such as
ligand-receptor pairs or the interplay of the generic interactions, a unique feature of this system is that the adhesion of GUVs is controlled by changing the temperature,
which may bring a new pathway of membrane technology.
We thank Prof. Toshihiro Kawakatsu (Tohoku University) for
valuable discussions. This work was supported by KAKENHI(Grant-in-Aid for Scientific Research) on Priority Area “SoftMatter Physics” from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.

References
1. R. Simson, E. Sackmann, in Physical Chemistry of Biological Interfaces (Marcel Dekker, Inc., New York, 2000).

413

2. L.J. Lis, M. McAlister, N. Fuller, R.P. Rand, V.A.
Parsegian, Biophys. J. 37, 657 (1982).
3. E. Evans, D. Needham, Faraday Discuss. Chem. Soc. 81,
267 (1986).
4. R. Lipowsky, S. Leibler, Phys. Rev. Lett. 56, 2541 (1986).
5. R.P. Rand, V.A. Parsegian, Biochem. Biophys. Acta 988,
351 (1989).
6. J.O. Rädler, T.J. Feder, H.H. Strey, E. Sackmann, Phys.
Rev. E 51, 4526 (1995).
7. T.J. McIntosh, S.A. Simon, Langmuir 12, 1622 (1996).
8. J.F. Nagel, S. Tristran-Nagel, Biochim. Biophys. Acta
1469, 159 (2000).
9. Although the properties of the binding-unbinding transition cannot be described by a simple superposition of thegeneric interactions, it gives a reasonable estimation of theeffective inter-membrane potential [4].
10. M.M. Kozlov, V.S. Markin, Biofizika 28, 255 (1983).
11. P. I. Kuzumin, J. Zimmerberg, Y.A. Chizmadzhev, F.S.
Cohen, Proc. Natl. Acad. Sci. U.S.A. 98, 7235 (2001).
12. V. Markin, J. Albanesi, Biophys. J. 82, 693 (2002).
13. S. May, Biophys. J. 83, 2969 (2002).
14. H. Noguchi, M. Takasu, J. Chem. Phys. 115, 9547 (2001).
15. S.J. Marrink, A.E. Mark, J. Am. Chem. Soc. 125, 11144(2003).
16. J. Heuvingh, F. Pincet, S. Cribier, Eur. Phys. J. E 14, 269(2004).
17. L. Yang, H.W. Huang, Science 297, 1877 (2002).
18. L. Yang, H.W. Huang, Biophys. J. 84, 1808 (2003).
19. J.P. Reeves, R.M. Dowben, J. Cell. Physiol. 73, 49 (1969).
20. D. Needham, E. Evans, Biochemistry 27, 8261 (1988).
21. S.L. Veatch, S.L. Keller, Biophys. J. 85, 3074 (2003).
22. R. Kwok, E.A. Evans, Biophys. J. 35, 637 (1981).
23. W. Rawicz, K.C. Olbrich, T. McIntosh, D. Needham, E.
Evans, Biophys. J. 79, 328 (2000).
24. W. Helfrich, Z. Naturforsch. 33a, 305 (1978).
25. H. Lindsey, N.O. Petersen, S.I. Chan, Biochim. Biophys.
Acta. 555, 147 (1979).
26. S.L. Veatch, S.L. Keller, Biophys. J. 85, 3074 (2003).
27. B.R. Lentz, Y. Barenholz, T.E. Thompson, Biochemistry15, 4529 (1976).
28. R. Lipowsky, Phys. Rev. Lett. 77, 1652 (1996).
29. S. Komura, D. Andelman, Europhys. Lett. 64, 844 (2003).
30. T.R. Weikl, R.R. Netz, R. Lipowsky, Phys. Rev. E 62, R45(2000).
31. T.R. Weikl, R. Lipowsky, Phys. Rev. E 64, 011903 (2001).
32. T.R. Weikl, D. Andelman, S. Komura, R. Lipowsky, Eur.
Phys. J. E 8, 59 (2002).
33. M. Nakano, M. Fukuda, T. Kudo, H. Endo, T. Handa,
Phys. Rev. Lett. 98, 238101 (2007).
34. H.P. Duwe, E. Sackmann, Physica A 163, 410 (1990).
35. D. Marsh, Chem. Phys. Lipids. 144, 146 (2006).
36. We estimated the bending modulus of DPPC in orderphase based on Milner and Safran method (S.T. Milner,
S.A. Safran, Phys. Rev. A 36, 4371 (1987)) by means ofa neutron spin echo and a small-angle neutron scattering
measurement.
37. R. Lipowsky, J. Phys. II 2, 1825 (1992).