Multitude of Morphological Dynamics of Giant ... - ACS Publications

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Multitude of Morphological Dynamics of Giant Multilamellar Vesicles in Regulated Nonequilibrium Environments Takuya Tomita,† Tadashi Sugawara,‡ and Yuichi Wakamoto†,‡,§,* †

Department of Basic Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902 Japan ‡ Research Center for Complex Systems Biology, The University of Tokyo, 3-8-1 Komaba, Meguro-ku Tokyo 153-8902 Japan § PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan

bS Supporting Information ABSTRACT: Lipid giant vesicles (GVs) exhibit biologically relevant morphological dynamics such as growth and division under certain conditions without any sophisticated molecular machineries employed by the current organisms. Nonequilibrium conditions are essential for the emergence of dynamic behaviors, which are normally generated by the addition of stimulating materials or by the change of some physical conditions. Therefore, an experimental method that allows flexible control of external conditions is desirable. Here we report a new and simple perfusion device for light microscopy observation that simultaneously realizes such control and tracking of individual phospholipid GVs for the long-term. We apply this device to the study of the morphological dynamics of POPC-based giant multilamellar vesicles (GMVs) under a monotonic and gradual increase of surfactant concentration; thereby we reveal the existence of multiple pathways in the slow solubilization processes, whose frequencies depend on the compositions of GMVs. This perfusion device would offer an unprecedented control of external conditions in the studies of GVs and might help us characterize the physicochemical origins of rich morphological dynamics of living cells.

’ INTRODUCTION Morphological changes of vesicles are inherently nonequilibrium phenomena driven by the influx and efflux of materials and energy. Different morphological patterns could potentially arise depending on slight differences of the environmental conditions. For instance, different dynamics may emerge in gradual and rapid environmental changes even between the identical initial and final equilibrium states. Giant vesicles (GVs) exhibit biologically relevant morphological dynamics such as growth and division,1
7 birthing,8
11 budding,12
15 fusion,16
23 and so on.24 Understanding the principles underlying these phenomena would provide fundamental insights into the physical and chemical origin of membrane dynamics underlying the establishment of the sophisticated molecular machineries employed by nature.25
28 The dynamics of GVs are tightly linked to the external conditions and driven, for example, by the addition of membrane molecules,1,6 membrane precursors,4,29,30 or membrane soluble surfactants,31
33 or by the change of physical conditions around GVs like osmotic pressure.34
38 Characterizing the morphological dynamics in well-defined environments freely controllable by experimentalists would be ideal for studying the mechanisms behind the vesicular dynamics. The necessity of such careful control of environmental conditions has not been fully addressed to date despite many studies on the designs and r 2011 American Chemical Society

properties of molecules constituting vesicles under steady state conditions.39
41 Another important aspect of the dynamics of GVs is the diversity of the behaviors in a single vesicle population; different dynamics could appear even under identical environmental changes depending on slight differences of initial states of GVs and other factors.31,32,34,42 The ability of GVs with nearly identical composition to exhibit qualitatively distinct patterns may form the primitive basis for the rich morphological dynamics exploited in living cells. Where the diversity exists, one must clarify which pathway is typical to link the molecular details of GVs to observed dynamics; the frequencies of different dynamics give an important clue for understanding the mechanism behind the dynamics. Diversity of morphology in a snapshot image is not a good indicator of transition pattern diversity because fluctuation may exist in the dynamics of individual GVs; an individual GV can potentially take different morphologies at different time points and the GVs that seem quite different at one point may eventually follow a qualitatively same pattern in the time course. Thus, one Received: May 17, 2011 Revised: June 21, 2011 Published: June 24, 2011 10106

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Langmuir must compare the full time-course transition of individual GVs in particular environments to unravel the actual transition pattern diversity. However, such long-term observation has been technically difficult especially under nonstationary conditions due to the drift of GVs with the flow in local environments. In this report, we introduce a simple perfusion device that allows the long-term microscopic tracking of individual GVs under well-controlled external conditions. Utilizing this device, we show the solubilization dynamics of three different kinds of POPC-based giant multilamellar vesicles (GMVs) under the conditions of a monotonic and gradual increase of surfactant (Triton X-100) concentration. We reveal the existence of multiple solubilization pathways characterized by different morphological patterns whose frequencies are modulated by the compositions of the GMVs. Furthermore, we quantitatively characterize the full time course transition of morphological changes of individual GMVs and show the existence of prominent morphological fluctuations before the occurrence of particular morphological transition events in the solubilization process. The systematic observations of qualitatively distinct morphological dynamics reported here might provide important information leading to theoretical analysis of GMV membrane dynamics.

’ EXPERIMENTAL SECTION Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-Palmitoyl-2-oleoyl-sn-glycero-3-[phosphor-rac-(1-glycerol)] (POPG) were purchased from NOF Corp. (Tokyo, Japan). Cholesterol, Triton X-100, monopotassium phosphate, and dipotassium phosphate were purchased from Wako Pure Chemical Industries, Inc. (Osaka, Japan). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-Lissamine Rhodamine B Sulfonyl, [1,2-Dioleoyl-sn-Glycero-3-], Ammonium Salt (Rho-DOPE) was purchased from Avanti polar lipids, Inc. (Alabaster, AL, U.S.). Fluorescein sodium salt was purchased from Sigma-Aldrich. Preparation of GMVs. GMVs were prepared by a film swelling method. Phospholipid mixtures dissolved in chloroform in a glass bottle were dried by N2 gas to form thin lipid films, and the remaining solvents were completely removed under reduced pressure for more than 2 h. The films were hydrated with pH 7.5 phosphate buffer (0.1 M) for the GMVs used in the pH alternation experiment shown in Supporting Infomation, SI, or with Milli-Q water for those used in the Triton X-100 solubilization experiment. The rehydrated GMV suspensions were incubated for overnight at room temperature and diluted 10-fold by the same phosphate buffer or Milli-Q water before observation. The final concentration of the lipids was adjusted to 0.1 mM in total (POPC + POPG + Cholesterol). For fluorescence microscopy, we also added 0.1 mol % Rho-DOPE in the preparation of GMVs. Three types of GMVs, denoted by “PC”, “PC:PG”, and “PC:PG: Chol”, were used in this work. The compositions of the GMVs (mol %) were 100% POPC for PC, 80% POPC and 20% POPG for PC:PG, and 80% POPC, 10% POPG, and 10% cholesterol for PC:PG:Chol. We confirmed that the GMVs prepared by this protocol encapsulate water-soluble dye (fluorescein). Thus, the dynamics shown below is that of GMVs, not of oil droplets. Design and Construction of Perfusion Device. We constructed a simple perfusion device by assembling glass slide (76  52 mm, thickness 0.8
1.0 mm, Matsunami), cellulose semipermeable membrane (SpectraPor 7, MWCO 25,000, Spectrum Laboratories, Inc.), two pieces of frame seal chamber (Frame-Seal, 25 μL (internal space, 9 9  0.3 mm), Bio-Rad Laboratories, Inc.) and polydimethylsiloxane (PDMS) pad connected with two silicon tubes for flowing solution (Figure 1(A)). The main flow chamber is composed of two compartments separated by cellulose semipermeable membrane (Figure 1(B)). Twenty-five μL

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Figure 1. Perfusion device. (A) Device structure. The perfusion device is constructed by assembling PDMS pad, two pieces of frame seal chamber, cellulose semipermeable membrane, and glass slide. Three silicone tubes protruding from the PDMS pad are used for flowing solution, one as the inlet and two as the outlet. The groove on the pad traps small bubbles entering the device with flowing solution. (B) Crosssection view. The internal space is separated by cellulose semipermeable membrane. GMVs are enclosed in the lower compartment, whereas solution is flown in the upper compartment. GMV dispersion was enclosed in the lower compartment during the setup of the device before sealing cellulose semipermeable membrane, and different buffers and stimulating solutions were flown in the upper compartment by peristaltic pump (SJ-1211 L, ATTO Corporation). We fixed the flow rate at 4.0 mL/h in this experiment. The internal volume of the both lower and upper compartments is only 25 μL, thus the solution in the chamber can be changed rapidly. Because of the large pore size of the semipermeable membrane (MWCO 25 000), small molecules can freely diffuse across the membrane, whereas large molecular complexes such as GMVs cannot pass. Therefore, GMVs can be stably trapped in the lower compartment. The cellulose semipermeable membrane used in this experiment was first washed in Milli-Q water extensively and cut to the size that can cover the whole internal area of the frame seal chamber. The membrane was soaked in methanol overnight and dried under reduced pressure sandwiched between two pieces of clean filter paper to avoid shrinkage. The dried membrane was stored at room temperature. Before the use in the experiment, the membrane was soaked in Milli-Q water and sealed on the frame seal chamber after removing excess water. For making PDMS pad, one piece of frame seal chamber was cut into three pieces and sealed on the base of a disposable plastic dish to use it as the mold for the bubble trap groove. Casting PDMS on this mold creates the pad with bubble trap groove (width 2 mm, depth 0.3 mm) as shown in Figure 1(A). The bubble trap groove prevents small bubbles in the flowing solution from coming into the central part of the chamber and allows the continuous microscopic observation without the interference by bubbles. The PDMS base and curing agents (Sylgard 184, Dow Corning Cooperation) were mixed at 10:1 weight ratio and cured at 65 °C for one hour. The pad was cut out and three holes were punched out at the positions shown in Figure 1(A). Silicon tubes (inner diameter 1 mm, outer diameter 2 mm, Kokugo, Co., Ltd.) were inserted into the holes. Small amount of mixed PDMS was put around the holes to fix the tubes and to completely close the holes. Microscopy. The dynamics of GMVs was continuously monitored by microscope (IX-70, Olympus) using a phase contrast 20  objective (LCPlanFl, Olympus). The images were acquired by color CCD camera (1129HMN1/3, Wraymer) and recorded on a hard drive of PC. The video images were analyzed using ImageJ (http://rsbweb. nih.gov/ij/).

Quantitative Characterization of Vesicle Morphology by Distortion Index. To quantitatively characterize the morphologies of

GMVs, we introduced “distortion index (D)”, a measure of deviation from the spherical shape (Figure 2). The distortion index was defined as 10107

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Hence, it is expected that the exchange rate of Triton X-100 across the cellulose membrane with MWCO 25 000 is fairly slow when 1.0 mM Triton X-100 was introduced in the upper compartment because Triton X-100 can diffuse across the membrane only when dissociated from the micelles. Our dialysis experiment showed that the exchange rate of Triton X-100 across the membrane, denoted k here, was estimated to be 3.4  10
2 cm h
1 (see SI for details). The concentration of Triton X-100 in the lower compartment of the perfusion device increases according to the following: ~

Figure 2. Distortion index for quantitative characterization of vesicle morphology. Distortion index can be determined for individual vesicles by measuring the perimeter and area on 2D image. (A) Distortion index for spherical GVs. (B) Distortion index for distorted GVs. (C) Representative images of GVs with different values of distortion index. The shape is nearly spherical when D e 0.5, whereas the GVs with distorted shapes have larger values of D. We used this index to quantitatively characterize the morphology of GVs. The bars in the images are 10 μm. follows: D¼

l2
4π S

where l is the perimeter (contour) and S is the area of a GMV on a 2D image. We measured l and S by ImageJ. D equals zero when the shape is the perfect sphere (Figure 2(A)), and becomes large as it deviates from the spherical shape (Figure 2(B)). Ideally, the distortion index must be defined based on the 3D structure of GMVs using the information of surface area and volume, but this index, which is simpler and easier to be measured, can also capture the differences of GMV shapes nicely as shown in Figure 2(C). Therefore, we used this simple index to quantify the 2D morphological dynamics of GVs in this work.

’ RESULTS AND DISCUSSION The perfusion device described in Figure 1 allows flexible environmental control and continuous monitoring of individual GMVs. For example, we can track the morphological changes of individual GMVs under periodically changing environment as shown in the SI (Figure S1 of the SI). In this report, we apply this device to the study of the morphological dynamics of GMVs under the conditions where the concentration of surfactant, Triton X-100, monotonically and gradually increases in the time-course. Triton X-100 is a nonionic surfactant that dissolves lipid membranes and known to induce morphological dynamics to GVs such as continuous-stepwise shrinkage with intermittent quake and pore formation.31,32,43 These reported morphological events proceed under constant and high concentration of Triton X-100 after the addition of the solution in the time scales of seconds to minutes. To study vesicle morphological dynamics in nonconstant environments, we arranged a monotonic and gradual increase of Triton X-100 concentration to a mild saturation level (1.0 mM) using the perfusion device; thereby we examined the morphological dynamics of the three POPC-based GMVs that proceed in the time scales of several hours before the complete dissolution. To induce a noticeable dissolution of GVs, the concentration of surfactants must exceed the critical micellization concentrations (CMCs), which is approximately 0.3 mM for Triton X-100.44 The molecular weight of the Triton X-100 micelle complex is ∼90 000 whereas that of one molecule is 650.44

CL ðtÞ ¼ CU ð1
e
kSt=VL Þ

ð1Þ

where CL (t) is the Triton X-100 concentration in the lower compartment at time t, CU is the constant Triton X-100 concentration in the upper compartment, ~S is the area of the cellulose membrane separating the two compartments and VL is the volume of the lower compartment (see SI). Incorporating the device configuration and experimental setting into the parameters indeed finds a slow monotonic increase curve of Triton X-100 concentration in the lower compartment; it exceeds the CMC in 19 min and the half saturation value (0.5 mM) in 38 min (Figure 3(A) and SI). We monitored the three types POPC-based vesicles, PC, PC: PG, and PC:PG:Chol, under the nonconstant Triton X-100 conditions described above. As a consequence, we found six different paths to the complete dissolution characterized by different morphological dynamics: disassembly by peeling, shrinkage by dissolution, burst, birthing, budding and fission, and open-and-close (Figure 3(B) and Movies S1
S7 of the SI). Furthermore, we determined the approximate frequencies of these different dynamics following many GMVs (Table 1). The below is the summary of the observed dynamics. Disassembly by Peeling (Movie S1 of the SI). The process by which GMVs are broken into many vesicles with low lamellarity. In this process, the outer layers were gradually peeled off from GMVs and formed new vesicles. The newly formed low lamellar vesicles stay intact for a short period, thereafter dissolved individually. This process was the major pathway for PC (Table 1). Shrinkage by Dissolution (Movies S2 and S3 of the SI). The process by which GMVs steadily decreases their size in the course of solubilization. Some GMVs maintained nearly spherical shape (Movie S2 of the SI), whereas the others deformed in the courses of shrinkage (Movie S3 of the SI). This process was observed only for PC:PG and PC:PG:Chol. Burst (Movie S4 of the SI). The process in which GMVs dissolve suddenly without premonitory morphological changes. This was the major pathway for the PC:PG. Birthing (Movie S5 of the SI). The process in which the internally encapsulated vesicles are released through the outer layers of the membrane. The released vesicles sometimes repeated the same birthing process a few times successively. This was the major pathway for the PC:PG:Chol. Budding and Fission (Movie S6 of the SI). The process in which a small bud gradually grows out on vesicles and is eventually detached from the mother vesicle. The sizes of the mother vesicles were nearly unchanged during this process. Open-and-Close (Movie S7 of the SI). The process in which a part of the vesicle is radically ruptured and the membrane returns to spherical shape thereafter. The size of the GMVs was smaller after rupture, which suggests that the internal content was released when the membrane was open. 10108

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Figure 3. Morphological dynamics of GMVs in monotonic increase of surfactant concentration. (A) Estimated kinetics of Triton X-100 concentration increase in lower compartment. The parameter values specified by the device configuration and experimental setting were incorporated into [eq 1] to determine the curve: CU = 1 mM, k = 3.4  10
2 cm h
1, ~S = 0.81 cm2, and VL = 2.5  10
2 cm3. (B) Multiple pathways to solubilization of POPC-based GMVs. The small micrographs show the representative behaviors of the vesicles following the different pathways. The constrast of the second three images in disassembly was enhanced to make the low lamellar vesicles visible. The thickness of the colored arrows indicates the approximate frequencies of the pathways. The different colors of the arrows, blue, red, and green, correspond to PC, PC:PG, and PC:PG:Chol vesicles, respectively. The bars in the micrographs are 20 μm.

Table 1. Frequencies of Different Morphological Patterns in the Solubilization Processes of Three POPC-Based GMVsa PC disassembly

97% (73/75)

shrinkage burst birthing budding open-and-close

3% (2/75)

PC:PG

PC:PG:Chol

4% (3/78)

5% (4/76)

23% (18/78)

17% (13/76)

62% (48/78)

32% (24/76)

8% (7/78) 3% (2/78)

38% (29/76) 5% (4/76) 3% (2/76)

a

The numbers in the parentheses show the actual sample numbers in the observations.

The frequency and multitude of the solubilization pathways were significantly affected by the composition of the vesicles (Figure 3(B) and Table 1). For example, only minor fraction of the PC:PG and PC:PG:Chol GMVs exhibited disassembly though it was the major pathway for the PC GMVs. The multitude of the pathways increased with the number of vesicle components. This might be due to the diversity of the membrane compositions in individual vesicles; even minor

deviations in the distribution of the membrane molecules in the lipid films before rehydration during the vesicle preparations might have produced the GMVs with different compositions. Inequality of the composition does not exist in the PC vesicles, but should become prominent for binary and tertiary mixed membranes (PC:PG and PC:PG:Chol). We implemented a statistical test for the dependence of the pathway choice on initial size of GMVs using the patterns with reasonable sample numbers: shrinkage and burst for PC:PG, and shrinkage, burst, and birthing for PC:PG:Chol (Table 1). We detected a statistically significant difference only between burst and birthing for PC:PG:Chol (Wilcoxon rank sum test for PC: PG, multiple-comparison test after Kruskal
Wallis test for PC: PG:Chol, p < 0.05). This result suggests that initial size of GMVs is not a strong indicator of pathway choice. The examination of the time-course transition of distortion index and size of individual vesicles revealed the differences in the premonitory behaviors before the drastic morphological events (Figure 4). In the disassembly and birthing processes, the shapes of the vesicles deformed and fluctuated significantly long before breaking into many vesicles or the release of internal vesicles (Figure 4(A),(D)). However, almost no notable premonitory 10109

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Figure 4. Representative time-course dynamics of distortion index and size of individual vesicles before drastic morphological events. In all panels, the upper and lower graphs show the temporal changes of distortion index and vesicle size (total area on 2D images, relative to initial size), respectively. The images above the graphs are the snapshots of the vesicles at the time points indicated by the gray arrows. The end time points in the graphs are just before proceeding to the drastic morphological dynamics, which are shown in the images after the thick black arrows. The resultant dynamics are disassembly by peeling (A), shrinkage by dissolution (B), burst (C), birthing (D), budding (E), and open-and-close (F). The types of GMVs used to make the plots are PC for (A), PC:PG for (B) and (E), and PC:PG:Chol for (C), (D), and (F). The qualitative behaviors in the same pathways were unchanged between different compositions. The bars in the images are all 20 μm.

deformation was observed in the burst, budding, and open-andclose processes (Figure 4(C),(E), and (F)). The shrinkage process was intermediate in terms that the deformation occurs only in some cases, but the specification of the GMVs to this pathway was readily detected as the distinct decrease of vesicle sizes (Figure 4(B)). The differences of the premonitory behaviors long before the final and drastic morphological events suggest that the fates of GMVs regarding which pathways they follow might be specified in relatively early stages. The difference of premonitory behaviors might allow the grouping of these six dynamics. For example, burst, budding, and open-and-close processes might be categorized into one group because there is no noticeable morphological change before the drastic events. However, disassembly, shrinkage, and birthing might be categirozed into another with the existence of premonitory morphological changes. It is of great interest to examine whether the difference of the groups might reflect the different internal processes causing the morphological dynamics. The rich morphological dynamics of simple lipid membranes and the multiple pathways to solubilization shown in Figures 3 and 4 have not been reported previously in similar experiments.32,33,38 The biggest difference in our experiment from these studies is that we used relatively large size multilamellar vesicles, not unilamellar. The diversities of internal states due to high lamellarity should be an important requirement for the emergence of multiple pathways. The importance of internal state diversity is also expected from that the number of possible pathways increased with the number of constituent molecules (Figure 3 and Table 1). The contributions of lamellarity and lipid compositions would probably be examined by, for example, electron microscopic structural analysis of GMVs.

Another important difference is that the concentration of Triton X-100 was increased very slowly in our experiment (Figure 3(A)). The mild solubilization conditions used in this report might have provided enough time for the GMVs to exhibit slow deformation before being dissolved rapidly. The importance of slow increase of Triton X-100 concentration for the multitude of the dynamics is also suggested by the fact that the GMVs were immediately dissolved when the GMV dispersion was simply mixed with 1 mM Triton X-100 solution. This confirms the importance of the speed of environmental changes for the membrane dynamics of GMVs; the different dynamics could potentially arise in gradual and rapid environmental changes even between the identical initial and final conditions. It is demonstrated that domain formation induces shape transformation to multicomponent vesicles;45
47 the sizes and distributions of domains on GMVs might also be relevant to the observed morphological dynamics. Understanding the roles of domains on multicomponent GMVs in different morphological changes should be crutial to gain insights into the mechanisms underlying the multitude of morphological pathways. We introduced a simple perfusion device that allows flexible environmental control and long-term tracking of individual vesicles in this report. The properties of vesicle dynamics depend on both internal factors such as the properties and organization of constituent molecules and external factors such as the existence of stimulating agents around GVs. Therefore, the flexible control of environmental conditions by this device should offer an ideal experimental tool to study the membrane dynamics of GVs. This perfusion device might also facilitate the exploration of the prebiotic conditions leading to protocells exhibiting a replication cycle. For example, it is suggested that cyclically changing environments such as tidal waves and thermal vents might have 10110

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Langmuir been key conditions for the emergence of early life forms and Darwinian evolution.48,49 It is difficult to observe vesicle behaviors in cyclically changing environments by conventional methods, but becomes feasible with this perfusion device. One of the most attractive applications of this device would be the study on growth and division of vesicles. The growth vesicles reported to date require cyclic addition of the solutions containing membrane molecules or precursors.1
4 The proof of the progress of growth has been based on the indirect evidence provided by population-based measurements; the perfusion device would enable us to directly verify growth and division progress at the individual vesicle level when appropriate cyclic environments are applied. The systematic observation of morphological dynamics reported here should provide important insights into the membrane dynamics of GMVs. The properties and dynamics of unior low-lamellar GVs have been studied intensively, whose relatively simple configuration have even allowed the formulation of theoretical models of their membrane dynamics such as areadifference-elasticity (ADE) models.50 The ADE model considers local curvature elasticity and elastic area difference stretching energy, which allows the prediction of the shapes of unilamellar vesicles.50
52 The dynamics provoked by Triton X-100 might also be predicted by this model with the information on the rate of insertion into membranes and the water exchange rate between the interior of vesicles and bulk. In the case of GMVs, the conditions of all inner membranes as well as the water volumes in different layers should affect the morphologies; theoretical analysis on the membrane dynamics of GMVs has been daunting because of this complexity and the lack of systematic experimental studies. However, the characterization of possible patterns of the dynamics and the quantative information on the pattern frequencies as we reported here might be an important stepping stone toward the theoretical analysis on the dynamics of GMVs.

’ ASSOCIATED CONTENT

bS

Supporting Information. Supporting Text, Figures S1 and S2, and Movies S1
S7. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Tel: +81-3-5454-4356; Fax: +81-3-5454-4356; E-mail: cwaka@ mail.ecc.u-tokyo.ac.jp.

’ ACKNOWLEDGMENT We thank Martin Hanczyc and Taro Toyota for critical reading of the manuscript and helpful comments. We thank Reiko Okura for technical support. This work was partially supported by KAKENHI 21684024. Y.W. was supported by the JST PRESTO program. ’ REFERENCES (1) Hanczyc, M. M.; Fujikawa, S. M.; Szostak, J. W. Experimental models of primitive cellular compartments: Encapsulation, growth, and division. Science 2003, 302 (5645), 618–622.

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(2) Hanczyc, M. M.; Szostak, J. W. Replicating vesicles as models of primitive cell growth and division. Curr. Opin. Chem. Biol. 2004, 8 (6), 660–664. (3) Zhu, T. F.; Szostak, J. W. Coupled growth and division of model protocell membranes. J. Am. Chem. Soc. 2009. (4) Takakura, K.; Sugawara, T. Membrane dynamics of a myelin-like giant multilamellar vesicle applicable to a self-reproducing system. Langmuir 2004, 20 (10), 3832–3834. (5) Staneva, G.; Seigneuret, M.; Koumanov, K.; Trugnan, G.; Angelova, M. I. Detergents induce raft-like domains budding and fission from giant unilamellar heterogeneous vesicles—A direct microscopy observation. Chem. Phys. Lipids 2005, 136 (1), 55–66. (6) Berclaz, N.; Muller, M.; Walde, P.; Luisi, P. L. Growth and transformation of vesicles studied by ferritin labeling and cryotransmission electron microscopy. J. Phys. Chem. B 2001, 105 (5), 1056–1064. (7) Zhou, Y. F.; Yan, D. Y. Real-time membrane fission of giant polymer vesicles. Angew. Chem., Int. Ed. 2005, 44 (21), 3223–3226. (8) Menger, F. M.; Gabrielson, K. Chemically-induced birthing and foraging in vesicle systems. J. Am. Chem. Soc. 1994, 116 (4), 1567–1568. (9) Menger, F. M.; Lee, S. J.; Keiper, J. S. Induced separation of a binate vesicle into two independent entities. Chem. Commun. 1998, No. 9, 957–958. (10) Menger, F. M.; Angelova, M. I. Giant vesicles: Imitating the cytological processes of cell membranes. Acc. Chem. Res. 1998, 31 (12), 789–797. (11) Takakura, K.; Toyota, T.; Yamada, K.; Ishimaru, M.; Yasuda, K.; Sugawara, T. Morphological change of giant vesicles triggered by dehydrocondensation reaction. Chem. Lett. 2002, No. 3, 404– 405. (12) Kaes, J.; Sackmann, E. Shape transitions and shape stability of giant phospholipid vesicles in pure water induced by area-to-volume changes. Biophys. J. 1991, 60 (4), 825–844. (13) Holopainen, J. M.; Angelova, M. I.; Kinnunen, P. K. J. Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophys. J. 2000, 78 (2), 830–838. (14) Berndl, K.; Kas, J.; Lipowsky, R.; Sackmann, E.; Seifert, U. Shape transformations of giant vesicles - extreme sensitivity to bilayer asymmetry. Europhys. Lett. 1990, 13 (7), 659–664. (15) Bernard, A. L.; Guedeau-Boudeville, M. A.; Jullien, L.; di Meglio, J. M. Raspberry vesicles. Biochim. Biophys. Acta-Biomembr. 2002, 1567 (1
2), 1–5. (16) Haluska, C. K.; Riske, K. A.; Marchi-Artzner, V.; Lehn, J. M.; Lipowsky, R.; Dimova, R. Time scales of membrane fusion revealed by direct imaging of vesicle fusion with high temporal resolution. Proc. Natl. Acad. Sci. U.S.A. 2006, 103 (43), 15841–15846. (17) Richard, A.; Marchi-Artzner, V.; Lalloz, M. N.; Brienne, M. J.; Artzner, F.; Gulik-Krzywicki, T.; Guedeau-Boudeville, M. A.; Lehn, J. M. Fusogenic supramolecular vesicle systems induced by metal ion binding to amphiphilic ligands. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (43), 15279–15284. (18) Nomura, F.; Inaba, T.; Ishikawa, S.; Nagata, M.; Takahashi, S.; Hotani, H.; Takiguchi, K. Microscopic observations reveal that fusogenic peptides induce liposome shrinkage prior to membrane fusion. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (10), 3420–3425. (19) Kahya, N.; Pecheur, E. I.; de Boeij, W. P.; Wiersma, D. A.; Hoekstra, D. Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion. Biophys. J. 2001, 81 (3), 1464–1474. (20) Maru, N.; Shohda, K. I.; Sugawara, T. Successive fusion of vesicles aggregated by DNA duplex formation in the presence of triton X-100. Chem. Lett. 2008, 37 (3), 340–341. (21) Sunami, T.; Caschera, F.; Morita, Y.; Toyota, T.; Nishimura, K.; Matsuura, T.; Suzuki, H.; Hanczyc, M. M.; Yomo, T. Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter. Langmuir 2010, 26 (19), 15098–15103. (22) Takiguchi, K.; Nomura, F.; Inaba, T.; Takeda, S.; Saitoh, A.; Hotani, H. Liposomes possess drastic capabilities for topological transformation. Chemphyschem 2002, 3 (7), 571–574. 10111

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Langmuir (23) Kulin, S.; Kishore, R.; Helmerson, K.; Locascio, L. Optical manipulation and fusion of liposomes as microreactors. Langmuir 2003, 19 (20), 8206–8210. (24) Luisi, P. L.; Walde, P., Giant Vesicles: Perspectives in Supramolecular Chemistry; Wiley-Interscience: New York, 2000. (25) Schrum, J. P.; Zhu, T. F.; Szostak, J. W. The origins of cellular life. Cold Spring Harb. Perspect. Biol. 2010, 2 (9), a002212. (26) Szostak, J. W.; Bartel, D. P.; Luisi, P. L. Synthesizing life. Nature 2001, 409 (6818), 387–390. (27) Luisi, P. L.; de Souza, T. P.; Stano, P. Vesicle behavior: In search of explanations. J. Phys. Chem. B 2008, 112 (46), 14655–14664. (28) Suzuki, K.; Toyota, T.; Takakura, K.; Sugawara, T. Sparkling morphological changes and spontaneous movements of self-assemblies in water induced by chemical reactions. Chem. Lett. 2009, 38 (11), 1010–1015. (29) Takakura, K.; Toyota, T.; Sugawara, T. A novel system of self-reproducing giant vesicles. J. Am. Chem. Soc. 2003, 125 (27), 8134– 8140. (30) Wick, R.; Walde, P.; Luisi, P. L. Light-microscopic investigations of the autocatalytic self-reproduction of giant vesicles. J. Am. Chem. Soc. 1995, 117 (4), 1435–1436. (31) Nomura, F.; Nagata, M.; Inaba, T.; Hiramatsu, H.; Hotani, H.; Takiguchi, K. Capabilities of liposomes for topological transformation. Proc. Natl. Acad. Sci. U.S.A. 2001, 98 (5), 2340–2345. (32) Hamada, T.; Hirabayashi, Y.; Ohta, T.; Takagi, M., Rhythmic pore dynamics in a shrinking lipid vesicle. Phys. Rev. E 2009, 80, (5). (33) Pata, V.; Ahmed, F.; Discher, D. E.; Dan, N. Membrane solubilization by detergent: Resistance conferred by thickness. Langmuir 2004, 20 (10), 3888–3893. (34) Hotani, H. Transformation pathways of liposomes. J. Mol. Biol. 1984, 178 (1), 113–120. (35) Hotani, H.; Nomura, F.; Suzuki, Y. Giant liposomes: From membrane dynamics to cell morphogenesis. Curr. Opin. Colloid Interface Sci. 1999, 4 (5), 358–368. (36) Ohno, M.; Hamada, T.; Takiguchi, K.; Homma, M. Dynamic behavior of giant liposomes at desired osmotic pressures. Langmuir 2009, 25 (19), 11680–11685. (37) Yanagisawa, M.; Imai, M.; Taniguchi, T., Shape deformation of ternary vesicles coupled with phase separation. Phys. Rev. Lett. 2008, 100, (14). (38) Sudbrack, T. P.; Archilha, N. L.; Itri, R.; Riske, K. A. Observing the solubilization of lipid bilayers by detergents with optical microscopy of GUVs. J. Phys. Chem. B 2011, 115 (2), 269–277. (39) Bhattacharya, S.; Bisivas, J. Understanding membranes through the molecular design of lipids. Langmuir 2010, 26 (7), 4642–4654. (40) Fukuda, H.; Kawata, K.; Okuda, H.; Regen, S. L. Bilayerforming ion-pair amphiphiles from single-chain surfactants. J. Am. Chem. Soc. 1990, 112 (4), 1635–1637. (41) Kaler, E. W.; Murthy, A. K.; Rodriguez, B. E.; Zasadzinski, J. A. N. Spontaneous vesicle formation in aqueous mixtures of singletailed surfactants. Science 1989, 245 (4924), 1371–1374. (42) Suzuki, K.; Toyota, T.; Sato, K.; Iwasaka, M.; Ueno, S.; Sugawara, T. Characteristic curved structure derived from collagencontaining tubular giant vesicles under static magnetic field. Chem. Phys. Lett. 2007, 286–290. (43) Hamada, T.; Miura, Y.; Ishii, K. I.; Araki, S.; Yoshikawa, K.; Vestergaard, M.; Takagi, M. Dynamic processes in endocytic transformation of a raft-exhibiting giant liposome. J. Phys. Chem. B 2007, 111, 10853–10857. (44) Yanagita, Y.; Kagawa, Y. Techniques for Analysis of Membrane Proteins; Chapmann & Hall: New York, 1986. (45) Baumgart, T.; Hess, S. T.; Webb, W. W. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 2003, 425 (6960), 821–824. (46) D€obereiner, H. G.; K€as, J.; Noppl, D.; Sprenger, I.; Sackmann, E. Budding and fission of vesicles. Biophys. J. 1993, 65 (4), 1396–1403. (47) Lipowsky, R. Domain-induced budding of fluid membranes. Biophys. J. 1993, 64 (4), 1133–1138.

ARTICLE

(48) Budin, I.; Bruckner, R. J.; Szostak, J. W. Formation of protocelllike vesicles in a thermal diffusion column. J. Am. Chem. Soc. 2009, 131 (28), 9628–9629. (49) Bywater, R. P.; Conde-Frieboes, K. Did life begin on the beach?. Astrobiology 2005, 5 (4), 568–574. (50) Miao, L.; Seifert, U.; Wortis, M.; D€obereiner, H. G. Budding transitions of fluid-bilayer vesicles: The effect of area-difference elasticity. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics 1994, 49 (6), 5389–5407. (51) Svetina, S.; Zeks, B. Shape behavior of lipid vesicles as the basis of some cellular processes. Anat Rec. 2002, 268 (3), 215–225. (52) Svetina, S. Vesicle budding and the origin of cellular life. Chemphyschem 2009, 10 (16), 2769–2776.

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