Deformation Modes of Giant Unilamellar Vesicles Encapsulating

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Deformation Modes of Giant Unilamellar Vesicles Encapsulating Biopolymers Taiji Okano, Koya Inoue, Kaoru Koseki, and Hiroaki Suzuki ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00460 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 3, 2018

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ACS Synthetic Biology

Deformation Modes of Giant Unilamellar Vesicles Encapsulating Biopolymers Taiji Okano‡, Koya Inoue‡, Kaoru Koseki, Hiroaki Suzuki* ‡

Equal contribution

* Corresponding Author

Faculty of Science and Engineering, Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan

ABSTRACT: The shapes of giant unilamellar vesicles (GUVs) enclosing polymer molecules at relatively high concentration, used as a model cytoplasm, significantly differ from those containing only small molecules. Here, we investigated the effects of the molecular weights and concentrations of polymers such as polyethylene glycol (PEG), bovine serum albumin (BSA), and DNA on the morphology of GUVs deflated by osmotic pressure. Although small PEG (MW < 1k) does not alter the mode of shape transformation even at > 10% (w/w), PEG with MW > 6k induces budding and pearling transformation at above 1% (w/w). Larger PEG frequently induced small buddings and tubulation from the membrane of mother GUVs. A similar trend was observed with BSA, indicating that the effect is irrelevant to the chemical nature of polymers. More surprisingly, long strands of DNA (> 105 bp) enclosed in GUVs induced budding transformation at concentrations as low as 0.01–0.1% (w/w). We expect that this molecular size dependency arises mainly from the depletion volume effect. Our results showed that curving, budding, and tubulation of lipid membranes, which are ubiquitous in living cells, can result from simple cell-mimics consisting of the membrane and cytosolic macromolecules, but without specific shape-determining proteins.

Cells and organelles, enclosed by phospholipid membranes, exhibit various shapes. Thus, the underlying principles that determine the morphology of the simplest membranous structures have been studied extensively using giant unilamellar vesicles (GUVs) composed of phospholipids.1−6 Because of its extremely low bending resistance, a vesicle composed of a phospholipid bilayer with extra membrane area can deform into many different shapes. Both analytical and computational studies have succeeded in predicting various shapes observed in experiments using

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GUVs, as a result of energy minimization under the given elastic moduli and the extent of excess membrane area. In addition to thermodynamically stable shapes, shapes such as lipid nanotubes, likely to be kinetically stable, are often observed in experiments.7−9 Although the morphology and dynamics of modern cells are mainly governed by the static and dynamic suprastructures of the cytoskeleton and other membrane-associated proteins,10−12 the relevance of the results of the GUV experiments to shapes observed in living cells are often discussed.8, 13−17 The static and dynamic shapes of GUVs could reflect the fundamental forms of cells, organelles, and their substructures. In addition, attempts to create an artificial cell by both bottom-up and top-down approaches are ongoing worldwide.18−28 Such entities, composed of minimum building blocks required to exhibit the desired cellular functions, are often constructed by enclosing active biopolymers into GUVs. Thus, shape studies of GUVs may contribute to the understanding of the physical and mechanical working principles of artificially engineered cells. Because, essentially, cells are filled with cytosolic biopolymers, it is important to understand the morphology of GUVs containing polymers29, 30 in addition to that of GUVs with dilute aqueous phase. We previously demonstrated that GUVs enclosing polymer molecules transform into budded shapes when they attain extra membrane area upon electrofusion.31 This transformation was found to be induced by the positive membrane curvature (inward bending) mediated by the depletion volume effect between the membrane and encapsulated crowding polymers. However, the concentration range of the crowding agent (polyethylene glycol (PEG) and dextran) was limited, and we only focused on whether the budding transformation occurred. Thus, GUV deformation in the parameter space including the polymer size and concentration needs to be uncovered in detail. In the present work, we examined the effect of the concentration and molecular weight of enclosed polymers on the GUV shapes. Excess membrane was created onto the originally spherical GUVs using osmotic deflation.

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Depending on the molecular weight and concentration of encapsulated PEG, GUVs exhibited varied shape deformation with apparent tendency. In addition to the synthetic polymer, we studied the effect of encapsulating a model protein and genome-size DNA. Interestingly, we found that the genomic DNA of λ-phage enclosed in a GUV was able to induce budding transformation at a concentration as low as ~100 µg/ml (~ 0.01% (w/w)). It was intriguing that crowding biopolymers in a cell membrane affects not only the association and reaction dynamics35, 36, but also the morphological characteristics of cellular entities.

RESULTS AND DISCUSSION Vesicle Formation and Osmotic Deflation. To produce GUV-containing polymers, the water-in-oil (W/O) emulsion transfer method37 was employed as described previously.31, 38 In short, an aqueous polymer solution was emulsified (PEG with MW of 400, 6k, or 20k, or λ-DNA together with 200 mM sucrose) into liquid paraffin oil containing 5 mg/ml of a phospholipid mixture (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/ -palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG)/cholesterol = 9:1:0.5 molar ratio) by vortexing. A small amount of negatively charged lipid (POPG) was included because it helps to obtain dispersed GUVs compared to those composed of POPC.38 After mounting the emulsion on another aqueous solution containing 200

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mM glucose in the test tube, centrifugation was applied to force the emulsion droplets to pass through the lipid monolayer formed at the oil/water interface. After a washing step, we obtained GUVs with a diameter range from one to several tens of micrometers (Figure 1a). It is well established that polymers can be encapsulated at concentrations identical to those of the original aqueous solution.37, 39 In addition, GUVs prepared by this method may contain the oil residue between bilayer leaflets, but in our preparation, no oil lump was seen under the microscope even after one week of storage. Moreover, GUVs prepared using identical conditions exhibited deformation dynamics similar to classical theories,40 and could sustain active membrane proteins.41-46 Thus, it was considered that GUVs prepared by the centrifugation method have minimal oil residue that can be used for studying vesicle shape similarly to GUVs formed by hydration-based methods. The osmotic deflation of vesicles was induced to produce an excess membrane area by simply mixing the GUV suspension and a high concentration glucose solution on a coverglass. In the initial trial stage, the effect of a sugar concentration difference ∆cs = 150 mM and 300 mM (350 and 500 mM final concentrations in the outer solutions, respectively) was tested. We found that both conditions produced similar trends (Figure S1). Thus, ∆cs = 300 mM, which generated more pronounced results, was employed in further experiments. The deflated GUVs were observed under a laser confocal microscope after approximately 10 min, at which point most GUVs had sedimented on the coverglass due to the density difference. Thus, the longest axis of the non-spherical GUV shapes lay nearly in parallel to the coverglass surface. Shape transformation induced by PEG. The internal volume and the membrane of GUVs were visualized with calcein (green fluorescence) and DiI (red fluorescence), respectively. The GUVs produced by the W/O emulsion transfer method were mostly spherical and unilamellar (Figure 1a).37, 38 After osmotic deflation, the outer shapes were found to be mostly circular, but often with one or several internalized hollow circles (Figure 1b) when GUVs contained no PEG. The three-dimensional construction of z-stack images revealed that GUVs with hollow inner circles were extreme stomatocytes (spheres with inward invaginations) (Figure 1c, d). Assuming that the osmotic difference is solely due to ∆cs, theoretically 60% water was excluded to attain isotonicity. This shrinkage corresponds to the reduced volume (vred = V/(4π/3R03) = 0.4, where V and R0 are the volume after shrinkage and the initial vesicle radius, respectively) at which the vesicle shape was predicted to become the stomatocyte shape in the spontaneous curvature (SC) model.2 GUVs with filled circular contours could be either spherical or oblate (disk shape), which cannot be distinguished from planar imaging. In either case, all the observed shapes are in accordance with the classical theories. Next, the same experiment was conducted with GUVs containing 2% (w/w) PEG 400 (50 mM). The resultant morphological changes (Figure 2 a to b) were very similar to those without PEG, indicating that PEG with a low molecular weight did not affect the mode of shape transformation. In contrast, when GUVs contained 2% (w/w) PEG 6k (3.3 mM), small buds often appeared around the mother GUVs in addition to transformation to the ordinal dumbbell (major budding) and pearl-chain shapes (Figure 2c). Finally, with 2% (w/w) PEG 20k (1 mM), various local and small buddings and tubulations of the membrane dominated (Figure 2d). Morphological Analysis. The effect of concentration of each PEG ranging from 1 to 12% (w/w) was

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investigated to obtain overall behavior. From the confocal microscopy images, GUVs between 5 and 20 µm in linear size were randomly selected and categorized into seven classes based on their morphological characteristics (Figure 3a): sphere, prolate (long spheroid), stomatocyte, pear, dumbbell (or pearl-chain), small buddings, and tubular. The dumbbell shape was defined as GUVs with neighboring daughter vesicular structure(s) larger than 2 µm in diameter, and the small buddings were determined as those with multiple small lipid humps smaller than 2 µm. The tubular shape was determined if the GUVs had protruded membrane structures longer than 3 µm. Comparison of shapes before and after deflation procedure is shown in Figure 3(b). With no PEG, most of the GUVs were spherical (86%), but with a small fraction of prolate shape (10%). After deflation, a significant portion of GUVs attained stomatocyte shape (26%). With PEG 20k, most of the GUVs were initially spherical, but 8% of them had small budding structures. However, after deflation, GUVs with small buddings increased to 39%. GUVs with dumbbell shape also increased to 15%. As mentioned below, we hypothesized that encapsulated PEG is responsible for budding transformations. Because a small portion of flaccid GUVs were present in the initial population, their extra membrane was deformed to become small buds even before osmotic deflation. The number of GUVs with small buds increased as they were deflated. The percentage of shape categories after deflation was plotted in Figure 3 (c) to (e) for each PEG. The morphology of PEG 400 was nearly identical to that without PEG among all the concentrations tested; the sphere and stomatocyte shapes were dominant, with small fractions of prolate, pear, and dumbbell shapes (Figure 3c). Here, note that PEG was only present inside GUVs and the polymer osmosis in the outer solution was not compensated, but we obtained nearly identical results. These shapes are a result of gentle transformations predicted by the energy-minimizing equilibrium of the membrane, confirming that PEG with a low molecular weight does not affect the morphology of a lipid membrane even at high concentrations. In contrast, although present at 1% (w/w) PEG 6k, the stomatocyte shape disappeared at concentrations above 2% (w/w) PEG 6k (Figure 3d). Moreover, the frequency of the dumbbell shape increased to 12% and 15% at 1 and 2% (w/w), respectively. Disappearance of stomatocyte shape as well as the appearance of the dumbbell shape is a result of the membrane obtaining a positive curvature, i.e., the membrane bending inward. At the same time, small buddings and tubulations constituted a large fraction of shapes observed above 2% (w/w). With PEG 20k, these protruded substructures became dominant even at 1% (w/w), and the trend of morphology change was almost identical among the concentrations tested (Figure 3d). Depletion Volume Effect. The increase in the dumbbell shapes above 1% (w/w) PEG 6k was similar to that in the budding transformation observed after GUV electrofusion as shown in our previous studies.31, 47 Thus, the same result was reproduced irrespective of the method of inducing excess membrane area. This phenomenon could be explained by the depletion volume effect of encapsulated PEG, which generates the positive curvature of the membrane.31, 48-51 Because PEG molecules have a defined size (3.1 nm for PEG 6k and 6.3 nm for PEG 20k in the gyration radius; calculated by rg = 0.02 Mw0.58 nm)52 and are essentially inert to the lipid membrane,53, 54 the center of the polymer coil cannot exist near the membrane closer than rg. Thus, polymers are depleted from the vicinity of the inner side of the membrane. Thermodynamically, reduction in the depletion volume Vdep is favored, because the volume of polymer solution in GUV in turn increases. This change results in an increase in the translational entropy

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of the polymer molecules (i.e., decrease in free energy described as ∆Gdep = −Π∆Vdep, where Π is the polymer osmotic pressure). Indeed, the decrease in Vdep can be accomplished via the membrane bending inward at the cost of bending energy. The stomatocyte shape disappeared at a higher concentration of larger PEG because the negative curvature (bending outward or invagination) had larger Vdep, which is entropically unfavored. Note again that this membrane curvature is induced by the asymmetry of polymer molecules across the membrane. When the polymer exists on both sides of the membrane, the positive curvature was not observed.8, 31, 55 Moreover, the generation of small buddings and tubulation can be explained by the strong depletion volume effect at higher concentrations and larger molecular weights of PEG. At the thermodynamic equilibrium, the local curvature of the membrane should be determined by the balance between the membrane bending energy and the entropy gain due to the reduction in Vdep. To test this hypothesis, we assumed a simple model in which an intrinsically flat membrane curls up to become a part of the sphere with radius of curvature Rs (see Supporting Information Part I). In this model, we derived Rs = 0.33 and 0.084 µm for PEG 6k and 20k, respectively, using Π ~ 10 mOsm (2% (w/w))56 and κb ~ 10 kBT. For a tubular structure, assuming that a flat membrane rolls up to become a part of cylinder wall with radius Rc, we obtained Rc ~ 0.16 and 0.042 µm, respectively, for PEG 6k and 20k. These scales are close to or at least not far from the observed structures. In addition, it is noteworthy that these small structures appeared close to the semi-dilute regime, where the polymer molecules start to overlap (overlapping concentration c* = Mw/(4/3πrg3NA), where NA is the Avogadro constant, for PEG 6k and 20k are 77 and 32 mg/ml, respectively). In this regime, the osmotic pressure and viscosity of the polymer solution increase with c2 (Flory-Huggins theory).57 Thus, it is reasonable to consider that the combination of the depletion volume effect, which bends the membrane inward, and the viscosity of macromolecular solution, which restricts the water flow into the budding structures, were responsible for the generation of small membrane structures. At concentrations higher than c*, Π is expected to increase with c9/4 scaling (the des Cloiseaux scaling).58 However, the thickness of the depletion volume is no longer represented by rg, but shrinks according to the correlation length, ξ = rg(c/c*)−0.75.59 Thus, it is reasonable that ∆Gdep and its resulting deformation mode only weakly depended on the polymer concentration above c*. Shape Transformation Induced by Protein. Next, we conducted the same deflating experiment with GUVs containing BSA as a model protein, instead of PEG. Indeed, as depicted in Figure 4, the mode of shape transformation was dependent on the BSA concentration, similarly to GUVs containing PEG 6k. In particular, stomatocyte shape, which consisted a major portion with 0 and 1% (w/w) BSA, decreased above 2% (w/w) BSA. In turn, the dumb-bell shape and small buddings increased significantly. This result is acceptable considering that the size (or Stokes radius) of folded BSA (~ 66.5 kDa) is ~ 3.4 nm,60 which is close to PEG 6k. The generation of small budding at high BSA concentration is in accordance with the previous study that reported deflating GUV enclosing 30% (w/w) BSA.29 Shape Transformation Induced by DNA. Next, the same deflating experiment was conducted with GUVs containing a large linear DNA. For this purpose, genomic DNA extracted from λ-phage (λDNA; 4.8 × 104 bp, 16 µm in contour length, 3.2 × 107 MW) was used. Typical confocal images of osmotically deflated vesicles (∆cs = 300 mM), containing 140 µg/ml (0.014% (w/w) or 4.5 nM) and 720 µg/ml (0.072% (w/w) or 22.5 nM) DNA are shown in

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Figure 5 (a) and (b), respectively. Although the weight concentration is two orders of magnitude smaller than that in typical PEG experiments, GUVs were transformed into dumbbell and pearl-chain shapes with 140 µg/ml DNA (Figure 5a). At 720 µg/ml, local small budding and tubulation were observed (Figure 5b), similar to the results obtained with PEG 6k above 2% (w/w) and PEG 20k (Figure 2d and 3d, 3e). To prove that this morphological alteration is not merely from the chemical nature of DNA, the same experiment was conducted with λDNA fragmented by sonication (Figure 5d). According to the gel electrophoresis pattern, λDNA was fragmented to around 103 bp (Figure 5e). Although the weight concentration was identical to the condition in Figure 5a (140 µg/ml), a significant portion of GUVs containing this fragmented DNA showed the spherical and stomatocyte shapes (Figure 5c), which are characteristic for polymer-free GUV and those containing only small molecules. Thus, the molecular size of DNA was again proven to be an important factor that affected the GUV morphology. The categorizing of GUV shapes (Figure 5d) clearly showed that the dumbbell shape was significantly present with 140 µg/ml λDNA (39%), while local small budding and tubulation comprised a major fraction observed with 720 µg/ml DNA (55%). Stomatocyte shape was only observed with fragmented λDNA. This apparent molecular weight dependence provides strong evidence that depletion volume effect is responsible for determining the shapes of GUVs. Again, the alteration of the mode of shape deformation was observed across c*, which is estimated to be ~ 0.1 mg/ml from rg,λDNA ~ 0.5 µm.61 Shape Transformation Induced by Internally Amplified DNA. Finally, we tested if the budding transformation can also be induced by genome-size DNA enzymatically amplified within GUVs, mimicking biologically relevant condition. We encapsulated φ29 DNA polymerase (500 U/ml) together with the T4 phage genomic DNA (hereafter T4 DNA, 169 kb, 1.1 × 108 MW) as a template. φ29 DNA polymerase isothermally amplifies the genomic DNA using a random primer (the whole genome amplification or WGA reaction, Figure 6a). We also included SYBR Green I dye in both the inner and outer solutions to visualize DNA. Since we used a buffer optimal to the polymerase, which contained higher concentration of salts (10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT, and 50 mM Tris-HCl (pH 7.5)) than that in the previous experiments, the formation efficiency of GUVs decreased significantly. However, a high GUV-formation efficiency was obtained when we added a PEGylated phospholipid (DSPE-PEG 2k) at 8 mol% concentration (Figure S2). Figure 6b depicts the fluorescence image of GUVs deflated by ∆cs = 300 mM before incubation at 30°C. The initial T4 DNA concentration (1.4 µg/ml) corresponds to 7.5 molecule/pL. Although some dumbbell or pearl-chain-like vesicles can be seen, stomatocyte vesicles, which are characteristic of vesicles containing no polymers, were frequently observed. Next, we performed the same deflating experiment after 25.5 h incubation. As shown in Figure 6c, an intense green fluorescence indicating DNA amplification can be seen within GUVs. Although it is difficult to quantify the DNA concentration in a GUV due to the non-linearity of SYBR Green fluorescence at high DNA concentrations, we confirmed that initially 1.4 µg/ml T4 DNA was amplified 310-fold (434 µg/ml) after 25.5 h incubation in the test tube (Figure S3). In this condition, small buddings and tubulation were dominant, while no stomatocyte shapes were observed (Figure 6c). This apparent morphological difference after deflation is considered to be the result of large DNA molecules amplified within the vesicles.

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Discussion. Our results clearly depicted that the molecular size as well as the concentration of enclosed inert polymers is important for determining the morphology of GUVs irrespective of the nature of polymers. It is informative to know that the colligative effect could affect the membrane shapes in addition to direct interaction of specific molecules.62-65 We showed that synthetic (PEG) and natural (BSA) polymers with the molecular size above a few tens of micrometers can induce similar morphological changes accompanied with positive curvature even at concentrations smaller than those found within the cytosol of living cells. Furthermore, the genome-size DNA, an extremely large biopolymer, can induce budding of the membrane at a concentration as low as 0.1 mg/ml. Here, let us compare the strength of the depletion volume effect, ∆Gdep = −Π∆Vdep. In Asakura & Oosawa’s model,66 in which polymer coil is considered as a hard sphere, ∆Vdep is expected to be proportional to rg2. Since rg of λDNA is nearly 100 times larger than that of PEG 6k, ∆Vdep is 104 times greater than that of λDNA. On the contrary, although there is dependence on the ionic strength of the solution, Π is roughly estimated to be 10−5−10−4 mOsm with 0.1 mg/ml DNA.61, 67 Since Π of 2% (w/w) PEG 6k is ~ 10 mOsm,56 Π is 10−6−10−5 times smaller in λDNA experiment. Altogether, our crude estimation indicates that ∆Gdep in the condition shown in Figure 5a is 0.1 to 0.01-fold smaller than that in Figure 2c. Currently, the reason for this discrepancy remains to be solved by the detailed measurement of the hydrodynamic size and colligative property of these polymers. One possibility is that the electrostatic repulsion among the DNA polymer chains and the negatively charged lipids in the membrane enhanced the depletion volume effect by increasing the apparent rg value. Overall, it is interesting that extremely large biopolymers at small weight concentrations drastically altered the mode of membrane deformation. Notably, the protein and genomic DNA concentrations in bacteria such as Escherichia coli is 200–300 and 5 mg/ml, respectively.68 Previously, Errington’s group showed that bacterial cells (Bacillus subtilis) devoid of the cell wall (L-form) were still able to proliferate by excess membrane synthesis.69 They suggested that the simple physical effect without protein-driven mechanism, including the present effect, might be driving the membrane blebbing and fission of the cell membrane. In addition, there are numerous examples in both prokaryotic and eukaryotic cells that cause budding,10, 12, 70, 71 blebbing,72 and tubulation.70, 73-75 Although specific membrane-bound proteins often govern the structures and dynamics of the lipid membrane, effect of non-specific crowding polymers could contribute to a significant extent. The present results may form the foundation of discussion about the molecular crowding effect on the membrane morphology of cells, organelles, and their substructures. Lastly, we would like to mention that this mechanism may be important in designing artificial cells for bioengineering in a bottom-up approach.

METHODS Formation of PEG- and BSA-containing GUVs. We prepared PEG-containing GUVs using the W/O emulsion transfer method. POPC (Avanti Polar Lipids), POPG (Avanti Polar Lipids), and cholesterol (Nacalai Tesque), dissolved in chloroform at the weight ratio of 9:1:0.5, were mixed with liquid paraffin (Wako) to a final concentration of 5 mg/ml. To visualize a lipid membrane, a lipophilic fluorescent dye (DiIC18(3); Thermo Fisher Sci.) dissolved in chloroform was also added to the liquid paraffin (final concentration, 2 µg/ml). After evaporating the chloroform completely by heating at 80°C for a few tens of minutes, 400 µl of this solution was transferred to a glass

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tube, to which 25 µl of inner solution was added. The inner solution consisted of 50 mM Tris-HCl (pH 8.0), 200 mM sucrose, 100 µM calcein (Dojindo), and PEG with a molecular weight of 400 (Wako, MW 360–440), 6k (Serva Electrophoresis, MW 5400–6600), or 20k (Wako, MW 15000–25000) at 1 to 12% (w/w). For the experiment with BSA, PEG was replaced by BSA (Sigma Aldrich) at 1 to 12% (w/w). The mixture of liquid paraffin and the inner solution was vortexed for 40 s to form a W/O emulsion. The emulsion was layered on 400 µl of outer solution, which was an aqueous buffer consisting of 50 mM Tris-HCl (pH 8.0) and 200 mM glucose, in a 1.5 ml tube. After centrifugation at 5,900 × g for 10 min at 23°C, the emulsion passed through the oil/water interface and was saturated by lipids to form a bilayer structure. Approximately 100 µl of the precipitated liposome suspension was collected through a hole opened at the bottom of the tube and was diluted with 400 µl of the outer solution. Centrifugation and collection processes were repeated to remove lipid aggregates. Formation of λDNA-containing GUV. λDNA-containing GUV was prepared with the W/O emulsion transfer method described above. The inner solution was prepared in 50 mM Tris-HCl (pH 8.0), 200 mM sucrose, 2× SYBR Green I (Thermo Fisher Sci.), and 140 µg/ml or 720 µg/ml λDNA (Takara Bio). For the inner solution with 720 µg/ml λDNA, we prepared concentrated DNA stock solution by evaporating water in a vacuum desiccator,29 because the original concentration of λDNA was 280 µg/ml. In the control experiment, fragmented λDNA, which was sonicated for 10 min at room temperature, was added at a concentration of 140 µg/ml to the inner solution instead of the intact λDNA. To prevent the dilution of the DNA stain in the inner solution, 2× SYBR Green I was also added to the outer solution consisting of 50 mM Tris-HCl (pH 8.0) and 200 mM glucose. Formation of GUV containing WGA reaction mixture. GUV containing the whole genome amplification (WGA) reaction mixture was prepared with the W/O emulsion transfer method described above. To enhance the formation efficiency of GUVs, we supplemented a PEGylated phospholipid (DSPE-PEG 2k, Avanti Polar Lipids) at 8 mol% concentration to a lipid mixture containing POPC, POPG, and cholesterol. The composition of the inner and outer solutions is shown in Table S1 in Supporting Information. We used a random hexamer, which has two 3′-terminal phosphorothioate modifications, as the primer to inhibit the 3′ to 5′ exonuclease activity of the polymerase. Prior to preparing the inner solution, the template DNA was denatured in alkaline solution (200 mM KOH and 5 mM EDTA) at room temperature for 3 min, and neutralized in 200 mM Tris-HCl. Experimental procedure. The observation chamber was constructed with two cover glasses and double-sided adhesive spacer with 160 µm thickness. The GUV suspension containing 200 mM glucose was mixed with 800 mM glucose solution at 1:1 volume ratio by gentle pipetting on a cover glass, resulting in a final concentration of 500 mM glucose. Then, the mixture was covered with another cover glass with the spacer between them. The difference of sugar concentration between the inner and outer solutions (∆cs = 300 mM) induced the osmotic deflation. For GUVs containing WGA reaction, this procedure was performed before and after incubation at 30°C. The GUVs were observed under a confocal laser scanning microscope (LSM 700; Carl Zeiss) equipped with the 60× /NA 1.4 oil immersion objective. Confocal images were obtained with 10 mW 488 nm laser for calcein or SYBR Green I (493−550 nm emission) and a 10 mW 555 nm laser for DiIC18(3) (560−800 nm emission). We categorized GUVs into

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seven classes based on their morphological characteristics, and the percentages of each GUVs were manually counted from the acquired images.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00250. Theoretical models, additional figures and tables (PDF).

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

ORCID Hiroaki Suzuki: 0000-0002-8899-0955

Author Contributions ‡These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant Numbers JP24770153, JP26115713, JP26289038, JP15KT0153. This work was also funded partly by ImPACT Program of Council for Science, Technology, and Innovation (Cabinet Office, Government of Japan). We thank Prof. K. Osada in the University of Tokyo for valuable discussion on polymer physics.

REFERENCES (1) Lipowsky, R. (1991) The Conformation of Membranes, Nature 349, 475−481. (2) Seifert, U., Berndl, K., and Lipowsky, R. (1991) Shape Transformations of Vesicles - Phase-Diagram for Spontaneous-Curvature and Bilayer-Coupling Models, Phys. Rev. A 44, 1182−1202. (3) Miao, L., Seifert, U., Wortis, M., and Dobereiner, H. G. (1994) Budding Transitions of Fluid-Bilayer Vesicles - the Effect of Area-Difference Elasticity, Phys. Rev. E 49, 5389−5407. (4) Lipowsky, R. (1995) The Morphology of Lipid-Membranes, Curr Opin Struc Biol 5, 531−540. (5) Dobereiner, H. G., Evans, E., Kraus, M., Seifert, U., and Wortis, M. (1997) Mapping vesicle shapes

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Figures

Figure 1. Confocal images of GUVs encapsulating no polymer (a) before and (b) after osmotic deflation. Red: lipid membrane stained by DiI. Green: internal aqueous core of the vesicle visualized by calcein. (c) Orthogonal projections of z-stacks of a stomatocyte-shape GUV. (d) 3D reconstruction image.

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Figure 2. Confocal images of GUVs containing 2% (w/w) PEG with different molecular weights (50 mM for PEG 400, 3.3 mM for PEG 6k, and 1 mM for PEG 20k). GUVs containing PEG 400 (a) before and (b) after osmotic deflation. GUVs containing (c) PEG 6k and (d) PEG 20k after osmotic deflation.

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Figure 3. (a) Representative images of the seven shape categories. Scale bar: 10 µm. (b) Comparison between seven shape categories of GUVs before and after deflation. (c) to (e) Percentages of the shape categories for various molecular weights and concentrations. GUVs containing (c) PEG 400, (d) PEG 6k, (e) PEG 20k at concentrations from 1 to 12% (w/w) were imaged after deflation and analyzed. More than 100 vesicle images in each condition were used for analysis.

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Figure 4. Percentages of the seven shape categories for various concentrations of BSA (N > 100 in each condition). Colors representing each category are identical to those used in Figure 3.

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Figure 5. Confocal images of deflated GUVs containing λDNA after osmotic deflation. (a) 140 µg/ml, (b) 720 µg/ml, (c) 140 µg/ml fragmented DNA. (d) Percentages of shape categories in each condition. (e) Gel electrophoresis result of λDNA. M: Size marker, Lane 1: intact DNA, Lane 2: 3 min sonication, and Lane 3: 1 h sonication.

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Figure 6. (a) Schematic representation of genome-size DNA amplification in GUV. (b, c) Images of GUVs containing φ29 polymerase and template genome-size DNA templates deflated by ∆cs = 300 mM. (b) Deflated GUVs before DNA amplification. (c) Deflated GUVs after DNA amplification. Right panels: magnified images of representative GUVs.

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Deformation Modes of Giant Unilamellar Vesicles Encapsulating

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