Vesicle Fission of Giant Unilamellar Vesicles of Liquid-Ordered-Phase

Vesicle fissions are very important processes of biomembranes in cells, but their mechanisms are not clear and are controversial. Using the single gia...
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Langmuir 2007, 23, 720-728

Vesicle Fission of Giant Unilamellar Vesicles of Liquid-Ordered-Phase Membranes Induced by Amphiphiles with a Single Long Hydrocarbon Chain Yasuyuki Inaoka† and Masahito Yamazaki*,†,‡ Department of Physics, Faculty of Science, Shizuoka UniVersity, Shizuoka, 422-8529, Japan, and Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka UniVersity, 422-8529, Japan ReceiVed July 18, 2006. In Final Form: October 19, 2006 Vesicle fissions are very important processes of biomembranes in cells, but their mechanisms are not clear and are controversial. Using the single giant unilamellar vesicle (GUV) method, we recently found that low concentrations (less than the critical micelle concentration (CMC)) of lysophosphatidylcholine (lyso-PC) induced the vesicle fission of GUVs of dipalmitoylphosphatidylcholine/cholesterol(6/4) (DPPC/chol(6/4)) membranes and sphingomyelin/ cholesterol membranes (6/4) in the liquid-ordered (lo) phase. In this report, to elucidate its mechanism, we have investigated the effect of low concentrations (much less than their CMC) of other amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) on DPPC/chol(6/4) GUVs as well as the effect of the membrane composition on the lyso-PC-induced vesicle fission. We found that low concentrations of single long chain amphiphiles (lyosophosphatidic acid, octylglucoside, and sodium dodecyl sulfate) induced the shape change from a prolate to two spheres connected by a very narrow neck, indicating that the single long chain amphiphiles can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. As the single long chain amphiphile concentrations were increased, all of them induced vesicle fission of DPPC/chol(6/4) GUVs above their threshold concentrations. To elucidate the role of cholesterol in the single long chain amphiphile-induced vesicle fission, we investigated the effect of lyso-PC on GUVs of dioleoyl-PC (DOPC)/chol(6/4) membranes in the LR phase; no vesicle fission occurred, indicating that cholesterol in itself did not play an important role in the vesicle fission. Finally, to elucidate the effect of the inclusion of DOPC in the lo-phase membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we investigated the effect of low concentrations of lyso-PC on GUVs of DPPC/ DOPC/chol membranes. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC increased. At and above 30 mol % DOPC, no vesicle fission occurred. On the basis of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of a GUV of a lo-phase membrane.

1. Introduction Vesicle fission is the process of the separation of one or more vesicles from a larger vesicle of biomembranes. In cells, many kinds of vesicle fissions occur on various occasions, such as endocytosis, budding of vesicles from the Golgi and endoplasmic reticulum apparatuses, division of mitochondria and chloroplast, and cytokinesis at cell division.1 They play vital roles in cells, but the molecular mechanisms of these vesicle fissions have not been revealed. It is very tough to elucidate their mechanisms using the cells, because the cells contain various kinds of proteins and lipids, most of which are not identified. Giant liposomes or giant unilamellar vesicles (GUVs) of lipid membranes with diameters greater than 10 µm have been used for investigations of the physical and biological properties of vesicle membranes such as elasticity and shape change.2-8 The shape of a single GUV and its physical properties in water can be measured in real time. Therefore, GUVs have a great advantage * To whom correspondence should be addressed. Address: Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, 422-8529, Japan. Tel and Fax: 81-54-238-4741. E-mail: [email protected]. † Faculty of Science. ‡ Graduate School of Science and Technology. (1) Alberts, B.; Johnson, A.; Lewis, J.; Raff, M.; Roberts, K.; Walter, P. Molecular Biology of the Cell, 4th ed.; Garland Science: New York, 2002. (2) Evans, E.; Rawicz, W. Phys. ReV. Lett. 1990, 64, 2094. (3) Sandre, O.; Moreaux, L.; Brochard-Wyart, F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10591. (4) Ka¨s, J.; Sackmann, E. Biophys. J. 1991, 60, 825.

over smaller liposomes such as large unilamellar vesicles (LUVs) and small unilamellar vesicles (SUVs) in investigating the physical properties and structural changes of liposomes. So far, almost all studies of liposomes have been carried out on a suspension of many small liposomes such as LUVs and SUVs using light scattering, fluorescence spectroscopy, electron spin resonance, and X-ray scattering. In these studies, the average values of the physical parameters of liposomes have been obtained from a large number of liposomes, and therefore a lot of information has been lost. In contrast, studies of single GUVs provide information on the structure and physical properties of single GUVs as a function of time and spatial coordinates, and the statistical analysis of the physical properties of many single GUVs will provide a great deal of new information on the structure and function of biomembranes and lipid membranes which cannot be obtained by the studies on suspensions of LUVs and SUVs (the single GUV method).9,10 For example, the single GUV method has successfully revealed the process of the pore formation of the antimicrobial peptide magainin 2 in a lipid membrane10 and the process of the membrane fusion.11 (5) Farge, E.; Devaux, P. F. Biophys. J. 1992, 61, 347. (6) Tanaka, T.; Tamba, Y.; Masum, S. M.; Yamashita, Y.; Yamazaki, M. Biochim. Biophys. Acta 2002, 1564, 173. (7) Yamashita, Y.; Masum, S. M.; Tanaka, T.; Yamazaki, M. Langmuir 2002, 18, 9638. (8) Saitoh, A.; Takiguchi, K.; Tanaka, Y.; Hotani, H. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 1026. (9) Yamazaki M.; Tamba, Y. e-J. Surf. Sci. Nanotech. 2005, 3, 218. (10) Tamba, Y.; Yamazaki, M. Biochemistry 2005, 44, 15823.

10.1021/la062078k CCC: $37.00 © 2007 American Chemical Society Published on Web 12/09/2006

Vesicle Fission of GUVs of lo-Phase Membranes

Using the single GUV method, we recently found that low concentrations (less than the critical micelle concentration (CMC)) of lysophosphatidylcholine (lyso-PC) induced the vesicle fission of GUVs of dipalmitoylphosphatidylcholine (DPPC)/cholesterol membranes and sphingomyelin (SM)/cholesterol membranes in the liquid-ordered (lo) phase.12 This is the first finding of the vesicle fission of GUVs of biological lipid membranes induced by a substance. This vesicle fission occurred in the GUVs of the lo-phase membrane, not in the GUVs of the liquid-crystalline (LR) phase membrane. Lipid membranes in the lo phase have intermediate properties between that of the LR phase and that of the gel phase; in the lo phase, acyl chains of lipids have high orientational order, but the lateral diffusion coefficient of lipids in the membrane is relatively high.13-15 In cells, microdomains (or rafts) in plasma membranes are considered to be in the lo phase and play important physiological roles.16 On the basis of our experimental results, we have proposed a hypothesis for the mechanism of the vesicle fission.12 At first, lyso-PC molecules in aqueous solution enter into the external monolayer membrane of the GUV, inducing the shape change from a prolate to two spheres connected by a very narrow neck. Further increase in lyso-PC concentration in the external monolayer induces the instabilization of the internal monolayer membrane at the region of the narrow neck, resulting in the vesicle fission. However, to elucidate the detailed mechanism of the vesicle fission, we need further experimental and theoretical studies. First, it is important to address the generality of this vesicle fission, because some experts may conclude that the specific interaction of lyso-PC with cholesterol in the GUV of the lophase membrane plays an important role in the vesicle fission. As we suggested in our previous paper,12 we have a hypothesis that any amphiphiles with a long hydrocarbon chain (i.e., single long chain amphiphiles) can induce similar vesicle fission. In this report, to clarify this point, we first investigated the effect of several single long chain amphiphiles on GUVs of the lophase membrane. For these amphiphiles, we used lysophosphatidic acid (lyso-PA), octylglucoside (OG), and sodium dodecyl sulfate (SDS). To eliminate the effects of the micelles on the GUVs, we investigated the effect of low concentrations (much less than their CMC) of these amphiphiles. As the GUV of the lo-phase membrane, we used the GUVs of a 60 mol % DPPC/40 mol % cholesterol (DPPC/chol(6/4)) membrane, which is well defined in the lo phase.12,14 We found that all the single long chain amphiphiles induced vesicle fission of DPPC/chol(6/4) GUVs above their threshold concentrations. To elucidate the role of cholesterol in the lyso-PC-induced vesicle fission of DPPC/ chol(6/4) GUVs, we investigated the effect of lyso-PC on GUVs of a dioleoyl-PC (DOPC)/chol(6/4) membrane in the LR phase and on GUVs of a dimyristoyl-PC (DMPC)/chol(6/4) membrane in the lo phase. Finally, to elucidate the effect of the inclusion of DOPC (which can form the LR-phase membrane) in the lophase membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we investigated the effect of low concentrations of lyso-PC (16:0) on GUVs of a DPPC/DOPC/ chol membrane. On the basis of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of the GUVs of the lo-phase membrane. (11) Tanaka, T.; Yamazaki, M. Langmuir 2004, 20, 5160. (12) Tanaka, T.; Sano, R.; Yamashita, Y.; Yamazaki, M. Langmuir 2004, 20, 9526. (13) Ipsen, J. H.; Karlstro¨m, G.; Mouritsen, O. G.; Wennersto¨m, H.; Zuckermann, M. J. Biochim. Biophys. Acta. 1987, 905, 162. (14) Vist, M. R.; Davis, J. H. Biochemistry 1990, 29, 451. (15) Bloom, M.; Evans, E.; Mouritsen, O. G. Q. ReV. Biophys. 1991, 24, 293. (16) Simons, K.; Ikonen, E. Science 2000, 290, 1721.

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2. Materials and Methods 2.1. Materials and Sample Preparation. 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (lyso-PA(18:1)) (sodium salt), 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (lyso-PC(14:0)), and 1-palmitoyl-2-hydroxy-sn-glycero3-phosphocholine (lyso-PC(16:0)) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Texas Red 1,2-dihexadecanoyl-snglycero-3-phosphoethanolamine (Texas Red-DHPE) was purchased from Molecular Probes, Inc. (Eugene, OR). OG was purchased from Dojindo Co. Cholesterol and SDS were purchased from Wako Chemical Co. (Tokyo, Japan). 2.2. Formation of GUVs and Observation of GUVs Using a Microscope. GUVs of lipid membranes were prepared in water by the natural swelling of a dry lipid film as follows.12 A 200 µL portion of 1 mM phospholipid mixtures in chloroform in a small glass bottle (5 mL) was dried by N2 gas to produce a thin, homogeneous lipid film, and then the solvent was completely removed by placing the bottle containing the dry lipid film in a vacuum desiccator connected to a rotary vacuum pump for more than 12 h. A 20 µL portion of water was added into this glass bottle, and it was incubated at 45 °C for 5-10 min (prehydration). Then, 1 mL of 0.1 M sucrose in water was added into the glass bottle, and it was incubated at 37 °C for 2 h for the formation of GUVs of two-component membranes, and at 60 °C for 2 h for the formation of GUVs of three-component membranes (i.e., DPPC/DOPC/chol). For the interaction of lyso-PC with the GUVs of three-component membranes, we used the GUVs that were incubated at room temperature (around 23 °C) for 30 min to 2 h after the incubation at 60 °C for the GUV formation. The results did not depend on the incubation time at room temperature. A 10 µL portion of GUV solution (0.1 M sucrose solution; internal solution) was diluted into 300 µL of 0.1 M glucose aqueous solution (external solution) and then transferred into a handmade microchamber. This chamber (1 × 1 cm wide and 3 mm high; internal volume, ∼0.3 mL) was formed on a slide glass by inserting a U-shaped silicone-rubber spacer between a cover glass and the slide glass. We observed GUVs using an inverted phase-contrast microscope (IX-70, Olympus, Tokyo, Japan) at 23 ( 2 °C. Phase-contrast images of GUVs were recorded through a charge-coupled device (CCD) camera (DCX-108, SONY, TOKYO, Japan), and fluorescence images of GUVs containing Texas Red-DHPE were recorded on a computer using an electron-multiplying charge-coupled device (EMCCD) camera (C9100-12, Hamamatsu Photonics, Hamamatsu, Japan), which is a very highly sensitive digital fluorescence camera. The fluorescence intensities of GUVs of lipid membranes containing 0.2 mol % Texas-Red DHPE were sufficiently strong to observe them using the EMCCD camera. 2.3. Shape Changes of GUVs by the Addition of Single Long Chain Amphiphiles. Various concentrations of single long chain amphiphiles such as lyso-PC in 0.1 M glucose solution were added slowly into the vicinity of a GUV through a 10-20 µm diameter glass micropipet, the position of which was controlled by a micromanipulator (MMW-23, Narishige, Tokyo, Japan).6,7 A glass micropipet was prepared as follows: first we pulled a glass tube with 1.0 mm diameter (G-1, Narishige, Japan) to a needle point using a puller (PP-83, Narishige, Japan) and then broke it by quick fracture at the desired tip diameter. The micropipet was filled with the external solution of GUVs containing various concentrations of single long chain amphiphiles using aspiration by a vacuum pump (DA-5D, ULVAC KIKO, Japan), and then it was held by the micromanipulator, enabling us to control the position of the tip of the micropipet. The injection pressure was controlled by changing the height of the vertical column of water in the U-shaped glass tube to which the micropipet was hydraulically connected.6,7

3. Results 3.1. Shape Changes and Vesicle Fission of DPPC/chol(6/4) GUVs Induced by Lyso-PA. We investigated the effect of low concentrations of lyso-PA (18:1) (sodium salt), a negatively

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Figure 1. Phase-contrast images (the bar in the picture corresponds to 10 µm). (A) Shape change of a DPPC/chol(6/4) GUV induced by the addition of 4.0 µM lyso-PA (18:1) at 23 °C. The prolate changed into a pear, and then into an asymmetrical two spheres connected by a very narrow neck. The time after starting the addition of 4.0 µM lyso-PA solution through the micropipet is (1) 0 s, (2) 14.7 s, (3) 15.6 s, and (4) 15.7 s for the pictures. After the addition of lyso-PA was stopped, the shape change was reversed. The time after stopping the addition of lyso-PA is (5) 0 s, (6) 32.0 s, (7) 36.0 s, and (8) 39.0 s. (B) Vesicle fission of a DPPC/chol(6/4) GUV induced by the addition of 50 µM lyso-PA (18:1) at 23 °C. The time after starting the addition of 50 µM lyso-PA solution is (1) 0 s, (2) 10.3 s, (3) 11.2 s, (4) 11.7 s, and (5) 43.2 s. After the addition of lyso-PA was stopped, the distance between the two spherical vesicles increased with time. The time after stopping the addition of lyso-PA is (6) 170 s.

charged lipid with a long acyl chain (CMC: 346 µM),17 on GUVs of an lo-phase membrane. Figure 1A shows the shape change of a DPPC/chol(6/4) GUV induced by the addition of 4.0 µM lyso-PA in a 0.1 M glucose aqueous solution through a 10-µm diameter micropipet near the GUV at 23 ( 2 °C. In Figure 1A, at first (in the absence of lyso-PA), the GUV was a prolate (Figure 1A(1)). After the addition of lyso-PA, the shape changed into a pear (Figure 1A(2)) and then into an asymmetrical two spheres connected by a narrow neck (Figure 1A(3)) (here, “asymmetric” means that the sizes of the two spheres are different). Finally the diameter of the neck became very small (we define this shape as vesiculation; see also the analysis of the shape change in the Discussion section) (Figure 1A(4)). We observed this shape change in 23 GUVs among 23 examined GUVs (n ) 23). The shape change from the two spheres connected by a narrow neck (Figure 1A(3)) to that connected to a very narrow neck (i.e., vesiculation) (Figure 1A(4)) was complete in less than 1 s, and therefore this shape change can be considered as a transition (i.e., vesiculation transition). In order to determine the reversibility of the shape change, the addition of lyso-PA was stopped after the shape change of the GUV was complete, and then we observed the shape change of the GUV. Figure 1A(5)-(8) shows the time course of the shape change of the GUV after the addition of lyso-PA was stopped. At first, the two spheres connected by a narrow neck changed into the pear (Figure 1A(7)), then into the prolate (Figure 1A(8)). After the addition was stopped, lyso-PA diffused away from the vicinity of the GUV into the bulk solution, inducing the decrease in lyso-PA concentration near the GUV, and then the partition of lyso-PA into the membrane decreased (i.e., lysoPA molecules in the external monolayer of the GUV transferred into the aqueous solution). This result indicates that the 4.0 µM (17) Li, Z.; Mintzer, E.; Bittman, R. Chem. Phys. Lipids 2004, 130, 197.

Inaoka and Yamazaki

lyso-PA-induced shape change of the DPPC/chol(6/4) GUV was reversible, indicating that no vesicle fission occurred. We observed the reversibility of this shape change in 21 GUVs among 23 GUVs in which shape change occurred (n ) 21). We investigated the effect of lyso-PA concentration on the shape change of the GUV. At a concentration of e1.0 µM, this type of shape change of the GUV did not occur, and the threshold concentration of lyso-PA for the shape change (i.e., the lyso-PA concentration at which the shape change occurred in 50% of the examined GUVs) was 3 µM. Next, we investigated the effect of higher concentrations (50 µM; less than its CMC) of lyso-PA on DPPC/chol(6/4) GUVs. In Figure 1B, at first (in the absence of lyso-PA), the GUV was a prolate (Figure 1B(1)). After the addition of 50 µM lyso-PA, the shape changed into a pear (Figure 1B(2)), then into an asymmetrical two spheres connected by a narrow neck (Figure 1B(3)), then into two spheres connected by a very narrow neck (Figure 1B(4)), and then finally two spherical vesicles were separated (Figure 1B(5)). In order to determine the reversibility of the shape change, the addition of lyso-PA was stopped after the shape change of the GUV was complete, and then we observed the shape of the GUV. The distance between the two spherical vesicles increased with time (Figure 1B(6)). This result indicated that the 50 µM-lyso-PA-induced shape change of DPPC/chol(6/4) GUVs was not reversible, that is, vesicle fission occurred. We observed this vesicle fission in 18 GUVs among 18 examined GUVs (n ) 18). The process from the two spheres connected by a very narrow neck to the separation of two vesicles (i.e., the vesicle fission) was complete in less than 1 s. The rate of the vesicle fission seems to be much higher than 1 s, but it was difficult to determine this value precisely because of the limited spatial resolution to distinguish the shape after the vesicle fission from the two spheres connected by a very narrow neck. We investigated the effect of lyso-PA concentration on the vesicle fission of the GUV. At a concentration of e10 µM, this type of vesicle fission of the GUV did not occur, and the threshold concentration of lyso-PA for the vesicle fission (i.e., the lyso-PA concentration at which the vesicle fission occurred in 50% of the examined GUVs) was 20 µM. 3.2. Shape Changes and Vesicle Fission of DPPC/chol(6/4) GUVs Induced by OG. First, we investigated the effect of low concentrations of OG with a long hydrocarbon chain (CMC: 22 mM)18 on GUVs of the lo-phase membrane. Figure S1A (Supporting Information) shows the shape change of a DPPC/ chol(6/4) GUV in the lo phase induced by the addition of 3.0 mM OG in a 0.1 M glucose aqueous solution through a 10-µm diameter micropipet near the GUV at 23 ( 2 °C. As shown in Figure S1A(1)-(4), 3.0 mM OG induced a shape changed from a prolate to two spheres connected by a very narrow neck (n ) 16). In order to determine the reversibility of the shape change, the addition of OG was stopped after the shape change of the GUV was complete, and then we observed the shape change of the GUV. Figure S1A(5)-(8) shows the time course of the shape change of the GUV after the addition of OG was stopped. As the OG concentration near the GUV decreased, the two spheres connected by a very narrow neck changed into the original shape, prolate (n ) 16). After the addition was stopped, OG diffused away from the vicinity of the GUV into the bulk solution, inducing a decrease in OG concentration near the GUV, and then the partition of OG into the membrane decreased. This result indicated that the 3.0 mM-OG-induced shape change of the DPPC/chol(6/4) GUV was reversible, indicating that no vesicle (18) Walter, A.; Kuehl, G.; Barnes, K.; VanderWaerdt, G. Biochim. Biophys. Acta 2000, 1508, 20.

Vesicle Fission of GUVs of lo-Phase Membranes

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Table 1. Threshold Concentrations of Single Long Chain Amphiphiles in Water That Induced Shape Changes in and Vesicle Fission of DPPC/chol(6/4) GUVs

lyso-PA (C18:1)-Na OG (C8:0) SDS (C12:0) lyso-PC (C10:0) lyso-PC (C12:0) lyso-PC (C14:0) lyso-PC (C16:0) a

threshold conca to induce the shape changes of GUVs: Cw,* (shape change)

threshold conca to induce vesicle fission of GUVs: Cw,* (vesicle fission)

CMC of single long chain amphiphiles

reference

3 µM 1 mM 2 µM 500 (5 × 102) µM 20 (2 × 101) µM 0.9 (9 × 10-1) µM 0.07 (7 × 10-2) µM

20 (2 × 10) µM 4 mM 50 (5 × 10) µM 1000 (1 × 103) µM 40 (4 × 101) µM 8 µM 0.5 (5 × 10-1) µM

346 µMb 22 mMc 8.1 mMd 7 × 103 µMd 7 × 102 µMd 7 × 101 µMd 7 µMd

this work this work this work Tanaka et al. (ref 12) Tanaka et al. (ref 12) Tanaka et al. (ref 12) Tanaka et al. (ref 12)

The number in the parenthesis shows the significant figure of the experimental value. b From ref 17. cFrom ref 18. dFrom ref 19.

fission occurred. We investigated the effect of OG concentration on the shape change of the GUV. At a concentration of e0.5 mM, this type of shape change of the GUV did not occur, and the threshold concentration of OG for the shape change was 1 mM. Next, we investigated the effect of higher concentrations (10 mM; less than its CMC) of OG on DPPC/chol(6/4) GUVs. As shown in Figure S1B(1)-(4) (Supporting Information), 10 mM OG induced the shape change from a prolate into an asymmetrical two spheres connected by a very narrow neck (Figure S1B(4)), and finally two spherical vesicles were separated (Figure S1B(5)). In order to determine the reversibility of the shape change, the addition of OG was stopped after the shape change of the GUV was complete, and then we observed the shape of the GUV. The distance between two spherical vesicles increased with time (Figure S1B(6)) (n ) 10). This result indicates that 10 mM OG induced the vesicle fission of DPPC/chol(6/4) GUVs. The process from the two spheres connected by a very narrow neck to the separation of two vesicles (i.e., the vesicle fission) was complete in less than 1 s. We investigated the effect of OG concentration on the vesicle fission of the GUV. At a concentration of e3.0 mM, this type of vesicle fission of the GUV did not occur, and the threshold concentration of OG for the vesicle fission was 4 mM. 3.3. Shape Changes and Vesicle Fission of DPPC/chol(6/4) GUVs Induced by SDS. First, we investigated the effect of low concentrations of SDS with a long hydrocarbon chain (CMC: 8.1 mM)19 on GUVs of the lo-phase membrane. We found that 8.0 µM SDS induced a shape change from a prolate to two spheres connected by a very narrow neck (data not shown) (n ) 10), which is almost the same as that induced by lyso-PA and OG. This shape change was reversible. At a concentration of e0.5 µM, this type of shape change of the GUV did not occur, and the threshold concentration of SDS for the shape change was 2 µM. Next, we investigated the effect of higher concentrations (100 µM; less than its CMC) of SDS on DPPC/chol(6/4) GUVs. We found that 100 µM SDS induced vesicle fission (data not shown) (n ) 10), which is almost the same as that induced by lyso-PA and OG. At a concentration of e8 µM, this type of vesicle fission of the GUV did not occur, and the threshold concentration of SDS for the vesicle fission was 50 µM. As described above and also in our previous paper,12 all the single long chain amphiphiles induced the same shape change from a prolate to two spheres connected by a very narrow neck above their threshold concentrations; also, higher concentrations of single long chain amphiphiles induced the vesicle fission of (19) Israelachvili, J. Intermolecular and Surface Forces, 2nd ed; Academic Press: San Diego, CA, 1992. (20) Sackmann, E. In Structure and Dynamics of Membranes; Lipowsky, R., Sackmann, E., Eds.; Elsevier Science: Amsterdam, The Netherlands, 1995; p 213.

GUVs of a DPPC/chol(6/4) membrane in the lo phase above their higher threshold concentrations. We summarized these threshold concentrations in Table 1. 3.4. Shape Changes and Vesicle Fission of DOPC/chol(6/4) GUVs and DMPC/chol(6/4) GUVs Induced by Lyso-PC. To elucidate the role of cholesterol in the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we first investigated the effect of low concentrations of lyso-PC (14:0) (CMC: 70 µM)19 on GUVs of a DOPC/chol(6/4) membrane in the LR phase.6 As shown in Figure S2A (Supporting Information), a shape change from a prolate (Figure S2A(1)) to two spheres connected by a very narrow neck (Figure S2A(4)) was induced by the addition of 30 µM lyso-PC in a 0.1 M glucose solution (n ) 10). This shape change was reversible; after the addition of lyso-PC was stopped, the two spheres connected by a very narrow neck (Figure S2A(5)) changed into the original prolate (Figure S2A(8)) (n ) 10). However, at higher concentrations of lyso-PC less than its CMC (e.g., 50 µM), vesicle fission did not occur (n ) 10). Next, we investigated the effect of low concentrations of lysoPC (14:0) on GUVs of a DMPC/chol(6/4) membrane in the lo phase.20 As shown in Figure S2B (Supporting Information), a shape change from a prolate (Figure S2B(1)) to two spheres connected by a very narrow neck (Figure S2B(4)) was induced by the addition of 10 µM lyso-PC in a 0.1 M glucose solution (n ) 15). This shape change was reversible; after the addition of lyso-PC was stopped, the two spheres connected by a very narrow neck (Figure S2B(5)) changed into the original prolate (Figure S2B(8)) (n ) 15). On the other hand, at higher concentrations (30 µM; less than its CMC), lyso-PC induced the vesicle fission of DMPC/chol(6/4) GUVs (Figure S2C (Supporting Information)) (n ) 16). The threshold concentration of lyso-PC for the vesicle fission was 20 µM. 3.5. Effect of DOPC on Lyso-PC-Induced Vesicle Fission. To elucidate the effect of the inclusion of DOPC (which can form the LR-phase membrane) in the lo-phase membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we investigated the effect of low concentrations of lysoPC (16:0) on GUVs of DPPC/DOPC/chol membranes. It is wellknown that the phase separation between the lo-phase domain and the LR-phase domain occurred in DPPC/DOPC/chol membranes and SM/DOPC/chol membranes under some conditions.21,22 To investigate the phase separation, a small amount (0.2 mol % in the membrane lipids) of a fluorescence probe, Texas red-DHPE, was included in the DPPC/DOPC/chol membranes. Figure 2A shows the fluorescence microscopic image of a GUV of a DOPC membrane containing 0.2 mol % Texas Red-DHPE. The fluorescence intensity was homogeneous at the entire surface, indicating that no phase separation occurred. In (21) Veatch, S. L.; Keller, S. L. Phys. ReV. Lett. 2002, 26, 268101. (22) Baumgart, T.; Hess, S. T.; Webb, W. W. Nature 2003, 425, 821.

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Inaoka and Yamazaki Table 2. Threshold Concentrations of Lyso-PC(16:0)-Induced Vesicle Fission of DPPC/DOPC/chol GUVs

Figure 2. Fluorescence microscope images (the bars in the pictures correspond to 10 µm). (A) DOPC GUV. The GUV was incubated at 23 °C for 30 min after an incubation at 60 °C for the GUV formation. The fluorescence intensity in the membrane was homogeneous, showing that no phase separation occurred. (B) DPPC/DOPC/chol(4.2/3.0/2.8)) GUV. The GUV was incubated at 23 °C for 22 min after the incubation at 60 °C for the GUV formation. Clear phase separation was observed, and the domains with high fluorescence intensity correspond to the LR phase. (C) Shape change of a DPPC/ DOPC/chol(5/1/4) GUV induced by the addition of 0.5 µM lyso-PC (16:0) at 23 °C. Before the addition, the GUV was incubated at 23 °C for 110 min after the incubation at 60 °C for the GUV formation. The prolate changed into a pear, and then into an asymmetrical two spheres connected by a narrow neck. The time after starting the addition of 0.5 µM lyso-PC solution is (1) 0 s, (2) 14.2 s, (3) 14.9 s, and (4) 15.2 s for the pictures. After the addition of lyso-PC was stopped, the shape did not change. The time after stopping the addition of lyso-PC is (5) 300 s. (D) Vesicle fission of a DPPC/DOPC/ chol(5/1/4) GUV induced by the addition of 2.0 µM lyso-PC (16:0) at 23 °C. Before the addition, the GUV was incubated at 23 °C for 32 min after the incubation at 60 °C for the GUV formation. The time after starting the addition of 2.0 µM lyso-PC solution is (1) 0 s, (2) 4.8 s, (3) 5.7 s, and (4) 23.3 s. After the addition of lyso-PC was stopped, the distance between the two spherical vesicles increased with time. The time after stopping the addition of lyso-PC is (5) 60.0 s. (E) Shape change of a DPPC/DOPC/chol(3/3/4) GUV induced by the addition of 4.0 µM lyso-PC (16:0) at 23 °C. Before the addition, the GUV was incubated at 23 °C for 39 min after the incubation at 60 °C for the GUV formation. The prolate changed into a pear, and then into an asymmetrical two spheres connected by a narrow neck. The time after starting the addition of 4.0 µM lyso-PC solution is (1) 0 s, (2) 6.5 s, (3) 7.0 s, and (4) 7.5 s for the pictures. After the addition of lyso-PC was stopped, the shape did not change. The time after stopping the addition of lyso-PC is (5) 300 s.

contrast, Figure 2B shows the fluorescence microscopic images of a 42 mol % DPPC/30 mol % DOPC/28 mol % chol (DPPC/ DOPC/chol(4.2/3.0/2.8)) GUV. Clear large phase separation was observed, and the domains with high fluorescence intensity correspond to the LR phase.21,22 These results indicate that the fluorescence microscopy system we used here can reveal large phase separation clearly. Using this system, we first investigated the effect of lyso-PC on a DPPC/DOPC/chol(5/1/4) GUV. As shown in Figure 2C, 0.5 µM lyso-PC induced a shape change from a prolate to two spheres connected by a narrow neck. At

components of lipid membranes of GUVs (molar ratio)

threshold concentration of vesicle fission (µM)

DPPC/chol(6/4) DPPC/DOPC/chol(5.5/0.5/4) DPPC/DOPC/chol(5/1/4) DPPC/DOPC/chol(4/2/4) DPPC/DOPC/chol (3/3/4) DOPC/chol (6/4) DOPC

0.5 0.7 1 3 no fission no fission no fission

higher concentrations of lyso-PC (e.g., 2.0 µM lyso-PC), after the same shape change, vesicle fission occurred (Figure 2D). In contrast, when we used a DPPC/DOPC/chol (3/3/4) GUV, the lyso-PC did not induce vesicle fission, even at high concentrations such as 4.0 µM (less than the CMC), although the same shape change from a prolate to two spheres connected by a narrow neck occurred (Figure 2E). In Table 2, the threshold concentrations of lyso-PC that induced the vesicle fission of DPPC/DOPC/chol GUVs are summarized. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC increased. At and above 30 mol % DOPC, no vesicle fission occurred. We could not observe large phase separation in DPPC/ DOPC/chol GUVs, which we used here for their interaction with lyso-PC (Table 2).

4. Discussion All the results in this report clearly show that various kinds of single long chain amphiphiles induced the shape change from a prolate to two spheres connected by a very narrow neck in the GUV of a DPPC/chol(6/4) membrane in the lo phase above their threshold concentrations, and, at higher concentrations, vesicle fission occurred above their higher threshold concentrations (Table 1). We also included the data on the lyso-PC-induced shape change and vesicle fission of the DPPC/chol(6/4) GUVs in Table 1.12 These results clearly show that the specific interaction of single long chain amphiphiles such as lyso-PC with cholesterol in the GUV of the lo-phase membrane does not play an important role in vesicle fission. The fact that lyso-PC could not induce the vesicle fission of a GUV of DOPC/chol(6/4) membranes in the LR phase (Figure S2A) indicates that cholesterol in itself does not play an important role in vesicle fission and that single long chain amphiphile-induced vesicle fission can occur only in GUVs of the lo-phase membrane. Moreover, in GUVs of other lo-phase membranes such as the DMPC/chol(6/4) membrane (this report) and the SM/chol(6/4) membrane,12 similar vesicle fissions were induced by lyso-PC, indicating that the specific interaction of lyso-PC with other lipids (DPPC, DMPC, SM) does not play an important role. On the basis of these results, we can conclude that single long chain amphiphiles induce the vesicle fission of the GUV of the lo-phase membrane owing to some physical mechanism. First, we consider the single long chain amphiphile-induced shape changes of the GUVs from a prolate to two spheres connected by a very narrow neck. These shape changes were induced in the GUVs of the lo-phase membrane and also in the GUVs of the LR-phase membrane. These shape changes can be explained by the same mechanism used for the lyso-PC-induced shape change.12 Generally, the shape of a GUV of lipid membranes is determined by the minimum of the elastic energy of the closed membrane of the GUV. In the area-difference-elasticity model (ADE model), the elastic energy of the GUV can be expressed as the sum of the membrane bending energy and the energy of

Vesicle Fission of GUVs of lo-Phase Membranes

the relative monolayer stretching.23,24 In the ADE model, the shape of the GUV is determined by the minimization of the membrane elastic energy for a given area A, a given volume V, and also a given equilibrium (i.e., nonstretched) area difference between the external monolayer (Aex 0 ) and the internal monoex in ) of the GUV, ∆A () A layer (Ain 0 0 0 - A0 ). The analysis based on the ADE model shows that, under the condition of a constant volume of the GUV, the shape changes as follows: with an increase in ∆A0, (1) a prolate f a pear (i.e., asymmetric prolate) f two spheres connected by a narrow neck, (2) a dumbbell (i.e., symmetric prolate) f two spheres connected by a narrow neck, and (3) a cylinder f pearls on a string. Further increase in ∆A0 induces the vesiculation where the neck diameter goes to zero at a critical value of ∆A0. However, above the critical value of ∆A0, the ADE model does not give us a good prediction, and it also cannot explain the vesicle fission because it involves topological change of the membrane. These shape changes predicted by the above analysis based on the ADE model are completely the same as the single long chain amphiphile-induced shape changes of the GUVs. Therefore, this analysis shows that the single long chain amphiphile entered into the external monolayer of the GUVs from the outside aqueous solution, increasing the ∆A0 of the GUVs. This is the main reason for the single long chain amphiphile-induced shape changes of the GUVs. Moreover, this analysis indicates that a monomer of a single long chain amphiphile in aqueous solution can enter into the lo-phase membrane as well as the LR-phase membrane and also into the microdomain in plasma membranes. We can estimate the amount of single long chain amphiphiles incorporated into the external monolayer to induce the shape change from a prolate to two spheres connected by a very narrow neck. Devaux and his colleagues estimated that the minimal asymmetry to induce this shape change is on the order of a 0.1% increase of the total surface area of the external monolayer.5,25 For simplicity, here we evaluate the minimum concentration of single long chain amphiphiles to induce this kind of shape change of a DPPC/chol(5/5) GUV. Since the temperature dependence of the area of the lo-phase membrane is very small,12 we can conclude that the average area per lipid molecule of the DPPC/ chol(5/5) membrane at 20 °C is 0.40 nm2.26 We also assume that the area of a single long chain amphiphile is 0.30 nm2 (minimum estimation), which is a little larger than a saturated hydrocarbon chain. In this case, the minimum single long chain amphiphile concentration in the external monolayer to induce the shape change is 0.0013, which is its molar ratio (i.e., DPPC/chol/single long chain amphiphile: 5/5/0.013) in the external monolayer of the GUV. Therefore, the minimum number of single long chain amphiphiles in the external monolayer required for this shape change is 4.2 × 106 molecules when the total surface area of the external monolayer is 1.26 × 109 nm2 (corresponding to that of a spherical GUV with 20 µm diameter). Therefore, a very small number of single long chain amphiphiles in the external monolayer can induce the shape change from a prolate to two spheres connected by a very narrow neck. Our experimental results show that, above the threshold concentrations of single long chain amphiphiles in aqueous solution, the vesicle fission of the GUV of the lo-phase membrane occurred. In our previous report,12 from the thermodynamic analysis, we indicated that the threshold concentrations of lyso(23) Heinrich, V.; Svetina, S.; Zeks, B. Phys. ReV. E. 1993, 48, 3112. (24) Miao, L.; Seifert, U.; Wortis, M.; Do¨bereiner, H.-G. Phys. ReV. E. 1994, 49, 5389. (25) Lopez-Montero, I.; Rodriguez, N.; Cribier, S.; Pohl, A.; Velez, M.; Devaux, P. F. J. Biol. Chem. 2005, 280, 25811. (26) Edholm, O.; Nagle, J. F. Biophys. J. 2005, 89, 1827.

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PC in the external monolayer membrane are required for vesicle fission. So, we can reasonably conclude that the vesicle fission of the GUV of the lo-phase membrane occurred above the threshold concentrations of all the single long chain amphiphiles in the external monolayer of the GUV. One more important thing is that the single long chain amphiphiles could not induce the vesicle fission of GUVs of the LR-phase membrane as well as that of GUVs containing large amounts (g30 mol %) of the LR-phase-forming lipid, DOPC. On the basis of these results, we have proposed a hypothesis for the mechanism of the single long chain amphiphile-induced vesicle fission of GUVs of the lophase membrane as follows. First, single long chain amphiphile molecules in aqueous solution enter into the external monolayer membrane of the GUV in order to decrease the Gibbs free energy of the total system. Then, the shape change of the GUV occurs until the elastic energy of the GUV (Wel) becomes minimum, and the shape becomes the two spheres connected by a very narrow neck (e.g., Figure 1A(4)) (Figure 3B(1)). Further insertion of single long chain amphiphiles into the external monolayer induces stretching of the internal monolayer membrane; especially at the region of the narrow neck, its stretching is very large due to the large curvature of the membrane (Figure 3B(2)), because we can reasonably conclude that no flip-flop occurs between the external and the internal monolayer on the basis of the reversibility of the single long chain amphiphile-induced shape changes below the threshold concentration. When the single long chain amphiphile concentration in the external monolayer increases and reaches the threshold value, the distance between the hydrocarbons of neighboring lipids suddenly increases, or defects in the hydrocarbon packing are suddenly formed in the lipid membrane at the region of the high curvature in the internal monolayer (Figure 3B(2)), because, in the lo phase and the gel phase, the hydrocarbon chains of lipids cannot take various conformations, and their main conformation is the all-trans conformation. Therefore, at these defects, some parts of the hydrocarbon chains would face the water. Israelachvili and his colleagues pointed out, on the basis of their surface force apparatus measurement of supported lipid bilayers, that such defects in the hydrocarbon chain region induced the attractive force between the opposing two bilayers.27 Therefore, at the region of the very narrow neck of the GUV, the opposing two bilayers attracted each other strongly to contact each other (Figure 3B(3)). Then, a breakdown of the internal monolayers occurs at the region of the narrow neck, and then they reseal to reduce the high curvature of internal monolayers (Figure 3B(4)). To decrease the elastic energy of the high-curvature region of the internal monolayer at the neck, the GUV becomes a shape with a low-curvature internal monolayer (Figure 3B(5)). However, in the GUV, there is still a high-energy interstitial hydrocarbon region at the narrow neck (indicated by blue triangles in Figure 3B(5)).28 To decrease the free energy of this shape, the merge of outer monolayer membranes occurs, and the vesicle fission is complete (Figure 3B(6), A(2)). This process for single long chain amphiphile-induced vesicle fission is a mirror image of the “stalk model” of vesicle fusion, which is one of the most popular models for the vesicle fusion between the influenza virus and the endosome inside cells.29-32 In this model, Figure 3B(2),(4),(5) corresponds to the fusion pore, the (27) Bentz, M.; Gutsmann, T.; Chen, N.; Tadmor, R.; Israelachvili, J. Biophys. J. 2004, 86, 870. (28) Kinoshita, K.; Li, S. J.; Yamazaki, M. Eur. Biophys. J. 2001, 30, 207. (29) Blumenthal, R.; Clague, M. J.; Durell, S. R.; Epand, R. M. Chem. ReV. 2003, 103, 53. (30) Cevc, G.; Richardsen, H. AdV. Drug DeliVery ReV. 1999, 38, 207. (31) Chernomordik, L.; Chanturiya, A.; Green, J.; Zimmerberg, J. Biophys. J. 1995, 69, 922. (32) Siegel, D. P.; Epand, R. M. Biophys. J. 1997, 73, 3089.

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Figure 3. (A,B) Scheme of our hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of GUVs of lo-phase membranes. (C,D) Scheme of our hypothesis of the mechanism of no vesicle fission of GUVs of LR-phase membranes induced by single long chain amphiphiles. A detailed description is provided in the text. Red arrows indicate the lateral pressure of the internal membranes induced by the shape change of the GUV. Blue triangles indicate the interstitial hydrocarbon region where the free energy of chain packing is very large.

hemifusion intermediate, and the stalk, respectively. For the vesicle fusion based on the stalk model, cone-shaped lipids are required to form a negative curvature,29-32 although, for lysoPC-induced vesicle fission, such kinds of lipids are not necessary. As expected from this mechanism, the single long chain amphiphiles that can make transverse diffusion (i.e., flip-flop) from the external to the internal monolayer of GUVs rapidly, such as ceramides,25 cannot induce the vesicle fission of the GUV of the lo-phase membrane. Next, we consider the interaction of a single long chain amphiphile with GUVs of lipid membranes in the LR phase. First, single long chain amphiphile molecules in aqueous solution enter into the external monolayer membrane of the GUV in order to decrease the Gibbs free energy of the total system. Then, a shape change of the GUV occurs until the elastic energy of the GUV (Wel) becomes minimum, and the shape becomes two spheres connected by a very narrow neck (Figure 3C(1)). This induces stretching of the internal monolayer membrane; especially at the region of the narrow neck, its stretching is very large due to the large curvature of the membrane (Figure 3D(1)). Even when the single long chain amphiphile concentration in the external monolayer increases and reaches the threshold value, the hydrocarbon chains can change their conformations so that the defects in the hydrocarbon packing cannot be formed at the region of the high curvature in the internal monolayer (Figure 3D(2)). Therefore, at the region of the very narrow neck of the GUV, the opposing two bilayers do not contact each other (Figure

3D(2)). Thus, in the GUVs of the LR-phase membrane, no vesicle fission occurs, although the single long chain amphiphiles induced the same shape change as that of GUVs of the lo-phase membrane. Our results in this paper and our previous one12 as well as the results of Roux et al.33 indicate that lo-phase membranes or lophase domains play an important role in vesicle fission. In cells, biomembranes are composed of lo-phase domains and LR-phase membranes. Roux et al. indicated that vesicle fission occurred at the boundary between lo-phase and ld-phase (i.e., LR-phase) domains in tubular membranes.33 On the other hand, our results suggest another possible mechanism of vesicle fission in cells such as endocytosis, divisions of organelles, and cytokinesis: vesicle fission occurs in the lo-phase domain by the insertion of single long chain lipids such as lyso-PA and lyso-PC. Figure 4 illustrates two examples of this kind of vesicle fission in the biomembranes composed of lo-phase domains and LR-phase membranes. In one case, the budded vesicle is composed of the lo-phase domain (Figure 4A), and, in the other case, only the fission site (where the connection of the membranes of two vesicles is broken) or the region of the very narrow neck is composed of the lo-phase domain (Figure 4B). As shown in Figure 3, in our hypothesis of the mechanism of vesicle fission, the minimum requirements of vesicle fission are the presence of (33) Roux, A.; Cuvelier, D.; Nassoy, P.; Prost, J.; Bassereau, P.; Goud, B. EMBO J. 2005, 24, 1537.

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Figure 4. Two possible examples of vesicle fission in the biomembranes composed of lo-phase domains (green) and LR-phase membranes (yellow) induced by the insertion of single long chain amphiphiles. (A) The budded vesicle is composed of the lo-phase domain. (B) Only the fission site (where the connection of the membranes of two vesicles is broken) or the region of the very narrow neck is composed of the lo-phase domain. In these examples, some specific proteins must play an important role in the localization of the lo-phase domain at the very narrow neck region.

the lo-phase domain in the internal monolayer at the very narrow neck and some driving force for the stretching of the internal monolayer membrane, such as the insertion of single long chain lipids into the external monolayer. For these examples, the localization of the lo-phase domain at the very narrow neck region is necessary, and some specific proteins must play an important role in this localization. Further investigation is needed on the role of the lo-phase domain in vesicle fission in cells.

Conclusion In this report, we clearly show that single long chain amphiphiles can induce the vesicle fission of GUVs of lo-phase membranes. Low concentrations of single long chain amphiphiles in aqueous solution induced the reversible shape change from a prolate to two spheres connected by a very narrow neck, indicating that single long chain amphiphiles can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. As the single long chain amphiphile concentrations were increased, all of them induced the vesicle fission of GUVs of lo-phase membranes above their threshold concentrations. Thermodynamic analysis indicates that the threshold concentrations of the single long chain amphiphiles in the external monolayer membrane of the GUVs are required for vesicle fission. When GUV membranes contain high concentrations of LR-phaseforming lipids such as DOPC or when GUV membranes are composed of 100% LR-phase-forming lipids, the single long chain amphiphiles can not induce the vesicle fission of the GUVs. We have proposed a hypothesis on the single long chain amphiphile-

induced vesicle fission of the GUVs of lo-phase membranes. After the GUV’s shape is transformed into the two spheres connected by a very narrow neck, further insertion of single long chain amphiphiles into the external monolayer induces stretching of the internal monolayer membrane; especially at the region of the narrow neck, its stretching is very large due to the large curvature of the membrane. When the single long chain amphiphile concentration in the external monolayer increases and reaches the threshold value, the distance between the hydrocarbons of neighboring lipids suddenly increases, or defects in the hydrocarbon packing are suddenly formed in the lipid membrane at the region of the high curvature in the internal monolayer because, in the lo phase and the gel phase, the hydrocarbon chains of lipids cannot take various conformations, and their main conformation is the all-trans conformation. This instability would induce the association of the opposing two bilayers, and the following rearrangement of the membranes would occur, resulting in vesicle fission. This hypothesis at present contains some speculations, and therefore, further experimental and theoretical studies are necessary. However, the novel fact that the single long chain amphiphiles can induce the vesicle fission of GUVs of lo-phase membranes reveals a new characteristic of lo-phase membranes. Moreover, this is the first finding of the vesicle fission of GUVs of biological lipid membranes induced by substances, and therefore, it can be used for the controlled vesicle fission of an artificial cell such as a GUV containing DNA (or RNA) and proteins. It will be very helpful to develop a de novo cell that can be divided into two daughter cells.

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Acknowledgment. This work was supported in part by a Grant-in-Aid for General Scientific Research (B) (No.17310071) and also by a Grant-in-Aid for Scientific Research in Priority Areas (System Cell Engineering by Multi-scale Manipulation) (No.18048020) from the Ministry of Education, Science, and Culture (Japan) to M.Y. We thank Drs. Masum Shah, MD and Yukihiro Tamba for their critical reading and comments.

Inaoka and Yamazaki

Supporting Information Available: Phase-contrast images illustrating the shape change and vesicle fission of DPPC/chol(6/4), DOPC/chol(6/4), and DMPC/chol(6/4) GUVs. This material is available free of charge via the Internet at http://pubs.acs.org. LA062078K