pubs.acs.org/JPCL
Biomimetic Microdroplet Membrane Interface: Detection of the Lateral Localization of Amyloid Beta Peptides Tsutomu Hamada, Masamune Morita, Yuko Kishimoto, Yuuki Komatsu, Mun'delanji Vestergaard, and Masahiro Takagi* School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan
ABSTRACT Lateral membrane organization into domains, such as lipid rafts, plays an important role in the selective association of biological and nonbiological materials on heterogeneous membrane surfaces. The localization of such materials has profound influence on cellular responses. We constructed a biomimetic water-in-oil microdroplet membrane to study the lateral localization of these materials at heterogeneous biological interfaces. As a case study, we studied selective association of amyloid β peptide on the constructed membrane surface. Amyloid β peptide has attracted much attention as one of these membraneassociating proteins because of its “role” in Alzheimer's disease pathology. Ternary lipid membranes covering microdroplets successfully produced lipid ordered structures, which mimicked biological lipid rafts. We revealed that amyloid β peptide selectively localizes within nonraft fluid membrane regions. The successful lateral organization in microdroplet membrane systems may lead to new opportunities for the study of molecular associations within heterogeneous membranes. SECTION Surfactants, Membranes
I
medium have many advantages as model membranes compared to conventional model membrane systems such as giant liposomes. For example, it is difficult to prepare liposomes with physiological aqueous solutions because of their high sensitivity to osmotic or mechanical stress. In contrast, since microdroplets offer good resistance to osmotic or physical stress, it is possible to conduct experiments under physiological ionic conditions, and biological macromolecules can be easily encapsulated without being denatured.20 To the best of our knowledge, there has been no previous report on the formation of raft-like domains within W/O droplet systems. Figure 1A shows typical fluorescence microscopy images of a W/O droplet membrane composed of ternary lipids of dioleoyl L-R phosphatidylcholine (DOPC), saturated phospholipid dipalmitoyl L-R phosphatidylcholine (DPPC), and cholesterol (Chol) (DOPC/DPPC/Chol = 20/60/20). They contain 0.5% of the fluorescent phospholipid rhodamine red-X dihexadecanoyl phosphoethanolamine (rho-PE), which preferentially partitions into the disordered phase, and the droplets were dispersed in olive oil. Microdroplets were prepared by a simple mixing procedure as described previously.21 The fluorescent images unequivocally show lateral phase separation; the heterogeneous membrane surface was covered by several raft-like domains. The thermal motion of the domains was much slower than that within liposomes due to the high
t is important to study molecular-binding events such as protein/peptide segregation on a cell membrane surface because of the possible effects on cellular signaling and molecular toxicity at the membrane interface. Lateral membrane compartmentalization plays an important role in the selective associations of proteins.1 Within plasma membranes, lipid clusters called rafts, which are composed largely of cholesterol and saturated lipids, are formed with high lipid order and slow dynamics.2,3 Rafts are considered to be a form of order-disorder phase separation that develops due to the interaction between lipid molecules (ordered and disordered phases correspond to rafts and the surrounding fluid membrane, respectively).4 One of these membrane-associating proteins, Alzheimer's amyloid β peptide (Aβ), has attracted much attention.5-8 Fundamental questions, such as how and where soluble Aβ is converted into “toxic” amyloid species, are still unanswered.9 Recently, it has been suggested that a raft-constituent lipid ganglioside (GM1) interacts with Aβ and accelerates Aβ aggregation.10-16 Here, we developed a technique for preparing microdroplet membranes that exhibit rafts. We used a biomimetic microdroplet interface to study the localization of Aβ peptide in this heterogeneous membrane in the presence and absence of GM1. Water-in-oil (W/O) microdroplets coated by phospholipids can be studied as a model of a membrane interface since phospholipid molecules are arranged on the surface with their hydrophilic moieties are oriented toward the inner aqueous phase, as a plasma membrane.17-19 Water droplets in an oil
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Received Date: October 8, 2009 Accepted Date: November 6, 2009 Published on Web Date: November 12, 2009
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Table 1. Percentage of Phase-Separated Microdroplets with Several Oil Speciesa oil
Figure 1. (A) Schematic illustration of a raft-exhibiting water-inoil microdroplet. (B) Typical fluorescent images of microdroplets surrounded by olive oil.
olive oil
38.5%
(n = 148)
dodecane
19.7%
(n = 152)
rape seed oil mineral oil
0% 0%
(n > 150) (n > 150)
squalene
0%
(n > 150)
a
The lipid monolayer consists of DOPC/DPPC/Chol = 20/60/20.
Next, we determined the membrane interaction of amyloid β peptide (Aβ) using fluorescence (HiLyte Fluor 488)-labeled Aβ (Aβ-488). Typical fluorescent images of W/O droplets interacting with prefibril Aβ without GM1 (Figure 2C) and with 1 mol % GM1 (Figure 2D) are shown. In contrast to the membrane interaction of CtxB, Aβ tended to localize on the disordered phase independent of the presence of GM1. Aβ oligomer with a shorter incubation time showed the same membrane interaction (see Supporting Information). Assuming that almost all of the Aβ peptides localize on the disordered phase, covering ∼75% of the membrane surface (see Supporting Information) of microdroplets of 10 μm radius, the peptide density per unit area is estimated to be ∼10000 μm-2. Notably, we confirmed this association using raft-exhibiting giant liposomes (see Supporting Information). This is the first report to visually show Aβ localized in a nonraft membrane and poses crucial questions regarding the role of GM1 in Aβ aggregation and assembly. GM1 has been reported to form Aβ/GM1 species, which is a seed for the self-assembly of Aβ peptide.11 However, it has also been reported that the concentration of GM1 molecules influences its interaction with Aβ.12 In agreement with our findings, Radovan et al. recently reported that another amyloidogenic peptide, islet amyloid polypeptide (IAPP), preferentially partitions into the disordered phase of phase-separated liposomes.28 It should be noted that the binding of membrane-associating proteins such as CtxB25,29 and Aβ12 to GM1-containing membranes possibly causes a change in membrane phase behaviors and/or the distribution of lipid molecules. Differences in mechanical properties, such as membrane tension, of the model membranes from those of the plasma membranes should be taken into consideration and investigated. Further studies of model and actual plasma membranes are awaited in order to fully understand interaction between membrane-associating peptides and heterogeneous lipid membranes. In conclusion, the microdroplet membrane system that we have reported behaves similarly to conventional raft-exhibiting giant liposomes. In addition, it has improved sample economy and detection time compared with those of liposome membrane systems since the detectable molecules are encapsulated in a micrometric vesicular space. The amount of sample solution needed is just enough to fill the inner pools of the microdroplets. In 10 μm microdroplets, the characteristic reaction time for molecules with a diffusion coefficient of
viscosity of the oil phase, where the collision and fusion of domains were not observed over the experimental time scale of a few hours. In other words, raft-like domains that formed within an oil-water interface show a stable structure against thermal agitation, similar to plasma membranes. This is in contrast to a liposomal bilayer membrane, the domains of which thermally collide and fuse.22 We found that the formation of raft-like domain structures in the droplet surface largely depends on the surrounding oil phase. The droplets dispersed in olive oil and dodecane exhibited lateral phase separation, whereas raft-like domain structures in the droplet surface were not observed in rape seed oil, mineral oil, or squalene (Table 1). Therefore, we selected olive oil for subsequent experimental work on membrane interactions. To qualitatively characterize the lipid phase organization of microdroplets in olive oil, we measured the area fraction occupied by the ordered phase. When we changed the molar ratio of the ternary components, there was no clear change in the domain phase structure (see Supporting Information). These results indicate that the bulk oil phase play a central role in lipid phase separation of microdroplets. Although the detailed mechanism is still unclear, we consider that the oil or some oil components insert within the lipid monolayer. It should be noted that there is no correlation between the viscosities of oils and the membrane phase separation because both olive oil (84 cP) and dodecane (1.3 cP) induced phase separation. Further experimental developments intended to unravel the possible effect of oils on the lipid organization behavior are underway. At plasma membranes, toxic proteins, such as cholera toxin, bind to sugar chains of the membrane lipid GM1 which is localized in raft regions.23,24 Along these lines, fluorescenttagged Alexa Fluor 488 conjugate cholera toxin subunit B (CtxB-488) is widely used to monitor rafts containing GM1 within cell and model membranes.25-27 It is reported that the heterogeneous bilayer membranes with 1 mol % GM1 tend to form GM1 clusters inside of the ordered phase even in the absence of CTxB.24 Figure 2A,B shows typical fluorescent images of multicolored W/O droplets that were simultaneously stained with rho-PE and CtxB-488 probes, where the droplets consisted of DOPC/DPPC/Chol = 35/35/30 without GM1 (Figure 2A) and with 1 mol % GM1 (Figure 2B). CtxB was distributed throughout the disordered phase of the heterogeneous membrane without GM1, whereas CtxB was localized in the ordered phase of the GM1-containing membrane. The clear intensity of the two separate phases indicates that the W/O droplets successfully produced biological raft structures that sort membrane lipid GM1.
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percentage of phase separated droplets
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Figure 2. Molecular association of cholera toxin (A, B) and amyloid β peptide (C, D) within raft-exhibiting microdroplet membranes. The W/O interface is composed of DOPC/DPPC/Chol = 35/35/30 without GM1 (A, C) or with 1 mol % GM1 (B, D). Fluorescent intensities (F.I.) for each dye along the dashed line are shown at the bottom of the images. The scale bar is 5 μm.
D ∼ 10-10 m2 s-1 is 1 s. The successful lateral phase separation in W/O droplet systems may lead to new opportunities for the study of molecular associations within heterogeneous membranes.
Preparation of Amyloid β.Fluorescence-labeled amyloid β and amyloid β were mixed in a molar ratio of 1:2 and incubated at 37 °C in 20 mM Tris buffer, pH 7.4, at 80 μM for various incubation periods. After incubation, Aβ was introduced to the oil phase containing the lipids at a final concentration of 5 μM by dilution in deionized water for the microdroplet experiments. Preparation and Observation of W/O Microdroplets. Phospholipids dissolved in chloroform/methanol (2:1, v/v) were poured into a glass test tube. The organic solvent was then evaporated under a nitrogen flow and dried under vacuum to make a dry film at the bottom of the test tube. Oil was then added to the test tube prior to ultrasonication for 60 min at 50 °C and vortex mixing (final lipid concentrations in oil were 0.1 mM for a ternary mixture of DOPC/DPPC/cholesterol with 0.5 mol % rho-PE). To obtain W/O droplets, we added 5 vol % of aqueous solutions containing sucrose, 100 μg/mL CtxB-488, or 5 μM Aβ to the oil phase containing the lipids and then emulsified the mixture by tapping. The structures of lipid organization in the droplet surface were observed using a laser scanning microscope (FV1000-D; Olympus) at room temperature (25 °C). Diode lasers (559 and 473 nm) were used to excite rho-PE and CtxB-488 or Aβ-488, respectively.
Experimental Methods Materials. Dioleoyl L-R phosphatidylcholine (DOPC), dipalmitoyl L-R phosphatidylcholine (DPPC), and cholesterol (Chol) were obtained from Avanti Polar Lipids. Bovine brain ganglioside ammonium salt (GM1) was purchased from Calbiochem. Fluorescent phospholipids, N-(rhodamine red-X)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (rho-PE, λex =560 nm, λem =580 nm) and Alexa Fluor 488 conjugate cholera toxin subunit B (CtxB-488, λex = 495 nm, λem=519 nm) were obtained from Invitrogen. Amyloid β protein (Human, 1-40) trifluoroacetate salts (Aβ) were purchased from Peptide Institute, and fluorescent (HiLyte Fluor 488)-labeled amyloid β (1-40) (Aβ-488, λex = 503 nm, λem = 528 nm) was obtained from Anaspec. Olive oil was purchased from Wako Pure Chemicals. Rape seed oil, mineral oil, squalene, and dodecane were purchased from Nacalai Tesque. Olive oil and rape seed oil are composed of triacylglycerols with less than 1 and 0.25% free fatty acids, respectively, and the main fat composition of the triacylglycerols is oleic acid. The viscosities of the oil phases are as follows: 84 cP for olive oil, 164 cP for rape seed oil, 1.9 cP for mineral oil, and 1.3 cP for dodecane. Deionized water was obtained using a Millipore Milli Q purification system.
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SUPPORTING INFORMATION AVAILABLE Characterization of phase-separated droplets, AFM observation of Aβ, lateral localization of oligomeric Aβ species, and Aβ localization detected using liposome membrane systems. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
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Corresponding Author: *To whom correspondence should be addressed. E-mail: takagi@ jaist.ac.jp. Phone: þ81-761-51-1650. Fax: þ81-761-51-1525.
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ACKNOWLEDGMENT This work was supported by a Grant-in-Aid
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for Scientific Research on Priority Areas “Soft Matter Physics”, “Bio Manipulation”, and “Life Surveyor” from the MEXT of Japan. M.V. gratefully acknowledges financial support from the Japan Society for the Promotion of Science.
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