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Liquid-Ordered/Liquid-Crystalline Phase Separation at a Lipid Bilayer Suspended over Microwells Koji Sumitomo, and Azusa Oshima Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b02156 • Publication Date (Web): 31 Oct 2017 Downloaded from http://pubs.acs.org on November 3, 2017
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Liquid-Ordered/Liquid-Crystalline Phase Separation at a Lipid Bilayer Suspended over Microwells Koji Sumitomo* ,†, Azusa Oshima NTT Basic Research Laboratories, NTT Corporation, 3-1 Morinosato Wakamiya, Atsugi, Kanagawa 243-0198, Japan
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Abstract
The localization of liquid-ordered (Lo) and liquid-crystalline (Lα) phase domains on a silicon substrate with a microwell array is investigated. Although the phase separation of the Lo and Lα phases on both a giant unilamellar vesicle (GUV) and a supported membrane remains stable for a long time, the lateral diffusion of lipids across each domain boundary occurs quickly. Since the phase separation and domain arrangement are governed by the stiffness and lateral tension of the lipid membrane, the phase separation is rearranged on a micropatterned substrate. Similar phase separation of the Lo and Lα phases is observed at a lipid membrane suspended over a microwell. However, the Lα phase is preferred at a suspended membrane, and saturated lipids and cholesterol are excluded towards the supporting membrane on the periphery. Since the Lo domain area is reduced by anisotropic diffusion through the boundary between the suspended and supported membranes, a very slow reduction rate with a linear functional relation is observed. Finally, a localized Lα phase domain is observed at a membrane suspended over a microwell, which is surrounded by an Lo phase supported membrane.
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1. INTRODUCTION
The phase diagram and lateral phase separation in a phospholipid membrane has been studied to obtain an understanding of the physical properties of biological membranes.1 Specifically, in the past two decades, extensive studies have been undertaken on the phase separation of the liquid-ordered (Lo) and liquid-crystalline (Lα) phases in artificial lipid membranes of giant unilamellar vesicles (GUVs) formed of ternary mixtures of saturated lipids, unsaturated lipids, and cholesterol.2-4 Since the domains on GUVs are much larger than the domains of a biological cell, they can be directly observed with a fluorescence microscope. It is considered that the lipiddriven lateral separation of liquid phases plays an important role in the formation of rafts in cell membranes. Therefore, phase separation in GUVs has been studied as a simplified model of rafts in cell membranes.5 The localization of membrane proteins or peptides in a specific liquid domain has also been reported,6 and it is expected that their functions will be studied. The physical properties of lipid membranes have also been quantitatively evaluated of thanks to the simplicity of the system. It has been reported that domain formation and phase separation depend on the tension of the membrane as well as the composition of the ternary mixture and the temperature.7 On supported membranes on substrates, the phase separation of liquid domains and their physical properties have also been studied both theoretically and experimentally.8,9 For a supported membrane, it is reported that the phase separation process depends strongly on the interplay between the substrate topography, the bending rigidity, and the line tension of the membrane domains. Domain patterning on a sub-micron scale has been demonstrated using an E-beam fabricated substrate.10 A polymerized lipid controlled by UV light has been used to propose that lateral diffusion was obstructed and resulted in phase separation control.11
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On the other hand, artificial lipid membranes have also been attracting attention as key components for device applications.12-15 We can expect nanobiodevices to be realized that will work with membrane proteins such as ion channels, ion pumps, and transporters. Lipid membranes are indispensable elements for functioning membrane proteins. A combined approach that incorporates silicon nanotechnology is promising in terms of realizing a nanobiodevice to analyze the membrane protein functions.15-18 In such a nanobiodevice, it is desirable that the artificial membrane is used not as a supported membrane on the substrate but as a suspended membrane over the nanostructures. This is because the membrane proteins do not interact with the substrate, which could potentially interfere with reconstitution, diffusion, and functions such as ion transaction.19 For ion channels, it is also preferable that there is a solution of sufficient volume under the lipid membrane. In previous work, we showed that we could monitor ion channel activity by using a microwell array on a Si substrate that was sealed with an artificial lipid bilayer.16,17 Fluorescent probes (ion indicators) were confined in the microwells, and ion flux through the ion channels in the suspended lipid bilayer could be monitored from the change in fluorescence intensity. One hurdle when fabricating such nanobiodevices relates to controlling the arrangement and function of the membrane proteins. It would help to overcome this hurdle if we could control the phase separation of the liquid domains in suspended lipid membranes, because it has been suggested that these domains play an important role in the localization and function of membrane proteins. Orth et al. reported that a lipid membrane suspended over a porous structure is a model system for mimicking a plasma membrane attached to a cytoskeleton.20 It is expected that phase separation and a raft-like domain will also be used for nanobiodevices. In addition, more recently, Schütte et al. reported the size and mobility of lipid domains in a lipid membrane suspended over a porous structure.21 By surface
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functionalization with a hydrophilic self-assembled monolayer on a gold surface, a continuous lipid membrane was formed between the suspended area and the supported area. In this paper, we investigate the phase separation and localization of the Lo and Lα phases in lipid bilayers with ternary lipid mixtures that are suspended over microwells on a Si substrate covered with a SiO2 layer to apply the raft-like domain to our nanobiodevice. The SiO2/Si substrate is one of the most stable substrates. It is formed by a simple process and is widely used for biodevices.15 We ruptured a phase-separated giant unilamellar vesicle (GUV) on a substrate to form a suspended membrane. We then examined the rearrangement of the phase-separated domains on a substrate with microwells. We investigated the stability of the local microdomains formed in the suspended membrane and the lateral diffusion across the boundary between the suspended and supported membranes. It is important to determine the behavior of the liquid domains in a lipid bilayer suspended over nanostructures both for understanding the physical properties and for fabricating nanobiodevices.
2. Experimental
2.1 GUV preparation The GUVs were prepared from ternary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol by the electroformation method.22 The typical conditions for GUV formation involved supplying an AC voltage of 1 V at 10 Hz for 2 h while heating to 50 °C in a sucrose solution (200 mM). The GUV suspension in the sucrose solution was cooled slowly to room temperature (RT) at a typical cooling
rate
of
10
deg/h.
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine
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rhodamine B sulfonyl) (Rhod-DOPE) was used at 0.05 mol % as a dye for labeling the Lα domain of the lipid bilayers. First, Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) was used at 0.5 mol% as a dye for labeling the Lo domain to confirm the phase separation when the GUVs were prepared. After that, only Rhod-DOPE was used for labeling the Lα domain to minimize the influence on the physical properties. All the lipids were obtained from Avanti Polar Lipids (Alabaster, AL). Laurdan was obtained from Thermo Fisher Scientific (Waltham, MA). Here, the DOPC/DPPC/Chol was mixed at a molar ratio of 30/40/30. To make it possible to dye each domain a different color, the effect of the fluorescence resonance energy transfer (FRET) from Laurdan to rhodamine was minimized by using a much lower concentration of Rhod-DOPE than of Laurdan. Rhod-DOPE had previously been found to partition strongly out of Lo phases coexisting with Lα phases.23,24 The Laurdan generalized polarization (GP) value is related to the lateral packing of the lipid bilayers,25,26 although Laurdan is distributed in both the Lα and Lo domains. The fluorescence intensity in the 430 to 455 nm range from Laurdan become higher in the Lo phase than in the Lα phase, because of the dense packing of lipids in the Lo phase.27 Therefore, a clear phase separation into red (Lα) and blue (Lo) regions was observed. Figure 1 shows a fluorescence image of a typical GUV with two liquid phase (Lα and Lo) domains. Phase separation throughout the GUV was clearly seen in a stack of 27 slices of the fluorescent image when the confocal plane was scanned with a ∆z = 3 µm interval. The two-piece divided GUVs were dominant although some GUVs had three or more domains.
[Fig. 1]
2.2 Microwell substrate
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Microwell patterns were fabricated on a silicon substrate, which was covered with a 120-nmthick thermal oxide layer, using a conventional photolithographic and dry etching technique. The microwells were circular with diameters of 1, 2, 4, or 8 µm, and were 1 µm deep. Microwells of the same size were arranged in 100-µm wide stripes and the four types of stripes were repeated, as shown in Fig. S1. In this work, we mainly used microwells with 1 or 4 µm apertures. To prevent the lipid membrane from falling into the microwells, an overhang was formed at the microwell apertures by selective wet etching with KOH (10 weight %). Because the Si substrate was selectively etched unlike the SiO2 overlayer, an overhang shape was formed. The process is described in detail elsewhere.16,17 The supported lipid membrane was stabilized by the attractive interaction with the substrate. However, the attractive interaction with the sidewall of the microwells works to draw the suspended lipid membrane into the microwells. The attractive interaction with the sidewall can be avoided by the overhang shape.16 The surface roughness of the SiO2 after the process was completed was estimated to be less than 0.3 nm (RMS) from AFM images measured in air.
2.3 Confocal scanning laser microscopy The samples were observed with a confocal laser scanning microscope BX51-FV1200 (Olympus Ltd, Japan) under a ×40 objective lens. We used laser light sources emitting at 405, 473 and 559 nm for excitation, and 430-455, 490-540, and 575-675 nm band-pass filters to detect the fluorescence from Laurdan, calcein, and rhodamine, respectively.
2.4 Florescence recovery after photobleaching analysis
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The determination of the diffusion coefficient by fluorescence recovery after photobleaching (FRAP) has been established and is commonly used.28,29 In this work, however, the diameter of the bleaching area was limited by the size of the Lα and Lo domains or microwells. For a small bleaching area, the spread of the laser beam used for bleaching and the diffusion of fluorescent dyes during the bleaching process are not negligible. Therefore, we adopted a numerical calculation for the diffusion equation to obtain the theoretical fluorescence recovery curves. In the calculation, we assume an isotropic diffusion and a circular bleaching area, and we represent the diffusion equation as follows, ∂C, 1 , , = + ∂t
(1) where C(r, t) is the concentration of unbleached fluorophores at position r and time t, and D is the diffusion coefficient. Bleaching was assumed to be undertaken in an area r
