Abstract
Model membrane systems are essential tools for biology, enabling study of biological processes in a simplified setting to reveal the underlying physicochemical principles. As cell-derived membrane systems, giant plasma membrane vesicles (GPMVs) constitute an intermediate model between native cellular plasma and artificial membranes. Certain applications, however, require planar membrane surfaces. Here, we report a novel approach for creating supported plasma membrane bilayers (SPMBs) by bursting cell-derived GPMVs using an ultrasonic pressure field generated within an acoustofluidic device. We show that the mobility of outer leaflet molecules is preserved in SPMBs, suggesting that they are accessible on the surface of the bilayers. Such model membrane systems will be useful for many applications requiring detailed characterization of plasma membrane dynamics.
Introduction
Artificial model membranes are useful tools for understanding cell membrane function and structure[1]. Their controllable composition facilitates study of the role of specific molecules. Commonly employed model membrane systems include giant unilamellar vesicles (GUVs), hanging black lipid bilayers, and supported lipid bilayers (SLBs) [2]. In SLBs, unlike free-standing membranes, a planar lipid bilayer of known composition is formed on a glass or mica surface[3]. For certain applications SLBs have important advantages over free-standing bilayers. For example, they are much more straightforward to use for near field optical microscopy or atomic force microscopy[4], since they are immobile, present a large surface area and can be imaged for a long time[5]. Consequently, by incorporating receptor-like molecules on bilayers, SLBs can be used to study cell-cell interactions [5] in a very convenient format.
A disadvantage of the aforementioned model membrane systems is that they fail to mimic the compositional complexity of the cell membrane, being made up of only a few different lipid species. Consequently, giant plasma membrane vesicles (GPMVs) obtained from the plasma membrane of living cells have been used as an intermediate model membrane system between purely artificial membranes and the living cell [6, 7]. This system has proven its usefulness in multiple fields ranging from membrane biophysics [8, 9] to developmental biology [10] and drug delivery [11]. An important property of GPMVs is their capacity to exhibit lipid phase separation, allowing this phenomena to be studied in a more biologically relevant context [12-18]. In analogy to SLBs, there have consequently also been efforts to create cell-derived (supported) planar model membrane systems. A common approach has so far been to disassemble cell-derived vesicles on glass surfaces by inducing the vesicle to burst with the help of additional artificial membrane patches [19-21], at air-water interfaces (thereby keeping the original cell membrane composition undisturbed) [22], or on polymer-supported lipid monolayers transferred from a Langmuir trough [23]. A common difficulty with all of these approaches is controlling which leaflet of the plasma membrane presents outwards (i.e. away from the support) which is essential when these systems are used study molecular interactions.
To address this challenge, we propose a novel and straightforward approach for creating supported plasma membrane bilayers (SPMBs), generated by bursting cell-derived GPMVs on a plasma-cleaned glass surface using acoustic radiation forces that originate from an ultrasonic standing wave field[24]. Upon testing the diffusion of lipids as well as proteins in these SPMBs, we confirmed that the outer leaflet molecules are diffusive.
Results
The procedure of creating supported plasma membrane bilayers (SPMBs) is shown schematically in Figure 1. We placed giant plasma membrane vesicles (GPMVs) on plasma-cleaned coverslips and induced their bursting (Figure 1A) by accelerating them towards the glass surface using acoustic radiation forces, within a custom-built acoustofluidic device. This device is based on a ‘thin-reflector’ resonator configuration, as described in references [24, 25]. It comprises a 1 mm thick ultrasound generator (transducer) coupled with a 0.8 mm thick ceramic carrier, a 0.2 mm thick fluid layer containing a suspension of GPMVs, and a 0.15 mm thick cover glass. The device is operated at the first thickness resonance of its layered structure (at a frequency of 0.75 MHz), resulting in acoustic pressure minima positioned at the solid-air boundaries (Figure 1B) [24]. The acoustic pressure within the fluid layer gradually decreases towards the glass surfaces (Figure 1B), and the suspended GPMVs are thus subject to an axial acoustic primary ration force that drives them towards the glass, where they eventually burst (Figure 1C).
Supported plasma membrane bilayers (SPMBs). A) Schematic of SPMB production from GPMVs. B) Normalised acoustic pressure across the different layers of the device (at an operating frequency of 0.75 MHz), obtained from 1-D transfer impedance modelling. The device consisted of a 1 mm thick ultrasound generator (transducer, blue), a 0.8 mm thick ceramic carrier (red), a 0.2 mm thick chamber for the GPMV-containing fluid (green), and a 0.15 mm thick plasma cleaned microscope cover glass (black). Operating the device at its first thickness resonance generated a primary acoustic radiation force onto the GPMVs within the fluid, which was directed towards the glass surface (red area). C) Representative confocal microscopy images of the equatorial plane of the GPMVs before and of the SPMBs on the cover glass after applying the acoustic pressure field. Membrane labeling was done by the green membrane dye DiO and the red fluorescently labeled fluorescent lipid analog Abberior-Star Red (AbStR) DPPE. Scale bar is 10 µm.
An important aspect is the topology of the resulting SPMBs, i.e. whether the upper accessible surface is the inner leaflet (inside-out bursting) or the outer leaflet (outside-out bursting) of the original GPMVs (see Figure 2A, B for both scenarios). This is crucial for molecular accessibility and diffusivity, and hence for studying receptor activities or cell-cell interactions. To assess this, we first performed fluorescence imaging experiments using three-dimensional confocal microscopy and GPMVs derived from CHO cells labelled with DiO. Besides complete bursting, we occasionally observed semi-burst vesicles (Figure 2C), which can only be possible when GPMVs are burst from the bottom and form a bilayer gradually instead of bursting from the top, suggesting an outside-out bursting scenario (i.e. the accessible leaflet of the SPMB is the outer leaflet of the GPMVs (Figure 2B)).
Bursting topology of SPMBs upon application of the acoustic field. A, B) Inside-out (A, inner leaflet is facing upwards and is thus accessible) and outside-out bursting (B, outer leaflet is facing upwards and is thus accessible) scenarios. C) Representative confocal image of a SPMB with a semi-burst GPMV (right: top x-y view, middle: side y-z view, left: oblique 3D-rendering). D) Confocal microscopy top view of a representative SPMB membrane-labeled with a fluorescent lipid analog (red, Abberior Star Red DPPE) and of cytoplasmic GFP expressed in cells and thus inside the GPMVs, indicating that the cytoplasmic GFP got captured underneath the bilayer upon bursting (i.e. outside-out bursting scenario). D) Diffusion coefficients of various outer leaflet molecules as determined by FCS on the SPMBs. Scale bars are 10 µm.
To further confirm this, we prepared GPMVs from CHO cells expressing cytoplasmic fluorescent green fluorescent proteins (GFP). After forming SPMBs from these vesicles, inside-out bursting (Figure 2A) would result in a loss of GFP fluorescence due to escape of GFP. However, we observed residual GFP fluorescence underneath the SPMB (Figure 2D), indicating capturing of cytosolic components by the bilayer, which is, again, possible in the case of the outside-out bursting (Figure 2B).
Finally, we tested the diffusion of outer leaflet molecules as well as of lipids coexisting in both leaflets. In the case of the inside-out bursting scenario (Figure 2A), the outer leaflet molecules would be expected to be immobile as they would get stuck between the SPMB patch and the glass coverslip, while they would remain mobile in the outside-out scenario (Figure 2B). We specifically measured the mobility of the following fluorescently tagged molecules in the SPMBs using fluorescence correlation spectroscopy (FCS): (i) Abberior Star Red-labelled Chol with a PEG linker as an outer leaflet lipid [26, 27], (ii, iii) GFP-labelled glycosylphosphatidylinositol (GPI, ii) [28] and Lypd6 (a full length GPI-anchored protein [10], iii) as two outer leaflet membrane proteins, (iv) Topfluor-labelled cholesterol as a lipid analog that partitions in both leaflets [27]. All of these components appeared to be mobile in the SPMBs (Figure 2E) confirming the outer leaflet molecules are accessible on the surface and not stuck between the bilayer and the glass slide.
Materials and Methods
Cells, Lipids and Proteins
We used Chinese Hamster Ovary (CHO) cells to create GPMVs. CHO cells were maintained in DMEM-F12 medium supplemented with 10% FBS medium and 1% L-glutamine.
We purchased 23-(dipyrrometheneboron difluoride)-24-norcholesterol (Topfluor Cholesterol; TF-Chol) from Avanti Polar Lipids. Abberior Star Red-PEG-Cholesterol was obtained from Abberior. GPI-GFP is the plasmid published in ref [29]. Lypd6 was a gift from Dr Gunes Ozhan as in ref [10]. Cells were transfected with the plasmids using Lipofectamine 3000 as described in manufacturer’s protocol.
Preparation of GPMVs
GPMVs were prepared as previously described [6]. Briefly, cells seeded out on a 60 mm petri dish (≈70 % confluent) were washed with GPMV buffer (150 mM NaCl, 10 mM Hepes, 2 mM CaCl2, pH 7.4) twice. 2 ml of GPMV buffer was added to the cells. 25 mM PFA and 2 mM DTT (final concentrations) were added in the GPMV buffer. The cells were incubated for 2 h at 37 °C. Then, GPMVs were collected by pipetting out the supernatant. GPMVs were labelled by adding the lipid analogues to a final concentration of 50 ng/mL.
Acoustofluidic Device
The acoustofluidic device design and manufacturing protocols were taken from ref [30], with minor adaptations. The ultrasound (US) source consisted of a 13.0 mm × 30 mm × 1.0 mm piezoelectric element (PZ26, Meggit PLC, UK), which was attached to a carrier layer using epoxy resin (RX771C/NC, Robnor Resins Ltd., UK) cured at 30°C for 24 h. The carrier layer consisted of a 0.8 mm thick machinable glass-ceramic material (Macor, Ceramic Substrates & Components Ltd, UK). A 0.2 mm × 12 mm (thickness × width) fluidic cavity was milled into the carrier layer, using a computerized numerical control (CNC) milling machine (VM10, Hurco Companies, Inc., USA). The lateral walls of this cavity were formed by a molded polydimethylsiloxane (PDMS) gasket (Sylgard® 184, Dow Corning Corporation, USA), which was manufactured by mixing PDMS precursor and curing agent (10:1 w/w), followed by degassing and curing at 90°C for 1 h. The fluid cavity was sealed by a glass layer, consisting of a 75 mm × 25 mm × 0.15 mm glass coverslip (Logitech Ltd., Scotland). A micro-milled Perspex® manifold was manufactured for integrating the device with inlet/outlet tubing, to deliver the suspension of GPMVs within the fluid cavity. A metal frame with a central cut-out was employed to achieve stable contact between the layers, whilst providing optical access for microscope imaging. A 1-dimensional (1-D) transfer impedance model implemented in MATLAB® (The MathWorks Inc., USA) was employed to design the thickness of each layer, in order to achieve the desired properties of the acoustic pressure field within the fluid cavity[31]. Since the thickness of the glass layer (0.15 mm) is significantly lower than the US wavelength (∼1.9 mm), a significant proportion of the incoming acoustic energy is reflected at the glass-air boundary, resulting in a minimum in the acoustic pressure at this location (Fig. 2C). Thus, the resulting primary acoustic radiation force acting on the GMPVs within the fluid cavity is directed towards the glass surface. The device was operated at its first thickness resonance of ∼0.75 MHz, at a driving voltage of 25 V peak-to-peak. A frequency sweep of ± 0.025 MHz (sweep period: 50 ms) centered on the resonance frequency was applied, to ensure stable operation of the device over time. Upon loading the device with GPMVs, the piezoelectric elements was actuated by a radio frequency (RF) power amplifier (55 dB, Electronics & Innovation, Ltd., USA) driven by a sine-wave from a signal generator (33220A, Agilent Technologies Inc., USA). An oscilloscope (HM2005, Hameg Instruments GmbH, Germany) was used to monitor the applied voltage and US frequency. A continuous ultrasound wave was applied until GPMVs bursting was observed, which took approximately 10-20 seconds.
Imaging and FCS
SPMBs were imaged in GPMV buffer. All imaging was done at room temperature (21-23 °C). All imaging was done on plasma-cleaned glass slides with thickness of 0.17 mm. Samples were imaged with a Zeiss LSM 780 (or 880) confocal microscope. Topfluor, DiO and GFP were excited with 488 nm and emission collected between 505-550 nm. Abberior Star Red was excited with 633 nm and emission collected with 650-700 nm.
FCS on SPMBs was performed using Zeiss LSM 780 (or 880) microscope, 40X water immersion objective (numerical aperture 1.2) as described before [29]. Briefly, before the measurement, the shape and the size of the focal spot was calibrated using Alexa 488 and Alexa 647 dyes in water in an 8-well glass bottom (#1.5) chamber. To measure the diffusion on the membrane, the laser spot was focused on the SPMBs by maximising the fluorescence intensity. Then, 3-5 curves were obtained for each spot (five seconds each). The obtained curves were fit using the freely available FoCuS-point software [32] using 2D and triplet model.
Discussion
We herein present a novel way of creating supported plasma membrane bilayers (SPMBs) employing acoustic radiation pressure to push GPMVs towards plasma-cleaned coverslips for bursting. Control experiments showed a likely bursting mechanism that keeps the outer leaflet molecules accessible and mobile. All outer leaflet probes we tested were still diffusing freely in the SPMBs. This is very useful property because such a system can present surface molecules and thus be used as a platform to study receptor-ligand interactions and signaling. Importantly, this methodology does not rely on extra synthetic lipid elements to trigger bursting, thus it preserves the native composition of the cell membrane. Moreover, the diffusivity of the molecules are comparable to the diffusivity in the native cell membrane. Therefore, we believe this system will be useful for several applications in cell and membrane biology.
Acknowledgement
We thank Umesh Sai Jonnalagadda and Elisabetta Bottaro for their effort on the acoustic devices. We thank the Wolfson Imaging Centre Oxford and the Micron Advanced Bioimaging Unit (Wellcome Trust Strategic Award 091911) for providing microscope facility and financial support. We acknowledge funding by the Wolfson Foundation, the Medical Research Council (MRC, grant number MC_UU_12010/unit programmes G0902418 and MC_UU_12025), MRC/BBSRC/EPSRC (grant number MR/K01577X/1), the Wellcome Trust (grant ref 104924/14/Z/14), the Deutsche Forschungsgemeinschaft (Research unit 1905 “Structure and function of the peroxisomal translocon”), Oxford-internal funds (John Fell Fund and EPA Cephalosporin Fund) and Wellcome Institutional Strategic Support Fund (ISSF). ES is funded by the Newton-Katip Celebi Institutional Links grant (352333122).
