Preserved Transmembrane Protein Mobility in Polymer-Supported

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Preserved Transmembrane Protein Mobility in PolymerSupported Lipid Bilayers Derived from Cell Membranes Hudson P. Pace, Lisa Simonsson Nyström, Anders Gunnarsson, Elizabeth Eck, Christopher F. Monson, Stefan Geschwindner, Arjan Snijder, and Fredrik Höök Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01449 • Publication Date (Web): 13 Aug 2015 Downloaded from http://pubs.acs.org on August 18, 2015

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Analytical Chemistry

Preserved Transmembrane Protein Mobility in Polymer-Supported Lipid Bilayers Derived from Cell Membranes Hudson Pace*†, Lisa Simonsson Nyström†, Anders Gunnarsson‡, Elizabeth Eck†, Christopher Monson§, Stefan Geschwindner‡, Arjan Snijder‡, Fredrik Höök*† †

Department of Applied Physics, Chalmers University of Technology, SE-41296 Gothenburg,

Sweden ‡

Discovery Sciences, AstraZeneca R&D Mölndal, SE-43183 Mölndal, Sweden

§

Department of Physical Science, Southern Utah University, Utah 84720 United States

ABSTRACT Supported lipid bilayers (SLBs) have contributed invaluable information about the physiochemical properties of cell membranes, but their compositional simplicity often limits the level of knowledge that can be gained about the structure and function of transmembrane proteins in their native environment. Herein, we demonstrate a generic protocol for producing polymer-supported lipid bilayers on glass surfaces that contain essentially all naturally occurring cell-membrane components of a cell line while still retaining transmembrane protein mobility and activity. This was achieved by merging vesicles made from synthetic lipids (PEGylated lipids and POPC lipids) with native cell-membrane vesicles to generate hybrid vesicles which readily rupture into a continuous polymer-supported lipid bilayer. To investigate the properties of these complex hybrid SLBs and particularly the behavior of their integral membrane-proteins we used total internal reflection fluorescence imaging to study a transmembrane protease, β-secratase 1 (BACE1), whose ectoplasmic and cytoplasmic domains could both be specifically targeted with fluorescent reporters. By selectively probing the two different orientations of BACE1 in the resulting hybrid SLBs, the role of the PEGcushion on transmembrane protein lateral mobility was investigated. The results reveal the necessity of having the PEGylated lipids present during vesicle adsorption to prevent immobilization of transmembrane proteins with protruding domains. The proteolytic activity of BACE1 was unadulterated by the sonication process used to merge the synthetic and native membrane vesicles; importantly it was also conserved in the SLB. The presented strategy could thus serve both fundamental studies of membrane biophysics and the production of surface-based bioanalytical sensor platforms. 1 ACS Paragon Plus Environment


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KEYWORDS: Supported lipid bilayer, transmembrane protein, mobility, biosensor, biomimetic surface, PEG cushion, TIRF microscopy, native membrane vesicle, hybrid vesicle

INTRODUCTION Supported lipid bilayers (SLBs) have been widely employed as bioanalytical sensor platforms1 and to investigate biomembrane structure/function due to their homology with the cell surface2,3 and compatibility with surface based analytical methods4. Historically, much of the work in the field has centered on simplified model systems in which the sensing component (receptor of interest) is one of only a few constituents used to produce the SLB architecture. However, the importance of SLB composition on the functionality of the sensing elements should not be underestimated. Indeed, studies have shown the essential roles of lipid composition and its ability to modulate the activity of many classes of transmembrane proteins5-7. In order to produce supramolecular architectures providing a closer mimic of the cell membrane researchers have been increasing the compositional complexity of SLBs. A popular approach has been the combination of various lipids and reconstituted proteins to build a more complex biomimetic interface8-10, which can be viewed as a bottom-up design principle. Alternatively, researchers are actively pursuing methodologies to create SLBs directly from native cell membranes in a top-down approach11-13. While the bottom-up approach allows additional control over composition that may aid data interpretation, the topdown approach provides a compositional complexity closer to the natural environment of the membrane

components,

eliminates

potentially

destructive

detergent-based

extraction/reconstitution steps, and enables the possibility to discover previously unknown interaction partners. A major barrier inhibiting the top-down strategy has been the vesicle-substrate rupture process to produce SLBs, which is generally inhibited by complex vesicle compositions (e.g. high cholesterol content/lipid diversity14-17 or high protein density17,18). Several strategies to induce vesicle-substrate fusion of such systems have been investigated19, including α-helical (AH) peptide-induced fusion14, bilayer edge-induced fusion16,17, and synthetic/native membrane vesicle co-adsorption11,12. Yet, in the few reports where native membrane vesicles have been used to produce SLBs the mobility of membrane proteins within the SLBs has been either very poor or non-disclosed11-13,20. The loss of membrane protein mobility is generally attributed to irreversible adsorption of proteins to the substrate which is most likely to occur at the critical stage of vesicle adsorption, at which point transmembrane proteins with protruding 2 ACS Paragon Plus Environment

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domains can be in direct contact with the substrate. Attempts to solve the problem of substrate-protein immobilization have been thoroughly investigated with the consensus that a hydrated polymer cushion is probably the most viable option21. There have been many reports studying various methods of applying polymer cushions to substrates for SLB formation21,22 (e.g., adsorption, substrate-tethering, and lipid-tethering); though, in most cases the studies are restricted to SLB formation and lipid mobility measurements. The few reports on the successful use of polymer cushions to create SLBs containing mobile transmembrane proteins remain hostage to bottom-up approaches8,23-27 (i.e., using simplified reconstituted systems). Additionally, many of the cushioning strategies studied rely on surface coatings/modifications which can lack long-range surface uniformity, batch reproducibility in terms of surface density, adaptability to different instrumental setups, and suffer relatively high production costs per substrate22. Alternatively, approaches in which the polymers are tethered to the vesicle via lipid-anchors prior to SLB formation allow freedom from these surface fabrication constraints. Particularly, using PEGylated-lipids the cushioning architecture can be introduced by the vesicles, thus requiring only a clean surface that would normally support SLB formation by synthetic lipids (e.g., 100% POPC). Furthermore, the presence of PEG moieties on both sides of the SLB formed from such vesicles provide not only a cushion to reduce transmembrane protein immobilization on the substrate8, but also a reduction in non-specific binding of proteins and cells from the bulk solution28 (an advantage not provided by surface fabrication approaches). Herein, we describe a sonication-based methodology to merge “native membrane vesicles” (NMVs) with ‘synthetic’ vesicles containing PEGylated-lipids and lipids which are known to promote SLB formation (e.g., POPC). The hybrid vesicles resulting from this merger spontaneously formed SLBs directly on glass substrates (Figure 1), while maintaining transmembrane protein mobility and activity. The NMVs were produced using standard detergent-free mechanical cell-lysis and centrifugation methods29,30 from the insect cell line Spodoptera frugiperda, here genetically engineered to overexpress β-secretase 1 (BACE1). BACE1 is a widely studied transmembrane protease which plays a critical role in the progression of Alzheimer’s disease31, in this work chosen because it is structurally well suited for investigating the importance of polymer cushions in SLBs to retain integral protein mobility and activity. Thanks to the asymmetry of its protruding regions and its mixed orientation within the hybrid SLB (Figure 1), site-specific fluorescent reporters could be directed to either the active site (Rho-PI) or the C-terminal 6×His-tag (TrisNTA488);

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allowing the lateral diffusion of both orientations of BACE1 to be probed independently. An additional benefit of BACE1 is the ability to probe protease activity during processing (e.g., sonication and SLB formation) which was used as a measure of protein integrity/structure conservation during the different preparation steps. The preserved mobility for both protein orientations is discussed in relation to the molecular architecture of the hybrid SLBs created.

Figure 1. A schematic of the merging of PEG_POPC vesicles with BACE1-containing native membrane vesicles to produce hybrid vesicles, which subsequently can undergo spontaneous SLB formation to produce polymer cushioned supported lipid bilayers on glass substrates. The two orientations of BACE1 in the SLB can be probed independently using either Rho-PI (green star) to target the active site or TrisNTA488 (blue star) to target the C-terminus. The red protein structure is an adaptation of the crystal structure of a truncated BACE1 ectoplasmic domain. Both PEG moieties and proteins protruding into the lumen of the vesicles are neglected for image clarity.

EXPERIMENTAL Preparation of Glass Substrates and PDMS wells. Borosilicate coverslips (No. 1; Brand, Germany) were boiled in 1% Liquinox (Alconox, USA) in water for 30 min, thoroughly 4 ACS Paragon Plus Environment

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rinsed and stored in 18 MΩ water. These substrates were stored under 18 MΩ water and used within two weeks. Wells of polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, Midland, MI) were made by casting a sheet 1-3 mm thick, which was cut to fit the cover slips used, and a hole-punch was used to create wells in the PDMS slabs. Preparation of Synthetic Vesicles. All vesicles, except the native membrane vesicles, were prepared from pure synthesized lipids (Avanti Polar Lipids, USA). Large unilamellar vesicles (LUVs) were prepared using the extrusion method.32,33 Briefly, lipids dissolved in chloroform/methanol were mixed in the desired mole ratios and the organic solvent was evaporated under a stream of nitrogen followed by complete solvent removal under vacuum overnight. The vesicle compositions used in these experiments were: 1 mol% Lissamine rhodamine B-DOPE (LRB-DOPE), 1 mol% NBD-DOPE, 98 mol% POPC (FRET_POPC); 100 mol% POPC (POPC); 0.5 mol% PEG5000-ceremide, 99.5 mol% POPC (PEG_POPC); 0.5 mol% PEG5000-ceremide, 1 mol% Lissamine rhodamine B-DOPE, 98.5 mol% POPC (PEG_Rho_POPC) ; 1 mol% Lissamine rhodamine B-DOPE, 98.5 mol% POPC (Rho_POPC). Desiccated lipid mixtures were rehydrated to 1 mg/ml with PBS (10 mM sodium phosphate buffer and 150 mM NaCl, pH 7.5). The vesicle solutions were extruded 10 times through a polycarbonate filter containing 100 nm pores (Whatman, USA) and stored at 4˚C until use. Vesicle size of each batch was characterized using Nanoparticle Tracking Analysis (NanoSight, UK). Specific size distributions are stated where relevant in this manuscript. Expression of full-length BACE1. Baculovirus containing full-length BACE1 was kindly provided by Prof. H. Danielsson, Uppsala University, Sweden (details on the generation of the viral stock)10. The virus stock was amplified in Sf21 cells grown in Sf900 II media supplemented with 10% fetal bovine serum (Life Technologies) at 27°C and 150 rpm. In brief, cells (500 ml, 1.5 × 106 cells/ml) were infected at a cell-to-virus ratio of 50:1. The cells were harvested (3400×g for 15 min at 4°C) one week post infection and the supernatant was collected with a virus titer of 2 × 108 c.f.u/ml. The virus stock was stored at -80°C. For protein expression, Sf9 (or Sf21) cells were infected at mid –log (2 × 106 cells/ml) using a cell-to-virus ratio of 1:2 and harvested 48 h post infection. The cell pellet was washed twice in PBS buffer and stored at -80°C. Protein expression was analyzed by SDS-PAGE using NuPAGE gel system (Life Technologies). For Coomassie staining, the gel was transferred to Instant Blue (Expedeon). For Western blot, the PDVF membrane was blocked with 2× PBS containing BSA before incubation with primary anti-His mouse IgG (Clontech) and secondary anti-mouse IgG-alkaline phosphatase (Promega). Unbound antibodies were removed by 5 ACS Paragon Plus Environment


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washing in 2× PBS before development with 1-step Ultra TMB blotting solution (Thermo Fischer). SDS-PAGE and Western blot results can be found in the Supplemental Information (Figure S1). Preparation of Native membrane Vesicles. Unless otherwise stated, all chemicals were purchased from Sigma Aldrich. The cell pellet was thawed on ice, dissolved in lysis buffer (2× PBS, 1 mM EDTA and protease inhibitor cocktail, Roche) and disrupted using an UltraTurrax T25 (IKA) at 13000 rpm for 2× 20 seconds. Unbroken cells were removed by centrifugation (1000×g for 10 min, Beckman Coulter). Nuclei and mitochondria were removed by centrifugation (9000×g for 10 min, Beckman Coulter). The soluble proteins were separated from the membrane fraction by ultracentrifugation (>100,000×g for 30 min, Beckman Coulter) and the membrane pellet was resuspended in 2× PBS with protease inhibitor cocktail, homogenized with a dounce homogenizer. The pellet was washed and collected once more by ultracentrifugation and dissolved in 2× PBS, 20% glycerol. Aliquots of the NMVs were snap frozen in liquid N2 and stored at -80°C. Vesicle Mixing via Sonication. A bath sonicator was used. (Ultrasonic frequency: 37 kHz, Ultrasonic power effective: 140 W) (Elmasonic S40H, Germany). FRET experiments were carried out using 1 µL of source vesicles (FRET_POPC) and varying volumes of drain vesicles (100% POPC or NMVs); stated specifically in figure captions. Fluorescence data to monitor the degree of mixing was acquired using a spectrofluorometer (QM-4/2005 spectrofluorometer, Photon Technology International Inc., USA). Sonication induced merging of NMVs with FRET_POPC, as described below, provided the rough estimation of the NMVs concentration to ~10 mg/mL; in short, 1 µL of NMVs provided a similar increase in intensity at 530 nm with a concurrent decrease in intensity at 590 nm as 10 µL of 1 mg/mL POPC lipids when mixed with FRET_POPC vesicles. As detailed in the discussion below, this spectral change can be correlated to the amount of membrane material diluting the FRET pair. For all SLB data and activity testing data, samples were made by mixing 2 µL of NMVs with 20 µL of PBS and 40 µL of either: POPC, PEG_POPC, or PEG_Rho_POPC vesicles in a 0.5 µL Eppendorf tube and sonicated at 20°C for 10 min. This specific recipe provides a 1:20 (NMVs:LUVs) volume mixing ratio, which is roughly a 1:2 (NMVs:LUVs) mass ratio. Despite similarities, our protocol for sonication based merging of NMVs and POPC vesicles to form SLBs was created independently from Granqvist et al.,20 and incorporates PEGylated lipids as a new component. BACE1 Activity Testing. The activity of BACE1 before and after sonication was assessed in bulk solution with the TruPoint β-secretase assay kit (PerkinElmer) which utilizes a FRET 6 ACS Paragon Plus Environment

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substrate to monitor the cleavage (FRET-APP). In short, sample (either sonicator or nonsonicated) was diluted 5 times in reaction buffer, mixed with an equal volume of substrate peptide (0.2 µM) in Reaction buffer and incubated for 15 min. The reaction as monitored at 615 nm (λem) and excited at 330 nm (λex) using a Safire2, microplate reader (Tecan, Switzerland). The activity of BACE1 in the hybrid SLB was assessed by using components of the BACE1 (β-Secretase) FRET Assay Kit (Life Technologies). In short, the hybrid SLBs were incubating with ~450 nM of the “BACE1 Substrate” (FRET-APP) for 3 hrs with pipettemixing every 20 min to mediate diffusion limitations. Then ~60% of the bulk solution (30 µL) was removed from the well containing the SLB, diluted with assay buffer up to 500 µL, and analyzed using a spectrofluorometer (QM-4/2005 spectrofluorometer, Photon Technology International Inc., USA). For the hybrid SLB BACE1 activity test, PDMS wells measured ~10 mm in diameter and ~1 mm in height (~50 µL total volume). The aspect ratio of these wells was chosen to maximize the substrate surface area to bulk volume ratio, since the enzyme was constrained to the SLB. TIRFM and Observing SLB Formation. TIRFM measurements were performed using an inverted Nikon Eclipse Ti-E microscope (Nikon Corporation, Japan) equipped with an Andor Ixon+ camera (Andor Technology, Ireland), a 60× magnification oil immersion objective, mercury lamp, and a perfect focus system. For SLB formation and FRAP measurements the wells were ~3 mm in diameter and in height. SLB formation was monitored through the use of “tracer vesicles”, either Rho_POPC, PEG_Rho_POPC (synthetic tracers) or from hybrid vesicles created by merging PEG_Rho_POPC vesicles with NMVs (hybrid tracers) as described above. The results section indicates when each specific tracer vesicle was used. The solution used to observe SLB formation was composed of 2 µL hybrid vesicle solution (exact concentration unknown, but estimated to be ~1 mg/mL), 1 µL of tracer vesicles (1 µg/mL), 7 µL PBS; mixed together prior to addition to the PDMS well (~3 mm diameter, ~15 µL total volume) on a freshly cleaned glass substrate. The dilution is important (~0.1-0.2 mg/mL final vesicle concentration) so that SLB formation can be observed microscopically. Wells are thoroughly rinsed with PBS after to remove all unbound vesicles. This technique was used to confirm SLB formation for each system mentioned in this report; however, SLBs used for FRAP studies were made without tracer vesicles. SLB Mobility Measurements. Fluorescence recovery after photo-bleaching (FRAP) experiments were carried out using a diode pumped solid-state 532 nm laser (B&W TEK inc., USA) with 100 mW output. SLBs were observed by using either a lipid-conjugated rhodamine (LRB-DOPE), a 6×Histidine-tag specific dye (TrisNTA488) (synthesized by Jacob 7 ACS Paragon Plus Environment


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Piehler’s group34), or using a water soluble, non-cleavable, rhodamine labeled BACE1 inhibitor peptide (Rho-PI) (custom synthesis by Thermo Fischer, Rho-NH-KTEEISEVN[sta]-VAEF-COOH35). The LRB-DOPE probe was introduced along with the other synthetic lipids for each system studied through the merging of PEG_Rho_POPC vesicles with NMVs as described above. The TrisNTA and Rho-PI were introduced after SLB formation. For TrisNTA: the wells were thoroughly rinsed with PBS buffer, then TrisNTA was added to the PDMS well (~250 nM final conc.), after 5 min incubation the wells were thoroughly rinsed with PBS again. For Rho-PI: the wells were thoroughly rinsed with acetate buffer (100 mM sodium acetate, 200 mM NaCl, pH 4.5), then Rho-PI was added to the PDMS well (~2 µM final conc.), after 5 min incubation the wells were thoroughly rinsed with acetate buffer again. A stock solution of Rho-PI (~4 µM in acetate buffer) was made fresh daily due to its low stability in solution. The low pH was required since BACE1 only binds its substrates under acidic conditions. FRAP analysis was done using the MATLAB software developed previously by Jönsson et al36.

RESULTS & DISCUSSION A crucial step in the process of making NMV-derived SLBs was the merging of the synthetic vesicles with the NMVs via bath sonication. To gain understanding of the boundary conditions (i.e., time and temperature) for sonication-facilitated fusion of NMVs and synthetic vesicles, pure synthetic vesicle systems were initially utilized together with a common FRET assay:37,38 As the lipid components of the source and drain vesicles merge the FRET pair becomes diluted, which increases their intermolecular distance and lowers the amount of energy transferred, resulting in a spectral change that was used to probe the extent of mixing. Source POPC vesicles containing a FRET pair (Rhodamine-DOPE and NBD-DOPE) were mixed with drain POPC vesicles lacking fluorophores, which resulted in an increase in emission intensity at 530 nm (donor, green) with the concerted decrease in emission intensity at 590 nm (acceptor, red) attributed to membrane fusion or lipid mixing (Figure 2a). Figure 2b shows the spectral change that occurred as the ratio of drain to source vesicles was increased from 1:1 to 40:1 (lipid mass ratio). By recording this spectral change as a function of sonication parameters, i.e., time and temperature, it was possible to identify the conditions required for complete mixing (Figure 2c). In general, higher temperatures were observed to facilitate mixing, allowing maximum mixing to be achieved in shorter time. Within the model systems studied, full mixing of source and drain vesicles occurred within 10 minutes for temperatures between 5°C and 30°C. The effect of sonication on vesicle size distribution was 8 ACS Paragon Plus Environment

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investigated using a NanoSight particle tracking (NTA) instrument. Interestingly the procedure produced an increase in mean vesicle diameter and polydispersity within the sample, even when applied to a single-component system (i.e., ~110±30 nm POPC vesicles) (Figure 2d). These observations suggest that under these specific sonication conditions, vesicles-vesicle fusion occurs predominately over vesicle fission. In contrast, the sonication conditions which have been widely reported for producing small unilamellar vesicles (SUVs)39,40 tend to use either high powered probe-tip sonication or bath sonication at higher temperatures and for longer periods than used in our protocol.

Figure 2. (a) Schematic illustration of the FRET-based vesicle fusion assay. Fusion of source and drain vesicles results in the dilution of the FRET-pair and thus decreased FRET signal in the resulting, larger fused vesicle. (b) Fluorescence spectra of the merged vesicles showing the reduction in FRET as the ratio of drain (POPC) to source (FRET_POPC) vesicles is increased. All samples were sonicated at 30°C for 20 min to attain full mixing. Ratios are expressed in mass of lipids. (c) Fluorescence emission at 530 nm as a function of sonication time for two different sonication bath temperatures using a source to drain ratio of 1 to 10. (d) Vesicle size distribution of POPC vesicles before and after sonication at 20°C for 10 min.

To investigate the efficiency by which synthetic lipid vesicles could be merged with BACE1-containing NMVs using the sonication method outlined above, the same FRET-based 9 ACS Paragon Plus Environment


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strategy was applied. The spectral changes observed as a function of increasing amounts of NMVs (drain vesicles) were similar to the synthetic model system (Figure 3a). Complete mixing was not observed to occur within 10 minutes of sonication at 20°C; however, the spectral changes indicate that these conditions were still sufficient for hybrid vesicle formation (Figure 3b). The increase in time and temperature conditions required for complete mixing of the NMVs and synthetic vesicles is attributed to the fact that native membranes are known to be more rigid than simple POPC vesicles due to their complex lipid composition and the presence of proteins17. An in-depth study on the role of vesicle composition on sonication-facilitated vesicle mixing is currently under further investigation. The specific activity of BACE1 was measured using the cleavage product of a water-soluble amyloid precursor peptide analog which has a rhodamine-quencher pair conjugated to either end (FRET-APP). The use of bath sonication (as opposed to probe sonication) under these operating conditions did not appear to debilitate our target protein as the proteolytic activity of BACE1 was fully preserved after sonication at 20°C for 10 minutes (Figure 3c). This gentle sonication protocol was also used on NMVs containing CXCR3, a G protein-coupled receptor, which retained its binding capacity for its specific ligands (to be published). The effect of these sonication conditions on vesicles size was investigated using NTA (Figure 3d). Size distributions for pure 0.5 mol% PEG_POPC vesicles, pure BACE1-containing NMVs, and a mixture of the two before and after sonication are shown in figure 3d. NMV preparations tend to show wide size distributions as compared to extruded synthetic vesicles. In analogy with the pure POPC vesicle case, the sonication procedure produces an increase in mean vesicle diameter and polydispersity within the mixture. These observations further support the belief that under these sonication conditions, vesicles-vesicle fusion occurs predominately over vesicle fission.


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Figure 3. (a) Fluorescence spectra of the merged hybrid vesicles showing the reduction in FRET as the ratio of drain (NMVs) to source (FRET_POPC) vesicles is increased. All samples were sonicated at 20°C for 10 min. (b) Fluorescence emission at 530 nm as a function of sonication time at a source to drain ratio of 1 to 8 (see Experimental Section). (c) Proteolytic activity of BACE1 NMVs before and after sonication at 20°C for 10 min. (d) Size distribution of vesicles: PEG_POPC before sonication (black), BACE1 NMVs before sonication (blue), the mixture of PEG_POPC and BACE1 NMVs before (red) and after (orange) sonication. Mix ratio of BACE1 NMVs to PEG_POPC, 1 to 20 (see Experimental Section). Sonication performed at 20°C for 10 min.

Once the sonication parameters required for mixing have been identified, the next step was to determine which ratio of NMVs to PEG_POPC vesicles would produce hybrid vesicles that would spontaneously form a SLB. Production of SLBs from hybrid vesicles via substratevesicle rupture was investigated using total internal reflection fluorescence microscopy (TIRFM) (Figure 4). By using a diluted solution (~0.1 mg/mL total membrane material) containing a small fraction of fluorescently labeled “tracer” vesicles and a significantly larger fraction of non-labeled hybrid vesicles (mass ratio of ~1:500) the formation of hybrid SLBs was observed over a period of ~3 min. Initially the substrate devoid of fluorophores was observed (t = -10 s) and then immediately after addition of the vesicle solution, both hybrid and tracer vesicles began adsorbing to the surface although only the latter were observed by 11 ACS Paragon Plus Environment


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TIRFM (t = 3 s). The number of vesicles on the surface continued to increase until the surface became saturated (t = 50 s). The dark area between the visible vesicles can at this stage be attributed to adsorbed non-labeled hybrid vesicles. The tracer vesicles then appeared to rapidly lose fluorescence intensity as the fluorophores diffused from their initial position, creating a camouflage pattern, within the newly formed continuous hybrid SLB (t = 50 to 130 s). The final micrograph (t = 600 s) shows the SLB after the weakly bound vesicles have been rinsed from the surface. Also shown in Figure 4 is a graph displaying the number of nonruptured vesicles on the surface during the SLB formation process, displaying a time evolution pattern in agreement with previous reports on substrate-induced SLB formation41. This means of following SLB formation was used to optimize the NMVs to PEG_POPC vesicle ratio used to produce hybrid vesicles that would spontaneously rupture to form SLBs. Due to limitations in the established techniques for accurately characterizing the protein and lipid content of NMVs, this ratio was simply expressed as volume of NMVs to volume of PEG_POPC vesicles (1 mg/mL), which for our preparation conditions was determined to be 1 to 20, respectively (see Experimental Section). We stress that this methodology of monitoring SLB formation was a very useful tool in order to find conditions that promoted SLB formation, because it is fast and requires no direct labeling of the hybrid vesicles; thus offering minor perturbations to the system under investigation. For instance, while fluorescence recovery after photobleaching (FRAP) is well suited to determine both SLB formation and the diffusivity of the labeled components; the ability to have a nearly unlabeled SLB after confirmed formation was particularly valuable for further studies of mobility and/or functionality of membrane proteins based on additional fluorescent probes. While the use of synthetic tracers alone accomplishes the goal of optimizing the NMV:PEG_POPC ratio to use for efficient formation of SLBs, the use of tracers made of fluorescently labeled hybrid vesicles (hybrid tracers) provides more accurate information about the kinetics of cell-derived SLB formation and the fraction of non-ruptured hybrid vesicles that remain on the surface after SLB formation. Tracer vesicles composed of purely synthetic lipids (synthetic tracers) and hybrid vesicles (hybrid tracers) display qualitatively similar behavior (Figure 4 graph), although there is a clear difference in the maximum number of adsorbed tracer vesicles prior to rupture and the fraction of non-ruptured vesicles remaining after SLB formation. The dissimilarity in the maximum vesicle coverage prior to SLB formation (known to correspond to approximately 30% surface coverage42) is attributed to differences in the concentration of the tracer vesicle suspensions; however, the difference at saturated binding is attributed to differences in the propensity of the different vesicles to 12 ACS Paragon Plus Environment

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rupture. If we consider that the coverage of non-ruptured vesicles approaches the maximum 54% obtained upon random sequential adsorption of spheres43, a conservative estimate of the fraction of non-ruptured hybrid vesicles would be ~15% (~190/650×50%) coverage. This number is in good agreement with the between 70 and 90% mobile fraction measured for BACE1 and lipids, respectively, using FRAP data (discussed further below). While the number of non-ruptured vesicles could have certainly been reduced using approaches such as an osmotic shock41, the aim was to create SLBs spontaneously from NMV hybrids at near physiological buffering conditions. Decreasing the number on non-ruptured vesicles on the surface is definitely of interest and future work will explore how to accomplish this while maintaining buffer conditions that do not hamper membrane-protein activity.

Figure 4. TIRFM micrographs showing the formation of a hybrid SLB as a function of time. A dilute solution containing Rho_POPC tracer vesicles and hybrid vesicles (~1:500), was added to a clean glass substrate to slowly form a hybrid SLB. Tracer vesicles began to adsorb 13 ACS Paragon Plus Environment


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(3 s), increasing in surface density until the surface was saturated (50 s). The dark areas of the surface were filled with non-labeled hybrid vesicles. Then the vesicles began to rupture, forming the continuous hybrid SLB (50-130 s). The surface is nearly free of non-ruptured vesicles after rinsing with buffer (600 s). The graph compares the number of tracer vesicles within a 273×230 µm2 field of view on the substrate as a function of time. Tracer vesicles were either only PEG_Rho_POPC (Synthetic tracers) or hybrid vesicles produced by merging NMVs with PEG_Rho_POPC vesicles (Hybrid tracers). The number of vesicles on the substrate was estimated as described by Gunnarsson et al44.

After confirming which ratio of NMVs to PEG_POPC vesicles produced hybrid vesicles which spontaneously form SLBs, the use of site-specific fluorescent reporters allowed for the unambiguous investigation of BACE1 in the presence of the numerous protein and lipid components of the native membranes. Additionally, by targeting motifs present explicitly on either the ectoplasmic or cytoplasmic domains of BACE1 we aimed at independently probing both of its orientations in the hybrid SLBs. The ectoplasmic domain, which protrudes ~5 nm from the membrane45, was targeted using a rhodamine-labeled peptide inhibitor35 (Rho-PI) which binds firmly to the proteolytic active site. The cytoplasmic domain, which protrudes less than 1 nm from the membrane, was targeted by genetically incorporating a 6×His-tag on the C-terminus, which could be specifically identified by addition of a TrisNTA-dye construct34. Since these probes are not membrane permeable, non-covalent labeling of the protein’s domains after SLB formation was anticipated to allow the probes to report the opposing domain’s mobility and diffusive properties. In other words, Rho-PI is expected to report the interaction of the cytoplasmic domain with the substrate (Figure 5a-c), while TrisNTA488 should report the interaction of the ectoplasmic domain with the substrate (Figure 5d-f). Additionally, a rhodamine-labeled lipid (LRB-DOPE) was used to provide insight into the lipid environment of the hybrid SLB (Figure 5g-i). LRB-DOPE was introduced to each system by using either PEG_Rho_POPC or Rho_POPC vesicles in place of either PEG_POPC or POPC vesicles during SLB preparation. Each of the fluorescent reporters were investigated in separate experiments to prevent spectral overlap and while SLB formation was verified using tracer vesicles as illustrated in Figure 4, the SLBs used for FRAP data collection did not contain tracer vesicles.


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Figure 5. Micrographs of the hybrid SLBs produced by either (Left column) a sonicated mixture of NMVs and PEG_POPC vesicles, (Central column) a sonicated mixture of NMVs and POPC vesicles, or (Right column) a non-sonicated mixture of NMVs and PEG_POPC vesicles. The hybrid SLBs were visualized using either (a-c) a Rho-PI fluorescent reporter that specifically binds the ectoplasmic domain of BACE1, (d-f) a TrisNTA488 fluorescent reporter that specifically binds the cytoplasmic domain of BACE1, or (g-i) a LRB-DOPE fluorescently-conjugated lipid that non-specifically reports the presence and quality of the continuous hybrid SLB. Inset illustrations convey the perceived hybrid SLB architecture and the location of the fluorescent reporters in each case (Rho-PI, green star; TrisNTA488, blue star; LRB-DOPE, red star). Each micrograph also contains the measured diffusion coefficient and percent mobility of the specific fluorescent reporter used in that system (n=6). Scale bars are all 30 µm.

TIRFM analysis of Rho-PI labeled SLBs (Figure 5a-c) show that incorporation of PEGylated lipids via sonication produces a more uniform (i.e., appearing less patchy) SLB with a higher mobile fraction (~76% vs ~62%) compared to the other hybrid SLBs; however, 15 ACS Paragon Plus Environment


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incorporation of PEG does not appear critical for the creation of a hybrid SLB or the free diffusion of BACE1 species whose ectoplasmic domain is facing the bulk solution. Particularly, the lack of PEGylated lipids does not greatly affect the free diffusion of this orientation of BACE1, most likely due to the small dimensions (~1 nm) of the cytoplasmic domain (Figure 5b). Note that even without sonication some membrane components originally contained in the NMVs becomes dispersed in the resulting SLB, albeit heterogeneously (Figure 5c). This is likely to occur due to interactions between NMVs adsorbed to the surface and the high energy edge of the SLB initially formed by rupture of the synthetic vesicles; it is basically the co-adsorption technique used previously for making similar hybrid SLBs11,12. The measured diffusion coefficient of BACE1 in these near-native SLBs is in good agreement with literature values reported for single α-helical transmembrane proteins in live cells (~0.2 µm2/s). Additionally it is worth noting that immobile fractions were reported between 10-20% for all proteins studied by Frick et al.46, indicating that the system produced with this protocol is a near-native membrane environment. In contrast to the Rho-PI data, imaging of TrisNTA (Figure 5d-f) which targets the BACE1 species whose ectoplasmic domain is facing the substrate showed a drastic difference in the mobility of this specific orientation of BACE1 for the different SLB preparation methods. In the PEG-free case (Figure 5e) the complete absence of mobility is attributed to the inability of the ectoplasmic domain (~5 nm in diameter) to fit in the ~1 nm hydration layer under the SLB. In the non-sonicated system (Figure 5f) it is thought that the BACE1 ectoplasmic domain adsorb irreversibly to the substrate before SLB formation, thus preventing the opportunity for the PEGylated lipids to protect the proteins from the substrate. In contrast, the incorporation of PEGylated lipids into the NMVs via sonication prior to SLB formation appears vital to maintaining transmembrane protein mobility (~70%) for species with large protruding domains that face the substrate (Figure 5d). Although the two orientations were probed under different pH conditions (i.e., Rho-PI at pH 4.5 and TrisNTA at pH 7.5), which is known to slightly influence lipid diffusivity, the 5-fold decrease in diffusion coefficient of this orientation of BACE1 (~0.04 µm2/s vs ~0.2 µm2/s) is believed to be due to the steric hindrance experienced by the ectoplasmic domain as it attempts to migrate through a mixture of PEG chains and large (>5 nm) immobilized non-fluorescently labeled membrane proteins sandwiched between the SLB and the substrate. Additionally, this orientation is most likely also experiencing reduced lateral diffusion speed due to weak interactions with, or transient pinning to, the substrate. While PEG chains and membrane proteins are also present on the SLB facing the bulk solution, the lack of substrate proximity provides them with one 16 ACS Paragon Plus Environment

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less dimension of confinement and thereby limits their role in the inhibition of BACE1 mobility. In order to improve the interpretation of the protein mobility data (e.g., non-ruptured vesicles vs. immobilized proteins) the lipid mobility was independently investigated. The use of a lipid probe confirmed the tracer vesicle data in Figure 4, verifying that a continuous SLB had been formed (Figure 5g-i). In comparison to control PEG_Rho_POPC SLBs (1.6±0.1 µm²/s), a reduction of LRB-DOPE’s diffusion coefficient by ~50% was observed in the hybrid SLBs created from sonicated vesicles containing PEG (0.76±0.11 µm²/s) (Figure 5g) and an ~70% reduction was observed in the hybrid SLBs created from sonicated vesicles devoid of PEG (0.49±0.2 µm²/s) (Figure 5h). These observations are in agreement with expectations since the presence of membrane proteins, gel-phase lipids, and sterols contributed from the native membranes should lower the mobility of the florescent lipid probe. Indeed, it has been reported that the systematic increase in native membrane content leads to a concerted decrease in the diffusion coefficient of a lipid-conjugated fluorophore13. The lower diffusion in the PEG-free system (Figure 5h) is perceived to be due to the large number of immobilized proteins (Figure 5e) which would create diffusion barriers leading to a decrease in the observed diffusion coefficient. In contrast, the SLBs produced from nonsonicated vesicle mixtures exhibited only a ~25% reduction in LRB-DOPE’s diffusion coefficient (Figure 5i), attributed to reduced mixing between the NMV’s and the synthetic vesicle’s constituents. The relatively high mobile fraction (~98%) observed in Figure 5i for non-sonicated NMVs and PEG-containing lipid vesicles shows that nearly all the synthetic vesicles containing the fluorescent reporter readily rupture, releasing the reporter into the continuous hybrid SLB in agreement with the visualization of tracer vesicles (Figure 4). This suggests that the ~10% immobile fraction observed for LRB-DOPE in the PEGylated hybrid-vesicle derived SLB in Figure 5g is likely to arise from LRB-DOPE trapped in non-ruptured hybrid vesicles, which from hybrid tracer vesicles was estimated to be ~15% (Figure 4). Since the fraction of non-ruptured hybrid vesicles that account for this 10% immobile fraction should contain BACE1 in both orientations, one plausible interpretation of the larger immobile fractions observed from the BACE1 data, i.e., ~25% (Figure 5a) and ~30% (Figure 5d); is that these non-ruptured vesicles are rich in protein making them less prone to fuse. Additionally, it cannot be excluded that a fraction of both BACE1 orientations are immobile due to domain intercalation in the SLB; however, in either case this logic does indicate that the mobile fraction of BACE1 in the SLB is considerably larger than ~70-75% observed. 17 ACS Paragon Plus Environment


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The final hybrid SLB contains BACE1 in both orientations, but since two different fluorescent probes were used to identify them, it was not possible to quantify the amount of respective orientations. It remains unclear which step in the process (sonication, NMV preparation protocol (i.e., cell lysis and membrane homogenization steps), or the vesiclesubstrate rupture process) that causes the mixed orientation. As details regarding the SLB formation mechanism are still under debate47 it is possible that the vesicle-substrate rupture process may introduce mixed orientations even from highly oriented NMVs (e.g., cell blebs12). Investigations are currently underway to determine if mixed orientations will always be produced from the vesicle-substrate rupture process. Despite these remaining questions, the results convincingly demonstrates successful formation of continuous SLBs derived from native cell membranes with sustained mobility of a transmembrane proteins with large protruding domains (≤5 nm), irrespective of orientation. To further validate the integrity of the transmembrane proteins contained in the hybrid SLB the retention of proteolytic activity in the SLB was probed (Figure 6). Due to a large uncertainty in determining the amount of BACE1 in the hybrid SLBs, the specific activity of BACE1 could not be quantified. Still, measuring the cleavage product of the water soluble amyloid precursor peptide analog (FRET-APP) confirmed that active BACE1 did remain present in the hybrid SLB as compared to a control SLB. Indeed, since the presence of BACE1 was verified using Rho-PI, a substrate analog that binds to the active site of BACE1 when in its active conformation and FRAP data using Rho-PI demonstrated that >70% of the active sites available to the bulk liquid are mobile, this suggests that a majority of the measured activity originates from the BACE1 in the SLB. The extended incubation time for this measurement relates to the low surface density of BACE1 in the SLB which translates into a low concentration of product in solution. Diffusion limitations were mediated by frequent mixing of the bulk solution during the incubation.


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Figure 6. The specific proteolytic activity of BACE1 measured on a hybrid SLB derived from hybrid vesicles in comparison to a PEG_POPC SLB (negative control). The proteolysis of the FRET-APP substrate was quantified by the fluorescence emission of the cleavage product (575 nm) in the bulk solution over the SLB after 3 hrs of incubation. The inset illustration depicts the cleavage of FRET-APP substrate by the ectoplasmic domain of BACE1 oriented towards the bulk solution in a hybrid SLB.

CONCLUSION The results reported herein provide insight into how biomimetic surfaces derived from native membranes can be created using a top-down approach. By simply merging lipid-anchored polymers and NMVs using sonication, we have shown the ability to produce hybrid polymersupported lipid bilayers which retain transmembrane protein mobility and enzymatic activity. Site specific fluorescent reporters were used to determine how membrane proteins which protruded from the SLB interacted with the underlying substrate. The results reveal the necessity of the PEGylated lipids presence in the vesicle during adsorption to prevent the immobilization of transmembrane proteins with protruding domains. While efforts to further optimize the mobility of all membrane proteins in the hybrid SLB are currently being investigated through the tuning of both synthetic vesicle composition and sonication conditions, it is important to note that this is the first report demonstrating the lateral mobility of a transmembrane protein in a cell-membrane derived SLB with a large (>1 nm) protruding region facing the substrate. The methodology of producing NMV-derived SLBs presented herein is at this point empirical, due to limitations in the established techniques for accurately quantifying the protein and lipid content of NMVs. However, we do not see this as a critical limitation, as verified from the fact that we have also been able to use the tools and empirical optimization strategies described in this report for creating SLBs from several other NMV sources (data not shown). Our current strategy is simple: the FRET assay is used for determining sonication parameters for creating hybrid vesicles, tracer vesicles are used to monitor the formation of hybrid SLBs from various ratios of synthetic vesicles to NMVs, specific fluorescent reporters are used to measure lipid and protein diffusion characteristics. This approach is thus likely to pave the way for a new generation of bioanalytical sensor interfaces derived directly from native membranes, being attractive due to the potential presence of undiscovered pharmaceutical and pathogenic receptors which have been marginalized by traditional extraction and reconstitution methodologies. Since all the required cushioning architecture is 19 ACS Paragon Plus Environment


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contained within the vesicle along with the native membrane’s protein and lipid components this approach makes the production of complex biomimetic surfaces amenable to many surface analytical techniques (e.g., QCM-D, SPR, AFM, ect.). Additionally, the generic nature of this methodology underscores the potential versatility of this approach to produce SLB-based biosensors from any cell line. Research is ongoing in using these surfaces to probe both host-pathogen interactions and cell-cell interactions. This work is also a key component in the further development of SLB electrophoresis48,49 and hydrodynamic17,50 technologies for the separation of native membrane components and complexes within their natural environment. The ability to carry out native polyacrylamide gel electrophoresis (native PAGE)-like separations in cellular membranes to study both protein-protein interactions and protein-lipid interactions without detergent solubilization could prove to be an invaluable tool in studying the mechanisms of disease and physiology. Separated components/complexes could be probed directly within the SLB using immunolabeling (similar to western blot) to look for interactions of known species or using mass spectrometry imaging (MSI) to discover new interaction species51.

AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected] ACKNOWLEDGEMENT SSF (#RMA11-0104) and the Swedish Research Council (2014-5557) are acknowledged for financial support. Dr. Jacob Piehler is acknowledged for the generous gift of the TrisNTA488 compound which played a critical role in producing the revelations described in this manuscript. The authors gratefully acknowledge Prof. Helena Danielsson for kindly providing baculovirus containing full-length BACE1.

SUPPORTING INFORMATION AVAILABLE SDS-PAGE and western blot data are provided. This material is available free of charge via the Internet at http://pubs.acs.org.


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TOC GRAPHIC


Preserved Transmembrane Protein Mobility in Polymer-Supported

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