Reconstitution of Fusion Proteins in Supported Lipid Bilayers for the

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Reconstitution of fusion proteins in supported lipid bilayers for the study of cell surface receptor–ligand interactions in cell-cell contact Ranjita Ghosh Moulick, Dzmitry Afanasenkau, Sung-Eun Choi, Jonas Albers, Wienke Lange, Vanessa Maybeck, Tillmann Utesch, and Andreas Offenhäusser Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04644 • Publication Date (Web): 17 Mar 2016 Downloaded from http://pubs.acs.org on March 22, 2016

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Figure 1 Upper part: Reconstitution of EA5-Fc chimera in a fluid SLB by fusion of SUV. Lower part: Eph A receptors expressed in cortical neurons recognize EA5-Fc embedded in a SLB. 209x236mm (180 x 180 DPI)

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Figure 2 Neuronal cell growth on different substrates studied in this work. (A): POPC SLB with incorporated EA5-Fc provided cell adhesion, (B): SLB containing only POPC did not support cell adhesion, (C): SLB with incorporated EA5-Fc that was blocked with EphA5-Fc did not support cell adhesion, (D): SLB containing only POPC did not support cell adhesion, even when preclustered EA5 was added in to medium. Inset on every image shows a schematic representation of SLB and placement proteins. 415x360mm (90 x 90 DPI)


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Figure 3 Quantification of neuronal cell growth after 6 DIV on various substrates. 100 neurons per mm² were cultured on each substrate and the number of live neurons was counted after DIV 6 193x158mm (90 x 90 DPI)


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Figure 4 Cortical neurons grown on POPC-EA5 SLB. The neurons are visualized in phase contrast mode, EA5 is labelled by DyLight (red). The overlay indicates an increased concentration of EA5 in the vicinity of the neurons. (B): Accumulation of the ligand EA5 (immunostained in green) under the cell body (with aggregated proteins) (C): Expression of EphA5 (red) in the cells in response to the EA5. 292x79mm (60 x 60 DPI)


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Figure 5 The expression of EphA5R, normalized to the housekeeping gene GAPDH, is higher in cultures grown on EA5-POPC-SLB than on PLL coated glass both at 2000 cells/cm2 and 8000 cells/cm2. Values shown are mean plus/minus standard deviation of the fluorescence ratio of EphA5R product / GAPDH product. Analysis by qRT-PCR with plating densities of 2000 cells/cm2 and 4000 cells/cm2 further corroborates an increase in EphA5R on EA5-POPC-SLB (2.3 and 2.2 respectively, crossing point analysis relative to GAPDH). 121x71mm (96 x 96 DPI)


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Figure 6 Analysis of electrophysiological activity of cortical neurons growing on EA5-POPC indicates an early synapse maturation process. The percentage of cells exhibiting excitatory postsynaptic potentials (red bars) is higher for EA5-POPC than for PLL coated glass (red bars). The percentage of cells with no synaptic activity (blue bars) is reversed. 115x124mm (300 x 300 DPI)


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Figure 7 Possible positions of EA5-Fc complex in the supported lipid bilayer. (A): the Fc-domain penetrates the SLB and is attached to the glass. (B): Fc-EA5 inserts upside-down in the SLB. (C): Fc-EA5 is only partially embedded into the SLB and floats. (D): EA5-Fc forms weak aggregates that attach to the glass surface in the absence of other forces. 146x163mm (96 x 96 DPI)


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Reconstitution of fusion proteins in supported lipid bilayers for the study of cell surface receptor–ligand interactions in cell-cell contact R Ghosh Moulick1, D Afanasenkau1, S-E Choi1, J Albers1, W Lange1, V Maybeck1, T Utesch2 and A Offenhäusser1,3 1

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Institute for Bioelectronics (ICS-8), Forschungszentrum Jülich, Wilhelm-Johnen Str., 52425 Jülich (Germany) Department of Chemistry, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin (Germany) Author to whom any correspondence should be addressed

Supplementary material for this article is available

Abstract. Bioactive molecules such as adhesion ligands, growth factors or enzymes play an important role in modulating cell behaviour like cell adhesion, spreading, and differentiation. Deciphering the mechanism of ligand-mediated cell adhesion and associated signalling is of great interest not only for fundamental biophysical investigations, but also for applications in medicine and biotechnology. In the presented work, we developed a new biomimetic platform that enables culturing primary neurons and testing cell surface-receptor ligand interactions in cell-cell contacts as e.g. in neuronal synapses. This platform consists of a supported lipid bilayer modified with incorporated neuronal adhesion proteins conjugated with the Fc domain of IgG (ephrin A5 Fc-chimera). We extensively characterized properties of these protein containing bilayers using Fluorescence Recovery after Photobleaching (FRAP), Quartz Crystal Microbalance with Dissipation (QCM-D) and immunostaining. We conclude that the Fcdomain is the part responsible for the incorporation of the protein into the bilayer. The biomimetic platform prepared by this new approach was able to promote neuronal cell adhesion and maintain growth as well as facilitate neuronal maturation as shown by electrophysiological measurements. We believe that our approach can be extended to insert other proteins to create a general culture platform for neurons and other cell types.


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1. Introduction Cell adhesion proteins play an important role in cell differentiation, guidance and communication. In particular, they influence neuronal synapses - specialized intercellular contacts formed between neurons. The extracellular domains of ligand proteins bind selectively to receptor proteins on partner cell surfaces, while their internal domains may initiate signalling cascades and mediate trans-cellular communication1. Numerous proteins have been recently identified to build the molecular basis of the neuronal synapse 2: members of the immunoglobulin (Ig) superfamily (N-CAM, L1, nectin, SynCAM), neurexins and neuroligins, the cadherin family of Ca2+-dependent proteins, and ephrin/Eph receptors (EphR). Ephrin-EphR interactions result in diverse biological responses, such as neuronal cell adhesion and guidance 3 leading to forward and reverse signalling 4 between synaptically coupled cells. There are two subclasses of ephrins and Eph receptors named A and B. Each of them includes several members: eight different EphA receptors interact with five ephrin As and six EphB receptors interact with three ephrin Bs 4. Among these pairs EphrinA5-EphA5 receptor interaction is important for early neuronal development 5. These receptors are also well known for their roles in regulating cell growth, migration, differentiation and axonal guidance 6. Functional and structural studies of receptor-ligand binding 7 in the absence of other binding proteins which may influence the binding properties are of increasing importance, but ideally require the conservation of the function and mobility of the membrane protein 8. It was shown that EphRs require clustering for phosphorylation and to trigger signal cascades 5. Thus in vitro systems for studying Ephrin-EphRs signaling should provide proper orientation and assembly of receptors. Supported lipid bilayers (SLBs), prepared on solid substrates, such as glass, have been used extensively as a model system to study the molecular interactions between proteins on cell membranes, and cell-cell interaction interfaces 9,10. SLBs have emerged as a compelling model system for natural cell membranes 11 where this synthetic membrane replaced an interacting cell and a local change in cell mortility or interaction at the membrane level was captured. Ideally, such a system must mimic the cell membrane by providing proper orientation and mobility of incorporated proteins 12. Presently, there are three strategies to make a mobile protein-lipid bilayer 13 : i) proteins are tethered to the membrane by GPI linkers 14,15 ii) proteins with 6-histidine (6-His) tags are attached to Ni2+-chelating phospholipids 16 and iii) proteins are attached using biotin-streptavidin binding to biotinylated lipid (biotin-CAP-phosphatidylethanolamine) 17. Here, we report a new approach that allows the insertion of a protein (ephrinA5) into the SLBs. The protein is conjugated to the Fc-part of the Ig-G-domain (IgG) which serves to anchor the protein to the bilayer. We use a common procedure of a surfactant-mediated protein incorporation using n-octyl-ß-Dglucopyranoside (NOG). The choice of an Fc chimera was governed by the wide availability of such proteins developed for many different applications. Unlike GPI linked proteins, Fc fusion proteins are commercially available. Fc fusion proteins are very useful for in vitro as well as in vivo investigations, for example immunotherapy and fundamental biophysical investigations 18. Fc-based fusion proteins are composed of an immunoglobin Fc domain that is directly linked to another peptide. The recombinant human ephrin-A5 (EA5) - Fc chimera consists of two EA5 molecules connected to one Fc domain. In this paper we show that the Fc-region can facilitate the incorporation of a protein into the SLB and that the protein doped bilayer can present specific molecules for neuronal receptors to bind to adhering neurons. We show that the embedded protein is accessible for specific recognition, allows aggregation and clustering of the protein by the cell that finally leads to cellular maturation. The procedure of protein incorporation into SLBs is a simple, one step, detergent mediated process that avoids multistep, time- consuming reactions and is possible to insert all kinds of Fc domain into


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the lipid membrane. Further, we discuss possible orientations of the proteins incorporated into the SLB.


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2. Materials and methods 2.1. Materials The lipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) in chloroform, 20 mg/ml and the powder detergent, n-octyl-ß-D-glucopyranoside (NOG) were purchased from Avanti Polar Lipids. Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE) was from Invitrogen GmbH. EphrinA5 (Recombinant Human Ephrin-A5 Fc Chimera, CF), goat primary antibody (Human Ephrin-A5 Antibody), Human IgG1 Fc region antibody, and recombinant mouse EphA5 were purchased from R&D systems, whereas mouse EphA5 antibody was from Cell Applications and His tag antibody was from Abcam. Rabbit polyclonal EphA4 antibody (sc922) and p-Tyr (PY99, sc-7020) used for staining were from Santa Cruz Biotechnology, Inc and all the secondary antibodies, Alexa Fluor 488 donkey anti-goat IgG (H+L), Alexa Fluor 568 rabbit anti mouse IgG (H+L), Alexa Fluor 633 goat anti rabbit IgG (H+L) and Alexa Fluor 488 donkey anti mouse IgG (H+L) were from Life technologies. Anti-human IgG (Fc specific)-FITC antibody produced in goat obtained from Sigma, was used for preclustering experiments. DylightTM microscale antibody labelling kits were supplied by Thermo Scientific. 2.2. Bilayer preparation and characterization 2.2.1.Formation of SLB Proteoliposomes were prepared by detergent mediated reconstitution 19,20. Briefly, POPC was dried onto glass under a stream of nitrogen and then left in vacuum for 1h to remove solvent traces. 12 mM of NOG, and 12 ug/ml of recombinant human Ephrin-A5 Fc chimera was mixed with 0.1 M PBS and added to the dried lipid. The final POPC concentration was 1 mg/ml. The mixture was vortexed and then sonicated for 30 min on ice, and extruded through the Avanti mini extruder (Avanti Polar Lipids, Inc) to form small unilamellar vesicles (SUVs). Clean and plasma activated glass coverslips (MenzelGlaeser, Gerhard Menzel GmbH, Germany) were used for the spreading and fusion of the SUVs to prepare SLBs 9,21,22 by incubation with the proteoliposomes for at least 20 min at room temperature (RT). Excess SUVs and NOG were washed away from the SLB by a continuous stream of PBS. 2.2.2. Characterization of bilayer formation by QCM-D Lipid bilayer formation was monitored by a Quartz crystal microbalance with dissipation (QCM-D, Qsense, Västra Frölunda, Sweden) device. 1 ml of lipid vesicle solution (diluted to 0.3 mg/ml in Dulbecco's Phosphate-Buffered Saline (DBPS) from Sigma) together with the 12 mM NOG and 12 µg/ml EA5-Fc was added onto the SiO2 coated sensor in a QCM-D open module. After lipid bilayer formation, excess lipid vesicles were washed away with PBS. Bilayer formation with vesicle solution and 12 mM NOG (without protein) was also monitored as a control experiment. 2.2.3. Bilayer characterization by fluorescence microscopy 0.2% Texas Red DHPE (Texas Red® 1,2- dihexadecanoyl – sn – glycerol – 3-phosphoethanolamine, triethylammonium salt) doped POPC was used for SLB preparation with or without protein. Oregon green labelled lipid (Oregon Green 488 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) was used for the Fluorescence Recovery After Photobleaching (FRAP) experiments. Bleaching was done by using a 473 nm laser from Rapp OptoElectronics coupled into a Zeiss Axio Scope for Oregon green doped lipids. A series of images were made to track the recovery of fluorescence. Analysis of recovery was done as described before 23, a method that provides the possibility to calculate the diffusion coefficient from images of the recovery without any assumption about initial shape of the fluorescence


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profile. The diffusion coefficient was determined by fitting the analytically calculated curves to the experimental data. Data processing was performed by custom developed MatLab based software. 2.2.4. Characterization of protein in the bilayer Antibody staining against the EA5 part of the chimera After washing with the blocking solution, first, the primary antibody (goat Human Ephrin-A5 Antibody, 1:100 in blocking buffer) was applied for 1 h at RT. Then the secondary antibody (Alexa Fluor 488 donkey anti-goat IgG (H+L), 1:250) was applied and incubated for 1 h at RT before samples were washed with PBS to remove the unbound antibodies. Antibody staining against Fc-part of the chimera Human IgG1 Fc region antibody was applied to EA5-Fc SLB and incubated 2 h at RT then washed with PBS. A secondary antibody (Alexa Fluor 488 donkey anti goat IgG (H+L)) was again applied for 1 h at RT to fluorescently label the Fc region of SLB embedded EA5-Fc. Labeling EA5-Fc-Chimera by DyLight The labeling of EA5-Fc was done using a commercial protein labeling kit (DylightTM Microscale Antibody Labeling Kits) according to the manufacturer’s protocol. In this procedure, the lyophilized ephrinA5 was diluted in 0.05 M borate buffer to prepare a concentration of 1 mg/ml. 100 µl of ephrinA5 was mixed with the DyLight reagent vial and incubated in the dark for 1 h. After preparation of the spin column as directed by the company, the reaction mixture was loaded and then centrifuged to collect the purified protein. The labeling ratio, dye/protein was found to be 0.537, considering molar extinction coefficients for EA5 and DyLight 633 to be 30710 M-1cm-1 (http://www.signalinggateway.org/molecule/query;jsessionid=563efedabc84e95837e44caf7b3365c844da5e2137fbdc1e1643 be3a4085ef1b?afcsid=A005632) and 170000 M-1cm-1 (http://www.piercenet.com/guide/overviewdylight-633-fluorophore) respectively. Protein – antibody binding measurement with QCM-D Lipid bilayer is prepared on the SiO2 coated sensors as mentioned before. After SLB formation, goat primary EA5 antibody, Human IgG1 Fc region Antibody, and Histag antibody (Anti-6X His tag® antibody [HIS.H8], ab18184) was applied respectively and incubated until the signal stopped changing. Excess antibodies were washed with PBS and the change of mass was calculated by Sauerbery equation.

Blocking EA5-POPC surface with EphA5-Fc receptor The EA5-Fc embedded SLB surface was treated with 12 µg/ml of EphA5 receptor for half an hour and was washed with PBS to get rid of the unbound EphA5-Fc receptor, before seeding the neurons. 2.3. Cortical neurons on SLBs 2.3.1 Neuronal cell culture E18 cortical rat neurons were obtained from pregnant Wistar rats as described previously 23,24 with slight modifications. Briefly, embryonic cortices were removed in Hanks balanced salt solution without Ca2+ and Mg2+ and supplemented with 0.035% sodium bicarbonate, 1 mM sodium pyruvate,


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10 mM HEPES and 20 mM glucose (HBSS-) (Sigma®). The tissue was triturated with a silanized, fire-polished pasteur pipette in 1 ml HBSS- per cortex. After dissociation, two volumes HBSS+ (with Ca2+ and Mg2+ and supplements) were added to the suspension and it was allowed to settle on ice for 3 minutes. The top half of the suspension, was collected and centrifuged for 2 minutes at 200 g, and subsequently resuspended in Neurobasal medium with 1% B-27® (Gibco®, USA), 0.5 mM glutamine and 50 µg/ml gentamicin. Cells were diluted in this medium and 10,000 cells per cm2 were plated on a 30 mm diameter glass coverslip coated with either SLB or 10 µg/ml Poly-L-Lysine (PLL) (Sigma Aldrich). Three hours after plating, the medium was replaced with 2 ml fresh medium. Subsequently, half the medium was changed every 3 to 4 days. Samples were kept at 37°C, 5% CO2 and 100% humidity. As control glass substrates coated by PLL were used, which were prepared by applying 1 µg/ml PLL solution on a clean and oxygen plasma activated glass surface for half an hour. After the reaction, the surface was washed with PBS buffer several times, to remove all the unbound PLL. In addition, glass substrates coated by preclustered EA5-Fc were used as described before 25. Preclustered EA5-Fc was prepared by incubation of EA5-Fc in the presence of Human IgG1 Fc region Antibody (Anti-human IgG (Fc specific)-FITC), produced in goat from Sigma, for 1 hour in a ratio of 1:10 (EA5-Fc: IgG1 Fc). This preclustered EA5-Fc was applied to clean glass coverslips and incubated for 30 min. The surface was washed with PBS before culturing neurons to remove the excess molecules. Alternatively, to check the influence of the soluble EA5-Fc on POPC SLB, a ratio of 1:10 EA5-Fc and IgG1 Fc was added directly to the cell culture medium during seeding of neurons on POPC-SLB, with the concentration of EA5-Fc matching the concentration of EA5-Fc used for proteoliposome preparation (12 µg/ml). In this case clustered EA5-Fc was also added after changing the medium. 2.3.2. Phase Contrast and Immunofluorescence-Microscopy Neuronal morphology and cell growth was studied by phase contrast microscopy. Protein expression of neurons was characterized by immunofluorescence microscopy. Briefly, cells were fixed with 4% paraformaldehyde in PBS for 20 min at 4ºC, washed with PBS, then blocked in 1% BSA, 2% goat serum in PBS (blocking buffer) and permeabilized in 0.1% TritonX-100 for 15 min at RT. Samples were washed again with PBS. Then the cultures were incubated with primary antibodies for 1h at RT. Goat polyclonal human Ephrin-A5-Ab, mouse EphA5-Ab, mouse monoclonal p-Tyr (PY99)-Ab or rabbit polyclonal EphA4-Ab were used at 1:200 dilution in blocking buffer. After washing with PBS, the secondary antibodies, Alexa Fluor 488 donkey anti-goat IgG (H+L), Alexa Fluor 568 rabbit anti mouse IgG, Alexa Fluor 633 goat anti rabbit IgG (H+L) or Alexa Fluor 488 donkey anti mouse IgG (H+L) were applied for 1h at a dilution of 1:500 in blocking buffer at RT then washed with PBS. Cells were analyzed with an Axio Imager Z.1 (Carl Zeiss, Oberkochen, Germany) using an Axiocam MRM and Axiovision software. 2.3.3. RT-PCR of EphA5 Total tissue RNA was extracted using the RNA extraction Kit (RNeasy Plus Micro Kit from Qiagen) supplemented with betamercaptoethanol. RNA was immediately transcribed into cDNA using Roche’s First Strand cDNA Synthesis kit with random hexamers. 2 µL of cDNA were used with SYBR Green (Invitrogen) and 4 µL of the primer pair specific for EphA5R, 5’-TCCACACACCTACGAAGATCC3’ and 5’-AAACTGCCCCATGATACTCG-3’ for RT-PCR. RT-PCR was carried out on a Roche LightCycler 2.0 with a 95°C pre-incubation for 10 min, followed by 50 cycles of 59°C, 72°C, 85°C for 5 sec each. Fluorescence was measured during the 85°C step. GAPDH was used as a housekeeping gene with primers 5’ GCAAGTTCAACGGCACA-3’ / 5’-CGCCAGTAGACTCCACGAC-3’ for


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reference expression levels. Samples showing melting point curves consistent with a single product of the appropriate size were analyzed using LightCycler auto CP finder (F” max). Standard curves were determined for the primer pair using dilutions of 1, 1:10, 1:100, and 1:1000. 2.3.4. Electrophysiology After 6 days in vitro (DIV) patch clamp recordings in whole cell configuration were performed with an EPC9/3 (HEKA Elektronik, Germany) in current clamp mode. Single cells among a group of neurons were randomly chosen and patched with a borosilicate glass micropipette (Sutter Instrument Co., USA). The pipettes were pulled with a micropipette puller P-2000 (Sutter Instrument Co., USA) to a tip resistance of 7-9 MΩ. The applied stimuli were controlled with the software TIDA 5.240 (HEKA Elektronik, Germany). To record excitatory postsynaptic potentials (EPSPs) the membrane current was adjusted to a value that led to a membrane potential of -70 mV. Changes in membrane potential were recorded for 10 sec. All measurements were performed in extracellular solution that contained (mM): NaCl 120, KCl 3, MgCl2 1, HEPES 10, CaCl2 2; pH value was adjusted to 7.3 with 1 M NaOH. The pipette was filled with intracellular solution containing (mM): NaCl 2, KCl 120, MgCl2 4, HEPES 5, EGTA 0.2, Mg-ATP 0.20. The pH value of the intracellular solution was adjusted to 7.3 with 1 M KOH. The osmolarity of the extracellular solution was adjusted with D-(+)-Glucose (Sigma) to a matching value if the osmolarity of the culture exceeded the osmolarity of the extracellular solution by more than 10 mOsm.


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3. Results 3.1 Properties of EA5-Fc-Supported Lipid Bilayer Recombinant Human Ephrin-A5 Fc Chimera (EA5-Fc) was incorporated into 1-palmitoyl-2oleoylphosphotidylcholine (POPC) small unilamellar vesicles (SUV) according to a detergent mediated reconstitution method 19,20, where EA5-Fc and the detergent n-octyl-ß-D-glucopyranoside (NOG) were diluted in buffer and added to the dried lipid at the hydration step. After vortex, sonication and extrusion, the EA5-Fc containing proteoliposomes were directly deposited on clean and O2-plasma activated glass cover slips (Figure 1). POPC was used for the SLB because of its fluidity at room temperature and similarity to the lipids of natural cell membranes. SLB formation was confirmed by QCM-D (see supplementary information (SI) Figure S1a) while fluidity of POPC in protein tied SLB was verified by FRAP (Figure S1b). Presence of EA5-Fc in the SLB was verified by immunohistochemistry (Figure S2a): while fluorescence was observed only in case of the SLB with EA5-Fc, pure POPC-SLB showed almost no fluorescence signal when stained for EA5. As a control both SLB systems showed fluorescence when doped with Texas Red DHPE. To further confirm the presence of EA5-Fc in the SLB, the protein was labelled by DyLight prior to the proteoliposome formation and the labelled proteoliposomes were then deposited on a clean glass coverslip as described before to form the SLB. The fluorescence from the SLB was observed due to the presence of fluorescently labelled EA5-Fc (Figure S2b). The presence of EA5-Fc was also verified by monitoring the binding of EA5 antibody on the SLB with QCM-D (Figure S2c). FRAP experiments could not reveal the mobility of the EA5-Fc. Fluorescence of labelled protein did not recovered after photobleaching, suggesting that the protein was anchored to the surface of the substrate. However, the fluidity of SLB itself was almost unchanged after protein incorporation of various concentration (observed by FRAP of labelled lipids) (Figure S1c), likely because of the protein’s overall low concentration (~1011 /cm²)). Further we investigated the role of NOG for the presence of EA5-Fc in the bilayer: the results clearly show that when the proteoliposomes are prepared without NOG no EA5-Fc could be detected by antibody binding experiment in QCM-D (see Table S1) indicating no presence of EA5-Fc in the SLB. Immunostaining with anti-Fc antibodies showed accessibility of the Fc-part of the chimera (Figure S2d). In addition, we performed anti-Fc antibody binding experiments in QCM-D (see Table S1), which also showed the accessibility of Fc-part to the antibody. However, antibodies against the his-tag located on the Fc-part opposite to the ephrin domains did not show any binding by optical detection nor by QCM-D, which suggests that this part of the protein is hidden in the bilayer. 3.2 Neuronal growth on SLB containing EA5-Fc Rat cortical neurons were cultured on SLBs with and without EA5 to test cell adhesion and outgrowth. The SLB made of POPC with EA5 (EA5-POPC) initiates adhesion and facilitates neuronal growth (Figure 2A and Figure 3 for quantification) whereas, as shown before 23, neurons do not grow on SLBs made of POPC only (Figure 2B). To identify EA5 as a key factor for neuronal attachment and growth, we blocked the surface of EA5POPC with a commercially available receptor EphA5R that binds EA5. The receptor was applied to the surface and the receptor binding sites of the EA5 present in the SLB were expected to be occupied


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by the applied receptor due to ligand–receptor interactions. When neurons were plated on that surface, it was found that the cells did not adhere and grow (Figure 2C), indicating a lack of free EA5 molecules available to the neurons' receptors for adhesion. Collocalization of the EphA5R to the EA5Fc presented in the SLB was confirmed by immunostaining with anti EphA5R antibody together with anti-EA5 antibody (Figure 4). As a control we checked neuronal growth (4 DIV) on the glass substrates which were prepared by adding preclustered EA5 (made with anti-human IgG) on PLL coated glass substrates 25. The cells showed very good adhesion to this kind of substrate: the number of alive neurons appeared to be larger than on glass coated with only PLL and on EA5-POPC bilayers (Figure 3). Contrarily, neurons did not grow on POPC-SLBs to which EA5-Fc or preclustered EA5 was added to the medium (Figure 2D), confirming that the functional activation of neurons by EA5 requires proper orientation and clustering of the ligand 8,25. We also checked if neurons can grow on surface coated EA5-Fc. The number of cells grown on EA5-Fc coated glass was found to be much less in number than EA5-Fc in SLB (Data not shown). To exclude the effect of NOG as an adhesion mediating reagent, bilayers were prepared with the detergent only (without protein) and used for neuronal cell culture. It was found that the surface behaved similarly to pure POPC: cells did not adhere on them. Neuronal cells did not grow on only Fc part emphasizing the fact EA5 is promoting cellular growth. Data not shown in both the cases. 3.3 Receptor expression along neuronal cell body Neurons (DIV 1) on the SLB were immunostained for the receptor EphA5 while the bilayer was stained for EA5. This allows us to evaluate the distribution of EA5 in the SLB after neuronal adherence simultaneously with the expression of the EphA5R in the cells. As a control experiment EA5 was labelled with DyLight before incorporation into the SLB to confirm the origin of the EA5, whether endogenous or exogenous. A high number of EA5 clusters was observed under the cell body in both cases (Figure 4A and B). EphA5 expressed in the cell was colocalized with the EA5 in the SLB (Figure 4B and C) Finally, RT-PCR was performed with the cortical neurons grown on EA5-POPC and PLL coated glass to compare the expression of the EphA5R on DIV 6. Both gel analysis and qRT-PCR show an increase in EphA5R when neurons were grown on EA5-POPC compared to PLL coated glass for both low and high density cultures (Figure 5).

3.4 Electrophysiology We investigated the influence of EA5-POPC on the synaptic activity of cortical neurons. Patch clamp technique in whole cell configurations was used to record excitatory postsynaptic potentials (EPSPs) from single neurons. Patch-clamp recordings were performed in current clamp mode. The percentage of cells exhibiting excitatory postsynaptic potentials and the percentage of cells with no synaptic activity was compared for neurons growing on EA5-POPC and on PLL coated surfaces (Figure 6). All measurements were performed on DIV 6, when these neurons usually have not yet formed mature synapses. The results indicate an early synapse maturation for neurons on EA5-POPC, as was shown in literature for solid substrates with EA5 5. Only 20% of cells on control substrates (PLL coated glass) showed spontaneous activity, whereas most cells (73%) on EA5-POPC expressed spontaneous EPSPs at this early stage.


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4. Discussion 4.1 Properties of EA5-Fc-Supported Lipid Bilayer In the present work we constructed SLBs containing EA5-Fc protein and used them as a platform for studying adhesion and development of rat cortical neurons. We performed a number of experiments to characterize the properties of the obtained SLB including QCM-D, FRAP and immunohistochemistry experiments. On the basis of these tests we propose the orientation of EA5-Fc in the SLB. The simplest picture one can draw is that the Fc-part is embedded into the SLB while EA5 domains are situated outside (Figure 7A)26. As the Fc-domain requires more space (see 27) than the thickness of the SLB it would be also partially exposed to the medium. Our antibody binding experiments show that both EA5 and the Fc part are accessible to the antibodies. However, as we assume a symmetric bilayer, the His-tag at the C-terminal should be also accessible for antibody binding if the individual Fc domains penetrate the bilayer (Figure 7B). However, this was not observed in our experiments. As an alternative, we can consider that a part of the Fc-domain is embedded in the SLB and that the histag is hidden in the bilayer (Figure 7C). If this is the correct picture then the protein should be mobile within the single leaflet. Yet, our FRAP experiments did not show mobility of the protein within the bilayer. If we take into account that there are also proteins on the other side of the bilayer in the opposing leaflet, we can imagine that their Fc-domains cluster inside of the SLB (Figure 7D). The lower protein can attach to the support glass would therefore not be mobile. The interaction between the proteins in the top and bottom leaflet would hold the upper one in place, making it immobile too. This is the picture that fits to all our SLB characterization experiments. The interaction between the Fc domains in the aggregate is most likely not very strong (van der Waals forces, hydrophobic interactions, etc.). Thus, detachments of a protein molecule from the aggregate can be possible and may happen from time to time. This could provide enough mobility for the protein to take proper position to be attached to the receptor in the cell membrane. Since the receptor binding is expected to be stronger than the His tag interactions, this would affect a protein capture by the cell. The EA5-Fc complex bound to the Eph receptor can then be pulled by the cell and be accumulated under the cell body as is seen in Figure 4 after random encounters bring new EA5-Fc in contact with free receptors. We also can imagine a possibility that parts of SLB together with the proteins can be remodelled by the cell. In this way, the cell could penetrate the bilayer and rearrange the pieces of the SLB. However, investigating the bilayer remodelling is beyond the scope of this work and we would leave it as well as a question about possible protein transfer from the cell to the SLB or vice versa for further studies. Although the Fc-domain is partially exposed to the medium it cannot provide cell adhesion to the SLB as seen from the experiment where the ephrin domains were blocked with Eph receptors, which left only the Fc-domains free for cell interactions (Figure 2). 4.2 Quantification of the neuronal development Quantification of the neuronal growth (number of alive neurons per area) presented in Figure 3 shows that EA5-POPC supports neuronal adhesion and growth as good as PLL coated glass (the usual control surface for neuronal experiments) and glass coated with PLL mixed with preclustered EA5. The number of neurons on the later surface seems to be a bit higher, which may be due to simultaneous action of both nonspecific adhesion to PLL (provided by its positive charge) and specific adhesion to EA5 mediated by Eph receptors. This does however indicate that clustered EA5 is not itself harmful to


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the cells. It then follows that since addition of preclustered EA5 into the cell culture medium while plating cells on pure POPC SLBs didn’t improve the adhesion of cells: (almost no cells adhere and stayed alive). Thus, only proper anchoring of the EA5 on the surface with the possibility for orientation change and clustering of proteins can provide cell adhesion and support neuronal development. When EA5 in the POPC SLB was blocked by Eph receptors neurons didn’t adhere at all (similar to pure POPC SLB). This result indicates that only interaction of the cell with EA5 in the SLB is responsible for the cell adhesion. This experiment also leads to a conclusion that the Fc-domain of the EA5-Fc complex (although likely to be partially exposed to the medium) cannot provide cell adhesion to the SLB. In this case Fc-domains of Eph receptors are also available for the cell and didn’t provide adhesion. 4.3 Receptor expression Eph receptor comes from the largest subfamily of Tyrosine kinase. Tyrosine kinases initiate signaling cascades by catalyzing the phosphorylation of Tyr residues. Cells in contact with EA5 in the SLB should generate these internal cues to regulate downstream functions. A higher amount of EphA5 receptor production by the cells indicates that the cascade was operating. The results of low density culture in comparison to the high density culture are essential to rule out the possibility of neighbouring cells inducing signalling via natively expressed EA5. Gene expression was normalized to GAPDH levels, which are expected to remain stable per cell on all surfaces, and all cell concentrations, to account for differences in the efficiency of RNA extraction from sample to sample. At both concentrations, cells grown on EA5-POPC showed similar ratiometric increases in EphR expression versus PLL, we therefore attribute the dominant factor for increasing EphR receptor production to receptor activation via the EA5-Fc incorporated bilayer rather than interactions between neighboring cells. Therefore, our finding indicates that EA5-POPC acts as a good surrogate for cellcell contacts. 4.4 Electrophysiology The spontaneous activity of dissociated cortical cultures develops over time as cells contact neighbouring cells. It is an indication of neuronal maturation and network formation. During network formation neurons undergo several steps of development. First, the axon needs to be formed and the initial target needs to be reached. At approximately seven days in vitro the dendritic spines are formed and the network of neurons is established 28. The cells on SLB with EA5 could feel the signals from incorporated Ephrin ligands similar to the situation when the cell reaches another cell. These signals could promote intracellular signals that speed up cell maturation. The results obtained from electrophysiological measurements confirmed this hypothesis: at matched cell densities, the majority of neurons on EA5 SLB exhibited spontaneous synaptic activity on DIV6, while only some neurons growing on PLL coated glass had matured enough to be active at that time (Figure 6). 5. Conclusion In summary, we report a new method to anchor proteins into a lipid bilayer without requiring complex synthesis or molecular assembly. EA5-Fc protein containing SLBs were prepared by incorporation of the protein into lipid vesicles which were then ruptured on glass. The presented approach using the ephrin-A5- Fc chimera was applied to study the interaction of EA5 ligands with EphRs in neuronal cells. The ligand not only enhances neuronal adhesion and growth (though several reports about


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repulsive effect of EA5 is reported 28,29we observe only the adhesion here compared to the pure POPC SLB substrates), but also activates EphA4 and EphA5 on the surface of cortical neurons, as shown by phosphorylation, gene expression, and the emergence of electrophysiological activity. Our results indicate the lack of observed mobility can be explained by the interaction between protein aggregates and the solid substrate. One approach to circumvent this problem is the use of polymer tethered bilayers 29,30 , polymer tethered lipid multi-bilayers 31 or lipids with longer hydrocarbon tails which could offer a better environment for the Fc-part. Alternatively, the protein could be inserted from solution containing detergent into a preformed bilayer (results). This method could ensure the insertion of the protein only from the upper leaflet of the SLB. However we found out that only a very small amount of protein could be inserted (preliminary results not shown). However, the presented system provides enough mobility and viscous environment for the protein to allow clustering by the cells. We believe that the concept is applicable to other membrane proteins conjugated to an Fc-domain including neurexins, NCAM, and N-cadherin, and may also add a new dimension in the field of immunosensors.

Acknowledgements The authors would like to thank Bettina Breuer and Rita Fricke for their support with neuronal cell preparation. This work was supported by the Deutsche Forschungsgemeinschaft Research Training Group (GRK) 1572 (Bionik). References (1) Dalva, M. B.; McClelland, A. C.; Kayser, M. S. Cell Adhesion Molecules: Signalling Functions at the Synapse. Nat. Rev. Neurosci. 2007, 8 (3), 206–220. (2) Yamada, S.; Nelson, W. J. Synapses: Sites of Cell Recognition, Adhesion, and Functional Specification. Annu Rev Biochem 2007, 76, 267–294. (3) Suetterlin, P.; Marler, K. M.; Drescher, U. Axonal ephrinA/EphA Interactions, and the Emergence of Order in Topographic Projections. Semin Cell Dev Biol 2012, 23, 1–6. (4) Murai, K. K. `Eph’ective Signaling: Forward, Reverse and Crosstalk. J. Cell Sci. 2003, 116 (14), 2823–2832. (5) Akaneya, Y.; Sohya, K.; Kitamura, A.; Kimura, F.; Washburn, C.; Zhou, R.; Ninan, I.; Tsumoto, T.; Ziff, E. B. Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development. PLoS One 2010, 5, e12486. (6) M, R.; R, H.; G., L. Eph Receptors and Ephrin Ligands in Axon Guidance. In Axon Growth Guid.; D, B., Ed.; Springer US: New York, 2007; pp 32–49. (7) Shi, P.; Scott, M. A.; Ghosh, B.; Wan, D.; Wissner-Gross, Z.; Mazitschek, R.; Haggarty, S. J.; Yanik, M. F. Synapse Microarray Identification of Small Molecules That Enhance Synaptogenesis. Nat. Commun. 2011, 2, 510. (8) Himanen, J. P.; Nikolov, D. B. Eph Signaling: A Structural View. Trends Neurosci 2003, 26, 46–51. (9) Sackmann, E. Supported Membranes: Scientific and Practical Applications. Science 1996, 271 (5245), 43–48. (10) Salafsky, J.; Groves, J. T.; Boxer, S. G. Architecture and Function of Membrane Proteins in Planar Supported Bilayers: A Study with Photosynthetic Reaction Centers †. Biochemistry 1996, 35 (47), 14773–14781. (11) Dutta, D.; Kam, L. C. Micropatterned, Multicomponent Supported Lipid Bilayers for


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Cellular Systems. Methods Cell Biol. 2014, 120, 53–67. (12) Shen, Y.; Saboe, P. O.; Sines, I. T.; Erbakan, M.; Kumar, M. Biomimetic Membranes: A Review. J. Memb. Sci. 2014, 454, 359–381. (13) Dustin, M. L.; Starr, T.; Varma, R.; Thomas, V. K. Supported Planar Bilayers for Study of the Immunological Synapse. Curr Protoc Immunol 2007, Chapter 18, Unit 18 13. (14) Fein, M.; Unkeless, J.; Chuang, F. Y. S.; Sassaroli, M.; da Costa, R.; Väänänen, H.; Eisinger, J. Lateral Mobility of Lipid Analogues and GPI-Anchored Proteins in Supported Bilayers Determined by Fluorescent Bead Tracking. J. Membr. Biol. 1993, 135 (1), 83–92. (15) Pautot, S.; Lee, H.; Isacoff, E. Y.; Groves, J. T. Neuronal Synapse Interaction Reconstituted between Live Cells and Supported Lipid Bilayers. Nat. Chem. Biol. 2005, 1, 283–289. (16) Xu, Q.; Lin, W.-C.; Petit, R. S.; Groves, J. T. EphA2 Receptor Activation by Monomeric Ephrin-A1 on Supported Membranes. Biophys. J. 2011, 101 (11), 2731–2739. (17) G, G. Liposome Technology; CRC Press, 1992. (18) Czajkowsky, D. M.; Hu, J.; Shao, Z.; Pleass, R. J. Fc-Fusion Proteins: New Developments and Future Perspectives. EMBO Mol. Med. 2012, 4 (10), 1015–1028. (19) Rigaud, J.-L.; Pitard, B.; Levy, D. Reconstitution of Membrane Proteins into Liposomes: Application to Energy-Transducing Membrane Proteins. Biochim. Biophys. Acta Bioenerg. 1995, 1231 (3), 223–246. (20) Seddon, A. M.; Curnow, P.; Booth, P. J. Membrane Proteins, Lipids and Detergents: Not Just a Soap Opera. Biochim. Biophys. Acta 2004, 1666 (1-2), 105–117. (21) Kiessling, V.; Domanska, M. K.; Murray, D.; Wan, C.; Tamm, L. K. Supported Lipid Bilayers : Development and Applications in Chemical Biology. Chem. Biol. 2008. (22) Cremer, P. S.; Boxer, S. G. Formation and Spreading of Lipid Bilayers on Planar Glass Supports. J. Phys. Chem. B 1999, 103 (13), 2554–2559. (23) Afanasenkau, D.; Offenhäusser, A. Positively Charged Supported Lipid Bilayers as a Biomimetic Platform for Neuronal Cell Culture. Langmuir 2012, 28 (37), 13387–13394. (24) Brewer, G. J.; Torricelli, J. R.; Evege, E. K.; Price, P. J. Optimized Survival of Hippocampal Neurons in B27-Supplemented Neurobasal, a New Serum-Free Medium Combination. J. Neurosci. Res. 1993, 35 (5), 567–576. (25) Zimmer, G.; Kastner, B.; Weth, F.; Bolz, J. Multiple Effects of Ephrin-A5 on Cortical Neurons Are Mediated by Src Family Kinases. J. Neurosci. 2007, 27 (21), 5643–5653. (26) Godoy, S.; Violot, S.; Boullanger, P.; Bouchu, M.-N.; Leca-Bouvier, B. D.; Blum, L. J.; Girard-Egrot, A. P. Kinetics Study of Bungarus Fasciatus Venom Acetylcholinesterase Immobilised on a Langmuir-Blodgett Proteo-Glycolipidic Bilayer. ChemBioChem 2005, 6 (2), 395–404. (27) Padlan, E. A. X-Ray Crystallography of Antibodies. 1996, 49, 57–133. (28) Arimura, N.; Kaibuchi, K. Neuronal Polarity: From Extracellular Signals to Intracellular Mechanisms. Nat. Rev. Neurosci. 2007, 8 (3), 194–205. (29) Egea, J.; Klein, R. Bidirectional Eph–ephrin Signaling during Axon Guidance. Trends Cell Biol. 2007, 17 (5), 230–238. (30) Purrucker, O.; Gönnenwein, S.; Förtig, A.; Jordan, R.; Rusp, M.; Bärmann, M.; Moroder, L.; Sackmann, E.; Tanaka, M. Polymer-Tethered Membranes as Quantitative Models for the Study of Integrin-Mediated Cell Adhesion. Soft Matter 2007, 3 (3), 333–336. (31) Lautscham, L. A.; Lin, C. Y.; Auernheimer, V.; Naumann, C. A.; Goldmann, W. H.;


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Fabry, B. Biomembrane-Mimicking Lipid Bilayer System as a Mechanically Tunable Cell Substrate. Biomaterials 2014, 35 (10), 3198–3207. Figure Captions: Figure 1: Upper part: Reconstitution of EA5-Fc chimera in a fluid SLB by fusion of SUV. Lower part: Eph A receptors expressed in cortical neurons recognize EA5-Fc embedded in a SLB. Figure 2: Neuronal cell growth on different substrates studied in this work. (A): POPC SLB with incorporated EA5-Fc provided cell adhesion, (B): SLB containing only POPC did not support cell adhesion, (C): SLB with incorporated EA5-Fc that was blocked with EphA5-Fc did not support cell adhesion, (D): SLB containing only POPC did not support cell adhesion, even when preclustered EA5 was added in to medium. Inset on every image shows a schematic representation of SLB and placement proteins. Figure 3: Quantification of neuronal cell growth after 6 DIV on various substrates. 100 neurons per mm² were cultured on each substrate and the number of live neurons was counted after DIV 6 Figure 4: (A): Cortical neurons grown on POPC-EA5 SLB. The neurons are visualized in phase contrast mode, EA5 is labelled by DyLight (red). The overlay indicates an increased concentration of EA5 in the vicinity of the neurons. (B): Accumulation of the ligand EA5 (immunostained in green) under the cell body (with aggregated proteins) (C): Expression of EphA5 (red) in the cells in response to the EA5. Figure 5: The expression of EphA5R, normalized to the housekeeping gene GAPDH, is higher in cultures grown on EA5-POPC-SLB than on PLL coated glass both at 2000 cells/cm2 and 8000 cells/cm2. Values shown are mean plus/minus standard deviation of the fluorescence ratio of EphA5R product / GAPDH product. Analysis by qRT-PCR with plating densities of 2000 cells/cm2 and 4000 cells/cm2 further corroborates an increase in EphA5R on EA5-POPC-SLB (2.3 and 2.2 respectively, crossing point analysis relative to GAPDH). Figure 6: Analysis of electrophysiological activity of cortical neurons growing on EA5-POPC indicates an early synapse maturation process. The percentage of cells exhibiting excitatory postsynaptic potentials (red bars) is higher for EA5-POPC than for PLL coated glass (red bars). The percentage of cells with no synaptic activity (blue bars) is reversed. Figure 7: Possible positions of EA5-Fc complex in the supported lipid bilayer. (A): the Fc-domain penetrates the SLB and is attached to the glass. (B): Fc-EA5 inserted upside-down in the SLB. (C): Fc-EA5 is only partially embedded into the SLB and floats. (D): EA5-Fc forms weak aggregates that attach to the glass surface in the absence of other forces.


Reconstitution of Fusion Proteins in Supported Lipid Bilayers for the

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