Reconstitution of Fusion Proteins in Supported Lipid Bilayers for the Study of Cell Surface Receptor–Ligand Interactions in Cell–Cell Contact

Abstract

Bioactive molecules such as adhesion ligands, growth factors, or enzymes play an important role in modulating cell behavior such as cell adhesion, spreading, and differentiation. Deciphering the mechanism of ligand-mediated cell adhesion and associated signaling 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 Fc-domain 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.

N-Cadherin modified lipid bilayers promote neural network formation and circuitry

“…Thus, the molecular ratio between lipid and antibody, and presumably lipid to N-cadherin, is around 1800:1, with is similar to the reported distribution and amount of ephrinA5 in the bilayer reported previously. 32 The orientation of the extracellular domain in the lipid bilayer and the accessibility of the Fc-par at the C-terminal end of SLB incorporated proteins was also reported in previous report using ephrinA5. 32 The detection and localization of ephrinA5 or N-cadherin in artificial membranes has been confirmed by labeling with DyLight (660nm) (S 1, B and C, Supplementary Information), whereas pure and untreated POPC membranes show only the fluorescence of labeled lipids stained by Texas Red DHPE (S 1, A).…”

“…32 The orientation of the extracellular domain in the lipid bilayer and the accessibility of the Fc-par at the C-terminal end of SLB incorporated proteins was also reported in previous report using ephrinA5. 32 The detection and localization of ephrinA5 or N-cadherin in artificial membranes has been confirmed by labeling with DyLight (660nm) (S 1, B and C, Supplementary Information), whereas pure and untreated POPC membranes show only the fluorescence of labeled lipids stained by Texas Red DHPE (S 1, A). Fluorescence analysis has shown that the lipid bilayer formation occurs after 30 min incubation.…”

“…We monitored the formation of the POPC bilayer with incorporated N-cadherin on a SiO2 coated sensor. The formation of ephrinA5 containing SLBs formation was evaluated by Ghosh Moulick et al 32 A change in mass (∆m) on the sensor surface is proportional to the change of the resonance frequency (∆f), according to the Sauerbrey relation (see Equation 1). With the change in energy dissipation (∆D) we could evaluate the change of frictional (viscous) loss stemming from the viscoelastic properties of SLB 33 .…”

“…Ghosh Moulick et al described that addition of EphrA5 after formation of ephrinA5-Fc doped POPC lipid membranes did not support an attachment of cortical neurons, confirming a proper orientation of ephrinA5-Fc and functional activation of the appropriate EphrA5. 32 Only a correct anchoring to ephrinA5 on the lipid bilayer with the possibility to change the protein orientation can provide cell adhesion and support neural network formation. Moreover, ephrinA5 is predominantly localized at axons as well as neurites, which exemplifies the importance of ephrinA5 in neurite guidances.…”

Patterning artificial lipid bilayer on nanostructured surfaces

“…The fluidity was checked using fluorescence recovery after photobleaching (FRAP), and neuronal adhesion was monitored on such structure using EphrinA5-tethered lipid bilayer. 4 EphrinA5 is a ligand that binds to the Eph receptors of rat cortical neurons and influences cellular adhesion. 5…”