1. Reconstitution of Fusion Proteins in Supported Lipid Bilayers for the Study of Cell Surface Receptor-Ligand Interactions in Cell-Cell Contact.
- Author
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Ghosh Moulick R, Afanasenkau D, Choi SE, Albers J, Lange W, Maybeck V, Utesch T, and Offenhäusser A
- Subjects
- Animals, Biomimetic Materials, Cell Adhesion, Cells, Cultured, Cerebral Cortex cytology, Cerebral Cortex metabolism, Ephrin-A5 chemistry, Ephrin-A5 genetics, Female, Humans, Immunoglobulin Fc Fragments chemistry, Immunoglobulin Fc Fragments genetics, Lipid Bilayers, Mice, Neurons cytology, Neurons physiology, Patch-Clamp Techniques, Phosphatidylcholines chemistry, Rats, Wistar, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Ephrin-A5 metabolism, Immunoglobulin Fc Fragments metabolism, Receptor, EphA5 metabolism, Recombinant Fusion Proteins metabolism
- 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.
- Published
- 2016
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