Preserved Transmembrane Protein Mobility in Polymer-Supported Lipid Bilayers Derived from Cell Membranes

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-secretase 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 PEG-cushion 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.

Single Virion Tracking Microscopy for the Study of Virus Entry Processes in Live Cells and Biomimetic Platforms

“…There has also been progress in using composite membranes made from cell-derived membrane components mixed with PEGylated liposomes to engender the SLB with both biologically relevant material (receptors, etc.) and a built-in cushion to maintain constituent mobility [51,52]. Other work has demonstrated the ability to form bilayers from cell plasma membrane blebs as a way to incorporate transmembrane proteinaceous receptors and complete native cell materials into planar geometries [13,53,54].…”

Affinity Capturing and Surface Enrichment of a Membrane Protein Embedded in a Continuous Supported Lipid Bilayer

“…The advent of two‐dimensional membrane protein affinity capturing in a lipid‐bilayer environment may potentially be expanded from local capturing and enrichment of a single type of membrane protein to isolation of specific membrane proteins in a complex matrix, once the concept is extended to supported membranes derived directly from live cells. Parallel activities to address the challenge of converting cell‐membrane material into a continuous SLBs are indeed ongoing . If such efforts are successfully combined with the concept presented herein, we believe that the approach may provide several new exciting opportunities for membrane protein analysis, such as identification of unknown membrane protein–protein or lipid–protein interactions as well as drug screening of membrane‐embedded receptors including kinetic profiling facilitated by the microfluidic setup.…”

“…Parallel activities to address the challenge of converting cell-membrane materiali nto ac ontinuous SLBs are indeed ongoing. [31] If such efforts are successfully combined with the concept presented herein, we believe that the approachm ay provide several new exciting opportunities for membrane protein analysis, such as identification of unknown membrane protein-protein or lipid-protein interactions as well as drug screening of membrane-embedded receptors including kinetic profiling facilitated by the microfluidic setup.…”

Interplays of Interfacial Forces Modulate Structure and Function of Soft and Biological Matters in Aquatic Environments

“…Although various polymer supports, such as chemically immobilized (or cross-linked) gels (Kühner et al, 1994;Kuhl et al, 1998), polymer brushes (Rehfeldt et al, 2006;Kaufmann et al, 2011), and polyelectrolyte multilayers (Wong et al, 1999;Delajon et al, 2005), have been reported, not all hydrophilic polymer films could act as good supports for membranes. Since it is even possible to spread "native cell membranes" uniformly on polymer cushions or spread cell membrane extracts mixed with lipopolymercontaining vesicles (Pace et al, 2015), such systems can be considered as a "1/2 model" of cell-cell contact (Figure 3A). One of the most important criterion in choosing the polymer material is that the polymer-supported membranes must be thermodynamically stable.…”