# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 4 Different Types of Tethered-Bilayer Lipid Membranes
### Vesicle Fusion Method to Form tBLMs: A \"Top-Down\" Approach
#### 4.5.1 Using PEG as Anchoring Spacer
The latter offer the possibility to reproduce liquid-disordered (I\({}_{\text{d}}\)) and liquid-ordered (I\({}_{\text{o}}\)) phase segregation within both leaflets [225]. In this approach, highly monodisperse very small unilamellar vesicles (VSUVs) generated by extraction detergent with cyclodextrin, including fluorescent lipids and transmembrane protein models, were first captured onto a dense PEG polymer brush functionalized with fatty acid moieties (i.e., palmitic acid); their fusion and the consecutive self-assembly of the membrane were induced by PEG solution. This liposome spreading on pre-functionalized surface gives the opportunity to mix vesicles containing different components on demand. Lipid and protein diffusion were characterized in details by FRAP, fluorescence correlation spectroscopy (FCS) and single molecule imaging (SMT). Full mobility of lipids and a high degree of protein mobility as well as homogeneous diffusion of both were observed. Additionally, quantitative ligand binding studies confirmed functional integrity of a transmembrane receptor reconstituted into these polymer-tBLMs [93]. It could be noticed that the lipopolymer used in this approach is constituted of only one fatty acid chain. It is possible that the smaller volume of the hydrophobic anchor interferes less with the diffusivity of the membrane compounds, unlike what was observed previously with the former polymer-tBLMs (see Section 4.1). The advantage of this method lies in the formation of VSUV by detergent extraction with cyclodextrin which is compatible with a broad spectrum of detergents used for the solubilization of membrane proteins and thus, allows rapid and automatized reconstitution of the latter [93]. Finally, a similar combined approach between the \"bottom-up\" and \"top-down\" method, has been recently proposed by [PERSON]'s group to form polymer-supported mammalian cell membranes directly created from plasma cell membrane vesicles [226]. These vesicles, also called cell blebs, that bud from the plasma membrane, are readily generated either by mechanical cell lysis or by chemically induced methods, and they correspond to small biopsies of the intact plasma membrane. Cell blebs, expressing either GPI (glycosphosphatidylinositol)-linked yellow fluorescent proteins or neon-green fused with a 6-pass \(\alpha\) helix transmembrane P2X2 receptor, were induced to fuse and rupture on glass surfaces using PEGylated lipid vesicles which both trigger the bleb rupture and provide some space between the glass surface and the lower leaflet of the planar bilayer to accommodate the cytosolic extramembranous domains. Single molecule tracking and moment scaling spectrum (MSS) analysis have revealed mobility of over 50% for multipass transmembrane proteins and over 90% for GPI-bound proteins, and enzymatic assays, that both proteins are oriented in the planar bilayer with extracellular domains facing toward the bulk, indicating that the dominant mode of bleb rupture is via the \"parachute\" mechanism [226]. In order to enhance the mobility of the transmembrane proteins over 75%, such membranes have been prepared on a surface-tethered tunable-length polyelectrolyte cushion (i.e., PMETAC: poly([(2-methoxylooxy)ethyl]trimethylammonium chloride)), involving direct electrostatic interaction between the naturally negatively charged cell plasma membrane vesicles and the positive charges polyelectrolyte brushes to induce vesicle rupture [92]. More recently, they have been formed on optically transparent and electrically conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (i.e., PEDOT:PSS) in order to monitor ATP (adenosine 5-triphosphate)-gated P2X2 ion channel activity through EIS [227]. In all cases, enzyme accessibility assays have confirmed that the protein orientation is preserved in these types of polymer-tBLMs, and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support.