Supported Cell‐Membrane Sheets for Functional Fluorescence Imaging of Membrane Proteins

Abstract

Cell‐membrane sheets suitable for in‐vitro functional fluorescence studies have been prepared by direct detachment from cell membranes using poly‐L‐lysine‐coated glass slides. The resulting transferred planar membranes conserve the composition as well as most properties of the original plasma membrane; in particular, both membrane leaflets remain fluid, allowing the investigation of diffusion properties of different cellular membrane components. Measurements on membrane sheets offer several advantages as compared to those on living cells. First, access to the intracellular leaflet is obtained, in particular to the intracellular part of membrane proteins and to cytoplasmic membrane‐associated proteins, opening the possibility of labeling them and modulating their properties with membrane impermeable compounds. Second, the cytosolic autofluorescence of the cells is absent, allowing ultrasensitive measurements to be performed down to the single‐molecule level. Third, the complexity of cellular processes occurring at the plasma membrane can be reduced, allowing the sequential investigation of selected events from complex biochemical networks. These advantages are illustrated by ligand‐binding studies on the α1b‐adrenergic receptor. Our results indicate that supported membrane sheets might find a broad application as an ideal in‐vitro system for the elucidation of complex signaling pathways.

Cell Type-specific β2-Adrenergic Receptor Clusters Identified Using Photoactivated Localization Microscopy Are Not Lipid Raft Related, but Depend on Actin Cytoskeleton Integrity

“…Preparation of Supported Membrane Sheets-Following the protocol of Perez et al (21), cells in a 6-well plate the day after transfection were washed three times with phosphate-buffered saline (PBS) and then covered with deionized water (pH 6 -7) for 2 min to induce osmotic swelling. In the next step, a poly-Llysine (0.1%)-coated coverslip was put on top of the cells for a few minutes and then removed, allowing the transfer of the membrane patches onto the glass coverslip.…”

“…h, schematic representation of the experimental procedure used to obtain the membrane sheets (patches) onto poly-L-lysine-coated coverslips. Following the protocol of Perez et al (21), a poly-L-lysine-coated coverslip was put on top of the cells for few minutes and then removed allowing the transfer of the membrane patches onto the glass coverslip.…”

“…Cell-membrane sheets can be prepared by detachment of the plasma membrane of living cells that are transferred to a poly-L-lysine-coated glass coverslip as observed by Perez at al. (21). These membranes maintain most of their functional properties as in the native conditions.…”

Identification of the factors affecting co-localization precision for quantitative multicolor localization microscopy

“…Experiment in cells were performed by fusing the double fluorophore chimeras to the plasma membrane shuttling short sequence SrcN15 (Annibale et al 2011a). In most of the cases we investigated plasma membrane patches, obtained according to a previously published protocol (Perez et al 2006) in order to minimize background autofluorescence.…”

Nano for bio: Nanopore arrays for stable and functional lipid bilayer membranes (Mini Review)

“…The lateral mobility of lipids in the bilayer is calculated from the recovery time of fluorescent dye-labeled lipids required to refill a small photobleached spot in the bilayer. 19 In order to achieve stable and fluid lipid membranes, chemists have synthesized new organic molecules to bind lipid bilayers on surfaces by molecular tethers. [20][21][22][23] Alternatively, thin hydrophilic polymer films have been spread on the surface to generate a small aqueous gap between the bilayer and the surface.…”