Dissecting the interactions established between proteins and membranes in a given type of cells is not an easy task. Using a cell-free system of large unilamellar vesicles (LUVs) to analyze these interactions may help decipher these interactions and identify potential membrane deformations induced by the proteins incubated with these LUVs. This article describes the protocols for 1) extraction of total lipids from eukaryotic cells using the method developed by Bligh and Dyer (1959), 2) the quantification of glycerophospholipids by gas chromatography after methanolysis, followed by 3) the formation of LUVs by extrusion, 4) protein-lipid binding assay, 5) analysis of the incubation product by transmission electron microscopy (TEM) and by flotation across a discontinuous sucrose gradient and finally, 6) analysis of the proteins by immunoblot and revelation of the glycerophospholipids by iodin fumigation.
[Background] Cell-free systems consisting in giant unilamellar vesicles (GUVs; vesicles composed of a single bilayer of phospholipids and with a diameter greater than 1 μm) or liposomes incubated with recombinant proteins may help understand these interactions. Depending on their diameter and number of lamellae, liposomes are classified into small unilamellar vesicles (SUVs; vesicles constituted of a single bilayer of phospholipids and with a diameter comprised between 20 and 100 nm), large unilamellar vesicles (LUVs; vesicles constituted of a single bilayer of phospholipids and with a diameter comprised between 100 and 400 nm), large multilamellar vesicles (MLVs; vesicles constituted of multiple phospholipid bilayers and with a diameter comprised between 200 nm and 3 μm) and multivesicular vesicles (MVVs; large vesicles composed of a single bilayer of phospholipids and containing several smaller vesicles, each composed of a single bilayer of phospholipids).
When a dried mix of lipids is dispersed in an aqueous solvent, large multilamellar vesicles (LMVs) form spontaneously. Smaller liposomes (SUVs or LUVs) may then be formed by sonication or extrusion. Here we report the formation of LUVs by extrusion of LMVs formed from complex lipids extracted from HeLa cells and their use to investigate Toxoplasma proteins/membrane interactions by flotation across a sucrose gradient and by TEM.
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