23.08.2021: Protocol Alert - Electroformation of Giant Unilamellar Vesicles and A Case Study of the Specific Host-Toxin Interaction of Lipid Membranes with Cassiicolin
Electroformation of Giant Unilamellar Vesicles and A Case Study of the Specific Host-Toxin Interaction of Lipid Membranes with Cassiicolin
An alternating electric field is applied to induce swelling of thin lipid films and generation of giant unilamellar vesicles (GUVs) on an indium tin oxide (ITO)-coated glass surface. The process is, hereafter, referred to as the electroformation of GUVs. Several important parameters such as lipid manipulation, temperature, osmolarity and ionic strength of the solutions involved, and the electric field (current (DC, AC), amplitude, frequency) should be optimal for the successful electroformation of GUVs. In our case study, GUVs composed of lipid mixtures available in plant cells provide many benefits for studying the lipid-dependent pathogenicity of cassiicolin (Cas) toxins and thereby deciphering the host-selective toxin interaction of Cas toxins with the specific lipid membranes of plant cells. GUVs gently maintained in the solution furnish perfectly suspended and intact lipid membranes similar to cytoplasmic membranes enabling us to examine the selective binding of GFP-Cas1 and GFP-Cas2 to the specific lipid membranes. In this protocol, we briefly explain the principle of electroformation method and provide the experimental conditions and the manipulation for successfully making GUVs composed of plant lipids (DPPC, DPPC/DPPA, DPPC/MGDG, DPPC/DGDG, DPPC/stigmasterol, DPPC/sitosterol, DPPC/DGTS-d9, and DPPC/DGTS).