Abstract
We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.
Highlights
We have previously developed a model system capable of specific, leakage-free, full fusion, which was inspired by SNARE-driven membrane fusion
There are a limited number of fusion studies which have been conducted with Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs), employing either natural SNARE proteins[40,41,42,43], or amphipathic, monomeric peptides based on viral fusion protein sequences[44,45]
Lipid mixing was detected simultaneously by dual-color fluorescence imaging of peptide-functionalized GUVs incorporating ATTO 488 dioleoyl-sn-glycero-3-p hosphoethanolamine (DOPE) and peptide-functionalized LUVs labeled with ATTO 633 DOPE
Summary
We have previously developed a model system capable of specific, leakage-free, full fusion, which was inspired by SNARE-driven membrane fusion. In previous studies with batch content-mixing experiments, it was discovered that mixing CP4K4 and the detergent Tween 20 increases the fusion efficiency of the fusogenic system, probably by softening the GUV lipid bilayer55, and breaking up lipopeptide aggregates56 that are formed in the target membrane (Fig. 4A,B).
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