Abstract
Giant unilamellar lipid vesicles (GUVs) are widely used as model membrane systems and provide an excellent basis to construct artificial cells. To construct more sophisticated artificial cells, proteins-in particular membrane proteins-need to be incorporated in GUVs. However, current methods for protein reconstitution have limited throughput or are not generally applicable for all proteins because they depend on detergent solubilization. This limitation is addressed here by introducing calcium-mediated membrane fusion to transfer proteins between negatively charged GUVs and cell-derived plasma membrane vesicles (CDVs), derived from HEK293T cells overexpressing a membrane receptor protein. Fusion conditions are optimized using large unilamellar vesicles and GUVs containing phosphatidylserines and fusogenic lipids. The approach is then applied to induce lipid mixing and subsequent transfer of the overexpressed membrane receptor from CDVs into GUVs. The membrane receptor is detected by immunofluorescence on GUVs that underwent lipid mixing with CDVs. Those GUVs also exhibit esterase activity because cytosolic esterases entrapped in the CDVs are transferred during membrane fusion. Thus, content mixing is demonstrated. Using CDVs circumvents the need to purify or solubilize proteins. Moreover, calcium-mediated fusion allows transfer of lipids, water-soluble and membrane bound proteins in one step, resulting in a semi-synthetic cell.
Highlights
Giant unilamellar lipid vesicles (GUVs) are widely used as cell membrane models or to reconstitute cellular reactions in a synthetic and simplified system
cell-derived plasma membrane vesicles (CDVs) accumulated on the artificial membranes through electrostatic interaction induced by Ca2+ which eventually lead to lipid mixing and membrane fusion
GUVs that were either not exposed to CDVs or Ca2+ were tested and we found that the fluorescence was decreased compared to the positive samples, confirming that the antibody assay was specific and calcium was required to induce protein reconstitution
Summary
Giant unilamellar lipid vesicles (GUVs) are widely used as cell membrane models or to reconstitute cellular reactions in a synthetic and simplified system. They protein reconstitution have limited throughput or are not generally applicable can be considered as a basic building block for all proteins because they depend on detergent solubilization. This limitation is addressed here by introducing calcium-mediated membrane fusion to transfer proteins between negatively charged GUVs and cell-derived plasma membrane vesicles (CDVs), derived from HEK293T cells overexpressing a membrane receptor protein. Integrating membrane proteins into the artificial membrane of GUVs remains challenging and tedious
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