Development of supported plasma membrane platforms from giant plasma membrane vesicles to study membrane protein functions
Date Issued
2016
Date
2016
Author(s)
Wang, Jou-Fang
Abstract
Processing and handling cell membrane proteins while maintaining their intact structural information remains one of the biggest bottlenecks to characterize and understand their structure-function behavior, even though membrane proteins are the major targets for therapeutic development. Most of the problem stems from the requirement of protecting the delicate membrane-embedded hydrophobic core from water during processing to prevent denaturation and loss of function. Here, we obtained giant plasma membrane vesicles (GPMVs) directly from Hela cells and used them to form supported lipid bilayer platforms, so that the membrane proteins can be processed in their native lipid bilayer environment. The data from fluorescence recovery after photobleaching technique show that the species in the supported GPMV membrane platform have fluidity. GPMVs deposited on polymer cushions, PEMA and polydopamine, were shown to have better fluidity than those deposited on bare glass coverslips. More importantly, since the two lipid leaflets and the membrane proteins across the cell membrane are asymmetric, controlling which side of the cell membrane faces the aqueous environment and which one faces the solid support is important for us to study the interested protein function. The anti-ADAM17 antibody experiment shows that the inner leaflet faced the aqueous environment when the GPMVs spontaneously broke on the solid surface. We developed a blotting method to transfer the formed supported GPMV membrane to another suitable support to make an outer-leaflet-facing out supported GPMV membrane. With the blotting method, we might be able to use supported GPMV membranes to study membrane proteins from either side of the cell plasma membrane.
Subjects
supported lipid bilayer
giant plasma membrane vesicle (GPMV)
rupturing mechanism
polymer cushion
cell membrane orientation
membrane blotting
proteoliposome
Type
thesis
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