Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Thursday Speaker Abstracts

Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs Mark Richards 2 , Chih-yun Hsia 2 , Rohit Singh 2 , Huma Haider 2 , Toshimitsu Kawate 2 . Susan Daniel 2 , 2 Cornell University, Ithaca, NY, USA. Membrane protein interactions with lipids are crucial for their native biological behavior, yet characterization methods are often carried out on purified protein in the absence of native lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer (SLB) platform. The SLB platform is planar and compatible with many surface characterization tools. However, its full potential has yet to be reached because of the challenges associated with integration of membrane proteins, namely maintaining protein fluidity, orientation, and function. We developed a method for the delivery of proteins to SLBs via cell blebs. Cell blebs are sections of the cell membrane that bud off into a proteoliposome during local detachment of the membrane from the cystoskeleton. Native membrane travels with the proteins to the SLB, so crucial lipid interactions can be preserved. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multi-pass transmembrane proteins and over 90% for GPI-linked proteins. Individual proteins were tracked and diffusion was characterized as free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the SLB. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing towards the bulk, and that the dominant mode of bleb rupture is via the “parachute” mechanism. This work is the first experimental confirmation of the parachute mechanism, to our knowledge.

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