Biophysical Society Thematic Meeting

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

, Chih-yun Hsia

, Rohit Singh

, Huma Haider

, Toshimitsu Kawate

Susan

Daniel

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.