Biophysical Society Thematic Meeting | Ascona, Switzerland

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

Poster Abstracts

59-POS Board 30 Single Particle Tracking in Cushioned, Blebbed Supported Lipid Bilayers Enables Studies of Transmembrane Protein Diffusion

Rohit R. Singh 1 , Martin I. Malgapo 2 , Maurine E. Linder 2 .Susan Daniel 1 , 1 Cornell University, Ithaca, NY, USA, 2 Cornell University, Ithaca, NY, USA.

Supported Lipid Bilayers (SLB’s) are effective models for studying some biomembrane phenomena. A thin layer of water between the substrate and the bilayer engenders 2D fluidity and enables studies of lipid diffusion and peripheral membrane protein diffusion. However, the water layer is not thick enough to prevent friction between most transmembrane proteins and the substrate. Because of this, it is difficult to study the diffusive properties of proteins that protrude significantly from the membrane. Here, we study several related cushioning strategies that are easy to construct and support the mobility of most transmembrane proteins. All cushions make use of PEGylated lipids to lift the bilayer away from the substrate. The concentration and length of the PEGylated lipids can be varied to maximize mobility for a protein of choice. The PEGylated lipids can also be biotinylated to allow for a double cushion strategy. In this approach, streptavidin is first used to passivate the substrate and will form bonds with the PEGylated lipids to anchor them in place. The bilayer can be formed by vesicle fusion, allowing us to incorporate membrane proteins from cell blebs without using detergents or other artifactual methods. The efficacy of the different cushioning strategies was assessed through single particle tracking (SPT) of fluorescently tagged DHHC20, a ~40 kDa acyltransferase with 4 transmembrane domains. We will discuss the biological implications of these results and the applications of this platform to studying cytoskeleton-mediated confinement of plasma membrane components. We will also briefly discuss a complementary method for carrying out Brownian dynamics simulations to model protein diffusion. The results of these simulations will be used to put forth a model for the mechanism of cytoskeletal confinement based on hydrodynamic interactions with immobilized membrane proteins.

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