Biophysical Society Thematic Meeting

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

, Martin I. Malgapo

, Maurine E. Linder

.Susan Daniel

Cornell University, Ithaca, NY, USA,

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.