Single Particle Tracking in Double Cushioned, Blebbed Supported Lipid Bilayers Enables Studies of Transmembrane Protein Diffusion

Abstract

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 describe a double cushioning SLB platform that is easy to construct and supports the mobility of most transmembrane proteins. The platform makes use of streptavidin to passivate the substrate and PEGylated lipids (with biotin attached) to act as a cushion. The bilayer can be formed by vesicle fusion, allowing us to incorporate membrane proteins from cell blebs without using detergents or other artifactual methods. Atomic force microscopy images of each layer in this SLB reveal heterogeneities in the lateral distribution and height of the cushioning components. Single particle tracking of fluorescently tagged DHHC20, a ∼40 kDa acyltransferase with 4 transmembrane domains, demonstrates fluidity of large multi-pass membrane proteins. Detailed analysis of trajectories shows the effects of the cushion heterogeneities on the diffusive properties of DHHC20. 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 briefly discuss a model for the mechanism of cytoskeletal confinement based on hydrodynamic interactions with immobilized membrane proteins.