Development of Curved Supported Lipid Bilayers for Atomic Force Microscopy Studies of Amyloid Aggregation on Membranes

Abstract

With the use of model lipid bilayer surfaces as substrates, atomic force microscopy (AFM) has become a useful method to obtain physical insights into the formation of toxic protein aggregates associated with amyloid diseases on membranes. The ability to directly image and measure changes in mechanical properties of lipid bilayers associated with exposure to amyloid-forming peptides in a “quasi” real-time fashion can provide new insights that are exceedingly difficult to obtain using other traditional methods. However, there are several limitations associated with using flat, supported bilayers as model surfaces. One of these limitations is the absence of membrane curvature, which can heavily influence the interaction of proteins at lipid interfaces. To remedy this, we have developed model lipid bilayer systems in which the underlying solid support is comprised of flat and curved regions to induce regions of curvature in the bilayer. To accomplish this, we deposited 50nm silica nanoparticles on a silicon substrate coated with poly-L-lysine. These substrates were exposed to total brain lipid extract (TBLE) vesicles, which can fuse on surfaces to form defect free lipid bilayers. This process was monitored with tapping mode AFM in solution by continuously imaging the same portion of the surface. An initial image prior to the addition of the vesicles was taken as a reference. From analysis of particle height, it was verified that the 50nm particles were fully covered by lipid bilayer, resulting in a supported bilayer on the surface that contained flat and curved regions. These supported lipid bilayers can be exposed to amyloid-forming proteins to determine the impact of membrane curvature on protein/lipid interactions associated with disease.