Nano-Domains of Cell Membrane Stiffness, Proteins Diffusion and Concentration Characterized by Thermal Noise Imaging

Abstract

The cell membrane is thought to contain transient spatial domains: cholesterol-stabilized lipid nano-domains and corrals formed by interactions with the membrane cytoskeleton. Due to their small size and transient nature, these cannot be visualized directly and are challenging to characterize in intact cells. One possibility is to measure the diffusion of membrane proteins interacting with these domains. However, the diffusion should be measured with nanometer spatial and microsecond temporal resolution to correctly plot the protein's path in the membrane. We show here that not only is the spatio-temporal resolution of thermal noise imaging (TNI) in an optical trap sufficient to plot the membrane protein's path, but the trapping also allows gathering sufficient data within one small membrane area to “image” the membrane. We create for high resolutions maps of the local diffusion, local attraction potentials and membrane stiffness by using TNI to confine a single membrane protein to diffuse for seconds in an area of 300nm x 300nm. Using a GPI-anchored green fluorescent protein (GFP), which is often used a marker for cholesterol-stabilized nano-domains, to probe the membrane of PtK2 cells we detect domains that are at the same time stiffer, concentrate the protein and show slower diffusion. These align along linear feature and show convex polygons shape. These domains are further stabilized by addition of Ganglioside cross-linking toxins and disappear after removal of the cholesterol. Another marker, GFP-labeled transferrin receptor molecule, detects linear features and linearly demarcated areas of increased protein concentration.