Combinatorial Live Cell Homo- and Hetero-FRET Microscopy of Membrane Proteins

Abstract

dimerization of the receptor-ligand complex and then oligomerization. Past research, through predominantly biochemical methods, have concluded that EphA2 signaling depends on the degrees of multimerization of the proteins and the topology of the ligand presentation. However, clustering mechanisms of EphA2 proteins are not well understood because these signaling molecules function in the cell membrane, which is an environment that is difficult to characterize and manipulate. Our hypothesis is that the multi-scale organization of EphA2 in the cell membrane regulates its biochemical function. To mimic the cell-cell junction, we use a supported lipid bilayer -cell membrane hybrid system. Breast cancer cells presenting EphA2 are cultured on a fluid lipid bilayer consisting of ligand fusion proteins, which can stably interact with a subset of capturing lipids within the bilayer. This interaction allows us to control the protein density, precisely image it, and maintain molecular mobility so ligand-induced receptor clustering can occur. Receptor cluster size is varied by changing the cluster size and degrees of oligomerization of its ligand. On the nanometer length scale, antibodies are used to cross link monomeric forms of ligand fusion proteins and thereby vary the degrees of oligomerization. On the micrometer scale, patterned chromium substrates are used to segregate ligands into corrals of variable cluster sizes. Our results suggest that the spatial organization of receptor plays a role in orchestrating the cascade of signaling switches.