Probing the Membrane Environment of Plasma Membrane Proteins: A Micropatterning Approach

Abstract

It is well established that lipids and proteins are not just independent components of the plasma membrane of eukaryotic cells but that their arrangement, dynamics and function are interdependent. Besides specific lipid-protein interaction, transmembrane proteins are thought to bind a shell of annular lipids, which are more or less tightly associated with the proteins. Furthermore, highly ordered nanoscopic membrane domains have been proposed to act to compartmentalize proteins and their interactions, but have thus far not been directly observed. Here, we use a combination of single molecule tracking and protein micropatterning to examine these interactions directly in the plasma membrane of living cells. In our experimental approach, different proteins of interest are immobilized within defined patterns in the plasma membrane, where they act as steric obstacles to the diffusion of lipid tracers and thus locally decrease their mobility. In the presence of lipid-protein interactions of any type, lipid mobility will be even further decreased within protein patterns. We used different types of proteins for patterning - single- and multi-spanning transmembrane proteins, a multi-subunit protein and a GPI-anchored protein. For all proteins we found that lipid tracer diffusion within protein patterns was slowed down only by steric hindrance since the size of the protein “sensed” by the diffusing lipid corresponded well with the size estimated from the protein crystal structure. These findings indicate that none of the examined proteins influence their membrane environment beyond their physical size, neither via tightly associated annular lipids nor via more ordered membrane domains.