Protein micropatterning as a tool to probe the membrane environment of transmembrane proteins in live-cells

Abstract

The plasma membrane of eukaryotic cells hosts a plethora of chemically distinct lipids and proteins which may interact with structural elements of the cytoskeleton. Studying their organization as well as their interactions therefore becomes particularly challenging. Often model systems, e.g. functionalized lipid bilayers or giant unilamellar vesicles, are used to assess the behaviour of lipids and proteins in the plasma membrane but may not account for certain, likely relevant aspects of plasma membrane function, i.e. membrane heterogeneity and asymmetry as well as cytoskeleton interactions. We use a micropatterning approach to determine the immediate membrane environment of transmembrane proteins in living cells. Here, GFP-tagged proteins are selectively immobilized in micropatterns posing as obstacles to freely diffusing lipids in the plasma membrane. The immediate nano-environment of studied proteins is then assessed by tracking fluorescently labelled lipids (tracers) and comparing their mobility in- and outside of created protein micropatterns. This approach allows us to discern if a given protein of interest is surrounded by nanofeatures, such as more viscous membrane phases or annular lipids, as they would alter the diffusion of the tracer lipid. We found no evidence of such nanofeatures for all studied transmembrane proteins.