Quantitative Mapping of the Lipid Nanoenvironment around Transmembrane Proteins in Living Cells

Weak and transient lipid-protein interactions are thought to shape plasma membrane organization and function but have largely eluded experimental characterization. While model systems can only capture certain aspects of these interactions, extraction of unambiguous data from live cell experiments is challenging. We here ask a simple question directed at a fundamental aspect of plasma membrane organization: To what extent does a transmembrane protein influence, by its mere presence, the fluidity of its immediate lipid nanoenvironment? By specifically immobilizing proteins of interest at various densities in the live cell plasma membrane, we were able to determine its apparent in-plane hydrodynamic radius via quantification of the mobility reduction of individual lipid tracer molecules. In this assay, tight adhesion of lipid layers with reduced fluidity would manifest as an increased effective protein radius. We compared these values with structural biology data and used simulations to map the parameter space of possible nanoenvironment architectures around four different transmembrane proteins. For three of the four proteins tested, our data rule out the presence of tightly associated boundary lipids, calling into question their role as a general membrane-organizing principle.