Sub-Resolution Lipid Domains in the Plasma Membrane Influence Diffusion and Clustering

Abstract

Ordered membrane lipid microdomains have been postulated to regulate protein signaling by influencing the diffusion and clustering properties of membrane proteins. However, direct observation of lipid domains in cell membrane has been difficult. Here, we used fluorescence lifetime imaging of the order-sensitive dye Laurdan and phasor analysis to show the plasma membrane comprises a sub-resolution mixture of ordered and disordered domains. Simultaneous measurements of membrane order and protein diffusion showed that the membrane protein LAT (Linker for Activation of T cells) transiently interacted with sub-resolution domains, which retarded its diffusion. Palmitoylation-deficient LAT (C-S LAT) did not interact with lipid domains. In contrast, depolymerization of cortical actin had no effect on the abundance of lipid domains but directly affected the diffusion of LAT. Using super-resolution photo-activated localization microscopy (PALM) and quantitative cluster analysis, we demonstrated that diffusion correlated with LAT clustering. Hence treatments that modulate the abundance of sub-resolution lipid domains indirectly affect LAT clustering by altering diffusion, while cytoskeletal fences have a direct effect on the protein behavior. In conclusion, our data provide first insights into the molecular organization of the plasma membrane and how its biophysical properties may be exploited in the assembly of protein complexes.