Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling. These have been suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access the sub-diffraction diffusion regime of different fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the cellular plasma membrane, and compare it to the diffusion regime of these molecules in cell-derived actin-free giant plasma membrane vesicles (GPMVs). We show that phospholipids and sphingomyelin, which undergo hindered diffusion in the live cell membrane, diffuse freely in the GPMVs. In contrast to sphingomyelin, which is transiently trapped on molecular-scale complexes in intact cells, diffusion of the ganglioside lipid GM1 suggests transient incorporation into nanodomains, which is less influenced by the actin cortex. Finally, our data on GPI-APs indicate two molecular pools in living cells, one pool showing high mobility with trapped and compartmentalized diffusion, and the other forming immobile clusters both of which disappear in GPMVs. Our data underlines the crucial role of the actin cortex in maintaining hindered diffusion modes of most but not all membrane molecules.

Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS