Long acyl chain ceramides govern cholesterol and cytoskeleton dependence of membrane outer leaflet dynamics

The spatiotemporal dynamics of the plasma membrane is a consequence of fine-tuned interactions between membrane components. However, the precise identity of molecular factors that maintain this delicate balance, which is lost even in cell membrane derived mimics, remains elusive. Here, we use two cell lines, CHO-K1 and RBL-2H3, which show differences in outer membrane organization, dynamics, and cytoskeleton coupling, to investigate the underlying factors. To our surprise, knock-down of the cytoskeleton-interacting Immunoglobulin E receptor, which is abundant in RBL-2H3 but not in CHO-K1 cells, is not responsible for lipid confinement or cytoskeleton coupling. A subsequent lipidomic analysis of the two cell membranes revealed differences in total membrane ceramide content (C16 to C24). Analysis of the dynamics and organization of ceramide treated live cell membranes by imaging fluorescence correlation spectroscopy demonstrates that C24 and C16 saturated ceramides uniquely alter membrane dynamics by promoting the formation of cholesterol-independent domains and by elevating the inter-leaflet coupling.