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

ABSTRACT The cellular plasma membrane composition and organization is crucial for the regulation of biological processes. Based on our earlier work showing that the same lipid probe, DiI, exhibits different dynamics in CHO-K1 and RBL-2H3 cells, we investigate the molecular factors that govern these differences. First, we determined that the cytoskeleton-interacting Immunoglobulin E receptor (FcεRI), which is abundant in RBL-2H3 but not in CHO-K1 cells, is not responsible for the DiI confinement found in RBL-2H3 cells. Second, lipid mass spectrometry of the plasma membrane of the two cells indicated differences in ceramide content, especially with long and very long acyl chains (C16 to C24). We, therefore, measure membrane dynamics by imaging total internal reflection fluorescence correlation spectroscopy in dependence on these ceramides. Our results show that C24 and C16 saturated ceramides uniquely alter the membrane dynamics by promoting the formation of cholesterol-independent domains and by elevating inter-leaflet coupling.