Tuning of Membrane Sphingolipid Content Influences the Links of Outer-Leaflet Membrane Lipid Dynamics to Cholesterol and Cytoskeleton

The plasma membrane (PM) compartmentalization is crucial for specific biochemical processes where lipid domains and actin cytoskeleton are regarded as the fundamental compartmentalization factors. The PM composition and organization vary across cell types. For instance, in some cell types, the connection of outer-leaflet lipid diffusion with cholesterol and cytoskeleton is stronger than in others. Transmembrane proteins interacting with the actin cytoskeleton are thought responsible for mediating these connections. There is less evidence to show that lipids can also be responsible for establishing these connections. Previously, in an ITIR-FCS based comparative study between CHO-K1 and RBL-2H3 cell membranes, we have shown that in RBL-2H3 cell membranes, DiI-C18 (an outer-leaflet liquid disordered phase marker) shows confined diffusion and is sensitive to cholesterol depletion and cytoskeleton disruption. In this study, we explore the differences in DiI-C18 diffusion in RBL-2H3 and CHO-K1 cells by comparing their PM lipid compositions using lipidomics. In addition, we investigate the influence of FcεRIα receptor (an abundant transmembrane protein in RBL-2H3 cells associated with the cytoskeleton) concentrations on the DiI-C18 diffusion in RBL-2H3 cells. Results, after FcεRIα knockdown, show that the FcεRIα does not link DiI-C18 diffusion to cytoskeleton and cholesterol. Subsequently, lipidomics data reveal a higher percentage of sphingomyelin and ceramides in PM of RBL-2H3 cells as compared to CHO-K1 cells. Therefore, we manipulated the sphingolipid content in RBL-2H3 cells and monitored the DiI-C18 diffusion. Results suggest that higher membrane ceramide content is responsible for cholesterol and cytoskeleton sensitive DiI-C18 diffusion. Furthermore, exogenous treatment of CHO-K1 cells with structurally distinct ceramides show DiI-C18 diffusion similar to RBL-2H3 cells. Finally, we conclude that a higher membrane ceramide content is sufficient to induce coupling of outer-leaflet lipid diffusion with cholesterol and cytoskeleton.