There is a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze-fracture replica-labelling immunoelectron microscopy in combination with lipid-binding proteins and a peptide to examine both transbilayer distribution and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells, such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBCs, lipid asymmetry was maintained both in resting and thrombin-activated platelets. However, the microparticles released from thrombin-activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine and/or phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell-type specificity of lipid asymmetry in the plasma membrane.
Keywords: Electron microscopy; Freeze fracture; Lipid asymmetry; Microdomain; Plasma membrane.
© 2015. Published by The Company of Biologists Ltd.