Distributing Aminophospholipids Asymmetrically Across Leaflets Causes Anomalous Membrane Stiffening

We studied the mechanical leaflet coupling of prototypic mammalian plasma membranes using neutron spin-echo spectroscopy. In particular, we examined a series of asymmetric phospholipid vesicles with phosphatidylcholine and sphingomyelin enriched in the outer leaflet and inner leaflets composed of phosphatidylethanolamine / phosphatidylserine mixtures. The bending rigidities of most asymmetric membranes were anomalously high, exceeding even those of symmetric membranes formed from their cognate leaflets. Only asymmetric vesicles with outer leaflets enriched in sphingolipid displayed bending rigidities in conformity with these symmetric controls. We performed complementary small-angle neutron and X-ray experiments on the same vesicles to examine possible links to structural coupling mechanisms, which would show up in corresponding changes in membrane thickness. Additionally, we estimated differential stress between leaflets originating either from a mismatch of their lateral areas or spontaneous curvatures. However, no correlation with asymmetry-induced membrane stiffening was observed. To reconcile our findings, we speculate that an asymmetric distribution charged or H-bond forming lipids may induce an intraleaflet coupling which increases the weight of hard undulatory modes of membrane fluctuations and hence the overall membrane stiffness. SIGNIFICANCE Biological membranes not only act as the outer barriers of cells and organelles, but host a delicate machinery of proteins, which is sensitive not only to the surrounding lipid composition, but also to the mechanical properties of the bilayer. Little is known about the effect of transbilayer lipid asymmetry, a hallmark of all plasma membranes, on bilayer structure and dynamics. The results of this study indicate that the composition of the plasma membrane leads to unique mechanical coupling mechanisms, suggesting that the regulation of lipid asymmetry through flipases/flopases and scramblases could be an important means to tune the mechanical stiffness of the membrane.