Lipid number asymmetry: The hidden dimension of mammalian plasma membranes

The transbilayer distribution of lipids in mammalian plasma membranes (PMs) is functionally important and incompletely understood. It is generally assumed that the two leaflets of lipid bilayers must contain similar numbers of phospholipids (PLs) due to the constraint that their areas must be evenly matched. Contrary to this assumption, our recent detailed lipidomics analysis of live human erythrocytes reveals a large phospholipid imbalance between PM leaflets, with the cytoplasmic leaflet possessing almost 2-fold more PLs than the exoplasmic one. This surprising finding challenges our understanding of living membrane organization and structure. Extensive atomistic simulations guided by the lipidomics data reveals that a large PL imbalance between PM leaflets can be sustained via highly asymmetric distribution of cholesterol. We confirm that cholesterol increases membrane tolerance for PL imbalances in model membranes and cells. Driven by preferential interactions with saturated lipids and its tendency to 'fill gaps', we show that cholesterol is poised for enrichment in the PM exoplasmic leaflet and confirm this prediction in live red blood cells using a novel FRET-based assay. The resulting lipid number asymmetries give rise to unique PM biophysical properties including differential permeability of the two bilayer leaflets and substantial differential stress. Thus, lipid number asymmetry of major membrane constituents presents a largely unexplored dimension of membrane organization.