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 has been generally assumed that the two PM leaflets contain similar numbers of phospholipids (PLs) due to the constraint that their areas must be evenly matched in a bilayer. Contrary to this widely held assumption, our recent detailed lipidomics analysis of 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 of a PM model guided by the lipidomics data reveal that a large PL imbalance between the leaflets can be sustained via highly asymmetric distribution of cholesterol. We also demonstrate 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 exoplasmic leaflet of the plasma membrane and confirm this prediction in live red blood cells using a novel FRET-based assay. The resulting PL and cholesterol number asymmetries in the PM give rise to unique biophysical properties including differential permeability of the two bilayer leaflets and a substantial differential stress. Being under tension, the cytoplasmic leaflet has deep-reaching hydrophobic defects and imaging of living mammalian cells confirms their role in recruiting lipidated proteins to the plasma membrane. This number asymmetry of major membrane constituents thus presents a new and largely unexplored dimension of membrane organization.