RT Journal Article SR Electronic T1 The Molecular and Structural Asymmetry of the Plasma Membrane JF bioRxiv FD Cold Spring Harbor Laboratory SP 698837 DO 10.1101/698837 A1 JH Lorent A1 L Ganesan A1 G Rivera-Longsworth A1 E Sezgin A1 KR Levental A1 E Lyman A1 I Levental YR 2019 UL http://biorxiv.org/content/early/2019/07/11/698837.abstract AB A fundamental feature of cellular plasma membranes (PM) is the asymmetric distribution of lipids between the bilayer leaflets. This asymmetry is central to cellular physiology, regulating signaling, apoptosis, coagulation, and cell-cell fusion. While the broad transbilayer distributions of some lipid types are well established, the detailed lipidomic asymmetry of the PM has not been characterized and thus the compositions of the individual leaflets remain poorly understood. Further, how these compositional differences contribute to structural asymmetries between PM leaflets in living cells is not defined. Here, we report the distinct lipidomes and biophysical properties of both monolayers in living mammalian PMs. Using mass spectrometry coupled to enzymatic digestion, we report the detailed compositions of PM leaflets of human erythrocytes. We find a dramatic asymmetry in phospholipid unsaturation, with the cytoplasmic leaflet being ~2-fold more unsaturated than the exoplasmic. Atomistic simulations of lipid mixtures compiled from the lipidomic observations suggested significant asymmetries in lipid order, packing, and dynamics between the two PM leaflets. These were probed directly in the PM of living cells by Fluorescence Lifetime Imaging Microscopy (FLIM) of leaflet-selective, environment-sensitive probes. The outer PM leaflet is highly packed and ordered, resembling a liquid ordered phase, whereas the inner leaflet is significantly more disordered. This biophysical asymmetry is maintained in the endocytic system. These observations reveal the detailed compositional and biophysical asymmetry of mammalian plasma membranes, elucidating fundamental design principles of living membranes.SIGNIFICANCE STATEMENT The asymmetric distribution of lipids between the two bilayer leaflets is a fundamental feature of mammalian plasma membranes. Here, we quantify the comprehensive lipidomes of the two PM leaflets in living mammalian cells and examine their consequences on leaflet physical properties. Using computer simulations, we predict distinct biophysical features for the two leaflets and confirm these with a variety of spectroscopic and microscopic techniques. We find that the outer leaflet of the membrane contains many more fully saturated lipids, which endows it with higher lipid packing and lower diffusivity.