Wednesday, April 24, 2024
4/24/2024
Project Summary Membranes play central and fundamental roles in cell biology. In addition to providing the physical and functional interface between cellular life and the extracellular world, membranes enable most intracellular compartmentalization in eukaryotes. Furthermore, close to a third of mammalian proteins are membrane embedded, with their organization and activity intrinsically coupled to the emergent properties resulting from the collective assembly of lipids and proteins into membranes. Despite this central importance, the structure and organization of living plasma membranes (PMs) remain poorly characterized. Most notably, living membranes are largely compositionally asymmetric; however, how those distinct leaflet compositions affect biophysical properties remains almost completely unexplored. This knowledge gap has persisted because robust technologies for exploring asymmetric membranes have not been available. However, recent methodological breakthroughs have enabled the construction and characterization of complex, biomimetic, asymmetric bilayers. In parallel, quantitative approaches have been developed to probe the biophysical asymmetry of living membranes. Here, we propose to extend these studies through an unprecedented integration of lipidomics, biophysical experiments, cryogenic transmission electron microscopy (cryoEM), and advanced molecular simulations, to test our central hypothesis that compositionally asymmetric membranes have unique biophysical properties resulting from robust coupling between lateral and transverse membrane organization. We will approach this goal through three independent yet complementary lines of inquiry. In Aim 1, we will investigate the biophysical coupling between leaflet asymmetry and membrane lateral organization in model membranes. We will use confocal microscopy, cryoEM, and atomistic simulations to probe the dependence of lipid composition on interleaflet coupling, thereby defining the compositional drivers and molecular mechanisms of leaflet coupling in asymmetric bilayers. Aim 2 will extend these studies into more complex systems to define the biophysical disparity between leaflets in compositionally biomimetic, asymmetric bilayers. We will compare symmetric membranes representative of the inner and outer leaflet of mammalian PMs to their asymmetric counterparts to directly identify the novel consequences arising from asymmetric lipid distributions. Finally, in Aim 3 we will extend our studies into membrane asymmetry in live cell membranes. Recently developed techniques to selectively probe individual leaflets of cultured mammalian cell PMs will be combined with manipulations of compositional asymmetry to determine the biophysical asymmetry of the resting PM and its perturbation by lipid scrambling. Finally, we will perform the first detailed cryoEM characterization of PMs in situ to determine membrane thickness and density distributions in asymmetric compared to scrambled living membranes. These studies comprise a comprehensive, integrated approach to characterize for the first time the consequences of leaflet asymmetry on the structure and organization of biological membranes.
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