Investigating the organisation of the asymmetric plasma membrane using a novel solvatochromic reporter

The plasma membrane (PM) of an animal cell is a heterogenous mixture of lipids and proteins that exhibits both lateral heterogeneity and transverse asymmetry in composition. The complex mixture of lipid species with a range of headgroups and degrees of acyl-chain unsaturation influences the properties of the bilayer such as ordering and consequently the function of embedded membrane proteins. In this study, we have probed the presence of liquid-ordered (lo) domains in each leaflet of the PM and their transbilayer registry in live cells by designing a membrane localised probe that is sensitive to lipid order. This solvatochromic probe, C3L, uses Laurdan as a template and reports on changes in lipid order of complex lipid compositions such as Giant Plasma Membrane Vesicles (GPMVs), while exhibiting a preference for lo-domains. C3L distributes across both leaflets in the live cell membrane, regulated by flippases and scramblases and may be selectively and robustly confined to the inner leaflet after back extraction. C3L indicates the inner leaflet to be significantly disordered compared to the outer leaflet wherein the depletion of sphingomyelin (SM) leads to a more outer-leaflet restricted effect but phosphatidylserine (PS) depletion leads to effects on both leaflets, especially the inner leaflet. This suggests that PS and SM play different roles in building and maintaining domains and coupling them across the bilayer. Further, C3L reported lipid organisation indicates domains of smaller scale and broader order distribution in the PM of live cells at the same temperature compared to homogenous and disordered domains in GPMVs derived from them providing support for active de-mixing in membranes of living cells. Using this tool, we intend to explore the impact of membrane composition and cytoskeletal activity in tuning cellular signalling by regulating transbilayer properties of the PM.