THE INFLUENCE OF CHOLESTEROL AND ORDERED PHOSPHATIDYLSERINE ON THE LIPID PACKING FOR MODELS OF THE CYTOPLASMIC LEAFLET OF THE PLASMA MEMBRANE.

The eukaryotic plasma membrane (PM) is a complex structure, with its functionality intricately associated with its asymmetry. The difference in lipid composition between the inner and outer leaflets of the lipid bilayer highlights this asymmetry. Interestingly, the cytoplasmic leaflet has a significant fraction of unsaturated lipids, conferring on the leaflet a high fluid character. Additionally, a key component of the PM is cholesterol, which affects lipid packing and could promote the organization of coexistence of liquid phases, depending on the lipid composition. To evaluate the impact of cholesterol on lipid packing, we used large unilamellar vesicles (LUVs) composed of lipid structures to mimic the cytoplasmic leaflet of the plasma membrane. We used a lipid packing sensor probe, Laurdan, to evaluate the lipid packing/ order of different lipid compositions. We examine the role of phosphatidylserine (PS), phosphatidylcholine (PE), and cholesterol in ordering or disordering the membrane. Here, we perform experiments using mono and di-unsaturated lipids. We analyzed the Generalized Polarization (GP) of the Laurdan to quantify membrane packing/order, and to compare the results. In addition, a deconvolution analysis of the Laurdan emission peaks (centered at 2.82 eV and 2.53 eV) corroborates the observed GP values. We also investigate how divalent ions affect lipid packing, hemifusion, and fusion. We monitor vesicle aggregation and fusion using optical and fluorescence microscopy, as well as dynamic light scattering.  We observe hemifusion and fusion events in Giant Unilamellar Vesicles (GUVs). From these results, we demonstrate how cholesterol fractions and divalent ions influence membrane packing. Finally, we investigate the sources of lipid ordering in models of the cytoplasmic leaflet, and we overestimate the ordering effect using saturated PS lipids. In these experiments, we evaluate whether ordered lipids in the cytoplasmic leaflet could induce phase separation. Our results elucidate the main interactions in the PM inner leaflet and may provide important insights in the study of asymmetric membranes.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2024/02663-1 and # 2022/04046-4).