Incorporation of Triacylglycerol and Cholesteryl Ester Droplets in Phase-Separated Giant Unilamellar Vesicles

Cytoplasmic lipid droplets form from the endoplasmic reticulum (ER). Because the ER membrane can undergo phase separation, the interaction of lipid droplets with phase-separated bilayers is of significant interest. In this study, we used fluorescence microscopy to investigate the incorporation of droplets composed of triolein, trilinolein, trimyristolein, trieicosenoin, and cholesteryl arachidonate in the bilayers of giant unilamellar vesicles (GUVs) consisting of a mixture of 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine (DPPC), and cholesterol. After the triacylglycerol droplets were incorporated, the DOPC/DPPC/cholesterol (3:3:2) GUVs, which exhibited liquid-disordered (L_d) and liquid-ordered (L_o) phase separation, retained their phase-separated state. The triacylglycerol droplets were predominantly partitioned in the L_d domains. To elucidate the basis of this preferential partitioning, we investigated the surface pressures of DOPC, DPPC, and cholesterol monolayers containing triolein at the air/water interface using a Langmuir trough. From these measurements, we determined the interfacial tension at the monolayer-covered triolein/water interface. The results showed that DOPC most effectively reduced the interfacial tension. Thus, the droplet sorting into the DOPC-enriched L_d domains likely arose from the difference in the abilities of the two phases to stabilize the droplet interface. In contrast, cholesteryl arachidonate had a profound effect on bilayer phase behavior. Fluorescence images of the DOPC/DPPC/cholesterol (3:3:2) GUVs showed that the domain structures disappeared after droplet incorporation. Additionally, surface pressure measurements of DOPC/DPPC/cholesterol (3:3:2) monolayers containing cholesteryl arachidonate at the air/water interface suggested that cholesteryl arachidonate weakened the lipid-lipid interaction. The results indicate that the cholesteryl arachidonate molecules diffused across the bilayer to hinder the bilayer phase separation.