Line Tension and Phase Separation of a Four-Component Phospholipid Bilayer

When phospholipid bilayers have coexisting liquid disordered (Ld) and liquid ordered (Lo) phases, Ld/Lo line tension has strong control over the size of the coexisting liquid domains. In order to minimize light-induced artifacts that involve lipid reactivity, we chose a lipid mixture with minimal double bonds, DSPC, diphytanoyl-PC, DLPC, and cholesterol. Giant unilamellar vesicles (GUVs) were generated using the electroswelling technique and visualized with an inverted fluorescent microscope. Line tension was determined from the boundary fluctuations between Ld and Lo domains using Flicker Spectroscopy. The Ld/Lo line tension is measured for different GUV compositions within the Ld + Lo coexistence region. The ratio of diphytanoyl-PC and DLPC is then adjusted to control the size of coexisting domains. Different fluorescent dyes are compared to find the minimal light-induced artifacts during the line tension measurements. The results presented in this study provide a more detailed picture of how a cell might control lipid raft size.