Pinning Cholesterol Chemical Potential Impacts the Miscibility Transition in Isolated Plasma Membrane Vesicles

Abstract

Giant plasma membrane vesicles (GPMVs) isolated from mammalian cell lines separate into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases below growth temperatures, and are often considered a model system for understanding lipid raft heterogeneity in intact cells. Past work shows GPMV transition temperature can be dependent on cell type, growth conditions, or the presence of small molecules, and the surface fraction of phases can be altered by modulating cholesterol levels in cells prior to GPMV isolation using methyl β cyclodextrin (MβCD). Past experiments have treated GPMVs as closed systems with fixed composition since lipids and membrane bound proteins are not soluble in the aqueous phase. It is possible to prepare GPMVs under conditions where cholesterol can exchange between vesicles and the aqueous phase, allowing us to examine vesicle thermodynamic behavior under conditions of fixed cholesterol chemical potential. Experimentally, this is accomplished by bathing vesicles in buffers containing MβCD and MβCD-cholesterol complexes. Recently published work has taken this approach to characterize and manipulate cholesterol chemical potential in intact cells (Ayuyan and Cohen BJ 114(4):904 2018) where it was shown that cholesterol chemical potential varies with cell density. Cell density also alters miscibility temperatures in GPMVs, leading us to speculate possible connections. This poster will present our ongoing work exploring the miscibility transition in GPMVs under conditions of fixed cholesterol chemical potential. We are exploring how the chemical potential of cholesterol within GPMVs varies with growth conditions, while examining how miscibility transition temperatures, surface fraction of phases, and the emergence of gel phase domains are impacted by varying cholesterol chemical potential. Our long term goal is to better understand how the miscibility transition couples to cholesterol chemical potential since this quantity governs binding between cholesterol and membrane bound proteins.