1 Single Lipid Extraction – The Anchoring Strength of Cholesterol in Liquid Ordered and Liquid Disordered Phases Running Head: Single Cholesterol Extraction from Lipid Membranes

Cholesterol is important for the formation of microdomains in lipid bilayers and is enriched in the liquid ordered phase. To understand the interactions leading to this enrichment, we developed an AFM based single lipid extraction approach, which allows to determine the anchorage strength of cholesterol in the two phases of a phase separated lipid membrane. As expected, the forces necessary for extracting a single cholesterol molecule from liquid ordered phases are significantly higher than for extracting it from the liquid disordered phases. Interestingly, application of the Bell model shows two energy barriers that correlate with the head and full length of the cholesterol molecule. The resulting lifetimes for complete extraction are 90 s and 11 s in the liquid ordered and liquid disordered phase, respectively. Molecular dynamics simulations of the very same experiment show similar force profiles and indicate that the stabilization of cholesterol in the liquid ordered phase is mainly due to non-polar contacts