Direct correlation of structures and nanomechanical properties of multicomponent lipid bilayers

Exploring the fine structures and physicochemical properties of physiologically relevant membranes is crucial to understanding biological membrane functions including membrane mechanical stability. We report a direct correlation of the self-organized structures exhibited in phase-segregated supported lipid bilayers consisting of dioleoylphosphatidylcholine/egg sphingomyelin/cholesterol (DEC) in the absence and presence of ceramide (DEC-Ceramide) with their nanomechanical properties using AFM imaging and high-resolution force mapping. Direct incorporation of ceramide into phase-segregated supported lipid bilayers formed ceramide-enriched domains, where the height topography was found to be imaging setpoint dependent. In contrast, liquid ordered domains in both DEC and DEC-Ceramide presented similar heights regardless of AFM imaging settings. Owing to its capability for simultaneous determination of the topology and interaction forces, AFM-based force mapping was used in our study to directly correlate the structures and mechanical responses of different coexisting phases. The intrinsic breakthrough forces, regarded as fingerprints of bilayer stability, along with elastic moduli, adhesion forces, and indentation of the different phases in the bilayers were systematically determined on the nanometer scale, and the results were presented as two-dimensional visual maps using a self-developed code for force curves batch analysis. The mechanical stability and compactness were increased in both liquid ordered domains and fluid disordered phases of DEC-Ceramide, attributed to the influence of ceramide in the organization of the bilayer, as well as to the displacement of cholesterol as a result of the generation of ceramide-enriched domains. The use of AFM force mapping in studying phase segregation of multicomponent lipid membrane systems is a valuable complement to other biophysical techniques such as imaging and spectroscopy, as it provides unprecedented insight into lipid membrane mechanical properties and functions.