Atomic Force Microscopy of Phase Separation on Ruptured, Giant Unilamellar Vesicles, and a Mechanical Pathway for the Co-Existence of Lipid Gel Phases

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Giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) are synthetic model systems widely used in biophysical studies of lipid membranes. Although SLBs are advantageous for biophysical analysis, phase separation behaviors of lipid species in these two model systems can differ due to the lipid-substrate interactions that are present only for SLBs. In the present study, we report that in binary systems, certain phase domains on GUVs retain their original shapes and patterns after the GUVs rupture on glass surfaces. This enabled atomic force microscopy (AFM) experiments on phase domains, a procedure difficult to perform and interpret when applied to GUVs. Unusual phase behavior was evident in binary GUVs containing DLPC and either DPPC or DSPC. These DLPC/DSPC and DLPC/DPPC GUVs both presented the thermodynamic anomaly of having two co-existing gel phases. One phase (a bright phase) included a relatively high concentration of DiI-C20 but excluded Bodipy-HPC, and the other (dark phase) excluded both probes. The bright phases are of interest because they seem to stabilize dark phases against coalescence. Results suggested that the gel phases labeled by DiIC20 in the DLPC/DSPC membrane, which surround the dark gel phase, is an extra layer of membrane, indicating a highly curved structure that might stabilize the interior dark domains, thereby enabling the co-existence of two different gel phases. Results show the utility of AFM on collapsed GUVs, and suggest a possible mechanism for stabilization of lipid domains.

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