Figure 4.

Effect of the ceramide analogs on sterol-enriched domains in complex bilayers. 7SLPC-induced fluorescence quenching of CTL as a function of temperature was measured in multilamellar vesicles with 50 μM final lipid concentration. The vesicles were composed of POPC/PSM/CHL (60/30/10 or 75/15/10 mol %) or POPC/PSM/Cer/CHL (60/15/15/10 mol %). F/F₀ was defined as the relative fluorescence intensity of CTL in the F samples that contained both 7SLPC (replacing 50% of POPC) and CTL (replacing 1 mol % of CHL) over the intensity in the F₀ samples that contained CTL (replacing 1 mol % of CHL) but no 7SLPC. CTL-quenching revealed that when PSM and 18:1- or 20:1-PCer existed in the bilayers at an equimolar amount, sterol was displaced from the PSM environment and no domain melting was observed. Displacement of sterol was verified by the existence of PSM/ceramide-rich, high-melting-temperature, gel-phase domains in those bilayers (Fig. S1 and Table S1). The POPC/PSM/CHL 75/15/10 molar composition is shown as a reference for bilayers with a reduced amount of PSM but no ceramide. As a reference to the relative changes in the F/F₀ between the different mixtures, the initial F/F₀ value is given for each measurement. Representative curves of at least three independently repeated experiments are shown.