Stable and Unstable Lipid Domains in Ceramide-Containing Membranes

We applied x-ray diffraction, calorimetry, and infrared spectroscopy to lipid mixtures of palmitoyl-oleoyl phosphatidylcholine, sphingomyelin, and ceramide. This combination of experimental techniques allowed us to probe the stability and structural properties of coexisting lipid domains without resorting to any molecular probes. In particular, we found unstable microscopic domains (compositional/phase fluctuations) in the absence of ceramide, and macroscopically separated fluid and gel phases upon addition of ceramide. We also observed phase fluctuations in the presence of ceramide within the broad phase transition regions. We compare our results with fluorescence spectroscopy data and complement the previously reported phase diagram. We also obtained electron paramagnetic resonance data to assess the possible limitations of techniques employing a single label. Our study demonstrates the necessity of applying a combination of experimental techniques to probe local/global structural and fast/slow motional properties in complex lipid mixtures.