Triton X-100-Resistant Bilayers: Effect of Lipid Composition and Relevance to the Raft Phenomenon

The molecular basis for the existence of the so-called “detergent-resistant membranes” has been explored. With that aim, vesicles composed of phosphatidylcholine, sphingolipid, and cholesterol were treated with the nonionic detergent Triton X-100 either at 4 °C or at 37 °C and tested for solubilization using turbidity and centrifugation methods. Bilayer fluidity was systematically measured as fluorescence anisotropy of a diphenylhexatriene derivative of phosphatidylcholine. Putative sphingomyelin−cholesterol interactions were explored using IR spectroscopy. The combined experimental evidence clearly indicates that these lipid mixtures are solubilized more easily at 4 °C than at 37 °C, that an increased membrane fluidity does not correlate with an easier solubilization, and that sphingomyelin−cholesterol interactions are essential for insolubility. Sphingolipids by themselves do not hinder detergent solubilization, and some of them, e.g., gangliosides, actually increase bilayer solubility in the presence of detergents. At least with some lipid compositions, there is a range of detergent concentrations at which partial solubilization occurs concomitantly with major changes in bilayer architecture (lysis and reassembly). Moreover, a nonsolubilized residue of composition phosphatidylcholine/sphingomyelin/cholesterol of ca. 1:1:1 (mole ratio) is recovered by centrifugation after detergent treatment of vesicles with very different original lipid compositions. These observations do not preclude the presence of liquid-ordered domains in the cell membrane but support the idea that the detergent-resistant membranes obtained after detergent treatments may well be the result of bilayer partial solubilization and reassembly, instead of corresponding precisely to structures pre-existing in the cell membrane.