Effect of cholesterol depletion and temperature on the isolation of detergent-resistant membranes from human erythrocytes

Transient lateral microdomains or lipid-rafts play important roles in many physiological membrane-mediated cell processes. Detergent-resistant membranes (DRMs) are good models for the study of lipid-rafts. Here we report that DRMs can be obtained by treating human erythrocytes with the non-ionic detergents Triton X-100 or C12E8 at 37°C, and by treatment at 4°C of cholesterol-depleted erythrocytes. Electron paramagnetic resonance with spin labels inserted at different membrane depths (5- and 16-doxyl stearic acids, 5-SASL and 16-SASL) were used to measure the order parameter (S) of the cell membranes and DRMs. We previously reported significantly higher S values in DRMs with respect to intact erythrocyte membranes. Here we show that higher S values were still measurable in DRMs prepared from intact erythrocytes at 37°C, or from cholesterol-depleted cells at 4°C, for both detergents. For 5-SASL only, increased S values were measured in 4°C-DRMs obtained from cholesterol-depleted vs intact erythrocytes. Flotillin-2, a protein marker of lipid-rafts, was found in DRMs from intact cells in trace amounts but it was sensitively increased in C12E8 DRMs prepared at 4 °C from cholesterol-depleted erythrocytes, while the membrane-skeletal proteins spectrin and actin were excluded from both Triton X-100 and C12E8 DRMs. However, contrary to the 4 °C treatment results, flotillin-2 and stomatin were not resistant to Triton X-100 and C12E8 treatment at physiological temperature. The role of cholesterol in DRMs formation is discussed and the results presented provide further support for the use of C12E8 to the study of DRMs.