Domain-specific lipid distribution in macrophage plasma membranes

Katharina Gaus, Macarena Rodriguez, Kalani R. Ruberu, Ingrid Gelissen, Timothy M. Sloane, Leonard Kritharides, Wendy Jessup
2005 Journal of Lipid Research  
Lipid rafts, defined as cholesterol-and sphingolipid-rich domains, provide specialized lipid environments understood to regulate the organization and function of many plasma membrane proteins. Growing evidence of their existence, protein cargo, and regulation is based largely on the study of isolated lipid rafts; however, the consistency and validity of common isolation methods is controversial. Here, we provide a detailed and direct comparison of the lipid and protein composition of plasma
more » ... rane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergentbased and detergent-free methods can generate raft fractions with similar lipid contents and a biophysical structure close to that previously found on living cells, even in cells not expressing caveolin-1, such as primary human macrophages. However, important differences between isolation methods are demonstrated. Triton X-100-resistant rafts are less sensitive to cholesterol or sphingomyelin depletion than those prepared by detergent-free methods. Moreover, we show that detergent-based methods can scramble membrane lipids during the isolation process, reorganizing lipids previously in sonication-derived nonraft domains to generate new detergent-resistant rafts. The role of rafts in regulating the biological activities of macrophage plasma membrane proteins may require careful reevaluation using multiple isolation procedures, analyses of lipids, and microscopic techniques. -Gaus, K., M. Rodriguez, K. R. Ruberu, I. Gelissen, T. M. Sloane, L. Kritharides, and W. Jessup. Domain-specific lipid distribution in macrophage plasma membranes. J. Lipid Res. 2005. 46: 1526-1538. Supplementary key words lipid rafts • detergent-resistant membranes • lipid order
doi:10.1194/jlr.m500103-jlr200 pmid:15863834 fatcat:zg7xkjgxrzgghah7n5m5cdii3a