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* Centre for Vascular Research at the School of Medical Sciences, University of New South Wales, and Department of Haematology, Prince of Wales Hospital, Sydney, NSW Australia
Department of Cardiology, Concord Hospital, Concord, NSW Australia
Published, JLR Papers in Press, May 1, 2005. DOI 10.1194/jlr.M500103-JLR200
1 To whom correspondence should be addressed. e-mail: k.gaus{at}unsw.edu.au
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 membrane "rafts" prepared from human macrophages by different methods, including several detergent-based isolations and a detergent-free method. We find that detergent-based 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.
Abbreviations: CH/PL ratio, cholesterol-phospholipid ratio; DRM, detergent-resistant membranes; GP, generalized polarization; HMDM, human monocyte-derived macrophage; mßCD, methyl-ß-cyclodextrin; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PMA, phorbol 12-myristate 13-acetate; PS, phosphatidylserine; SM, sphingomyelin; SMase, sphingomyelinase; SR-BI, scavenger receptor class B type I; TfR, transferrin receptor
Supplementary key words lipid rafts • detergent-resistant membranes • lipid order
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