Abstract
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.
Highlights
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
Protein analysis of plasma membrane lipid rafts prepared by different methods We first compared the ability of sonication and various detergents to separate protein markers of raft and nonraft membrane domains from macrophage membranes
Others have shown that caveolin-1 is expressed and associated with lipid rafts in mouse macrophages [15, 49] and differentiated human THP-1 macrophages [37] but that caveolin-1 is undetectable in undifferentiated THP-1 “monocytes” [37] and primary human monocyte-derived macrophage (HMDM) [12]
Summary
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. Lipid rafts have been implicated in a range of macrophage functions, including endotoxin-mediated activation and cytokine production, major histocompatibility complex (MHC) class II antigen presentation [10], phagocytosis [11], and cholesterol export [12]. These cells are subject to large variations in cholesterol status during normal phagocytic clearance of damaged, apoptotic, and foreign cells as well as to pathological accumulation of lipoprotein-derived sterol in atherosclerotic lesions. Surprisingly little is known about the lipid and protein composition of membrane domains in human macrophages and which factors govern the formation and maintenance of their lipid rafts
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