Isolation of lipid rafts (detergent-resistant microdomains) and comparison to extracellular vesicles (exosomes)

Abstract

Lipid rafts (LRs) represent cellular microdomains enriched in sphingolipids and cholesterol which may fuse to form platforms in which signaling molecules can be organized and regulated (Simons and Ikonen, Nature 387:569–572, 1997; Pike, Biochem J 378:281–292, 2004; Grassme et al., J Immunol 168: 300–307, 2002; Cheng et al., J Exp Med 190:1549–1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62–75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 190:1549–1550, 1999) the LR has a well-ordered central core composed mainly of cholesterol and sphingolipids that is surrounded by a zone of decreasing lipid order. Detergents such as Triton X-100 can solubilize the core (and a significant amount of phosphoglyceride), but the LRs will be insoluble at 4 °C and be enriched in a well-characterized set of biomarkers. Model 2 proposes that the LRs are homogeneous, but there is selectivity in the lipids (and proteins) extracted by the 1% Triton X-100. Model 3 proposes LRs with distinct lipid compositions are highly structured and can be destroyed by binding molecules such as beta-methylcyclodextrin or filipin. These may be Caveolin in some cell types but not in brain. Since it is unlikely that two LR preparations will be exactly the same this review will concentrate on LRs defined as “small (50 nm) membranous particles which are insoluble in 1% Triton X-100 at 4 °C and have a low buoyant density (Simons and Ikonen, Nature 387:569–572, 1997; Pike, Biochem J 378:281–292, 2004; Grassme et al., J Immunol 168: 300–307, 2002; Cheng et al., J Exp Med 190:1549–1550, 1999; Kilkus et al., J Neurosci Res 72(1):62–75, 2003; Testai et al., J Neurochem 89:636–644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1–6].