Domain-specific lipid distribution in macrophage plasma membranes

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 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. Membranes of eukaryotic cells comprise an immense diversity of lipid species whose purpose and function are poorly understood. The simple model of biological membranes as two-dimensional lipid bilayers has recently been modified to recognize that the self-organizing properties of some lipids drive the formation of specialized domains within cellular membranes (1). Although biological membranes are typically in a fluid (liquid-disordered) state at physiological temperatures, cholesterol and sphingolipids self-associate to form condensed, liquid-ordered domains, or "lipid rafts," within the more fluid "sea" of the rest of the membrane (2-4).Recent interest in lipid rafts comes from the observation that some membrane proteins appear to preferentially partition into raft domains, whereas others are excluded from them. Hence, the structure of lipid rafts, their distribution, and their abundance could control key biological events dependent on the functional organization of the plasma membrane, such as signaling cascades (2), protein and lipid sorting and trafficking (5), cell adhesion and migration (6), entry of viruses (7), bacteria, and toxins (8), and immune responses (9). 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 sta...

Agrin elicits membrane lipid condensation at sites of acetylcholine receptor clusters in C2C12 myotubes

“…Conversely, the nonraft proteins tubulin and transferrin receptors are present almost exclusively in the heavier fractions of the gradient. Consistent with other reports (43,44), we also observed that the protein-detergent (Triton X-100) ratio was critical for AChR partitioning in DRMs. A minimal protein-detergent ratio of 1:1 (w/w) was required to recover AChRs within light fractions of the gradients as DRMs.…”

“…Although raft biochemical extraction has raised controversies (52,53), the demonstration by Gaus et al (44) that THP-1 macrophages treated with either 0.2% Triton X-100 or sonication yield membranes of similar lipid structure prompted us to biochemically investigate rafts from C2C12 myotubes. We show here that three different biochemical preparations of DRM fractions using either cold Triton X-100 or detergent-free extraction procedures (Figs.…”

Francisella Targets Cholesterol-Rich Host Cell Membrane Domains for Entry into Macrophages

“…Caveolin-1 is expressed and associated with lipid rafts in mouse macrophages (31,35). Caveolae demarcate cholesterol and sphingolipid-rich microdomains of the host cell plasma membrane (36).…”

The ontogeny of P-glycoprotein in the developing human blood–brain barrier: implication for opioid toxicity in neonates

“…It has been shown that when these two proteins interact, P-gp activity is inhibited (6). However the localization of P-gp in caveolae and its interaction with caveolin-1 remains controversial (7) and could depend on the specific cell line and detergent used in caveolae purification methods (8,9). At present, no studies have looked at caveolin-1 expression at the developing blood-brain barrier (BBB) or how its expression may modulate P-gp function at the developing BBB.…”