Markers for Detergent-resistant Lipid Rafts Occupy Distinct and Dynamic Domains in Native Membranes

Abstract

Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, Fc⑀RI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with Fc⑀RI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of Fc⑀RI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked Fc⑀RI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes. INTRODUCTIONOrdered regions of membrane, known as microdomains, lipid rafts, detergent-resistant membranes (DRMs), and other abbreviations are thought to be critical sites of signal propagation and membrane trafficking (Edidin, 1997(Edidin, , 2001Simons and Ikonen, 1997;Anderson, 1998; London, 1998, 2000;Jacobson and Dietrich, 1999;Langlet et al., 2000;Anderson and Jacobson, 2002). Analysis of these regions typically begins with detergent solubilization of whole cells followed by sucrose density gradient centrifugation and the recovery of detergent-resistant membranes from the light fractions of the gradient. The DRMs are enriched for caveolin, glycosylphosphatidylinositol-linked (GPI-linked) proteins, glycosphingolipids, GM1 ganglioside, and cholesterol, suggesting that these components are associated in the liquid-ordered (l o ) phase of the lipid bilayer (Schroeder et al., 1994;Ahmed et al., 1997). The interpretation of gradient centrifugation experiments remains controversial. There is evidence that detergents may force associations between components that are not colocalized in intact cells (Mayor and Maxfield, 1995), and fractionation results are known to be dramatically altered by varying the concentration of Triton X-100 (Field et al., 1999;Parolini et al., 1999), by use of alternative detergents (Montixi et al., 1998;Surviladze et al., 1998) or by omission of detergent altogether (Ilangumaran et al., 1999;Harder and Kuhn, 2000;Surviladze et al., 2001). Methods to observe membrane segregation in situ have also generated controversy. Results based on light and fluorescence microscopy of proteins and lipids, including refinements of the established fluorescence recovery after photobleaching methods and new single particle tracking methods, have led some investigators to co...

Topography of plasma membrane microdomains and its consequences for mast cell signaling

“…3A-D). Furthermore, we confirmed recent data [15] that aggregation of Thy-1 does not lead to its enhanced colocalization with FceRI (Fig. 3E, F).…”

“…However, EM observations failed to discern any significant increase in association between Thy-1 and aggregated FceRI [12,15]. To elucidate these discrepancies, we further investigated the topography of Thy-1 and FceRI by means of FRET, a method with 1-10 nm resolution.…”

“…These studies showed that aggregated FceRI accumulate in osmiophilic membrane regions together with several signaling molecules but with no preference for colocalization with Thy-1 or other proteins considered to be localized in lipid rafts [14,15]. Based on these results, Wilson and co-authors suggested that the observed FceRI aggregates could represent the true signal transduction organizing units [14].…”

Lipid rafts, cholesterol, and the brain

“…In some instances, clustering of raft proteins in microdomains can be detected by electron microscopy. The best studied examples include Ras, influenza hemagglutinin, and components of the high affinity IgE receptor signaling pathway (Wilson et al 2002;Parton and Hancock 2004;Wilson et al 2004;Hess et al 2005) (Takeda et al 2003). In addition, large-scale lipid phase separation can be observed in plasma membrane blebs (Baumgart et al 2007).…”

Markov Random Field Modeling of the Spatial Distribution of Proteins on Cell Membranes

“…To observe the topographical events associated with cell signaling, several groups have generated high resolution spatial maps of colloidal gold particles marking receptors and signaling proteins and lipids in native membranes (Wilson et al 2000(Wilson et al , 2001(Wilson et al , 2002(Wilson et al , 2004Prior et al, 2003;Volna et al, 2004;Kim et al, 2005;Plowman et al, 2005;Xue et al, 2007). The technique involves labeling membrane sheets stripped from the dorsal cell surface with ligand-or antibody-coated electron-dense 2-10 nm gold nanoprobes, imaging the labeled sheets by transmission electron microscopy (TEM), and extracting probe coordinates from the digitized images.…”