Use of detergents to study membrane rafts: the good, the bad, and the ugly

Eukaryotic cell membranes contain microdomains called lipid rafts, which are cholesterol-rich domains in which lipid acyl chains are tightly packed and highly extended. A variety of proteins associate preferentially with rafts, and this raft association is important in a wide range of functions. A powerful and widely-used method for studying lipid rafts takes advantage of their insolubility in non-ionic detergents. Here we describe the basis of detergent insolubility, and review strengths, limitations, and unresolved puzzles regarding this method.     

Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation

Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 degrees C. They have been studied in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains from the midgut epithelial membrane of Heliothis virescens and Manduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-beta-cyclodextrin differentially disrupted the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed that several putative Bacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately 2-3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation and pore formation.     

Lipid rafts prepared by different methods contain different connexin channels, but gap junctions are not lipid rafts

Cell extraction with cold nonionic detergents or alkaline carbonate prepares an insoluble membrane fraction whose buoyant density permits its flotation in discontinuous sucrose gradients. These lipid "rafts" are implicated in protein sorting and are attractive candidates as platforms that coordinate signal transduction pathways with intracellular substrates. Gap junctions form a direct molecular signaling pathway by end-to-end apposition of hemichannels containing one (homomeric) or more (heteromeric) connexin isoforms. Residency of channels composed of Cx26 and/or Cx32 in lipid rafts was assessed by membrane insolubility in alkaline carbonate or different concentrations of Triton X100, Nonidet P40 and Brij-58 nonionic detergents. Using Triton X100, insoluble raft membranes contained homomeric Cx32 channels, but Cx26-containing channels only when low detergent concentrations were used. Results were similar using Nonidet P40, except that Cx26-containing channels were excluded from raft membranes at all detergent concentrations. In contrast, homomeric Cx26 channels were enriched within Brij-58-insoluble rafts, whereas Cx32-containing channels partitioned between raft and nonraft membranes. Immunofluorescence microscopy showed prominent colocalization only of nonjunctional connexin channels with raft plasma membrane; junctional plaques were not lipid rafts. Rafts prepared by different extraction methods had considerable quantitative and qualitative differences in their lipid compositions. That functionally different nonjunctional connexin channels partition among rafts with distinct lipid compositions suggests that unpaired Cx26 and/or Cx32 channels exist in membrane domains of slightly different physicochemical character. Rafts may be involved in trafficking of plasma membrane connexin channels to gap junctions.     

Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)

The insolubility of lipids in detergents is a useful method for probing the structure of biological membranes. Insolubility in detergents like Triton X-100 is observed in lipid bilayers that exist in physical states in which lipid packing is tight. The Triton X-100-insoluble lipid fraction obtained after detergent extraction of eukaryotic cells is composed of detergent-insoluble membranes rich in sphingolipids and cholesterol. These insoluble membranes appear to arise from sphingolipid- and cholesterol-rich membrane domains (rafts) in the tightly packed liquid ordered state. Because the degree of lipid insolubility depends on the stability of lipid-lipid interactions relative to lipid-detergent interactions, the quantitative relationship between rafts and detergent-insoluble membranes is complex, and can depend on lipid composition, detergent and temperature. Nevertheless, when used conservatively detergent insolubility is an invaluable tool for studying cellular rafts and characterizing their composition.     

Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles

Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase.     

Two-dimensional electrophoretic analysis reveals that lipid rafts are intact at physiological temperature

Different proteins are found in lipid rafts depending on the isolation method. For example, insulin receptor was predominantly found in lipid raft fractions prepared from HepG2 cells with Brij 35, but were not present in lipid rafts isolated with Triton X-100. In order to assess the effect of detergent type and temperature on raft isolation, raft proteins from HepG2 cells were analyzed by two-dimensional (2-D) electrophoresis. More raft protein spots appeared when rafts were isolated by Brij 35 than by Triton X-100. In addition, more raft proteins were found when isolated at 37 degrees C than at 4 degrees C, indicating that lipid rafts are much more stable at physiological temperature (37 degrees C) in the presence of detergents. Indeed, lipid-modified proteins, such as Src and Lyn, were found in raft fractions even when detergent-resistant rafts were isolated at room or physiological temperature. The 2-D gel profile of raft proteins isolated with detergent-free (high-pH/carbonate) method was considerably similar to that of detergent-resistant raft proteins but contained a greater number of distinct protein spots. Whereas many detergent-resistant raft proteins disappeared upon cellular exposure to methyl-beta-cyclodextrin, high pH/carbonate-resistant raft proteins did not, suggesting that many of proteins isolated by high pH/carbonate could be contaminants. Considering these data, we conclude that liquid-ordered state of detergent-resistant lipid rafts is not destroyed at physiological temperature.     

MHC class II molecules traffic into lipid rafts during intracellular transport

There have been many studies demonstrating that a portion of MHC class II molecules reside in detergent-insoluble membrane domains (commonly referred to as lipid rafts). We have proposed that the function of raft association is to concentrate specific MHC class II-peptide complexes in plasma membrane microdomains that can facilitate efficient T cell activation. We now show that MHC class II becomes lipid raft associated before binding antigenic peptides. Using pulse-chase radiolabeling techniques, we find that newly synthesized MHC class II and MHC class II-invariant chain complexes initially reside in a detergent-soluble membrane fraction and acquire detergent insolubility as they traffic to lysosomal Ag processing compartments. Monensin, an inhibitor of protein transport through the Golgi apparatus, blocks association of newly synthesized MHC class II with lipid rafts. Treatment of cells with leupeptin, which inhibits invariant chain degradation, leads to the accumulation of MHC class II in lipid rafts within the lysosome-like Ag-processing compartments. Raft fractionation of lysosomal membranes confirmed the presence of MHC class II in detergent-insoluble microdomains in Ag-processing compartments. These findings indicate that newly synthesized MHC class II complexes are directed to detergent-insoluble lipid raft microdomains before peptide loading, a process that may facilitate the loading of similar peptides on MHC class II complexes in these microdomains.     

The CD20 calcium channel is localized to microvilli and constitutively associated with membrane rafts: antibody binding increases the affinity of the association through an epitope-dependent cross-linking-independent mechanism

CD20 is a B cell-specific membrane protein that functions in store-operated calcium entry and serves as a useful target for antibody-mediated therapeutic depletion of B cells. Antibody binding to CD20 induces a diversity of biological effects, some of which are dependent on lipid rafts. Rafts are isolated as low density detergent-resistant membranes, initially characterized using Triton X-100. We have previously reported that CD20 is soluble in 1% Triton but that antibodies induce the association of CD20 with Triton-resistant rafts. However, by using several other detergents to isolate rafts and by microscopic co-localization with a glycosylphosphatidylinositol-linked protein, we show in this report that CD20 is constitutively raft-associated. CD20 was distributed in a punctate pattern on the cell surface as visualized by fluorescence imaging and was also localized to microvilli by electron microscopy. The mechanism underlying antibody-induced association of CD20 with Triton-resistant rafts was investigated and found not to require cellular ATP, kinase activity, actin polymerization, or antibody cross-linking but was dependent on the epitope recognized. Thus, antibody-induced insolubility in 1% Triton most likely reflects a transition from relatively weak to strong raft association that occurs as a result of a conformational change in the CD20 protein.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.     

Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review).

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.     

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 °C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.     

Mesoscale organization of domains in the plasma membrane – beyond the lipid raft

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid–protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the “lipid raft” concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms “membrane raft” or “membrane nanodomain” are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.     

An update on plant membrane rafts

The dynamic segregation of membrane components within microdomains, such as the sterol-enriched and sphingolipid-enriched membrane rafts, emerges as a central regulatory mechanism governing physiological responses in various organisms. Over the past five years, plasma membrane located raft-like domains have been described in several plant species. The protein and lipid compositions of detergent-insoluble membranes, supposed to contain these domains, have been extensively characterised. Imaging methods have shown that lateral segregation of lipids and proteins exists at the nanoscale level at the plant plasma membrane, correlating detergent insolubility and membrane-domain localisation of presumptive raft proteins. Finally, the dynamic association of specific proteins with detergent-insoluble membranes upon environmental stress has been reported, confirming a possible role for plant rafts as signal transduction platforms, particularly during biotic interactions.     

Epidermal growth factor receptors are localized to lipid rafts that contain a balance of inner and outer leaflet lipids: a shotgun lipidomics study

The epidermal growth factor (EGF) receptor partitions into lipid rafts made using a detergent-free method, but is extracted from low density fractions by Triton X-100. By screening several detergents, we identified Brij 98 as a detergent in which the EGF receptor is retained in detergent-resistant membrane fractions. To identify the difference in lipid composition between those rafts that harbored the EGF receptor (detergent-free and Brij 98-resistant) and those that did not (Triton X-100-resistant), we used multidimensional electrospray ionization mass spectrometry to perform a lipidomics study on these three raft preparations. Although all three raft preparations were similarly enriched in cholesterol, the EGF receptor-containing rafts contained more ethanolamine glycerophospholipids and less sphingomyelin than did the non-EGF receptor-containing Triton X-100 rafts. As a result, the detergent-free and Brij 98-resistant rafts exhibited a balance of inner and outer leaflet lipids, whereas the Triton X-100 rafts contained a preponderance of outer leaflet lipids. Furthermore, in all raft preparations, the outer leaflet phospholipid species were significantly different from those in the bulk membrane, whereas the inner leaflet lipids were quite similar to those found in the bulk membrane. These findings indicate that the EGF receptor is retained only in rafts that exhibit a lipid distribution compatible with a bilayer structure and that the selection of phospholipids for inclusion into rafts occurs mainly on the outer leaflet lipids.     

Lipid rafts: bringing order to chaos

Lipid rafts are subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids. They exist as distinct liquid-ordered regions of the membrane that are resistant to extraction with nonionic detergents. Rafts appear to be small in size, but may constitute a relatively large fraction of the plasma membrane. While rafts have a distinctive protein and lipid composition, all rafts do not appear to be identical in terms of either the proteins or the lipids that they contain. A variety of proteins, especially those involved in cell signaling, have been shown to partition into lipid rafts. As a result, lipid rafts are thought to be involved in the regulation of signal transduction. Experimental evidence suggests that there are probably several different mechanisms through which rafts control cell signaling. For example, rafts may contain incomplete signaling pathways that are activated when a receptor or other required molecule is recruited into the raft. Rafts may also be important in limiting signaling, either by physical sequestration of signaling components to block nonspecific interactions, or by suppressing the intrinsic activity of signaling proteins present within rafts. This review provides an overview of the physical characteristics of lipid rafts and summarizes studies that have helped to elucidate the role of lipid rafts in signaling via receptor tyrosine kinases and G protein-coupled receptors.     

Cholesterol modulation of membrane resistance to Triton X-100 explored by atomic force microscopy

Biomembranes are not homogeneous, they present a lateral segregation of lipids and proteins which leads to the formation of detergent-resistant domains, also called "rafts". These rafts are particularly enriched in sphingolipids and cholesterol. Despite the huge body of literature on raft insolubility in non-ionic detergents, the mechanisms governing their resistance at the nanometer scale still remain poorly documented. Herein, we report a real-time atomic force microscopy (AFM) study of model lipid bilayers exposed to Triton X-100 (TX-100) at different concentrations. Different kinds of supported bilayers were prepared with dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM) and cholesterol (Chol). The DOPC/SM 1:1 (mol/mol) membrane served as the non-resistant control, and DOPC/SM/Chol 2:1:1 (mol/mol/mol) corresponded to the raft-mimicking composition. For all the lipid compositions tested, AFM imaging revealed that TX-100 immediately solubilized the DOPC fluid phase leaving resistant patches of membrane. For the DOPC/SM bilayers, the remaining SM-enriched patches were slowly perforated leaving crumbled features reminiscent of the initial domains. For the raft model mixture, no holes appeared in the remaining SM/Chol patches and some erosion occurred. This work provides new, nanoscale information on the biomembranes' resistance to the TX-100-mediated solubilization, and especially about the influence of Chol.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

How principles of domain formation in model membranes may explain ambiguities concerning lipid raft formation in cells

Sphingolipid and cholesterol-rich liquid ordered lipid domains (lipid rafts) have been studied in both eukaryotic cells and model membranes. However, while the coexistence of ordered and disordered liquid phases can now be easily demonstrated in model membranes, the situation in cell membranes remains ambiguous. Unlike the usual situation in model membranes, under most conditions, cell membranes rich in sphingolipid and cholesterol may have a "granular" organization in which the size of ordered and/or disordered domains is extremely small and domains may be of borderline stability. This review attempts to explain the origin of the divergence between of our understanding of rafts in model membranes and in cells, and how the physical properties of model membranes can help explain many of the ambiguities concerning raft formation and properties in cells. How physical principles of ordered domain formation relate to limitations of detergent insolubility and cholesterol depletion methods used to infer the presence of rafts in cells is also discussed. Possible modifications of these techniques that may increase their reliability are considered. It will be necessary to study model membrane systems more closely approximating cell membranes in order gain a complete understanding of raft properties in cells. Very high concentrations of membrane cholesterol and proteins may explain key physical characteristics of domains in cellular membranes, and are the two of the most obvious factors requiring additional study.