Differential sensitivity to detergents of actin cytoskeleton from nerve endings

Detergent-resistant membranes (DRM), an experimental model used to study lipid rafts, are typically extracted from cells by means of detergent treatment and subsequent ultracentrifugation in density gradients, Triton X-100 being the detergent of choice in most of the works. Since lipid rafts are membrane microdomains rich in cholesterol, depletion of this component causes solubilization of DRM with detergent. In previous works from our group, the lack of effect of cholesterol depletion on DRM solubilization with Triton X-100 was detected in isolated rat brain synaptosomes. In consequence, the aim of the present work is to explore reasons for this observation, analyzing the possible role of the actin cytoskeleton, as well as the use of an alternative detergent, Brij 98, to overcome the insensitivity to Triton X-100 of cholesterol-depleted DRM. Brij 98 yields Brij-DRM that are highly dependent on cholesterol, since marker proteins (Flotillin-1 and Thy-1), as well as actin, appear solubilized after MCD treatment. Pretreatment with Latrunculin A results in a significant increase in Flotillin-1, Thy-1 and actin solubilization by Triton X-100 after cholesterol depletion. Studies with transmission electron microscopy show that combined treatment with MCD and Latrunculin A leads to a significant increase in solubilization of DRM with Triton X-100. Thus, Triton-DRM resistance to cholesterol depletion can be explained, at least partially, thanks to the scaffolding action of the actin cytoskeleton, without discarding differential effects of Brij 98 and Triton X-100 on specific membrane components. In conclusion, the detergent of choice is important when events that depend on the actin cytoskeleton are going to be studied.     

Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding

Cholesterol depletion has been shown to increase mitogen-activated protein kinase activation in response to stimulation with epidermal growth factor (EGF) (Furuchi, T., and Anderson, R. G. W. (1998) J. Biol. Chem. 273, 21099-21104). However, the underlying mechanisms are unknown. We show that cholesterol depletion increases EGF binding, whereas cholesterol loading lowers EGF binding. Based on binding analyses, we demonstrate that the observed changes in EGF binding are caused by alterations in the number of EGF receptors available for ligand binding, whereas the affinity of the receptor for EGF remains unaltered. We also show by immunofluorescence that in unstimulated cells the EGF receptor is localized in non-caveolar lipid rafts containing the ganglioside GM1 and that patching of these rafts by cholera toxin B-chain causes co-patching of EGF receptors. Experiments with solubilization in different detergents at 4 degrees C show that the association of the EGF receptor with these rafts is sensitive to Triton X-100 extraction but insensitive to extraction with another non-ionic detergent, Brij 58. Furthermore, experiments with cholesterol-depleted cells show that the association is cholesterol-dependent. We propose that non-caveolar lipid rafts function as negative regulators of EGF receptor signaling by sequestering a fraction of the EGF receptors in a state inaccessible for ligand binding.     

GM1-containing lipid rafts are depleted within clathrin-coated pits

Recent studies show that markers for lipid rafts are among the plasma membrane components most likely to be internalized independently of clathrin-coated pits, and there is evidence to suggest that lipid rafts may play a functional role in endocytic trafficking [1-5]. However, lipid rafts themselves are commonly defined purely in biochemical terms, by resistance to detergent extraction. The existence of rafts in live-cell membranes remains controversial [6-8], and their distribution relative to endocytic machinery has not been investigated. This study employs fluorescence resonance energy transfer (FRET) to show that in the plasma membrane (PM) of living cells the glycosphingolipid GM1, labeled with cholera toxin B subunit (CTB) [9,10], is found at least in part within clusters that also include GPI-linked proteins. These clusters are cholesterol-dependent and exclude non-raft proteins such as transferrin receptor and so possess predicted properties of lipid rafts. This type of lipid raft is largely excluded from clathrin-positive regions of the PM. They are found within Caveolin-positive regions at the same concentration as at the rest of the cell surface. The data provide evidence for a model in which lipid rafts are distributed uniformly across most of the PM of nonpolarized cells but are prevented from entering clathrin-coated pits.     

Synaptic proteins associate with a sub-set of lipid rafts when isolated from nerve endings at physiological temperature

Although the high presence of cholesterol in nerve terminals is well documented, specific roles of this lipid in transmitter release have remained elusive. Since cholesterol is a highly enriched component in the membrane microdomains known as lipid rafts, it is probable that these domains are very important in synaptic function. The extraction of lipid rafts using Brij 98 at 37 degrees C avoids the formation of nonspecific membrane aggregates at low temperature, allowing the isolation of more physiologically relevant lipid rafts. In the present work, we examine, by means of buoyancy analysis in sucrose gradients after solubilization of the membranes with Brij 98 or with Lubrol WX, the presence of proteins involved in exocytosis in detergent-resistant membranes (DRM) using rat brain synaptosomes as a neurological model. Significant proportions of the proteins tested in the present work, which are involved in neurotransmitter release, are found in Brij 98 raft fractions, demonstrating that significant pools of synaptic proteins are segregated in specific parts of the membrane at physiological temperature. On the other hand, Lubrol WX is unable to solubilize the major fraction of the proteins tested. Treatment of synaptosomes with methyl-beta-cyclodextrin (mbetaCD) causes alteration in the buoyancy properties of proteins initially present in Brij- as well as in Lubrol-resistant membranes, indicating the cholesterol-dependency of both kinds of microdomains. Finally, we detect the depolarization-induced enhancement of the cholesterol-dependent association of syntaxin 1 with Brij 98-rafts, under the same conditions in which prolonged neurotransmitter release is stimulated.     

Cytokine secretion via cholesterol-rich lipid raft-associated SNAREs at the phagocytic cup

Lipopolysaccharide-activated macrophages rapidly synthesize and secrete tumor necrosis factor alpha (TNFalpha) to prime the immune system. Surface delivery of membrane carrying newly synthesized TNFalpha is controlled and limited by the level of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 4 and SNAP-23. Many functions in immune cells are coordinated from lipid rafts in the plasma membrane, and we investigated a possible role for lipid rafts in TNFalpha trafficking and secretion. TNFalpha surface delivery and secretion were found to be cholesterol-dependent. Upon macrophage activation, syntaxin 4 was recruited to cholesterol-dependent lipid rafts, whereas its regulatory protein, Munc18c, was excluded from the rafts. Syntaxin 4 in activated macrophages localized to discrete cholesterol-dependent puncta on the plasma membrane, particularly on filopodia. Imaging the early stages of TNFalpha surface distribution revealed these puncta to be the initial points of TNFalpha delivery. During the early stages of phagocytosis, syntaxin 4 was recruited to the phagocytic cup in a cholesterol-dependent manner. Insertion of VAMP3-positive recycling endosome membrane is required for efficient ingestion of a pathogen. Without this recruitment of syntaxin 4, it is not incorporated into the plasma membrane, and phagocytosis is greatly reduced. Thus, relocation of syntaxin 4 into lipid rafts in macrophages is a critical and rate-limiting step in initiating an effective immune response.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

CD38 is associated with lipid rafts and upon receptor stimulation leads to Akt/protein kinase B and Erk activation in the absence of the CD3-zeta immune receptor tyrosine-based activation motifs.

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other cell surface structures, including the CD38 co-receptor molecule. Here, we show that in TCR+ T cells that express a CD3-zeta lacking the cytoplasmic domain, cross-linking with CD38- or CD3-specific monoclonal antibodies induces tyrosine phosphorylation of CD3-epsilon, zeta-associated protein-70, linker for activation of T cells, and Shc. Moreover, in these cells, anti-CD38 or anti-CD3 stimulation leads to protein kinase B/Akt and Erk activation, suggesting that the CD3-zeta-immunoreceptor tyrosine-based activation motifs are not required for CD38 signaling in T cells. Interestingly, in unstimulated T cells, lipid rafts are highly enriched in CD38, including the T cells lacking the cytoplasmic tail of CD3-zeta. Moreover, CD38 clustering by extensive cross-linking with an anti-CD38 monoclonal antibody and a secondary antibody leads to an increased resistance of CD38 to detergent solubilization, suggesting that CD38 is constitutively associated with membrane rafts. Consistent with this, cholesterol depletion with methyl-beta-cyclodextrin substantially reduces CD38-mediated Akt activation while enhancing CD38-mediated Erk activation. CD38/raft association may improve the signaling capabilities of CD38 via formation of protein/lipid domains to which signaling-competent molecules, such as immunoreceptor tyrosine-based activation motif-bearing CD3 molecules and protein-tyrosine kinases, are recruited.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.     

Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme

Evidence is now accumulating that the plasma membrane is organized in different lipid and protein subdomains. Thus, glycosylphosphatidylinositol (GPI)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and sphingolipids, called rafts.By a detergent-mediated method, alkaline phosphatase, a GPI-anchored enzyme, was efficiently inserted into the membrane of sphingolipids- and cholesterol-rich liposomes as demonstrated by flotation in sucrose gradients. We have determined the enzyme extraluminal orientation. Using defined lipid components to assess the possible requirements for GPI-anchored protein insertion, we have demonstrated that insertion into membranes was cholesterol-dependent as the cholesterol addition increased the enzyme incorporation in simple phosphatidylcholine liposomes.     

The C-terminal domain of perfringolysin O is an essential cholesterol-binding unit targeting to cholesterol-rich microdomains.

There is much evidence to indicate that cholesterol forms lateral membrane microdomains (rafts), and to suggest their important role in cellular signaling. However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin, whose chemically modified form has been recently shown to bind selectively to rafts. BIAcore and structural analyses demonstrate that the C-terminal domain (domain 4) of the toxin is the smallest functional unit that has the same cholesterol-binding activity as the full-size toxin with structural stability. Cell membrane-bound recombinant domain 4 was detected in the floating low-density fractions and was found to be cofractionated with the raft-associated protein Lck, indicating that recombinant domain 4 also binds selectively to cholesterol-rich rafts. Furthermore, an enhanced green fluorescent protein-domain 4 fusion protein stains membrane surfaces in a cholesterol-dependent manner in living cells. Therefore, domain 4 of theta-toxin is an essential cholesterol-binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells.     

Binding and internalization of Helicobacter pylori VacA via cellular lipid rafts in epithelial cells

In this study we investigated the roles of lipid rafts and glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the process of VacA binding and internalization into epithelial cells. Vacuolating activity analysis in AGS, CHO cells, and a CHO-derived line that highly expresses GPI-linked fasI proteins indicated the significance of cholesterol and GPI-APs for VacA activity. Flow cytometric analysis along with VacA-cholesterol co-extraction experiments showed a cholesterol-dependent manner for VacA cell-binding activity, while GPI-APs were not related to it. Differential detergent extraction and fractionation in sucrose density gradient showed co-association of VacA and fasI with rafts on cell membranes. Subcellular distribution of fasI visualized by confocal microscope suggested that fasI trafficked via a newly defined endocytic pathway for GPI-APs in the derived line. Upon VacA intoxication, VacA was visualized to co-migrate along with fasI and finally induced vacuolation coupled with dramatic redistribution of fasI molecules. These results suggest that VacA exploits rafts for docking and entering the cell via the endocytic pathway of GPI-APs.     

Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.     

Shedding of collagen XVII ectodomain depends on plasma membrane microenvironment

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-alpha-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-beta-cyclodextrin (MbetaCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MbetaCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.     

Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.     

The secretory pathway Ca-ATPase 1 is associated with cholesterol-rich microdomains of human colon adenocarcinoma cells

Lipid rafts are often considered as microdomains enriched in sphingomyelin and cholesterol, predominantly residing in the plasma membrane but which originate in earlier compartments of the cellular secretory pathway. Within this pathway, the membranes of the Golgi complex represent a transition stage between the cholesterol-poor membranes of the endoplasmic reticulum (ER) and the cholesterol-rich plasma membrane. The rafts are related to detergent-resistant membranes, which because of their ordered structure are poorly penetrated by cold non-ionic detergents and float in density gradient centrifugation. In this study the microdomain niche of the Golgi-resident SPCA Ca²⁺/Mn²⁺ pumps was investigated in HT29 cells by Triton X-100 detergent extraction and density-gradient centrifugation. Similarly to cholesterol and the raft-resident flotillin-2, SPCA1 was found mainly in detergent-resistant fractions, while SERCA3 was detergent-soluble. Furthermore, cholesterol depletion of cells resulted in redistribution of flotillin-2 and SPCA1 to the detergent-soluble fractions of the density gradient. Additionally, the time course of solubilization by Triton X-100 was investigated in live COS-1 and HT29 cells expressing fluorescent SERCA2b, SPCA1d or SPCA2. In both cell types, the ER-resident SERCA2b protein was gradually solubilized, while SPCA1d resisted to detergent solubilization. SPCA2 was more sensitive to detergent extraction than SPCA1d. To investigate the functional impact of cholesterol on SPCA1, ATPase activity was monitored. Depletion of cholesterol inhibited the activity of SPCA1d, while SERCA2b function was not altered. From these results we conclude that SPCA1 is associated with cholesterol-rich domains of HT29 cells and that the cholesterol-rich environment is essential for the functioning of the pump.     

Visualization of detergent solubilization of membranes: implications for the isolation of rafts

Although different detergents can give rise to detergent-resistant membranes of different composition, it is unclear whether this represents domain heterogeneity in the original membrane. We compared the mechanism of action of five detergents on supported lipid bilayers composed of equimolar sphingomyelin, cholesterol, and dioleoylphosphatidylcholine imaged by atomic force microscopy, and on raft and nonraft marker proteins in live cells imaged by confocal microscopy. There was a marked correlation between the detergent solubilization of the cell membrane and that of the supported lipid bilayers. In both systems Triton X-100 and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) distinguished between the nonraft liquid-disordered (l_d) and raft liquid ordered (l_o) lipid phases by selectively solubilizing the l_d phase. A higher concentration of Lubrol was required, and not all the l_d phase was solubilized. The solubilization by Brij 96 occurred by a two-stage mechanism that initially resulted in the solubilization of some l_d phase and then progressed to the solubilization of both l_d and l_o phases simultaneously. Octyl glucoside simultaneously solubilized both l_o and l_d phases. These data show that the mechanism of membrane solubilization is unique to an individual detergent. Our observations have significant implications for using different detergents to isolate membrane rafts from biological systems.     

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.     

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association

EF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein-protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils.     

Requirements for CEACAMs and cholesterol during murine coronavirus cell entry

Previous reports have documented that cholesterol supplementations increase cytopathic effects in tissue culture and also intensify in vivo pathogenicities during infection by the enveloped coronavirus murine hepatitis virus (MHV). To move toward a mechanistic understanding of these phenomena, we used growth media enriched with methyl-beta-cyclodextrin or cholesterol to reduce or elevate cellular membrane sterols, respectively. Cholesterol depletions reduced plaque development 2- to 20-fold, depending on the infecting MHV strain, while supplementations increased susceptibility 2- to 10-fold. These various cholesterol levels had no effect on the binding of viral spike (S) proteins to cellular carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, rather they correlated directly with S-protein-mediated membrane fusion activities. We considered whether cholesterol was indirectly involved in membrane fusion by condensing CEACAMs into "lipid raft" membrane microdomains, thereby creating opportunities for simultaneous binding of multiple S proteins that subsequently cooperate in the receptor-triggered membrane fusion process. However, the vast majority of CEACAMs were solubilized by cold Triton X-100 (TX-100), indicating their absence from lipid rafts. Furthermore, engineered CEACAMs appended to glycosylphosphatidylinositol anchors partitioned with TX-100-resistant lipid rafts, but cells bearing these raft-associated CEACAMs were not hypersensitive to MHV infection. These findings argued against the importance of cholesterol-dependent CEACAM localizations into membrane microdomains for MHV entry, instead suggesting that cholesterol had a more direct role. Indeed, we found that cholesterol was required even for those rare S-mediated fusions taking place in the absence of CEACAMs. We conclude that cholesterol is an essential membrane fusion cofactor that can act with or without CEACAMs to promote MHV entry.