Relation between the organization of spectrin and of membrane lipids in lymphocytes

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.     

Peculiar behaviour of rabbit thymocytes in interaction with liposomes of different compositions shown by fluorescence polarization studies,lipid analysis,and uptake of vesicle-entrapped carboxyfluorescein

In order to obtain more information on membrane phenomena occurring at the cell surface of rabbit thymocytes we have performed experiments aimed at altering the lipid composition of the plasma membrane. Thymocytes were incubated at 37°C with phospholipid vesicles of different compositions. Vesicle-cell interaction was followed by measuring the degree of fluorescence polarization and the uptake of vesicle-entrapped carboxyfluorescein. Neutral and negatively charged liposomes prepared from egg phosphatidylcholine are currently used in investigations of vesicle-cell interaction. In this report we show that these liposomes do not interact with rabbit thymocytes as is evident from unaltered lipid fluidity measured in whole cells and in isolated plasma membranes. This was confirmed by experiments with vesicle-entrapped carboxyfluorescein showing hardly any uptake of the fluorophor from neutral and negatively charged egg phosphatidylcholine liposomes. Using both techniques substantial interaction was found with positively charged egg phosphatidylcholine liposomes and with liposomes prepared from soybean lecithin which is composed of a variety of phospholipids. The results of these experiments were supported by lipid analysis of cells treated with soybean lecithin liposomes. Increase in phosphatidylcholine contents of mixed phospholipid vesicles was further shown to result in decreased vesicle-cell interaction. From measurements of the quantity of carboxyfluorescein inside cells and the total amount of cell-associated carboxyfluorescein it is concluded that adsorption plays a prominent role in interaction between liposomes and rabbit lymphocytes. The grade of maturation of lymphocytes was also found to affect vesicle-cell interaction. The more mature thymocytes took up more vesicle-entrapped carboxyfluorescein from soybean liposomes than immature thymocytes. Mesenteric lymph node cells exhibited a still stronger interaction. The role of vesicle and cell surface charge and membrane fluidity of both vesicles and cells in interaction between liposomes and rabbit thymocytes is discussed.     

Shedding of gangliosides from calf thymocytes

In the course of our work on calf thymus gangliosides [Dyatlovitskaya, Zablotskaya, Azizov & Bergelson (1980) Eur. J. Biochem. 110, 475-483] we studied the gangliosides exfoliated from the cell surface of thymocytes. It was shown that calf thymocytes shed gangliosides both in vivo and in vitro. Various gangliosides were found to be present in high amounts both in extracellular plasma membrane vesicles and in the 64000 X g supernatant. The extracellular membrane fragments were comparatively higher in disialosyllactosylceramide and the 64000 X g supernatant was higher in sialosyllactosylceramide than the cells. Comparison of the ganglioside composition of extracellular membrane fragments, thymocytes and lymphocytes led us to suggest that the shedding of gangliosides from the surface of thymocytes may be involved in the transformation of immunologically incompetent cortical thymocytes into immunocompetent virgin T-cells.     

Surface glycoproteins of resting and activated human T lymphocytes

Summary This review summarizes some recent studies on the surface glycoproteins of human thymocytes and T lymphocytes. Purified cells were surface labeled by the galactose oxidase-NaB³H₄ or periodate-NaB³H₄ techniques. The radioactive membrane glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. Thymocytes and T lymphocytes show characteristic surface glycoprotein profiles which are easily distinguishable from those of the other main groups of human leukocytes. We observed specific changes in the surface glycoprotein patterns which correlate with the degree of maturation and functional activation of T cells. Surface molecules carrying T cell specific antigens were identified by immune-precipitation from lysates of surface labeled thymocytes and T lymphocytes using rabbit anti-human T cell antibodies. Finally we describe a leukocyte membrane glycoprotein which is a precursor of serum ₁ acid glycoprotein (orosomucoid).     

Loss of net negative surface charge during MLC stimulation of human T lymphocytes: correlation to "stable" E-rosette formation and natural attachment to normal and malignant target cells.

Activation of human blood T lymphocytes in mixed lymphocyte culture (MLC) causes a reduction in the net negative surface charge, as indicated by the reduction in the electrophoretic mobility. Concomitantly, the activated cells acquire new properties, including the ability to form “stable” E rosettes, and attach to normal and malignant cells of the same species (natural attachment (NA)). These properties were found to be expressed by lymphocytes within the low electrophoretic fractions (cells with low negative charge) of the MLC populations. The formation of stable E rosettes and natural attachment capacities of human thymocytes were also found to correlate with the amount of surface negative charge. The slowly migrating (less negative charged) cortical thymocytes, reported earlier as being able to form stable E rosettes, were found also to exhibit NA activity. Medullary thymocytes carrying a high net negative surface charge lack these characteristics. We consider it likely that the reduction of negative charge during activation of peripheral T cells, facilitates cell-to-cell contacts, and thus prepares the (activated) cells to perform cooperative interactions with other cell types, and express the lytic activity of T cells.     

Isolation and identification of the major concanavalin A binding glycoproteins from murine-lymphocytes.

The major glycoproteins which bind concanavalin A have been isolated and identified from murine spleen cells, thymocytes,and purified thymus-derived (T) lymphocytes, and from the spleen cells of congenitally athymic (nude) mice. The cells were radiolabeled by lactoperoxidase catalyzed 125I iodination or by culturing the cells in media containing [3H]leucine or [3H]fucose. The cell membrane was solubilized with Nonidet P-40 and the concanavalin A binding proteins were isolated by affinity chromatography and analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major proteins from various lymphocyte preparations were identified by immunoprecipitation with specific antisera. The molecules coded by the histocompatibility-2 complex acted as concanavalin A binding proteins H-2K and H-2D were isolated from T lymphocytes, thymocytes, and bone marrow derived (B) lymphocytes. The Ia antigens were identified from B lymphocytes and tentatively identified from T lymphocytes. In addition to these H-2 complex proteins, immunoglobulin M and D on B lymphocytes also bound concanavalin A binding. All these glycoproteins have previously been identified as cell surface molecules. The presence of certain minor unidentified concanavalin A binding proteins on lymphoid cells is indicated.     

Changes in the cellular membrane surface coat of lymphocytes and thymocytes after incubation in vitro with cystein as revealed with electronmicroscopy.

Changes in the cellular membrane surface coat of lymphocytes and thymocytes after incubation with cystein in vitro were revealed with electronmicroscope, while performing the reaction with Ruthenium Red and Concanavaline A. Lymphocytes and thymocytes not incubated with cystein to which reaction with Ruthenium red and Cocanavaline A was applied have shown a well developed and preserved surface coat of the cellular membrane. Contrary to this finding when lymphocytes and thymocytes were incubated with cystein and thereafter treated with Ruthenium Red and Concanavaline A no reaction product on the surface of the cellular membrane was observed. The experimental results could indicate on the influence of cystein on the glycoside bonds.     

Glycoproteins and antigens of membranes prepared from rat thymocytes after lysis by shearing or with the detergent Tween-40.

In purification of cell surface antigens an efficient method for preparing membrane from large numbers of cells is needed. Such a method is described for preparing membranes from rat thymocytes after lysis in the non-ionic detergent Tween-40. Cell surface antigens were recovered at a yield of 30-50%, and a purification of 30-40-fold. By contrast enzyme markers for the other cell organelles were present in the membrane fraction in very low yield. The membrane obtained with the detergent method was compared with that resulting from the best of previously describes methods involving cell lysis by shearing. The detergent method compared favourably for simplicity as well as for yield and purification, and both membrane preparations contained similar protein and glycoprotein constituents. The main glycoprotein bands of membranes from thymocytes and thoracic duct lymphocytes were identified after polyacrylamide gel electrophoresis in dodecyl sulphate. In thymocyte membrane, three main bands at apparent molecular weights of 150 000, 84 000 and 25 000 were seen, and of these the 84 000 glycoprotein did not bind to the lentil lectin. In thoracic duct lymphocyte membrane the 25 000 glycoprotein was absent and a band at 95 000 was intensified in comparison with thymocytes.     

Glycoproteins and antigens of membranes prepared from rat thymocytes after lysis by shearing or with the detergent Tween-40

In purification of cell surface antigens an efficient method for preparing membrane from large numbers of cells is needed. Such a method is described for preparing membranes from rat thymocytes after lysis in the non-ionic detergent Tween-40. Cell surface antigens were recovered at a yield of 30–50%, and a purification of 30–40-fold. By contrast enzyme markers for the other cell organelles were present in the membrane fraction in very low yield.The membrane obtained with the detergent method was compared with that resulting from the best of previously described methods involving cell lysis by shearing. The detergent method compared favourably for simplicity as well as for yield and purification, and both membrane preparations contained similar protein and glycoprotein constituents.The main glycoprotein bands of membranes from thymocytes and thoracic duct lymphocytes were identified after polyacrylamide gel electrophoresis in dodecyl sulphate. In thymocyte membrane, three main bands at apparent molecular weights of 150 000, 84 000 and 25 000 were seen, and of these the 84 000 glycoprotein did not bind to the lentil lectin. In thoracic duct lymphocyte membrane the 25 000 glycoprotein was absent and a band at 95 000 was intensified in comparison with thymocytes.     

Human autologous rosette-forming cells. III. Binding of erythrocytes from different species to the T-cell receptors for autologous red blood cells

Spontaneous rosette formation in humans is restricted to a subpopulation of the circulating T cells. We have previously shown that the interaction between lymphocytes and autologous red blood cells (auto-RBC) is not mediated by a self-recognition mechanism, since allogeneic (allo-) RBC bind to T cells through the same receptors. In this work, we have extended these observations to thymocytes. Using a mixed-rosette assay in which one type of erythrocyte was identified by FITC labeling, we have shown that almost all the thymocytes which attached auto-RBC could also fix allo-RBC. However, as for the peripheral blood lymphocytes (PBL), binding of human RBC to thymocytes occurred with varying affinities according to the erythrocyte's origin. In order to further study the specificity of the erythrocyte to lymphocyte binding in rosette formation, PBL were mixed with auto-RBC and erythrocytes of xenogeneic (xeno-) origin. Although very disparate incidences of rosettes were found according to the species from which the RBC were derived, most of the autorosetting lymphocytes also had receptors for xeno-RBC. In addition, preincubation of PBL with monoclonal antibody OKT11A (directed against the sheep RBC receptors on T cells) completely abrogated rosette formation with all the erythrocytes tested (human auto- and allo-, sheep, pig, and rabbit) except mouse RBC. Taken together these data strongly suggest that human auto- or allo-, as well as sheep or some other xeno-RBC, bind to T lymphocytes by a single receptor and that the combining sites are expressed with different densities or varying affinities depending upon the RBC's origin. Therefore, spontaneous autorosettes may represent T lymphocytes having high-affinity receptors for sheep RBC.     

Characterization of a monoclonal antibody to guinea pig T cells that inhibits rosette formation of the cells with rabbit erythrocytes: similarity of the antigen to E-receptor on human T cells

A monoclonal antibody, GPT-1, was prepared by fusion of the splenic cells of mice immunized with guinea pig thymocytes with a mouse myeloma cell line. GPT-1 completely inhibited spontaneous rosette formation of T cells with papain-treated rabbit erythrocytes. GPT-1 reacted with 90% of thymocytes, 70% of peripheral blood lymphocytes, and 45% of splenic lymphocytes, but not with B cells. These results indicate that GPT-1 has pan-T reactivity. The antibody specifically bound to a single polypeptide chain with a molecular size of 50-65 kD. The surface density of the antigen was higher on thymocytes than on peripheral T cells, suggesting that the antigen is a certain differentiation antigen on T cells. Phytohemagglutinin-activated T cells expressed more antigen molecules than resting T cells. In addition, GPT-1 suppressed the proliferation of T cells induced by the mitogen, indicating that GPT-1 recognizes a T cell-specific surface antigen which is associated with T cell activation. Based on these results, it was concluded that GPT-1 reacts with a guinea pig T cell surface antigen which is similar to the E-receptor protein on human T cells (CD2 molecule).     

CD69 molecule in human neutrophils: its expression and role in signal-transducing mechanisms.

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes and it is constitutively expressed on thymocytes and platelets. Here we report its presence on neutrophils and on bone marrow-derived myeloid precursors. Indeed, promyelocytic cells are CD69+ on the cell membrane, while in resting neutrophils this molecule is located inside the cell. However, intracellular CD69 molecules are rapidly mobilized to the cell surface upon activation by PMA or fMLP. This translocation is independent on a new protein synthesis, as it is not inhibited by cycloheximide; furthermore, CD69 molecules are likely stored in a trans-Golgi structure since their expression is not affected by brefeldin A, a drug that blocks molecular trafficking from ER to Golgi vesicles. Immunoprecipitation of CD69 molecules either from activated neutrophils or from bone marrow cells showed that this protein has the same molecular size (28-34 kDa) as observed in platelets, T and B lymphocytes, and thymocytes. This similarity is reflected also in the functional role played by this molecule: in neutrophils as well as in lymphocytes and platelets, CD69 stimulation induced Ca2+ influx through cellular membrane; furthermore, the perturbation of the CD69 antigen on PMA-activated neutrophils enhances the lysozyme release, suggesting a role of this molecule in the regulation of granule exocytosis, probably through a Ca(2+)-dependent mechanism.     

Plasma membrane lipid organization and the adherence of differentiating lymphocytes to macrophages

Changes in the packing of phospholipids in the plasma membrane of lymphocytes occur during differentiation within primary and secondary lymphoid organs. As they differentiate, lymphocytes interact with a variety of reticuloendothelial cells, including macrophages. To investigate a possible relation between these two phenomena, the strength of the interactions between lymphocytes and macrophages was measured in vitro as a function of the tightness of packing of phospholipids on the lymphocyte surface. Strength of adherence was measured by the ability of lymphocytes to remain adherent to macrophages when subjected to increasing centrifugal forces. Phospholipid packing was assessed using the fluorescent lipophilic probe merocyanine 540 (MC540), which preferentially binds to bilayers in which the lipids are more loosely packed. Three subpopulations of murine thymocytes were resolved with respect to strength of adherence to peritoneal or thymic macrophages. To determine whether these subpopulations corresponded with the three classes of cells distinguishable by MC540 fluorescence, populations enriched for staining or non-staining cells, and cells sorted on the basis of MC540 fluorescence intensity, were examined. The least fluorescent cells were the least strongly adherent; the most fluorescent cells were the most strongly adherent; and cells of intermediate fluorescence had intermediate adherence. When splenic lymphocytes were examined with respect to adherence to peritoneal or splenic macrophages, similar patterns of fluorescence and adherence were seen. These results suggest that the organization of the plasma membrane lipid bilayer of lymphocytes may be involved in their interactions with macrophages during primary and secondary differentiation. The adherence signal for lymphocytes thus may be similar to that proposed for other blood cells.     

Cytofluorometric analysis of R-Thy-1. antigens in various rat lymphocytes with different electrophoretic mobility and organ distribution.

Small bone marrow lymphocytes, which had been previously enriched by velocity sedimentation, thymocytes, lymph node cells and spleen cells were electrophoretically separated, stained with fluorescein conjugated rabbit a-rat-Thy-1. globulin and their fluorescence intensities analyzed with a flow cytophotometer. Thy-1. antigens were found in 80% of the bone marrow small lymphocytes showing low electrophoretic mobility (EPM), in all thymocytes, about 80% of which show low and the rest medium to high EPM, and in a few lymph node cells of high EPM. Thy-1. positive cells were not observed in the spleen. All fluorescence intensity histograms obtained were modal and could be properly fitted with normal curves showing coefficients of variation (C.V.) in the range of 20% to 30%. It was observed that the thymocytes of low EPM had an antibody binding affinity significantly different from that of the other stained lymphocytes. Moreover the surface antigen density decreased in the sequence: thymocytes of low EPM, bone marrow lymphocytes of low EPM and thymocytes of high EPM. The fluorescence intensity of stained lymph node cells of high EPM appeared similar to that of thymocytes of high EPM but was not evaluated precisely. Thus the two dimensional cell analysis provided by a combination of EPM and surface fluorescence of Thy-1.+ cells, allows the characterization of different lymphocyte populations which cannot be clearly identified with normal one dimensional techniques. The biological significance of the results is discussed briefly.     

Macrophages and the functional differentiation of thymocytes (a review of the authors' own data)

Cellular, humoral, and genetic mechanisms of induction of T-effectors of the transplant vs. host reaction (THR) have been studied in two-cell culture of phagocyte mononuclears and thymocytes. A direct physical contact and similarity of H-2K locus of major histocompatibility complex between the cooperating cells in culture is required for successful induction of T-effectors of THR. Contact interaction of macrophages with thymocytes leads to accumulation of a 67 KDa humoral factor in the culture medium. Incubation of intact thymocytes with this factor leads to functional transformation of immature thymocytes into corresponding effector cells. Similarity of H-2K locus of the factor producers and intact lymphocytes is also required for successful humoral induction of the T-effectors. The surface H-2K antigen is able to induce formation of THR t-effectors from non-reactive thymocytes. The H2-K-specific mediator, affinity-isolated from the supernatant of the macrophage-thymocyte culture can also cause this induction.     

Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.

During normal tissue remodeling, macrophages remove unwanted cells, including those that have undergone programmed cell death, or apoptosis. This widespread process extends to the deletion of thymocytes (negative selection), in which cells expressing inappropriate Ag receptors undergo apoptosis, and are phagocytosed by thymic macrophages. Although phagocytosis of effete leukocytes by macrophages has been known since the time of Metchnikoff, only recently has it been recognized that apoptosis leads to surface changes that allow recognition and removal of these cells before they are lysed. Our data suggest that macrophages specifically recognize phosphatidylserine that is exposed on the surface of lymphocytes during the development of apoptosis. Macrophage phagocytosis of apoptotic lymphocytes was inhibited, in a dose-dependent manner, by liposomes containing phosphatidyl-L-serine, but not by liposomes containing other anionic phospholipids, including phosphatidyl-D-serine. Phagocytosis of apoptotic lymphocytes was also inhibited by the L isoforms of compounds structurally related to phosphatidylserine, including glycerophosphorylserine and phosphoserine. The membranes of apoptotic lymphocytes bound increased amounts of merocyanine 540 dye relative to those of normal cells, indicating that their membrane lipids were more loosely packed, consistent with a loss of membrane phospholipid asymmetry. Apoptotic lymphocytes were shown to express phosphatidylserine (PS) externally, because PS on their surfaces was accessible to derivatization by fluorescamine, and because apoptotic cells expressed procoagulant activity. These observations suggest that apoptotic lymphocytes lose membrane phospholipid asymmetry and expose phosphatidylserine on the outer leaflet of the plasma membrane. Macrophages then phagocytose apoptotic lymphocytes after specific recognition of the exposed PS.     

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.     

Serological detection of H-2K- and H-2D-gene products

Using the fluorescence-activated cell sorter (FACS II), we have analyzed the expression of H-2K- and H-2D-gene products on the membrane of various cellular components of the murine immune system. Using this serological technique we show a basic difference between T and B lymphocytes. Whereas all cellular components analyzed — hydrocortisone-resistant thymocytes, splenic T and B lymphocytes, macrophages and bone-marrow cells — expressed H-2K-subregion-encoded alloantigens at a high density, it seems that the high density expression of H-2D-encoded alloantigens is restricted mainly to B cells and to macrophages. Hydrocortisone-resistant thymocytes, splenic T lymphocytes and bone-marrow cells, on the other hand, showed significant expression of the H-2D alloantigens only at low membrane density. These results, then, provide evidence for the existence of an imbalance in serologically detectable expression of H-2K- and H-2D-region-gene products on the cell membrane of various cells comprising the murine immune system.Abbreviations usedin this paper DTH delayed type hypersensitivity - FCS fetal calf serum - FITC fluorescein isothiocyanate - HrT hydrocortisone-resistant thymocytes - Ig immunoglobulins P. De Baetselier is an EMBO and Euratom postdoctoral fellow     

A surface membrane determinant shared by subpopulations of thymocytes and B lymphocytes.

Utilizing a quantitative fluorescence assay with the fluorescence-activated cell sorter (FACS), we have demonstrated that a rabbit antiserum obtained by immunization with cells of a mouse IgM-producing plasma cell tumor (MOPC104E) is reactive with at least two surface determinants, designated Th-B and ML2, on subpopulations of normal murine lymphocytes. The ML2 determinant is restricted to B lymphocytes. The Th-B determinant is shared by splenic B lymphocytes and a large subpopulation of thymocytes, the latter of which express a 3-fold higher density of Th-B on their surface than do the B lymphocytes. Neither Th-B nor ML2 were found on peripheral T cells or on brain, liver, or kidney cells. The available evidence suggesting that Th-B may be a stem cell determinant that is lost upon maturation is discussed.     

An essential role for membrane rafts in the initiation of Fas/CD95-triggered cell death in mouse thymocytes

Fas, a member of the tumor necrosis factor receptor family, can upon ligation by its ligand or agonistic antibodies trigger signaling cascades leading to cell death in lymphocytes and other cell types. Such signaling cascades are initiated through the formation of a membrane death-inducing signaling complex (DISC) that includes Fas, the Fas-associated death domain protein (FADD) and caspase-8. We report here that a considerable fraction of Fas is constitutively partitioned into sphingolipid- and cholesterol-rich membrane rafts in mouse thymocytes as well as the L12.10-Fas T cells, and Fas ligation promotes a rapid and specific recruitment of FADD and caspase-8 to the rafts. Raft disruption by cholesterol depletion abolishes Fas-triggered recruitment of FADD and caspase-8 to the membrane, DISC formation and cell death. Taken together, our results provide the first demonstration for an essential role of membrane rafts in the initiation of Fas-mediated cell death signaling.