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© The Rockefeller University Press,
0021-9525/2000//1131 $5.00
The Journal of Cell Biology, Volume 149, Number 5,
, 2000 1131-1142
Original Article |
Observing Fc
ri Signaling from the Inside of the Mast Cell Membrane
bwilson{at}thor.unm.edu
We have determined the membrane topography of the high-affinity IgE receptor, Fc
RI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017–1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcRI and Lyn are distributed as small clusters (2–9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcRI clusters contain Lyn. In contrast, there is essentially no FcRI-Syk colocalization in resting cells. 2 min after FcRI cross-linking, 
10% of Lyn colocalizes with small and medium-sized FcRI clusters (up to 20 gold particles), whereas 16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20–100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcRI signaling.
Key Words: microdomains • Lyn • Syk • rafts
© 2000 The Rockefeller University Press
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Introduction |
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 TopAbstract Introduction Materials and MethodsResultsDiscussionReferences |
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The high-affinity IgE receptor, FcRI, is a tetrameric complex consisting of an IgE-binding 
subunit, a tetraspan β subunit and two disulfide-linked 
subunits (Blank et al. 1989). It is a member of a larger family of multi-chain immune recognition receptors (MIRRs), which also includes the T cell receptor (TCR), the B cell receptor (BCR), and two IgG receptors, FcRI and FcRIII (Cambier 1995). These receptors have no intrinsic catalytic activity. Instead, signaling is initiated when Src-related kinases phosphorylate ITAMs (immunoreceptor tyrosine-based activation motifs) within the cytoplasmic tails of receptor subunits, creating phospho-ITAM–binding sites for the binding of Syk-related kinases. These interactions result in the activation and phosphorylation of Syk, that in turn recruit and phosphorylate SH2 domain–containing downstream signaling molecules (reviewed in Tamir and Cambier 1998). Previous biochemical studies identified Lyn and Syk as the principal FcR1-associated kinases of RBL-2H3 mast cells, showed that Lyn's primary substrates are the FcR1β and ITAMs, and linked Syk activation to FcR1 signal propagation (Eiseman and Bolen 1992; Hutchcroft et al. 1992; Oliver et al. 1994; Yamashita et al. 1994; Rivera and Brugge 1995; Zhang et al. 1996; Kinet 1999).
It has been suggested that FcRI signaling may occur in microdomains. Field et al. 1995, Field et al. 1997, Field et al. 1999 reported that the myristoylated src family member, Lyn, associates in RBL-2H3 cells with large detergent-resistant membrane domains characterized by the presence of the GPI-linked protein, Thy-1, glycosphingolipids, gangliosides, and cholesterol. Cross-linked, but not monomeric, FcRI were also detected in these membranes, leading to the hypothesis that aggregated FcRI might move into Lyn-containing ordered lipid microdomains and thereby initiate FcRI signaling. However, contrasting biochemical evidence suggests that the association of FcRI with Lyn relies primarily upon defined protein–protein interactions. In particular, Vonakis et al. 1997 demonstrated that a portion of Lyn binds to monomeric FcRI via interactions of its unique NH2-terminal SH4 domain with non-ITAM regions in the FcRI β subunit COOH terminus. Furthermore, fluorescence microscopic experiments by Stauffer and Meyer 1997 suggested that aggregated FcRI may colocalize with Syk rather than Lyn in ordered lipid microdomains. These investigators showed that FcRI redistribute after cross-linking to ganglioside GM1-enriched membrane domains that also recruit Syk and PLC- by SH2 domain interactions. Peptide inhibitors of Lyn that blocked FcRI phosphorylation also blocked Syk recruitment to the membrane (Stauffer et al. 1997).
In this report, signaling molecules were localized on native membrane sheets from the dorsal surface of RBL-2H3 mast cells by immunogold labeling and high-resolution TEM analysis. Our results show that a portion of monomeric FcRI and Lyn are colocalized in small clusters in unstimulated cells and that Lyn is dramatically excluded from the large receptor clusters that form after FcRI cross-linking. Moreover, cross-linked FcRI encounter Syk and coated pits in topographically distinct membrane domains.
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Materials and Methods |
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RI β mAb was a generous gift from Dr. Juan Rivera (NIH, Bethesda, MD). Affinity-purified mouse anti-DNP IgE and rabbit anti–mouse IgE Abs were prepared as described (Liu et al. 1980; Seagrave et al. 1991). Colloidal gold particles (3–10 nm in diameter) conjugated with anti–rabbit IgG, anti–mouse IgG, and streptavidin were from Nanoprobes and Amersham Pharmacia Biotech. Biotin-phalloidin and DNP-BSA were from Molecular Probes.
Cell Activation and Membrane Labeling
RBL-2H3 cells were allowed to settle overnight onto 15-mm round, clean glass coverslips in the presence of anti-DNP IgE (1 µg/ml) to prime cell surface FcRI. After washing to remove excess IgE, FcRI was cross-linked by incubation with DNP-BSA (0.1–1 µg/ml), with rabbit polyclonal anti-IgE (1 µg/ml) or with gold conjugates of these reagents (prepared as in Seagrave et al. 1991). Plasma membrane sheets were prepared by a modification of a procedure described by Sanan and Anderson 1991. The coverslips were rapidly chilled by immersion in ice-cold Hepes buffer (25 mM Hepes, pH 7, 25 mM KCl, and 2.5 mM MgAcetate) and inverted onto nickel EM grids that had been coated with formvar and carbon and, on the day of the experiment, glow-discharged and floated on poly-L-lysine (0.8 mg/ml for 30 min, followed by 10 s dH2O rinse and air drying). Pressure was applied to the coverslip for 20 s by bearing down with a cork. The coverslips were lifted, leaving sections of the upper cell surface adherent to the poly-L-lysine–coated grid. Membranes were rinsed in 4°C Hepes buffer and fixed in 2% paraformaldehyde for 10 min. For experiments using anti–IgE-gold to label FcRI in the resting state, cells were fixed with 0.5% paraformaldehyde for 5 min and then incubated with anti–IgE-gold before inversion onto EM grids. All membranes were labeled from the inside by inverting the grids onto droplets containing primary antibodies or biotin-phalloidin (5 units/ml) followed by gold-conjugated secondary reagents. Incubations were for 30 min. Intermediate washes in PBS were performed by inverting the grids onto droplets. Primary antibodies were diluted in PBS, 0.1% BSA at the following concentrations: Syk, 10 µg/ml; FcRI β, 28 µg/ml; Lyn, 2 µg/ml. Gold-conjugated secondary reagents were diluted 1:20 from commercial stocks in PBS-BSA. The samples were post-fixed in 2% glutaraldehyde in PBS and held overnight in PBS. Next, samples were stained for 10 min with 1% OsO4 prepared in 0.1 M cacodylate buffer and washed 5 min with cacodylate buffer and twice for 5 min in dH2O. Samples were then processed for 10 min in 1% aqueous tannic acid, followed by two 5-min rinses with dH2O, 10 min with 1% aqueous uranyl acetate and two 1-min rinses with dH2O. Grids were air-dried and examined using an Hitachi 600 transmission electron microscope.
Quantifying Gold Particle Distributions
Micrographs from up to four separate experiments were sorted into six groups of at least 25 according to the following treatment and labeling conditions: labeled only with 5- or 10-nm gold particles marking FcRI β; labeled with 5-nm gold particles marking Lyn plus 10-nm particles marking FcRI β; labeled with 5-nm gold particles marking Syk plus 10-nm particles marking FcRI β; resting and activated. A minimum of 3,000 gold particles per set of micrographs were counted for (a) numbers of gold particles marking FcRI β distributed as singlets or as clusters (with cluster size), (b) numbers of gold particles marking Lyn distributed as singlets or as clusters (with cluster size), and (c) numbers of gold particles marking Lyn that were or were not colocalized with FcRI β clusters (distinguishing, in activated cells, between gold particles marking Lyn that occurred within FcRI clusters versus gold particles marking Lyn that instead surrounded these clusters). Our procedure simply involved highlighting singlets and clusters and recording their composition until no unmarked particle or group remained on the micrograph. Numbers of gold particles marking Syk were counted over 55 µm2 of membrane and scored for colocalization or not with FcRI β.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI on Resting and Activated Mast CellsRI β in resting cells is illustrated in Fig. 1 A and quantified in Fig. 2 A. The micrograph shows that gold particles marking FcRI β are distributed as a mixture of singlets and small dispersed clusters that rarely exceed a diameter of 100 nm (the length of the scale bar in Fig. 1 A). No gold particles associate with clathrin-coated vesicles that are major features of the cytoplasmic face of the plasma membrane. Particle counting (Fig. 2 A) established that 33% of gold particles marking FcRI β are distributed as singlets and that the rest are in clusters that most commonly contain 2–3 gold particles and only rarely exceed 9 gold particles.
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RI β is found in clusters that are moderately or substantially larger than the clusters on resting cells (Fig. 1 B). These clusters are particularly associated in activated cells with distinct membrane regions that stain darkly with the mixture of osmium, tannic acid, and uranyl acetate used to provide contrast to these samples. Clathrin-coated pits typically occur at the periphery of these osmiophilic membrane regions. Further particle counting, also reported in Fig. 2 A, showed that 25% of 5-nm gold particles marking FcRI β in activated cells are in clusters of 10–20 (intermediate clusters) and that 40% are in clusters of greater than 20 (and sometimes more than 100; large clusters). The internalization of cross-linked receptors by coated pit-mediated endocytosis was confirmed by activating cells with DNP-BSA 10-nm colloidal gold and localizing the gold particles within clathrin-caged vesicles (Fig. 1 C, arrow).
To establish the reliability of the membrane sheet technique for demonstrating topographical relationships between membrane-associated proteins, we labeled FcRI from the extracellular as well as the cytoplasmic faces of the membrane and looked for colocalization of gold particles. In Fig. 1 D, IgE-primed cells were fixed lightly, then labeled from the outside with 10-nm rabbit anti–IgE-gold particles and subsequently, from the inside, with 5-nm anti-FcRI β particles. 5-nm gold particles marking FcRI β are again distributed in the singlets and small dispersed clusters that characterize unstimulated cells. The small clusters now also contain 10-nm gold particles marking FcRI . In Fig. 1 E, cells were activated for 2 min at 37°C with 10-nm rabbit anti–IgE-gold particles before labeling from the inside with 5-nm anti-FcRI β particles. This time both sizes of gold colocalize in the larger receptor clusters surrounding coated pits (arrows) that characterize activated cells. We interpret the very regular distance between the 5-nm particles marking FcRI β and the 10-nm gold particles marking FcRI as strong evidence that the same receptor is being labeled, although on different subunits and from different sides of the membrane.
Previous studies using the lower resolution techniques of fluorescence and scanning electron microscopy had not revealed FcRI clusters in resting cells. Therefore, we performed further control experiments to ensure that the small clusters were not induced in the fixed samples by the labeling Abs. In one control experiment, membrane sheets were fixed with 2% paraformaldehyde for either the standard 10 min or for only 5 min, then labeled for 30 min with monoclonal anti–FcRI β and polyclonal anti-Lyn followed by gold secondary antibodies for 30 min. In another, membrane sheets were fixed for 10 minutes with 0.5, 2, and 4% paraformaldehyde and labeled as above. As an additional control, sheets were fixed for 10 min with 2% paraformaldehyde and labeled with anti–FcRI β, anti-Lyn, and gold secondary antibodies and held for up to 12 h before fixation with glutaraldehyde. Gold particle labeling densities and distributions were not altered by reducing the fixation time or fixative concentration. They also were not altered by increasing the holding time before the glutaraldehyde post-fixation step, that could conceivably promote reagent-induced clusters to form in lightly fixed samples.
The Topography of Lyn and Its Association with FcRI
Fig. 3 demonstrates the relative distributions of FcRI and Lyn, the kinase that phosphorylates the β and subunits of the cross-linked FcRI (Oliver et al. 1994; Yamashita et al. 1994), in resting cells. In these images, either 3- (Fig. 3 A) or 10-nm (Fig. 3 B) gold particles mark the location of FcRI β, while Lyn is labeled with 5-nm gold particles. Two inferences can be drawn from these micrographs. First, gold particles marking Lyn are frequently in clusters. Second, Lyn clusters frequently, but not always, colocalize with FcRI β in resting mast cells. Fig. 3 C is included to show there is essentially no background binding of either 5- or 10-nm anti–mouse IgG gold particles to membrane sheets; backgrounds are similarly low using all of the colloidal gold reagents in this study (not shown).
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20% of total Lyn gold particles were in close proximity to gold particle(s) marking FcRI β. If counted from the complementary perspective of total FcRI gold particles, the percentage of these in close proximity to Lyn was 25%. These quantitative data confirm the visual observation that a significant percentage of Lyn and FcRI are colocalized in resting cells.
The redistribution of Lyn that follows FcRI cross-linking with multivalent antigen or anti-IgE is demonstrated by the series of micrographs in Fig. 4. Fig. 4 A is a relatively low-power field to show multiple associations of Lyn, marked by 5-nm gold, and FcRI β, marked by 3-nm gold, in membrane sheets. Lyn has three principal distributions in this micrograph. First, there are multiple Lyn particles and clusters that do not associate with receptor. Second, a portion of Lyn and FcRI β are colocalized within receptor clusters of small to intermediate size (circles). Finally Lyn surrounds, but is very rarely intercalated within, the large clusters of cross-linked FcRI β that form on osmiophilic membrane regions in activated cells (arrows). Fig. 4B–D, provide additional examples of the receptor-rich osmiophilic membrane regions (arrows) of activated cells to demonstrate both the absence of intercalated Lyn and the presence of Lyn in strings and clusters surrounding the receptor clusters (rectangular boxes).
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RI cross-linking so that >50% of clusters now contain >6 gold particles (Fig. 2 B). This observation implies that the population of Lyn as a whole has altered properties in activated cells. Second, 74% of gold particles marking Lyn were in clusters that showed no apparent association with receptor. Importantly, 10% of total Lyn was distributed within small or medium clusters of FcRI β gold particles, whereas 16% of total Lyn was localized at the periphery of the large clusters of FcRI β on osmiophilic membrane.
The segregation of Lyn from highly clustered receptors was further demonstrated by comparing the topography of FcRI and Lyn with the topography of clathrin and actin. As already noted, cross-linked FcRI can be internalized through clathrin-coated pits that often bud from the edges of these clusters. These pits consistently label with gold particles marking the clathrin adaptin, AP-2, but they almost never label with gold particles marking Lyn (data not shown). Conversely, strings of Lyn are seen on fibrous structures that also label with phalloidin, a marker for F-actin (Fig. 4 F, boxed). Phalloidin-gold particles do not associate with FcRI β clusters (not shown).
Based principally on sucrose density fractionation of detergent-solubilized membranes, previous investigators have proposed that Lyn associates in resting cells with cholesterol and ganglioside GM-1–enriched microdomains that also accumulate GPI-linked proteins including Thy-1 (Surviladze et al. 1998). However, the membrane distribution of Thy-1, labeled from the outside of lightly fixed resting cells, is rarely coincident with either Lyn (Fig. 5 A) or FcRI β (Fig. 5 B) labeled from the inside of the same cells.
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RIRI to downstream responses (Hutchcroft et al. 1992; Oliver et al. 1994; Zhang et al. 1996). Lyn-mediated tyrosine phosphorylation of paired ITAMs within the cytoplasmic tail of the cross-linked FcRI subunit creates sites for the binding and activation of Syk (Shiue et al. 1995).
The distribution of Syk was determined in membrane sheets prepared from resting (Fig. 6) and antigen-stimulated (Fig. 7) RBL-2H3 cells. In resting cells, gold particles marking Syk occurred in singlets and small clusters with essentially no interaction with FcRI (Fig. 6, circles). In contrast, the majority of Syk label is found intercalated into both small and large receptor clusters in activated cells (Fig. 7, arrows). A few clusters of isolated Syk persist in the membrane sheets of activated cells (Fig. 7, circles).
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RI cross-linking induced only a modest increase in total Syk labeling at the membrane, the topography of Syk was dramatically altered. Specifically, in membranes from resting cells, <10% of Syk label is found in close proximity to gold particles marking FcRI β. In contrast, >70% of gold particles marking Syk are found in receptor clusters after activation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI signaling in which receptor cross-linking leads to the Lyn-mediated tyrosine phosphorylation of ITAMs within the cytoplasmic tails of the β and subunits of the FcRI. FcRI phosphorylation in turn provides binding sites for the binding and activation of Syk. Our goal was to determine the membrane topography of the high-affinity IgE receptor, FcRI, and of its associated tyrosine kinases, Lyn and Syk, during this biochemical cascade. We used a modification of the method of Sanan and Anderson 1991 to generate membrane sheets from the dorsal surface of RBL-2H3 mast cells. Signaling molecules were localized on these native membranes by immunogold labeling and high-resolution TEM analysis.
We found that the majority of gold particles marking FcRI (>65%) and Lyn (>85%) in resting cells occur as clusters and that a significant proportion (20–25%) of these are mixed clusters, containing both FcRI and Lyn. Neither FcRI nor Lyn associated with clathrin-coated membrane in resting cells. The colocalization of Lyn and monomeric receptor is consistent with previous evidence from the Metzger laboratory (Vonakis et al. 1997; Metzger 1999) showing that a portion of Lyn associates with the resting FcRI via interactions of its SH4 domain with determinants on the FcRI β subunit. In contrast, cell fractionation studies by Field et al. 1995, Field et al. 1997, Field et al. 1999 identified Lyn in detergent-insoluble, glycosphingolipid-enriched membrane microdomains that were also enriched in cholesterol, sphingomyelin, and glycosylphosphatidylinositol (GPI)-anchored proteins, including Thy-1, but specifically did not include monomeric receptor. To explain this discrepancy, we suggest that the interaction of monomeric FcRI with myristoylated Lyn may be disrupted when cells are treated with Triton X-100 and subjected to sucrose gradient centrifugation. In short, our results support the concept that Lyn occurs in mast cells in microdomains but show clearly that these microdomains do not exclude unactivated FcRI.
While the unique composition of DRMs has provided strong evidence for the concept of membrane microdomains, previous investigators have also described discrepancies between the protein compositions of low-density sucrose fractions and the protein composition of microdomains in the native plasma membrane. In particular, Chang et al. 1994 demonstrated that isolated caveolae are enriched in a complex profile of proteins and that Triton X-100 selectively extracts a subset of these proteins, including acylated G proteins. Adding to the complexity, the recovery of membrane proteins in the light fractions of density gradients is highly dependent on the choice of detergent (Surviladze et al. 1998) and on the temperature during preparative steps (Harder et al. 1998). This variability emphasizes the need to complement cell fractionation studies with direct analyses of microdomains in native membranes, made possible here by high-resolution electron microscopy.
Previous studies by fluorescence, transmission, and scanning electron microscopy (Menon et al. 1984; Pfeiffer et al. 1985; Stump et al. 1988; Seagrave et al. 1991; Mao et al. 1993; Santini and Keen 1996; Stauffer and Meyer 1997) have all failed to reveal the inherently clustered distribution of FcRI that we now readily demonstrate using 3- and 5-nm gold particles to mark receptors. We speculate that these small clusters are below the resolution of the light microscope (250 nm; Jacobson and Dietrich 1999) and depend on the substantially higher resolution achieved here by TEM, which readily resolves gold particles as small as 3 nm. Interestingly, our own previous studies of FcRI topography by immunogold labeling with 15-nm gold probes and SEM analysis initially suggested an inherently random distribution of the FcRI (Seagrave et al. 1991). However, subsequent quantitative analysis of gold particle distributions on digitized negatives from these SEM experiments showed a nonrandom distribution of the 15-nm particles on resting cells (Anderson 2000). Thus, the more obviously nonrandom distributions of monomeric FcRI observed here on membrane sheets is probably the result of the higher resolution achieved with smaller (3–10 nm) gold particles and is not an artifact of the method of observation.
FcRI cross-linking causes the formation of large receptor clusters seen previously by fluorescence, transmission, and scanning electron microscopy (Menon et al. 1984; Pfeiffer et al. 1985; Stump et al. 1988; Seagrave et al. 1991; Mao et al. 1993; Santini and Keen 1996; Stauffer and Meyer 1997). Remarkably, Lyn, the kinase that phosphorylates ITAMs within the β and subunits of the cross-linked FcRI (Yamashita et al. 1994) colocalizes with FcRI in clusters of small and intermediate size but is dramatically segregated to the periphery of the large clusters. This segregation may be regulated in part by interactions of Lyn with components of the actin cytoskeleton in activated cells. Supporting this suggestion, early biochemical and flow cytometric studies showed that increased F-actin assembly is an immediate response of RBL-2H3 mast cells to FcRI cross-linking (Pfeiffer et al. 1985), whereas the current work shows that Lyn associates with fibrous structures that also label with phalloidin, a marker for F-actin. One important unanswered question is where Lyn-mediated FcRI tyrosine phosphorylation actually occurs. We suspect the clusters of intermediate size of serving this function.
Syk, the cytoplasmic kinase that couples the cross-linked FcRI to downstream responses (Hutchcroft et al. 1992; Oliver et al. 1994; Rivera and Brugge 1995; Zhang et al. 1996), is not colocalized with the receptor in resting cells. Nevertheless some Syk is resident at the membrane of resting cells. Unlike Lyn, Syk has no membrane localization domain. However, it may well interact in resting mast cells with other receptor tyrosine kinases, for example c-Kit, or with binding proteins like tubulin and Cbl that may in turn interact with membranes (Ota and Samelson 1997; Fernandez et al. 1999).
Syk is dramatically recruited into the large receptor clusters that form on osmiophilic membrane patches within minutes after cross-linking. This recruitment is likely to occur both by the translocation of Syk from the cytosol to the membrane and by the recruitment of Syk from other sites of membrane association. We propose the Syk-enriched, Lyn-negative FcRI clusters as the probable sites of downstream signaling. The identities of other components within these putative signaling domains are not yet known. However, their distinct appearance as darkened membrane patches suggests that they are not merely the products of the aggregation of smaller clusters but may instead contain a unique subset of membrane lipids and proteins that stain differently from bulk membrane with the combination of osmium, tannic acid, and uranyl acetate used here to provide contrast to the membrane sheets.
Because the Syk-enriched clusters are sufficiently large to be observed readily by fluorescence microscopy, they are very likely to correspond to the microdomains seen previously by Stauffer and Meyer 1997 that labeled with fluorescent cholera toxin B (a ligand for the GM1 ganglioside) and accumulated cross-linked FcRI and GFP-conjugated SH2 domains of both Syk and PLC-1. Although the clusters seen by TEM exclude Lyn, they are typically surrounded by Lyn in an F-actin-associated (presumably detergent-insoluble) form. Thus the detergent-insoluble, FcRI- and Lyn-containing membranes isolated by Field et al. 1995, Field et al. 1997, Field et al. 1999 from activated mast cells could possibly represent cross-linked receptors surrounded by, rather than intercalated with, Lyn.
We observed that clathrin-coated vesicles internalize cross-linked receptors from membrane adjacent to the Syk-FcRI complexes. Previous thin section TEM studies established that cross-linked FcRI are internalized through coated pits that often have long necks and can support several clathrin-coated buds (Pfeiffer et al. 1985). These structures are major features of the membrane sheets described here. In membrane sheets from resting cells, their distribution seems unrelated to particular topographical features of the membrane. In membrane sheets from activated cells, they appear particularly at the edges of the darkened patches proposed to represent signaling domains. Gold particles marking Syk were seen at the necks of coated pits but not in clathrin cages, suggesting that Syk dissociates before cross-linked FcRI are internalized. Lyn appears to be actively excluded from membrane adjacent to coated pits in both resting and activated cells.
Previous thin section TEM studies in mast cells have not so far demonstrated clear increases in coated pit density induced by FcRI cross-linking (Mao et al. 1993; Santini and Keen 1996). However, we previously localized the majority of coated pits to smooth membrane near the bases of microvilli or lamellae in both resting and activated mast cells (Pfeiffer et al. 1985; Mao et al. 1993). Additionally, we established by statistical analyses of thin section TEM micrographs that coated pits are distributed in dispersed clusters, and not randomly, on a range of immune cells and that events that polarize cell shape, for example capping or chemotaxis, further polarize coated pits (Pfeiffer et al. 1980; Oliver and Berlin 1983). Recently, Gaidarov et al. 1999 presented further evidence that coated pits may assemble as clusters in specific locations on membranes. These indications of the nonrandom distribution of coated pits raises the possibility that their assembly at the periphery of signaling domains is regulated.
There is substantial evidence that efficient TCR signaling occurs by the segregation of the TCR and coreceptors like CD4 and CD28 to detergent-resistant membrane domains that accumulate GPI-linked proteins, Src kinases, Zap70, scaffolding proteins like LAT and molecules implicated in signal propagation such as PLC- (Montixi et al. 1998; Xavier et al. 1998; Zhang et al. 1998; Ilangumaran et al. 1999). Once signaling is initiated there is evidence that the signaling domains can additionally recruit cytoskeletal and adhesive proteins that may stabilize an "immunological synapse" between T cells and APCs (Monks et al. 1998; Grakoui et al. 1999; Penninger and Crabtree 1999). Because TCR and FcRI signaling are similar, it is possible that closer microscopic analysis in T cells will show that the TCR actually encounters Src and Syk kinases in distinct domains. Indeed, Monks et al. 1998 used fluorescence techniques to identify discrete organization of the TCR and accessory molecules at the sites of T cell and APC contact. They placed the TCR in the center of an activation cluster, which they termed the central supramolecular activation cluster (c-SMAC), surrounded by proteins such as LFA-1 and talin in a more peripheral ring (p-SMAC). Further studies in both systems will be required to localize at high resolution the other constituents involved in signal propagation via the TCR and FcRI, including important scaffolding proteins, such as LAT and SLP76.
In summary, we have discovered that FcRI encounters Lyn, Syk, and coated pits in topographically distinct membrane domains. The sequence of events revealed to date is shown schematically in Fig. 8. Studies are in progress to determine where FcRI β and subunit phosphorylation occurs, to identify other components of the small and large receptor clusters, to determine how FcRI cross-linking induces the segregation of Lyn from FcRI and to explore mechanisms regulating the topography of coated pit formation.
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Acknowledgments |
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This study was supported in part by the National Institutes of Health grants RO1 GM49814 and PO1 HL56384.
Submitted: 11 January 2000
Revised: 17 April 2000
Accepted: 18 April 2000
Abbreviations used in this paper: BCR, B cell receptor; DIGs, detergent-insoluble glycolipid-enriched domains; DIMs, detergent-insoluble membranes; DRMs, detergent-resistant membranes; GEMs, glycolipid-enriched membranes; GPI, glycosyl-phosphatidylinositol; ITAMs, immunoreceptor tyrosine-based activation motifs; MIRRs, multi-chain immune recognition receptors; TCR, T cell receptor; TEM, transmission electron microscopy.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Anderson R.G.W.. The caveolae membrane system, Annu. Rev. Biochem., 67, 1998, 199–225.[Medline]
Anderson A., Ph.D. thesis, 2000, University of New Mexico, Albuquerque, NM..
Blank U., Ra C., Miller L., White K., Metzger H. & Kinet J.-P.. Complete structure and expression in transfected cells of high-affinity IgE receptor, Nature, 337, 1989, 187–189.[Medline]
Brown D.A. & London E.. Functions of lipid rafts in biological membranes, Annu. Rev. Cell Dev. Biol., 14, 1998, 111–136.[Medline]
Cambier J.C.. Antigen and Fc receptor signalling, J. Immunol., 15, 1995, 3281–3285.
Chang W.J., Ying Y.-S., Rothberg K.G., Hooper N.M., Turner A.J., Bambliel H.A., De Gunzburg J., Mumby S.M., Gilman A.G. & Anderson R.G.W.. Purification and characterization of smooth muscle cell caveolae, J. Cell Biol., 126, 1994, 127–138.
Edidin M.. Lipid microdomains in cell-surface membranes, Curr. Opin. Struct. Biol., 7, 1997, 528–532.[Medline]
Eiseman E. & Bolen J.B.. Engagement of the high affinity IgE receptor activates src protein-related tyrosine kinases, Nature., 355, 1992, 78–80.[Medline]
Fernandez J.A., Keshvara L.M., Peters J.D., Furlong M.T., Harrison M.L. & Geahlen R.L.. Phosphorylation- and activation-independent association of the tyrosine kinase Syk and the tyrosine kinase substrates Cbl and Vav with tubulin in B-cells, J. Biol. Chem., 274, 1999, 1401–1406.
Field K.A., Holowka D. & Baird B.. FcRI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling, Proc. Natl. Acad. Sci. USA., 92, 1995, 9201–9205.
Field K.A., Holowka D. & Baird B.. Compartmentalized activation of the high affinity immunoglobulin E receptor within membrane domains, J. Biol. Chem, 272, 1997, 4276–4280.
Field K.A., Holowka D. & Baird B.. Structural aspects of the association of FcRI with detergent-resistant membranes, J. Biol. Chem., 274, 1999, 1753–1758.
Friedrichson T. & Kurzchalia T.V.. Microdomains of GPI-anchored proteins in living cells revealed by cross-linking, Nature., 394, 1998, 802–805.[Medline]
Gaidarov I., Santini F., Warren R.A. & Keen J.H.. Spatial control of coated-pit dynamics in living cells, Nat. Cell Biol., 1, 1999, 1–7.[Medline]
Grakoui A., Bromley S.K., Sumen C., Davis M.M., Shaw A.S., Allen P.M. & Dustin M.L.. The immunological synapsea molecular machine controlling T cell activation, Science., 285, 1999, 221–227.
Harder T., Scheiffele P., Verkade P. & Simons K.. Lipid domain structure of the plasma membrane revealed by patching of membrane components, J. Cell Biol., 4, 1998, 929–942.[Medline]
Hutchcroft J.E., Geahlen R.L., Deanin G.G. & Oliver J.M.. FcRI-mediated tyrosine phosphorylation and activation of the 72 kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells, Proc. Natl. Acad. Sci. USA., 89, 1992, 9107–9111.
Ilangumaran S., Arni S., van Echten-Deckert G., Borisch B. & Hoessli D.C.. Microdomain-dependent regulation of Lck and Fyn protein-tyrosine kinases in T lymphocyte plasma membranes, Mol. Biol. Cell., 10, 1999, 891–905.
Jacobson K. & Dietrich C.. Looking at lipid rafts?, Trends Cell Biol., 9, 1999, 87–91.[Medline]
Kenworthy A.K. & Edidin M.. Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of angstrom using imaging fluorescence resonance energy-transfer, J. Cell Biol., 142, 1998, 69–84.
Kinet J.P.. The high-affinity IgE receptor (FcRI)from physiology to pathology, Annu. Rev. Immunol, 17, 1999, 931–972.[Medline]
Liu F.T., Bohn J.W., Ferry E.L., Yamamoto H., Molinaro C.A., Sherman L.A., Klinman N.R. & Katz D.H.. Monoclonal dintrophenyl-specific murine IgE antibodypreparation, isolation and characterization, J. Immunol, 24, 1980, 2728–2737.
Mao S.-Y., Pfeiffer J.R., Oliver J.M. & Metzger H.. Effect of subunit mutation on the coated pit localization and internalization of cross-linked IgE-receptor complexes, J. Immunol, 151, 1993, 2760–2764.[Abstract]
Menon A.K., Holowka G. & Baird B.. Small oligomers of immunoglobulin E (IgE) cause large-scale clustering of IgE receptors on the surface of rat basohilic leukemia cells, J. Cell Biol., 98, 1984, 577–583.
Metzger H.. It's spring, and thoughts turn to...allergies, Cell., 97, 1999, 287–290.[Medline]
Mineo C., Anderson R.G.W. & White M.A.. Physical association with Ras enhances activation of membrane-bound Raf (Raf-CAAX), J. Biol. Chem., 272, 1997, 10345–10348.
Monks C.R.F., Freiberg B.A., Kupfer H., Sciaky N. & Kupfer A.. Three-dimensional segregation of supramolecular activation clusters in T cells, Nature., 395, 1998, 82–86.[Medline]
Montixi C., Langlet C., Bernard A.-M., Thimonier J., Dubois C., Wurbel M.-A., Chauvin J.-P., Pierres M. & He H.-T.. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains, EMBO (Eur. Mol. Biol. Organ.) J., 17, 1998, 5334–5348.[Medline]
Oliver J.M. & Berlin R.D.. Microtubules, microfilaments and the regulation of membrane functions, Symp. Soc. Exp. Biol., 33, 1979, 277–298.[Medline]
Oliver J.M. & Berlin R.D.. Surface and cytoskeletal events regulating leukocyte membrane topography, Semin. Hematol., 20, 1983, 282–304.[Medline]
Oliver J.M., Ukena T.E. & Berlin R.D.. Effects of phagocytosis and colchicine on the distribution of lectin-binding sites on cell surfaces, Proc. Natl. Acad. Sci. USA., 71, 1974, 394–398.
Oliver J.M., Burg D.L., Wilson B.S., McLaughlin J. & Geahlen R.L. Inhibition of mast cell FcRI-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol, J. Biol. Chem., 269, 1994, 29697–29703.
Ota Y. & Samelson L.E.. The product of the protooncogene c-cbla negative regulator of the Syk tyrosine kinase, Science., 276, 1997, 418–420.
Penninger J.M. & Crabtree G.R.. The actin cytoskeleton and lymphocyte activation, Cell., 96, 1999, 9–12.[Medline]
Pfeiffer J.R., Oliver J.M. & Berlin R.D.. Topographical distribution of coated pits, Nature., 286, 1980, 727–729.[Medline]
Pfeiffer J.R., Deanin G.G., Seagrave J.C., Davis B.H. & Oliver J.M.. Membrane and cytoskeletal changes associated with IgE-mediated serotonin release in rat basophilic leukemia cells, J. Cell Biol., 101, 1985, 2145–2155.
Rivera V.M. & Brugge J.S.. Clustering of Syk is sufficient to induce tyrosine phosphorylation and release of allergic mediators from rat basophilic leukemia cells, Mol. Cell. Biol., 15, 1995, 1582–1590.[Abstract]
Sanan D.A. & Anderson R.G.W.. Simultaneous visualization of LDL receptor distribution and clathrin lattices on membranes torn from the upper surface of cultured cells, J. Histochem. Cytochem., 39, 1991, 1017–1024.[Abstract]
Santini F. & Keen J.H.. Endocytosis of activated receptors and clathrin-coated pit formationdeciphering the chicken or egg relationship, J. Cell Biol., 132, 1996, 1025–1036.
Seagrave J.C., Pfeiffer J.R., Wofsy C. & Oliver J.M.. The relationship of IgE receptor topography to secretion in RBL-2H3 mast cells, J. Cell Phys, 148, 1991, 139–151.[Medline]
Shiue L., Zoller M.J. & Brugge J.S.. Syk is activated by phosphotyrosine-containing peptides representing the tyrosine-based activation motifs of the high affinity receptor for IgE, J. Biol. Chem, 270, 1995, 10498–10502.
Simons K. & Ikonen E.. Functional rafts in cell membranes, Nature., 387, 1997, 569–572.[Medline]
Surviladze A., Dráberová L., Kubinová L. & Dráber P.. Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells, Eur. J. Immunol., 28, 1998, 1847–1858.[Medline]
Stauffer T.P. & Meyer T.. Compartmentalized IgE receptor-mediated signal transduction in living cells, J. Cell Biol., 139, 1997, 1447–1454.
Stauffer T.P., Martinson C.H., Rider J.E., Kay B.K. & Meyer T.. Inhibition of Lyn function in mast cell activation by SH3 domain binding peptide, Biochemistry., 36, 1997, 9388–9394.[Medline]
Stulnig T.M., Berger M., Sigmund T., Stockinger H., Horejsi V. & Waldhaus W.. Polyunsaturated fatty-acids inhibit T-cell signal-transduction by modification of detergent-insoluble membrane domains, J. Cell Biol, 143, 1998, 637–644.
Stump R.F., Pfeiffer J.R., Seagrave J.C. & Oliver J.M.. Mapping gold-labeled IgE receptors on mast cells by scanning electron microscopyreceptor distribution revealed by silver enhancement, backscattered electron imaging and digital image analysis, J. Histochem. Cytochem., 36, 1988, 493–502.[Abstract]
Tamir I. & Cambier J.C.. Antigen receptor signalingintegration of protein-tyrosine kinase functions, Oncogene, 17, 1998, 1353–1364.[Medline]
Tsan M.F. & Berlin R.D.. Effect of phagocytosis on membrane transport of nonelectrolytes, J. Exp. Med., 134, 1971, 1016–1035.[Abstract]
Varma R. & Mayor S.. PI-anchored proteins are organized in submicron domains at the cell surface, Nature., 394, 1998, 798–801.[Medline]
Vonakis B.M., Chen H., Haleem-Smith H. & Metzger H.. The unique domain as a site on Lyn kinase for its constitutive association with the high affinity receptor for IgE, J. Biol. Chem, 272, 1997, 24072–24080.
Xavier R., Brennan T., Li Q., McCormack C. & Seed B.. Membrane compartmentation is required for efficient T cell activation, Immunity., 8, 1998, 723–732.[Medline]
Yamashita T., Mao S.-Y. & Metzger H.. Aggregation of the high-affinity IgE receptor and enhanced activity of p53/56lyn protein-tyrosine kinase, Proc. Natl. Acad. Sci. USA., 91, 1994, 11251–11255.
Zhang J., Berenstein E.H., Evans R.L & Siraganian R.P.. Transfection of syk tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a syk-negative variant of rat basophilic leukemia RBL-2H3 cells, J. Exp. Med., 184, 1996, 71–79.
Zhang W., Trible R.P. & Samelson L.E.. LAT palymitoylationits essential role in membrane microdomain targeting and tyrosine phosphorylation during T cell activation, Immunity., 9, 1998, 239–246.[Medline]
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