Correspondence to: Bridget S. Wilson, Department of Pathology, University of New Mexico Health Sciences Center, CRF 205, 2325 Camino de Salud, Albuquerque, NM 87131. Tel:(505) 272-8852 Fax:(505) 272-1435 E-mail:bwilson{at}thor.unm.edu.
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Abstract |
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We have determined the membrane topography of the high-affinity IgE receptor, FcRI, 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. 39:10171024) 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 Fc
RI and Lyn are distributed as small clusters (29 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of Fc
RI clusters contain Lyn. In contrast, there is essentially no Fc
RI-Syk colocalization in resting cells. 2 min after Fc
RI cross-linking, ~10% of Lyn colocalizes with small and medium-sized Fc
RI clusters (up to 20 gold particles), whereas ~16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20100 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 Fc
RI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of Fc
RI signaling.
Key Words: microdomains, Lyn, Syk, rafts
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Introduction |
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The discovery that different membrane proteins partition away from or into membrane that is internalized during phagocytosis provided early evidence that proteins can segregate nonrandomly in the plasma membrane and implicated cytoskeletal proteins in the segregation (
The high-affinity IgE receptor, FcRI, is a tetrameric complex consisting of an IgE-binding
subunit, a tetraspan ß subunit and two disulfide-linked
subunits (
RI and Fc
RIII (
R1-associated kinases of RBL-2H3 mast cells, showed that Lyn's primary substrates are the Fc
R1ß and
ITAMs, and linked Syk activation to Fc
R1 signal propagation (
It has been suggested that FcRI signaling may occur in microdomains.
RI were also detected in these membranes, leading to the hypothesis that aggregated Fc
RI might move into Lyn-containing ordered lipid microdomains and thereby initiate Fc
RI signaling. However, contrasting biochemical evidence suggests that the association of Fc
RI with Lyn relies primarily upon defined proteinprotein interactions. In particular,
RI via interactions of its unique NH2-terminal SH4 domain with non-ITAM regions in the Fc
RI ß subunit COOH terminus. Furthermore, fluorescence microscopic experiments by
RI may colocalize with Syk rather than Lyn in ordered lipid microdomains. These investigators showed that Fc
RI 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 Fc
RI phosphorylation also blocked Syk recruitment to the membrane (
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 Fc
RI cross-linking. Moreover, cross-linked Fc
RI encounter Syk and coated pits in topographically distinct membrane domains.
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Materials and Methods |
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Reagents and Cell Culture
RBL-2H3 cells were grown in MEM (GIBCO BRL) supplemented with 15% fetal calf serum, penicillin-streptomycin, and L-glutamine. Rabbit anti-Lyn (44) and anti-Syk (N-19) Abs were from Santa Cruz Biotechnology, Inc. Mouse anti-FcRI ß mAb was a generous gift from Dr. Juan Rivera (NIH, Bethesda, MD). Affinity-purified mouse anti-DNP IgE and rabbit antimouse IgE Abs were prepared as described (
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, Fc
RI was cross-linked by incubation with DNP-BSA (0.11 µg/ml), with rabbit polyclonal anti-IgE (1 µg/ml) or with gold conjugates of these reagents (prepared as in
RI in the resting state, cells were fixed with 0.5% paraformaldehyde for 5 min and then incubated with antiIgE-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; Fc
RI ß, 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 ß; labeled with 5-nm gold particles marking Lyn plus 10-nm particles marking Fc
RI ß; labeled with 5-nm gold particles marking Syk plus 10-nm particles marking Fc
RI ß; resting and activated. A minimum of 3,000 gold particles per set of micrographs were counted for (a) numbers of gold particles marking Fc
RI ß 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 Fc
RI ß clusters (distinguishing, in activated cells, between gold particles marking Lyn that occurred within Fc
RI 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 Fc
RI ß.
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Results |
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The Distribution of FcRI on Resting and Activated Mast Cells
The distribution of 5-nm gold particles marking FcRI ß in resting cells is illustrated in Fig 1 A and quantified in Fig 2 A. The micrograph shows that gold particles marking Fc
RI ß 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 Fc
RI ß are distributed as singlets and that the rest are in clusters that most commonly contain 23 gold particles and only rarely exceed 9 gold particles.
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After cross-linking for 2 min at 37°C with DNP-BSA (0.1 µg/ml), most FcRI ß 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 Fc
RI ß in activated cells are in clusters of 1020 (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 antiIgE-gold particles and subsequently, from the inside, with 5-nm anti-Fc
RI ß particles. 5-nm gold particles marking Fc
RI ß 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 Fc
RI
. In Fig 1 E, cells were activated for 2 min at 37°C with 10-nm rabbit antiIgE-gold particles before labeling from the inside with 5-nm anti-Fc
RI ß 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 Fc
RI ß and the 10-nm gold particles marking Fc
RI
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 antiFc
RI ß 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 antiFc
RI ß, 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 ß and
subunits of the cross-linked Fc
RI (
RI ß, 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 Fc
RI ß in resting mast cells. Fig 3 C is included to show there is essentially no background binding of either 5- or 10-nm antimouse 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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Counting gold particle numbers and distributions on replicate micrographs confirmed that indeed the majority (at least 85%) of gold particles marking Lyn are in clusters (Fig 2 B). Clusters of 23 particles are most common and clusters of >9 particles are rare. We found that ~20% of total Lyn gold particles were in close proximity to gold particle(s) marking FcRI ß. If counted from the complementary perspective of total Fc
RI 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 Fc
RI 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 Fc
RI ß, 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 Fc
RI ß 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 Fc
RI ß that form on osmiophilic membrane regions in activated cells (arrows). Fig 4BD, 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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Counting gold particle numbers and distributions emphasized the topographical differences in Lyn distribution between resting and activated cells. First, Lyn clusters increase in size in response to 2 min of FcRI 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 Fc
RI ß gold particles, whereas 16% of total Lyn was localized at the periphery of the large clusters of Fc
RI ß 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 Fc
RI 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 Fc
RI ß 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-1enriched microdomains that also accumulate GPI-linked proteins including Thy-1 (RI ß (Fig 5 B) labeled from the inside of the same cells.
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The Topography of Syk and Its Association with FcRI
Previous biochemical studies have shown that a second tyrosine kinase, Syk, couples the cross-linked FcRI to downstream responses (
RI
subunit creates sites for the binding and activation of Syk (
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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Results of counting gold particles specific for Syk are shown in Table 1. The total number of gold particles marking Syk over 55 µm2 of resting plasma membrane was 978, compared with 1,218 gold particles over the same area of activated plasma membrane. Thus, at 2 min of continuous receptor cross-linking, there was a 24% increase in Syk labeling. Although FcRI 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 Fc
RI ß. In contrast, >70% of gold particles marking Syk are found in receptor clusters after activation.
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Discussion |
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Results of previous biochemical studies have suggested a model for FcRI signaling in which receptor cross-linking leads to the Lyn-mediated tyrosine phosphorylation of ITAMs within the cytoplasmic tails of the ß and
subunits of the Fc
RI. Fc
RI
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, Fc
RI, and of its associated tyrosine kinases, Lyn and Syk, during this biochemical cascade. We used a modification of the method of
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 (2025%) of these are mixed clusters, containing both Fc
RI and Lyn. Neither Fc
RI 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 (
RI via interactions of its SH4 domain with determinants on the Fc
RI ß subunit. In contrast, cell fractionation studies by
RI 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 Fc
RI.
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,
Previous studies by fluorescence, transmission, and scanning electron microscopy (RI 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;
RI topography by immunogold labeling with 15-nm gold probes and SEM analysis initially suggested an inherently random distribution of the Fc
RI (
RI observed here on membrane sheets is probably the result of the higher resolution achieved with smaller (310 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 (
subunits of the cross-linked Fc
RI (
RI 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 Fc
RI cross-linking (
RI 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 (
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 RI 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, Fc
RI- and Lyn-containing membranes isolated by
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 Fc
RI are internalized through coated pits that often have long necks and can support several clathrin-coated buds (
RI 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 (
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- (
RI 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,
RI, 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 Fc
RI ß and
subunit phosphorylation occurs, to identify other components of the small and large receptor clusters, to determine how Fc
RI cross-linking induces the segregation of Lyn from Fc
RI and to explore mechanisms regulating the topography of coated pit formation.
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Footnotes |
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1 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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Acknowledgements |
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We thank Graham MacKay and Zurab Surviladze for valuable discussion.
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
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References |
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