Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces.

Fc epsilonRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of Fc epsilonRI to other cellular components at the synapse, such as actin, cholesterol, and linker for activation of T cells, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells, with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that Fc epsilonRI cross-linking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low-level degranulation upon ligand recognition.

Small, mobile FcepsilonRI receptor aggregates are signaling competent.

Crosslinking of IgE-bound FcepsilonRI triggers mast cell degranulation. Previous fluorescence recovery after photobleaching (FRAP) and phosphorescent anisotropy studies suggested that FcepsilonRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods were used for defining the relationship between receptor mobility and signaling. QD-IgE-FcepsilonRI aggregates of at least three receptors remained highly mobile over extended times at low concentrations of antigen that induced Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remained mobile at low doses that supported secretion. FcepsilonRI immobilization was marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocked secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates and a trigger for receptor internalization, but is not required for tyrosine kinase activation leading to secretion.

Actin restricts FcepsilonRI diffusion and facilitates antigen-induced receptor immobilization.

The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcepsilonRI motion and GFP-tagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganization of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time-dependent. Multiple FcepsilonRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcepsilonRI signalling is activated by crosslinking with multivalent antigen. We show that receptors become immobilized within seconds of crosslinking. Disruption of the actin cytoskeleton results in delayed immobilization kinetics and increased diffusion of crosslinked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their long-range mobility, sequestration and response to ligand binding.

Pivotal advance: IgE accelerates in vitro development of mast cells and modifies their phenotype.

Antigen-dependent activation of IgE-bound mast cells is critical for immediate hypersensitivity and other allergic disorders. Recent studies have revealed the effects of monomeric IgEs on mast cell survival and activation. Furthermore, IgE molecules exhibit a wide range of heterogeneity in the ability to induce mast cell activation in the absence of antigen. Highly cytokinergic (HC) IgEs can induce a variety of activation events including cell survival, degranulation, cytokine production, and migration, whereas poorly cytokinergic (PC) IgEs can do so inefficiently. Here, we show that culture of bone marrow cells in the presence of monomeric IgEs results in an increased number of mast cells compared with cultures grown without IgE. Furthermore, time in culture required to generate > or =80% pure mast cells is decreased. IgE molecules can directly influence mast cell progenitors to differentiate into mast cells. mRNA expression of several mast cell proteases and mast cell-related transcription factors is higher in mast cells cultured with an HC IgE than those cultured with a PC IgE or without IgE. Expression of early growth response factor-1, a transcription factor that is involved in the production of TNF-alpha in mast cells, is enhanced in cultures containing high and low concentrations of HC IgE and a high concentration of PC IgE. Consistent with this, expression of TNF-alpha is higher in mast cells cultured with HC IgE than PC IgE. Therefore, our results suggest that monomeric IgEs, especially HC IgEs, not only promote mast cell development but also modulate the mast cell phenotype.

Visualizing clathrin-mediated IgE receptor internalization by electron and atomic force microscopy.

A significant step in the immunoglobulin E (IgE) receptor signaling pathway in mast cell membranes is receptor internalization by clathrin-coated vesicles. Visualization in native membrane sheets of the emerging clathrin lattice structures containing the IgE receptor and associated signaling partners has been accomplished with high-resolution transmission electron microscopy (TEM). More recently, membrane sheets with labeled clathrin have also been characterized with atomic force microscopy (AFM) in combination with fluorescence imaging. We discuss here the procedure for creating fixed, native cell membrane sheets, labeling with immunogold or fluorescent labels, and utilization for TEM or AFM/fluorescence imaging of clathrin-mediated IgE internalization.

Activated N-formyl peptide receptor and high-affinity IgE receptor occupy common domains for signaling and internalization.

Immune cells display multiple cell surface receptors that integrate signals for survival, proliferation, migration, and degranulation. Here, immunogold labeling is used to map the plasma membrane distributions of two separate receptors, the N-formyl peptide receptor (FPR) and the high-affinity IgE receptor (FepsilonRI). We show that the FPR forms signaling clusters in response to monovalent ligand. These domains recruit Gi, followed by the negative regulatory molecule arrestin2. There are low levels of colocalization of FPR with FcepsilonRI in unstimulated cells, shown by computer simulation to be a consequence of receptor density. Remarkably, there is a large increase in receptor coclustering when cells are simultaneously treated with N-formyl-methionyl-leucyl-phenylalanine and IgE plus polyvalent antigen. The proximity of two active receptors may promote localized cross-talk, leading to enhanced inositol-(3,4,5)-trisphosphate production and secretion. Some cointernalization and trafficking of the two receptors can be detected by live cell imaging, but the bulk of FPR and FcepsilonRI segregates over time. This segregation is associated with more efficient internalization of cross-linked FcepsilonRI than of arrestin-desensitized FPR. The observation of receptors in lightly coated membrane invaginations suggests that, despite the lack of caveolin, hematopoietic cells harbor caveolae-like structures that are candidates for nonclathrin-mediated endocytosis.

Exploring membrane domains using native membrane sheets and transmission electron microscopy.

The flow of information in cells requires the constant remodeling of cell signaling and trafficking networks. To observe the remodeling events associated with activation of receptors on the cell surface, the authors have generated and analyzed high-resolution topographical maps of colloidal gold nanoprobes (3-10 nm) marking receptors, signaling proteins, and lipids in native membranes. The technology involves sandwiching of cells between glass cover slips and electron microscopy (EM) grids, followed by ripping. Membrane sheets on EM grids are fixed, labeled with functionalized nanoprobes, and imaged by transmission electron microscopy. Probe coordinates are extracted from digitized images and the distributions of the probes are analyzed with respect to each other and to membrane features like clathrin-coated pits, caveolae, and the cortical cytoskeleton.

Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton.

Although much evidence suggests that the plasma membrane of eukaryotic cells is not homogenous, the precise architecture of this important structure has not been clear. Here we use transmission electron microscopy of plasma membrane sheets and specific probes to show that most or all plasma membrane-associated proteins are clustered in cholesterol-enriched domains ("islands") that are separated by "protein-free" and cholesterol-low membrane. These islands are further divided into subregions, as shown by the localization of "raft" and "non-raft" markers to specific areas. Abundant actin staining and inhibitor studies show that these structures are connected to the cytoskeleton and at least partially depend on it for their formation and/or maintenance.

Characterizing the topography of membrane receptors and signaling molecules from spatial patterns obtained using nanometer-scale electron-dense probes and electron microscopy.

The flow of information through a cell requires the constant remodeling of cell signaling networks. Thus, spatially and temporally resolved microscopy of signaling components is needed to understand the behavior of normal cells as well as to uncover abnormal behavior leading to human disease. Nanoprobe labeling and transmission electron microscopy of cytoplasmic face-up sheets of cell membrane have been developed as a high-resolution approach to map the interactions of proteins and lipid during cell signaling. Membrane sheets are labeled with 3-15 nm electron-dense probes for receptors, signaling proteins and lipids and micrographs record the distributions of the probes relative to each other and to surface features. Here, we establish computational methods to extract spatial coordinates of probes from micrographs, to analyze and statistically validate the clustering and co-clustering of these probes and to integrate results between experiments in order to establish the relative spatial distributions of single and multiple probes. Our analyses, and the resulting programs for automating data collection and for carrying out statistical and clustering analyses provide toolboxes specialized for the spatiotemporal analysis and modeling of signal transduction pathways.

Membrane receptor mapping: the membrane topography of Fc(epsilon)RI signaling.

Ligand binding to membrane receptors initiates cascades of biochemical events leading to physiological responses. Hundreds of proteins and lipids are implicated in signaling networks and programs in genomics and proteomics are continuously adding new components to the signaling "parts lists". Here, we generate high resolution maps of signaling networks using cytoplasmic face-up membrane sheets that can be labeled with immunogold probes (3-10 nm) and imaged in the transmission electron microscope. Our model system is the mast cell and we focus on mapping the topography of the high affinity IgE receptor, Fc(epsilon)RI, its associated tyrosine kinases, Lyn and Syk, and the signaling proteins that propagate signals from these kinases. Crosslinked receptors and their signaling partners segregate during signaling to multiple, dynamic membrane domains, including a transient Fc(epsilon)RI-Lyn domain and at least two other distinct domains, one characterized by the presence of receptor, Syk and multiple signaling proteins, but not Lyn (primary signaling domains), and one characterized by the presence of LAT and PLCgamma1 but not receptor (secondary signaling domains). PI 3-kinase associates with both primary and secondary signaling domains and may help to recruit specific signaling proteins through the local remodeling of inositol phospholipids. The lipid raft markers, GM1 and Thy-1, fail to localize in native membrane sheets either with each other or with signaling domains. We introduce new probes to localize multiple signaling molecules on the same membrane sheet and new computational tools to capture and analyze their topographical relationships. In the future, we expect that high resolution maps of signaling networks will be integrated with chemical kinetic analyses, with cell fractionation data and with a range of real-time fluorescence measurements, into mathematical models with power to predict mechanisms that regulate the efficiency, specificity, amplitude and duration of signaling pathways.

Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes.

Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, FcepsilonRI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with FcepsilonRI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of FcepsilonRI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked FcepsilonRI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes.

Communication between the maternal testa and the embryo and/or endosperm affect testa attributes in tomato.

Two tomato (Lycopersicon esculentum) mutants with dark testae displaying poor germination rate and percentage on both water and 100 microM gibberellin(4 + 7) were recovered. The mutants were allelic (black seed1-1; bks1-1 and bks1-2), inherited in Mendelian fashion as a recessive gene residing on chromosome 11. They are not allelic to bs (brown seed) -1, -2, or -4, which impair seed germination and possess dark testae. The bks/bs mutants accumulated dark pigment in the cell layers of the testa above the endothelium, which itself accumulated proanthocyanidins similar to wild type. The poor germination performance of bks mutant seeds was because of impediment of the mutant testae to radicle egress. Imbibition on gibberellin(4 + 7) did not ameliorate germination percentage or rate. The toughening of the bks testa and associated poor germination were partially overcome when seeds were not dried before germination or were dried under N(2). The seeds of the bks mutant have elevated activity of at least one enzyme responsible for the detoxification of reactive oxygen species. The bks mutant is epistatic to 12 anthocyaninless mutants of tomato. Bio- and physicochemical analysis of the bks testa determined that it accumulated a melanic substance. Inheritance of bks/bs mutations contrasts with that of the anthocyaninless mutants, which are inherited according to the genotype of the maternally derived testa. This suggests that the testa manufactures components before its demise that can maximize testa strength, whereas the endosperm/embryo produces factors that are conveyed to the testa, mitigating this process.

Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells.

Antigen-mediated activation of mast cells results in Ca2+-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca2+ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response.

FcepsilonRI signaling observed from the inside of the mast cell membrane.

Crosslinking the high affinity IgE receptor, FcrepsilonRI, on basophils and mast cells initiates cascades of biochemical events leading to degranulation, membrane ruffling and other physiological responses. Downstream of FcepsilonRI and its coupled tyrosine kinases, Lyn and Syk, scores of different proteins and lipids are implicated in these signaling cascades and new players are being identified continuously. Here, we use immunogold probes to label receptors and signaling proteins on the cytoplasmic face of membrane sheets prepared from RBL-2H3 mast cells and transmission electron microscopy to examine their distributions in relationship to each other and to features of the membrane. New topographical data are integrated with existing knowledge of the biochemistry of FcepsilonRI signaling and of cell shape during signaling to implicate at least two distinct membrane domains in FcepsilonRI signaling. "Primary signaling domains", also called osmiophilic patches, are recognized by their dark staining with osmium, adjacency to coated pits (previously mapped to planar membrane between lamellae) and by the characteristic presence of receptor, Syk and PLCgamma2, but not Lyn. "Secondary signaling domains" are characterized by the presence of large elliptical linker for activation of T cells (LAT) rafts and of PLCgamma1 (previously mapped to lamellae) but not receptor. The signaling proteins, Vav, Grb2, Cbl and Gab2, and the endocytic proteins, AP2 and clathrin, all map to the primary domains, while the p85 regulatory subunit of phosphatidylinositol 3 (PI 3)-kinase maps to both domains. Recognition that FcepsilonRI signaling is controlled not only by which chemical species are available for interaction, but also by where the interactions occur, may provide new opportunities for the modeling of signaling cascades and new targets for the development of drugs to treat allergies and asthma.