Single-cell measurements of IgE-mediated FcεRI signaling using an integrated microfluidic platform.

Heterogeneity in responses of cells to a stimulus, such as a pathogen or allergen, can potentially play an important role in deciding the fate of the responding cell population and the overall systemic response. Measuring heterogeneous responses requires tools capable of interrogating individual cells. Cell signaling studies commonly do not have single-cell resolution because of the limitations of techniques used such as Westerns, ELISAs, mass spectrometry, and DNA microarrays.

Insights into cell membrane microdomain organization from live cell single particle tracking of the IgE high affinity receptor FcϵRI of mast cells.

Current models propose that the plasma membrane of animal cells is composed of heterogeneous and dynamic microdomains known variously as cytoskeletal corrals, lipid rafts and protein islands. Much of the experimental evidence for these membrane compartments is indirect. Recently, live cell single particle tracking studies using quantum dot-labeled IgE bound to its high affinity receptor FcϵRI, provided direct evidence for the confinement of receptors within micrometer-scale cytoskeletal corrals.

Using hierarchical clustering and dendrograms to quantify the clustering of membrane proteins.

Cell biologists have developed methods to label membrane proteins with gold nanoparticles and then extract spatial point patterns of the gold particles from transmission electron microscopy images using image processing software. Previously, the resulting patterns were analyzed using the Hopkins statistic, which distinguishes nonclustered from modestly and highly clustered distributions, but is not designed to quantify the number or sizes of the clusters. Clusters were defined by the partitional clustering approach which required the choice of a distance.

Spatio-temporal signaling in mast cells.

This chapter summarizes the evidence for localized signaling domains in mast cells and basophils, with a particular focus on the high affinity IgE receptor, FcεRI and its crosstalk with other membrane proteins. It is noteworthy that a literature spanning 30 years established the FcεRI as a model receptor for studying activation-induced changes in receptor diffusion and lipid raft association. Now a combination of high resolution microscopy methods, including immunoelectron microscopy and sophisticated fluorescence-based techniques, provide new insight into the nanoscale spatial and temporal aspects of receptor topography on the mast cell plasma membrane.

Roles for the High Affinity IgE Receptor, FcεRI, of Human Basophils in the Pathogenesis and Therapy of Allergic Asthma: Disease Promotion, Protection or Both?

The role of basophils, the rarest of blood granulocytes, in the pathophysiology of allergic asthma is still incompletely understood. Indirect evidence generated over many decades is consistent with a role for basophils in disease promotion. Recent improvements in procedures to purify and analyze very small numbers of human cells have generally supported this view, but have also revealed new complexities.

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).

Reduced FcepsilonRI-mediated release of asthma-promoting cytokines and chemokines from human basophils during omalizumab therapy.

Background: Treating asthmatics with the humanized IgE-scavenging antibody, omalizumab (rhuMAb-E25, Xolair, reduces airways inflammation and asthma symptoms. Previously, omalizumab was shown to cause a dramatic and reversible loss of cell surface high-affinity IgE receptors, FcepsilonRI, from the peripheral blood basophils of asthmatics. The consequences of receptor loss for the FcepsilonRI-mediated synthesis and release of cytokines implicated in allergic asthma have not been examined.

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.

Differential expression of Ikaros isoforms in monozygotic twins with MLL-rearranged precursor-B acute lymphoblastic leukemia.

Infant leukemia associated with rearrangement of the MLL gene typically presents with high-risk clinical features. Relapse is common despite aggressive therapy and perturbations in signaling pathways may contribute to disease resistance. We evaluated twin 4-month-old monozygotic baby boys who presented with MLL-rearranged precursor-B acute lymphoblastic leukemia.

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.

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.

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.

A comparison of inflammatory mediators released by basophils of asthmatic and control subjects in response to high-affinity IgE receptor aggregation.

Background: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to secretion of inflammatory mediators and cytokine production. Basophils are known to play an important role in the pathogenesis of asthma but there has been no comprehensive examination of the effectors these cells produce. Here a study of the transcription and release of a selection of chemokines and cytokines from basophils was undertaken.

Markov random field modeling of the spatial distribution of proteins on cell membranes.

Cell membranes display a range of receptors that bind ligands and activate signaling pathways. Signaling is characterized by dramatic changes in membrane molecular topography, including the co-clustering of receptors with signaling molecules and the segregation of other signaling molecules away from receptors. Electron microscopy of immunogold-labeled membranes is a critical technique to generate topographical information at the 5-10 nm resolution needed to understand how signaling complexes assemble and function.

Mapping ErbB receptors on breast cancer cell membranes during signal transduction.

Distributions of ErbB receptors on membranes of SKBR3 breast cancer cells were mapped by immunoelectron microscopy. The most abundant receptor, ErbB2, is phosphorylated, clustered and active. Kinase inhibitors ablate ErbB2 phosphorylation without dispersing clusters.

Histamine release from the basophils of control and asthmatic subjects and a comparison of gene expression between "releaser" and "nonreleaser" basophils.

Most human blood basophils respond to FcepsilonRI cross-linking by releasing histamine and other inflammatory mediators. Basophils that do not degranulate after anti-IgE challenge, known as "nonreleaser" basophils, characteristically have no or barely detectable levels of the Syk tyrosine kinase. The true incidence of the nonreleaser phenotype, its relationship (if any) to allergic asthma, and its molecular mechanism are not well understood.

Analysis of proteins binding to the ITAM motif of the beta-subunit of the high-affinity receptor for IgE (FcepsilonRI).

Aggregation of the multichain (alphabetagamma2) high-affinity IgE receptor (Fcepsilon RI) initiates a signaling cascade that results in the release of allergic mediators. The cytoplasmic tails of the FcepsilonRI-beta and -gamma subunits contain immunoreceptor tyrosine-based activation motifs (ITAMs). Phosphorylation of the gammaITAM mediates activation of Syk kinase and is sufficient for triggering the responses induced by Fcepsilon RI crosslinking.

Kinetic proofreading of ligand-FcepsilonRI interactions may persist beyond LAT phosphorylation.

Cells may discriminate among ligands with different dwell times for receptor binding through a mechanism called kinetic proofreading in which the formation of an activated receptor complex requires a progression of events that is aborted if the ligand dissociates before completion. This mechanism explains how, at equivalent levels of receptor occupancy, a rapidly dissociating ligand can be less effective than a more slowly dissociating analog at generating distal cellular responses. Simple mathematical models predict that kinetic proofreading is limited to the initial complex; once the signal passes to second messengers, the dwell time no longer regulates the signal.

Water-soluble germanium(0) nanocrystals: cell recognition and near-infrared photothermal conversion properties.

Surfactant-passivated germanium nanocrystals (Ge(0) NCs) 3-5 nm in diameter were synthesized and encapsulated with functionalized phospholipids to yield water-soluble Ge(0) NCs. Upon encapsulation, the NCs retained their cubic crystalline phase and displayed good resistance to oxidation, as determined by transmission electron microscopy and X-ray photoelectron spectroscopy. As a test of their cell compatibility, the ability of carboxyfluorescein (CF)-labeled dinitrophenyl (DNP)-functionalized Ge(0) NCs to crosslink dinitrophenol-specific immunoglobulin E antibodies on the surface of mast cells (RBL-2H3) was examined in vitro.

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.

Synthesizing biofunctionalized nanoparticles to image cell signaling pathways.

This minireview outlines the synthetic efforts, from our research group, to produce nanomaterials for use as imaging agents to study cell signaling pathways. An overview of our approach to the synthesis and biofunctionalization of metal, semiconductor, and ceramic nanomaterials is presented. The probes investigated include coinage metals, Cd-based, Ge(o), naturally occurring fluorescent (NOF) minerals, and Ln-based nanoparticles which were synthesized from novel metal alkoxide, amide, and alkyl precursors.

Increased expression of genes linked to FcepsilonRI Signaling and to cytokine and chemokine production in Lyn-deficient mast cells.

Cross-linking the high-affinity IgE receptor, FcepsilonRI, on mast cells activates signaling pathways leading to the release of preformed inflammatory mediators and the production of cytokines and chemokines associated with allergic disorders. Bone marrow-derived mast cells (BMMCs) from Lyn-deficient (Lyn-/-) mice are hyperresponsive to FcepsilonRI cross-linking with multivalent Ag. Previous studies linked the hyperresponsive phenotype in part to increased Fyn kinase activity and reduced SHIP phosphatase activity in the Lyn-/- BMMCs in comparison with wild-type (WT) cells.

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 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.