Abstract Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has proposed a mechanism persistence this microorganism in certain infections. Therefore, we investigated whether polymorphonuclear neutrophils (PMN) contributes to pathogenesis S. Our data demonstrate that PMN isolated from site infection contain viable intracellular organisms and these infected are...

Although much evidence suggests that the plasma membrane of eukaryotic cells is not homogenous, precise architecture this important structure has been clear. Here we use transmission electron microscopy sheets and specific probes to show most or all membrane-associated proteins are clustered in cholesterol-enriched domains ("islands") separated by "protein-free" cholesterol-low membrane. These islands further divided into subregions, as shown localization "raft" "non-raft" markers areas....

In RBL-2H3 rat tumor mast cells, antigens that cross-link the high affinity cell surface IgE receptor, Fc epsilon R1, activate at least two receptor-associated protein-tyrosine kinases, Lyn and Syk, cause tyrosine phosphorylation of receptor beta gamma subunits, PLC 1, Vav, other proteins. Cross-linking also induce increased phosphatidylinositol turnover, Ca2+ mobilization, secretion, actin polymerization, spreading, membrane ruffling. We have used kinase inhibitor, piceatannol...

We have determined the membrane topography of high-affinity IgE receptor, FcεRI, and its associated tyrosine kinases, Lyn Syk, by immunogold labeling transmission electron microscopic (TEM) analysis sheets prepared from RBL-2H3 mast cells. The method Sanan Anderson (Sanan, D.A., R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017–1024) was modified to generate dorsal surface Signaling molecules were localized on cytoplasmic face these native membranes high-resolution TEM analysis. In...

Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, ganglioside GM1, palmitoylated LAT, cross-linked IgE receptors, FcepsilonRI. This study addresses relationship of data to organization raft markers in native membranes. Immunogold labeling electron microscopy shows there is little or no colocalization LAT with each other FcepsilonRI on membrane sheets prepared unstimulated...

In mast cells, cross-linking the high-affinity IgE receptor (FcϵRI) initiates Lyn-mediated phosphorylation of ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling membrane sheets showed that resting FcϵRI colocalize loosely with Lyn, whereas cross-linked redistribute into specialized domains (osmiophilic patches) exclude accumulate Syk, and are often bordered by coated pits. Here, distribution β is mapped relative to linker activation T cells (LAT), Grb2-binding...

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 displayed good resistance oxidation, as determined by transmission electron microscopy X-ray photoelectron spectroscopy. As a test of cell compatibility, ability carboxyfluorescein (CF)-labeled dinitrophenyl (DNP)-functionalized crosslink...

We have investigated the effects of wortmannin, an inhibitor phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in RBL-2H3 mast cell model. In cells, cross-linking high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, stimulates secretion, membrane ruffling, spreading, pinocytosis, formation actin plaques implicated increased cell-substrate adhesion. addition, Fc R1 PI 3-kinase. It was previously shown...

Engagement of the Fcε receptor I (FcεRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation two transmembrane adaptor proteins, linker for T (LAT) non–T cell (NTAL; also called LAB; a product Wbscr5 gene). Previous studies showed that secretory response was partially inhibited in bone marrow–derived (BMMCs) from LAT-deficient mice. To clarify role NTAL degranulation, we compared FcεRI-mediated BMMCs...

The Golgi apparatus is a dynamic organelle whose structure sensitive to vesicular traffic and cell cycle control. We have examined the potential role for rab1a, GTPase previously associated with ER intra-Golgi transport, in formation maintenance of structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis morphology fluorescence electron microscopy. Three proteins were tested: wild-type rab, mutant rab1a(S25N),...

Abstract Studies in B cells from Lyn-deficient mice have identified Lyn as both a kinetic accelerator and negative regulator of signaling through the BCR. The properties bone marrow-derived mast Lyn−/− (Lyn−/− BMMCs) also been explored, but their phenotype remains controversial. We confirm that BMMCs release more β-hexosaminidase than wild-type following FcεRI cross-linking show multiple cell responses to (the phosphorylation receptor subunits other proteins, activation phospholipase Cγ...

We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms to follow three-dimensional molecular motion. This has substantial advantages over methods based upon charge-coupled device cameras, including increased Z-tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used the ability perform time-resolved spectroscopy (such as...

Often considered to be a "dead" kinase, erbB3 is implicated in escape from erbB-targeted cancer therapies. Here, heregulin stimulation shown markedly upregulate kinase activity immunoprecipitates. Intact, activated phosphorylates tyrosine sites an exogenous peptide substrate, and this abolished by mutagenesis of lysine 723 the catalytic domain. Enhanced linked heterointeractions with catalytically active erbB2, since it largely blocked cells pretreated lapatinib or pertuzumab. erbB2...

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% NSCLC. The molecular mechanisms by these confer constitutive activity remain unresolved. Using multiple...

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In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P 3 production. Using immune complex phospholipase assays, we show that FcεRI both PLCγ1 PLCγ2. Activation is accompanied by increased phosphorylation of PLCγ isoforms on serine in antigen-treated cells. We also two have distinct subcellular localizations. primarily resting low levels plasma membrane association. After stimulation,...

Antigen‐mediated activation of mast cells results in Ca 2+ ‐dependent exocytosis preformed mediators the inflammatory response. To investigate role secretory vesicle motility this response, we have performed time‐lapse confocal microscopy on RBL‐2H3 transfected with a green fluorescent protein‐Fas ligand fusion protein (GFP‐FasL). Green protein‐labeled vesicles exhibit rapid, bidirectional movement both resting and activated can be localized adjacent to microtubules. Colchicine treatment...

Distributions of ErbB receptors on membranes 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. Modest co-clustering EGFR, even after EGF treatment, suggests that both are predominantly involved in homointeractions. Heregulin leads to dramatic clusters ErbB3 contain some EGFR PI 3-kinase. Other docking proteins, such as Shc...