Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging.

Simultaneous atomic force microscopy (AFM) and confocal fluorescence imaging were used to observe in aqueous buffer the three-dimensional landscape of the inner surface of membrane sheets stripped from fixed tumor mast cells. The AFM images reveal prominent, irregularly shaped raised domains that label with fluorescent markers for both resting and activated immunoglobin E receptors (FcepsilonRI), as well as with cholera toxin-aggregated GM1 and clathrin. The latter suggests that coated pits bud from these regions. These features are interspersed with flatter regions of membrane and are frequently surrounded and interconnected by cytoskeletal assemblies. The raised domains shrink in height by approximately 50% when cholesterol is extracted with methyl-beta-cyclodextrin. Based on composition, the raised domains seen by AFM correspond to the cholesterol-enriched dark patches observed in transmission electron microscopy (TEM). These patches were previously identified as sites of signaling and endocytosis based on their localization of activated FcepsilonRI, at least 10 associated signaling molecules, and the presence of clathrin-coated pits. Overall the data suggest that signaling and endocytosis occur in mast cells from raised membrane regions that depend on cholesterol for their integrity and may be organized in specific relationship with the cortical cytoskeleton.

Overcoming the signaling defect of Lyn-sequestering, signal-curtailing FcepsilonRI dimers: aggregated dimers can dissociate from Lyn and form signaling complexes with Syk.

Clustering the tetrameric (alphabetagamma(2)) IgE receptor, FcepsilonRI, on basophils and mast cells activates the Src-family tyrosine kinase, Lyn, which phosphorylates FcepsilonRI beta and gamma subunit tyrosines, creating binding sites for the recruitment and activation of Syk. We reported previously that FcepsilonRI dimers formed by a particular anti-FcepsilonRI alpha mAb (H10) initiate signaling through Lyn activation and FcepsilonRI subunit phosphorylation, but cause only modest activation of Syk and little Ca(2+) mobilization and secretion. Curtailed signaling was linked to the formation of unusual, detergent-resistant complexes between Lyn and phosphorylated receptor subunits. Here, we show that H10-FcepsilonRI multimers, induced by adding F(ab')(2) of goat anti-mouse IgG to H10-treated cells, support strong Ca(2+) mobilization and secretion. Accompanying the recovery of signaling, H10-FcepsilonRI multimers do not form stable complexes with Lyn and do support the phosphorylation of Syk and phospholipase Cgamma2. Immunogold electron microscopy showed that H10-FcepsilonRI dimers colocalize preferentially with Lyn and are rarely within the osmiophilic "signaling domains" that accumulate FcepsilonRI and Syk in Ag-treated cells. In contrast, H10-FcepsilonRI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of detergent-extracted cells, H10-treated cells show a more complete redistribution of FcepsilonRI beta from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with FcepsilonRI multimers. We hypothesize that restraints imposed by the particular orientation of H10-FcepsilonRI dimers traps them in signal-initiating Lyn microdomains, and that converting the dimers to multimers permits receptors to dissociate from Lyn and redistribute to separate membrane domains that support Syk-dependent signal propagation.

High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT.

In mast cells, cross-linking the high-affinity IgE receptor (Fc(epsilon)RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc(epsilon)RI colocalize loosely with Lyn, whereas cross-linked Fc(epsilon)RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc(epsilon)RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCgamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCgamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCgamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCgamma2, Gab2, and a portion of p85 colocalize with Fc(epsilon)RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCgamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, Fc(epsilon)RI cross-linking increases PI3-kinase activity in anti-LAT, anti-Fc(epsilon)RIbeta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc(epsilon)RIbeta and from secondary domains, including one organized around LAT.

p110beta and p110delta phosphatidylinositol 3-kinases up-regulate Fc(epsilon)RI-activated Ca2+ influx by enhancing inositol 1,4,5-trisphosphate production.

Fc(epsilon)RI-induced Ca2+ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase Cgamma2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca2+ responses after Fc(epsilon)RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110beta or p110delta catalytic isoforms of PI 3-kinase, but not antibodies to p110alpha, lengthens the lag time to release of Ca2+ stores and blunts the sustained phase of the calcium response. Ca2+ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, a competitive inhibitor of the Ins(1,4,5)P3 receptor. This indicates a requirement for Ins(1,4,5)P3 to initiate and sustain Ca2+ responses even when PI 3-kinase is fully active. Antigen-induced cell ruffling, a calcium-independent event, is blocked by injection of p110beta and p110delta antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110alpha antibodies. These results suggest that the p110beta and p110delta isoforms of PI 3-kinase support Fc(epsilon)RI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel.

Differential sensitivity to acute cholesterol lowering of activation mediated via the high-affinity IgE receptor and Thy-1 glycoprotein.

Lateral cross-linking of transmembrane high-affinity IgE receptors (FcepsilonRI) or glycosylphosphatidylinositol-anchored Thy-1 glycoproteins on the surface of rat mast cells and rat basophilic leukemia (RBL) cells triggers the signaling pathways that lead to the release of allergy mediators. Although both of these pathways are initiated by an increased activity of Lyn kinase, the exact mechanism by which Lyn kinase interacts with aggregated FcepsilonRI and Thy-1 is not completely understood. Here we demonstrate that pretreatment of RBL cells with methyl-beta-cyclodextrin (MBCD) resulted in a dose- and time-dependent decrease in cellular cholesterol, increased detergent solubilization of Thy-1 and Lyn kinase, and a transient increase in tyrosine phosphorylation of several proteins. Acute lowering of cholesterol suppressed the activation through Thy-1, as determined by tyrosine phosphorylation of Syk kinase and some other proteins, and modulation of free cytoplasmic calcium. In contrast, the FcepsilonRI-mediated activation events were more resistant. Thy-1 and FcepsilonRI in MBCD-pretreated cells also differed in the extent of aggregation after cross-linking: Thy-1 formed large caps, whereas FcepsilonRI accumulated in small patches. MBCD treatment induced an increased release of secretory components in both Thy-1- and FcepsilonRI-activated cells. The combined data indicate that cholesterol depletion does not merely block receptor signaling but has more complex consequences.

Protein tyrosine kinase p53/p56(lyn) forms complexes with gamma-tubulin in rat basophilic leukemia cells.

The aggregation of receptors with high affinity for IgE (FcepsilonRI) on the surface of mast cells and basophils initiates a chain of biochemical events culminating in the release of allergy mediators. Although microtubules have been implicated in the activation process, the molecular mechanism of their interactions with signal transduction molecules is poorly understood. Here we show that in rat basophilic leukemia cells large amounts of alphabeta-tubulin dimers ( approximately 70%) and gamma-tubulin ( approximately 85%) are found in a soluble pool which was released from the cells after permeabilization with saponin, or extraction with non-ionic detergents. Soluble tubulins were found in large complexes with other molecules. Complexes of soluble gamma-tubulin released from activated cells contained tyrosine-phosphorylated proteins of relative mol. wt approximately 25, 50, 53, 56, 60, 75, 80, 97, 115 and 200 kDa. Increased tyrosine phosphorylation of proteins associated with the cytoskeleton, i.e. around centrosomes, was detected by immunofluorescence microscopy. In vitro kinase assays revealed increased tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from activated cells. Two of the tyrosine phosphorylated proteins in these complexes were identified as the p53/56(lyn) kinase. Furthermore, gamma-tubulin bound to the N-terminal fragment of recombinant Lyn kinase and its binding was slightly enhanced in activated cells. Pretreatment of the cells with Src family-selective tyrosine kinase inhibitor, PP1, decreased the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, as well as the amount of gamma-tubulin in Lyn kinase immunocomplexes. The combined data suggest that gamma-tubulin is involved in early stages of mast cell activation.

Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells.

Antibody-mediated cross-linking of Thy-1 glycoprotein on the surface of rat mast cells and rat basophilic leukemia (RBL) cells initiates biochemical events which culminate in secretion of allergy mediators. Thy-1, like some other glycosylphosphatidylinositol (GPI)-anchored proteins, forms detergent-insoluble complexes containing protein tyrosine kinases (PTK) and some other molecules which are implicated in the signaling pathway. On the surface of a rat mast cell there are more than 10(6) Thy-1 molecules; however, it is not known which fraction of them is involved in transmembrane signaling, and what exactly is the heterogeneity of Thy-1 complexes. Using sucrose density gradient ultracentrifugation of detergent-lysed RBL cells we found that the density of Thy-1 complexes depended on the detergent used and the lysis conditions employed. Sepharose 4B gel chromatography fractionation followed by density gradient ultracentrifugation revealed both size and density heterogeneity of Thy-1 and Lyn PTK complexes. Cross-linking of surface Thy-1 caused significant changes in the density of these complexes, and an increase in Lyn kinase activity in low/medium-density fractions. Thy-1 in low-density fractions was relatively resistant to cleavage with phosphatidylinositol-specific phospholipase C (PI-PLC). Interestingly, removal of only a small fraction of surface Thy-1 by PI-PLC abolished the cell activation as determined by tyrosine phosphorylation of certain proteins. When Triton X-100 lysates were fractionated at 12000 x g, about 50 % of Thy-1 remained associated with the nuclear/cytoskeleton pellet; this fraction of Thy-1 exhibited an increased sensitivity to PI-PLC. Confocal laser scanning microscopy on fixed cells revealed that the total Thy-1 was relatively homogeneously distributed over the plasma membrane, whereas the PI-PLC-resistant Thy-1 was found mostly in small clusters. The combined data suggest that specialized membrane microdomains enriched in Thy-1 with increased sensitivity to PI-PLC are directly involved in coupling Thy-1 aggregation to transmembrane signaling.

[Steady state kinetics of DNA degradation with pancreatic deoxyribonuclease A in the presence of Mg2+]. / Statsionarnaia kinetika passhchepleniia DNK pankreaticheskoi dezoksiribonukleazoi A v prisutstvii Mg2+.

Steady state kinetics of DNA depolymerisation in the presence of the DNAase A and Mg2+ ions were investigated at pH 5.5 and wide region of the enzyme, substrate and metal ion concentrations. A model, which is consistent with experimental results obtained is suggested. According to the model catalytically active form of the DNAase A should be a metal-bound enzyme. That species reacts with the metal-free DNA to form the Michaelis complex. The kinetics observed can be described in terms of mechanism which involves covalent enzyme-substrate intermediate formation. It was shown that the second Mg2+ ion binding to the complex Mg2+ DNAase -- DNA (KD - 2.2 . 10(-3) M) enhances the kinetic parameters of the reaction. To rationalise the effect one has to assume that the rate of the intermediate formation was accelerated as a result of the second Mg2+ binding.