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A Sobota

Publications and source records attributed to A Sobota.

At least 19 recordsLinked to original sources
PubMed2002

Sequential translocation of tyrosine kinases Lyn and Syk to the activated Fcgamma receptors during phagocytosis.

Tyrosine phosphorylation of numerous proteins is one of the earliest events detectable during Fcgamma receptor-mediated phagocytosis. We demonstrate that IgG-coated particles associated with the surface of macrophages are enriched with numerous tyrosine-phosphorylated proteins. During particle internalization the proteins are still associated with particles but their phosphorylation is reduced. Lyn kinase is phosphorylated both at particle binding and internalization steps. The phosphorylated Syk kinase is the major kinase associated with engulfed particles. Imnunofluorescent studies confirm spatial and temporal distribution of Lyn and Syk kinases at different stages of phagocytosis. Our data indicate that ligation of Fcgamma receptors activates Lyn followed by Syk kinase and in the result multimolecular complex of the kinases and several accompanying tyrosine phosphorylated proteins with Fcgamma receptors is organized leading to local reorganization of actin-based skeleton and particle uptake.

Animals↗
PubMed2001

The clustered Fcgamma receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner.

Phosphorylation of clustered Fcgamma receptor II (FcgammaRII) by Src family tyrosine kinases is the earliest event in the receptor signaling cascade. However, the molecular mechanisms for the interaction between FcgammaRII and these kinases are not elucidated. To asses this problem we isolated high molecular weight complexes of cross-linked FcgammaRII from non-ionic detergent lysates of U937 monocytic cells. CD55, a glycosylphosphatidylinositol-anchored protein, a ganglioside GM1 and Lyn, a Src family tyrosine kinase, were also located in these complexes. Gradient centrifugation demonstrated that the complexes containing cross-linked FcgammaRII displayed a low buoyant density. The FcgammaRII present in the complexes underwent tyrosine phosphorylation. Cross-linked FcgammaRII and Lyn occupied common 100-200 nm detergent-resistant membrane fragments, as demonstrated by immunoprecipitation and microscopy studies. Pretreatment of the cells with beta-cyclodextrin, a cholesterol acceptor, depleted membrane cholesterol and released CD55, GM1 and Lyn from the detergent-resistant complexes. In parallel, the association of Lyn with cross-linked FcgammaRII was disrupted and phosphorylation of the receptor inhibited. Reincorporation of cholesterol evoked the relocation of Lyn into the detergent-resistant membrane fraction and restored both Lyn association with cross-linked FcgammaRII and tyrosine phosphorylation of the receptor. Our data demonstrate that cholesterol-enriched membrane rafts can facilitate tyrosine phosphorylation of clustered FcgammaRII by Lyn kinase.

Animals↗
PubMed1999

Engagement of spectrin and actin in capping of FcgammaRII revealed by studies on permeabilized U937 cells.

Plasma membrane receptors can undergo translocation in the plane of plasma membrane after binding of polyvalent ligands. Ligand/receptor clusters, named patches, can collect into a polar cap, presumably due to their association with the submembrane actin-based cytoskeleton. We found that the assembly of Fcgamma receptor II caps in human monocytic U937 cells was accompanied by the accumulation of spectrin and actin in the cap region. Permeabilization of cells with streptolysin O rendered capping sensitive to inhibition by phalloidin, an actin filament stabilizing agent. A rabbit antibody directed against the chicken erythrocyte alpha-subunit of spectrin, an actin- and membrane-binding protein, also blocked the capping in a dose dependent manner. The inhibition reached approximately 50% after 20 minutes of cell treatment with the antibody. Anti-alpha-spectrin targeted specifically its submembrane antigen, in contrast to unspecific antibodies which remained dispersed in the cell interior and had no influence on the cap assembly. Our results indicate an active engagement of spectrin and actin filaments in the capping of Fcgamma receptor II.

Actins↗
PubMed1999

Tyrosine phosphorylation/dephosphorylation controls capping of Fcgamma receptor II in U937 cells.

In the capping of cell-surface receptors two stages can be distinguished: 1) clustering of the receptors (patching) induced by cross-linking with specific antibodies and 2) subsequent assembly of patches into a cap which is driven by the actin-based cytoskeleton. We found that patching of Fcgamma receptor II in U937 cells was correlated with tyrosine phosphorylation of certain proteins, most prominently those of 130, 110, 75 and 28 kDa. The phosphotyrosine-bearing proteins were accumulated at the receptor patches. Formation of the receptor caps was coincident with dephosphorylation of these proteins. Inhibition of protein tyrosine kinases with herbimycin A and genistein attenuated the protein tyrosine hyperphosphorylation and blocked capping in a dose-dependent manner. Phenylarsine oxide and pervanadate, inhibitors of protein tyrosine phosphatases, also suppressed capping of Fcgamma receptor II in a concentration-dependent fashion. Simultaneously, tyrosine hyperphosphorylation of proteins occurred. In the presence of the tyrosine kinase and phosphatase inhibitors the receptors were arrested at the patching stage. In contrast, okadaic acid, a serine/threonine phosphatase blocker, did not affect assembly of the receptor caps. The inhibitory effect of phenylarsine oxide was rapidly reversed by dithiols, 2,3-dimercapto-1-propanoldithiol and dithiotreitol, and was coincident with dephosphorylation of protein tyrosine residues. Extensive washing of pervanadate-exposed cells also resulted in progressive restoration of the cap assembly. Using streptolysin O-permeabilized cells we confirmed regulatory function played by dephosphorylation of tyrosine residues in capping of Fcgamma receptor II. Exogenous phosphatases, applied to permeabilized cells in which activity of endogenous tyrosine phosphatases was blocked, evoked dephosphorylation of protein tyrosine residues that was accompanied by recovery of capping ability in the cells.

Arsenicals↗
PubMed1999

Signaling pathways in phagocytosis.

Phagocytosis is an uptake of large particles governed by the actin-based cytoskeleton. Binding of particles to specific cell surface receptors is the first step of phagocytosis. In higher Eucaryota, the receptors able to mediate phagocytosis are expressed almost exclusively in macrophages, neutrophils, and monocytes, conferring immunodefence properties to these cells. Receptor clustering is thought to occur upon particle binding, that in turn generates a phagocytic signal. Several pathways of phagocytic signal transduction have been identified, including the activation of tyrosine kinases and (or) serine/threonine kinase C in pivotal roles. Kinase activation leads to phosphorylation of the receptors and other proteins, recruited at the sites of phagocytosis. Monomeric GTPases of the Rho and ARF families are likely to be engaged downstream of activated receptors. The GTPases, in cooperation with phosphatidylinositol 4-phosphate 5-kinase and phosphatidylinositol 3-kinase lipid modifying enzymes, can modulate locally the assembly of the submembranous actin filament system leading to particle internalization.

Actins↗
PubMed1999

The role of cholesterol and sphigomyelin in tyrosine phosphorylation of proteins and capping of Fcgamma receptor II.

Cross-linking of cell surface receptors by multivalent ligands, e.g. by antibodies, evokes their clustering -- patching. Subsequently, these clusters can be translocated by the acto-myosin machinery toward one pole of the cell and assembly cap. Patching of FcgammaRII in U937 cells correlates with tyrosine phosphorylation of several proteins while cap assembly correlates with their dephosphorylation. To study the mechanism of activation of tyrosine kinases during FcgammaRII activation we disturbed the organization of the putative plasma membrane microdomains by depletion of membrane cholesterol and sphingomyelin. Cholesterol was removed with the use of beta-cyclodextrin while sphingomyelin was decomposed by exogenous sphingomyelinase. Cyclodextrin at 5-10 mM removed about 70% of cholesterol from the cells and abolished the assembly of FcgammaRII caps thereby arresting the receptors at the patching stage. Similarly, 70 mU/ml sphingomyelinase inhibited cap formation by 60%. Cholesterol and sphingomyelin depletion also suppressed the tyrosine phosphorylation of proteins which accompanied cross-linking of FcgammaRII. The observations indicate that cholesterol and sphingomyelin can control the interactions of tyrosine kinases with clustered FcgammaRII.

Cell Membrane↗
PubMed1997

Tyrosine phosphorylation and Fcgamma receptor-mediated phagocytosis.

Phagocytosis of IgG-opsonized particulate material in hematopoietic cells is mediated by Fcgamma receptors (FcgammaRs). Interaction of the receptors with Fc domains of IgG triggers transduction of phagocytic signal in which a key role is played by phosphorylation of tyrosine residues of the receptors. These residues are arranged into a specific motif (immunoreceptor tyrosine-based activation motif; ITAM) which is located either in the cytoplasmic part of FcgammaRIIA or in gamma chains associated with FcgammaRI and FcgammaRIIIA. The conserved tyrosine residues are phosphorylated by, and associate with, tyrosine kinases of Src and Syk families. Coordinated action of these components initiates numerous intracellular events leading finally to local rearrangement of the actin-based cytoskeleton and internalization of the particles.

Phagocytosis↗
PubMed1997

Local accumulation of alpha-spectrin-related protein under plasma membrane during capping and phagocytosis in Acanthamoeba.

During capping and phagocytosis the interaction between cluster cell surface receptors and the submembraneous actin-based skeleton may be mediated by spectrin-like proteins. To test this possibility we examined the localization of an alpha-spectrin immunoanalogue, that had been previously identified in whole extracts of Acanthamoeba, during capping of Con A receptors and during phagocytosis of Con A-coated yeast. During capping alpha-spectrin and filamentous actin co-migrated with the Con A receptors and accumulated in the region of cap formation, as demonstrated by double immunofluorescence studies. Immunoelectron microscopy revealed submembraneous location of alpha-spectrin in cells exposed to Con A, both at the time of initial cross-linking and during accumulation of alpha-spectrin in the region of the cap. Phagocytosis studies showed that alpha-spectrin and actin filaments were concentrated around phagocytic cups that enclosed ConA-coated yeast upon internalization. The proteins also surrounded nascent phagosomes present in the vicinity of the plasma membrane but were absent at the later time point of phagosome maturation. These data demonstrate a correlation between clustering of cell surface receptors and submembraneous localization of alpha-spectrin, suggesting an involvement of spectrin-like proteins in mediating the interaction of receptor clusters with the actin cytoskeleton.

Acanthamoeba↗
PubMed1997

Syk kinase, tyrosine-phosphorylated proteins and actin filaments accumulate at forming phagosomes during Fcgamma receptor-mediated phagocytosis.

Phagocytosis mediated by Fcgamma receptors (FcgammaRs) is thought to be regulated by a cascade of tyrosine phosphorylation events that finally leads to the rearrangement of submembranous actin-based cytoskeleton and internalization of particles. Suggestions concerning the functional relationship between protein tyrosine kinases, their substrates, and actin filament reorganization prompted us to determine cellular distribution of these elements during uptake of IgG-coated particles in murine thio-macrophages. We found that the onset of uptake of the particles was accompanied by tyrosine phosphorylation of several proteins, among which 90, 50, 40, 30, and 25 kDa polypeptides were distinguished. In most of the proteins the tyrosine hyperphosphorylation persisted up to 3 min of the uptake; however, kinetics of the phosphorylation of individual proteins varied. Immunofluorescence data showed that the phosphotyrosine-bearing proteins were localized in regions of the particle uptake, being concentrated at phagocytic cups and nascent phagosomes. The local enrichment in tyrosine phosphorylated proteins was correlated with accumulation of actin filaments at these early stages of phagosome formation. During phagosome maturation, both tyrosine phosphorylated proteins and microfilaments disappeared from the periphagosomal regions. Syk, one of the tyrosine kinases, was translocated to the regions where FcgammaR-mediated phagocytosis had started. On the contrary, no enrichment in phosphatidylinositol 3-kinase was detected in these places.

Actins↗
PubMed1996

Phagocytosis occurs in Acanthamoeba castellanii after electroporation.

Acanthamoeba cells treated with an electric discharge were porated and their cytoplasm became accessible to exogenous molecules. Over a broad range of electric field densities low molecular weight markers (trypan blue, ruthenium red), normally unable to penetrate a plasma membrane, gained access to cytoplasm of 80-90% of the cells. Macromolecules (albumin-FITC and IgG-FITC) penetrated into 63-86% of the cells when electroporation was carried out over the range of 1500V/25 microF-400V/250 microF. Pulse labeling with fluorescent markers evidenced that even 3 hrs. after an electric pulse the plasma membrane was still permeable to exogenous fluorescent probes. Following this stage, the pores were gradually closed. The cells electroporated at 400 V/250 microF were able to ingest yeast particles. The uptake of the particles seems to be an active process since it was inhibited by azide and phalloidin. Therefore, the electroporation of Acanthamoeba makes possible the introduction of macromolecules into the cells and subsequent analysis of their effect on active motile processes such as phagocytosis. This should greatly facilitate characterization of the mechanisms by which such processes do occur.

Acanthamoeba↗
PubMed1993

The effect of annexin IV and VI on the fluidity of phosphatidylserine/phosphatidylcholine bilayers studied with the use of 5-deoxylstearate spin label.

An effect of annexin IV and VI on the fluidity of phosphatidylserine/phosphatidylcholine (PS/PC) membranes was studied by spin labeling technique with the use of 5-doxylstearic acid. It was found that calcium ions at micromolar concentrations induced a marked decrease in the order parameter of PS/PC membranes. This effect was completely abolished by both annexins. The influence of annexins on the mobility of fatty acid chains in the studied region in PE/PC membranes was insignificant.

Annexin A4↗
PubMed1992

Annexins IV (p32) and VI (p68) interact with erythrocyte membrane in a calcium-dependent manner.

Purification of annexin IV and VI from porcine liver was achieved by Mono Q ion exchange chromatography at pH 8.9 and pH 7.5, respectively. The isolated proteins interacted with erythrocyte membrane as function of calcium ion and the protein concentration. Half-maximal binding of annexin VI to erythrocyte membrane was found to occur at 8 microM Ca2+. The maximal binding was estimated as 2 micrograms of annexin VI per 1 microgram or erythrocyte membrane protein, in the presence of 100 microM Ca2+. The property of erythrocyte membrane to interact with annexins was utilized in preparation of a affinity-column with polyacrylamide-immobilized erythrocyte membrane.

Animals↗
PubMed1991

Actin-binding proteins involved in the capping of epidermal growth factor receptors in A431 cells.

A capping process of epidermal growth factor receptors (EGF-Rs) was used for the study of the relation between the receptors and the actin-binding proteins (spectrin, vinculin, annexin I) that may be involved in EGF-R-cytoskeleton interaction. In intact, adherent A431 cells, EGF-Rs were diffusively distributed on the cell surface. Spectrin, vinculin, and annexin I were located beneath the plasma membrane. An abundance of EGF-Rs as well as submembrane proteins was observed in regions of membrane ruffles and cell-cell contacts. Annexin I was localized also in cytoplasm being attached to filamentous structures surrounding the nucleus and extending to the cell periphery. Under polyvalent ligand treatment, EGF-Rs of adherent cells were aggregated on one side of the cell. Spectrin, vinculin, and annexin I dislocated together with EGF-Rs and were concentrated under plasma membrane at regions where cap formation took place. In suspended A431 cells only spectrin was located under the plasma membrane whereas annexin I and vinculin were diffusively distributed through the cells. During cap formation only spectrin was colocalized with EGF-Rs. The results confirmed the major role of spectrin as a receptor-microfilament linking protein.

Actins↗
PubMed1991

Participation of membrane skeleton proteins in aggregation of epidermal growth factor receptors in A431 cells.

Polyclonal antibody against alpha-spectrin of chicken erythrocytes was prepared. This antibody as well as anti-vinculin and anti-annexin I and II, were used for localization of the antigens in A431 cells during translocation of epidermal growth factor receptors (EGF-Rs) on cell surface. During aggregation of EGF-Rs only spectrin and actin aggregates colocalized with the "capped" receptors in adherent as well as in suspended cells. Physiological implication of spectrin involvement in EGF-Rs redistribution in A431 cells is discussed.

Actins↗
PubMed1990

Identification and purification of calpactins from cardiac muscle and their effect on Na+/Ca2+ exchange activity.

Calpactins were purified from bovine cardiac muscle by a slightly modified Glenney et al. procedure (J. Cell. Biol. 104, 503-511, 1987). Two major proteins (apparent MW of 36 and 68 kDa) able to bind phospholipids in a Ca2(+)-dependent manner were identified. These proteins completely reversed the inhibition of Na+/Ca2+ exchange activity of cardiac sarcolemmal vesicles consequent to EGTA-treatment. A modulation of cardiac Na+/Ca2+ exchange activity by calpactins is suggested.

Animals↗
PubMed1990

Alpha-spectrin immunoanalog in Acanthamoeba cells.

A monospecific, affinity purified antibody was prepared against chicken erythrocyte alpha-spectrin. The antibody cross-reacted with only one high molecular weight polypeptide (235 kDa) from whole Acanthamoeba cells. The localization of alpha-spectrin-related antigen in Acanthamoeba cells was examined using immunofluorescence and postembedding cytochemical techniques. Three patterns of distribution of alpha-spectrin immunoanalog were distinguished: as submembranous layer, cytoplasmic aggregates and uniform dispersion through the cytoplasm. Immunoelectron microscopic studies showed that the colloidal gold label was located in the cytoplasm in the vicinity of the plasma membrane. The gold particles were also aggregated around unidentified cytoplasmic filamentous structures. The presence of spectrin-related protein in protozoan cells of Acanthamoeba is in accordance with previous assumptions of the widespread occurrence of spectrin-related proteins. The heterogenous distribution of the immunoanalog of alpha-spectrin protein in Acanthamoeba cells is discussed.

Acanthamoeba↗
PubMed1988

A comparative study: perfusion of the micro- and macrocirculation as a function of the hematocrit value.

The fluidity (the inverse of viscosity) of red blood cell (RBC) suspensions in vivo was estimated by means of microcirculatory measurements such as RBC flow velocity, micropressure in arterioles and venules, and vessel geometry, and by means of simultaneous macrocirculatory measurements such as volume flow and perfusion pressure in the whole organ. These in vivo data were compared to in vitro data obtained by viscometry. The experiments were performed on the isolated rat mesentery perfused with a nonaggregating and an aggregating suspension: Human RBC were either suspended in Ringer's or Ficoll 400 solution. The tissue was perfused at various perfusion pressures and hematocrit values. The microcirculatory perfusion was recorded by means of microscopy using a video TV system. Five major results have been identified: First, microfluidities calculated from velocity data in capillaries of the mesenteric membrane were almost identical to the fluidities calculated from macroflow data (volume flow rates and perfusion pressure in the whole organ). Second, in vivo fluidities of both suspensions decrease with increasing hematocrit value but seem to be independent of driving pressure between 4 and 10 kPa. Third, the fluidity of the Ficoll suspensions is lower than the fluidity of the Ringer's suspensions by the same ratio as the continuous phases. Fourth, the in vivo fluidities of the Ringer's suspensions and the Ficoll suspensions for hematocrit values from 20 to 70% and a range of wall shear stresses from 0.6 to 1 Pa were higher than those measured in vitro. Finally, it has been quantified that an increasing number of vessels became stagnant (that is packed by red blood cells) at high hematocrit values, low perfusion pressure, and increased aggregability of RBC.

Animals↗