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Topography of signaling molecules on the plasma membrane in the course of mast cell activation
Lebduška, Pavel ; Dráber, Petr (advisor) ; Šebo, Peter (referee) ; Benada, Oldřich (referee) ; Tučková, Ludmila (referee)
presented technique of plasma membrane sheet preparation from nonadherent cells may facilitate research in this field. It must be, however, mentioned that a plastic view ofsignal transduction across the plasma membrane can be achieved only by combination of various mutually complementary approaches. Conclusions Three techniques of lsoladon of plasmr m€mbrane sh$ts from nonadherent BMMC mast cells have bmn developed. one of them, based on edsorption ofl€ukocýes to glass suďace, turned out to be very promlsing md provided many scientifrc datr(article E). Actlvation of RBL m9st c€l|s by FGRI raeptor dimerintion led to increme of Grb2 adaptor content in the Plasma membrane. Ilowever' by contřast to the case of receptor mu|t|merintion, this Grb2 did not sign|ficantly colocallz€ w|th FERI' and' by |mmuno|rbeling of membrane she€ts, distribution of FC5RI wrs not d|stinguishabl€ from the disfibution on nonact|vrted ce|ls (article A). BMMC, In contrast to RBL cells, after multimerization of FaRI did not form larger aggregat€s ofthis r€c€ptor thrn nonact|vat€d cells did. FGRI muldmer|ation led to lts int€rna|iation of comparable intensity rnd overa|l dynemics ln BMMC end RBL cel|s' but loce| redistribut|on of FaRI fundamentďly differed betwcn these two c€|| wes (article E). Established mode| oflrrg€ (8pproxim8t€|y...
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New aspects of the cell submembrane signaling
Heneberg, Petr ; Dráber, Petr (advisor) ; Bilej, Martin (referee) ; Folk, Petr (referee)
This dissertation contributes to elucidation of some mechanisms of the mammalian cell submembrane signaling. Major part of the research was conducted on mast cells and basophils activated via the high affinity IgE receptor, FcεRI, or via the cell surface glycoprotein Thy-1. New roles of actin cytoskeleton in mast cell signaling via FcεRI and Thy-1 are described. Discovery of new transmembrane adaptor protein non-T cell activation linker, NTAL, short time before the initiation of work on the thesis led to the increased attention paid to this protein. Dramatic changes of signaling in mast cells deficient in NTAL, or with up- or down-regulated expression of this protein are described. NTAL was also found to be one of proteins phosphorylated following the Thy-1 aggregation. Spatiotemporal distribution of surface glycoprotein Thy-1 at different levels of resolution and some biochemical properties of cells activated via Thy-1 are depicted. Screen for nonreceptor hitherto unknown protein tyrosine phosphatases in mast cells and basophils was conducted and initial analysis of spatiotemporal distribution and function of phosphatase PTP20 in mast cell signaling was performed. Next, the role of reactive oxygen and nitrogen species in the regulation of mast cell protein tyrosine phosphatases was summarized. New...
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FcεRI and Kit signal to actin cytoskeleton via different pathways
Šimíček, Michal ; Drbal, Karel (referee) ; Dráber, Petr (advisor)
Mast cells are key effector cells of the immune system whose exact physiological functions have been the subject of much debate. They have been mentioned mostly as the drivers of allergic and inflammatory reactions via allergens triggering their high affinity receptors for IgE (FcεRI). However, recent findings implicate their important role also in other innate immune functions like host protection from bacterial and viral infections. Agent recognized by surface receptor initialize various intracellular signaling pathways leading to cell response. Thus, better understanding of signal transduction in mast cells is important in development of new therapeutic approaches. Early phases of mast cell activation mediated by FcεRI and/or Kit, a receptor for stem cell factor (SCF), involve phosphorylation of the transmembrane adaptor protein Non-T cell activation linker (NTAL) and mast cell spreading. Morphological studies of mast cells derived from bone marrow of mice deficient in NTAL (NTAL knock out, NTAL -/-) revealed markedly reduced spreading on fibronectin after stimulation with antigen (Ag) alone or in combination with SCF, when compared to wild type (WT) cells. Subsequent quantification of cell area and analysis of other mophological parameters confirmed these observations. Mast cell activation was...
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Dual role of CD9 protein in mast cell activation
Machyna, Martin ; Dráber, Petr (advisor) ; Černý, Jan (referee)
Mast cells are well known effector cells in immune system. They have been implicated in such important processes as host defense against bacteria, toxins or parasites. However, in some cases they can develop improper reaction against harmless environmental antigens and thus causing allergies. It is therefore essential to understand signaling events that lead to activation of these cells in order to develop new treatment strategies. Newly prepared rat monoclonal antibody of IgG1 subtype raised against murine mast cells was characterized and found suitable for flow cytometry, immunoblotting and immunoprecipitation. Employing of optimized procedure for immunopurification in combination with mass spectrometry led to identification of its target cluster of differentiation (CD)9 protein. CD9 is a member of large protein family called tetraspanins. Functional studies showed that binding of this antibody to mast cells induced degranulation and early activation events such as increased tyrosine phosphorylation and enhanced levels of free cytoplasmic calcium. Interestingly, subsequent activation of these cells via antigen-mediated aggregation of the high-affinity IgE receptor (FcεRI) led to decreased degranulation, calcium response and tyrosine phosphorylation of several substrates. Importantly, anti-CD9 antibody did...
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New signaling pathways involved in mast cell activation and cell membrane repair
Shaik, Gouse Mohiddin ; Dráber, Petr (advisor) ; Černý, Jan (referee) ; Hašek, Jiří (referee)
RESULTS 54 RESULTS AND DISCUSSION Vacuolin-1-induced changes in mast cells morphology In pilot experiments we determined the effect of vacuolin-1 on mast cell morphology. Incubation of RBL-2H3 cells or BMMCs with 10 μM vacuolin-1 in complete culture media for 3 h resulted in appearance of numerous vacuoles in cytosolic compartment of the cells (Fig 1A-D). Size of the vacuolin-1-treated cells rose (Fig 1E and F) as reflected by mean increase in diameter of RBL-2H3 cells from 14.8 ± 0.2 μm (mean ± S.D., n = 3) to 18.8 ± 0.3 μm, and BMMCs from 14.6 ± 0.2 μm to 17.3 ± 0.2 μm. To decide whether formation of vacuoles depends on specific metabolic pathways we examined cells exposed to various concentrations of pharmacological inhibitors and 10 μM vacuolin-1. Data presented in Table 1 show that most of the drugs tested, including Cl- and/or K+ channel blockers [indanyloxyacetic acid 94 (IAA-94), (dihydroindenyl)oxy/alkanoic acid (DIOA), 5- Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'- disulphonate (DIDS) and glybenclamide], protein kinase C (PKC) inhibitor (tamoxifen), phosphoinositid-3 kinase (PI3K) inhibitor (wortmannin), Src and Syk family kinase inhibitors (PP2 and piceatannol) and non-muscle myosin (NMM) II ATPase activity inhibitor (blebbistatin)...
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