Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

• Klíčová slova
• FRAP, FcεRI signaling, STIM1-ORAI1 coupling, alkanol, flow-FRET, heat shock response, membrane fluidizer, store-operated calcium entry,
• MeSH
• cholesterol MeSH
• cytokiny MeSH
• heptanol MeSH
• mastocyty * MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cholesterol MeSH
• cytokiny MeSH
• heptanol MeSH

The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET) efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients K(D,A) > 10) for the determination of pore sizes with radii comparable to the Förster radius R(0). On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.

• MeSH
• fluorescenční barviva chemie   farmakokinetika   MeSH
• iontové kanály chemie   metabolismus   MeSH
• lipidové dvojvrstvy chemie   metabolismus   MeSH
• membránové mikrodomény chemie   metabolismus   MeSH
• metoda Monte Carlo MeSH
• rezonanční přenos fluorescenční energie * MeSH
• teoretické modely MeSH
• tkáňová distribuce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva MeSH
• iontové kanály MeSH
• lipidové dvojvrstvy MeSH

Transmembrane adaptor proteins are membrane-anchored proteins consisting of a short extracellular part, a transmembrane domain, and a cytoplasmic part with various protein-protein interaction motifs but lacking any enzymatic activity. They participate in the regulation of various signaling pathways by recruiting other proteins to the proximity of cellular membranes where the signaling is often initiated and propagated. In this work, we show that LST1/A, an incompletely characterized protein encoded by MHCIII locus, is a palmitoylated transmembrane adaptor protein. It is expressed specifically in leukocytes of the myeloid lineage, where it localizes to the tetraspanin-enriched microdomains. In addition, it binds SHP-1 and SHP-2 phosphatases in a phosphotyrosine-dependent manner, facilitating their recruitment to the plasma membrane. These data suggest a role for LST1/A in negative regulation of signal propagation.

• MeSH
• buněčná membrána metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• hlavní histokompatibilní komplex fyziologie   MeSH
• intracelulární signální peptidy a proteiny MeSH
• Jurkat buňky MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myeloidní buňky cytologie   metabolismus   MeSH
• plakiny metabolismus   MeSH
• primární buněčná kultura MeSH
• pseudopodia metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce fyziologie   MeSH
• terciární struktura proteinů fyziologie   MeSH
• transport proteinů fyziologie   MeSH
• tyrosinfosfatasa nereceptorového typu 11 metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 6 metabolismus   MeSH
• U937 buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• intracelulární signální peptidy a proteiny MeSH
• LST1 protein, human MeSH Prohlížeč
• membránové proteiny MeSH
• plakiny MeSH
• PTPN11 protein, human MeSH Prohlížeč
• PTPN6 protein, human MeSH Prohlížeč
• tyrosinfosfatasa nereceptorového typu 11 MeSH
• tyrosinfosfatasa nereceptorového typu 6 MeSH

In spite of a comprehensive understanding of the schematics of T cell receptor (TCR) signaling, the mechanisms regulating compartmentalization of signaling molecules, their transient interactions, and rearrangement of membrane structures initiated upon TCR engagement remain an outstanding problem. These gaps in our knowledge are exemplified by recent data demonstrating that TCR triggering is largely dependent on a preactivated pool of Lck concentrated in T cells in a specific type of membrane microdomains. Our current model posits that in resting T cells all critical components of TCR triggering machinery including TCR/CD3, Lck, Fyn, CD45, PAG, and LAT are associated with distinct types of lipid-based microdomains which represent the smallest structural and functional units of membrane confinement able to negatively control enzymatic activities and substrate availability that is required for the initiation of TCR signaling. In addition, the microdomains based segregation spatially limits the interaction of components of TCR triggering machinery prior to the onset of TCR signaling and allows their rapid communication and signal amplification after TCR engagement, via the process of their coalescence. Microdomains mediated compartmentalization thus represents an essential membrane organizing principle in resting T cells. The integration of these structural and functional aspects of signaling into a unified model of TCR triggering will require a deeper understanding of membrane biology, novel interdisciplinary approaches and the generation of specific reagents. We believe that the fully integrated model of TCR signaling must be based on membrane structural network which provides a proper environment for regulatory processes controlling TCR triggering.

• Klíčová slova
• Fyn, Lck, TCR triggering, compartmentalization, heavy and light DRMs, membrane microdomains, spatio-temporal regulation,
• Publikační typ
• časopisecké články MeSH

Formation of the immunological synapse between an antigen-presenting cell (APC) and a T cell leads to signal generation in both cells involved. In T cells, the lipid raft-associated transmembrane adaptor protein LAT plays a central role. Its phosphorylation is a crucial step in signal propagation, including the calcium response and mitogen-activated protein kinase activation, and largely depends on its association with the SLP76 adaptor protein. Here we report the discovery of a new palmitoylated transmembrane adaptor protein, termed SCIMP. SCIMP is expressed in B cells and other professional APCs and is localized in the immunological synapse due to its association with tetraspanin-enriched microdomains. In B cells, it is constitutively associated with Lyn kinase and becomes tyrosine phosphorylated after major histocompatibility complex type II (MHC-II) stimulation. When phosphorylated, SCIMP binds to the SLP65 adaptor protein and also to the inhibitory kinase Csk. While the association with SLP65 initiates the downstream signaling cascades, Csk binding functions as a negative regulatory loop. The results suggest that SCIMP is involved in signal transduction after MHC-II stimulation and therefore serves as a regulator of antigen presentation and other APC functions.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• aktivace lymfocytů MeSH
• antigen prezentující buňky imunologie   MeSH
• B-lymfocyty imunologie   metabolismus   MeSH
• C-terminální Src kinasa MeSH
• fosfoproteiny metabolismus   MeSH
• HEK293 buňky MeSH
• imunologické synapse chemie   MeSH
• lidé MeSH
• malá interferující RNA MeSH
• membránové mikrodomény chemie   metabolismus   MeSH
• membránové proteiny chemie   genetika   imunologie   metabolismus   MeSH
• MHC antigeny II. třídy imunologie   metabolismus   MeSH
• mitogenem aktivované proteinkinasy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• prezentace antigenu MeSH
• RNA interference MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu metabolismus   MeSH
• src homologní domény MeSH
• T-lymfocyty imunologie   MeSH
• tyrosinkinasy chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• B cell linker protein MeSH Prohlížeč
• C-terminální Src kinasa MeSH
• CSK protein, human MeSH Prohlížeč
• fosfoproteiny MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• malá interferující RNA MeSH
• membránové proteiny MeSH
• MHC antigeny II. třídy MeSH
• mitogenem aktivované proteinkinasy MeSH
• SCIMP protein, human MeSH Prohlížeč
• skupina kinas odvozených od src-genu MeSH
• SLP-76 signal Transducing adaptor proteins MeSH Prohlížeč
• tyrosinkinasy MeSH

Bordetella adenylate cyclase toxin (CyaA) binds the alpha(M)beta(2) integrin (CD11b/CD18, Mac-1, or CR3) of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC) enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+) influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

• MeSH
• adenylátcyklasový toxin chemie   metabolismus   MeSH
• antigeny CD11b metabolismus   MeSH
• antigeny CD18 metabolismus   MeSH
• Bordetella enzymologie   MeSH
• buněčná membrána enzymologie   mikrobiologie   MeSH
• cholesterol metabolismus   MeSH
• cytosol enzymologie   MeSH
• extracelulární prostor metabolismus   MeSH
• lidé MeSH
• makrofágový antigen 1 metabolismus   MeSH
• makrofágy metabolismus   mikrobiologie   MeSH
• membránové mikrodomény enzymologie   mikrobiologie   MeSH
• myši MeSH
• talin metabolismus   MeSH
• terciární struktura proteinů MeSH
• U937 buňky MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasový toxin MeSH
• antigeny CD11b MeSH
• antigeny CD18 MeSH
• cholesterol MeSH
• ITGAM protein, human MeSH Prohlížeč
• makrofágový antigen 1 MeSH
• talin MeSH
• vápník MeSH

The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes, this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (Fc epsilonRI). However, the exact molecular mechanism of this phosphorylation step is incompletely understood. In this study, we tested the hypothesis that changes in activity and/or topography of protein-tyrosine phosphatases (PTPs) could play a major role in the Fc epsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, H(2)O(2) or pervanadate, induced phosphorylation of the Fc epsilonRI subunits, similarly as Fc epsilonRI triggering. Interestingly, and in sharp contrast to antigen-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of Fc epsilonRI with detergent-resistant membranes and in the topography of Fc epsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine of several PTPs was detected. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with the actin cytoskeleton. Several PTPs (SHP-1, SHP-2, hematopoietic PTP, and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data, we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the Fc epsilonRI and other multichain immune receptors.

• MeSH
• aktivace enzymů účinky léků   genetika   imunologie   MeSH
• antigeny imunologie   metabolismus   farmakologie   MeSH
• fosforylace účinky léků   genetika   imunologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu Rattus MeSH
• mastocyty imunologie   metabolismus   MeSH
• membránové mikrodomény genetika   imunologie   metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce účinky léků   MeSH
• oxidancia farmakologie   MeSH
• peroxid vodíku farmakologie   MeSH
• receptory IgE genetika   imunologie   metabolismus   MeSH
• skupina kinas odvozených od src-genu genetika   imunologie   metabolismus   MeSH
• transport proteinů účinky léků   genetika   imunologie   MeSH
• tyrosinfosfatasy antagonisté a inhibitory   genetika   imunologie   metabolismus   MeSH
• vanadáty farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• inhibitory enzymů MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• oxidancia MeSH
• peroxid vodíku MeSH
• pervanadate MeSH Prohlížeč
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinfosfatasy MeSH
• vanadáty MeSH