Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

• Klíčová slova
• G protein-coupled receptor kinase-interacting protein 2 (GIT2), centrosome, mast cells, microtubule nucleation, protein kinase C (PKC),
• MeSH
• centrozom metabolismus   MeSH
• kostní dřeň * MeSH
• mastocyty * metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• myši MeSH
• proteiny aktivující GTPasu * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Git2 protein, mouse MeSH Prohlížeč
• proteiny aktivující GTPasu * MeSH

ER distribution depends on microtubules, and ER homeostasis disturbance activates the unfolded protein response resulting in ER remodeling. CDK5RAP3 (C53) implicated in various signaling pathways interacts with UFM1-protein ligase 1 (UFL1), which mediates the ufmylation of proteins in response to ER stress. Here we find that UFL1 and C53 associate with γ-tubulin ring complex proteins. Knockout of UFL1 or C53 in human osteosarcoma cells induces ER stress and boosts centrosomal microtubule nucleation accompanied by γ-tubulin accumulation, microtubule formation, and ER expansion. C53, which is stabilized by UFL1, associates with the centrosome and rescues microtubule nucleation in cells lacking UFL1. Pharmacological induction of ER stress by tunicamycin also leads to increased microtubule nucleation and ER expansion. Furthermore, tunicamycin suppresses the association of C53 with the centrosome. These findings point to a novel mechanism for the relief of ER stress by stimulation of centrosomal microtubule nucleation.

• Klíčová slova
• CDK5RAP3, ER stress, UFL1, microtubule nucleation, γ-tubulin,
• MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• nádorové supresorové proteiny metabolismus   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• stres endoplazmatického retikula fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK5RAP3 protein, human MeSH Prohlížeč
• nádorové supresorové proteiny MeSH
• proteiny buněčného cyklu MeSH

Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.

• MeSH
• aktiny metabolismus   MeSH
• Caco-2 buňky MeSH
• centrozom metabolismus   MeSH
• forminy metabolismus   MeSH
• genový knockout MeSH
• lidé MeSH
• melanom experimentální metabolismus   patologie   MeSH
• mikrofilamentové proteiny metabolismus   MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• nádory kůže metabolismus   patologie   MeSH
• polymerizace MeSH
• profiliny genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• transfekce MeSH
• tubulin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aktiny MeSH
• forminy MeSH
• mikrofilamentové proteiny MeSH
• PFN1 protein, human MeSH Prohlížeč
• Pfn1 protein, mouse MeSH Prohlížeč
• profiliny MeSH
• tubulin MeSH

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

• Klíčová slova
• SHP-1 tyrosine phosphatase, bone marrow-derived mast cells, cell activation, microtubule nucleation, γ-tubulin complexes,
• MeSH
• degranulace buněk MeSH
• HEK293 buňky MeSH
• kinasa Syk metabolismus   MeSH
• lidé MeSH
• mastocyty cytologie   metabolismus   MeSH
• MFC-7 buňky MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• tubulin metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 6 antagonisté a inhibitory   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kinasa Syk MeSH
• Ptpn6 protein, mouse MeSH Prohlížeč
• Syk protein, mouse MeSH Prohlížeč
• tubulin MeSH
• tyrosinfosfatasa nereceptorového typu 6 MeSH

Mast cells play an effector role in innate immunity, allergy, and inflammation. Antigen-mediated activation of mast cells initiates signaling events leading to Ca2+ response and the release of inflammatory and allergic mediators from granules. Diseases associated with deregulated mast cell functions are hard to treat and there is an increasing demand for new therapeutic strategies. Miltefosine (hexadecylphosphocholine) is a new candidate for treatment of mast cell-driven diseases as it inhibits activation of mast cells. It has been proposed that miltefosine acts as a lipid raft modulator through its interference with the structural organization of surface receptors in the cell membrane. However, molecular mechanisms of its action are not fully understood. Here, we report that in antigen-activated bone marrow-derived mast cells (BMMCs), miltefosine inhibits degranulation, reorganization of microtubules, as well as antigen-induced chemotaxis. While aggregation and tyrosine phosphorylation of IgE receptors were suppressed in activated cells pre-treated with miltefosine, overall tyrosine phosphorylation levels of Lyn and Syk kinases, and Ca2+ influx were not inhibited. In contrast, lipid raft disruptor methyl-β-cyclodextrin attenuated the Ca2+ influx. Tagged-miltefosine rapidly localized into the cell interior, and live-cell imaging of BMMCs with labeled intracellular granules disclosed that miltefosine inhibited movement of some granules. Immunoprecipitation and in vitro kinase assays revealed that miltefosine inhibited Ca2+- and diacylglycerol-regulated conventional protein kinase C (cPKC) isoforms that are important for mast cell degranulation. Inhibition of cPKCs by specific inhibitor Ly333531 affected activation of BMMCs in the same way as miltefosine. Collectively, our data suggest that miltefosine modulates mast cells both at the plasma membrane and in the cytosol by inhibition of cPKCs. This alters intracellular signaling pathway(s) directed to microtubules, degranulation, and migration.

• Klíčová slova
• bone marrow-derived mast cells, cell activation, microtubules, miltefosine, protein kinase C,
• Publikační typ
• časopisecké články MeSH

UNLABELLED: Transformation of rodent cells with avian Rous sarcoma virus (RSV) opened new ways to studying virus integration and expression in nonpermissive cells. We were interested in (i) the molecular changes accompanying fusion of RSV-transformed mammalian cells with avian cells leading to virus rescue and (ii) enhancement of this process by retroviral gene products. The RSV-transformed hamster RSCh cell line was characterized as producing only a marginal amount of env mRNA, no envelope glycoprotein, and a small amount of unprocessed Gag protein. Egress of viral unspliced genomic RNA from the nucleus was hampered, and its stability decreased. Cell fusion of the chicken DF-1 cell line with RSCh cells led to production of env mRNA, envelope glycoprotein, and processed Gag and virus-like particle formation. Proteosynthesis inhibition in DF-1 cells suppressed steps leading to virus rescue. Furthermore, new aberrantly spliced env mRNA species were found in the RSCh cells. Finally, we demonstrated that virus rescue efficiency can be significantly increased by complementation with the env gene and the highly expressed gag gene and can be increased the most by a helper virus infection. In summary, Env and Gag synthesis is increased after RSV-transformed hamster cell fusion with chicken fibroblasts, and both proteins provided in trans enhance RSV rescue. We conclude that the chicken fibroblast yields some factor(s) needed for RSV replication, particularly Env and Gag synthesis, in nonpermissive rodent cells. IMPORTANCE: One of the important issues in retrovirus heterotransmission is related to cellular factors that prevent virus replication. Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, is able to infect and transform mammalian cells; however, such transformed cells do not produce infectious virus particles. Using the well-defined model of RSV-transformed rodent cells, we established that the lack of virus replication is due to the absence of chicken factor(s), which can be supplemented by cell fusion. Cell fusion with permissive chicken cells led to an increase in RNA splicing and nuclear export of specific viral mRNAs, as well as synthesis of respective viral proteins and production of virus-like particles. RSV rescue by cell fusion can be potentiated by in trans expression of viral genes in chicken cells. We conclude that rodent cells lack some chicken factor(s) required for proper viral RNA processing and viral protein synthesis.

• MeSH
• fúze buněk MeSH
• genové produkty env genetika   metabolismus   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• křečci praví MeSH
• kur domácí MeSH
• nemoci drůbeže virologie   MeSH
• ptačí sarkom virologie   MeSH
• testy genetické komplementace MeSH
• transformované buněčné linie MeSH
• virová transformace buněk MeSH
• virus Rousova sarkomu genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env MeSH
• genové produkty gag MeSH

Mast cell activation mediated by the high affinity receptor for IgE (FcεRI) is a key event in allergic response and inflammation. Other receptors on mast cells, as c-Kit for stem cell factor and G protein-coupled receptors (GPCRs) synergistically enhance the FcεRI-mediated release of inflammatory mediators. Activation of various signaling pathways in mast cells results in changes in cell morphology, adhesion to substrate, exocytosis, and migration. Reorganization of cytoskeleton is pivotal in all these processes. Cytoskeletal proteins also play an important role in initial stages of FcεRI and other surface receptors induced triggering. Highly dynamic microtubules formed by αβ-tubulin dimers as well as microfilaments build up from polymerized actin are affected in activated cells by kinases/phosphatases, Rho GTPases and changes in concentration of cytosolic Ca(2+). Also important are nucleation proteins; the γ-tubulin complexes in case of microtubules or Arp 2/3 complex with its nucleation promoting factors and formins in case of microfilaments. The dynamic nature of microtubules and microfilaments in activated cells depends on many associated/regulatory proteins. Changes in rigidity of activated mast cells reflect changes in intermediate filaments build up from vimentin. This review offers a critical appraisal of current knowledge on the role of cytoskeleton in mast cells signaling.

• Klíčová slova
• actins, intermediate filaments, mast cell activation, microfilaments, microtubules, signal transduction, tubulins, vimentin,
• Publikační typ
• časopisecké články MeSH

γ-Tubulin is the key protein for microtubule nucleation. Duplication of the γ-tubulin gene occurred several times during evolution, and in mammals γ-tubulin genes encode proteins which share ∼97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that γ-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that γ-tubulin 1 represents ubiquitous γ-tubulin, while γ-tubulin 2 may have some specific functions and cannot substitute for γ-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between γ-tubulins remains unknown. Here we show that exogenous γ-tubulin 2 is targeted to centrosomes and interacts with γ-tubulin complex proteins 2 and 4. Depletion of γ-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in γ-tubulin 1-depleted cells is restored by expression of exogenous mouse or human γ-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that γ-tubulin 2 cannot rescue γ-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that γ-tubulin 2 is able to nucleate microtubules and substitute for γ-tubulin 1. We propose that mammalian γ-tubulins are functionally redundant with respect to the nucleation activity.

• MeSH
• časové faktory MeSH
• down regulace * MeSH
• embryonální vývoj genetika   MeSH
• implantace embrya MeSH
• intracelulární prostor metabolismus   MeSH
• lidé MeSH
• mikrotubuly metabolismus   MeSH
• mitóza genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protein - isoformy nedostatek   genetika   metabolismus   MeSH
• transport proteinů MeSH
• tubulin nedostatek   genetika   metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein - isoformy MeSH
• tubulin MeSH

We studied the expression and distribution of the microtubule-severing enzyme spastin in 3 human glioblastoma cell lines (U87MG, U138MG, and T98G) and in clinical tissue samples representative of all grades of diffuse astrocytic gliomas (n = 45). In adult human brains, spastin was distributed predominantly in neuronsand neuropil puncta and, to a lesser extent, in glia. Compared with normal mature brain tissues, spastin expression and cellular distribution were increased in neoplastic glial phenotypes, especiallyin glioblastoma (p < 0.05 vs low-grade diffuse astrocytomas). Overlapping punctate and diffuse patterns of localization wereidentified in tumor cells in tissues and in interphase and mitotic cells ofglioblastoma cell lines. There was enrichment of spastin in the leading edges of cells in T98G glioblastoma cell cultures and in neoplastic cell populations in tumor specimens. Real-time polymerase chain reaction and immunoblotting experiments revealed greater levels of spastin messenger RNA and protein expression in theglioblastoma cell lines versus normal human astrocytes. Functional experiments indicated that spastin depletion resulted in reduced cell motility and higher cell proliferation of T98G cells. Toour knowledge, this is the first report of spastin involvement incellmotility. Collectively, our results indicate that spastinexpression in glioblastomas might be linked to tumor cell motility, migration, and invasion.

• MeSH
• adenosintrifosfatasy genetika   metabolismus   MeSH
• dítě MeSH
• glioblastom enzymologie   patologie   MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mikrotubuly MeSH
• mladý dospělý MeSH
• mozek enzymologie   patologie   MeSH
• nádorové buněčné linie MeSH
• nádory mozku enzymologie   patologie   MeSH
• počet buněk metody   MeSH
• pohyb buněk fyziologie   MeSH
• proliferace buněk * MeSH
• regulace genové exprese u nádorů fyziologie   MeSH
• spastin MeSH
• věkové faktory MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• messenger RNA MeSH
• SPAST protein, human MeSH Prohlížeč
• spastin MeSH

BACKGROUND: The function of the cortical microtubules, composed of alphabeta-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited. RESULTS: Using homology modeling we have generated an Arabidopsis thaliana microtubule protofilament model that served for the prediction of surface exposure of five beta-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (beta1-10), TUB2.1 (beta426-435) and TU-14 (beta436-445). Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of beta-tubulins (behind beta425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that beta-tubulins could be one of the targets for tyrosine kinases. CONCLUSIONS: We predicted surface exposure of five beta-tubulin epitopes, as well as tyrosine residues, on the surface of A. thaliana microtubule protofilament model, and validated the obtained results by immunofluorescence microscopy on cortical microtubules in cells.The results suggest that prediction of epitope exposure on microtubules by means of homology modeling combined with site-directed antibodies can contribute to a better understanding of the interactions of plant microtubules with associated proteins.

• MeSH
• Arabidopsis imunologie   MeSH
• epitopy imunologie   MeSH
• fluorescenční mikroskopie MeSH
• mapování epitopu metody   MeSH
• mikrotubuly imunologie   MeSH
• molekulární modely MeSH
• monoklonální protilátky imunologie   MeSH
• proteiny huseníčku imunologie   MeSH
• tubulin imunologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• epitopy MeSH
• monoklonální protilátky MeSH
• proteiny huseníčku MeSH
• tubulin MeSH