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

The development of drug resistance is a major problem which often occurs during anticancer chemotherapies. Photodynamic therapy (PDT) has been studied as an alternative treatment modality for drug-resistant tumors, however the question of resistance to PDT and potential cross-resistance with chemotherapy has yet to be fully answered. To investigate the mechanism of resistance to PDT, we developed an in vitro experimental model system in a mouse mammary carcinoma cell line 4T1. We used two ethylene glycol derivatives of tetraphenylporphyrin, and tetraphenylchlorin derivative, temoporfin, as photosensitizers (PS). PDT-resistant clones were obtained by exposure to a set concentration of PS followed by irradiation with increasing light doses. PDT resistance to soluble glycol porphyrins was mediated mainly by increased drug efflux through ABCB1 (P-glycoprotein) as we demonstrated by specific ABCB1 knockdown experiments, which in turn rescued the sensitivity of resistant cells to PDT. In contrast, resistance raised to temoporfin, which is generally more lipophilic than glycol porphyrins, elicited mechanism based on sequestration of the drug to lysosomes. The resistance that is acquired from a particular PS could be overcome by using a different PS, which is not susceptible to the same mechanism(s) of resistance. Elucidation of the underlying mechanisms in various types of resistance might facilitate improvements in PDT treatment design.

• MeSH
• chemorezistence genetika   MeSH
• ethylenglykoly aplikace a dávkování   chemie   MeSH
• fotochemoterapie MeSH
• fotosenzibilizující látky aplikace a dávkování   chemie   MeSH
• genový knockdown MeSH
• glykoly chemie   MeSH
• lidé MeSH
• mesoporfyriny aplikace a dávkování   chemie   MeSH
• MFC-7 buňky MeSH
• myši MeSH
• nádory mléčné žlázy u zvířat farmakoterapie   genetika   patologie   MeSH
• P-glykoprotein genetika   MeSH
• paclitaxel škodlivé účinky   MeSH
• porfyriny aplikace a dávkování   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ethylenglykoly MeSH
• fotosenzibilizující látky MeSH
• glykoly MeSH
• mesoporfyriny MeSH
• P-glykoprotein MeSH
• paclitaxel MeSH
• porfyriny MeSH
• temoporfin MeSH Prohlížeč

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

Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I. Nucleolar localization of Pontin was confirmed by its detection in nucleolar extracts and by electron microscopy, which revealed Pontin accumulation specifically in the nucleolar fibrillar centers. Pontin localization in the nucleolus was dynamic and Pontin accumulated in large nucleolar dots mainly during S-phase. Pontin concentration in the large nucleolar dots correlated with reduced transcriptional activity of nucleoli. In addition, Pontin was found to associate with RNA polymerase I and to interact in a complex with c-Myc with rDNA sequences indicating that Pontin is involved in the c-Myc-dependent regulation of rRNA synthesis.

• MeSH
• ATPázy spojené s různými buněčnými aktivitami MeSH
• buněčné jadérko enzymologie   ultrastruktura   MeSH
• DNA-helikasy metabolismus   MeSH
• genetická transkripce MeSH
• HeLa buňky MeSH
• lidé MeSH
• RNA ribozomální biosyntéza   MeSH
• RNA-polymerasa I metabolismus   MeSH
• transkripční iniciační komplex Pol1 - proteiny metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• transportní proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ATPázy spojené s různými buněčnými aktivitami MeSH
• DNA-helikasy MeSH
• RNA ribozomální MeSH
• RNA-polymerasa I MeSH
• RUVBL1 protein, human MeSH Prohlížeč
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH
• transportní proteiny MeSH

gamma-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by gamma-tubulin and the regulation of this process are not fully understood. Here we show that there are two gamma-tubulin forms in the brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of a high salt concentration. Both gamma-tubulins co-polymerized with tubulin dimers, and multiple gamma-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against gamma-tubulin and alpha-tubulin revealed interactions of both gamma-tubulin forms with tubulin dimers, irrespective of the size of complexes. We suggest that, besides small and large gamma-tubulin complexes, other molecular gamma-tubulin form(s) exist in brain extracts. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin in both brain extracts and microtubule protein preparations. Post-translational modification(s) of gamma-tubulins might therefore have an important role in the regulation of microtubule nucleation in neuronal cells.

• MeSH
• dimerizace MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• frakcionace buněk MeSH
• mozek - chemie * MeSH
• prasata MeSH
• protein - isoformy MeSH
• tkáňové extrakty chemie   metabolismus   MeSH
• tubulin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein - isoformy MeSH
• tkáňové extrakty MeSH
• tubulin MeSH

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and delta 2-tubulin variants were detected on alpha-tubulin subunits; polyglutamylation was also found on beta-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and delta 2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and delta 2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of alpha- and beta-tubulin molecules, respectively, revealed that 11 isoforms belonged to the alpha-subunit and 11 isoforms to the beta-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several alpha-tubulin isoforms, antibodies against nontyrosinated and delta 2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism.

• MeSH
• acetylace MeSH
• chromatografie iontoměničová MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• genetická variace MeSH
• imunoblotting MeSH
• isoelektrická fokusace MeSH
• jedovaté rostliny * MeSH
• kultivované buňky MeSH
• mikrotubuly metabolismus   ultrastruktura   MeSH
• monoklonální protilátky MeSH
• myši MeSH
• posttranslační úpravy proteinů * MeSH
• tabák metabolismus   MeSH
• tubulin analýza   izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• monoklonální protilátky MeSH
• tubulin MeSH

The expression of vimentin and the phosphorylated variant of high molecular weight neurofilament protein (NF-H) was studied in developing human fetal dorsal root ganglia and spinal cord. The technique used for examination of cryosections was double-label fluorescence with monoclonal antibodies. Both proteins were present in the nerve fibres inside the ganglia of 6- and 8-week-old embryos. During further development the expression of vimentin continued to increase in the satellite cells, but was found to be decreasing in the ganglion cells. Phosphorylated NF-H was found in the processes of ganglion cells, as well as in the perikarya at all developmental stages. In the spinal cord of 6- and 8-week-old embryos, phosphorylated NF-H protein was found in the longitudinal fibres of the marginal layer and in processes of the mantle zone; some of the fibres also contained vimentin. Later the co-expression of the two proteins ceased and vimentin was found only in glial and mesenchymal derivatives. Phosphorylated NF-H was located, at all developmental stages, in the axons of both white and grey matter, but not in the neuronal perikarya. The results indicate that phosphorylation of the NF-H in human dorsal root ganglia starts in the perikarya of the ganglion cells while in the ganglion cells of the spinal cord it takes place in the axons.

• MeSH
• fosforylace MeSH
• gestační stáří MeSH
• lidé MeSH
• mícha embryologie   metabolismus   MeSH
• molekulová hmotnost MeSH
• monoklonální protilátky MeSH
• neurofilamentové proteiny chemie   imunologie   metabolismus   MeSH
• proteiny intermediálních filament metabolismus   MeSH
• specificita protilátek MeSH
• spinální ganglia embryologie   metabolismus   MeSH
• vimentin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• monoklonální protilátky MeSH
• neurofilament protein H MeSH Prohlížeč
• neurofilamentové proteiny MeSH
• proteiny intermediálních filament MeSH
• vimentin MeSH

Expression of intermediate filament proteins was studied in human developing spinal cord using immunoperoxidase and double-label immunofluorescence methods with monoclonal antibodies to vimentin and glial fibrillary acidic protein (GFAP). Vimentin was found in the processes of radial glial cells in 6-week embryos, while GFAP appeared in vimentin-positive astroglial cells at 8-10 weeks. GFAP and vimentin were present in approximately equal amounts in differentiating astrocytes in 23-week spinal cord. In 30-week fetuses, astrocytes reacted strongly for GFAP, while both the reaction intensity and the number of vimentin-positive cells fluctuated predominantly in the grey matter. No clear-cut transition from vimentin to GFAP was noticed during the development of astrocytes. The majority of ependymal cells in 23-week fetuses contained vimentin but only a few of them reacted for GFAP. The expression of vimentin continued during the whole development of the ependymal layer, in contrast to the reactivity for GFAP which disappeared between the 30th week and term.

• MeSH
• barvení a značení MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• exprese genu MeSH
• fluorescenční mikroskopie MeSH
• fluorescenční protilátková technika MeSH
• gestační stáří MeSH
• gliový fibrilární kyselý protein genetika   imunologie   MeSH
• lidé MeSH
• mícha embryologie   metabolismus   MeSH
• monoklonální protilátky MeSH
• těhotenství MeSH
• vimentin genetika   imunologie   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• gliový fibrilární kyselý protein MeSH
• monoklonální protilátky MeSH
• vimentin MeSH

In unfixed cryostat sections of the brains of early postnatal and adult rats, we screened for cells containing vimentin-positive intermediate filaments (VI+-IFs) by applying a panel of four monoclonal antibodies (Mabs VI-01, VI-02, VI-05 and VI-5B3) using indirect immunofluorescence. All of the Mabs stained VI+-IFs in the stromal part of the choroid plexus, in endothelial cells of blood vessels and in meninges in both adult and immature brains, although with varying strength (VI-5B3 and VI-01 stained more strongly than VI-05 and VI-02). In the brain parenchyma of adults, intense staining was mainly localized in ventricular ependymal cells (VI-5B3/VI-01 greater than VI-02/VI-05) and fibrous astrocyte-like cells (FAs). In the immature brain, the ependymal cells were activated in appearance, with evidence of cell enlargement, greater spreading of VI+-IFs within the cytoplasm and more pronounced VI+ cytoplasmic protrusions into the brain parenchyma. VI+-FAs were found near the ependymal and meningeal borders as well as in the white matter tracts of adult brain (VI-5B3/VI-01 greater than VI-05 greater than VI-02). In immature animals, VI+-FAs were less frequently encountered in the forebrain regions, except in and near the subepenydmal layer (in the adjacent parenchyma) as well as in submeningeal layers. Weaker staining was usually elicited by Mabs VI-02 and VI-05. In the cerebellum, Bergmann cell fibers were stained in both age groups. In adults, the most intense fluorescence usually occurred in segments close to the pia (VI-5B3/VI-01 greater than VI-05 greater than VI-02).(ABSTRACT TRUNCATED AT 250 WORDS)

• MeSH
• astrocyty metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• imunohistochemie MeSH
• inbrední kmeny potkanů MeSH
• krysa rodu Rattus MeSH
• monoklonální protilátky MeSH
• mozek anatomie a histologie   metabolismus   MeSH
• vimentin imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• monoklonální protilátky MeSH
• vimentin MeSH