RESUMO
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.
Assuntos
Medula Óssea , Proteínas Ativadoras de GTPase , Mastócitos , Microtúbulos , Animais , Camundongos , Centrossomo/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Mastócitos/metabolismo , Microtúbulos/metabolismoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Microtúbulos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , HumanosRESUMO
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.
Assuntos
Mastócitos/metabolismo , Microtúbulos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Quinase Syk/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Degranulação Celular , Células HEK293 , Humanos , Células MCF-7 , Mastócitos/citologia , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidoresRESUMO
γ-Tubulins are highly conserved members of the tubulin superfamily essential for microtubule nucleation. Humans possess 2 γ-tubulin genes. It is thought that γ-tubulin-1 represents a ubiquitous isotype, whereas γ-tubulin-2 is found predominantly in the brain, where it may be endowed with divergent functions beyond microtubule nucleation. The molecular basis of the purported functional differences between γ-tubulins is unknown. We report discrimination of human γ-tubulins according to their electrophoretic and immunochemical properties. In vitro mutagenesis revealed that the differences in electrophoretic mobility originate in the C-terminal regions of the γ-tubulins. Using epitope mapping, we discovered mouse monoclonal antibodies that can discriminate between human γ-tubulin isotypes. Real time quantitative RT-PCR and 2-dimensional-PAGE showed that γ-tubulin-1 is the dominant isotype in fetal neurons. Although γ-tubulin-2 accumulates in the adult brain, γ-tubulin-1 remains the major isotype in various brain regions. Localization of γ-tubulin-1 in mature neurons was confirmed by immunohistochemistry and immunofluorescence microscopy on clinical samples and tissue microarrays. Differentiation of SH-SY5Y human neuroblastoma cells by all-trans retinoic acid, or oxidative stress induced by mitochondrial inhibitors, resulted in upregulation of γ-tubulin-2, whereas the expression of γ-tubulin-1 was unchanged. Fractionation experiments and immunoelectron microscopy revealed an association of γ-tubulins with mitochondrial membranes. These data indicate that in the face of predominant γ-tubulin-1 expression, the accumulation of γ-tubulin-2 in mature neurons and neuroblastoma cells during oxidative stress may denote a prosurvival role of γ-tubulin-2 in neurons.-Dráberová, E., Sulimenko, V., Vinopal, S., Sulimenko, T., Sládková, V., D'Agostino, L., Sobol, M., Hozák, P., Kren, L., Katsetos, C. D., Dráber, P. Differential expression of human γ-tubulin isotypes during neuronal development and oxidative stress points to γ-tubulin-2 prosurvival function.
Assuntos
Neurogênese/fisiologia , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Tubulina (Proteína)/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Microtúbulos/metabolismo , Neuroblastoma/metabolismoRESUMO
Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (ßPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, ßPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of ßPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and ßPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and ßPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, ßPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and ßPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of ßPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/ßPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Microtúbulos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Transformada , Linhagem Celular Tumoral , Centrossomo/metabolismo , Células HEK293 , Humanos , Immunoblotting , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Transdução de Sinais , Tubulina (Proteína)/metabolismo , Quinases Ativadas por p21/genéticaRESUMO
The expression, cellular distribution, and subcellular sorting of the microtubule (MT)-nucleating γ-tubulin small complex (γTuSC) proteins, GCP2 and GCP3, were studied in human glioblastoma cell lines and in clinical tissue samples representing all histologic grades of adult diffuse astrocytic gliomas (n = 54). Quantitative real-time polymerase chain reaction revealed a significant increase in the expression of GCP2 and GCP3 transcripts in glioblastoma cells versus normal human astrocytes; these were associated with higher amounts of both γTuSC proteins. GCP2 and GCP3 were concentrated in the centrosomes in interphase glioblastoma cells, but punctate and diffuse localizations were also detected in the cytosol and nuclei/nucleoli. Nucleolar localization was fixation dependent. GCP2 and GCP3 formed complexes with γ-tubulin in the nucleoli as confirmed by reciprocal immunoprecipitation experiments and immunoelectron microscopy. GCP2 and GCP3 depletion caused accumulation of cells in G2/M and mitotic delay but did not affect nucleolar integrity. Overexpression of GCP2 antagonized the inhibitory effect of the CDK5 regulatory subunit-associated tumor suppressor protein 3 (C53) on DNA damage G2/M checkpoint activity. Tumor cell GCP2 and GCP3 immunoreactivity was significantly increased over that in normal brains in glioblastoma samples; it was also associated with microvascular proliferation. These findings suggest that γTuSC protein dysregulation in glioblastomas may be linked to altered transcriptional checkpoint activity or interaction with signaling pathways associated with a malignant phenotype.
Assuntos
Neoplasias Encefálicas/metabolismo , Nucléolo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Anuros , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/ultraestrutura , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Galinhas , Dano ao DNA/genética , Feminino , Glioblastoma/patologia , Glioblastoma/ultraestrutura , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Pessoa de Meia-Idade , Transporte Proteico , Tubulina (Proteína)/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto Jovem , Peixe-ZebraRESUMO
Ag-mediated activation of mast cells initiates signaling events leading to Ca(2+) response, release of allergic mediators from cytoplasmic granules, and synthesis of cytokines and chemokines. Although microtubule rearrangement during activation has been described, the molecular mechanisms that control their remodeling are largely unknown. Microtubule nucleation is mediated by complexes that are formed by γ-tubulin and γ-tubulin complex proteins. In this study, we report that, in bone marrow-derived mast cells (BMMCs), γ-tubulin interacts with p21-activated kinase interacting exchange factor ß (ßPIX) and G protein-coupled receptor kinase-interacting protein (GIT)1. Microtubule regrowth experiments showed that the depletion of ßPIX in BMMCs stimulated microtubule nucleation, whereas depletion of GIT1 led to the inhibition of nucleation compared with control cells. Phenotypic rescue experiments confirmed that ßPIX and GIT1 represent negative and positive regulators of microtubule nucleation in BMMCs, respectively. Live-cell imaging disclosed that both proteins are associated with centrosomes. Immunoprecipitation and pull-down experiments revealed that an enhanced level of free cytosolic Ca(2+) affects γ-tubulin properties and stimulates the association of GIT1 and γ-tubulin complex proteins with γ-tubulin. Microtubule nucleation also was affected by Ca(2+) level. Moreover, in activated BMMCs, γ-tubulin formed complexes with tyrosine-phosphorylated GIT1. Further experiments showed that GIT1 and ßPIX are involved in the regulation of such important physiological processes as Ag-induced chemotaxis and degranulation. Our study provides for the first time, to our knowledge, a possible mechanism for the concerted action of tyrosine kinases, GIT1/ßPIX proteins, and Ca(2+) in the propagation of signals leading to the regulation of microtubule nucleation in activated mast cells.
Assuntos
Células da Medula Óssea/citologia , Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Mastócitos/citologia , Microtúbulos/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Camundongos , Camundongos Endogâmicos BALB CRESUMO
γ-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that γ-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed γ-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. γ-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear γ-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of γ-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to γ-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of γ-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between γ-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of γ-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2) /M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, γ-tubulin may also have nuclear-specific function(s).
Assuntos
Nucléolo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Tubulina (Proteína)/metabolismo , Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Imunofluorescência , Genes Supressores de Tumor , Glioblastoma/metabolismo , Humanos , Imunoprecipitação , Espectrometria de Massas , Microscopia Imunoeletrônica , Microtúbulos/metabolismo , Transporte Proteico/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Imagem com Lapso de Tempo , Proteínas Supressoras de TumorRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Neoplasias Encefálicas/enzimologia , Movimento Celular/fisiologia , Proliferação de Células , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/enzimologia , Adenosina Trifosfatases/genética , Fatores Etários , Encéfalo/enzimologia , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Contagem de Células/métodos , Linhagem Celular Tumoral , Criança , Feminino , Glioblastoma/patologia , Humanos , Lactente , Masculino , Microtúbulos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Espastina , Adulto JovemRESUMO
BACKGROUND: Literature includes evidence of initial predominance of inflammation and later development of neurodegeneration in the pathogenesis of multiple sclerosis (MS). OBJECTIVE: To search for differences in inflammatory and degenerative cerebrospinal fluid (CSF) markers between relapsing-remitting MS (RRMS) and the clinical isolated syndrome (CIS). METHODS: A total of 148 subjects were evaluated, 65 MS patients (45 RRMS and 20 CIS) and 83 controls. The evaluated parameters included albumin quotient and prealbumin, transferrin, C3 and C4 complement factors, haptoglobin, beta-2-microglobulin, orosomucoid, alpha-1-antitrypsin, apolipoprotein A-I, apolipoprotein B, cystatin C, neuron-specific enolase, tau protein, beta-amyloid, oligoclonal IgG band (OCB), and IgG quotient (QIgG). RESULTS: No differences were found in the inflammatory and degenerative CSF markers between patients with RRMS and CIS. QIgG was higher in RRMS than that in CIS but the number of OCB was higher after CIS. Cystatin C levels were significantly lower in RRMS compared to the other groups. It can be considered an indicator of the demyelination degree. Normal values of beta-amyloid were less frequent in RRMS compared to those in controls.
Assuntos
Doenças Desmielinizantes/líquido cefalorraquidiano , Doenças Desmielinizantes/diagnóstico , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/diagnóstico , Degeneração Neural/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Biomarcadores/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Doenças Desmielinizantes/patologia , Feminino , Humanos , Imunoglobulinas/líquido cefalorraquidiano , Mediadores da Inflamação/líquido cefalorraquidiano , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/patologia , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Valor Preditivo dos Testes , Adulto JovemRESUMO
In previous studies, we have shown overexpression and ectopic subcellular distribution of gamma-tubulin and betaIII-tubulin in human glioblastomas and glioblastoma cell lines (Katsetos et al., 2006, J Neuropathol Exp Neurol 65:455-467; Katsetos et al., 2007, Neurochem Res 32:1387-1398). Here we determined the expression of gamma-tubulin in surgically excised medulloblastomas (n = 20) and in the human medulloblastoma cell lines D283 Med and DAOY. In clinical tissue samples, the immunohistochemical distribution of gamma-tubulin labeling was pervasive and inversely related to neuritogenesis. Overexpression of gamma-tubulin was widespread in poorly differentiated, proliferating tumor cells but was significantly diminished in quiescent differentiating tumor cells undergoing neuritogenesis, highlighted by betaIII-tubulin immunolabeling. By quantitative real-time PCR, gamma-tubulin transcripts for TUBG1, TUBG2, and TUBB3 genes were detected in both cell lines but expression was less prominent when compared with the human glioblastoma cell lines. Immunoblotting revealed comparable amounts of gamma-tubulin and betaIII-tubulin in different phases of cell cycle; however, a larger amount of gamma-tubulin was detected in D283 Med when compared with DAOY cells. Interphase D283 Med cells exhibited predominantly diffuse cytoplasmic gamma-tubulin localization, in addition to the expected centrosome-associated distribution. Robust betaIII-tubulin immunoreactivity was detected in mitotic spindles of DAOY cells. Our data indicate that overexpression of gamma-tubulin may be linked to phenotypic dedifferentiation (anaplasia) and tumor progression in medulloblastomas and may potentially serve as a promising tumor marker.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Meduloblastoma/metabolismo , Tubulina (Proteína)/metabolismo , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Ciclo Celular/fisiologia , Desdiferenciação Celular/fisiologia , Linhagem Celular Tumoral , Centrossomo/metabolismo , Criança , Pré-Escolar , Citoplasma/metabolismo , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Lactente , Masculino , Meduloblastoma/genética , Meduloblastoma/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estudos Retrospectivos , Fuso Acromático/metabolismo , Tubulina (Proteína)/genéticaRESUMO
AIMS: The aim of this study was to assess the correlation between IgG index values and the number of the oligoclonal IgG bands (OCB) in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS). MATERIAL AND METHODS: The set of 150 patients consisted of 41 males and 109 females (aged 18-68, mean 36.6 +/- 10.1 years). The CSF collected by a lumbar puncture was examined evaluating intrathecal synthesis using the IgG index and determining OCB. The number of alkaline OCB in the CSF was assessed using the method of isoelectric focusing. Pearson's correlation analysis, and homogeneity chi(2) test, Mann-Whitney test, paired-sample t-test (parametric) and Wilcoxon signed-ranks test (nonparametric) were used to evaluate the statistical significance of the results. RESULTS: No positive correlation between the IgG index and the number of OCB was found. Mann-Whitney test also failed to demonstrate any significant difference of the IgG index values in patients both with the OCB number > or = 2 and < 2. CONCLUSION: This study did not confirm any correlation between the IgG index values and the OCB number in the CSF of MS patients.