RESUMO
A central issue in stem cell biology is a better understanding of the molecular mechanisms that regulate self-renewal of human hematopoietic stem cells (HSCs). Control of the specific function of HSCs like self-renewal and differentiation might be regulated by a common set of critical genes. However, the regulation among these genes is yet to be elucidated. Here, we show that activation by a novel human GPI-linked glycoprotein ACA at the surface of human peripheral blood progenitor cells induces via PI3K/Akt/mTor/PTEN upregulation of WNT, Notch1, Bmi-1 and HoxB4 genes thus, promoting self-renewal and generation of primitive HSCs. ACA-generated self-renewing cells retained their lympho-myeloid repopulating potential in NOD/SCID mouse xeno-transplantation model with long term functional capacity. We conclude that ACA is an essential regulator of the genes involved in maintaining hematopoiesis and its use in clinical praxis could overcome many of the barriers present so far in transplantation medicine.
Assuntos
Proteínas Sanguíneas/fisiologia , Hematopoese , Glicoproteínas de Membrana/fisiologia , Animais , Antígenos CD34/metabolismo , Proliferação de Células , Células Cultivadas , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Xenoenxertos , Humanos , Leucócitos Mononucleares/fisiologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Processamento de Proteína Pós-Traducional , Regulação para Cima , Via de Sinalização WntRESUMO
The coiled body is a distinct subnuclear domain enriched in small nuclear ribonucleoprotein particles (snRNPs) involved in processing of pre-mRNA. Although the function of the coiled body is still unknown, current models propose that it may have a role in snRNP biogenesis, transport, or recycling. Here we describe that anti-coilin antibodies promote a specific disappearance of the coiled body in living human cells, thus providing a novel tool for the functional analysis of this structure. Monoclonal antibodies (mAbs) were raised against recombinant human coilin, the major structural protein of the coiled body. Four mAbs are shown to induce a progressive disappearance of coiled bodies within approximately 6 h after microinjection into the nucleus of HeLa cells. After their disappearance, coiled bodies are not seen to re-form, although injected cells remain viable for at least 3 d. Epitope mapping reveals that the mAbs recognize distinct amino acid motifs scattered along the complete coilin sequence. By 24 and 48 h after injection of antibodies that promote coiled body disappearance, splicing snRNPs are normally distributed in the nucleoplasm, the nucleolus remains unaffected, and the cell cycle progresses normally. Furthermore, cells devoid of coiled bodies for approximately 24 h maintain the ability to splice both adenoviral pre-mRNAs and transiently overexpressed human beta-globin transcripts. In conclusion, within the time range of this study, no major nuclear abnormalities are detected after coiled body disappearance.
Assuntos
Proteínas Nucleares/metabolismo , Anticorpos Monoclonais/farmacologia , Ciclo Celular/fisiologia , Nucléolo Celular/metabolismo , Mapeamento de Epitopos , Imunofluorescência , Globinas/genética , Células HeLa , Humanos , Microinjeções , Proteínas Nucleares/imunologia , Precursores de RNA/metabolismo , Splicing de RNA/genética , Proteínas Recombinantes/imunologia , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Transcrição Gênica/genéticaRESUMO
Structure determination of macromolecules in solution by nuclear magnetic resonance (NMR) spectroscopy involves the fitting of atomic models to the observed nuclear Overhauser effect (NOE) data. Complete cross-validation has been used to define reliable and unbiased criteria for the quality of solution NMR structures. The method is based on the partitioning of NOE data into test sets and the cross-validation of statistical quantities for each of the test sets. A high correlation between cross-validated measures of fit, such as distance bound violations and NMR R values, and the quality of solution NMR structures was observed. Less complete data resulted in poorer satisfaction of the cross-validated measures of fit. Optimization of cross-validated measures of fit will likely produce solution NMR structures with maximal information content.
Assuntos
Espectroscopia de Ressonância Magnética , Conformação Proteica , Proteínas/química , Inibidores da Tripsina , Proteínas de Bactérias/química , Matemática , Proteínas de Plantas/química , SoluçõesRESUMO
Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro, whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo, that is CD31(+) vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Neovascularização Patológica/metabolismo , Neoplasias Pancreáticas/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/metabolismo , Actinas/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antineoplásicos/farmacologia , Benzamidas , Bevacizumab , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Cetuximab , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Mesilato de Imatinib , Lentivirus/genética , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Nus , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Piperazinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Fator de Crescimento Derivado de Plaquetas/metabolismo , Pirimidinas/farmacologia , Esferoides Celulares/patologia , Transplante Heterólogo , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
BACKGROUND: The oskar (osk) gene encodes a determinant of posterior identity in Drosophila, and the localization of osk RNA to the pole plasm at the posterior pole of the oocyte is essential for development of the embryo. The mechanisms by which osk RNA is localized are unknown. RESULTS: To study the mechanisms underlying localization of osk RNA, we have injected fluorescently labelled RNA into oocytes at stages 9, 10 and 11. Injected osk RNA localizes to the pole plasm, reproducing localization of the endogenous RNA. In oocytes at stages 10 and 11, the long-range movement of injected osk RNA is promoted by a vigorous, microtubule-dependent cytoplasmic flow, or ooplasmic streaming. Treatment with colchicine, a microtubule-destabilizing drug, inhibits ooplasmic streaming and prevents localization of the RNA from an injection site distal to the posterior pole. If the RNA is injected close to the posterior pole, however, it localizes even in the presence of colchicine. Similarly, in small oocytes, such as stage 9 oocytes, localization of injected osk RNA is insensitive to colchicine. CONCLUSIONS: These results reveal that microtubule-dependent cytoplasmic flows could contribute to the long-range transport of osk RNA, whereas microtubule-independent processes could mediate short-range transport. These results also highlight the role of the osk RNA anchor in the localization process.
Assuntos
Proteínas de Drosophila , Proteínas de Insetos/biossíntese , Oócitos/fisiologia , Animais , Colchicina/farmacologia , Drosophila melanogaster , Genes de Insetos , Proteínas de Insetos/fisiologia , Cinética , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oogênese , RNA/metabolismo , Transcrição GênicaRESUMO
c-Jun activation by mitogen-activated protein kinases has been implicated in various cellular signal responses. We investigated how JNK and c-Jun contribute to neuronal differentiation, cell survival, and apoptosis. In differentiated PC12 cells, JNK signaling can induce apoptosis and c-Jun mediates this response. In contrast, we show that in PC12 cells that are not yet differentiated, the AP-1 family member ATF-2 and not c-Jun acts as an executor of apoptosis. In this context c-Jun expression protects against apoptosis and triggers neurite formation. Thus, c-Jun has opposite functions before and after neuronal differentiation. These findings suggest a model in which the balance between ATF-2 and Jun activity in PC12 cells governs the choice between differentiation towards a neuronal fate and an apoptotic program. Further analysis of c-Jun mutants showed that the differentiation response requires functional dimerization and DNA-binding domains and that it is stimulated by phosphorylation in the transactivation domain. In contrast, c-Jun mutants incompetent for DNA binding or dimerization and also mutants lacking JNK binding and phosphorylation sites that cannot elicit neuronal differentiation efficiently protect PC12 cells from apoptosis. Hence, the protective role of c-Jun appears to be mediated by an unconventional mechanism that is separable from its function as a classical AP-1 transcription factor.
Assuntos
Apoptose/fisiologia , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/metabolismo , Fator 2 Ativador da Transcrição , Animais , Tamanho Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Marcação In Situ das Extremidades Cortadas , Proteínas Quinases JNK Ativadas por Mitógeno , Microscopia Confocal , Neurônios/fisiologia , Células PC12 , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Ratos , Fator de Transcrição AP-1/genética , Fatores de Transcrição/metabolismoRESUMO
Although USF-1 and -2 are the major proteins that bind to Myc-regulated E-box (CACGTG) elements in many cells, there is no clear role for USF during Myc-dependent gene regulation. Using dominant negative alleles of USF-1 we now show that DNA binding by USF at a Myc-regulated E-box limits the ability of another E-box binding factor, TFE-3, to activate a target gene of Myc in vivo and to stimulate S phase entry in resting fibroblasts. Similarly, dominant negative alleles of USF-1 relieve the restriction that prevents activation of the IgH enhancer by TFE-3 in non B-cells. DNA binding activity of USF complexes is abundant in primary human B-cells and is significantly downregulated during B-cell immortalization. Re-expression of USF-1 in immortalized B-cells retards proliferation. Our data establish an essential role for USF in restricting E-box dependent gene activation in vivo and suggest that this control is relaxed during cellular immortalization.
Assuntos
Proteínas de Ligação a DNA , DNA/metabolismo , Genes myc , Fatores de Transcrição/metabolismo , Células 3T3 , Animais , Sítios de Ligação/genética , DNA/genética , Regulação da Expressão Gênica , Células HeLa , Sequências Hélice-Alça-Hélice , Humanos , Zíper de Leucina , Camundongos , Ligação Proteica , Fatores de Transcrição/genética , Ativação Transcricional , Fatores Estimuladores UpstreamRESUMO
Activation of Myc triggers a rapid induction of cyclin E/cdk2 kinase activity and degradation of p27. Overt degradation of p27 is preceded by a specific dissociation of p27 from cyclin E/cdk2, but not from cyclin D/cdk4 complexes. We now show that cyclin E/cdk2 phosphorylates p27 at a carboxy-terminal threonine residue (T187) in vitro; mutation of this residue to valine stabilises cyclin E/cdk2 complexes. This reaction is not significantly inhibited by high concentrations of p27, suggesting that cdk2 bound to p27 is catalytically active. In vivo, p27 bound to cyclins E and A, but not to D-type cyclins is phosphorylated. Myc-induced release of p27 from cdk2 requires cdk2 kinase activity and is delayed in a T187V mutant of p27. After induction of Myc, p27 phosphorylated at threonine 187 transiently accumulates in a non cdk2 bound form. Our data suggest a mechanism in which p27 is released from cyclin E/cdk2 upon phosphorylation; in Myc-transformed cells, release is efficient as phosphorylated p27 is transiently bound in a non-cdk2 containing complex and subsequently degraded.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclina E/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Genes myc/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor , Animais , Células Cultivadas , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Camundongos , Fosforilação , CoelhosRESUMO
The 1H-NMR spectrum of the neuropeptide head activator in aqueous solution has been completely assigned by two-dimensional NMR spectroscopy and selective deuteration. The apparent pseudo-first-order exchange rate, kex, of the backbone amide protons and the correspondent activation enthalpies, delta H not equal to, were determined. The exchange rates decrease and the activation enthalpies increase from the N-terminal to the C-terminal part of the peptide. The exchange rates vary from 21 to 0.3 s-1 at 274 K, the activation enthalpies from 60 to 75 kJ.mol-1. The pK values of the terminal carboxyl group and of the lysine amino group have been estimated as 3.3 and 10.3, respectively. The NMR results are in line with a dimeric structure in an antisymmetric arrangement of the subunits, forming an antiparallel beta-pleated sheet between C-terminal segments. The peptide bonds between pGlu-1, Pro-2 and Pro-3 are predominantly in trans-configuration, in fact no cis-isomers can be observed spectroscopically. The structure appears to be very stable; in the temperature and pH range studied, i.e., from 274 to 338 K and from pH 0.8 to pH 11.6, there are no spectroscopic indications for a global structural change.
Assuntos
Neuropeptídeos , Temperatura Alta , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Proteica , Ácido Pirrolidonocarboxílico/análiseRESUMO
The trafficking of intracellular membranes requires the coordination of membrane-cytoskeletal interactions. Rab proteins are key players in the regulation of vesicular transport, while Rho family members control actin-dependent cell functions. We have previously identified a rho protein, rhoD, which is localized to the plasma membrane and early endosomes. When overexpressed, rhoD alters the actin cytoskeleton and plays an important role in endosome organization. We found that a rhoD mutant exerts its effect on early endosome dynamics through an inhibition in organelle motility. In these studies, the effect of rhoD on endosome dynamics was evaluated in the presence of a constitutively active, GTPase-deficient mutant of rab5, rab5Q79L. As rab5Q79L itself stimulates endosome motility, rhoD might counteract this stimulation, without itself exerting any effect in the absence of rab5 activation. We have now addressed this issue by investigating the effect of rhoD in the absence of co-expressed rab5. We find that rhoDG26V alone alters vesicular dynamics. Vesicular movement, in particular the endocytic/recycling circuit, is altered during processes such as cell motility. Due to the participation of vesicular motility and cytoskeletal rearrangements in cell movement and the involvement of rhoD in both, we have addressed the role of rhoD in this process and have found that rhoDG26V inhibits endothelial cell motility.
Assuntos
Citoesqueleto de Actina/metabolismo , Compartimento Celular/fisiologia , Movimento Celular/fisiologia , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Transporte Proteico/fisiologia , Proteínas/metabolismo , Proteínas rho de Ligação ao GTP , Animais , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Cricetinae , Endossomos/ultraestrutura , Endotélio/citologia , Endotélio/metabolismo , Expressão Gênica/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde , Indicadores e Reagentes/farmacocinética , Membranas Intracelulares/ultraestrutura , Proteínas Luminescentes/farmacocinética , Lisossomos/metabolismo , Microscopia de Vídeo , Proteínas/genética , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , TransfecçãoRESUMO
Investigating targeting of the 22 kDa peroxisomal membrane protein (Pmp22p) to the peroxisomal membrane we have confined the targeting signal to amino acid residues 16-37 located in the N-terminal cytoplasmic tail. Comparison of Pmp22p orthologous sequences revealed a conserved motif Y3xL3xP3x(KQN) which might represent the core of this targeting signal not found so far in other Pmps. Fusion of the Pmp22p N-terminal tail to the C-terminal portion of Pmp22p which per se is not targeted to peroxisomes, conveys peroxisomal targeting. These data suggest that Pmp22p is targeted to peroxisomes by a new membrane targeting signal which is necessary and sufficient to target a polypeptide containing two transmembrane spans to peroxisomes.
Assuntos
Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Transporte Biológico , Células CHO , Cricetinae , Proteínas de Fluorescência Verde , Membranas Intracelulares/metabolismo , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , TransfecçãoRESUMO
The flavoenzyme glutathione reductase catalyses electron transfer reactions between two major intracellular redox buffers, namely the NADPH/NADP+ couple and the 2 glutathione/glutathione disulfide couple. On this account, microcrystals of the enzyme were tested as redox probes of intracellular compartments. For introducing protein crystals into human fibroblasts, different methods (microinjection, particle bombardment and optical tweezers) were explored and compared. When glutathione reductase crystals are present in a cytosolic environment, the transition of the yellow Eox form to the orange-red 2-electron reduced charge transfer form, EH2, is observed. Taking into account the midpoint potential of the Eox/EH2 couple, the redox potential of the cytosol was found to be < -270 mV at pH 7.4 and 37 degrees C. As a general conclusion, competent proteins in crystalline--that is signal-amplifying--form are promising probes for studying intracellular events.
Assuntos
Glutationa Redutase/administração & dosagem , Glutationa Redutase/metabolismo , Células Cultivadas , Cor , Cristalização , Citosol/metabolismo , Transporte de Elétrons , Fibroblastos/metabolismo , Glutationa Redutase/isolamento & purificação , Humanos , Indicadores e Reagentes , Microinjeções , OxirreduçãoAssuntos
Microcorpos/fisiologia , Microtúbulos/fisiologia , Animais , Células CHO , Cricetinae , Movimento (Física)RESUMO
Fluorescent probes are of increasing interest in medicinal and biological applications for the elucidation of the structures and functions of healthy as well as tumour cells. The quality of these investigations is determined by the intensity of the fluorescence signal. High dye/carrier ratios give strong signals. However, these are achieved by the occupation of a high number of derivatisation sites and therefore are accompanied by strong structural alterations of the carrier. Hence, polyvalent substances containing a high number of fluorescent dyes would be favourable because they would allow the introduction of many dyes at one position of the compound to be labelled.A large number of different dyes have been investigated to determine the efficiency of coupling to a dendrimer scaffold and the fluorescence properties of the oligomeric dyes, but compounds that fulfil the requirements of both strong fluorescence signals and reactivities are rare. Herein we describe the synthesis and characterisation of dye oligomers containing dansyl-, 7-nitro-2,1,3-benzoxadiazol-4-yl- (NBD), coumarin-343, 5(6)-carboxyfluorescein and sulforhodamine B2 moieties based on polyamidoamine (PAMAM) dendrimers. The PAMAM dendrimers were synthesised by an improved protocol that yielded highly homogeneous scaffolds with up to 128 conjugation sites. When comparing the fluorescent properties of the dye oligomers it was found that only the dansylated dendrimers met the requirements of enhanced fluorescence signals. The dendrimer containing 16 fluorescent dyes was conjugated to the anti-epidermal-growth-factor receptor (EGFR) antibody hMAb425 as a model compound to show the applicability of the dye multimer compounds. This conjugate revealed a preserved immunoreactivity of 54%.We demonstrate the applicability of the dye oligomers to the efficient and applicable labelling of proteins and other large molecules that enables high dye concentrations and therefore high contrasts in fluorescence applications.
Assuntos
Marcadores de Afinidade/química , Anticorpos Monoclonais/química , Corantes Fluorescentes/química , Poliaminas/química , Marcadores de Afinidade/síntese química , Sítios de Ligação , Linhagem Celular Tumoral , Dendrímeros , Receptores ErbB/imunologia , Fluorescência , Corantes Fluorescentes/síntese química , Humanos , Estrutura Molecular , Poliaminas/síntese química , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos , Estereoisomerismo , Fatores de TempoRESUMO
Genetic modification of human bone marrow mesenchymal stem cells (MSC) is highly valuable for their exploitation in basic science and therapeutic applications, for example in cancer. We present here a new, fast and easy-to-use method to enrich a functional population of lentiviral (LV)-transduced MSC expressing enhanced green fluorescent protein (eGFP). We replaced the eGFP gene by a fusion gene of puromycin acetyltransferase and eGFP. Upon LV gene transfer and puromycin selection, we quickly obtained a pure transduced MSC population, in which growth, differentiation capacity and migration preferences were not compromised. Furthermore, we are the first to report the migration velocity of MSC among which 30% were moving and velocity of about 15 mum h(-1) was not altered by LV transduction. Manipulated MSC underwent senescence one passage earlier than non-transduced cells, suggesting the use for therapeutic intervention in early passage numbers. Upon tail vein application in nude mice, the majority of LV-transduced MSC could be detected in human orthotopic pancreatic tumor xenografts and to a minor extent in mouse liver, kidney and lung. Together, LV transduction of genes to MSC followed by puromycin selection is a powerful tool for basic research and improves the therapeutic prospects of MSC as vehicles in gene therapy.
Assuntos
Lentivirus/genética , Células-Tronco Mesenquimais/citologia , Neoplasias Pancreáticas/terapia , Transdução Genética , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/patologiaRESUMO
The two MAP kinases JNK and ERK direct distinct cellular activities even though they share a number of common substrates, including several transcription factors. Here we have compared JNK and ERK signalling during PC12 cell differentiation and investigated how activation of c-Jun by the MAPKs contributes to this cellular response. Exposure to nerve growth factor, or expression of constitutively active MEK1-two treatments which cause differentiation of PC12 cells into a neuronal phenotype-result in activation of ERK-type MAP kinases and phosphorylation of c-Jun on several sites including Ser63 and Ser73. Constitutively activated c-Jun, which mimics the MAPK-phosphorylated form of the protein, can induce neuronal differentiation of PC12 cells independently of upstream signals. Conversely, expression of dominant-negative c-JunbZIP prevents neurite outgrowth induced by activated MEK1. Activation of MEKK1, which stimulates the JNK pathway, is not sufficient for PC12 cell differentiation but can induce apoptosis. However, neurite outgrowth is triggered when c-Jun is co-expressed with activated MEKK1 or SEK1. Consistently, MEK-induced ERK activation in PC12 cells induces c-Jun expression, while JNK signalling does not. Therefore, dual input of expression and phosphorylation of c-Jun provided by the ERK pathway is required to direct neuronal differentiation in PC12 cells.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteínas Quinases Ativadas por Mitógeno , Células PC12/enzimologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Diferenciação Celular/fisiologia , Ativação Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno , Neuritos/enzimologia , Neuritos/fisiologia , Neurônios/citologia , Neurônios/enzimologia , Células PC12/metabolismo , Células PC12/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/fisiologia , Ratos , Transdução de SinaisRESUMO
Mature glomerular visceral epithelial cells, or podocytes, are unique cells with a complex cell architecture. Characteristically, they possess a highly branched array of major processes and foot processes, which are essential for glomerular filtration in the kidney. A podocyte cell line with the potential to exhibit many features of differentiated podocytes, particularly the formation of cell processes, was recently established. In this study, it is shown that directed membrane transport is involved in process formation in cultured podocytes. The well-characterized vesicular stomatitis virus G was used as a marker protein for the biosynthetic pathway in these cells. It seems that newly synthesized vesicular stomatitis virus G is preferentially delivered into the cell processes of the podocytes, where it is colocalized with known regulators of vesicular transport from the Golgi apparatus to the plasma membrane, such as the small GTPase rab8 and the sec6/sec8 complex. To determine the role of vesicular transport in process formation, cells were treated with brefeldin A, a drug that disrupts the trafficking of post-Golgi transport vesicles. As a result, the podocytes reversibly lost their ability to form processes. These findings suggest that podocytes are dependent on a constant fresh source of lipids and proteins to form their processes.
Assuntos
Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Vírus da Estomatite Vesicular Indiana/metabolismo , Proteínas Virais/farmacocinética , Proteínas rab de Ligação ao GTP , Animais , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/fisiologia , Membranas Intracelulares/metabolismo , Glomérulos Renais/fisiologia , Proteínas de Membrana , Camundongos , Camundongos TransgênicosRESUMO
Neuronal differentiation of PC12 cells is achieved by stimulation with nerve growth factor (NGF) but not by epidermal growth factor (EGF). However, features of differentiation such as neurite outgrowth are observable at the earliest after several hours. Using actin staining of the cells, we show here that NGF stimulation leads to lamellipodia formation within only 3 min at the periphery of the PC12 cells. EGF stimulation or microinjection of differentiation-inducing c-Crk I protein does not cause lamellipodia. The actin reorganization after NGF stimulation is blocked by microinjecting dominant negative Rac protein. The lamellipodia formation is also abolished by inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY 294002 in a concentration-dependent manner. Phase-contrast time-lapse microscopy was used to analyze membrane dynamics in real time and to confirm the induction of lamellipodia by NGF and their inhibition by pretreatment with both wortmannin and LY 294002. The results indicate that NGF, but not EGF, leads to rapid lamellipodia formation in PC12 cells via phosphatidylinositol 3-kinase and the small GTPase Rac, thereby defining a novel role for these factors in early NGF signaling.
Assuntos
Citoplasma/fisiologia , Fator de Crescimento Neural/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Androstadienos/farmacologia , Animais , Cromonas/farmacologia , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Dimetil Sulfóxido/farmacologia , Inibidores Enzimáticos/farmacologia , Microinjeções , Morfolinas/farmacologia , Células PC12 , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-crk , Ratos , Proteínas Recombinantes/metabolismo , WortmaninaRESUMO
Cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. In human cells, cdc25 proteins are encoded by a multigene family, consisting of cdc25A, cdc25B and cdc25C. While cdc25A plays a crucial role at the G1/S phase transition, cdc25C is involved in the dephosphorylation and activation of the mitotic kinase, cdc2/cyclinB. In addition, cdc25C itself is regulated by cdc2/cyclinB which then creates a positive feedback loop that controls entry into mitosis. In this study we show that the activity of cdc25B appears during late S phase and peaks during G2 phase. Both in vitro and in vivo cdc25B is activated through phosphorylation during S-phase. Using a cell duplication, microinjection assay we show that ablation of cdc25B function by specific antibodies blocks cell cycle progression in Hs68 cells by inhibition of entry into mitosis. Cdc25B function neither plays a role in later stages of mitosis nor for the inititation of DNA replication. These results indicate that cdc25B is a mitotic regulator that might act as a 'starter phosphatase' to initiate the positive feedback loop at the entry into M phase.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiologia , Mitose/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Fosfatases cdc25 , Anticorpos/administração & dosagem , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/imunologia , Linhagem Celular , Ativação Enzimática , Retroalimentação , Células HeLa , Humanos , Técnicas In Vitro , Microinjeções , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/imunologia , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In NIH3T3 fibroblasts, the ubiquitous helix-loop-helix (HLH) protein E2A (E12/E47) and the myogenic HLH proteins MyoD, MRF4 and myogenin are growth-inhibitory, while two ubiquitous Id proteins lacking the basic region are not. The dimerization domain mediates inhibition. However, in addition to the HLH region, E2A contains two inhibitory regions over-lapping with the main transcriptional activation domains. The growth-suppressive activity of the intact E47 as well as MyoD was counteracted by the Id proteins. When E47 lacking the HLH domain was overexpressed, Id could no longer reverse growth inhibition. By increasing the amount of E47 with an inducible system or neutralizing the endogenous Id with microinjected anti-Id antibodies, withdrawal from the cell cycle occurred within hours before the G1-S transition point. The combined results suggest that the Id proteins are required for G1 progression. The antagonism between the E2A and Id proteins further suggests that both are involved in regulatory events prior to or near the restriction point in the G1 phase of the cell cycle.