RESUMEN
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.
Asunto(s)
Proteínas Sanguíneas/fisiología , Hematopoyesis , Glicoproteínas de Membrana/fisiología , Animales , Antígenos CD34/metabolismo , Proliferación Celular , Células Cultivadas , Sangre Fetal/citología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/fisiología , Xenoinjertos , Humanos , Leucocitos Mononucleares/fisiología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Procesamiento Proteico-Postraduccional , Regulación hacia Arriba , Vía de Señalización WntRESUMEN
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.
Asunto(s)
Marcadores de Afinidad/química , Anticuerpos Monoclonales/química , Colorantes Fluorescentes/química , Poliaminas/química , Marcadores de Afinidad/síntesis química , Sitios de Unión , Línea Celular Tumoral , Dendrímeros , Receptores ErbB/inmunología , Fluorescencia , Colorantes Fluorescentes/síntesis química , Humanos , Estructura Molecular , Poliaminas/síntesis química , Sensibilidad y Especificidad , Coloración y Etiquetado/métodos , Estereoisomerismo , Factores de TiempoRESUMEN
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.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Neovascularización Patológica/metabolismo , Neoplasias Pancreáticas/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/metabolismo , Actinas/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Antineoplásicos/farmacología , Benzamidas , Bevacizumab , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Cetuximab , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Mesilato de Imatinib , Lentivirus/genética , Masculino , Trasplante de Células Madre Mesenquimatosas , Ratones , Ratones Desnudos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Piperazinas/farmacología , Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Pirimidinas/farmacología , Esferoides Celulares/patología , Trasplante Heterólogo , Venas Umbilicales/citología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Lentivirus/genética , Células Madre Mesenquimatosas/citología , Neoplasias Pancreáticas/terapia , Transducción Genética , Animales , Diferenciación Celular , Línea Celular Tumoral , Movimiento Celular , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Ratones Desnudos , Neoplasias Pancreáticas/patologíaRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Compartimento Celular/fisiología , Movimiento Celular/fisiología , Endosomas/metabolismo , Membranas Intracelulares/metabolismo , Transporte de Proteínas/fisiología , Proteínas/metabolismo , Proteínas de Unión al GTP rho , Animales , Células Cultivadas/citología , Células Cultivadas/metabolismo , Cricetinae , Endosomas/ultraestructura , Endotelio/citología , Endotelio/metabolismo , Expresión Génica/fisiología , Vectores Genéticos , Proteínas Fluorescentes Verdes , Indicadores y Reactivos/farmacocinética , Membranas Intracelulares/ultraestructura , Proteínas Luminiscentes/farmacocinética , Lisosomas/metabolismo , Microscopía por Video , Proteínas/genética , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , TransfecciónRESUMEN
Sphingolipids are abundant constituents of neuronal membranes that have been implicated in intracellular signaling, neurite outgrowth and differentiation. Differential localization and trafficking of lipids to membrane domains contribute to the specialized functions. In non-neuronal cultured cell lines, plasma membrane short-chain sphingomyelin and glucosylceramide are recycled via endosomes or sorted to degradative compartments. However, depending on cell type and lipid membrane composition, short-chain glucosylceramide can also be diverted to the Golgi complex. Here, we show that NBD-labeled glucosylceramide and sphingomyelin are transported from the plasma membrane to the Golgi complex in cultured rat hippocampal neurons irrespective of the stage of neuronal differentiation. Golgi complex localization was confirmed by colocalization and Golgi disruption studies, and importantly did not result from conversion of NBD-glucosylceramide or NBD-sphingomyelin to NBD-ceramide. Double-labeling experiments with transferrin or wheat-germ agglutinin showed that NBD-sphingolipids are first internalized to early/recycling endosomes, and subsequently transported to the Golgi complex. The internalization of these two sphingolipid analogs was energy and temperature dependent, and their intracellular transport was insensitive to the NBD fluorescence quencher sodium dithionite. These results indicate that vesicles mediate the transport of internalized NBD-glucosylceramide and NBD-sphingomyelin to the Golgi complex.
Asunto(s)
Endocitosis , Aparato de Golgi/metabolismo , Neuronas/metabolismo , Esfingolípidos/biosíntesis , Animales , Antineoplásicos/farmacología , Brefeldino A/farmacología , Diferenciación Celular , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía en Capa Delgada , Citoplasma/metabolismo , Endosomas/metabolismo , Glucosilceramidas/biosíntesis , Hipocampo/citología , Hipocampo/metabolismo , Lisosomas/metabolismo , Microscopía de Contraste de Fase , Nocodazol/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Esfingomielinas/metabolismo , Temperatura , Factores de Tiempo , Transferrina/metabolismoRESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción Activador 2 , Animales , Tamaño de la Célula , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Etiquetado Corte-Fin in Situ , Proteínas Quinasas JNK Activadas por Mitógenos , Microscopía Confocal , Neuronas/fisiología , Células PC12 , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Factor de Transcripción AP-1/genética , Factores de Transcripción/metabolismoRESUMEN
The c-myc gene has been implicated in three distinct genetic programs regulating cell proliferation: control of cyclin E-cdk2 kinase activity, E2F-dependent transcription and cell growth. We have now used p27(-/-) fibroblasts to dissect these downstream signalling pathways. In these cells, activation of Myc stimulates transcription of E2F target genes, S-phase entry and cell growth without affecting cyclin E-cdk2 kinase activity. Both cyclin D2 and E2F2, potential direct target genes of Myc, are induced in p27(-/-) MycER cells. Ectopic expression of E2F2, but not of cyclin D2, induces S-phase entry, but, in contrast to Myc, does not stimulate cell growth. Our results show that stimulation of cyclin E-cdk2 kinase, of E2F-dependent transcription and of cell growth by Myc can be genetically separated from each other.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas Portadoras , Proteínas de Ciclo Celular , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/biosíntesis , Proteínas de Unión al ADN , Genes myc , Proteínas Serina-Treonina Quinasas/biosíntesis , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Células 3T3 , Animales , Apoptosis , División Celular , Células Cultivadas , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Factores de Transcripción E2F , Inducción Enzimática , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína 1 de Unión a Retinoblastoma , Retroviridae/genética , Factor de Transcripción DP1 , Transcripción Genética , TransfecciónRESUMEN
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.
Asunto(s)
Citoplasma/fisiología , Factor de Crecimiento Nervioso/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Androstadienos/farmacología , Animales , Cromonas/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/ultraestructura , Dimetilsulfóxido/farmacología , Inhibidores Enzimáticos/farmacología , Microinyecciones , Morfolinas/farmacología , Células PC12 , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-crk , Ratas , Proteínas Recombinantes/metabolismo , WortmaninaRESUMEN
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.
Asunto(s)
Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células CHO , Cricetinae , Proteínas Fluorescentes Verdes , Membranas Intracelulares/metabolismo , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , TransfecciónRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN , ADN/metabolismo , Genes myc , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Sitios de Unión/genética , ADN/genética , Regulación de la Expresión Génica , Células HeLa , Secuencias Hélice-Asa-Hélice , Humanos , Leucina Zippers , Ratones , Unión Proteica , Factores de Transcripción/genética , Activación Transcripcional , Factores Estimuladores hacia 5'RESUMEN
Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27(kip1) with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2-Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad-Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.
Asunto(s)
Proteínas de Ciclo Celular , Ciclo Celular/fisiología , Ciclinas/genética , Regulación de la Expresión Génica/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , Proteínas Supresoras de Tumor , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , Ciclina D2 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Cartilla de ADN , Humanos , Ratones , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
The regulation of peroxisomal motility was investigated both in CHO cells and in cells derived from human umbilical vein endothelium (HUE). The cells were transfected with a construct encoding the green fluorescent protein bearing the C-terminal peroxisomal targeting signal 1. Kinetic analysis following time-lapse imaging revealed that CHO cells respond to simultaneous stimulation with ATP and lysophosphatidic acid (LPA) by reducing peroxisomal movements. When Ca(2+) was omitted from the extracellular medium or the cells were incubated with inhibitors for heterotrimeric G(i)/G(o) proteins, phospholipase C, classical protein kinase C isoforms (cPKC), mitogen-activated protein kinase kinase (MEK) or phospholipase A(2) (PLA(2)), this signal-mediated motility block was abolished. HUE cells grown to confluency on microporous membranes responded similarly to ATP-LPA receptor co-stimulation, but only when the ligands had access to the basolateral membrane region. These data demonstrate that peroxisomal motility is subject to specific modulation from the extracellular environment and suggest a receptor-mediated signaling cascade comprising Ca(2+) influx, G(i)/G(o) proteins, phospholipase C, cPKC isoforms, MEK and PLA(2) being involved in the regulation of peroxisomal arrest.
Asunto(s)
Peroxisomas/fisiología , Receptores de Superficie Celular/fisiología , Receptores Acoplados a Proteínas G , Receptores Purinérgicos P2/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Células CHO , Polaridad Celular , Células Cultivadas , Cricetinae , Citosol/fisiología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Activación Enzimática/efectos de los fármacos , Humanos , Lisofosfolípidos/farmacología , Movimiento/efectos de los fármacos , Movimiento/fisiología , Peroxisomas/efectos de los fármacos , Fosfolipasas A/metabolismo , Receptores de Superficie Celular/efectos de los fármacos , Receptores del Ácido Lisofosfatídico , Receptores Purinérgicos P2/efectos de los fármacos , Transducción de SeñalRESUMEN
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.
Asunto(s)
Glomérulos Renales/citología , Glomérulos Renales/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismo , Proteínas Virales/farmacocinética , Proteínas de Unión al GTP rab , Animales , Transporte Biológico/fisiología , Proteínas Portadoras/metabolismo , Células Cultivadas , Proteínas de Unión al GTP/metabolismo , Aparato de Golgi/fisiología , Membranas Intracelulares/metabolismo , Glomérulos Renales/fisiología , Proteínas de la Membrana , Ratones , Ratones TransgénicosRESUMEN
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.
Asunto(s)
Glutatión Reductasa/administración & dosificación , Glutatión Reductasa/metabolismo , Células Cultivadas , Color , Cristalización , Citosol/metabolismo , Transporte de Electrón , Fibroblastos/metabolismo , Glutatión Reductasa/aislamiento & purificación , Humanos , Indicadores y Reactivos , Microinyecciones , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Proteínas Nucleares/metabolismo , Anticuerpos Monoclonales/farmacología , Ciclo Celular/fisiología , Nucléolo Celular/metabolismo , Mapeo Epitopo , Técnica del Anticuerpo Fluorescente , Globinas/genética , Células HeLa , Humanos , Microinyecciones , Proteínas Nucleares/inmunología , Precursores del ARN/metabolismo , Empalme del ARN/genética , Proteínas Recombinantes/inmunología , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Transcripción Genética/genéticaRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/fisiología , Proteínas Quinasas Activadas por Mitógenos , Células PC12/enzimología , Proteínas Proto-Oncogénicas c-jun/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Diferenciación Celular/fisiología , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos , Neuritas/enzimología , Neuritas/fisiología , Neuronas/citología , Neuronas/enzimología , Células PC12/metabolismo , Células PC12/patología , Fosforilación , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Proteínas Proto-Oncogénicas c-jun/fisiología , Ratas , Transducción de SeñalRESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiología , Mitosis/fisiología , Fosfoproteínas Fosfatasas/metabolismo , Fosfatasas cdc25 , Anticuerpos/administración & dosificación , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/inmunología , Línea Celular , Activación Enzimática , Retroalimentación , Células HeLa , Humanos , Técnicas In Vitro , Microinyecciones , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/inmunología , Fosforilación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A new approach is presented which allows the in vivo visualization of individual chromosome territories in the nuclei of living human cells. The fluorescent thymidine analog Cy3-AP3-dUTP was microinjected into the nuclei of cultured human cells, such as human diploid fibroblasts, HeLa cells and neuroblastoma cells. The fluorescent analog was incorporated during S-phase into the replicating genomic DNA. Labelled cells were further cultivated for several cell cycles in normal medium. This well-known scheme yielded sister chromatid labelling. Random segregation of labelled and unlabelled chromatids into daughter nuclei resulted in nuclei exhibiting individual in vivo detectable chromatid territories. The territories were composed of subcompartments with diameters ranging between approximately 400 and 800 nm which we refer to as subchromosomal foci. Time-resolved in vivo studies demonstrated changes of positioning and shape of territories and subchromosomal foci. The hypothesis that subchromosomal foci persist as functionally distinct entities was supported by double labelling of chromatin with CldU and IdU, respectively, at early and late S-phase and subsequent cultivation of corresponding cells for 5-10 cell cycles before fixation and immunocytochemical detection. This scheme yielded segregated chromatid territories with distinctly separated subchromosomal foci composed of either early- or late-replicating chromatin. The size range of subchromosomal foci was similar after shorter (2 h) and longer (16 h) labelling periods and was observed in nuclei of both living and fixed cells, suggesting their structural identity. A possible functional relevance of chromosome territory compartmentalization into subchromosomal foci is discussed in the context of present models of interphase chromosome and nuclear architecture.
Asunto(s)
Cromosomas Humanos/metabolismo , Interfase/genética , Bromodesoxiuridina/metabolismo , Carbocianinas/metabolismo , Núcleo Celular/metabolismo , Mapeo Cromosómico , Replicación del ADN , Nucleótidos de Desoxiuracil/metabolismo , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Fibroblastos , Colorantes Fluorescentes/metabolismo , Células HeLa , Humanos , Idoxuridina/metabolismo , Microscopía Fluorescente/métodos , Neuroblastoma , Timidina/análogos & derivados , Timidina/metabolismo , Células Tumorales CultivadasRESUMEN
Centromere protein CENP-A is a histone H3-like protein associated specifically with the centromere and represents one of the human autoantigens identified by sera taken from patients with the CREST variant of progressive systemic sclerosis. Injection of whole human autoimmune serum to the centromere into interphase cells disrupts some mitotic events. It has been assumed that this effect is due to CENP-E and CENP-C autoantigens, because of the effects of injecting monospecific sera to those proteins into culture cells. Here we have used an antibody raised against an N-terminal peptide of the human autoantigen CENP-A to determine its function in mitosis and during cell cycle progression. Affinity-purified anti-CENP-A antibodies injected into the nucleus during the early replication stages of the cell cycle caused cells to arrest in interphase before mitosis. These cells showed highly condensed small nuclei, a granular cytoplasm and loss of their division capability. On the other hand, microinjection of nocodazole-blocked HeLa cells in mitosis resulted in the typical punctate staining pattern of CENP-A for centromeres during different stages of mitosis and apparently normal cell division. This was corroborated by time-lapse imaging microscopy analysis of mid-interphase-injected cells, revealing that they undergo mitosis and divide properly. However, a significant delay throughout the progression of mitotic stages was observed. These results suggest that CENP-A is involved predominantly in an essential interphase event at the centromere before mitosis. This may include chromatin assembly at the kinetochore coordinate with late replication of satellite DNA to form an active centromere.