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BACKGROUND: Normative values for different morphometric parameters of muscle fibres during paediatric development, i.e. from 0 to 18 years, are currently unavailable. They would be of major importance to accurately evaluate pathological changes and could be used as reference biomarkers for evaluating treatment response in clinical trials, or physiological adjustments in sports or ageing. METHODS: Data were derived from 482 images with a total of 33 094 fibres from 10 µm cross-sections of snap-frozen muscle from 83 deltoid muscle biopsies from patients, 0-18 years, without neuromuscular pathology stained with ATPase 9.4. Data was acquired and analysed with patented image analysis algorithms from "CARPACCIO.cloud". Several parameters were extracted or calculated, including cross-sectional area (CSA), fibre type, circularity, as well as the Minimum diameter of Feret (MinFeret). FINDINGS: This study illustrates changes in quantitative parameters for muscle morphology over the course of paediatric development and the pivotal changes occurring around puberty. Only fibre size parameters (MinFeret, CSA) are dependent on gender, and only after puberty. All other parameters vary in a similar manner for females and males. The proportion of type 1 fibres is essentially constant from birth to age 10, decreasing to ≈40% by age 18. Circularity decreases with age, to plateau after age 10 for both fibre types. INTERPRETATION: Normative values and reference charts for muscle fibre types in this age range have been generated to allow comparison of data from patients in pathology laboratories working on neuromuscular diseases. FUNDING: BPI FRANCE, PULSALYS, Association de l'Institut de Myologie, French National Research Agency (ANR), LABEX CORTEX of Université de Lyon.
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Desarrollo de Músculos , Fibras Musculares Esqueléticas , Masculino , Femenino , Humanos , Niño , Adolescente , Estudios Transversales , Biopsia , Envejecimiento , Músculo EsqueléticoRESUMEN
Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.
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Caveolina 1/metabolismo , Núcleo Celular/metabolismo , Microdominios de Membrana/metabolismo , Factor de Crecimiento Nervioso/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Anticuerpos Monoclonales/inmunología , Proteína de Unión a CREB/metabolismo , Caveolina 1/antagonistas & inhibidores , Caveolina 1/genética , Caveolina 2/antagonistas & inhibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Ratones , Proteínas del Tejido Nervioso , Células PC12 , Fosforilación/efectos de los fármacos , Unión Proteica , Transporte de Proteínas/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/química , Receptor trkA/inmunología , Receptor trkA/metabolismo , Receptores de Factores de Crecimiento , Receptores de Factor de Crecimiento Nervioso/química , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5-16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60-80%) and low toxicity (>60% survival) were achieved with robust differentiation in response to Nerve growth factor (NGF). Moreover, 3-50 times fewer cells are needed (6 × 10(4)) compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures.
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BACKGROUND: The reliability and reproducibility of experimental procedures is a cornerstone of scientific practice. There is a pressing technological need for the better representation of biomedical protocols to enable other agents (human or machine) to better reproduce results. A framework that ensures that all information required for the replication of experimental protocols is essential to achieve reproducibility. To construct EXACT2 we manually inspected hundreds of published and commercial biomedical protocols from several areas of biomedicine. After establishing a clear pattern for extracting the required information we utilized text-mining tools to translate the protocols into a machine amenable format. We have verified the utility of EXACT2 through the successful processing of previously 'unseen' (not used for the construction of EXACT2)protocols. METHODS: We have developed the ontology EXACT2 (EXperimental ACTions) that is designed to capture the full semantics of biomedical protocols required for their reproducibility. RESULTS: The paper reports on a fundamentally new version EXACT2 that supports the semantically-defined representation of biomedical protocols. The ability of EXACT2 to capture the semantics of biomedical procedures was verified through a text mining use case. In this EXACT2 is used as a reference model for text mining tools to identify terms pertinent to experimental actions, and their properties, in biomedical protocols expressed in natural language. An EXACT2-based framework for the translation of biomedical protocols to a machine amenable format is proposed. CONCLUSIONS: The EXACT2 ontology is sufficient to record, in a machine processable form, the essential information about biomedical protocols. EXACT2 defines explicit semantics of experimental actions, and can be used by various computer applications. It can serve as a reference model for for the translation of biomedical protocols in natural language into a semantically-defined format.
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Ontologías Biológicas , Minería de Datos , Programas Informáticos , Procesamiento Automatizado de Datos , Lenguaje , Reproducibilidad de los Resultados , SemánticaRESUMEN
We previously identified a peptide aptamer (named R5G42) via functional selection for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using R5G42 as "bait," allowed the identification of two binding proteins with very different functions: calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized for its role in the immune response, and NS5A-TP2/HD domain containing 2, a much less studied protein induced subsequent to hepatitis C virus non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective in the present study was to dissect the dual target specificity of R5G42 in order to have tools with which to better characterize the actions of the peptide aptamers toward their individual targets. This was achieved through the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity toward CnA and NS5A-TP2 and analyzing their sequence. An interdisciplinary approach involving biomolecular computer simulations with integration of the sequence data and yeast two-hybrid binding phenotypes of these mutants yielded two structurally distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA- versus NS5A-TP2-specific peptide aptamers indicated that although both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate the nuclear translocation of nuclear factor of activated T cells, indicative of the activation of endogenous CnA. By dissecting the target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is capable of directly activating CnA independent of binding to NS5A-TP2 and will be an important tool in studying the role of CnA activation in the regulation of different signaling pathways, whereas Apta-E1 will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signaling pathways.
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Aptámeros de Péptidos/metabolismo , Calcineurina/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Aptámeros de Péptidos/genética , Línea Celular Tumoral , Células HeLa , Humanos , Mutagénesis Sitio-Dirigida , Ratas , Técnicas del Sistema de Dos HíbridosRESUMEN
This study examined the isolation and differentiation of dorsal root ganglion progenitor cells for therapeutic use in neurodegenerative diseases. Rat embryonic dorsal root ganglia progenitors were isolated and purified using the differential adhesion method combined with cytosine arabinoside treatment. After culture in serum-free medium supplemented with B27, basic fibroblast growth factor and epidermal growth factor, these cells remained viable and survived for more than 18 months in vitro. Most cells differentiated to neurons that were immunoreactive for gamma-aminobutyric acid and choline acetyltransferase as detected by immunohistochemical staining. In addition, nerve growth factor and neurotrophic tyrosine kinase receptor expression were also observed in dorsal root ganglion progenitors and differentiated cells. K252a, an inhibitor that blocks nerve growth factor-induced signaling, inhibited cell survival, suggesting the possible existence of a nerve growth factor autocrine loop in these proliferating cells.
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Peptide aptamers have primarily been used as research tools to manipulate protein function and study regulatory networks. However, they also find multiple applications in therapeutic research, from target identification and validation to drug discovery. Because of their unbiased combinatorial nature, peptide aptamers interrogate the biological significance of numerous molecular surfaces on target proteins. Their use enables the identification and validation of some of these surfaces as interesting therapeutic targets to pursue. Peptide aptamers can subsequently be used to guide the discovery of small molecule drugs specific for these molecular surfaces.Here, we present a high-throughput screening assay that identifies small molecules that displace interactions between proteins and their cognate peptide aptamers. AptaScreen is a duplex yeast two-hybrid assay featuring two luciferase reporter genes. It can be performed in 96- or 384-well plates and can be fully automated.
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Aptámeros de Péptidos/metabolismo , Aptámeros de Péptidos/uso terapéutico , Descubrimiento de Drogas , Bibliotecas de Moléculas Pequeñas/metabolismo , Aptámeros de Péptidos/genética , Humanos , Luminiscencia , Unión Proteica , Mapeo de Interacción de Proteínas , Técnicas del Sistema de Dos HíbridosRESUMEN
The study of protein function mostly relies on perturbing regulatory networks by acting upon protein expression levels or using transdominant negative agents. Here we used the Escherichia coli global transcription regulator Fur (ferric uptake regulator) as a case study to compare the perturbations exerted by a gene knock-out, the expression of a dominant negative allele of a gene, and the expression of peptide aptamers that bind a gene product. These three perturbations caused phenotypes that differed quantitatively and qualitatively from one another. The Fur peptide aptamers inhibited the activity of their target to various extents and reduced the virulence of a pathogenic E. coli strain in Drosophila. A genome-wide transcriptome analysis revealed that the "penetrance" of a peptide aptamer was comparable to that of a dominant negative allele but lower than the penetrance of the gene knock-out. Our work shows that comparative analysis of phenotypic and transcriptome responses to different types of perturbation can help decipher complex regulatory networks that control various biological processes.
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Alelos , Aptámeros de Péptidos/química , Genes Dominantes , Animales , Secuencia de Bases , Cartilla de ADN , Drosophila , Técnicas del Sistema de Dos HíbridosRESUMEN
The actin cytoskeleton of mature osteoclasts (OCs) adhering to nonmineralized substrates is organized in a belt of podosomes reminiscent of the sealing zone (SZ) found in bone resorbing OCs. In this study, we demonstrate that the belt is composed of two functionally different actin-based domains: podosome cores linked with CD44, which are involved in cell adhesion, and a diffuse cloud associated with beta3 integrin, which is involved in cell adhesion and contraction. Wiskott Aldrich Syndrome Protein (WASp) Interacting Protein (WIP)-/- OCs were devoid of podosomes, but they still exhibited actin clouds. Indeed, WIP-/- OCs show diminished expression of WASp, which is required for podosome formation. CD44 is a novel marker of OC podosome cores and the first nonintegrin receptor detected in these structures. The importance of CD44 is revealed by showing that its clustering restores podosome cores and WASp expression in WIP-/- OCs. However, although CD44 signals are sufficient to form a SZ, the presence of WIP is indispensable for the formation of a fully functional SZ.
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Actinas/metabolismo , Receptores de Hialuranos/metabolismo , Integrina beta3/metabolismo , Osteoclastos/metabolismo , Animales , Resorción Ósea , Membranas Intracelulares/metabolismo , Ratones , Ratones Noqueados , Transducción de Señal , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
The nerve growth factor (NGF)-tyrosine kinase receptor TrkA plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation, and proliferation. In breast cancer cells, TrkA stimulation results in the activation of cellular growth, but downstream signaling largely remains to be described. Here we used a proteomics-based approach to identify partners involved in TrkA signaling in breast cancer cells. Wild type and modified TrkA chimeric constructs with green fluorescent protein were transfected in MCF-7 cells, and co-immunoprecipitated proteins were separated by SDS-PAGE before nano-LC-MS/MS analysis. Several TrkA putative signaling partners were identified among which was the DNA repair protein Ku70, which is increasingly reported for its role in cell survival and carcinogenesis. Physiological interaction of Ku70 with endogenous TrkA was induced upon NGF stimulation in non-transfected cells, and co-localization was observed with confocal microscopy. Mass spectrometry analysis and Western blotting of phosphotyrosine immunoprecipitates demonstrated the induction of Ku70 tyrosine phosphorylation upon NGF stimulation. Interestingly no interaction between TrkA and Ku70 was detected in PC12 cells in the absence or presence of NGF, suggesting that it is not involved in the initiation of neuronal differentiation. In breast cancer cells, RNA interference indicated that whereas Ku70 depletion had no direct effect on cell survival, it induced a strong potentiation of apoptosis in TrkA-overexpressing cells. In conclusion, TrkA signaling appears to be proapoptotic in the absence of Ku70, and this protein might therefore play a role in the long time reported ambivalence of tyrosine kinase receptors that can exhibit both anti- and eventually proapoptotic activities.
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Antígenos Nucleares/metabolismo , Apoptosis , Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Secuencia de Aminoácidos , Antígenos Nucleares/genética , Apoptosis/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Autoantígeno Ku , Datos de Secuencia Molecular , Interferencia de ARN , Receptor trkA/genética , Transducción de SeñalRESUMEN
NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.
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Comunicación Autocrina , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Meiosis/efectos de los fármacos , Factor de Crecimiento Nervioso/metabolismo , Comunicación Paracrina , Espermatocitos/metabolismo , Espermatogénesis/efectos de los fármacos , Animales , Comunicación Autocrina/efectos de los fármacos , Carbazoles/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Relación Dosis-Respuesta a Droga , Alcaloides Indólicos , Masculino , Factor de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/metabolismo , Comunicación Paracrina/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Receptor trkA/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Túbulos Seminíferos/citología , Túbulos Seminíferos/efectos de los fármacos , Túbulos Seminíferos/metabolismo , Células de Sertoli/efectos de los fármacos , Espermatocitos/efectos de los fármacos , Factores de TiempoRESUMEN
Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.
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Aptámeros de Péptidos/farmacología , Calcineurina/biosíntesis , Proliferación Celular/efectos de los fármacos , Animales , Aptámeros de Péptidos/genética , Línea Celular Tumoral , Vectores Genéticos , Humanos , Biblioteca de Péptidos , Monoéster Fosfórico Hidrolasas/metabolismo , Ratas , Virus de la Inmunodeficiencia de los Simios/genética , Técnicas del Sistema de Dos Híbridos , Regulación hacia ArribaRESUMEN
Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.
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Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Transducción de Señal/fisiología , Animales , Diferenciación Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , MAP Quinasa Quinasa 1/metabolismo , Ratones , Factor de Crecimiento Nervioso/genética , Neuronas/metabolismo , Células PC12 , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptor trkA/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
In all systems examined so far, the G2/M phase transition is controlled by the M-phase promoting factor (MPF), a complex of cdc2 (CDK1) and cyclin B1. Histone H1 kinase activity and MPF components are present in pachytene spermatocytes (PS). However, it has not been demonstrated yet that direct inhibition of MPF activity prevents the G2/M transition in these cells. When roscovitine, a potent inhibitor of CDK1, CDK2, and CDK5 activities, was added to cocultures of PS with Sertoli cells, the number of both secondary spermatocytes and round spermatids formed were lower than in control cultures, despite similar cell viability. This effect of roscovitine was reversible, did not involve the Sertoli cells, and was dependent on the concentration of the inhibitor. Roscovitine did not modify the amount of MPF in these germ cells but inhibited the CDK1- or CDK2-associated histone H1 kinase activity of PS. Hence a functional relationship between cyclin-dependent kinase activity and the spontaneous processing of the first meiotic division and, for the first time, of the second meiotic division of male germ cells is shown.
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Proteína Quinasa CDC2/fisiología , Quinasas CDC2-CDC28/fisiología , Quinasas Ciclina-Dependientes/fisiología , Espermatocitos/citología , Espermatocitos/enzimología , Animales , Proteína Quinasa CDC2/metabolismo , Quinasas CDC2-CDC28/metabolismo , Tamaño de la Célula , Supervivencia Celular , Células Cultivadas , Técnicas de Cocultivo , Quinasa 2 Dependiente de la Ciclina , Quinasa 5 Dependiente de la Ciclina , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Masculino , Factor Promotor de Maduración/metabolismo , Meiosis/fisiología , Concentración Osmolar , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Purinas/administración & dosificación , Purinas/farmacología , Ratas , Roscovitina , Células de Sertoli , Espermátides/citología , Espermátides/fisiología , Espermatocitos/fisiologíaRESUMEN
A chimera of the nerve growth factor (NGF) receptor, TrkA, and green fluorescent protein (GFP) was engineered by expressing GFP in phase with the carboxyl terminus of TrkA. TrkA-GFP becomes phosphorylated on tyrosine residues in response to NGF and is capable of initiating signaling cascades leading to prolonged MAPK activation and differentiation in PC12 nnr5 cells. TrkA constructs, progressively truncated in the carboxyl-terminal domain, were prepared as GFP chimerae in order to identify which part of the receptor intracellular domain is involved in its trafficking. Immunofluorescence observations show that TrkA-GFP is found mainly in cell surface membrane ruffles and in endosomes. Biochemical analysis indicated that the cytoplasmic domain of TrkA is not necessary for correct maturation and cell surface translocation of the receptor. An antibody against the extracellular domain of TrkA (RTA) was used as ligand to stimulate internalization and phosphorylation of TrkA. Co-localization studies with anti-phosphorylated TrkA antibodies support a role for such complexes in the propagation of signaling from the cell surface, resulting in the activation of TrkA in areas of the endosome devoid of receptor-ligand complexes. Confocal time-lapse analysis reveals that the TrkA-GFP chimera shows highly dynamic trafficking between the cell surface and internal locations. TrkA-positive vesicles were estimated to move 0.46 +/- 0.09 microm/s anterograde and 0.48 +/- 0.07 microm/s retrograde. This approach and the fidelity of the biochemical properties of the TrkA-GFP demonstrate that real-time visualization of trafficking of tyrosine kinase receptors in the presence or absence of the ligand is feasible.
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Diferenciación Celular/fisiología , Factor de Crecimiento Nervioso/fisiología , Receptor trkA/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Secuencia de Bases , Membrana Celular/metabolismo , Cartilla de ADN , Regulación hacia Abajo/fisiología , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Fosforilación , Transporte de Proteínas , RatasRESUMEN
A growing body of evidence indicates a close relationship between tyrosine kinase receptor trafficking and signaling. Biochemical and molecular analyses of the expression, fate, and kinetics of membrane trafficking of the nerve growth factor (NGF) receptor TrkA were performed in PC12 cells. Pulse-chase experiments indicate that TrkA is synthesized as a 110-kDa N-glycosylated precursor that leads to the mature 140-kDa form of the receptor with a half-life of conversion of approximately 24 +/- 0.5 min. Neuraminidase digestion shows that modification of the carbohydrate moiety of the receptor by sialylation occurs during maturation. The 140-kDa form is rapidly translocated to the cell surface as assessed by cell surface biotinylation performed on intact PC12 cells. Mature receptor half-life is approximately 138 +/- 4 min and is shortened to 86 +/- 8 min by NGF treatment. Flow cytometric analysis indicates that NGF induces clearing of this receptor from the cell surface within minutes of treatment. The addition of NGF decreases the half-life of cell surface gp140(TrkA) from 100 to 35 min and leads to enhanced lysosomal degradation of the receptor. The process of NGF-induced TrkA internalization is clearly affected by interfering with ligand binding to p75(NTR). An analysis of receptor activation kinetics also shows that receptor signaling primarily takes place from an intracellular location. Together, these data show that the primary effect of NGF treatment is a p75(NTR)-modulated decrease in TrkA transit time at the cell surface.