RESUMEN
The axonal surface glycoproteins neuronglia cell adhesion molecule (NgCAM) and axonin-1 promote cell-cell adhesion, neurite outgrowth and fasciculation, and are involved in growth cone guidance. A direct binding between NgCAM and axonin-1 has been demonstrated using isolated molecules conjugated to the surface of fluorescent microspheres. By expressing NgCAM and axonin-1 in myeloma cells and performing cell aggregation assays, we found that NgCAM and axonin-1 cannot bind when present on the surface of different cells. In contrast, the cocapping of axonin-1 upon antibody-induced capping of NgCAM on the surface of CV-1 cells coexpressing NgCAM and axonin-1 and the selective chemical cross-linking of the two molecules in low density cultures of dorsal root ganglia neurons indicated a specific and direct binding of axonin-1 and Ng-CAM in the plane of the same membrane. Suppression of the axonin-1 translation by antisense oligonucleotides prevented neurite outgrowth in dissociated dorsal root ganglia neurons cultured on an NgCAM substratum, indicating that neurite outgrowth on NgCAM substratum requires axonin-1. Based on these and previous results, which implicated NgCAM as the neuronal receptor involved in neurite outgrowth on NgCAM substratum, we concluded that neurite outgrowth on an NgCAM substratum depends on two essential interactions of growth cone NgCAM: a trans-interaction with substratum NgCAM and a cis-interaction with axonin-1 residing in the same growth cone membrane.
Asunto(s)
Moléculas de Adhesión Celular Neurona-Glia/fisiología , Moléculas de Adhesión Celular Neuronal/fisiología , Neuritas/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Secuencia de Bases , Células COS , Moléculas de Adhesión Celular Neurona-Glia/química , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/inmunología , Agregación Celular , Línea Celular , Membrana Celular/química , Membrana Celular/fisiología , Embrión de Pollo , Contactina 2 , ADN , Dimerización , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Neural cell adhesion molecules of the immunoglobulin superfamily mediate cellular interactions via homophilic binding to identical molecules and heterophilic binding to other family members or structurally unrelated cell-surface glycoproteins. Here we report on an interaction between axonin-1 and Nr-CAM/Bravo. In search for novel ligands of axonin-1, fluorescent polystyrene microspheres conjugated with axonin-1 were found to bind to peripheral glial cells from dorsal root ganglia. By antibody blockage experiments an axonin-1 receptor on the glial cells was identified as Nr-CAM. The specificity of the interaction was confirmed with binding studies using purified axonin-1 and Nr-CAM. In cultures of dissociated dorsal root ganglia antibodies against axonin-1 and Nr-CAM perturbed the formation of contacts between neurites and peripheral glial cells. Together, these results implicate a binding between axonin-1 of the neuritic and Nr-CAM of the glial cell membrane in the early phase of axon ensheathment in the peripheral nervous system.
Asunto(s)
Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular , Neuroglía/citología , Neuronas/citología , Animales , Especificidad de Anticuerpos , Axones/fisiología , Secuencia de Bases , Adhesión Celular/fisiología , Moléculas de Adhesión Celular Neuronal/inmunología , Embrión de Pollo , Contactina 2 , Cartilla de ADN/química , Ganglios Espinales/citología , Ganglios Espinales/embriología , Integrinas/fisiología , Microesferas , Datos de Secuencia Molecular , Neuritas/ultraestructura , Neuroglía/ultraestructura , Neuronas/ultraestructura , Unión Proteica/fisiologíaRESUMEN
Neural cell adhesion molecules composed of immunoglobulin and fibronectin type III-like domains have been implicated in cell adhesion, neurite outgrowth, and fasciculation. Axonin-1 and Ng cell adhesion molecule (NgCAM), two molecules with predominantly axonal expression exhibit homophilic interactions across the extracellular space (axonin- 1/axonin-1 and NgCAM/NgCAM) and a heterophilic interaction (axonin-1-NgCAM) that occurs exclusively in the plane of the same membrane (cis-interaction). Using domain deletion mutants we localized the NgCAM homophilic binding in the Ig domains 1-4 whereas heterophilic binding to axonin-1 was localized in the Ig domains 2-4 and the third FnIII domain. The NgCAM-NgCAM interaction could be established simultaneously with the axonin-1-NgCAM interaction. In contrast, the axonin-1-NgCAM interaction excluded axonin-1/axonin-1 binding. These results and the examination of the coclustering of axonin-1 and NgCAM at cell contacts, suggest that intercellular contact is mediated by a symmetric axonin-12/NgCAM2 tetramer, in which homophilic NgCAM binding across the extracellular space occurs simultaneously with a cis-heterophilic interaction of axonin-1 and NgCAM. The enhanced neurite fasciculation after overexpression of NgCAM by adenoviral vectors indicates that NgCAM is the limiting component for the formation of the axonin-12/NgCAM2 complexes and, thus, neurite fasciculation in DRG neurons.
Asunto(s)
Moléculas de Adhesión Celular Neurona-Glia/química , Moléculas de Adhesión Celular Neurona-Glia/fisiología , Moléculas de Adhesión Celular Neuronal/química , Moléculas de Adhesión Celular Neuronal/fisiología , Ganglios Espinales/fisiología , Neuritas/fisiología , Conformación Proteica , Animales , Animales Recién Nacidos , Sitios de Unión , Moléculas de Adhesión Celular Neurona-Glia/genética , Moléculas de Adhesión Celular Neuronal/genética , Pollos , Contactina 2 , Espacio Extracelular/fisiología , Ratones , Ratones Endogámicos ICR , Modelos Moleculares , Mutagénesis , Neuronas/citología , Neuronas/fisiología , Técnicas de Cultivo de Órganos , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , TransfecciónRESUMEN
An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.
Asunto(s)
Axones/fisiología , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular , Inducción Embrionaria , Animales , Sitios de Unión , Adhesión Celular/fisiología , Moléculas de Adhesión Celular Neurona-Glia/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Células Cultivadas , Embrión de Pollo , Contactina 2 , Conos de Crecimiento/fisiología , Familia de Multigenes , Vías Nerviosas/embriología , Unión Proteica , Proteínas Recombinantes/metabolismo , Médula Espinal/citología , Médula Espinal/embriología , Médula Espinal/cirugíaRESUMEN
BACKGROUND: Growth cones at the tips of growing axons move along predetermined pathways to establish synaptic connections between neurons and their distant targets. To establish their orientation, growth cones continuously sample for, and respond to, guidance information provided by cell surfaces and the extracellular matrix. To identify specific guidance cues, growth cones have sensor molecules on their surface, which are expressed differentially during the temporospatial progress of axon outgrowth, at levels that depend on the pattern of neural activity. However, it has not been elucidated whether a change in gene expression can indeed change the molecular composition and, hence, the function of the sensor apparatus of growth cones. RESULTS: We have constructed adenoviral gene transfer vectors of the chicken growth cone sensor molecules axonin-1 and Ng-CAM. Using these vectors, we initiated the expression of axonin-1 and Ng-CAM in rat dorsal root ganglia explants during ongoing neurite outgrowth. Using specific surface immunodetection at varying time points after infection, we found that axonin-1 and Ng-CAM are transported directly to the growth cone and inserted exclusively in the growth cone membrane and not in the axolemma of the axon shaft. Furthermore, we found that axonin-1 and Ng-CAM do not diffuse retrogradely, suggesting that the sensor molecules are integrated into multimolecular complexes in the growth cone. CONCLUSIONS: During axon outgrowth, the pathway sensor apparatus of the growth cone is continuously updated by newly synthesized sensor molecules that originate directly from the transcription/translation machinery. Changes in the expression of sensor molecules may have a direct impact, therefore, on the exploratory function of the growth cone.
Asunto(s)
Axones , Neuritas , Animales , Axones/metabolismo , Moléculas de Adhesión Celular Neurona-Glia/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Embrión de Pollo , Contactina 2 , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Inmunohistoquímica , RatasRESUMEN
beta 2- and beta 3-subunits of GABAA receptors purified from the brains of 5-10-day-old rats were investigated in the intact or completely N-deglycosylated state using the beta-subunit-specific monoclonal antibody bd-17 and polyclonal antibodies directed against synthetic amino acid sequences specific for the GABAA receptor beta 2- or beta 3-subunits. The present results seem to indicate the existence of two different isoforms of the beta 3-subunit and several different isoforms of the beta 2-subunit of the GABAA receptor which probably are produced by alternative splicing.
Asunto(s)
Química Encefálica , Receptores de GABA-A/química , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Western Blotting , Epítopos/inmunología , Glicosilación , Técnicas de Inmunoadsorción , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Ratas , Receptores de GABA-A/inmunología , Receptores de GABA-A/aislamiento & purificaciónRESUMEN
Directed growth cone movement is crucial for the correct wiring of the nervous system. This movement is governed by the concerted actions of cell surface receptors, signaling proteins, cytoskeleton-associated molecules, and molecular motors. In order to investigate the molecular basis of growth cone motility, we applied a new technique to functionally inactivate proteins: micro-scale Chromophore-Assisted Laser Inactivation [Diamond et al. (1993) Neuron 11:409-421]. Micro-CALI uses laser light of 620 nm, focused through microscope optics into a 10-microm spot. The laser energy is targeted via specific Malachite green-labeled, non-function-blocking antibodies, that generate short-lived protein-damaging hydroxyl radicals [Liao et al. (1994) Proc Natl Acad Sci USA 91:2659-2663]. Micro-CALI mediates specific loss of protein function with unachieved spatial and temporal resolution. Combined with time-lapse video microscopy, it offers the possibility to induce and observe changes in growth cone dynamics on a real time base. We present here the effects of the acute and localized inactivation of selected growth cone molecules on growth cone behavior and morphology. Based on our observations, we propose specific roles for these proteins in growth cone motility and neurite outgrowth.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/fisiología , Rayos Láser , Proteínas del Tejido Nervioso/fisiología , Actinas/fisiología , Animales , Calcineurina/fisiología , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/fisiología , Moléculas de Adhesión Celular Neuronal/efectos de la radiación , Colorantes , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/efectos de la radiación , Daño del ADN , Conos de Crecimiento/química , Humanos , Complejo de Antígeno L1 de Leucocito , Glicoproteínas de Membrana/fisiología , Microscopía por Video/instrumentación , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas Asociadas a Microtúbulos/efectos de la radiación , Proteínas Motoras Moleculares/genética , Proteínas Motoras Moleculares/fisiología , Proteínas Motoras Moleculares/efectos de la radiación , Miosinas/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/efectos de la radiación , Moléculas de Adhesión de Célula Nerviosa/fisiología , Neuritas/fisiología , Colorantes de Rosanilina , Transducción de Señal , Talina/fisiología , Vinculina/fisiologíaRESUMEN
Cell adhesion molecules expressed on the axonal membrane have been thought to be involved in the guidance of axons to their target area. In the chick, axonin-1 and NgCAM have been shown to promote, through reciprocal interactions, neurite outgrowth in vitro. We have recently shown that chick retinal ganglion cells (RGC) express both proteins as early as the axonal elongation begins. Their expression continues throughout the development of the retinotectal system synchronously with the chronotopic spread of axons. To further investigate the spatiotemporal distribution of axonin-1 and NgCAM in the retina, we have analysed the expression of their mRNAs in the present study. From stage 36 (E10) until hatching photoreceptors express axonin-1 but not NgCAM. In the inner nuclear layer groups of amacrine cells were strongly labelled with both probes but they seemed to belong to different subgroups. These patterns of expression might indicate a differential influence of the two proteins on the development of the local neural circuits of the retina.
Asunto(s)
Axones/fisiología , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular , Retina/fisiología , Animales , Embrión de Pollo , Contactina 2 , Regulación del Desarrollo de la Expresión Génica/fisiología , Hibridación in Situ , Interneuronas/fisiología , Células Fotorreceptoras/fisiología , ARN Mensajero/metabolismo , Retina/citología , Retina/embriologíaRESUMEN
GABAA receptors purified from the brains of 5- to 10-day-old rats were completely N- and O-deglycosylated using N-glycanase and/or neuraminidase plus O-glycanase. Intact or completely deglycosylated receptors were subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis. Polyclonal antibodies directed against synthetic aminoacid sequences specific for the GABAA receptor alpha 1-, alpha 2- or alpha 3-subunits each identified an apparently single protein of about 51 kDa, 53 kDa or 59 kDa, respectively, in the intact receptors. In the deglycosylated receptors, however, three different proteins were identified by antibodies directed against the alpha 3-subunit and at least two different proteins were identified by antibodies directed against the alpha 2- or alpha 1-subunit.
Asunto(s)
Química Encefálica/fisiología , Receptores de GABA-A/química , Animales , Anticuerpos/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida , Flunitrazepam/farmacología , Glicósido Hidrolasas , Desnaturalización Proteica , Ratas , Receptores de GABA-A/inmunología , Receptores de GABA-A/aislamiento & purificación , Espectrometría de FluorescenciaAsunto(s)
Axones/fisiología , Moléculas de Adhesión Celular Neurona-Glia/fisiología , Moléculas de Adhesión Celular Neuronal/fisiología , Neuronas/fisiología , Animales , Moléculas de Adhesión Celular Neurona-Glia/química , Moléculas de Adhesión Celular Neuronal/química , Comunicación Celular/fisiología , Contactina 2 , Humanos , Modelos Moleculares , Modelos Neurológicos , Conformación ProteicaRESUMEN
The understanding of the molecular mechanisms of cellular function, growth, and proliferation is based on the accurate identification, isolation, and finally characterization of a specific single cell or a population of cells and its subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within one single experiment as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin are required. Sample preparation is therefore a very crucial step preceding the functional characterization of specific cell populations. Laser microdissection and laser pressure catapulting (LMPC) enables pure and homogeneous sample preparation resulting in an increased specificity of molecular analyses. With LMPC, the force of focused laser light is utilized to excise selected cells or large tissue areas from object slides down to individual single cells and subcellular components like organelles or chromosomes. After microdissection, the sample is directly catapulted into an appropriate collection vial. As this process works entirely without mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross-contamination. LMPC has been successfully applied to isolate and catapult cells from, for example, histological tissue sections, from forensic evidence material, and also from tough plant matter, supporting biomedical research, forensic science, and plant physiology studies. Even delicate living cells like stem cells have been captured for recultivation without affecting their viability or stem cell character, an important feature influencing stem cell research, regenerative medicine, and drug development. The combination of LMPC with microinjection to inject drugs or genetic material into individual cells and to capture them for molecular analyses bears great potential for efficient patient-tailored medication.
Asunto(s)
Rayos Láser , Microdisección/métodos , Animales , Caenorhabditis elegans/citología , Línea Celular Tumoral , Humanos , Microdisección/instrumentación , Células VegetalesRESUMEN
Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the alpha3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice.
Asunto(s)
Colágeno Tipo IV/deficiencia , Fallo Renal Crónico/patología , Fallo Renal Crónico/terapia , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/fisiología , Trasplante de Células Madre , Animales , Autoantígenos/genética , Proteína Morfogenética Ósea 7 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Movimiento Celular , Proliferación Celular , Colágeno Tipo IV/genética , Progresión de la Enfermedad , Células Endoteliales/citología , Células Endoteliales/fisiología , Femenino , Fibrosis/terapia , Riñón/metabolismo , Riñón/patología , Fallo Renal Crónico/genética , Células Madre Mesenquimatosas/citología , Ratones , Ratones Mutantes , Células Madre Multipotentes/citología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the gamma-aminobutyric acidA (GABAA) receptor alpha 5-subunit. These anti-peptide alpha 5(2-10) or anti-peptide alpha 5(427-433) antibodies reacted specifically with GABAA receptors purified from the brains of 5-10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABAA receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABAA receptors with N-Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABAA receptors with neuraminidase and O-Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide alpha 5(2-10) and the anti-peptide alpha 5(427-433) antibody. These results indicate the existence of at least two different alpha 5-subunits of the GABAA receptor that differ in their carbohydrate content. In contrast to other alpha- or beta-subunits of GABAA receptors so far investigated, at least one of these two alpha 5-subunits contains O-linked carbohydrates.