RESUMEN
Surface plasmon resonance (SPR) allows examination of protein-protein interactions in real time, from which both binding affinities and kinetics can be directly determined. We have used the SPR technique to search for proteins in heart tissue that would be candidate binding partners for the cardiac gap junction protein, connexin43 (Cx43). Heart lysate showed a strong, pH-dependent binding to the carboxyl terminus (CT) of Cx43 (amino acids 254-382) covalently linked to an SPR cuvette. Binding was inhibited by the presence of v-src transfected 3T3 cell lysate, suggesting that binding partners in these two lysates may compete for overlapping epitopes on Cx43CT. The combined application of proteomic and functional studies is expected to identify which proteins within heart tissue interact with Cx43 and what roles they may play in gap junction function.
Asunto(s)
Conexina 43/metabolismo , Miocardio/química , Resonancia por Plasmón de Superficie , Células 3T3 , Animales , Conexina 43/genética , Uniones Comunicantes/química , Uniones Comunicantes/metabolismo , Genes src , Concentración de Iones de Hidrógeno , Ratones , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ovinos , Resonancia por Plasmón de Superficie/instrumentación , Resonancia por Plasmón de Superficie/métodos , Extractos de Tejidos/química , Extractos de Tejidos/metabolismoRESUMEN
Vertebrate tissues use multiple junctional types to establish and maintain tissue architecture, including gap junctions for cytoplasmic connectivity and tight junctions (TJs) for paracellular and/or cell polarity barriers. The integral membrane proteins of gap junctions are connexins, whereas TJs are a complex between occludin and members of a recently characterized multigene family, the claudins. In normal brain, astrocytes are coupled by gap junctions composed primarily of connexin43 (Cx43), whereas TJs have not been detected in these cells. We now show that treatment of primary human astrocytes with the cytokine interleukin-1beta (IL-1beta) causes rapid induction of claudin-1, with an expression pattern reciprocal to loss of Cx43. Treatment also led to protracted downregulation of occludin but no change in expression of zonula occludens proteins ZO-1 and -2. Immunofluorescence staining localized claudin-1 to cell membranes in IL-1beta-treated astrocytes, whereas freeze-fracture replicas showed strand-like arrays of intramembranous particles in treated cells resembling rudimentary TJ assemblies. We conclude that in human astrocytes, IL-1beta regulates expression of the claudin multigene family and that gap and tight junction proteins are inversely regulated by this proinflammatory cytokine. We suggest that in pathological conditions of the human CNS, elevated IL-1beta expression fundamentally alters astrocyte-to-astrocyte connectivity.
Asunto(s)
Astrocitos/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Interleucina-1/metabolismo , Proteínas de la Membrana/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Claudina-1 , Técnica del Anticuerpo Fluorescente , Técnica de Fractura por Congelación , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/farmacología , Proteínas de la Membrana/genética , Ocludina , Fosfoproteínas/metabolismo , ARN Mensajero/biosíntesis , Receptores de Interleucina-1/metabolismo , Sialoglicoproteínas/metabolismo , Sialoglicoproteínas/farmacología , Proteínas de la Zonula Occludens , Proteína de la Zonula Occludens-1 , Proteína de la Zonula Occludens-2RESUMEN
In white matter regions of the brain and spinal cord of adult mammals, gap junctions previously were observed linking astrocytes to astrocytes, as well as to oligodendrocytes and ependymacytes. The resulting "functional syncytium" was proposed to modulate the ion fluxes that occur during electrical activity of the associated axons. Gap junctions also have been reported linking neurons with glia, and functional neuronal-glial coupling has been postulated. To investigate the glial syncytium and the neuron-to-glial coupling hypotheses, we used "grid-mapped freeze fracture," conventional thin-section electron microscopy, and light microscope immunocytochemistry to examine and characterize neurons and glia in gray and white matter of adult rat brain and spinal cord. We have obtained quantitative evidence for the abundance and widespread distribution of gap junctions interlinking the three primary types of macroglia throughout both gray and white matter of the mammalian central nervous system (CNS), thereby extending the concept to that of a functional panglial syncytium. In contrast to previous reports, we show that of more than 400 gap junctions in which both participating cells were identified, none were between neurons and glia. Thus, neuronal coupling and glial coupling involved separate and distinct pathways. Finally, putative water channels (i.e., "square arrays") were confirmed to be abundant and in close association with gap junctions in astrocytes and ependymacytes. Because the astrocyte "intermediaries" extend cytoplasmic conduits throughout gray and white matter of brain and spinal cord, from the ependymal layer to the pia-glial limitans, and from oligodendrocytes surrounding axons to astrocyte endfeet surrounding capillaries, the proposed panglial syncytium, with its abundance of water channels and intercellular ion channels, is optimally positioned and equipped to modulate water and ion fluxes across broad regions of the CNS.
Asunto(s)
Encéfalo/ultraestructura , Uniones Comunicantes/ultraestructura , Células Gigantes/ultraestructura , Neuroglía/ultraestructura , Neuronas/ultraestructura , Médula Espinal/ultraestructura , Animales , Astrocitos/ultraestructura , Encéfalo/citología , Epéndimo/citología , Epéndimo/ultraestructura , Femenino , Técnica de Fractura por Congelación , Inmunohistoquímica , Masculino , Microscopía Electrónica , Neuroglía/citología , Oligodendroglía/ultraestructura , Ratas , Ratas Sprague-Dawley , Médula Espinal/citologíaRESUMEN
Previously, synaptic activity in the spinal cord of adult mammals was attributed exclusively to chemical neurotransmission. In this study, evidence was obtained for the existence, relative abundance, and widespread distribution of "mixed" (chemical and electrical) synapses on neurons throughout the spinal cords of adult mammals. Using combined confocal microscopy and "grid-mapped freeze fracture," 36 mixed synapses containing 88 "micro" gap junctions (median = 45 connexons) were found and mapped to 33 interneurons and motor neurons in Rexed laminae III-IX in cervical, thoracic, and lumbosacral spinal cords of adult male and female rats. Gap junctions were adjacent to presumptive active zones, where even small gap junctions would be expected to increase synaptic efficacy. Two morphological types of mixed synapse were discerned. One type contained distinctive active zones consisting of "nested" concentric toroidal deformations of pre- and postsynaptic membranes, which, because of their unusual topology, were designated as "synaptic sombreros." A second type had gap junctions adjacent to active zones consisting of broad, flat, shallow indentations of the plasma membrane. Morphometric analysis indicates that mixed synapses correspond to 3-5% of all synapses on the somata and proximal dendrites, but, because of their subcellular location and morphology, they could represent 30-100% of excitatory synapses. The relative abundance of mixed synapses on several classes of neurons in spinal cords of adult rats suggests that mixed synapses provide important but previously unrecognized pathways for bidirectional communication between neurons in the mammalian central nervous system.