RESUMO
The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Neuropeptídeos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Caderinas/metabolismo , Diferenciação Celular , Células Cultivadas , Cromograninas/metabolismo , Hipocampo/citologia , Hipocampo/embriologia , Hipocampo/metabolismo , Fusão de Membrana/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Transporte Proteico/fisiologia , Proteínas Qa-SNARE , Ratos , Ratos Sprague-Dawley , Vesículas Secretórias/metabolismo , Proteína 25 Associada a SinaptossomaRESUMO
Piccolo is a novel component of the presynaptic cytoskeletal matrix (PCM) assembled at the active zone of neurotransmitter release. Analysis of its primary structure reveals that Piccolo is a multidomain zinc finger protein structurally related to Bassoon, another PCM protein. Both proteins were found to be shared components of glutamatergic and GABAergic CNS synapses but not of the cholinergic neuromuscular junction. The Piccolo zinc fingers were found to interact with the dual prenylated rab3A and VAMP2/Synaptobrevin II receptor PRA1. We show that PRA1 is a synaptic vesicle-associated protein that is colocalized with Piccolo in nerve terminals of hippocampal primary neurons. These data suggest that Piccolo plays a role in the trafficking of synaptic vesicles (SVs) at the active zone.
Assuntos
Proteínas de Transporte , Proteínas do Citoesqueleto/genética , Proteínas do Tecido Nervoso/genética , Neurônios/química , Neuropeptídeos/genética , Terminações Pré-Sinápticas/química , Receptores de Superfície Celular , Dedos de Zinco/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Éxons/genética , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP , Ácido Glutâmico/fisiologia , Hipocampo/citologia , Humanos , Íntrons/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Neurônios/citologia , Neurônios/metabolismo , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas R-SNARE , Coelhos , Ratos , Proteínas de Transporte Vesicular , Ácido gama-Aminobutírico/fisiologia , Proteína rab3A de Ligação ao GTP/metabolismoRESUMO
In this study, we describe a novel 420 kDa protein, called Piccolo, found at a wide variety of adult rat brain synapses. High protein levels in the cerebellum, the olfactory bulb and the hippocampus were frequently observed to be associated with asymmetric type 1 synapses. Piccolo is selectively enriched in presynaptic terminals, but is not a component of synaptic vesicles (SVs). Immunogold electron microscopy revealed that Piccolo localizes to the amorphous material among SVs at the presynaptic plasma membrane. Biochemical studies showed that it is very tightly bound to this structure. Thus, we speculate that Piccolo is a structural component of the presynaptic cytomatrix which anchors SVs to the presynaptic plasmalemma.
Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismo , Animais , Anticorpos/imunologia , Encéfalo/ultraestrutura , Ratos , Vesículas Sinápticas/metabolismoRESUMO
A novel synapse-associated protein, SAP90, accumulates around the axon hillock of Purkinje cells in rat cerebellum. By immuno-electron microscopy, SAP90 has been localized to the presynaptic termini of basket cells forming inhibitory, gamma-aminobutyric acid (GABA)ergic synapses onto Purkinje cell axon hillocks. The amino acid sequence for SAP90 has been deduced from the nucleotide sequence of a series of overlapping cDNA clones. SAP90 is related to the gene product encoded by the Drosophila tumor suppressor gene dlg-A. SAP90 and the dlg-A product share an overall sequence identity of 54%. Three distinct domains can be identified: (i) a potential cytoskeletal region consisting of three repeats of 90 amino acids in length, (ii) a domain with similarity to SH3, a putative regulatory motif found in the src family of non-receptor protein tyrosine kinases and several proteins associated with the cortical cytoskeleton, and (iii) a carboxyl-terminal domain homologous to yeast guanylate kinase. These features suggest a possible role for SAP90 in a guanine nucleotide-mediated signal transduction pathway at a subset of GABAergic synapses in the rat cerebellum.
Assuntos
Proteínas de Drosophila , Genes Supressores de Tumor , Hormônios de Inseto/genética , Proteínas do Tecido Nervoso/genética , Sinapses/metabolismo , Proteínas Supressoras de Tumor , Sequência de Aminoácidos , Animais , Sequência de Bases , Cerebelo/metabolismo , DNA , Drosophila , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Mapeamento por Restrição , Proteínas Associadas SAP90-PSD95 , Homologia de Sequência de AminoácidosRESUMO
Synapses are highly specialized sites of cell-cell contact involved in signal transfer. The molecular mechanisms modulating the assembly and stability of synapses are unknown. We previously reported the identification of a 90 kDa synapse-associated protein, SAP90, that is localized at the presynaptic termini of inhibitory GABAergic synapses. SAP90 is a mosaic protein composed of three 90 amino acid residue repeats, an SH3 domain and a region homologous to guanylate kinases. SAP90 shares domain specific homology with a family of proteins involved in the assembly and possibly stability of sites of cell contact. These include the product of the lethal(1) discs-large-1 (dlgA) tumor suppressor gene and the zonula occludens proteins ZO-1, ZO-2. The further characterization of cDNA clones encoding components of synaptic junctions has lead to the identification of a 97 kDa protein, called SAP97, that exhibits a strong overall sequence similarity to SAP90. The present study was undertaken to determine the spatial distribution of SAP97, and to reveal further clues to the possible roles of these proteins in synapses. Light and immunoelectron microscopic analysis of the rat hippocampal formation revealed that SAP97 is localized in the presynaptic nerve termini of excitatory synapses. In other brain regions, SAP97 is found in and along bundles of unmyelinated axons. SAP97 is not restricted to the CNS, but is also present at the basal lateral membrane between a variety of epithelial cells. In cultured T84 cells, it is restricted to the cytoplasmic surface of the plasma membranes between adjacent cells, but not at the edges of cells lacking cell-cell contact suggesting a role for SAP97 in cell adhesion. These data suggest that members of the SAP90/SAP97 subfamily may be involved in the site specific assembly, stability or functions of membrane specialization at sites of cell-cell contact.