RESUMO
At presynaptic active zones, arrays of large conserved scaffold proteins mediate fast and temporally precise release of synaptic vesicles (SVs). SV release sites could be identified by clusters of Munc13, which allow SVs to dock in defined nanoscale relation to Ca2+ channels. We here show in Drosophila that RIM-binding protein (RIM-BP) connects release sites physically and functionally to the ELKS family Bruchpilot (BRP)-based scaffold engaged in SV recruitment. The RIM-BP N-terminal domain, while dispensable for SV release site organization, was crucial for proper nanoscale patterning of the BRP scaffold and needed for SV recruitment of SVs under strong stimulation. Structural analysis further showed that the RIM-BP fibronectin domains form a "hinge" in the protein center, while the C-terminal SH3 domain tandem binds RIM, Munc13, and Ca2+ channels release machinery collectively. RIM-BPs' conserved domain architecture seemingly provides a relay to guide SVs from membrane far scaffolds into membrane close release sites.
Assuntos
Proteínas de Transporte/química , Sistema Nervoso Central/metabolismo , Proteínas do Citoesqueleto/química , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas rab3 de Ligação ao GTP/química , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistema Nervoso Central/ultraestrutura , Clonagem Molecular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Larva/genética , Larva/metabolismo , Larva/ultraestrutura , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinapses/ultraestrutura , Transmissão Sináptica , Vesículas Sinápticas/ultraestrutura , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismoRESUMO
Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D. melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.
Assuntos
Proteínas de Drosophila/metabolismo , Reguladores de Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Endocitose , Exocitose , Reguladores de Proteínas de Ligação ao GTP/genética , Proteínas Ativadoras de GTPase/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMO
Synaptic vesicles (SVs) fuse at a specialized membrane domain called the active zone (AZ), covered by a conserved cytomatrix. How exactly cytomatrix components intersect with SV release remains insufficiently understood. We showed previously that loss of the Drosophila melanogaster ELKS family protein Bruchpilot (BRP) eliminates the cytomatrix (T bar) and declusters Ca(2+) channels. In this paper, we explored additional functions of the cytomatrix, starting with the biochemical identification of two BRP isoforms. Both isoforms alternated in a circular array and were important for proper T-bar formation. Basal transmission was decreased in isoform-specific mutants, which we attributed to a reduction in the size of the readily releasable pool (RRP) of SVs. We also found a corresponding reduction in the number of SVs docked close to the remaining cytomatrix. We propose that the macromolecular architecture created by the alternating pattern of the BRP isoforms determines the number of Ca(2+) channel-coupled SV release slots available per AZ and thereby sets the size of the RRP.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Vesículas Sinápticas/metabolismo , AnimaisRESUMO
The molecular machinery mediating the fusion of synaptic vesicles (SVs) at presynaptic active zone (AZ) membranes has been studied in detail, and several essential components have been identified. AZ-associated protein scaffolds are viewed as only modulatory for transmission. We discovered that Drosophila Rab3-interacting molecule (RIM)-binding protein (DRBP) is essential not only for the integrity of the AZ scaffold but also for exocytotic neurotransmitter release. Two-color stimulated emission depletion microscopy showed that DRBP surrounds the central Ca(2+) channel field. In drbp mutants, Ca(2+) channel clustering and Ca(2+) influx were impaired, and synaptic release probability was drastically reduced. Our data identify RBP family proteins as prime effectors of the AZ scaffold that are essential for the coupling of SVs, Ca(2+) channels, and the SV fusion machinery.
