RESUMO
The secretory pathway in neurons requires efficient targeting of cargos and regulatory proteins to their release sites. Tomosyn contributes to synapse function by regulating synaptic vesicle (SV) and dense-core vesicle (DCV) secretion. While there is large support for the presynaptic accumulation of tomosyn in fixed preparations, alternative subcellular locations have been suggested. Here we studied the dynamic distribution of tomosyn-1 (Stxbp5) and tomosyn-2 (Stxbp5l) in mouse hippocampal neurons and observed a mixed diffuse and punctate localization pattern of both isoforms. Tomosyn-1 accumulations were present in axons and dendrites. As expected, tomosyn-1 was expressed in about 75% of the presynaptic terminals. Interestingly, also bidirectional moving tomosyn-1 and -2 puncta were observed. Despite the lack of a membrane anchor these puncta co-migrated with synapsin and neuropeptide Y, markers for respectively SVs and DCVs. Genetic blockade of two known tomosyn interactions with synaptotagmin-1 and its cognate SNAREs did not abolish its vesicular co-migration, suggesting an interplay of protein interactions mediated by the WD40 and SNARE domains. We hypothesize that the vesicle-binding properties of tomosyns may control the delivery, pan-synaptic sharing and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane.
Assuntos
Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Axônios/metabolismo , Sítios de Ligação , Western Blotting , Células Cultivadas , Dendritos/metabolismo , Hipocampo/citologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/ultraestrutura , Neuropeptídeo Y/metabolismo , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas R-SNARE/química , Proteínas R-SNARE/genética , Vesículas Secretórias/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismoRESUMO
Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2(KO/KO)) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders.
Assuntos
Atividade Motora/genética , Placa Motora/metabolismo , Junção Neuromuscular/citologia , Proteínas R-SNARE/deficiência , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Biofísica , Diafragma/fisiologia , Estimulação Elétrica , Embrião de Mamíferos , Regulação da Expressão Gênica/genética , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp , Desempenho Psicomotor/fisiologia , Proteínas R-SNARE/genética , Receptores Colinérgicos/metabolismo , Estatísticas não Paramétricas , Potenciais Sinápticos/genéticaRESUMO
Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASγ (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASγ. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASγ-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment.
