RESUMO
The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.
Assuntos
Endocitose/fisiologia , Exocitose/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Quelantes/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Exocitose/efeitos dos fármacos , Feminino , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Hidrazonas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Técnicas de Patch-Clamp , Fosforilação , Estatísticas não Paramétricas , Sinapses/efeitos dos fármacos , Sinapses/genética , Vesículas Sinápticas/efeitos dos fármacos , Toxina Tetânica/farmacologia , Transfecção/métodosRESUMO
Photophysical properties of any fluorophore are governed by the chemical nanoenvironment. In the context of imaging biological samples, this translates to different photophysical properties for different labels and probes. Here, we demonstrate that the nanoenvironment of fluorophores within a probe can be advantageously used to induce particular properties such as light-induced photoswitching. We demonstrate efficient photoswitching and single-molecule super-resolution imaging for various fluorophore-phalloidin conjugates in aqueous buffer without the addition of further chemicals. We further demonstrate the utility of two-color imaging of fluorophore-phalloidin and a photoactivatable fluorescent protein in presynaptic nerve terminals.