RESUMO
RNAi screening has gained popularity in recent years, due to its usefulness in systematic investigations of biological pathways. Combined with high-content screening and advances in imaging and analysis methods, it can enable detailed genetic characterization of cellular processes such as protein glycosylation, a major function of the Golgi apparatus. Glycosylation concerns about one third of all human proteins and regulates various cellular behaviors. Yet the methods available to study it are limited and not easily accessible. In this chapter, we detail a step-by-step method to systematically and quantitatively investigate glycosylation using fluorescent lectin staining, following high-throughput RNAi-based downregulation of gene activities. We also provide a workflow for downstream analysis of the data generated.
Assuntos
Complexo de Golgi/metabolismo , Ensaios de Triagem em Larga Escala , Interferência de RNA , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Técnicas de Silenciamento de Genes , Glicosilação , Humanos , Processamento de Imagem Assistida por Computador , Lectinas/genética , Lectinas/metabolismo , Microscopia de Fluorescência , Ligação ProteicaRESUMO
The amount of neurotransmitter stored in a single synaptic vesicle can determine the size of the postsynaptic response, but the factors that regulate vesicle filling are poorly understood. A proton electrochemical gradient (Δµ(H+)) generated by the vacuolar H(+)-ATPase drives the accumulation of classical transmitters into synaptic vesicles. The chemical component of Δµ(H+) (ΔpH) has received particular attention for its role in the vesicular transport of cationic transmitters as well as in protein sorting and degradation. Thus, considerable work has addressed the factors that promote ΔpH. However, synaptic vesicle uptake of the principal excitatory transmitter glutamate depends on the electrical component of Δµ(H+) (Δψ). We found that rat brain synaptic vesicles express monovalent cation/H(+) exchange activity that converts ΔpH into Δψ, and that this promotes synaptic vesicle filling with glutamate. Manipulating presynaptic K(+) at a glutamatergic synapse influenced quantal size, indicating that synaptic vesicle K(+)/H(+) exchange regulates glutamate release and synaptic transmission.
Assuntos
Potássio/metabolismo , Terminações Pré-Sinápticas/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas de Artrópodes , Ácido Aspártico/farmacocinética , Transporte Biológico , Fenômenos Biofísicos/efeitos dos fármacos , Encéfalo/citologia , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Cátions/metabolismo , Relação Dose-Resposta a Droga , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Gluconatos/farmacologia , Ácido Glutâmico/farmacocinética , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Ionóforos/farmacologia , Potencial da Membrana Mitocondrial , Monensin/farmacologia , Oligopeptídeos/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Cintilografia , Ratos , Ratos Wistar , Isótopos de Sódio/farmacocinética , Sinapses/diagnóstico por imagem , Sinapses/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Sinaptossomos/ultraestrutura , Trítio/farmacocinéticaRESUMO
Dopamine neurons in the ventral tegmental area (VTA) play an important role in the motivational systems underlying drug addiction, and recent work has suggested that they also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate corelease, we disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. The conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, apparently through a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes monoamine storage by increasing the pH gradient that drives vesicular monoamine transport. Remarkably, low concentrations of glutamate acidify synaptic vesicles more slowly but to a greater extent than equimolar Cl(-), indicating a distinct, presynaptic mechanism to regulate quantal size.
