IN VITRO ANALYSIS OF SECRETORY VESICLE DOCKING

这个项目涉及到对如何对接和 参与后高尔基体相互作用的融合过程 囊泡和靶质膜导致结构性或 调节分泌。这些过程似乎共享分子 成分，通过分离突触小泡和 突触前质膜蛋白参与的成分 结构性高尔基体内转运(NEM敏感因子，NSF)。我们 假设，作为一个工作假说，基本的分子结构 负责构成对接和聚变过程的采购 关闭它的其他元素；在休息条件下和在 由外部刺激触发的条件，取决于细胞的类型。 这项研究的具体目标是对分子的分析 同源和异源调控胞吐的要求 体外重建系统。为了探索这一假说和 提出的实验策略包括：a)体外重建 免疫组织化学中标记突触小泡的对接和去对接 分离的突触前神经末梢，b)特异性分析 在不同神经元后高尔基体衍生的囊泡与 活动区和c)寻找高尔基体后抑制物 重组穿孔酵母菌的结构性分泌物 带有突触蛋白的酿酒细胞。

This project is concerned with the understanding of how docking and fusion processes involved in the interactions between post-Golgi derived vesicles and target plasma membrane lead to either constitutive or regulated secretion. These processes appear to share molecular components, as suggested by the isolation of synaptic vesicle and presynaptic plasma membrane proteins by means of components involved in constitutive intra-Golgi transport (NEM-sensitive factor, NSF). We assume, as a working hypothesis, that the basic molecular machinery responsible for constitutive docking and fusion processes acquires additional elements that turn it off; in resting conditions and "on" in conditions triggered by external stimuli, depending on the kind of cell. The specific goal of this study is the analysis of the molecular requirements of regulated exocytosis using homologous and heterologous in vitro reconstitution systems. To explore this hypothesis an experimental strategy is proposed involving: a)in vitro reconstitution of the docking and undocking of labeled synaptic vesicles in immuno- isolated presynaptic nerve terminals, b) the analysis of the specificity in the binding of different neuronal post-Golgi derived vesicles to active zones and c) the search for inhibitors of the post Golgi constitutive secretion using reconstituting perforated Saccharomyces cerevisiae cells with synaptic proteins.