Secretion from individual vesicles

描述（由申请人提供）：胞吐作用是在细胞内囊泡与细胞膜融合后发生的钙触发的分泌过程，随后囊泡内容物被挤出到细胞外空间。在含有容易氧化的化学信使的细胞中，这一过程可以在单个细胞中用邻近细胞放置的碳纤维电极进行监测。在之前的资助期间，我们证明了在平均含有30，000个多巴胺分子的神经元中检测分泌事件。多巴胺神经元的鉴定能够与组织从一个突变的小鼠，表达人蛋白质的儿茶酚胺能细胞。因此，多巴胺细胞可以在含有许多细胞类型的神经元制剂中被荧光标记。此外，我们使用荧光指示剂来监测这些神经元中的细胞内钙离子。拟议的研究建立在这些工具的基础上，并将研究胞吐作用的基本控制点，具体目标如下： 1.评价多巴胺能神经元囊泡储存对随后释放的作用。囊泡在非神经元细胞中释放的量和时间过程反映了它们先前的储存。我们将研究神经元是否也是如此。 2.检查细胞内钙离子被控制的机制，并影响神经元释放的频率和持续时间。胞吐作用的触发物，细胞内钙离子，在许多细胞中被线粒体隔离。我们将测试的假设，这种机制终止释放多巴胺能神经元。 3.检查突触蛋白在决定释放囊泡的可用性中的作用。突触蛋白是一种丰富的神经元蛋白，在控制囊泡运输中起重要作用。将通过评估突触蛋白敲除小鼠中的释放来检验该假设。 4.比较细胞体和末梢释放的儿茶酚胺。现在有充分的证据表明，胞吐作用可以发生在神经元索马和终末。使用可视化的多巴胺神经元，我们将测试这一假设，即神经元的两个不同部分的释放调节是相似的。

DESCRIPTION (provided by applicant): Exocytosis is the calcium-triggered, secretory process that occurs at cells upon fusion of an intracellular vesicle with the cell membrane followed by extrusion of the vesicular contents into the extracellular space. At cells that contain easily oxidized chemical messengers, this process can be monitored at individual cells with carbon-fiber electrodes placed adjacent to the cell. In the prior funding period we demonstrated detection of secretory events at neurons that contained on average 30,000 dopamine molecules. Identification of dopamine neurons was enabled with tissue from a mutant mouse that expresses a human protein in catecholaminergic cells. Thus, dopamine cells can be fluorescently tagged in neuronal preparations that contained many cell types. In addition we used a fluorescent indicator to monitor intracellular calcium ions in these neurons. The proposed research builds upon these tools and will investigate basic control points of exocytosis with the following specific aims: 1. Evaluate the role of vesicular storage in dopaminergic neurons on subsequent release. The amount and time course of release of vesicles at nonneuronal cells reflect their prior storage. We will examine whether this is also the case in neurons. 2. Examine the mechanisms by which intracellular calcium ions are controlled and affect the frequency and duration of release in neurons. The trigger for exocytosis, intracellular calcium ions, are sequestered by mitochondria in many cells. We will test the hypothesis that this mechanism terminates release in dopaminergic neurons. 3. Examine the role of synapsin in determining the availability of vesicles for release. Synapsin, an abundant neuronal protein, appears to be important in controlling vesicular trafficking. This hypothesis will be test by evaluating release in synapsin knock-out mice. 4. Compare catecholamine release from cell bodies and terminals. It is now well documented that exocytosis can occur at neuronal soma as well as at terminals. Using visualized dopamine neurons, we will test the hypothesis that the regulation of release is similar in the two different parts of the neuron.