Secretion from individual vesicles

描述（由申请人提供）：胞吐作用是细胞内囊泡与细胞膜融合，然后将囊泡内容物挤出到细胞外空间时在细胞中发生的钙触发的分泌过程。对于含有容易氧化的化学信使的细胞，可以通过在细胞附近放置碳纤维电极来监测各个细胞的这一过程。在之前的资助期间，我们展示了对平均含有 30,000 个多巴胺分子的神经元的分泌事件的检测。利用在儿茶酚胺能细胞中表达人类蛋白质的突变小鼠的组织，可以鉴定多巴胺神经元。因此，可以在含有多种细胞类型的神经元制剂中对多巴胺细胞进行荧光标记。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。拟议的研究以这些工具为基础，并将研究胞吐作用的基本控制点，具体目标如下： 1. 评估多巴胺能神经元中囊泡储存对随后释放的作用。非神经元细胞中囊泡释放的量和时间过程反映了它们之前的储存。我们将检查神经元中是否也是如此。 2. 检查细胞内钙离子的控制机制以及影响神经元释放频率和持续时间的机制。胞吐作用的触发因素是细胞内钙离子，在许多细胞中被线粒体隔离。我们将检验这一机制终止多巴胺能神经元释放的假设。 3. 检查突触蛋白在确定囊泡释放可用性方面的作用。突触蛋白是一种丰富的神经元蛋白，似乎对于控制囊泡运输很重要。该假设将通过评估突触蛋白敲除小鼠的释放来检验。 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胞体和末梢。使用可视化的多巴胺神经元，我们将检验神经元两个不同部分的释放调节相似的假设。

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.