Secretion from individual vesicles

描述（由申请人提供）：胞吐是一种钙触发的分泌过程，发生在细胞内囊泡与细胞膜融合后，然后将囊泡内容物挤压到细胞外空间。在含有易氧化化学信使的细胞中，这一过程可以通过放置在细胞附近的碳纤维电极在单个细胞上进行监测。在之前的资助期内，我们展示了对平均含有30,000个多巴胺分子的神经元分泌事件的检测。多巴胺神经元的鉴定是通过在儿茶酚胺能细胞中表达人类蛋白的突变小鼠组织实现的。因此，多巴胺细胞可以在含有多种细胞类型的神经元制剂中进行荧光标记。此外，我们使用荧光指示剂监测细胞内钙离子在这些神经元。拟议的研究建立在这些工具的基础上，并将研究胞吐的基本控制点，具体目标如下：

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.