Secretion from individual vesicles

描述(申请人提供)：胞吐作用是细胞内的囊泡与细胞膜融合，随后囊泡内容物被挤出到细胞外空间时，细胞内钙触发的分泌过程。在含有容易氧化的化学信使的细胞中，这一过程可以通过放置在细胞旁边的碳纤维电极在单个细胞上进行监测。在之前的资助期间，我们展示了在平均含有30,000个多巴胺分子的神经元中检测到分泌事件。通过在儿茶酚胺能细胞中表达人类蛋白的突变小鼠的组织，可以鉴定多巴胺神经元。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，我们还使用了一个荧光指示剂来监测这些神经元的细胞内钙离子。拟议的研究建立在这些工具的基础上，将调查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的囊泡储存在随后释放时的作用。囊泡在非神经细胞中释放的数量和时间进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.研究细胞内钙离子的调控机制以及影响神经元内钙离子释放的频率和持续时间。胞吐的触发物，细胞内的钙离子，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.研究突触素在决定囊泡释放的可用性中的作用。突触素是一种丰富的神经元蛋白，似乎在控制囊泡运输方面发挥着重要作用。这一假说将通过评估突触素基因敲除小鼠的释放来检验。 4.比较胞体和终末的儿茶酚胺释放。现在已有充分的文献记载胞吐作用可发生在神经元胞体和终末。利用可视化的多巴胺神经元，我们将检验这一假设，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.