Analytical Techniques for Exocytosis

拟议研究的目标是开发和应用分析方法， 环境来探测调节和调节胞吐作用的特定机制。其中一个关键方面是 工作将是使用电流测量同时与荧光成像，以测试 假设钙稳态控制和胞吐作用是雌激素 是神经保护，使用小体积分离，以确定囊泡内容物的程度， 在胞吐事件期间释放，并使用安培实验和共聚焦荧光 成像检查一个新的和潜在的有争议的假设，脂质纳米管发挥作用， 调节囊泡状态和释放。这项工作将在培养的嗜铬细胞瘤（PC 12）细胞中进行。 因此，这里提出的实验旨在从分子水平上理解胞吐作用， 理解细胞是如何以一种可能在细胞生物学中更普遍的方式“连接”的。 该提案的具体目标是：1）使用电流法和钙成像来检查 通过测量儿茶酚胺释放和钙离子进入雌激素的神经保护作用; 2）发展 在小毛细血管中进行电化学检测的电泳，以确定神经信使的水平 并通过与细胞处的安培测量进行比较来确定释放的分数 3）通过荧光识别细胞中的脂质纳米管结构，并研究胞吐过程中的脂质纳米管结构。 膜运输到囊泡和从囊泡运输的机制;和4）使用电化学和荧光， 检查并开发在完全胞吐之前通过融合孔释放递质的模型， “吻了就跑“释放的替代假设。 这项提议抓住了我们设想的真正理解囊泡功能所必需的工作 以及神经传递和突触可塑性中的胞吐作用。我们获得的非常初步的数据 这表明脂质纳米管的存在将囊泡连接到其他结构，也许是彼此连接。如果这是 正确，它代表了细胞生物学的一个新想法，可能非常重要。总的来说，使用 电流分析法，荧光和分离，提出了研究细胞化学和新的想法， 与神经元通讯有关的细胞生物学。

The goal of the proposed research is to develop and apply analytical methods in extremely small environments to probe specific mechanisms that regulate and modulate exocytosis. A key aspect of this work will be to use amperometric measurements simultaneously with fluorescence imaging to test the hypothesis that control of calcium homeostasis and exocytosis are part of the mechanism by which estrogen is neuroprotective, to use small-volume separations to determine the extent to which the vesicle contents are released during an exocytosis event, and to use amperometric experiments and confocal fluorescence imaging to examine a new and potentially controversial hypothesis that lipid nanotubes play a role in regulating vesicle state and release. This work will be done with pheochromocytoma (PC12) cells in culture. Thus, the experiments proposed here are targeted at understanding exocytosis at the molecular level and understanding how cells are "wired" in a way that might be more general in cell biology. The specific aims of the proposal are:1) to use amperometry and calcium imaging to examine the neuroprotective effects of estrogen by measuring catecholamine release and calcium entry; 2) to develop electrophoresis with electrochemical detection in small capillaries to determine the level of neuromessenger in vesicles and by comparison to amperometric measurements at cells to determine the fraction released during exocytosis; 3) to identify lipid nanotube structures in cells by fluorescence and to investigate the mechanism of membrane trafficking to and from vesicles; and 4) to use electrochemistry and fluorescence to examine and develop models of transmitter release via the fusion pore prior to full exocytosis and an alternative hypothesis for "kiss and run"release. This proposal captures the work that we envision as necessary to truly understand the function of vesicles and exocytosis in neurotransmission and synaptic plasticity. The highly preliminary data we have obtained suggest that lipid nanotubes exist connecting vesicles to other structures, perhaps each other. If this is correct, it represents a new idea in cell biology and could be incredibly important. Overall, the use of amperometry, fluorescence and separations is proposed to investigate cellular chemistry and new ideas in cell biology related to neuronal communication.