Analytical Techniques for Exocytosis

描述（由申请人提供）：拟议研究的目标是在极小的环境中开发和应用分析方法，以探测调节和调节胞吐作用的特定机制。这项工作的一个关键方面将是同时使用电流测量和荧光成像来测试钙稳态和胞吐控制是雌激素神经保护机制的一部分的假设，使用小体积分离来确定囊泡内容物在胞吐事件期间释放的程度，并使用电流实验和共聚焦荧光成像来检查一个新的和潜在的有争议的假设，即脂质纳米管在调节囊泡状态和释放中起作用。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了解分子水平上的胞吐作用，并了解细胞如何以一种可能在细胞生物学中更普遍的方式“连接”。该提案的具体目标是：1）使用电流分析法和钙成像法，通过测量儿茶酚胺释放和钙进入来检查雌激素的神经保护作用; 2）在小毛细管中开发具有电化学检测的电泳，以确定囊泡中神经信使的水平，并通过与细胞中的电流分析法测量进行比较，以确定胞吐期间释放的分数; 3）通过荧光鉴定细胞中的脂质纳米管结构，并研究膜运输到囊泡和从囊泡运输的机制;和4）使用电化学和荧光来检查和开发在完全胞吐之前通过融合孔释放递质的模型和“吻和跑”释放的替代假设。这个建议抓住了我们设想的工作，作为必要的，以真正了解囊泡和胞吐作用在神经传递和突触可塑性。我们获得的高度初步的数据表明，存在脂质纳米管将囊泡连接到其他结构，也许是彼此。如果这是正确的，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，电流分析法，荧光和分离的使用，提出了研究细胞化学和新的想法，在细胞生物学与神经元通信。

DESCRIPTION (provided by applicant): 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.