Analytical Techniques for Exocytosis

拟议研究的目标是在极小的范围内开发和应用分析方法 探索调节和调节胞吐的特定机制的环境。这其中的一个关键方面 工作将是同时使用安培测量和荧光成像来测试 认为控制钙稳态和胞吐作用是雌激素作用机制的一部分 是神经保护的，使用小容量分离来确定水泡内容物被 在胞吐作用过程中释放，并使用安培实验和共聚焦荧光 检查一个新的和可能有争议的假说的成像，该假说认为脂质纳米管在 调节囊泡状态和释放。这项工作将在培养的嗜铬细胞瘤(PC12)细胞中完成。 因此，这里提出的实验目的是在分子水平上理解胞吐作用和 理解细胞是如何以一种在细胞生物学中可能更普遍的方式被“连接”的。 该提案的具体目的是：1)使用安培和钙成像来检查 通过测定儿茶酚胺释放和钙内流来研究雌激素的神经保护作用 电泳法测定小毛细血管内神经信使水平 并通过与细胞中的安培测量相比较来确定释放的部分 3)用荧光法鉴定细胞中的脂质纳米管结构，并研究其在细胞内的作用 膜转运到和离开囊泡的机制；以及4)利用电化学和荧光来 研究和开发在充分胞吐之前通过融合孔释放递质的模型和 《接吻就跑》释放的另一种假说。 这一建议抓住了我们所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。我们已经获得的高度初步的数据 这表明脂质纳米管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中的一种新思想，可能非常重要。总体而言， 提出了安培法、荧光法和分离法来研究细胞化学和新的思路 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.