Analytical Techniques for Exocytosis

胞吐作用的分析技术

• 批准号: 7098910
• 负责人: ANDREW G EWING
• 金额: $ 46.5万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7098910
• 关键词: calcium; catecholamines; estrogens; exocytosis; fluorescence; lipids; measurement

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.

描述(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，以探索调节和调节胞吐的具体机制。这项工作的一个关键方面将是同时使用安培测量和荧光成像来检验这一假说，即控制钙稳态和胞吐是雌激素神经保护机制的一部分，使用小体积分离来确定胞吐过程中囊泡内容物释放的程度，并使用安培实验和共聚焦荧光成像来检验一个新的、可能有争议的假说，即脂质纳米管在调节囊泡状态和释放方面发挥作用。这项工作将在培养的嗜铬细胞瘤(PC12)细胞中完成。因此，这里提出的实验的目标是在分子水平上了解胞吐作用，并了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。建议的具体目的是：1)通过测量儿茶酚胺的释放和钙离子的进入，使用安培和钙成像来检测雌激素的神经保护作用；2)发展具有电化学检测的小毛细管电泳法，以确定囊泡中神经信使的水平，并通过与细胞的安培测量相比较，来确定胞吐过程中释放的部分；3)通过荧光识别细胞中的脂质纳米管结构，并研究膜向囊泡和从囊泡转运的机制；以及4)使用电化学和荧光来检查和建立在完全胞吐之前通过融合孔释放递质的模型，并提出一种可供选择的“亲吻”释放假说。这一提议抓住了我们设想的真正理解囊泡和胞吐在神经传递和突触可塑性中的功能所必需的工作。我们已经获得的高度初步的数据表明，脂质纳米管存在将囊泡连接到其他结构，也许是彼此连接。如果这是正确的，它代表了细胞生物学的一个新观点，可能非常重要。综上所述，我们建议使用安培法、荧光法和分离法来研究细胞化学和细胞生物学中与神经元通讯相关的新思想。