Analytical Techniques for Exocytosis

胞吐作用的分析技术

• 批准号: 7230431
• 负责人: ANDREW G EWING
• 金额: $ 21.77万
• 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/7230431
• 关键词: Ablation; Area; Attention; Behavior; Blood capillaries; Brain; Calcium; Caliber; Capillary Electrophoresis; Catecholamines; Categories; Cell membrane; Cells; Cellular biology; Characteristics; Charge; Chemistry; Communication; Coupled; Cytolysis; Data; Detection; Development; Disease; Dyes; Electrochemistry; Electrophoresis; Environment; Estrogens; Event; Exocytosis; Fluorescence; Fluorescence Microscopy; Goals; Homeostasis; Hormones; Image; Journals; Laboratories; Lasers; Lead; Lipids; Measurement; Measures; Membrane; Membrane Protein Traffic; Methodology; Methods; Modeling; Molecular; Nanotubes; Neuromodulator; Neurons; Neurosciences; Neurotransmitters; Numbers; Organelles; PC12 Cells; Parkinson Disease; Pharmacological Treatment; Pheochromocytoma; Play; Process; Progress Reports; Publishing; Rate; Regulation; Reporting; Research; Role; Running; Source; Structure; Synaptic plasticity; System; Techniques; Technology; Testing; Transmembrane Transport; Vesicle; Work; analog; analytical method; base; capillary; falls; fluorescence imaging; fluorophore; interest; nanometer; neurotransmission; neurotransmitter release; organelle movement; research study; size

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（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，以探索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控制和胞吐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胞吐事件中囊泡内容物释放的程度。并利用安培实验和共聚焦荧光成像来检验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发挥作用。这项工作将在培养的嗜铬细胞瘤（PC12）细胞中进行。因此，本文提出的实验旨在从分子水平上理解胞泌作用，并以一种在细胞生物学中可能更普遍的方式理解细胞是如何“连接”的。该建议的具体目的是：1)通过测量儿茶酚胺的释放和钙的进入，使用电流测量和钙成像来检查雌激素的神经保护作用；2)在小毛细血管中发展电泳电化学检测，以确定囊泡中神经信使的水平，并通过与细胞的电流测量相比较，确定胞吐过程中释放的部分；3)利用荧光技术鉴定细胞内的脂质纳米管结构，研究细胞膜进出囊泡的转运机制；4)利用电化学和荧光技术研究和开发在细胞完全分泌之前通过融合孔释放递质的模型，以及“吻和跑”释放的另一种假设。这一建议抓住了我们设想的必要工作，以真正理解囊泡和胞吐在神经传递和突触可塑性中的功能。我们已经获得的高度初步的数据表明，脂质纳米管存在连接囊泡和其他结构，可能彼此连接。如果这是正确的，它代表了细胞生物学的一个新想法，可能是非常重要的。总之，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路。