CAPACITANCE & FLUORESCENCE MONITORING OF EXO/ENDOCYTOSIS

电容

• 批准号: 6187944
• 负责人: WILLIAM J BETZ
• 金额: $ 20.29万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6187944
• 关键词: biological signal transduction; calcium flux; calcium indicator; chromaffin cells; confocal scanning microscopy; dialysis; dielectric property; electron microscopy; endocytosis; exocytosis; fluorescent dye /probe; guanosinetriphosphatases; image processing; ionophores; membrane fusion; vesicle /vacuole; voltage /patch clamp

DESCRIPTION (from applicant's abstract) Exocytosis is one of the processes by which cells secrete substance to the external world. The complex physiological steps underlying the secretory cycle are only partly understood, but are of fundamental importance to all cells. Most early information about exo-and endocytosis came from studies of neuronal synapses. However, the patch clamp capacitance technique (Neher and Marty, 1982) shifted emphasis increasingly to isolated secretory cells that could be patch clamped, criteria that preclude most nerve terminals from such studies owing to their small size. Today, the prototypical cell for studying the physiology of secretion is the adrenal chromaffin cell; more is known about the mechanism of secretion in this cell than any other. The applicants propose to study exocytosis in chromaffin cells - particularly questions concerning exocytic 'hot spots' and 'kiss and run' exocytosis - using a technique that they developed - FM1-43 fluorescence - in combination with patch clamp capacitance and with electron microscopy. The technique of FM1-43 fluorescence has proven useful for studying exo- and endocytosis in neurons, and more recently, in chromaffin cells (Smith and Betz, 1996). Exocytosis can be monitored because FM1-43 fluorescence increases more than 300 times when it binds to a membrane. Thus, when a secretory granule undergoes exocytosis and its membrane binds FM1-43, the overall fluorescence of the cell increases. The applicants will optimize the spatial resolution of their measurements to detect single exocytic and endocytic sites. They will then address questions like: Are the sites distributed randomly over the surface of the chromaffin cell, or are they clustered in 'hot spots'? Does the distribution change with different amounts of stimulation? In addition to studying the spatial nature of exo- and endocytosis, they will also study the events that occur immediately after exocytosis. For example, in neurons synaptic vesicles collapse after they undergo exocytosis and become part of the cell surface membrane (they are retrieved by endocytosis later at remote sites). In chromaffin cells, recent evidence suggests that granules do not ordinarily collapse after exocytosis, but pinch back directly, a phenomenon known colloquially as 'kiss and run' exocytosis. The applicants have developed a method to monitor the post-exocytic fate of granules, and by injecting drugs, peptides, and other agents into a cell through a patch pipette, they will study the normal regulatory processes that determine the fate of a secretory granule.

描述（来自申请人的摘要）胞吐作用是其中一个过程 细胞通过它向外界分泌物质。 复杂 分泌周期的生理步骤只是部分 这是一个基本的概念，但对所有细胞都至关重要。 最早 关于胞吞和胞吞作用的信息来自于对神经细胞的研究， 突触 然而，膜片钳电容技术（Neher和Marty， 1982）将重点越来越多地转移到分离的分泌细胞， 被膜片钳，排除大多数神经末梢的标准， 研究由于其规模小。 今天， 研究分泌生理的是肾上腺嗜铬细胞;更多的是 比其他任何细胞都更了解这种细胞的分泌机制。 的 申请人提出研究嗜铬细胞中的胞吐作用-特别是 关于胞吐“热点”和“吻和运行”胞吐作用的问题- 使用他们开发的技术-FM 1 -43荧光-结合 用膜片钳电容和电子显微镜。 FM 1 -43荧光技术已被证明可用于研究外消旋和内消旋。 内吞作用在神经元，最近，在嗜铬细胞（史密斯和 贝茨，1996年）。 可以监测胞吐作用，因为FM 1 -43荧光 当它与细胞膜结合时会增加300倍以上。 当一个 分泌颗粒经历胞吐作用，其膜结合FM 1 -43， 细胞的总荧光增加。 申请人将优化其测量的空间分辨率， 检测单个胞吐和胞吞位点。 然后他们将解决 问题是：网站是否随机分布在表面上？ 嗜铬细胞，还是它们聚集在“热点”？ 是否 分布随刺激量的不同而变化？ 除了研究外吞和内吞作用的空间性质外， 还将研究胞吐作用后立即发生的事件。 为 例如，在神经元中，突触囊泡在经历胞吐作用后塌陷 并成为细胞表面膜的一部分（它们被 内吞作用稍后在远程位点）。 在嗜铬细胞中，最近的证据 表明颗粒在胞吐作用后通常不会塌陷，但 直接掐回去，这种现象俗称“吻了就跑” 胞吐作用 申请人已经开发了一种方法来监测 颗粒的胞吐后命运，以及通过注射药物、肽和其他 通过贴片吸管将药物注入细胞，他们将研究正常的 决定分泌颗粒命运的调节过程。