CAPACITANCE & FLUORESCENCE MONITORING OF EXO/ENDOCYTOSIS

描述(摘自申请者的摘要)胞吐是其中一个过程 细胞通过它向外部世界分泌物质。情结 作为分泌周期基础的生理步骤只有一部分 理解，但对所有细胞都是至关重要的。最早 有关外吞和内吞的信息来自对神经元的研究 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上 被膜片钳夹住，这是阻止大多数神经末梢发生这种情况的标准 由于书房规模较小，这些书房的面积较小。今天，人类的原型细胞 研究分泌生理学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个细胞的分泌机制。这个 申请者建议研究嗜铬细胞的胞吐作用--特别是 关于胞吐“热点”和“接吻即跑”胞吐的问题- 使用他们开发的一种技术-FM1-43荧光-组合 具有膜片钳电容和电子显微镜。 FM1-43荧光技术已被证明是研究外源和外源基因的有效方法。 神经元的内吞作用，以及最近嗜铬细胞的内吞作用(Smith和 贝茨，1996)。胞吐作用可以监测，因为FM1-43荧光 当它结合到膜上时，会增加300多倍。因此，当一个 分泌颗粒经历胞吐作用，其膜结合FM1-43。 细胞的整体荧光增强。 申请者将优化他们的测量的空间分辨率以 检测单个胞外和内吞部位。然后他们将发表演讲 像这样的问题：这些站点是否随机分布在 嗜铬细胞，还是它们聚集在“热点”中？这是不是 分布随刺激量的不同而变化？ 除了研究外吞和内吞的空间性质外，他们还 还将研究胞吐后立即发生的事件。为 例如，在神经元中，突触小泡在经历胞吐后崩溃。 并成为细胞表面膜的一部分(它们通过 稍后在偏远部位的内吞作用)。在嗜铬细胞中，最近的证据 这表明颗粒通常不会在胞吐后坍塌，但 直接捏回去，这一现象俗称为“接吻就跑” 胞吐。申请者已经开发出一种方法来监控 颗粒的胞吐后命运，以及通过注射药物、多肽和其他 药剂通过移液管进入细胞，它们将研究正常 决定分泌颗粒命运的调控过程。

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.