Capacitance & Fluorescence Monitoring of Exo/Endocytosis

描述（由申请人提供）： 本建议的长期目标是阐明机制， 神经内分泌细胞中的分泌颗粒经历胞吐作用， 随后通过内吞作用回收。我们将结合使用 电生理学（细胞附着和全细胞膜片钳电容）和 光学（FM染料荧光）技术来检查单个胞吐事件， 大鼠垂体催乳素我们先前表明， 催乳素使它们对于这样的研究特别有利。在这些 细胞，可以用FM实时观察单个胞吐事件 使用普通的落射荧光显微镜染色。我们发现融合孔 在这些细胞中，颗粒-颗粒融合 （复合胞吐）是一种常见现象，其调节独立于 颗粒-质膜融合，以及溶解和释放 致密核是一个受调控的过程。我们建议继续进行这些研究。 在使用细胞附着电容记录的初步实验中，我们有 发现了一种额外的、全新类型的胞吐行为，即 融合孔的自发的、有节奏的、脉动的开口。这种现象是 常见（在近1/4的斑块中明显）和稳健（持续不变） 十分钟）。单个的融合孔开口是非常规则的， 时间（频率约为1 Hz），并且融合孔开口的持续时间为 非常恒定（CA）。50 ms）。我们建议检查这种行为，其 潜在的机制，以及在未来调节它的细胞过程 补助期。 对人类健康的影响。所有的细胞都参与胞外分泌，这是一种重要的 一个复杂的过程，它只被部分理解，如果被破坏， 会导致细胞功能受损和健康不佳。虽然我们的工作重点是 在这个基本的细胞过程中，而不是一个特定的疾病实体， 了解调节这些重要功能的基本机制 可能会严重影响我们对抗各种疾病的能力。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to elucidate the mechanisms by which secretory granules in neuroendocrine cells undergo exocytosis and are retrieved subsequently by endocytosis. We will use a combination of electrophysiological (cell-attached and whole cell patch clamp capacitance) and optical (FM dye fluorescence) techniques to examine single exocytic events in rat pituitary lactotrophs. We showed previously that the secretory behavior of lactotrophs makes them particularly advantageous for such studies. In these cells, it is possible to observe single exocytic events in real-time with FM dyes using an ordinary epifluorescence microscope. We showed that fusion pores in these cells explode open upon stimulation, that granule-granule fusion (compound exocytosis) is a common phenomenon and is regulated independently of granule-plasma membrane fusion, and that the solubilization and release of dense cores is a regulated process. We propose to continue these studies. In preliminary experiments using cell-attached capacitance recordings, we have identified an additional, altogether new type of exocytic behavior, namely spontaneous, rhythmic, pulsatile openings of fusion pores. The phenomenon is common (evident in nearly 1/4 of patches) and robust (persisting unaltered for tens of minutes). The individual fusion pore openings are remarkably regular in time (frequency about 1 Hz) and the duration of the fusion pore openings is remarkably constant (ca. 50 ms). We propose to examine this behavior, its underlying mechanisms, and the cellular processes that regulate it in the next grant period. Implications for human health. All cells engage in exocytic secretion, a vital and complex process that is only partly understood, and which, if disrupted, can lead to impaired cellular function and ill health. While our work focuses on this basic cellular process, and not a specific disease entity, a better understanding of the fundamental mechanisms that regulate these vital functions could significantly impact our ability to combat a variety of diseases.