Capacitance & Fluorescence Monitoring of Exo/Endocytosis

描述(由申请人提供)： 这项建议的长期目标是通过以下方式阐明这些机制 神经内分泌细胞中的哪些分泌颗粒经历胞吐作用并 随后通过内吞作用被取回。我们将结合使用 电生理(细胞附着和全细胞膜片钳电容)和 光学(FM染料荧光)技术检测单个胞外事件 大鼠脑下垂体促乳素细胞。我们之前已经证明了 乳酸菌使它们对这类研究特别有利。在这些 细胞，可以用FM实时观察单个胞外事件 用普通的荧光显微镜观察染料。我们发现了融合的毛孔 在这些细胞中，在刺激下爆炸，颗粒-颗粒融合 (复合胞吐)是一种普遍现象，并独立于 颗粒-质膜融合，增溶和释放 致密的核心是一个受监管的过程。我们建议继续进行这些研究。 在使用电池连接的电容记录的初步实验中，我们有 发现了另一种全新的胞外行为，即 自发的、有节奏的、搏动性的融合毛孔开口。这一现象是 常见(在近1/4的补丁中很明显)和健壮(保持不变 几十分钟)。单个融合孔的开口非常规则。 时间(频率约为1赫兹)，并且融合孔开口的持续时间为 非常恒定(约50毫秒)。我们建议检查这一行为，其 潜在的机制，以及下一步调节它的细胞过程 授权期。 对人类健康的影响。所有细胞都参与胞外分泌，这是一种至关重要的 以及复杂的过程，这一过程只被部分了解，如果被破坏， 会导致细胞功能受损和健康状况不佳。当我们的工作集中在 在这个基本的细胞过程上，而不是特定的疾病实体，更好 对调节这些重要功能的基本机制的理解 可能会显著影响我们抗击各种疾病的能力。

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.