Capacitance & Fluorescence Monitoring of Exo/Endocytosis

电容

• 批准号: 6647663
• 负责人: WILLIAM J BETZ
• 金额: $ 32.98万
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6647663
• 关键词: adenosine triphosphate; biological signal transduction; bromocriptine; calcium flux; calcium indicator; cell membrane; chromaffin cells; cyclic AMP; dielectric property; dopamine; electrophysiology; endocytosis; exocytosis; fluorescence microscopy; fluorescent dye /probe; laboratory rat; membrane fusion; monensin; neuroendocrine system; pituitary gland; protein kinase A; protein kinase C; secretion; tissue /cell culture; vesicle /vacuole; voltage /patch clamp

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.

描述（由申请人提供）： 该提案的长期目标是通过以下方式阐明机制： 神经内分泌细胞中的哪些分泌颗粒进行胞吐作用并且是 随后通过内吞作用恢复。我们将使用以下组合 电生理学（细胞贴壁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。我们之前表明，分泌行为 泌乳素细胞使它们特别有利于此类研究。在这些 细胞，可以通过 FM 实时观察单个胞吐事件 使用普通落射荧光显微镜进行染料染色。我们证明了融合孔 在这些细胞中，受到刺激后会爆炸，颗粒间融合 （复合胞吐作用）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 致密核心是一个受监管的过程。我们建议继续这些研究。 在使用细胞附着电容记录的初步实验中，我们有 确定了另一种全新的胞吐行为，即 融合孔自发的、有节奏的、脉动的开口。现象是 常见（在近 1/4 的补丁中很明显）并且稳健（持续不变） 几十分钟）。各个融合孔的开口非常规则 时间（频率约1 Hz）和融合孔开口的持续时间为 非常恒定（约 50 毫秒）。我们建议检查这种行为及其 潜在的机制，以及在接下来的过程中调节它的细胞过程 授予期。 对人类健康的影响。所有细胞都参与胞吐分泌，这是一种重要的 以及仅被部分理解的复杂过程，如果被破坏， 可能导致细胞功能受损和健康状况不佳。虽然我们的工作重点是 基于这个基本的细胞过程，而不是特定的疾病实体，更好的 了解调节这些重要功能的基本机制 可能会极大地影响我们对抗多种疾病的能力。