SecreTrap - a tool to measure peptide hormone secretion at the single cell level

SecreTrap - 在单细胞水平上测量肽激素分泌的工具

• 批准号: 10317887
• 负责人: Carsten Schultz
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10317887
• 关键词: Address; Adenoviruses; Affinity; Behavioral; Beta Cell; Binding; Biological Assay; Blood; Blood Glucose; Blood specimen; Brain; Cell membrane; Cell surface; Cells; Cellular biology; Chemicals; Chimeric Proteins; Confocal Microscopy; Cultured Cells; Data; Development; Devices; Diffusion; Drug Screening; Energy Transfer; Engineering; Ensure; Enzyme-Linked Immunosorbent Assay; Female; Fluorescence; Fluorescent Probes; Free Will; Future; Glucagon; Glucose; Hormone secretion; Hormones; Image; Imaging Techniques; Insulin; Investigation; Islet Cell; Islets of Langerhans; Learning; Light; Lighting; Measurement; Measures; Medicine; Metabolic; Methods; Microscope; Monitor; Mus; Organ; Organism; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Pharmacology Study; Physiology; Play; Procedures; Proteins; Reporter; Research; Role; Sampling; Signal Transduction; Structure of beta Cell of islet; Surface; System; Techniques; Time; Tissues; Validation; Viral; Virus; Western Blotting; Work; adeno-associated viral vector; base; cell type; design; detection platform; drug candidate; established cell line; experimental study; extracellular; fluorophore; hormone regulation; improved; insulin secretion; interest; islet; male; nanomolar; neuropeptide Y; novel; optogenetics; pancreatic juice; pancreatic secretory trypsin inhibitor I; peptide hormone; prevent; promoter; ratiometric; receptor; tissue culture; tool; tool development; vector

Project Summary The secretion of peptide hormones regulates many of the metabolic and behavioral functions of higher organisms. Therefore, monitoring of peptide hormones has been widely used in physiology and medicine, most prominently for measuring blood insulin levels. However, there is currently no method available to measure peptide hormone release continuously, neither in patients nor during experimental work. In this project, we propose to develop a fluorescent probe that monitors the secretion of peptides hormones such as glucagon and insulin on the microscope stage in real time. The method is based on fluorescent protein-peptide fusions that are co-secreted with the endogenous peptide hormones. While those fusions have been introduced earlier, a major problem is the rapid diffusion away from the secreting cell after the release happened. Here, we introduce a novel system called SecreTrap which is designed to catch the secreted peptide hormone construct and accumulate it on the surface of the secreting cell. SecreTrap will produce an accumulating signal that will overcome the above mentioned diffusion and the resulting very short-lived signal. SecreTrap further features an optogenetic component that clears the trap on the cell surface by a flash of light. This feature is required to prevent saturation of the trapping device on the cell surface. The trap and the optogenetic tool is based on the nanomolar interaction of the well-known LOV2 domain with a short designed peptide (Zdk1). Upon illumination, the LOV2-Zdk1 interaction is massively weakened thus releasing the accumulated peptide. The optogenetic part has two important roles to play. First, it helps freeing the trap of any accumulated peptide fusion before the start of the experiment. Second, at the end of the experiment, all secreted peptide fusion during the time of the experiment can be released within a few seconds giving quantitative data about the overall amount of peptide released during a given period. By Western blot this value can be correlated with secreted endogenous peptide levels. Initially, we will focus on glucagon and insulin secretion from pancreas cells. This will allow us to use established cell lines as well as primary mouse cells in intact islets. While cultured cells will be transfected by standard procedures, we will pack the constructs into an adenovirus for delivery into islet cells. Because in mouse islets, the α-cells reside predominantly on the perimeter of the islet, we expect excellent expression of the constructs and an accumulation of the glucagon fusion protein on the outside of the islet. We will expand the technique to neuropeptide Y (NPY) release from pancreatic β-cells to demonstrate general feasibility. NPY secretion is important in many tissues and a reporter system will have broad applications. This project is fully focusing on tool development and will leave major applications of the new technique to future studies. Obviously, a continuous imaging technique such as SecreTrap is well suited for cell biology and pharmacological studies as well as screening of drug candidates.

项目摘要 多肽激素的分泌调节高等动物的许多新陈代谢和行为功能。 有机体。因此，多肽激素的监测在生理学和医学上得到了广泛的应用。 主要用于测量血液中的胰岛素水平。然而，目前还没有可用的方法来测量 无论是在病人体内还是在实验过程中，多肽激素都会持续释放。在这个项目中，我们 建议开发一种荧光探针，用于监测胰高血糖素等多肽激素的分泌 和胰岛素实时出现在显微镜的舞台上。该方法是基于荧光蛋白-多肽融合。 与内源性多肽荷尔蒙共同分泌。虽然这些融合已经被引入 早些时候，一个主要问题是在释放发生后，分泌细胞迅速扩散。这里, 我们介绍了一种名为SecreTrap的新系统，该系统旨在捕获分泌的多肽激素 构建并积累在分泌细胞的表面。SecreTrap将产生累积信号 这将克服上述扩散和由此产生的非常短暂的信号。进一步设置安全陷阱 具有光遗传组件，通过一道闪光清除细胞表面的陷阱。此功能是 以防止电池表面上的捕集装置饱和。陷阱和光遗传工具是 基于众所周知的LOV2结构域与设计的短肽(Zdk1)的纳摩尔相互作用。 在光照下，LOV2-Zdk1的相互作用被大量削弱，从而释放积累的多肽。 光遗传部分有两个重要的作用。首先，它有助于释放任何积聚的多肽 在实验开始之前进行核聚变。第二，在实验结束时，所有分泌的多肽融合 在实验期间可以在几秒钟内释放，从而提供关于 在给定时间段内释放的多肽总量。通过Western印迹分析，该值可以与 分泌内源性多肽水平。首先，我们将关注胰腺分泌的胰升糖素和胰岛素。 细胞。这将允许我们使用已建立的细胞系以及完整的胰岛中的原代小鼠细胞。而当 培养的细胞将通过标准程序进行转基因，我们将把构建的载体包装成腺病毒，用于 进入胰岛细胞。因为在小鼠胰岛中，α细胞主要驻留在胰岛的周边， 我们期待着这些构建物的出色表达和胰升糖素融合蛋白在 在小岛之外。我们将把从胰腺β细胞释放神经肽Y的技术扩展到 论证总体上的可行性。NPY分泌在许多组织中都很重要，一个报告系统将具有 应用广泛。该项目完全专注于工具开发，并将留下 为未来的研究提供新的技术。显然，像SecreTrap这样的连续成像技术非常适合 用于细胞生物学和药理学研究以及筛选候选药物。