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.

项目概要 肽激素的分泌调节高等动物的许多代谢和行为功能 因此，肽类激素的监测已广泛应用于生理学和医学领域。 主要用于测量血液胰岛素水平，但目前尚无可用的方法来测量。 在这个项目中，无论是在患者体内还是在实验工作期间，肽激素都会持续释放。 提议开发一种荧光探针来监测胰高血糖素等肽类激素的分泌 该方法基于荧光蛋白-肽融合。 与内源性肽激素共同分泌，而这些融合物已经被引入。 早些时候，一个主要问题是释放发生后迅速扩散离开分泌细胞， 我们推出了一种名为 SecreTrap 的新型系统，旨在捕获分泌的肽激素 构建并积累在分泌细胞表面会产生积累信号。 这将进一步克服上述扩散和由此产生的非常短暂的信号。 具有光遗传学成分，可通过闪光清除细胞表面的陷阱。 需要防止细胞表面上的捕获装置饱和。 基于众所周知的 LOV2 结构域与短设计肽 (Zdk1) 的纳摩尔相互作用。 光照后，LOV2-Zdk1 相互作用大大减弱，从而释放积累的肽。 光遗传学部分有两个重要作用，首先，它有助于释放任何积累的肽的陷阱。 实验开始前融合 二、实验结束时，所有分泌肽融合。 在实验期间可以在几秒钟内发布有关 通过蛋白质印迹，该值可以与给定时间内释放的肽总量相关。 首先，我们将重点关注胰腺的胰高血糖素和胰岛素分泌。 这将使我们能够使用已建立的细胞系以及完整胰岛中的原代小鼠细胞。 培养的细胞将通过标准程序转染，我们将构建体包装到腺病毒中 因为在小鼠胰岛中，α细胞主要分布在胰岛的周边， 我们期望构建体的良好表达以及胰高血糖素融合蛋白在 我们将把这项技术扩展到从胰腺 β 细胞释放神经肽 Y (NPY)。 证明 NPY 分泌在许多组织中很重要，并且报告系统将具有 该项目完全专注于工具开发，并将留下主要应用程序。 显然，SecreTrap 等连续成像技术非常适合未来的研究。 用于细胞生物学和药理学研究以及候选药物的筛选。