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: 10472659
• 负责人: Carsten Schultz
• 金额: $ 19.25万
• 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 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10472659
• 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 胡椒恐怖的分泌可以调节更高的代谢和行为功能 有机体。因此，对肽激素的监测已被广泛用于生理和医学，大多数 显着测量血液胰岛素水平。但是，目前尚无可用的测量方法 肽马酮不断释放，在患者中和实验性工作中均不连续释放。在这个项目中，我们 提出开发荧光探针，以监测肽激素（如谷子）的分泌 和胰岛素实时在显微镜阶段。该方法基于荧光蛋白肽融合 与内源性肽激素共分泌。这些融合已引入 早些时候，一个主要的问题是释放发生后，从分泌细胞中迅速扩散。这里， 我们介绍了一个名为SecretTrap的新型系统，该系统旨在捕获分泌的肽本人 在分泌细胞的表面构建和丙烯酸。秘诀将产生丙烯酸信号 这将克服上述扩散和产生的非常短的信号。进一步秘诀 具有光遗传学成分，可通过光线闪烁清除细胞表面上的陷阱。此功能是 需要防止细胞表面捕获装置饱和。陷阱和光遗传学工具是 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。 照明后，LOV2-ZDK1相互作用大大削弱，从而释放了加速肽。 光遗传学部分具有两个重要的角色。首先，它有助于释放任何累积肽的陷阱 实验开始之前的融合。第二，在实验结束时，所有分泌的肽融合 在实验期间，可以在几秒钟内发布有关该实验的定量数据 在给定期间释放的胡椒的总数。通过Western blot，这个值可以与 分泌的内源性肽水平。最初，我们将专注于胰腺的胰高血糖素和胰岛素分泌 细胞。这将使我们能够在完整胰岛中使用已建立的细胞系以及原代小鼠细胞。尽管 培养的单元将通过标准程序翻译，我们将将构建体包装到腺病毒中 输送到胰岛细胞中。因为在小鼠胰岛中，α细胞主要驻留在胰岛的周长上 我们期望构建体的出色表达和谷子融合蛋白的积累 在胰岛之外。我们将将技术扩展到神经肽Y（NPY）从胰腺β细胞释放到 证明一般可行性。 NPY分泌在许多组织中很重要，并且记者系统将具有 广泛的应用。该项目完全专注于工具开发，并将留下 未来研究的新技术。显然，诸如SecretTrap之类的连续成像技术非常适合 用于细胞生物学和药物研究以及候选药物的筛查。