Probes and techniques for biological imaging applications

用于生物成像应用的探针和技术

基本信息

批准号：
10448466
负责人：
Wen-hong Li
金额：
$ 42.31万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10448466
关键词：
Address Affect Affinity Alpha Cell Alpha Granule B-Lymphocytes Beta Cell Binding Binding Sites Biochemical Process Biological Biological Assay Biological Process Biology Cells Cytoplasmic Granules Cytosol D Cells Development Diabetes Mellitus Endocrine Engineering Family Flow Cytometry Future Genes Goals Homeostasis Human Human Genome Image Imaging Techniques Imidazole Investigation Ions Islet Cell Islets of Langerhans Knock-in Mouse Ligands Measurement Measures Monitor Mutation Non-Insulin-Dependent Diabetes Mellitus Organelles Organic Synthesis Organism Physiological Processes Play Predisposition Protein Isoforms Regulation Role Secretory Cell Secretory Vesicles Signal Transduction Single Nucleotide Polymorphism Specificity Techniques Testing Transition Elements Variant Zinc autocrine base biological systems cell type design experimental study extracellular genome wide association study human disease imaging probe improved innovation insight interest islet live cell imaging paracrine ratiometric sensor subcellular targeting tool

项目摘要

The long term goal of this project is to engineer imaging probes and techniques to enable biological discovery and biomedical innovation. This proposal aims to develop fluorescent Zn2+ sensors and cell based assays for characterizing Zn2+ levels in different subcellular compartments including secretory granules and cytosol. Zinc ion is the second most abundant transition metal in living organisms and plays numerous important roles in diverse biochemical, biological, and physiological processes. Malfunction of Zn2+ signaling or homeostasis is implicated in a number of human diseases, and there have been increasing interests and efforts to study the regulation and the function of Zn2+ dynamics in living cells. Fluorescent Zn2+ sensors are invaluable tools for such investigations. Despite progress over the past two decades in Zn2+ probe development, there are still pressing needs for engineering new Zn2+ sensors with improved subcellular targeting specificity, dynamic range, and suitable Zn2+ affinity tailored for specific cell organelles. In this proposal, we aim to develop two classes of Zn2+ sensors for imaging Zn2+ activity in the cytosol and in the Zn2+-rich secretory granules; and to combine these sensors with flow cytometry to investigate variations in Zn2+ levels in these two cellular compartments of pancreatic islet cells expressing different isoforms of a granule specific Zn2+ transporter, ZnT8. Human single nucleotide polymorphisms (SNPs) of ZnT8 have been associated with type 2 diabetes (T2D), yet the underlying mechanisms remain elusive, and how ZnT8 affect Zn2+ activity in islet cells needs to be determined. In aim 1, we plan to develop and apply fluorescent Zn2+ sensors to assay Zn2+ activity in secretory granules of pancreatic islet cells including a-cell, b-cell, and d-cell. In aim 2, we plan to develop fluorescent Zn2+ sensors for imaging cytosolic Zn2+ activity by improving the cytosol targeting specificity and Zn2+ binding affinity. In aim 3, we will combine Zn2+ sensors developed in aim 1 and aim 2 with flow cytometry to develop an assay that can simultaneously analyze cytosolic and granular Zn2+ activity in subsets of islet cells. We will apply the assay to compare variations in cytosolic and granular Zn2+ activity in islet cells expressing ZnT8 transporters that control T2D susceptibility. This proposal represents an integral approach by combining sensor design, organic synthesis, flow cytometry analysis, and pancreatic islet biology. We choose pancreatic islets as the biological system for testing, validating, and applying these tools, though probes and techniques developed here should have broad applications in other biological systems.

该项目的长期目标是设计成像探针和技术以实现生物发现和生物医学创新。该提案旨在开发荧光 Zn2+ 传感器和基于细胞的检测方法表征不同亚细胞区室（包括分泌颗粒和细胞质）中的 Zn2+ 水平。锌离子是生物体中第二丰富的过渡金属，发挥着许多重要作用在不同的生化、生物和生理过程中。 Zn2+信号传导或稳态失灵是与许多人类疾病有关，人们对研究它的兴趣和努力日益增加活细胞中 Zn2+ 动力学的调节和功能。荧光 Zn2+ 传感器是非常有价值的工具此类调查。尽管 Zn2+ 探针开发在过去二十年中取得了进展，但仍然存在一些问题迫切需要设计具有改进的亚细胞靶向特异性、动态性的新型 Zn2+ 传感器范围和适合特定细胞器的 Zn2+ 亲和力。在本提案中，我们的目标是开发两种用于对细胞质和富含 Zn2+ 的分泌颗粒中 Zn2+ 活性进行成像的 Zn2+ 传感器类别；并到将这些传感器与流式细胞术结合起来研究这两种细胞中 Zn2+ 水平的变化表达颗粒特异性 Zn2+ 转运蛋白 ZnT8 不同亚型的胰岛细胞区室。 ZnT8 的人类单核苷酸多态性 (SNP) 与 2 型糖尿病 (T2D) 相关，但潜在的机制仍然难以捉摸，ZnT8 如何影响胰岛细胞中的 Zn2+ 活性需要进一步研究决定。在目标1中，我们计划开发并应用荧光Zn2+传感器来测定分泌中的Zn2+活性胰岛细胞颗粒，包括 a 细胞、b 细胞和 d 细胞。在目标2中，我们计划开发荧光 Zn2+ 传感器通过改善细胞质靶向特异性和 Zn2+ 结合来成像细胞质 Zn2+ 活性亲和力。在目标 3 中，我们将目标 1 和目标 2 中开发的 Zn2+ 传感器与流式细胞术结合起来，开发一种可以同时分析胰岛细胞亚群中细胞质和颗粒 Zn2+ 活性的测定。我们将应用该测定法来比较表达 ZnT8 的胰岛细胞中胞质和颗粒 Zn2+ 活性的变化控制 T2D 易感性的转运蛋白。该提案代表了一种结合传感器的整体方法设计、有机合成、流式细胞术分析和胰岛生物学。我们选择胰岛作为尽管开发了探针和技术，但用于测试、验证和应用这些工具的生物系统这里应该在其他生物系统中具有广泛的应用。