Probes and techniques for biological imaging applications

该项目的长期目标是设计成像探针和技术，使生物发现成为可能 和生物医学创新。这项提议旨在开发荧光锌离子传感器和基于细胞的检测方法 不同亚细胞室中锌离子水平的特征，包括分泌颗粒和胞浆。 锌离子是生物体中含量第二丰富的过渡金属，具有多种重要作用。 在不同的生化、生物和生理过程中。锌离子信号失灵或动态平衡 与许多人类疾病有关，人们越来越有兴趣和努力研究这种 活体细胞内锌离子动力学的调控和功能。荧光锌离子传感器是非常有价值的工具 这样的调查。尽管在过去的二十年里，锌离子探针的开发取得了进展，但仍然有 迫切需要设计具有改进的亚细胞靶向性、动态化的新型锌离子传感器 范围，以及为特定细胞细胞器量身定做的合适的锌亲和力。在这项提案中，我们的目标是开发两个 用于对胞浆和富含锌离子的分泌颗粒中的锌离子活性进行成像的锌离子传感器类别；以及 将这些传感器与流式细胞术结合起来，研究这两种细胞中锌离子水平的变化 表达不同亚型颗粒特异性锌离子转运体的胰岛细胞的隔室。 人类锌转运蛋白8的单核苷酸多态(SNPs)与2型糖尿病(T2D)相关，但 潜在的机制仍然难以捉摸，而锌离子是如何影响胰岛细胞中锌离子活性的，还需要进一步研究。 下定决心。在目标1中，我们计划开发和应用荧光锌离子传感器来检测分泌物中的锌离子活性 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 通过提高胞浆靶向性和锌离子结合来成像胞浆锌离子活性的锌传感器 亲和力。在目标3中，我们将把目标1和目标2中开发的锌离子传感器与流式细胞术相结合，以开发一种 一种可以同时分析胰岛细胞亚群中胞浆和颗粒锌离子活性的方法。我们会 应用该方法比较表达ZnT8的胰岛细胞胞浆和颗粒锌的活性变化 控制T2D易感性的转运蛋白。该方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 生物系统用于测试、验证和应用这些工具的生物系统，尽管开发了探测器和技术 这在其他生物系统中应该有广泛的应用。

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.