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，以实现生物发现 和生物医学创新。该提案旨在开发荧光Zn 2+传感器和基于细胞的测定， 表征不同亚细胞区室（包括分泌颗粒和胞质溶胶）中的Zn 2+水平。 锌离子是生物体内第二丰富的过渡金属离子，具有重要的生物学功能 在不同的生化、生物和生理过程中。Zn 2+信号传导或稳态功能障碍是 与许多人类疾病有关，并且已经有越来越多的兴趣和努力来研究 调节和活细胞中Zn 2+动力学的功能。荧光Zn 2+传感器是非常宝贵的工具， 这样的调查。尽管在过去二十年中在Zn 2+探针开发方面取得了进展，但仍然存在 迫切需要工程新的Zn 2+传感器具有改进的亚细胞靶向特异性，动态 范围内，和合适的Zn 2+亲和力定制特定的细胞器。在这份提案中，我们的目标是开发两个 用于对胞质溶胶和富含Zn 2+的分泌颗粒中的Zn 2+活性进行成像的Zn 2+传感器的类别;以及 联合收割机将这些传感器与流式细胞术相结合，以研究这两种细胞中Zn 2+水平的变化。 表达不同亚型的颗粒特异性Zn 2+转运蛋白ZnT 8的胰岛细胞的隔室。 人类ZnT 8的单核苷酸多态性（SNP）与2型糖尿病（T2 D）相关，但 潜在的机制仍然是难以捉摸的，ZnT 8如何影响胰岛细胞中的Zn 2+活性需要被研究。 测定目的一：开发和应用荧光锌离子传感器，检测分泌细胞中锌离子的活性， 胰岛细胞的颗粒，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 通过改善胞质溶胶靶向特异性和Zn 2+结合来成像胞质溶胶Zn 2+活性的Zn 2+传感器 亲和力在目标3中，我们将联合收割机将目标1和目标2中开发的Zn 2+传感器与流式细胞术相结合，以开发一种流式细胞仪。 可以同时分析胰岛细胞亚群中胞质和颗粒状Zn 2+活性的测定。我们将 应用该测定来比较表达ZnT 8的胰岛细胞中胞质和颗粒Zn 2+活性的变化 控制T2 D易感性的转运蛋白。该建议代表了一种集成的方法， 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 测试、验证和应用这些工具的生物系统，尽管开发了探针和技术， 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.