Probes and techniques for biological imaging applications - Revision - 3

生物成像应用的探针和技术 - 修订版 - 3

• 批准号: 10581074
• 负责人: Wen-hong Li
• 金额: $ 4.48万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 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/10581074
• 关键词: Affect; Affinity; Alpha Granule; Binding; Biochemical Process; Biological; Biological Assay; Biological Process; Biology; Cells; Cytosol; Development; Diabetes Mellitus; Engineering; Flow Cytometry; Goals; Homeostasis; Human; Image; Imaging Techniques; Investigation; Ions; Islet Cell; Islets of Langerhans; Non-Insulin-Dependent Diabetes Mellitus; Organelles; Organic Synthesis; Organism; Physiological Processes; Play; Predisposition; Protein Isoforms; Regulation; Role; Secretory Vesicles; Signal Transduction; Single Nucleotide Polymorphism; Specificity; Techniques; Testing; Transition Elements; Variant; Zinc; base; biological systems; design; human disease; imaging probe; improved; innovation; interest; sensor; subcellular targeting; tool

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 -cell, -cell, and -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 该提案旨在开发荧光 Zn2+ 传感器和基于细胞的检测。 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 Zn2+ 信号或稳态的功能障碍。 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是非常有价值的工具。 尽管过去二十年在 Zn2+ 探针开发方面取得了进展，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； 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 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α细胞、β细胞和β细胞在目标2中，我们计划开发荧光细胞。 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 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，开发出一种亲和力传感器。 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。 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。

项目成果

Functional analysis of islet cells in vitro, in situ, and in vivo.

胰岛细胞的体外、原位和体内功能分析。

• 发表时间: 2020
• 影响因子: 7.3
• 作者: Li; Wen
• 通讯作者: Wen

Targeting Ceramides and Adiponectin Receptors in the Islet of Langerhans for Treating Diabetes.

靶向朗格汉斯岛的神经酰胺和脂联素受体来治疗糖尿病。

• 发表时间: 2022-09-19
• 作者: Li; Wen
• 通讯作者: Wen

A role and mechanism for redox sensing by SENP1 in β-cell responses to high fat feeding.

SENP1 氧化还原传感在β细胞对高脂肪喂养反应中的作用和机制。

• 发表时间: 2024-01-06
• 影响因子: 16.6
• 作者: Lin, Haopeng;Suzuki, Kunimasa;Smith, Nancy;Li, Xi;Nalbach, Lisa;Fuentes, Sonia;Spigelman, Aliya F;Dai, Xiao;Bautista, Austin;Ferdaoussi, Mourad;Aggarwal, Saloni;Pepper, Andrew R;Roma, Leticia P;Ampofo, Emmanuel;Li, Wen;MacDonald
• 通讯作者: MacDonald