Optical Tools to Study Purinergic Signaling - Administrative Supplement

研究嘌呤能信号传导的光学工具 - 行政补充

• 批准号: 10580281
• 负责人: Mathew Tantama
• 金额: $ 24.1万
• 依托单位: WELLESLEY COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10580281
• 关键词: Address; Administrative Supplement; Affect; Affinity; Biological; Brain; Cell Communication; Cell Volumes; Cell physiology; Cell surface; Cells; Chemotaxis; Communication; Complex; Coupling; Disease; Elements; Energy Metabolism; Event; Immune; Immune response; Infection; Injury; Learning; Long-Term Effects; Measurement; Measures; Mediating; Mediator of activation protein; Metabolic; Methods; Microglia; Molecular; Neuroglia; Neurons; Nucleotides; Optics; Organ; Pain; Pathology; Protein Engineering; Purinoceptor; Receptor Activation; Regulation; Sampling; Sepsis; Series; Signal Transduction; Specimen; System; Tissues; autocrine; cell type; extracellular; imaging study; insight; multiplexed imaging; novel; paracrine; sensor; tool; virtual

Project Summary/Abstract Extracellular ATP and ADP are mediators of purinergic signaling in cell-to-cell communication in virtually every tissue. Autocrine and paracrine purinergic signaling contributes to normal cellular functions such as immune cell chemotaxis and cell volume regulation, and purinergic signaling also contributes to pathology in injury, infection, sepsis, and a number of diseases. Notably, extracellular ATP and ADP mediate purinergic signaling between cells with both short-term and long-term effects. However, it has been a challenge to study the cellular and molecular mechanisms by which ATP and ADP control distinct signaling events because their levels are spatially heterogeneous and purinergic signaling can vary significantly from cell-to-cell. Clearly, direct measurements of extracellular nucleotides are needed, but current methods are not well adapted to measuring fluctuations in the low extracellular nucleotide concentrations expected in live specimens. To overcome this barrier, we will use protein engineering to develop high affinity cell-surface sensors that can be used to resolve the complex spatial and temporal dynamics of ATP and ADP release, clearance, and purinergic receptor activation. To demonstrate the application of our sensors and also guide their optimization, we will study neuron-glia purinergic signaling in the brain. In particular, we will use our sensors to learn how metabolic state affects the function of microglia, the resident immune cells in the brain, in regulating the regional purinergic dynamics that impact local neuronal activity. The proposed series of multiplexed imaging studies that pair our purinergic sensors with sensors of downstream signaling should directly visualize core elements of neuron-glia purinergic communication in an integrated manner. This should provide valuable insight into novel mechanisms responsible for the coupling between brain energy metabolism and excitability. Furthermore, the genetically-encoded tools developed through this proposal should be broadly applicable to deeper study of any purinergic system.

项目概要/摘要 细胞外 ATP 和 ADP 是几乎所有细胞间通讯中嘌呤能信号传导的介质 组织。自分泌和旁分泌嘌呤能信号传导有助于正常细胞功能，例如免疫细胞 趋化性和细胞体积调节以及嘌呤能信号也有助于损伤、感染、 败血症和许多疾病。值得注意的是，细胞外 ATP 和 ADP 介导之间的嘌呤能信号传导 细胞具有短期和长期效应。然而，研究细胞和 ATP 和 ADP 控制不同信号事件的分子机制，因为它们的水平在空间上是不同的 细胞与细胞之间的异质性和嘌呤能信号传导可能存在显着差异。显然，直接测量 需要细胞外核苷酸，但目前的方法并不能很好地适应测量细胞外核苷酸的波动 活体样本中预期细胞外核苷酸浓度较低。为了克服这个障碍，我们将使用 蛋白质工程开发高亲和力细胞表面传感器，可用于解决复杂的空间问题 ATP 和 ADP 释放、清除和嘌呤受体激活的时间动态。展示 我们的传感器的应用并指导其优化，我们将研究神经元-胶质细胞嘌呤能信号传导 大脑。特别是，我们将使用我们的传感器来了解代谢状态如何影响小胶质细胞的功能，小胶质细胞是 大脑中的常驻免疫细胞，调节影响局部神经元的区域嘌呤能动态 活动。拟议的一系列多重成像研究将我们的嘌呤能传感器与 下游信号传导应直接可视化神经元-胶质细胞嘌呤能通讯的核心要素 综合方式。这应该为负责耦合的新机制提供有价值的见解 大脑能量代谢和兴奋性之间的关系。此外，开发的基因编码工具 通过这个建议应该广泛适用于任何嘌呤能系统的更深入研究。