Optical Tools to Study Purinergic Signaling

研究嘌呤能信号传导的光学工具

基本信息

批准号：
10727035
负责人：
Mathew Tantama
金额：
$ 9.02万
依托单位：
WELLESLEY COLLEGE
依托单位国家：
美国
项目类别：
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/10727035
关键词：
Acetoacetates Acute Address Affect Affinity Astrocytes Biochemical Biological Biosensor Blood Vessels Brain Cell Communication Cell Volumes Cell physiology Cell surface Cells Chemotaxis Chronic Coculture Techniques Communication Complex Coupling Cyclic AMP Disease Drug resistance Elements Energy Metabolism Ensure Event Fasting Genetic Engineering Goals Health Hydroxybutyrates Hyperactivity Image Immune Immune response In Vitro Infection Injury Ketone Bodies Kinetics Knowledge Learning Length Ligands Lipids Long-Term Effects Measurement Measures Mediating Mediator Metabolic Metabolism Methods Microglia Mitochondria Molecular Monitor Neuroglia Neurons Nucleotides Optics Organ Organism Pain Pathology Pharmaceutical Preparations Pharmacology Phenotype Physiological Platelet Aggregation Inhibitors Plavix Preparation Protein Engineering Purinergic P1 Receptors Purinoceptor Receptor Activation Regulation Resolution Sampling Seizures Sepsis Series Signal Transduction Specificity Specimen Stroke Structure Surface Synaptic Transmission System Technology Testing Therapeutic Tissues Traumatic Brain Injury Visualization Work autocrine brain metabolism brain tissue cell type childhood epilepsy design,build,test ecto-nucleotidase effective therapy experimental study extracellular imaging study insight interest ketogenic diet live cell imaging model organism multiplexed imaging nanomolar neural network neuroprotection novel paracrine sensor small molecule success tool virtual

项目摘要

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。