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：
10364329
负责人：
Mathew Tantama
金额：
$ 35.57万
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/10364329
关键词：
Acetoacetates Acute Address Affect Affinity Animal Model 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 of activation protein 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 Work autocrine brain metabolism brain tissue cell type childhood epilepsy design-build-test effective therapy experimental study extracellular imaging study insight interest ketogenic diet live cell imaging 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.

项目摘要/摘要细胞外ATP和ADP是几乎每个细胞之间通信中嘌呤能信号传导的介体组织。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传导也有助于损伤，感染，败血症和许多疾病。值得注意的是，细胞外ATP和ADP介导了嘌呤能信号传导具有短期和长期作用的细胞。但是，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异质和嘌呤能信号传导与细胞之间的信号差异很大。显然，直接测量需要细胞外核苷酸，但是当前方法不能很好地适应测量的波动在活标本中预期的低细胞外核苷酸浓度。为了克服这个障碍，我们将使用蛋白质工程以开发高亲和力细胞表面传感器，可用于解决复杂的空间以及ATP和ADP释放，清除和嘌呤能受体激活的时间动力学。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 - 葡聚糖信号传导大脑。特别是，我们将使用传感器来了解代谢状态如何影响小胶质细胞的功能，在调节影响局部神经元的区域性嘌呤能动力学方面活动。提出的一系列多路复用成像研究将我们的嘌呤能传感器与传感器配对下游信号应直接可视化神经元 - 葡萄膜能通信的核心元素综合方式。这应该提供对负责耦合的新型机制的宝贵见解在大脑能量代谢和兴奋性之间。此外，开发了遗传编码的工具通过该建议，应广泛地适用于对任何嘌呤能系统的更深入研究。