Optical Tools to Study Purinergic Signaling

项目摘要/摘要 细胞外的ATP和ADP是细胞间通讯中的嘌呤能信号的介体。 组织。自分泌和旁分泌的嘌呤能信号有助于正常的细胞功能，如免疫细胞 趋化性和细胞体积调节，以及嘌呤能信号也在损伤、感染、 败血症，以及一些疾病。值得注意的是，胞外ATP和ADP介导的嘌呤能信号转导 既有短期影响又有长期影响的细胞。然而，对细胞和细胞的研究一直是一个挑战 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 异质和嘌呤能信号在不同的细胞之间有很大的不同。显然，直接测量 需要细胞外核苷酸，但目前的方法不能很好地适应测量细胞内 活标本中预期的低细胞外核苷酸浓度。为了克服这一障碍，我们将使用 蛋白质工程开发高亲和力细胞表面传感器，可用于解析复杂的空间 以及ATP和ADP释放、清除和嘌呤能受体激活的时间动力学。为了证明 我们的传感器的应用也指导它们的优化，我们将研究神经元-胶质细胞嘌呤能信号在 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 脑内常驻免疫细胞在调节影响局部神经元的局部嘌呤能动态中的作用 活动。拟议的一系列多路成像研究将我们的嘌呤能传感器与 下游信号应直接显示神经元-神经胶质细胞嘌呤能通讯的核心要素 整合的方式。这应该对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，开发出的遗传编码工具 通过这一建议应该广泛适用于对任何嘌呤能系统的深入研究。

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.