Optical Tools to Study Purinergic Signaling

项目概要/摘要 细胞外ATP和ADP是细胞间通讯中嘌呤能信号传导的介质，几乎在每一个细胞中， 组织.自分泌和旁分泌嘌呤能信号有助于正常细胞功能，如免疫细胞 趋化性和细胞体积调节以及嘌呤能信号传导也有助于损伤，感染， 脓毒症和许多疾病。值得注意的是，细胞外ATP和ADP介导嘌呤能信号传导， 具有短期和长期效果的细胞。然而，研究细胞和细胞外基质是一个挑战， 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 异质和嘌呤能信号传导在细胞与细胞之间可以显著变化。显然，直接测量 细胞外核苷酸是必需的，但目前的方法并不适合测量细胞外核苷酸的波动。 预期活标本中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的细胞表面传感器，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。证明 我们的传感器的应用，并指导他们的优化，我们将研究神经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， 在调节影响局部神经元的区域嘌呤能动力学中， 活动建议的一系列多路成像研究将我们的嘌呤能传感器与 下游信号应直接可视化核心元件的神经元胶质细胞嘌呤能通讯， 综合方式。这应该提供有价值的洞察到新的机制负责耦合 大脑能量代谢和兴奋性之间的联系此外，基因编码的工具开发 通过这一提议，应该广泛适用于任何嘌呤能系统的更深入研究。

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.