Imaging Spatiotemporal Regulation of Acetyl-CoA

项目总结 细胞代谢是高度分隔的，代谢物的亚细胞定位决定了 使用。一种中心代谢物乙酰辅酶A参与了许多生化反应，包括脂肪的产生。 酸、类固醇、酮体和乙酰化反应，取决于细胞代谢状态和亚细胞 本地化。因此，乙酰辅酶A的生产和使用受到营养可获得性和 调节新陈代谢的信号网络。因此，乙酰辅酶A代表了细胞新陈代谢和 信号转导。虽然大量测量表明乙酰-辅酶A是高度分隔的，但Dy-CoA- 在单细胞水平上，乙酰辅酶A在亚细胞水平上的动态调节仍然难以捉摸。一个 克服这些限制并阐明乙酰辅酶A亚细胞动力学的方法是活细胞成像 使用基于基因编码的荧光蛋白生物传感器。这些工具提供了投资的机会- 在保持细胞环境的同时，以高空间和时间分辨率实时地进行门细胞动力学- 环境。生物传感器传统上被用来研究激酶或神经递质，而现在才刚刚开始。 用来研究新陈代谢。由于生物传感器能够实时、动态地可视化细胞过程 单细胞，乙酰-辅酶A生物传感器将阐明乙酰-辅酶A的动力学，并支持增强的Under. 这一关键代谢物的地位。在这项建议中，我们将开发一种使用蛋白质引擎的乙酰-辅酶A生物传感器。 隐形和定向进化。然后，乙酰-CoA生物传感器将被用于研究区隔的乙酰-CoA生物传感器。 细胞信号转导乙酰辅酶A时空调控的辅酶A动力学和信号机制 网络。这一提议的成功将导致对中枢代谢物乙酰- CoA，并将一种新的工具引入该领域，进一步建立了在真实情况下研究新陈代谢的新范式 在单个细胞中使用生物传感器的时间。

PROJECT SUMMARY Cellular metabolism is highly compartmentalized, and subcellular localization of metabolites determines use. A central metabolite, acetyl-CoA, is involved in many biochemical reactions, including the production of fatty acids, steroids, ketone bodies, and acetylation reactions, dependent on cellular metabolic state and subcellular localization. Accordingly, the production and use of acetyl-CoA is tightly regulated by nutrient availability and signaling networks regulating metabolism. Therefore, acetyl-CoA represents a nexus for cellular metabolism and signal transduction. While bulk measurements have shown acetyl-CoA to be highly compartmentalized, the dy- namic regulation of acetyl-CoA across subcellular compartments at the single cell level remains elusive. An approach for overcoming these limitations and illuminating subcellular dynamics of acetyl-CoA is live cell imaging using genetically encoded fluorescent protein-based biosensors. These tools provide an opportunity to investi- gate cellular dynamics in real-time with high spatial and temporal resolution while maintaining the cellular envi- ronment. Biosensors have traditionally been used to study kinases or neurotransmitters and have only just begun to be used to study metabolism. As biosensors enable dynamic visualization of cellular processes in real-time in single cells, a biosensor for acetyl-CoA would illuminate acetyl-CoA dynamics and support an enhanced under- standing of this key metabolite. In this proposal we will develop a biosensor for acetyl-CoA using protein engi- neering and directed evolution. Then, the acetyl-CoA biosensor will be used to study compartmentalized acetyl- CoA dynamics and interrogate mechanisms for the spatiotemporal regulation of acetyl-CoA by cellular signaling networks. The success of this proposal will result in an enhanced understanding of the central metabolite acetyl- CoA and introduce a new tool into the field, further establishing a new paradigm for studying metabolism in real- time in single cells using biosensors.