Imaging Spatiotemporal Regulation of Acetyl-CoA

项目摘要 细胞代谢是高度区室化的，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参与许多生化反应，包括脂肪酸的产生， 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的产生和使用受到营养素可用性的严格调节， 调节新陈代谢的信号网络。因此，乙酰辅酶A代表了细胞代谢的联系， 信号转导虽然批量测量显示乙酰辅酶A是高度区室化的，但染料， 乙酰辅酶A在单细胞水平跨亚细胞区室的动力学调节仍然是难以捉摸的。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 使用基因编码的荧光蛋白生物传感器。这些工具提供了一个投资机会， 以高空间和时间分辨率实时门控细胞动态，同时保持细胞环境， 是的。生物传感器传统上被用于研究激酶或神经递质，这才刚刚开始 来研究新陈代谢。由于生物传感器能够实时动态可视化细胞过程， 单细胞，乙酰辅酶A的生物传感器将照亮乙酰辅酶A的动力学，并支持增强下， 这一关键代谢物的地位。本研究将利用蛋白质工程技术开发乙酰辅酶A生物传感器。 定向进化然后，乙酰辅酶A生物传感器将被用于研究区室化的乙酰辅酶A。 辅酶A动力学和细胞信号对乙酰辅酶A时空调控的询问机制 网络.这一提议的成功将导致对中枢代谢物乙酰基- CoA并将一种新的工具引入该领域，进一步建立了研究真实的代谢的新范式- 时间在单细胞使用生物传感器。

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.