使用 FluoSTEP-ICUE 测量内源性信号隔室内的时空 cAMP 动态。
Measuring Spatiotemporal cAMP Dynamics Within an Endogenous Signaling Compartment Using FluoSTEP-ICUE.
机构信息
Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
Department of Pharmacology, University of California San Diego, La Jolla, CA, USA.
出版信息
Methods Mol Biol. 2022;2483:351-366. doi: 10.1007/978-1-0716-2245-2_22.
cAMP is a ubiquitous second messenger involved in the regulation of diverse cellular processes. Spatiotemporal regulation of cAMP through compartmentalization within various subcellular microdomains is essential to ensure specific signaling. In the following protocol, we describe a method for directly visualizing signaling dynamics within cAMP microdomains using fluorescent sensors targeted to endogenous proteins (FluoSTEPs). Instead of overexpressing a biosensor-tagged protein of interest to target a microdomain, FluoSTEP Indicator of cAMP using Epac (FluoSTEP-ICUE) utilizes spontaneously complementing split GFP and CRISPR-Cas9 genome editing to localize a FRET-based cAMP biosensor to an endogenously expressed protein of interest. Utilizing this approach, FluoSTEP-ICUE can be used to measure cAMP levels within endogenous signaling compartments, thus providing a powerful tool for studying the spatiotemporal regulation of cAMP signaling.
cAMP 是一种普遍存在的第二信使，参与调节多种细胞过程。通过在各种亚细胞微域内进行区室化来对 cAMP 进行时空调节对于确保特定的信号转导至关重要。在以下方案中，我们描述了一种使用针对内源性蛋白质的荧光传感器（FluoSTEPs）直接可视化 cAMP 微域内信号转导动态的方法。与过表达生物传感器标记的感兴趣的蛋白质以靶向微域不同，使用 Epac 的 cAMP 荧光传感器指示剂（FluoSTEP-ICUE）利用自发互补的分裂 GFP 和 CRISPR-Cas9 基因组编辑将基于 FRET 的 cAMP 生物传感器定位到内源性表达的感兴趣的蛋白质上。利用这种方法，FluoSTEP-ICUE 可用于测量内源性信号转导隔室内的 cAMP 水平，从而为研究 cAMP 信号转导的时空调节提供了有力工具。
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