White Carl W, Kilpatrick Laura E, Pfleger Kevin D G, Hill Stephen J
Cell Signalling and Pharmacology Research Group, Division of Physiology, Pharmacology & Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK.
Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, The Midlands, UK.
iScience. 2020 Dec 30;24(1):102011. doi: 10.1016/j.isci.2020.102011. eCollection 2021 Jan 22.
Secreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time.
分泌型趋化因子是细胞通讯的关键介质，通过与膜结合受体结合引发细胞内信号传导。在此，我们展示了一种灵敏的实时方法的开发与应用，用于量化活细胞中天然趋化因子的分泌及受体结合情况，以更好地理解它们的分子相互作用和功能。利用CRISPR/Cas9基因组编辑技术，用纳米荧光素酶片段HiBiT标记趋化因子CXCL12。随后通过发光输出监测并量化CXCL12的分泌。由于纳米荧光素酶互补的空间位阻限制，可以监测标记的CXCL12与趋化因子受体或膜糖胺聚糖的结合。此外，天然CXCL12-HiBiT与AlexaFluor488标记的CXCR4趋化因子受体的结合也能与糖胺聚糖结合区分开来，并使用生物发光共振能量转移进行药理学分析。这些活细胞方法将纳米荧光素酶的灵敏度与CRISPR/Cas9基因组编辑相结合，以实时检测、量化和监测低水平天然分泌蛋白的结合情况。
