A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding

White, Carl W.; Kilpatrick, Laura E.; Pfleger, Kevin D.G.; Hill, Stephen J.

Abstract

© 2020 The Author(s) 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.

Citation

White, C. W., Kilpatrick, L. E., Pfleger, K. D., & Hill, S. J. (2021). A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding. iScience, 24(1), Article 102011. https://doi.org/10.1016/j.isci.2020.102011

• Journal Article Type: Article
• Acceptance Date: Dec 28, 2020
• Online Publication Date: Dec 30, 2020
• Publication Date: 2021-01
• Deposit Date: Apr 13, 2021
• Publicly Available Date: Apr 20, 2021
• Journal: iScience
• Electronic ISSN: 2589-0042
• Publisher: Cell Press
• Peer Reviewed: Peer Reviewed
• Volume: 24
• Issue: 1
• Article Number: 102011
• DOI: https://doi.org/10.1016/j.isci.2020.102011
• Keywords: Multidisciplinary
• Public URL: https://nottingham-repository.worktribe.com/output/5251722
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S2589004220312086
• Additional Information: This article is maintained by: Elsevier; Article Title: A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding; Journal Title: iScience; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.isci.2020.102011; Content Type: article; Copyright: © 2020 The Author(s).