The use of fluorescence correlation spectroscopy to monitor cell surface β2‐adrenoceptors at low expression levels in human embryonic stem cell‐derived cardiomyocytes and fibroblasts

Goulding, Jo�lle; Kondrashov, Alexander; Mistry, Sarah J; Melarangi, Tony; Vo, Nguyen T N; Hoang, Duc M; White, Carl W; Denning, Chris; Briddon, Stephen J; Hill, Stephen J

Abstract

The importance of cell phenotype in determining the molecular mechanisms underlying β2- adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged b2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface b2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of embryonic stem cell technology in combination with FCS allowed us to demonstrate that cell surface b2ARs internalise in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalisation is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting b2AR agonists.

Citation

Goulding, J., Kondrashov, A., Mistry, S. J., Melarangi, T., Vo, N. T. N., Hoang, D. M., White, C. W., Denning, C., Briddon, S. J., & Hill, S. J. (2021). The use of fluorescence correlation spectroscopy to monitor cell surface β2‐adrenoceptors at low expression levels in human embryonic stem cell‐derived cardiomyocytes and fibroblasts. FASEB Journal, 35(4), Article e21398. https://doi.org/10.1096/fj.202002268r

• Journal Article Type: Article
• Acceptance Date: Jan 12, 2021
• Online Publication Date: Mar 12, 2021
• Publication Date: Apr 1, 2021
• Deposit Date: Jan 27, 2021
• Publicly Available Date: Mar 15, 2021
• Journal: The FASEB Journal
• Print ISSN: 0892-6638
• Electronic ISSN: 1530-6860
• Publisher: Federation of American Society of Experimental Biology (FASEB)
• Peer Reviewed: Peer Reviewed
• Volume: 35
• Issue: 4
• Article Number: e21398
• DOI: https://doi.org/10.1096/fj.202002268r
• Keywords: Biotechnology; Genetics; Biochemistry; Molecular Biology
• Public URL: https://nottingham-repository.worktribe.com/output/5271766
• Publisher URL: https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202002268R