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Flow cytometric reporter assays provide robust functional analysis of signaling complexes

Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay...

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Autores principales: Muusse, Timothy W., Lee, Morris Y.L., Kim, Hyoyoung, Parat, Marie-Odile, Nanson, Jeffrey D., Kobe, Bostjan, Vajjhala, Parimala R., Stacey, Katryn J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9747584/
https://www.ncbi.nlm.nih.gov/pubmed/36334634
http://dx.doi.org/10.1016/j.jbc.2022.102666
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author Muusse, Timothy W.
Lee, Morris Y.L.
Kim, Hyoyoung
Parat, Marie-Odile
Nanson, Jeffrey D.
Kobe, Bostjan
Vajjhala, Parimala R.
Stacey, Katryn J.
author_facet Muusse, Timothy W.
Lee, Morris Y.L.
Kim, Hyoyoung
Parat, Marie-Odile
Nanson, Jeffrey D.
Kobe, Bostjan
Vajjhala, Parimala R.
Stacey, Katryn J.
author_sort Muusse, Timothy W.
collection PubMed
description Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB–responsive promoter driving mScarlet-I expression. The assay enables exclusion of nontransfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by lipopolysaccharide, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways.
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spelling pubmed-97475842022-12-15 Flow cytometric reporter assays provide robust functional analysis of signaling complexes Muusse, Timothy W. Lee, Morris Y.L. Kim, Hyoyoung Parat, Marie-Odile Nanson, Jeffrey D. Kobe, Bostjan Vajjhala, Parimala R. Stacey, Katryn J. J Biol Chem Methods and Resources Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB–responsive promoter driving mScarlet-I expression. The assay enables exclusion of nontransfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by lipopolysaccharide, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways. American Society for Biochemistry and Molecular Biology 2022-11-02 /pmc/articles/PMC9747584/ /pubmed/36334634 http://dx.doi.org/10.1016/j.jbc.2022.102666 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Methods and Resources
Muusse, Timothy W.
Lee, Morris Y.L.
Kim, Hyoyoung
Parat, Marie-Odile
Nanson, Jeffrey D.
Kobe, Bostjan
Vajjhala, Parimala R.
Stacey, Katryn J.
Flow cytometric reporter assays provide robust functional analysis of signaling complexes
title Flow cytometric reporter assays provide robust functional analysis of signaling complexes
title_full Flow cytometric reporter assays provide robust functional analysis of signaling complexes
title_fullStr Flow cytometric reporter assays provide robust functional analysis of signaling complexes
title_full_unstemmed Flow cytometric reporter assays provide robust functional analysis of signaling complexes
title_short Flow cytometric reporter assays provide robust functional analysis of signaling complexes
title_sort flow cytometric reporter assays provide robust functional analysis of signaling complexes
topic Methods and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9747584/
https://www.ncbi.nlm.nih.gov/pubmed/36334634
http://dx.doi.org/10.1016/j.jbc.2022.102666
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