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High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation

The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel...

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Autores principales: Koraïchi, Faten, Gence, Rémi, Bouchenot, Catherine, Grosjean, Sarah, Lajoie-Mazenc, Isabelle, Favre, Gilles, Cabantous, Stéphanie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Company of Biologists Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818064/
https://www.ncbi.nlm.nih.gov/pubmed/29192060
http://dx.doi.org/10.1242/jcs.210419
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author Koraïchi, Faten
Gence, Rémi
Bouchenot, Catherine
Grosjean, Sarah
Lajoie-Mazenc, Isabelle
Favre, Gilles
Cabantous, Stéphanie
author_facet Koraïchi, Faten
Gence, Rémi
Bouchenot, Catherine
Grosjean, Sarah
Lajoie-Mazenc, Isabelle
Favre, Gilles
Cabantous, Stéphanie
author_sort Koraïchi, Faten
collection PubMed
description The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains.
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spelling pubmed-58180642018-02-28 High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation Koraïchi, Faten Gence, Rémi Bouchenot, Catherine Grosjean, Sarah Lajoie-Mazenc, Isabelle Favre, Gilles Cabantous, Stéphanie J Cell Sci Tools and Resources The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains. The Company of Biologists Ltd 2018-01-01 /pmc/articles/PMC5818064/ /pubmed/29192060 http://dx.doi.org/10.1242/jcs.210419 Text en © 2018. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
spellingShingle Tools and Resources
Koraïchi, Faten
Gence, Rémi
Bouchenot, Catherine
Grosjean, Sarah
Lajoie-Mazenc, Isabelle
Favre, Gilles
Cabantous, Stéphanie
High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
title High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
title_full High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
title_fullStr High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
title_full_unstemmed High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
title_short High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
title_sort high-content tripartite split-gfp cell-based assays to screen for modulators of small gtpase activation
topic Tools and Resources
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818064/
https://www.ncbi.nlm.nih.gov/pubmed/29192060
http://dx.doi.org/10.1242/jcs.210419
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