Faten Koraïchi: Cancer Research Center of Toulouse, INSERM U1037, 31037 Toulouse, France. ORCID
Rémi Gence: Cancer Research Center of Toulouse, INSERM U1037, 31037 Toulouse, France.
Catherine Bouchenot: Cancer Research Center of Toulouse, INSERM U1037, 31037 Toulouse, France.
Sarah Grosjean: Cancer Research Center of Toulouse, INSERM U1037, 31037 Toulouse, France. ORCID
Isabelle Lajoie-Mazenc: Cancer Research Center of Toulouse, INSERM U1037, 31037 Toulouse, France.
Gilles Favre: Cancer Research Center of Toulouse, INSERM U1037, 31037 Toulouse, France stephanie.cabantous@inserm.fr favre.gilles@iuct-oncopole.fr. ORCID
Stéphanie Cabantous: Cancer Research Center of Toulouse, INSERM U1037, 31037 Toulouse, France stephanie.cabantous@inserm.fr favre.gilles@iuct-oncopole.fr. ORCID
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.
