Visualizing the subcellular localization of RHOB-GTP and GTPase-Effector complexes using a split-GFP/nanobody labelling assay

• DOI: 10.1016/j.ejcb.2023.151355
• Publication Date: 2023-08-21T15:30:26Z
• Authors: Sebastian Castillo; Rémi Gence; Delphine Pagan; Faten Koraïchi; Catherine Bouchenot; Benoit J. Pons; Betty Boëlle; Aurélien Olichon; Isabelle Lajoie-M...; Gilles Favre; Jean-Denis Pédelacq; Stéphanie Cabantous
• Keywords: 570; Small GTPase; QH573-671; [SDV]Life Sciences [q-bio]; Cell Membrane; 610; RHOB; GTP Phosphohydrolases; Localization; Nanobody; Endosome; Split-GFP; Guanosine Triphosphate; Cytology; rhoB GTP-Binding Protein; rhoA GTP-Binding Protein; Signal Transduction

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.