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Visualizing the subcellular localization of RHOB-GTP and GTPase-Effector complexes using a split-GFP/nanobody
Sebastian Castillo1, Rémi Gence1, Delphine Pagan1
1Centre de Recherche en Cancérologie de Toulouse (CRCT), INSERM, Université de Toulouse, UPS, CNRS, 31037 Toulouse, France.
European Journal of Cell Biology
|August 28, 2023
Summary
This study introduces a novel assay for precisely locating small GTPase-effector complexes, revealing RHOB
Area of Science:
- Cell Biology
- Molecular Biology
- Signal Transduction
Background:
- Small GTPases are key regulators of cellular signaling pathways.
- RHOB, a member of the RHOA subfamily, is implicated in endocytic trafficking of signaling proteins.
Purpose of the Study:
- To develop and apply a high-resolution assay for localizing GTPase-effector complexes.
- To investigate the localization dynamics of RHOB-RBD complexes in response to serum stimulation.
Main Methods:
- Utilized tripartite split-GFP complementation for high-resolution localization of GTPase-effector complexes.
- Employed recombinant GFP1-9 and a GFP nanobody for in vitro detection of RHOB-RBD interactions.
- Combined localization profiles with immunostaining and live cell imaging.
Main Results:
- Demonstrated serum-dependent reorganization of the endosomal and membrane-associated active RHOB pool.
- Successfully applied the technology to visualize the subcellular localization of various RHO-effector complexes with high resolution.
Conclusions:
- The developed assay provides high-resolution insights into GTPase-effector complex localization.
- RHOB plays a role in regulating endosomal trafficking, with its activity dynamically reorganized by serum stimulation.
