title: Multiplex imaging of Rho family GTPase activities in living cells.
abstract: Here, we provide procedures for imaging the Rho GTPase biosensors in both single and multiplex acquisition modes. The multiplex approach enables the direct visualization of two biosensor readouts from a single living cell. Here, we take as an example a combination of the RhoA biosensor based on a CFP/YFP FRET modality and the Cdc42 biosensor based on organic dyes that change fluorescence as a function of the local solvent polarity. We list the required optical components as well as cellular manipulation techniques necessary to successfully image these two ratiometric biosensors in a single living cell.
authors.affiliation: Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY, USA.
authors.forename: Désirée
authors.initials: D
authors.lastname: Spiering
authors.forename: Louis
authors.initials: L
authors.lastname: Hodgson
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004789:Enzyme Activation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D013194:Staining and Labeling; D014161:Transduction, Genetic; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins