title: Ratiometric Imaging Using a Single Dye Enables Simultaneous Visualization of Rac1 and Cdc42 Activation.
abstract: Biosensors that report endogenous protein activity in vivo can be based on environment-sensing fluorescent dyes. The dyes can be attached to reagents that bind selectively to a specific conformation of the targeted protein, such that binding leads to a fluorescence change. Dyes that are sufficiently bright for use at low, nonperturbing intracellular concentrations typically undergo changes in intensity rather than the shifts in excitation or emission maxima that would enable precise quantitation through ratiometric imaging. We report here mero199, an environment-sensing dye that undergoes a 33 nm solvent-dependent shift in excitation. The dye was used to generate a ratiometric biosensor of Cdc42 (CRIB199) without the need for additional fluorophores. CRIB199 was used in the same cell with a FRET sensor of Rac1 activation to simultaneously observe Cdc42 and Rac1 activity in cellular protrusions, indicating that Rac1 but not Cdc42 activity was reduced during tail retraction, and specific protrusions had reduced Cdc42 activity. A novel program (EdgeProps) used to correlate localized activation with cell edge dynamics indicated that Rac1 was specifically reduced during retraction.
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Christopher J
authors: CJ
authors: MacNevin
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Alexei
authors: A
authors: Toutchkine
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Daniel J
authors: DJ
authors: Marston
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Chia-Wen
authors: CW
authors: Hsu
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Denis
authors: D
authors: Tsygankov
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Li
authors: L
authors: Li
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Bei
authors: B
authors: Liu
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Timothy
authors: T
authors: Qi
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Dan-Vinh
authors: DV
authors: Nguyen
authors: Department of Pharmacology, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors: Klaus M
authors: KM
authors: Hahn
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D038761:Photobleaching; D011726:Pyridinium Compounds / Q000737:chemistry*; D020764:cdc42 GTP-Binding Protein / Q000032:analysis*; D020830:rac1 GTP-Binding Protein / Q000032:analysis*
chemical_list: D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein