Department of Pharmacology, University of California, San Diego, San Diego, United States; Department of Biomedical Engineering, Johns Hopkins University, Baltimore, United States
Brian Tenner
Department of Pharmacology, University of California, San Diego, San Diego, United States; Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, United States
Michele L Markwardt
Department of Physiology, University of Maryland Baltimore, Baltimore, United States
Adam Zviman
Department of Physiology, University of Maryland Baltimore, Baltimore, United States
Guoli Shi
Department of Orthopaedics, University of Maryland Baltimore, Baltimore, United States
Jaclyn P Kerr
Department of Orthopaedics, University of Maryland Baltimore, Baltimore, United States
Nicole E Snell
Department of Physiology, University of Maryland Baltimore, Baltimore, United States
Jennifer J McFarland
Department of Physiology, University of Maryland Baltimore, Baltimore, United States
Joseph R Mauban
Department of Physiology, University of Maryland Baltimore, Baltimore, United States
Christopher W Ward
Department of Orthopaedics, University of Maryland Baltimore, Baltimore, United States
Department of Pharmacology, University of California, San Diego, San Diego, United States; Program in Molecular Biophysics, Johns Hopkins University School of Medicine, Baltimore, United States; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, United States
Genetically encoded fluorescent biosensors have revolutionized the study of signal transduction by enabling the real-time tracking of signaling activities in live cells. Investigating the interaction between signaling networks has become increasingly important to understanding complex cellular phenomena, necessitating an update of the biosensor toolkit to allow monitoring and perturbing multiple activities simultaneously in the same cell. We therefore developed a new class of fluorescent biosensors based on homo-FRET, deemed FLuorescence Anisotropy REporters (FLAREs), which combine the multiplexing ability of single-color sensors with a quantitative, ratiometric readout. Using an array of color variants, we were able to demonstrate multiplexed imaging of three activity reporters simultaneously in the same cell. We further demonstrate the compatibility of FLAREs for use with optogenetic tools as well as intravital two-photon imaging.
