Single-color, ratiometric biosensors for detecting signaling activities in live cells

Brian L Ross,Nicole E Snell,Brian Tenner,Megan A Rizzo,Jin Zhang,Guoli Shi,Joseph R Mauban,Michele L Markwardt,Christopher W Ward,Jennifer J Mcfarland,Adam Zviman,Jaclyn P Kerr

doi:10.7554/elife.35458

Abstract

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.

Highlights

Encoded biosensors have revolutionized the study of cell signaling by allowing the realtime monitoring of signaling activities, such as enzymatic activity or the release of second messengers, in live cells
To create our FLuorescence Anisotropy REporters (FLAREs) probes, we adapted existing Forster Resonance Energy Transfer (FRET)-based biosensors for homo-FRET measurements by replacing the traditional FRET pair with two fluorescent proteins (FPs) of the same color
To test the effect of FP circular permutation on these biosensors, we developed two FLARE-AKAR variants based on the yellow FP mVenus: one in which the C-terminal FP was circularly permuted at position 172, consistent with the hetero-FRET A Kinase Activity Reporter 4 (AKAR4) sensor, and one without circular permutation

Summary

Introduction

Encoded biosensors have revolutionized the study of cell signaling by allowing the realtime monitoring of signaling activities, such as enzymatic activity or the release of second messengers, in live cells. We expressed mVenus-cp172Venus FLARE-AKAR in HEK293T cells and captured a time-course using fluorescence polarization microscopy. We observed a slight positive correlation between intensity and anisotropy change for Venus-cp172Venus FLARE AKAR; the expression level does not significantly impact the reporting ability of these sensors in general (Figure 1—figure supplement 3a).

Results

Conclusion