Biosensor Architectures for High-Fidelity Reporting of Cellular Signaling

Omer Dushek,Annemarie C Lellouch,David J Vaux,Vahid Shahrezaei

doi:10.1016/j.bpj.2014.06.021

Abstract

Understanding mechanisms of information processing in cellular signaling networks requires quantitative measurements of protein activities in living cells. Biosensors are molecular probes that have been developed to directly track the activity of specific signaling proteins and their use is revolutionizing our understanding of signal transduction. The use of biosensors relies on the assumption that their activity is linearly proportional to the activity of the signaling protein they have been engineered to track. We use mechanistic mathematical models of common biosensor architectures (single-chain FRET-based biosensors), which include both intramolecular and intermolecular reactions, to study the validity of the linearity assumption. As a result of the classic mechanism of zero-order ultrasensitivity, we find that biosensor activity can be highly nonlinear so that small changes in signaling protein activity can give rise to large changes in biosensor activity and vice versa. This nonlinearity is abolished in architectures that favor the formation of biosensor oligomers, but oligomeric biosensors produce complicated FRET states. Based on this finding, we show that high-fidelity reporting is possible when a single-chain intermolecular biosensor is used that cannot undergo intramolecular reactions and is restricted to forming dimers. We provide phase diagrams that compare various trade-offs, including observer effects, which further highlight the utility of biosensor architectures that favor intermolecular over intramolecular binding. We discuss challenges in calibrating and constructing biosensors and highlight the utility of mathematical models in designing novel probes for cellular signaling.

Highlights

The major challenge in studying cellular signaling is no longer to identify new signaling proteins but to understand the interaction networks that the identified signaling proteins form
We find that the canonical biosensor architecture, which works primarily through intramolecular reactions, produces biosensor response curves that depend on the biosensor concentration and that can be highly ultrasensitive, and canonical biosensors may be providing highly nonlinear reporting
Biosensors are emerging as important tools for the quantitative study of cellular signaling

Summary

Introduction

The major challenge in studying cellular signaling is no longer to identify new signaling proteins but to understand the interaction networks that the identified signaling proteins form. This is important in various pathologies, such as cancer, where mutations to specific signaling proteins perturb the cellular signaling network, resulting in dysregulated cell proliferation. Fluorescent biosensors, either genetically encodable or synthetic peptide/protein molecular probes, are emerging as important tools for the direct study of signaling protein activity in both healthy and disease cells [1,2,3,4,5]. A widely employed architecture is a single-chain intramolecular Forster

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