Genetically encoded fluorescent biosensors for live-cell visualization of protein phosphorylation

Abstract

Fluorescence-based, genetically encodable biosensors are widely used tools for real-time analysis of biological processes. Over the last few decades, the number of available genetically encodable biosensors and the types of processes they can monitor have increased rapidly. Here, we aim to introduce the reader to general principles and practices in biosensor development and highlight ways in which biosensors can be used to illuminate outstanding questions of biological function. Specifically, we focus on sensors developed for monitoring kinase activity and use them to illustrate some common considerations for biosensor design. We describe several uses to which kinase and second-messenger biosensors have been put, and conclude with considerations for the use of biosensors once they are developed. Overall, as fluorescence-based biosensors continue to diversify and improve, we expect them to continue to be widely used as reliable and fruitful tools for gaining deeper insights into cellular and organismal function.

Figure 1

The pipeline for kinase activity reporter development. (A) Design. The majority of two-fluorescent-protein reporter blueprints position the fluorescent protein pair around a molecular switch, either endogenous or engineered. (B) Development is typically an iterative process. (C ) Initial characterization of a reporter usually involves measuring its response to a known stimulus of the signal of interest; here, “a” represents an earlier version of a sensor with lower dynamic range, whereas “b” represents activity of an optimized version. (D) Optimization of a reporter may involve changing the fluorescent proteins, linker, or length/identity of the specific protein components that recognize the signal. After such optimization, new versions of a probe are compared to earlier versions. (E) Application of a probe, in cell lines (typically to investigate kinase activity and spatiotemporal regulation, or crosstalk with other pathways); primary cells (in order to explore kinase involvement in cell-type-specific behaviors); and live animals (to explore the Involvement of a kinase in specific processes or pathologies in vivo) can be both an end goal of probe development, and a spur to further optimization.

Figure 2

Visualizing a protein's conformational change using an engineered polypeptide sequence. (A) Sensing unit: a molecular switch, usually based on endogenous sequence of a kinase or its substrate. On/off “switch” may occur endogenously, as in a conformational change or a binding event; or be engineered as in a unimolecular adaptation of a binding event or a pseudoligand displacement probe.” (B) The reporter unit generates optical readout, either by single-color fluorescence change, or by a change in resonance energy transfer.