Fluorescent biosensors of protein function

Fluorescent biosensors allow researchers to image and quantify protein activity and small molecule signals in living cells with high spatial and temporal resolution. Genetically encoded sensors are coded by a DNA sequence and hence constructed entirely out of amino acids. These biosensors typically utilize light-emitting proteins, such as derivatives of the green fluorescent protein (GFP), and have been developed for a wide range of small molecules and enzyme activities. Fluorescent biosensors can be genetically targeted to distinct locations within cells, such as organelles and membranes. This feature facilitates elucidation of how protein activities and cellular signals are modulated in different regions of the cell. Improvements in the dynamic range and robustness of sensors have enabled high throughput screening for molecules that act as agonists or antagonists of protein function.

Introduction

With the expanding list of completely sequenced genomes, scientists in the ‘post-genomic’ era are charged with identifying the function of each gene product and determining how each acts in time and space to orchestrate cellular functions. The primary challenge is to build a map detailing how proteins interact with each other and with other cellular cues, such as signaling molecules or ions. A complete map of such interactions would illuminate crosstalk between signaling cascades to build an integrated network of how proteins function synergistically and antagonistically to control cellular phenotypes. This systems approach is crucial if we as a scientific community are to convert information gleaned from genome sequencing into information that will be useful for treating human disease. While genomics and proteomics approaches provide insight into protein functions and enable quantification of protein levels under different cellular conditions, they are incapable of addressing when and where a protein is active. Yet protein localization and protein function are intricately linked [1]. Moreover, compartmentalized signaling reactions may lead to different cellular phenotypes. Thus, imaging protein activity and the cellular cues that stimulate activity in living cells can provide an important complement to classical genomic and proteomic approaches. The advantage of genetically encoded fluorescent biosensors is that they enable the researcher to study a specific protein or cellular signal within the complex environment of the living cell. This approach preserves the spatial and temporal control of protein function and signaling cascades. Thus, the researcher can assess when and where a protein is active in a cell; as well as identify how a stimulus influences the dynamics of signaling cues, which in turn dictate protein function.

Genetically encoded sensors are generated by translation of a nucleic acid sequence, and are incorporated into cells, tissues, and whole organisms by transfection of plasmid DNA or transgenic technologies. In order to convert protein activity or ligand-binding into an optical response, these sensors are usually comprised of one or more fluorescent proteins. There are a number of key advantages of genetically encoded sensors. (1) They can be easily incorporated into cells, tissues, and even organisms. Once they are loaded, the sensors permit long-term imaging over days or longer (in the case of stable incorporation). (2) The sensors can be explicitly targeted to defined regions in the cell either by attaching signal sequences or by fusing the sensor to a protein that localizes to a specific domain. This feature is critical for monitoring the spatial heterogeneity of the cellular signals and protein activities being interrogated. (3) The concentration of sensor can be regulated if the sensor is put under control of an inducible promoter (such as the Tet-on or Tet-off promoter). This enables variation of the sensor concentration and critical evaluation of the extent to which the sensor perturbs the cellular environment.

This review focuses on recent developments in genetically encoded fluorescent sensors of cellular signals and protein activities, highlighting the insights provided by localized sensors. We comment on approaches for the design and optimization of fluorescent biosensors, as these ultimately are key to creating robust sensors for use in high-throughput assays. We suggest these sensors can be very useful in screening whether a chemical influences the activity of a specific protein, or a signaling pathway, and as such, can be an important complement to genomics and proteomics.