Next-Generation Genetically Encoded Fluorescent Biosensors Illuminate Cell Signaling and Metabolism

Abstract

Genetically encoded fluorescent biosensors have revolutionized the study of cell signaling and metabolism, as they allow for live-cell measurements with high spatiotemporal resolution. This success has spurred the development of tailor-made biosensors that enable the study of dynamic phenomena on different timescales and length scales. In this review, we discuss different approaches to enhancing and developing new biosensors. We summarize the technologies used to gain structural insights into biosensor design and comment on useful screening technologies. Furthermore, we give an overview of different applications where biosensors have led to key advances over recent years. Finally, we give our perspective on where future work is bound to make a large impact.

Keywords: biosensor design; biosensor multiplexing; fluorescence microscopy; hybrid biosensors; in vivo imaging; super-resolution microscopy.

Figure 1:

Schematic of the two most common biosensor designs. (a) FRET-based biosensors consist of two fluorophores (i.e., FRET-donor and FRET-acceptor) fused to a sensing unit. (b) Intensiometric biosensor based on a single fluorophore inserted into a bipartite sensing unit. The two most common topologies are illustrated, though other configurations are possible.

Figure 2:

Naturally occurring and synthetic sensing units. (a) PBPs show a large hinge-like motion upon analyte binding, which is well conserved among different family members. (b) GPCRs, a diverse class of structurally conserved transmembrane receptors, exhibit a conformational change in the 3^rd intracellular loop upon analyte binding. (c) VSDs respond to changes in membrane polarization with a movement of the fourth transmembrane helix. (d) CNBDs are composed of a flexible α-helix and a β-sandwich with a conserved phosphate binding cassette. Binding/dissociation of the cyclic nucleotide triggers rearrangement of the α-helices. (e) Synthetic switch that works as an affinity clamp. (f) Analyte binding leads to dissociation of the two protein domains in mutually exclusive binding designs. Arrows indicated potential insertion sites for reporting units. Alternatively reporting units can also be attached to the termini.

Figure 3:

Crystal structures of reporting units along with chemical structures of the corresponding chromophore/fluorophore. (a) FPs based on matured chromophores, illustrated by mNeonGreen (PDB ID: 5LTR). (b) NIR FPs such as miRFP670nano (PDB ID: 6MGH) are dependent on the cofactor biliverdin. miRFP670nano forms a covalent bond with biliverdin via Cys86. (c) SLP tags such as HaloTag7 (PDB ID: 6Y7A) can be labeled with a variety of different fluorophores. Tetramethylrhodamine is illustrated here. (d) The FAP FAST (PDB ID: 7AVA) non-covalently binds 4-hydroxybenzylidine rhodanines and its derivatives such as N871b, as indicated in the crystal structure.

Figure 4:

Methods used to streamline biosensor engineering. Structural and computational information are used to help rational biosensor engineering and inform targeted screening campaigns. At the same time, high-throughput methods are adapted to help biosensor screening.

Figure 5:

Biosensors contribute to various application areas. (a) Biosensor multiplexing can help to investigate intertwined signaling pathways. Strategies generally rely on spectral, spatial, or temporal information to distinguish the signals from different biosensors. (b-c) Super-resolution compatible biosensors. Reversibly switchable Ca²⁺ biosensor generated from rsEGFP, CaM, and the M13 peptide(90). Only in the Ca²⁺ bound state can the biosensor be photo-switched using blue and UV light. The biosensor was paired with RESOLFT imaging to generate super-resolved maps of Ca²⁺ in the endoplasmic reticulum(90) (b). FLINC kinase activity biosensor for imaging via SOFI (92). The proximity of Dronpa to TagRFP-T induces fluorescence fluctuations in the emission of the latter (multiple circles) (c). (d) Biosensors compatible with far-red or NIR excitation help to perform in vivo imaging as the depth penetration of red-shifted light is greater. (e) In vivo imaging using 2P excitation offers increased penetration depth due to longer-wavelength illumination, while the smaller excitation area produced in 2P illumination helps reject out of focus light and obtain higher signal-to-background ratio.