Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors

Abstract

Cells rely on a complex, interconnected network of signaling pathways to sense and interpret changes in their extracellular environment. The development of genetically encoded fluorescent protein (FP)-based biosensors has made it possible for researchers to directly observe and characterize the spatiotemporal dynamics of these intracellular signaling pathways in living cells. However, detailed information regarding the precise temporal and spatial relationships between intersecting pathways is often lost when individual signaling events are monitored in isolation. As the development of biosensor technology continues to advance, it is becoming increasingly feasible to image multiple FP-based biosensors concurrently, permitting greater insights into the intricate coordination of intracellular signaling networks by enabling parallel monitoring of distinct signaling events within the same cell. In this review, we discuss several strategies for multiplexed imaging of FP-based biosensors, while also underscoring some of the challenges associated with these techniques and highlighting additional avenues that could lead to further improvements in parallel monitoring of intracellular signaling events.

Fig. 1

General designs of FP-based biosensors. (a) Schematic of a translocation-based biosensor reporting on changes in the level of a signaling molecule at the plasma membrane through redistribution of fluorescence from the cytosol (left) to the membrane (right). (b) A single-FP based biosensor featuring a molecular switch inserted into a circularly permuted FP. Upon detection of a specific biochemical event (orange triangle), a conformational change in the molecular switch results in increased fluorescence intensity. (c) A FRET-based biosensor with an engineered molecular switch. As depicted here, the conformational change results in increased FRET

Fig. 2

Representative illustrations of co-imaging strategies using FRET-based biosensors. (a) Spatial separation of spectrally similar biosensors to co-image signaling dynamics in distinct cellular compartments, e.g. plasma membrane and nucleus. (b) Spectrally separated biosensors can be used to co-image multiple signaling events in the same region of the cell. One strategy involves using biosensors with distinct FRET donors and a shared acceptor

Fig. 3

Excitation/emission spectra for various FRET pairs used in co-imaging. (a) CFP and YFP excitation spectra are sufficiently separated, minimizing cross-excitation, while their emission spectra overlap the excitation spectrum of RFP, which serves as a common FRET acceptor. (b) Despite having distinct excitation and emission maxima, the spectra for OFP and RFP overlap significantly, potentially leading to cross-excitation and contamination of the FRET channel, resulting in a decreased signal-to-noise ratio. OFP is also not well separated from YFP, which may also lead to contamination of CY FRET measurements. (c) Although the emission spectrum for mAmetrine is virtually identical to that of Citrine, its long Stokes-shift allows it to serve as the donor for tdTomato, while Citrine serves as the acceptor for mTFP. However, overlap between the excitation spectra of the two donors leads to significant cross-excitation. In all panels, boxes are shown to illustrate the excitation (ex) and emission (em) filters that were used to image these FRET pairs.