Multiplexed visualization of dynamic signaling networks using genetically encoded fluorescent protein-based biosensors
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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.
KeywordsCo-imaging FRET Signal transduction Microscopy Biosensor
We wish to thank members of the Zhang lab for helpful discussions, in particular Fabian Hertel for comments on the manuscript. This work is supported by the National Institutes of Health (R01DK073368 and DP1CA174423 to J.Z.).
Conflict of interest
The authors declare they have no competing financial interests.
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