Article

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

Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Pflügers Archiv - European Journal of Physiology (Impact Factor: 4.1). 11/2012; 465(3). DOI: 10.1007/s00424-012-1175-y
Source: PubMed

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.

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    • "Type, density, and timing of labeling can be controlled by tissue-specific expression of the recombinase. Genetically encoded fluorescent protein-based biosensors can be used to study a broad assortment of signaling molecules and networks (Depry et al., 2013). For example, fluorescent proteins can be used to monitor protein-protein interactions in living cells using fluorescent resonance energy transfer (FRET) microscopy (Stepanenko et al., 2011; Day and Davidson, 2012). "
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    • "Genetically encoded probes have become powerful tools for fluorescent analysis of the function and concentration of multiple intracellular ions and proteins (Bregestovski and Arosio, 2012; Depry et al., 2013; Perron et al., 2012). GFP derivatives with different colors have been successfully used to monitor Ca 2+ (Miyawaki et al., 1997; Ohkura et al., 2012), pH (Kneen et al., 1998; Llopis et al., 1998; Miesenbock et al., 1998; Li and Tsien, 2012) and protein–protein interactions (Heim, 1999). "
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