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.
SourceAvailable from: Saumya Saurabh
Chapter: Targeting Dyes for BiologyCell Membrane Nanodomains: From Biochemistry to Nanoscopy, Edited by Alessandra Cambi, Diane S. Lidke, 10/2014: chapter 15: pages 341; CRC Press.
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ABSTRACT: The cardiac action potential (AP) and the consequent cytosolic Ca(2+) transient are key indicators of cardiac function. Natural developmental processes, as well as many drugs and pathologies change the waveform, propagation, or variability (between cells or over time) of these parameters. Here we apply a genetically encoded dual-function calcium and voltage reporter (CaViar) to study the development of the zebrafish heart in vivo between 1.5 and 4 days post fertilization (dpf). We developed a high-sensitivity spinning disk confocal microscope and associated software for simultaneous three-dimensional optical mapping of voltage and calcium. We produced a transgenic zebrafish line expressing CaViar under control of the heart-specific cmlc2 promoter, and applied ion channel blockers at a series of developmental stages to map the maturation of the action potential in vivo. Early in development, the AP initiated via a calcium current through L-type calcium channels. Between 90 and 102 h post fertilization (hpf), the ventricular AP switched to a sodium-driven upswing, while the atrial AP remained calcium driven. In the adult zebrafish heart, a sodium current drives the AP in both the atrium and ventricle. Simultaneous voltage and calcium imaging with genetically encoded reporters provides a new approach for monitoring cardiac development, and the effects of drugs on cardiac function.Frontiers in Physiology 09/2014; 5:344. DOI:10.3389/fphys.2014.00344
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ABSTRACT: Protein-based fluorescent biosensors have emerged as key bio-analytical tools to visualize and quantify a wide range of biological substances and events in vitro, in cells, and even in vivo. On the basis of the construction method, the protein-based fluorescent biosensors can be principally classified into two classes: 1) genetically encoded fluorescent biosensors harnessing fluorescent proteins (FPs), and 2) semisynthetic biosensors comprised of protein scaffolds and synthetic fluorophores. Recent advances in protein engineering and chemical biology not only allowed the further optimization of conventional biosensors, but also facilitated the creation of novel biosensors based on unique strategies. In this review, we survey the recent studies in the development and improvement of protein-based fluorescent biosensors, and highlight the successful applications to live cell and in vivo imaging. Furthermore, we provide perspectives on possible future directions of the technique.ACS Chemical Biology 10/2014; 9(12). DOI:10.1021/cb500661v · 5.36 Impact Factor