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.
"The Ca2+ reporter used here, could readily be replaced by any other GFP-based genetically encoded reporter. Such tools exist to probe pH, calcium, ATP, NADH, cAMP, glutamate, reactive oxygen species, several redox potentials, activity of kinases and phosphatases, and many other modalities (Hung et al., 2011; Mehta and Zhang, 2011; Depry et al., 2013; Tantama et al., 2013). "
[Show abstract][Hide abstract] 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 · 3.53 Impact Factor
"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). "
[Show abstract][Hide abstract] ABSTRACT: Corticospinal motor neurons (CSMN) have a unique ability to receive, integrate, translate, and transmit the cerebral cortex's input toward spinal cord targets and therefore act as a "spokesperson" for the initiation and modulation of voluntary movements that require cortical input. CSMN degeneration has an immense impact on motor neuron circuitry and is one of the underlying causes of numerous neurodegenerative diseases, such as primary lateral sclerosis (PLS), hereditary spastic paraplegia (HSP), and amyotrophic lateral sclerosis (ALS). In addition, CSMN death results in long-term paralysis in spinal cord injury patients. Detailed cellular analyses are crucial to gain a better understanding of the pathologies underlying CSMN degeneration. However, visualizing and identifying these vulnerable neuron populations in the complex and heterogeneous environment of the cerebral cortex have proved challenging. Here, we will review recent developments and current applications of novel strategies that reveal the cellular and molecular basis of CSMN health and vulnerability. Such studies hold promise for building long-term effective treatment solutions in the near future.
Frontiers in Neuroanatomy 03/2014; 8:16. DOI:10.3389/fnana.2014.00016 · 3.54 Impact Factor
"Novel strategies present an avenue for multiplex visualization of several enzymes at a time. For example, three-fluorescentprotein FRET (reviewed by Depry et al., 2013) enables simultaneous visualization of two FRET-based biosensors. Likewise, multicolor bimolecular fluorescence complementation (Waadt et al., 2008) may allow characterization of complexes that regulate kinase signaling. "
[Show abstract][Hide abstract] ABSTRACT: Fluorescence-based, genetically encodable biosensors are widely used tools for real-time analysis of biological processes. Over the last few decades, the number of available genetically encodable biosensors and the types of processes they can monitor have increased rapidly. Here, we aim to introduce the reader to general principles and practices in biosensor development and highlight ways in which biosensors can be used to illuminate outstanding questions of biological function. Specifically, we focus on sensors developed for monitoring kinase activity and use them to illustrate some common considerations for biosensor design. We describe several uses to which kinase and second-messenger biosensors have been put, and conclude with considerations for the use of biosensors once they are developed. Overall, as fluorescence-based biosensors continue to diversify and improve, we expect them to continue to be widely used as reliable and fruitful tools for gaining deeper insights into cellular and organismal function.
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